Efficient green preparation method of hydrazide polypeptide and application thereof

Abstract

The application provides a green and efficient preparation method and application of hydrazide polypeptides, and belongs to the technical field of polypeptide preparation and biological medicine. In view of the safety risk and environmental pollution problems caused by the fact that DMF is widely used as a solid-phase polypeptide synthesis solvent in the current polypeptide industry, a kind of efficient green preparation method of hydrazide polypeptides is designed and developed by replacing DMF with a green solvent. The green solvent is successfully applied to the construction of hydrazine resin and solid-phase polypeptide synthesis, two key links, so that hydrazide polypeptide products with the same quality as DMF system can be obtained, the health hazards and environmental pollution problems of DMF are effectively avoided, and the application value is good.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and disease treatment technology, specifically relating to an efficient and green preparation method and application of acylhydrazide peptides. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Peptides are crucial in the biopharmaceutical field, representing a class of medium-molecular-weight drugs with broad application prospects. Compared to small-molecule drugs, peptides offer superior targeting, stronger receptor affinity, and higher biocompatibility. Furthermore, compared to large biomolecules like antibodies, they possess advantages such as better tissue penetration, lower immunogenicity, ease of synthesis and modification, and lower production costs. Currently, there are over 100 marketed peptide drugs globally, with over 500 peptide drug candidates in clinical trials. Peptide drugs broadly cover major disease areas such as diabetes, malignant tumors, cardiovascular diseases, metabolic diseases, and bacterial infections, and the global market size exceeded $40 billion in 2024.

[0004] Solid-phase peptide synthesis (SPPS) is a common method for preparing peptides and their analogues. This technique was developed by Professor Merrifield in 1963. Compared to traditional liquid-phase synthesis, it reduces the workload by approximately 50 times when synthesizing the same target peptide. For his contributions to peptide synthesis, Merrifield was awarded the 1984 Nobel Prize in Chemistry. Currently, SPPS is widely used in the synthesis of peptides, peptide analogues, and proteins. Furthermore, hydrazide groups, as core linkage groups for peptide fragments, play a crucial role in protein chemical synthesis. C-terminal hydrazideation of the peptide backbone is an important structural modification that can prevent protease degradation, extend the peptide's half-life, and improve its biological activity. In addition, C-terminal hydrazideation can undergo various chemical modifications and linkage reactions, endowing peptides with a structurally editable coupling unit. Therefore, C-terminal hydrazideation not only improves peptide stability but also gives it the potential to be further spliced ​​into long-chain peptides or proteins. Currently, SPPS remains the most common method for obtaining hydrazidelated peptides.

[0005] Amide condensing agents, catalysts, and solvents are key factors in solid-phase peptide synthesis. Currently, DMF (N,N-dimethylformamide) is almost universally used as a solvent for solid-phase peptide synthesis in both laboratory and industrial production. Furthermore, the construction of hydrazine resins and the preparation of hydrazide-modified peptides also require DMF as a solvent for solid-phase peptide synthesis. However, while DMF is highly efficient in peptide synthesis, it also pollutes the environment and poses health risks to laboratory operators. To date, the use of DMF has been regulated and restricted by the European Chemicals Agency (ECHA) and the European Union. Therefore, developing green solvents that can replace DMF for peptide and hydrazide-modified peptide synthesis is of significant application value in order to reduce environmental pollution and minimize health risks to laboratory operators. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention develops an efficient and green method for the preparation of hydrazide-modified peptides and their applications. This invention uses the green solvent GVL as the solvent for the amino acid condensation process in hydrazine resin construction and peptide synthesis, and the green solvent TEP as the solvent for the removal of the 9-fluorenemethoxycarbonyl (Fmoc) protecting group in peptide synthesis. Using an N,N'-diisopropylcarbodiimide (DIC) / ethyl 2-oxime cyanoacetate (Oxyma) condensation system, a series of C-terminal hydrazide-modified antitumor peptides were efficiently synthesized in an air bath at 50°C. Compared with using DMF as a solvent, the hydrazide-modified peptides synthesized in this invention exhibit comparable chromatographic purity and yield, as well as tumor cell proliferation inhibition ability.

[0007] In a first aspect, the present invention provides a method for synthesizing the aforementioned polypeptide, the method comprising synthesizing the polypeptide using a solid-phase polypeptide synthesis method; specifically, the aforementioned polypeptide is synthesized using a 9-fluorenemethoxycarbonyl solid-phase polypeptide synthesis method (Fmoc-SPPS). To achieve the above-mentioned technical objective, the technical solution adopted by the present invention is as follows:

[0008] An efficient and green method for preparing acylhydrazide-modified peptides includes the following steps:

[0009] 1) Weigh a certain amount of 2-Cl-(Trt)-Cl resin (1 equivalent, molar ratio) into a polypeptide synthesis tube, wash once with standard (wash twice with GVL, twice with DCM, twice with GVL), and dry under vacuum; add 3 mL of GVL, soak and swell in an air bath at 28℃ for 2 hours, wash once with standard, add 3 mL of GVL / DCM (dichloromethane) (v / v = 1:1) mixed solution, activate the resin in an air bath at 28℃ for 30 min, wash once with standard, and dry under vacuum for later use;

[0010] 2) Add a 10% hydrazine hydrate GVL solution (v / v), react at 28°C in an air bath constant temperature shaker for 30 min, and wash three times with GVL; add a 10% hydrazine hydrate GVL solution (v / v), react at 28°C in an air bath constant temperature shaker for 30 min, and wash three times with GVL; add a 10% methanol GVL solution (v / v), react at 28°C in an air bath constant temperature shaker for 5 min, and wash three times with GVL; add a 10% methanol GVL solution (v / v), react at 28°C in an air bath constant temperature shaker for 10 min, wash once with standard, and dry for later use;

