A solid phase synthesis method for peptides

Abstract

The present invention provides a solid phase synthesis method of peptides, wherein: 1) peptide resin is used for synthesis, peptide chains are condensed sequentially from the first amino acid at the C-terminus, and peptide resin is obtained by washing and drying; 2) peptide resin is subjected to cleavage reaction, concentrated, precipitated, and dried to obtain a crude peptide product; 3) crude peptide product is pretreated and purified by high performance liquid chromatography; the pretreatment process includes extracting the crude peptide product with 0.1%-10% alcohol solution, preparing a pre-purified solution after ultrafiltration, and separating and purifying the pre-purified solution by high performance liquid chromatography; the alcohol solution includes one or more of C1~C5 monosubstituted and / or polysubstituted alcohols and / or sugar alcohols. The present invention can remove unwanted protein contaminants through extraction, ultrafiltration and other processes, thereby reducing the toxic effects on the chromatographic column in subsequent high performance liquid chromatography purification, improving the column separation state, further improving production efficiency, and saving production costs.

Description

Technical Field

[0001] The present invention relates to a method for synthesizing peptides, in particular to a solid phase synthesis method for peptides. Background Art

[0002] The special physiological functions of peptide compounds make them valuable for application in the field of biotechnology, such as protein engineering and preparation of biological agents. Currently, peptide products are widely used in medicine, cosmetics, agriculture and other fields. In the field of medicine, the development of peptide drugs provides a new strategy for the treatment of various diseases; in the field of cosmetics, peptide products provide an effective way for human anti-aging and beauty; in the field of agriculture, peptide substances can be used to improve the quality and safety of feed, thereby improving the quality of livestock and poultry products.

[0003] Traditional peptide synthesis mainly relies on chemical methods, but this method has obvious disadvantages, such as racemization, side chain functional groups need to be protected, overall low efficiency, use of large amounts of toxic solvents and high costs. Currently, chemical methods are only used for laboratory research or the production of high-priced peptides. With the development of enzyme catalysis technology, new synthetic methods such as chemical enzyme method and enzymatic method have gradually developed in the field of peptide synthesis. These methods may have advantages in efficiency, safety, cost and other aspects compared to traditional chemical methods.

[0004] Currently, peptide compounds can be synthesized through solid phase or chemical methods. These synthetic peptide methods can generate polypeptides with clear sequences and stable structures. These synthetic peptides can be used as immunogens in vaccine development or in other biotechnology research.

[0005] Research on solid-phase synthesis methods of peptides mainly focuses on improving synthesis efficiency, reducing costs, and improving purification processes. There are still many difficult problems to solve: 1) Solid-phase peptide synthesis usually involves complex protection / deprotection steps, which may cause problems in the purification process; 2) Traditional solid-phase synthesis methods may have cleavage reagents and side-protected byproducts mixed together when removing the resin, which makes the next step of purification more difficult; 3) In the synthesis of long-sequence peptides, accumulation of side reactions may occur, which will also cause additional challenges in the purification process; 4) The correct treatment and removal of protecting groups may introduce undesirable impurities during the purification process, thereby affecting the purity and quality of the final product; 5) The cost and efficiency of solid-phase peptide synthesis are also factors that need to be considered in the purification process. For example, the use of expensive special carriers may have poor feasibility in the scale-up process, which may affect the scale and efficiency of purification; 6) Traditional purification methods such as high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (MS) are currently commonly used purification technologies. However, these methods have high operating costs, serious equipment damage, require professional operation and equipment maintenance, and may require structural analysis of the final product, etc.

[0006] Therefore, the solid phase synthesis method of peptides has many problems, including the limitations of traditional methods, the impact of side reactions, protection group treatment, cost and efficiency considerations, and challenges of subsequent purification technology. In response to these problems, researchers need to continuously explore new synthesis strategies and purification technologies to improve synthesis efficiency and product quality. Summary of the invention

[0007] In order to overcome the defects of the prior art, the present invention provides a solid phase synthesis method of peptides, after peptide resin synthesis and cracking process to obtain crude peptides, the crude peptides are purified by high performance liquid phase method after pretreatment. The pretreatment process can not only improve protein-protein interaction, change the solution viscosity to inhibit the speed of physicochemical reactions, improve protein stability and / or increase protein solubility, and improve the transport of required protein active ingredients, but also remove unnecessary protein contaminants through extraction, ultrafiltration and other processes, thereby reducing the toxic effect on the chromatographic column in the subsequent high performance liquid chromatography purification, improving the column separation state, further improving production efficiency, and saving production costs.