[0011] 3) The condensation system used for amino acids other than arginine, histidine, and serine was a 3 mL GVL solution containing Fmoc-amino acids (3 equivalents, molar ratio), Oxyma (3 equivalents, molar ratio), and DIC (6 equivalents, molar ratio). The Fmoc-amino acids used included both L-configuration and D-configuration amino acids. The first amino acid was condensed three times under isothermal shaking conditions in a 50°C air bath: the first reaction for 15 min, the second for 20 min, and the third for 25 min. Other amino acids were condensed twice under isothermal shaking conditions in a 50°C air bath: the first reaction for 15 min and the second for 20 min. Then, under constant temperature oscillation in an air bath at 50°C, the Fmoc protecting group was removed using a 20% piperidine TEP solution. The first reaction lasted 3 min, and the second reaction lasted 5 min. After all amino acid condensation and protecting group removal, the resin color development was detected under heating conditions using Kaiser reagent (solution A: 80% phenol in ethanol solution (w / v); solution B: 5% ninhydrin in ethanol solution (w / v)).

[0012] 4) The condensation system used for Fmoc-protected arginine, histidine, and serine contained 3 mL of GVL solution of Fmoc-amino acids (3 equivalents, molar ratio), Oxyma (3 equivalents, molar ratio), and DIC (6 equivalents, molar ratio). Condensation was performed under isothermal shaking in an air bath at 28°C for 60 min for the first reaction and 90 min for the second. Then, under isothermal shaking in an air bath at 28°C, the Fmoc protecting groups were removed using a 20% piperidine TEP solution for 5 min for the first reaction and 10 min for the second reaction.

[0013] 5) Peptide cleavage: After one standard wash, remove the Fmoc protecting group using a 20% piperidine TEP solution under constant temperature shaking conditions in an air bath at 28°C. The first reaction is 5 min, followed by three TEP washes. Then, add another 20% piperidine TEP solution and react for 10 min, followed by one standard wash. Take a small amount of resin and test the color development with Kaiser reagent under heating conditions. Rinse the resin 5 times with DCM, vacuum with a water pump for 5 min, vacuum with an oil pump for 5 min, and add a peptide cleavage reagent (trifluoroacetic acid / phenol / water / anisole / 1,2-ethylenedithiol, 85:2.5:5:5:2.5, v / v / v / v / v) that has been pre-cooled to below 5°C in an ice-salt bath. React in the dark for 2.5 hours in a constant temperature shaker in an air bath at 28°C.

[0014] 6) Peptide precipitation: Concentrate the peptide cleavage reagent to 2-3 mL using a high-purity nitrogen bubbling concentration method, precipitate with pre-cooled diethyl ether, centrifuge at 3000 r / min for 3 min, discard the supernatant and retain the precipitate; resuspend the precipitate in pre-cooled diethyl ether by sonication, centrifuge, discard the supernatant, and repeat this step three times; let the peptide precipitate stand in a fume hood until dry.

[0015] 7) Separation and Purification: The crude peptide product was dissolved in an acetonitrile-water mixture containing 0.1% TFA, and then freeze-dried at low temperature to remove all residual solvent. The crude peptide product was then dissolved again in an acetonitrile-water mixture containing 0.1% TFA, and purified using semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The purified product was then freeze-dried at low temperature to obtain the target hydrazide-substituted peptide sample. The purity, molecular weight, and amino acid sequence of the crude target hydrazide-substituted peptide product were analyzed using analytical reversed-phase HPLC and high-resolution mass spectrometry (ESI-MS). The crude peptide product was purified using semi-preparative reversed-phase HPLC.

[0016] The GVL mentioned is γ-valerolactone (GVL).

[0017] The TEP mentioned is triethyl phosphate (abbreviated as TEP).

[0018] The method for preparing the 10% hydrazine hydrate GVL solution is as follows: take 471 μL of 85% hydrazine hydrate and add GVL to make up to 4 mL.

[0019] The method for preparing the 10% methanol GVL solution is as follows: take 400 μL of methanol and add GVL to make up to 4 mL.

[0020] The standard wash consists of two GVL washes, two DCM washes, and two GVL washes.

[0021] A second aspect of the present invention provides a class of acylhydrazide-modified polypeptides, said polypeptides comprising the following amino acid residue sequence:

[0022] MCQ-1 H-KKWWKKW(Dip)K-NHNH2

[0023] MCQ-2 H-kkwwkkw(dip)k-NHNH2

[0024] MCQ-3 H-GIGAVLKVLTTGLPALISWIKRKRQQ-NHNH2

[0025] MCQ-4 H-LAVISWKCQEWNSLWKKRKRKT-NHNH2

[0026] MCQ-5 H-KGWFKAMKSIAKFIAKEKLKEHL-NHNH2

[0027] The amino acids in the example peptide MCQ-1 structure are L-configured.

[0028] The amino acids in the example peptide MCQ-2 structure are in the D configuration.

[0029] The amino acids in the example peptide MCQ-3 structure are L-configured.

[0030] The amino acids in the example peptide MCQ-4 are L-configured.

[0031] The amino acids in the example peptide MCQ-5 structure are L-configured.

[0032] The Dip mentioned is L-3,3-diphenylalanine.

[0033] The dip mentioned is D-3,3-diphenylalanine.

[0034] The acylhydrazide peptides MCQ-1, MCQ-2, MCQ-3, MCQ-4, and MCQ-5 interfere with the normal physiological activities of tumor cells by damaging the cell membrane and mitochondrial membrane structure, causing tumor cell death. Tumor cells are less likely to develop drug resistance.

[0035] A third aspect of the present invention provides the use of the above-described acylhydrazide polypeptide in the preparation of polypeptide drugs for the prevention or treatment of tumor-related diseases.

[0036] It should also be noted that the term "tumor" is used in this invention as is known to those skilled in the art, and includes benign tumors and / or malignant tumors. A benign tumor is defined as the excessive proliferation of cells that cannot form an invasive, metastatic tumor in the body. Conversely, a malignant tumor is defined as cells with various cellular and biochemical abnormalities that can cause systemic disease (e.g., tumor metastasis in distant organs).

[0037] A fourth aspect of the present invention provides a method for preventing or treating tumors, the method comprising administering to a subject a therapeutically effective dose of the aforementioned polypeptide.

[0038] A fifth aspect of the invention provides the use of the above-mentioned polypeptide as a non-therapeutic tumor cell proliferation inhibitor. According to the invention, "non-therapeutic" means inhibiting tumor cell proliferation and promoting tumor cell death in vitro. Applying the polypeptide of the invention to tumor cells (such as HeLa cervical cancer cells and 4T1 breast cancer cells) facilitates the study of tumor growth-related signaling pathways and gene expression interactions, thereby providing original materials and laying the foundation for further research on tumor-related diseases.

[0039] A sixth aspect of the present invention provides a method for inhibiting tumor cell proliferation in vitro, the method comprising treating in vitro cultured tumor cells with the aforementioned polypeptide.

[0040] The beneficial technical effects of the above technical solution are as follows:

[0041] 1. Enhanced safety. DMF is widely used in solid-phase peptide synthesis; however, DMF poisoning can cause serious acute and chronic diseases, and it is difficult to degrade naturally in the environment, causing long-term pollution. The green solvent described in this invention can effectively replace DMF in the construction of hydrazine resins and in solid-phase peptide synthesis, effectively reducing the threats to the safety of experimental personnel and environmental pollution during peptide synthesis.

[0042] 2. High efficiency in hydrazine resin preparation. Hydrazine resins are generally prepared using DMF as a solvent. The green solvent method developed in this invention can effectively construct hydrazine resins and ultimately synthesize long-chain and short-chain acylhydrazide peptides with good purity comparable to that of DMF synthesis.

[0043] 3. High efficiency in solid-phase peptide synthesis. The method of this invention utilizes GVL as the solvent for the construction of hydrazine resins and the amino acid condensation process in peptide synthesis, and TEP as the solvent for removing Fmoc during peptide synthesis. The acylhydrazide peptides synthesized using this method exhibit good chromatographic purity, even showing a significant purity advantage over DMF in the synthesis of long-chain acylhydrazide peptides.

[0044] 4. Highly effective tumor cell proliferation inhibitory ability. The acylhydrazide peptides in this invention damage the normal physiological structure of tumor cells by disrupting the cell membrane and mitochondrial membrane, inducing cell death. Tumor cells are less likely to develop drug resistance. Specifically, each peptide exhibits an IC50 (increase / decrease) against HeLa cervical cancer cells. 50 The effective values ​​were 29.63±1.10 µM (MCQ-1, DMF), 25.56±0.44 µM (MCQ-1, this invention), 20.27±3.11 µM (MCQ-2, DMF), 21.26±3.60 µM (MCQ-2, this invention), 0.25±0.01 µM (MCQ-3, DMF), 0.20±0.06 µM (MCQ-3, this invention), 16.65±1.72 µM (MCQ-4, DMF), 16.94±1.27 µM (MCQ-4, this invention), 23.15±2.52 µM (MCQ-5, DMF), and 21.64±1.96 µM (MCQ-5, this invention). The IC50 values ​​of each peptide against 4T1 breast cancer cells were: 29.63±1.10 µM (MCQ-1, DMF), 25.56±0.44 µM (MCQ-1, this invention), 20.27±3.11 µM (MCQ-2, DMF), 21.26±3.60 µM (MCQ-2, this invention), 0.25±0.01 µM (MCQ-3, DMF), 0.20±0.06 µM (MCQ-3, this invention), 16.65±1.72 µM (MCQ-4, DMF), 16.94±1.27 µM (MCQ-4, this invention), 23.15±2.52 µM (MCQ-5, DMF), and 21.64±1.96 µM (MCQ-5, this invention). 50 The values ​​were 30.00±1.79 µM (MCQ-1, DMF), 25.36±0.66 µM (MCQ-1, this invention), 27.83±2.91 µM (MCQ-2, DMF), 24.59±0.91 µM (MCQ-2, this invention), 2.46±0.18 µM (MCQ-3, DMF), 2.80±0.61 µM (MCQ-3, this invention), 20.37±1.22 µM (MCQ-4, DMF), 21.81±1.08 µM (MCQ-4, this invention), 26.40±0.82 µM (MCQ-5, DMF), and 26.36±1.72 µM (MCQ-5, this invention).

[0045] In summary, the above technical solutions can effectively replace DMF in the amino acid condensation process of hydrazine resin construction and solid-phase peptide synthesis using the green solvent GVL, and can replace DMF in the removal of Fmoc protecting groups in solid-phase peptide synthesis using the green solvent TEP. The method of this invention has significant advantages in the efficient construction of hydrazine resins and its efficient application in solid-phase peptide synthesis. The chromatographic purity of the crude acylhydrazide peptides synthesized by this method is comparable to that synthesized by DMF, and even exhibits a significant purity advantage in the synthesis of long-chain acylhydrazide peptides. The acylhydrazide peptides synthesized by this invention have a highly efficient ability to inhibit tumor cell proliferation, with activity similar to that of corresponding peptides synthesized by classical DMF. Therefore, it has good prospects for practical application. Attached Figure Description

[0046] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0047] Figure 1 This is a schematic diagram of the solid-phase polypeptide synthesis method of the present invention.

[0048] Figure 2 The present invention provides the structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of MCQ-1.

[0049] Figure 3 The present invention provides the structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of MCQ-2.

[0050] Figure 4 The present invention provides the structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of MCQ-3.

[0051] Figure 5 The present invention provides the structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of MCQ-4.

[0052] Figure 6 The present invention provides the structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of MCQ-5.

[0053] Figure 7 A comparison of high-performance liquid chromatograms of the example peptide MCQ-1 synthesized using the method of the present invention and synthesized using DMF.

[0054] Figure 8 A comparison of high-performance liquid chromatograms of the example peptide MCQ-2 synthesized using the method of the present invention and synthesized using DMF.

[0055] Figure 9 A comparison of high-performance liquid chromatograms of the example peptide MCQ-3 synthesized using the method of the present invention and synthesized using DMF.

[0056] Figure 10 A comparison of high-performance liquid chromatograms of the example peptide MCQ-4 synthesized using the method of the present invention and synthesized using DMF.

[0057] Figure 11 A comparison of high-performance liquid chromatograms of the example peptide MCQ-5 synthesized using the method of the present invention and synthesized using DMF.

[0058] Figure 12Circular dichroism chromatograms of acylhydrazide peptides MCQ-1, MCQ-2, MCQ-3, MCQ-4, and MCQ-5 synthesized using the method of the present invention and using DMF.

[0059] Figure 13 The inhibitory effects of DMF and the acylhydrazide peptides MCQ-1, MCQ-2, MCQ-3, MCQ-4 and MCQ-5 synthesized using the method of the present invention on the proliferation of tumor cells 4T1 and HeLa cells. A) Inhibitory effect of DMF and MCQ-1 and MCQ-2 synthesized using the method of this invention on the proliferation of 4T1 cells; B) Inhibitory effect of DMF and MCQ-3 synthesized using the method of this invention on the proliferation of 4T1 cells; C) Inhibitory effect of DMF and MCQ-4 and MCQ-5 synthesized using the method of this invention on the proliferation of 4T1 cells; D) Inhibitory effect of DMF and MCQ-1 and MCQ-2 synthesized using the method of this invention on the proliferation of HeLa cells; E) Inhibitory effect of DMF and MCQ-3 synthesized using the method of this invention on the proliferation of HeLa cells; F) Inhibitory effect of DMF and MCQ-4 and MCQ-5 synthesized using the method of this invention on the proliferation of HeLa cells; G) Inhibitory effect of DMF and MCQ-1, MCQ-2, MCQ-3, MCQ-4 and MCQ-5 synthesized using the method of this invention on the IC50 of 4T1 cells and HeLa cells. 50 (μM) value. Detailed Implementation

[0060] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and specific examples. In the specific embodiments, all original reagents and raw materials are commercially available. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0061] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0062] In one specific embodiment of the present invention, a method for synthesizing the above-mentioned hydrazide-based polypeptides is provided. In this embodiment, all hydrazide-based polypeptides are prepared using Fmoc-SPPS. Unless otherwise specified, all amino acids used in the Fmoc-SPPS process are Fmoc-protected L- or D-type amino acids. Resin selection: The preparation of hydrazide resin requires a 2-Cl-(Trt)-Cl resin base; therefore, 2-Cl-(Trt)-Cl resin is selected as the synthesis support. The scale of the synthesized polypeptide is generally 0.2 mmol. Figure 1 As shown.

[0063] The present invention will now be described in further detail with reference to the accompanying drawings.

[0064] Weigh a certain amount of 2-Cl-(Trt)-Cl resin (1 equivalent, molar ratio) into a polypeptide synthesis tube, wash once with standard (wash twice with GVL, twice with DCM, and twice with GVL), and dry under vacuum; add 3 mL of GVL, soak and swell in an air bath at 28℃ for 2 hours, wash once with standard, add 3 mL of GVL / DCM (v / v = 1:1) mixed solution, activate the resin in an air bath at 28℃ for 30 min, wash once with standard, and dry under vacuum for later use.

[0065] Add 10% hydrazine hydrate GVL solution (v / v), react at 28°C in an air bath constant temperature shaker for 30 min, and wash three times with GVL; add 10% hydrazine hydrate GVL solution (v / v), react at 28°C in an air bath constant temperature shaker for 30 min, and wash three times with GVL; add 10% methanol GVL solution (v / v), react at 28°C in an air bath constant temperature shaker for 5 min, and wash three times with GVL; add 10% methanol GVL solution (v / v), react at 28°C in an air bath constant temperature shaker for 10 min, wash once with standard, and dry for later use.

[0066] The condensation system used for amino acids other than arginine, histidine, and serine was a 3 mL GVL solution containing Fmoc-amino acids (3 equivalents, molar ratio), Oxyma (3 equivalents, molar ratio), and DIC (6 equivalents, molar ratio). The first amino acid was condensed three times under isothermal shaking in an air bath at 50°C: the first reaction was 15 min, the second 20 min, and the third 25 min. Subsequent amino acids were condensed twice under isothermal shaking in an air bath at 50°C: the first reaction was 15 min, and the second 20 min. Then, under isothermal shaking in an air bath at 50°C, the Fmoc protecting group was removed using a 20% piperidine TEP solution: the first reaction was 3 min, and the second was 5 min. After all amino acid condensation and after the protecting group removal, the resin color development was detected under heating conditions using Kaiser's reagent (solution A: 80% phenol in ethanol (w / v); solution B: 5% ninhydrin in ethanol (w / v)).

[0067] Arginine, histidine, and serine were condensed at 28°C. The condensation system used for Fmoc-protected arginine and serine contained 3 mL of GVL solution of Fmoc-amino acids (3 equivalents, molar ratio), Oxyma (3 equivalents, molar ratio), and DIC (6 equivalents, molar ratio). Condensation was carried out under isothermal shaking at 28°C for 60 min for the first reaction and 90 min for the second. Then, under isothermal shaking at 28°C, the Fmoc protecting group was removed using a 20% piperidine TEP solution (v / v) for 5 min for the first reaction and 10 min for the second reaction.

[0068] Peptide cleavage: After one standard wash, remove the Fmoc protecting group using a 20% piperidine TEP solution (v / v) under constant temperature shaking conditions in an air bath at 28°C. The first reaction is 5 min, followed by three TEP washes. Then, add another 20% piperidine TEP solution (v / v) and react for 10 min. After one standard wash, take a small amount of resin and test the color development using Kaiser reagent (containing an ethanol solution (w / v) of 80% phenol and an ethanol solution (w / v) of 5% ninhydrin) under heating conditions. Rinse the resin 5 times with DCM, vacuum with a water pump for 5 min, vacuum with an oil pump for 5 min, add pre-cooled peptide cleavage reagent (trifluoroacetic acid / phenol / water / anisole / 1,2-ethylenedithiol, 85:2.5:5:5:2.5, v / v / v / v / v), and react in a constant temperature shaker in an air bath at 28°C in the dark for 2.5 hours.

[0069] Peptide precipitation: The peptide cleavage reagent was concentrated to 2-3 mL using a high-purity nitrogen bubbling concentration method. Precipitate with pre-cooled diethyl ether, centrifuge at 3000 r / min for 3 min, discard the supernatant, and retain the precipitate. Resuspend the precipitate in ice-cold diethyl ether by sonication, centrifuge, and discard the supernatant. Repeat this step three times. The crude peptide product precipitate was allowed to stand in a fume hood until dry.

[0070] Separation and purification: The crude peptide product was dissolved in an acetonitrile-water mixture containing 0.1% TFA, and the residual solvent was removed by low-temperature freeze drying. The crude peptide product was then dissolved again in an acetonitrile-water mixture containing 0.1% TFA, and purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC), followed by low-temperature freeze drying to obtain the target acylhydrazide peptide sample. The purity, molecular weight, and amino acid sequence of the target acylhydrazide peptide sample were analyzed using analytical reversed-phase HPLC and high-resolution mass spectrometry (HPLC-MS).

[0071] In another typical embodiment of the present invention, a class of acylhydrazine-modified polypeptides is provided, wherein the acylhydrazine-modified polypeptides comprise the following amino acid residue sequence:

[0072] MCQ-1 H-KKWWKKW(Dip)K-NHNH2

[0073] MCQ-2 H-kkwwkkw(dip)k-NHNH2

[0074] MCQ-3 H-GIGAVLKVLTTGLPALISWIKRKRQQ-NHNH2

[0075] MCQ-4 H-KGWFKAMKSIAKFIAKEKLKEHL-NHNH2

[0076] MCQ-5 H-LAVISWKCQEWNSLWKKRKRKT-NHNH2

[0077] The aforementioned acylhydrazide-modified peptides can interfere with the normal physiological activities of tumor cells and cause tumor cell death by damaging the cell membrane and mitochondrial membrane structure. Tumor cells are less likely to develop drug resistance.

[0078] In another specific embodiment of the present invention, the use of the above-mentioned acylhydrazine-modified polypeptide in the preparation of polypeptide drugs for the prevention or treatment of tumor-related diseases is provided.

[0079] It should also be noted that the term "tumor" is used in this invention as is known to those skilled in the art, and includes benign tumors and / or malignant tumors. A benign tumor is defined as the excessive proliferation of cells that cannot form an invasive, metastatic tumor in the body. Conversely, a malignant tumor is defined as cells with various cellular and biochemical abnormalities that can cause systemic disease (e.g., tumor metastasis in distant organs).

[0080] In another specific embodiment of the present invention, the polypeptide of the present invention can be used to treat malignant tumors. Examples of malignant tumors that can be treated with the polypeptide of the present invention include solid tumors. Solid tumors can be tumors of the breast, bladder, bone, brain, colon, endometrium, germ cells, head and neck, liver, lung, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, testis, stomach, skin (such as melanoma), vagina, and vulva. Malignant tumors include hereditary cancers such as retinoblastoma and nephroblastoma. Furthermore, malignant tumors include primary tumors in the said organs and corresponding secondary tumors (tumor metastases) in distant organs.

[0081] In another specific embodiment of the present invention, a method for preventing or treating tumors is provided, the method comprising administering a therapeutically effective dose of the aforementioned polypeptide to a subject. The subject refers to an animal, preferably a mammal, and most preferably a human, that is already the subject of treatment, observation, or experimentation. The "therapeutically effective dose" refers to an amount of active compound or agent, including the compound of the present invention, that can elicit a biological or medical response in an tissue system, animal, or human sought by the researcher, veterinarian, physician, or other medical professional, including the reduction or partial reduction of symptoms of the treated disease, syndrome, symptom, or disorder. It must be recognized that the optimal dosage and interval of the polypeptide of the present invention are determined by its properties and external conditions such as the form, route, and site of administration, and the specific mammal being treated, and this optimal dosage can be determined using conventional techniques. It must also be recognized that the optimal course of treatment, i.e., the daily dose of the compound over a specified period, can be determined using methods known in the art.

[0082] In another specific embodiment of the present invention, the above-mentioned polypeptide is provided for use as a non-therapeutic tumor cell proliferation inhibitor. According to the present invention, "non-therapeutic" refers to inhibiting tumor cell proliferation and promoting tumor cell death in vitro. Applying the polypeptide of the present invention to tumor cells (such as HeLa cervical cancer cells and 4T1 breast cancer cells) facilitates the study of tumor growth-related signaling pathways and gene expression interactions, thereby providing original materials and laying the foundation for further research on tumor-related diseases.

[0083] In another specific embodiment of the present invention, a method for inhibiting tumor cell proliferation in vitro is provided, the method comprising treating tumor cells cultured in vitro with the aforementioned polypeptide.

[0084] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0085] Example 1

[0086] In this embodiment, all hydrazide-modified peptides were prepared using Fmoc-SPPS. Unless otherwise specified, all amino acids used in the Fmoc-SPPS process were Fmoc-protected L- or D-amino acids. Resin selection: The preparation of hydrazide resins requires a 2-Cl-(Trt)-Cl resin base; therefore, 2-Cl-(Trt)-Cl resin was chosen as the synthetic support. The scale of synthesized peptides was generally 0.2 mmol. Figure 1 As shown.

[0087] The synthesized target peptides were analyzed and identified using analytical reversed-phase high-performance liquid chromatography and high-resolution mass spectrometry, such as... Figure 2-6As shown. The chromatographic conditions for MCQ-1 were: Grace Vydac “Protein C18”, 250 × 4.6 mm, 5 μm particle size; first, 15% acetonitrile (containing 0.1% TFA) for 2 min, then 15%-55% acetonitrile (containing 0.1% TFA) for 30 min; flow rate: 1.0 mL / min; λ = 214 nm. Theoretical molecular weight: 1454.84; actual determined molecular weight: 1453.87. The chromatographic conditions for MCQ-2 were: Grace Vydac “Protein C18”, 250 × 4.6 mm, 5 μm particle size; first, 15% acetonitrile (containing 0.1% TFA) for 2 min, then 15%-55% acetonitrile (containing 0.1% TFA) for 30 min; flow rate: 1.0 mL / min; λ = 214 nm. Theoretical molecular weight: 1454.84, actual measured molecular weight: 1453.87. Chromatographic analysis conditions for MCQ-3: Grace Vydac "Protein C18", 250 × 4.6 mm, 5 μm particle size, first 15% acetonitrile (containing 0.1% TFA) for 2 min, then 15%-65% acetonitrile (containing 0.1% TFA) for 30 min, flow rate 1.0 mL / min, λ = 214 nm. Theoretical molecular weight: 2861.53, actual measured molecular weight: 2860.80. Chromatographic analysis conditions for MCQ-4: Grace Vydac "Protein C18", 250 × 4.6 mm, 5 μm particle size, first 15% acetonitrile (containing 0.1% TFA) for 2 min, then 15%-55% acetonitrile (containing 0.1% TFA) for 30 min, flow rate 1.0 mL / min, λ = 214 nm. Theoretical molecular weight: 2746.41, actual measured molecular weight: 2745.66. Chromatographic analysis conditions for MCQ-5: Grace Vydac “Protein C18”, 250 × 4.6 mm, 5 μm particle size, first 15% acetonitrile (containing 0.1% TFA) for 2 min, then 15%-55% acetonitrile (containing 0.1% TFA) for 30 min, flow rate 1.0 mL / min, λ = 214 nm. Theoretical molecular weight: 2803.38, actual measured molecular weight: 2802.60. Reversed-phase high-performance liquid chromatography showed that the purity of the target peptides was higher than 95%. Mass spectrometry data confirmed that the molecular weights of the synthesized peptides were correct compared to the theoretically calculated molecular weights.

[0088] A comparison of the high-performance liquid chromatography (HPLC) chromatograms of the hydrazide-synthesized peptides synthesized with green solvents and those synthesized with DMF in this invention reveals that the chromatographic purity of the hydrazide-synthesized peptides synthesized using the combined green solvents GVL and TEP is comparable to that synthesized with DMF. Figure 7-11As shown. Therefore, this invention has good practical application value.

[0089] For MCQ-1, using green solvent and DMF as solvents, the chromatographic purities of the crude peptide were 28.2% and 31.8%, respectively (e.g., ...). Figure 7 (As shown).

[0090] For MCQ-2, using green solvent and DMF as solvents, the chromatographic purities of the crude peptide were 28.6% and 35.4%, respectively (e.g., ...). Figure 8 (As shown).

[0091] For MCQ-3, using green solvent and DMF as solvents, the chromatographic purities of the crude peptide were 52.0% and 29.5%, respectively (e.g., ...). Figure 9 (As shown).

[0092] For MCQ-4, using green solvent and DMF as solvents, the chromatographic purities of the crude peptide were 55.8% and 39.6%, respectively (e.g., ...). Figure 10 (As shown).

[0093] For MCQ-5, using green solvent and DMF as solvents, the chromatographic purities of the crude peptide were 44.6% and 37.9%, respectively (e.g., ...). Figure 11 (As shown).

[0094] Circular dichroism spectroscopy was used to detect the secondary conformations of example peptides MCQ-1, MCQ-2, MCQ-3, MCQ-4, and MCQ-5 synthesized using green solvent or DMF as solvent in this invention. It was found that MCQ-1 and MCQ-2 synthesized using green solvent exhibited mirror symmetry and a random coil conformation. MCQ-3, MCQ-4, and MCQ-5 synthesized using green solvent all possessed good α-helix structures and had similar conformations and helicity to the corresponding peptides synthesized using DMF (e.g., ...). Figure 12 (As shown). Therefore, this invention has good application value.

[0095] The MTT assay was used to evaluate the inhibitory effects of the acylhydrazide peptides synthesized in the green solvent and the corresponding acylhydrazide peptides synthesized in DMF on the proliferation of 4T1 breast cancer cells and HeLa cervical cancer cells. The cells were subjected to a 10... 4Cells were seeded at a density of [number] cells / well in 96-well plates and cultured overnight at 37°C. For the MCQ-1, MCQ-2, MCQ-4, and MCQ-5 treatment groups, different volumes of high-concentration peptide solution were added to each well to achieve a peptide concentration gradient of 3.125, 6.25, 12.5, 25, 50, and 100 µM in the cell culture medium. For the MCQ-3 treatment group, different volumes of high-concentration peptide solution were added to each well to achieve a peptide concentration gradient of 0.001, 0.01, 0.1, 1, 10, and 100 µM in the cell culture medium. After adding the peptide solution, each group was cultured for 24 h, and then 15 μL of MTT (5 mg / mL) was added to each well, and the cells were incubated at 37°C for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well. After incubation for 1 h, the absorbance of each well was measured at 492 nm using a microplate reader. The IC values ​​obtained from three independent parallel experiments were calculated using IBM SPSS Statistics 27 software. 50 Dose-response curves were generated using GraphPad Prism 10.1.2 software, and all IC50 values ​​were analyzed. 50 The values ​​are expressed as mean ± SEM.

[0096] A comparison of the antitumor effects of the hydrazide-synthesized peptides synthesized with green solvents and the corresponding hydrazide-synthesized peptides synthesized with DMF in this invention: Both the target peptides synthesized by the method of this invention and the peptides synthesized with DMF exhibit concentration-dependent antitumor effects, and it can be found that the antitumor effects of the hydrazide-synthesized peptides synthesized by the method of this invention are comparable to those synthesized with DMF. Figure 13 As shown in AG. The target peptide synthesized by the method of this invention and the peptide synthesized by DMF show their effects on the IC50 of 4T1 cells and HeLa cells. 50 The values ​​are equivalent. For example... Figure 13 As shown, the three acylhydrazide peptides (MCQ-1, MCQ-2, MCQ-3, MCQ-4, MCQ-5) synthesized using the green solvent of this invention are effective against 4T1 cells (IC50, 1000-1000 mmol / L). 50 : 25.36±0.66 µM, 24.59±0.91 µM, 2.80±0.61 µM, 21.81±1.08 µM, 26.36±1.72 µM) and HeLa cells (IC50, ... 50 The DMF-synthesized peptides (25.56±0.44 µM, 21.26±3.60 µM, 0.20±0.06 µM, 16.94±1.27 µM, and 21.64±1.96 µM) showed good inhibitory effects, which is consistent with the inhibitory effects of DMF-synthesized peptides on 4T1 cells (IC50, 25.56±0.44 µM, 21.26±3.60 µM, 0.20±0.06 µM, 16.94±1.27 µM, and 21.64±1.96 µM). 50: 30.0±1.79 µM, 27.83±2.91 µM, 2.46±0.18 µM, 20.37±1.22 µM, 26.40±0.82 µM) and HeLa cells (IC50, ... 50 The inhibitory effects of DMF at concentrations of 29.63±1.10 µM, 20.27±3.11 µM, 0.25±0.01 µM, 16.65±1.72 µM, and 23.15±2.52 µM were comparable. Therefore, this invention can replace DMF in the chemical synthesis of hydrazide-substituted peptides, and the resulting peptides exhibit good antitumor effects and significant practical application value.

[0097] In summary, this invention uses GVL as a solvent for amino acid condensation in hydrazine resin preparation and solid-phase peptide synthesis, and TEP as a solvent for the removal of Fmoc protecting groups in solid-phase peptide synthesis. This invention not only achieves efficient construction of hydrazine resin and efficient synthesis of acylhydrazide peptides, but also achieves chromatographic purity comparable to that obtained using DMF as a solvent, even exhibiting a purity advantage in the synthesis of long-chain acylhydrazide peptides. Furthermore, the five acylhydrazide peptides synthesized using this method showed comparable inhibitory effects on the proliferation of 4T1 breast cancer cells and HeLa cervical cancer cells as those synthesized using DMF. In addition, this method, while efficiently preparing hydrazine resin and synthesizing solid-phase peptides, uses a green solvent instead of traditional DMF as the reaction solvent, significantly reducing the safety risks to laboratory personnel and potential environmental hazards. Therefore, this invention has good practical application value.

[0098] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A highly efficient and green method for preparing acylhydrazide-modified peptides, characterized in that, Includes the following steps: 1) Weigh a certain amount of 2-Cl-(Trt)-Cl resin (1 equivalent, molar ratio) into a polypeptide synthesis tube, wash once with standard, and dry under vacuum; add 3 mL GVL, soak and swell in an air bath at 28℃ for 2 hours, wash once with standard, add 3 mL GVL / DCM (v / v = 1:1) mixed solution, activate the resin in an air bath at 28℃ for 30 min, wash once with standard, and dry under vacuum for later use; 2) Add a 10% hydrazine hydrate GVL solution (v / v), react at 28°C in an air bath constant temperature shaker for 30 min, and wash three times with GVL; add a 10% hydrazine hydrate GVL solution (v / v), react at 28°C in an air bath constant temperature shaker for 30 min, and wash three times with GVL; add a 10% methanol GVL solution (v / v), react at 28°C in an air bath constant temperature shaker for 10 min, wash once with standard, and dry for later use; 3) The condensation system used for amino acids other than arginine, histidine, and serine was a 3 mL GVL solution containing Fmoc-amino acids (3 equivalents, molar ratio), Oxyma (3 equivalents, molar ratio), and DIC (6 equivalents, molar ratio). The Fmoc-amino acids used included L-configured and D-configured amino acids. The first amino acid was condensed three times under isothermal shaking in a 50°C air bath, with the first reaction lasting 15 min, the second 20 min, and the third 25 min. Subsequent amino acids were condensed twice under isothermal shaking in a 50°C air bath, with the first reaction lasting 15 min and the second 20 min. Then, under isothermal shaking in a 50°C air bath, the Fmoc protecting group was removed using a 20% piperidine TEP solution, with the first reaction lasting 3 min and the second 5 min. min; After all amino acid condensation and after the protecting group was removed, the resin color development was detected under heating conditions using Kaiser reagent (solution A: 80% phenol in ethanol (w / v); solution B: 5% ninhydrin in ethanol (w / v)); 4) The condensation system used for Fmoc-protected arginine, histidine, and serine contained 3 mL of GVL solution of Fmoc-amino acids (3 equivalents, molar ratio), Oxyma (3 equivalents, molar ratio), and DIC (6 equivalents, molar ratio). Condensation was carried out under constant temperature shaking conditions in an air bath at 28°C for 60 min for the first reaction and 90 min for the second reaction. Then, under constant temperature shaking conditions in an air bath at 28°C, the Fmoc protecting group was removed using a 20% piperidine TEP solution for 5 min for the first reaction and 10 min for the second reaction. 5) Peptide cleavage: After one standard wash, remove the Fmoc protecting group using a 20% piperidine TEP solution under constant temperature shaking conditions in an air bath at 28°C. The first reaction is 5 min, followed by three TEP washes. Then, add another 20% piperidine TEP solution and react for 10 min. After one standard wash, take a small amount of resin and test the color development using Kaiser reagent under heating conditions. Rinse the resin 5 times with DCM, followed by 5 min of vacuum pumping and 5 min of vacuum pumping. Add the peptide cleavage reagent (trifluoroacetic acid / phenol / water / anisole / 1,2-ethylenedithiol, 85:) pre-cooled to below 5°C in an ice bath. 2.5:5:5:2.5 (v / v / v / v / v), reacted in a constant temperature oscillator in an air bath at 28°C for 2.5 hours in the dark; 6) Peptide precipitation: Concentrate the peptide cleavage reagent to 2-3 mL using a high-purity nitrogen bubbling concentration method. Precipitate the peptide using pre-cooled diethyl ether, centrifuge at 3000 r / min for 3 min, discard the supernatant, and retain the precipitate. Resuspend the precipitate in pre-cooled ice-cold diethyl ether by sonication, centrifugation, and discarding the supernatant, repeating this process three times. Allow the peptide precipitate to stand in a fume hood until dry. 7) Dissolve the crude peptide product in a mixed solution of acetonitrile and water containing 0.1% TFA, and freeze-dry it using a low-temperature freeze dryer to remove residual solvent; The crude peptide product was then dissolved again in a mixed solvent of acetonitrile and water containing 0.1% TFA, and purified by semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The product was then freeze-dried at low temperature to obtain the target acylhydrazide peptide sample. The purity, molecular weight, and amino acid sequence of the crude target acylhydrazide peptide product were analyzed by analytical reversed-phase HPLC and high-resolution mass spectrometry (ESI-MS). The crude peptide product was then purified by semi-preparative reversed-phase HPLC.

2. A class of acylhydrazide-modified polypeptides, characterized in that, The amino acid residue sequence of the acylhydrazide polypeptide is as follows: MCQ-3 H-GIGAVLKVLTTGLPALISWIKRKRQQ-NHNH2 MCQ-4 H-LAVISWKCQEWNSLWKKRKRKT-NHNH2 MCQ-5 H-KGWFKAMKSIAKFIAKEKLKEHL-NHNH 2。 3. The method for preparing an acylhydrazide-modified polypeptide according to claim 1, characterized in that, The GVL mentioned is γ Gamma-Valerolactone (abbreviated as GVL) is an omega-valerolactone.

4. The method for preparing an acylhydrazide-modified polypeptide according to claim 1, characterized in that, The TEP mentioned is triethyl phosphate (abbreviated as TEP).

5. The method for preparing an acylhydrazide-modified polypeptide according to claim 1, characterized in that, The standard washing procedure is as follows: two GVL washes, two DCM washes, and two GVL washes.

6. The method for preparing an acylhydrazide-modified polypeptide according to claim 1, characterized in that, The method for preparing the 10% hydrazine hydrate GVL solution is as follows: take 471 µL of 85% hydrazine hydrate and dilute it to 4 mL with GVL.

7. The use of the acylhydrazide polypeptide of claim 2 in the prevention or treatment of tumor-related diseases.

8. A method for preventing or treating tumors, characterized in that, The method includes administering a therapeutically effective dose of the polypeptide of claim 2 to a subject.

9. Use of the polypeptide of claim 2 as a non-therapeutic tumor cell inhibitor.

10. A method for inhibiting tumor cell proliferation in vitro, characterized in that, The method includes administering the polypeptide of claim 2 to tumor cells cultured in vitro.

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