[0008] The invention provides a solid phase synthesis method of peptides, wherein: 1) peptide resin is used for synthesis, peptide chains are condensed in sequence starting from the first amino acid at the C-terminus, and the peptide resin is obtained by washing and drying; 2) the peptide resin is subjected to a cleavage reaction, and a crude peptide product is obtained after concentration, precipitation and drying; 3) the crude peptide product is pretreated and purified by high performance liquid chromatography; the pretreatment process comprises extracting the crude peptide product with a 0.1%-10% alcohol solution, preparing a pre-purified solution after ultrafiltration, and separating and purifying the pre-purified solution by high performance liquid chromatography; the alcohol solution comprises one or more of C1-C5 mono-substituted and / or poly-substituted alcohols and / or sugar alcohols.

[0009] The present invention adopts peptide resin synthesis, and sequentially condenses the peptide chain starting from the first amino acid at the C-terminus, thereby reducing the generation of by-products. Through continuous exploration by the inventors, it is found that the use of alcohol solutions of C1~C5 monosubstituted and / or polysubstituted alcohols and / or sugar alcohols to extract crude peptides not only reduces the content of by-products at different solubilities, but also changes the viscosity of the crude peptide solution, which can inhibit the speed of physicochemical reactions, improve protein-protein interactions, and improve the transport of protein active ingredients. At the same time, the pre-purified solution prepared after extraction and ultrafiltration reduces the introduction of protein impurities for the subsequent high-efficiency liquid phase purification process, and further reduces the toxic effects on the chromatographic column, prolongs the service life and number of chromatographic columns, improves the column separation state, further improves production efficiency, and greatly reduces the possibility of repeated purification.

[0010] Preferably, in the solid phase synthesis method provided by the present invention, the peptides include pentapeptide-18, hexapeptide-9, nonapeptide-1, acetyl tetrapeptide-2, acetyl tetrapeptide-5 and / or acetyl tetrapeptide-9. The above-mentioned peptide products, as determined by the inventors through experimental studies, can improve the purity of the product and simplify the purification process by using the above-mentioned reaction process and alcohol solution extraction and ultrafiltration.

[0011] Preferably, the solid phase synthesis method provided by the present invention, wherein the pretreatment process is extracted with 0.2%-5% methanol, ethanol, isopropanol, glycerol and / or sugar alcohol solution, and the sugar alcohol is selected from xylitol, maltitol, erythritol, lactitol, sorbitol and / or mannitol; the ultrafiltration is selected from cellulose 1~10kD ultrafiltration membrane package, preferably regenerated cellulose 1~5kD ultrafiltration membrane package. After repeated verification by the inventors, the above-mentioned alcohol solution has a better effect on achieving the purpose of the present invention, and the above-mentioned ultrafiltration membrane package can not only reduce the adsorption of polypeptides, but also meet the efficiency and effect requirements of ultrafiltration. Through continuous exploration, the inventors found that after the above-mentioned extraction and ultrafiltration, entering the high-performance liquid phase purification process, the purity and yield of the product can be effectively improved, and the daily loss of the high-performance liquid phase column can be reduced, and the purification efficiency can be improved, and unexpected results have been achieved.

[0012] Preferably, in the solid phase synthesis method provided by the present invention, the resin adopts CTC Resin with a substitution value ranging from 1.0 mmol / g to 1.4 mmol / g; in the peptide resin synthesis, the condensing agent is selected from one or more of carbodiimide type, phosphorus cation type, urea cation type, imine cation type, organic phosphorus condensing agent and / or other types of condensing agents. The use of CTC Resin with a substitution value ranging from 1.0 mmol / g to 1.4 mmol / g and the corresponding condensing agent can achieve the purpose of solid phase synthesis of peptides in the present invention.

[0013] Preferably, in the solid phase synthesis method provided by the present invention, the condensing agent is selected from one or more of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), chloro-1-hydroxybenzotriazole (CI-HOBt), 1-hydroxybenzotriazole (HOBT), and / or 1,3-diisopropylcarbodiimide (DIC). After repeated experiments by the inventors, the side reactions of the above condensing agents are fewer and the yield and purity of the overall reaction can be improved.

[0014] Preferably, in the solid phase synthesis method provided by the present invention, the peptide resin synthesis includes a coupling step, in which the molar ratio of Fmoc-protected amino acid to resin is (1.0-10.0):1; the coupling step is carried out in an organic solution, the coupling temperature is 10-40°C, and the time is more than 100 min.

[0015] Preferably, in the solid phase synthesis method provided by the present invention, the molar ratio of Fmoc-protected amino acid to resin is (2.0-5.0):1, the coupling step is carried out in N,N-dimethylformamide solution, the coupling temperature is 15-30°C, and the coupling time is 200-800 min.

[0016] Preferably, in the solid phase synthesis method provided by the present invention, the mass-volume ratio of the peptide resin to the cleavage agent is 1 g: 5 ml to 20 ml; the cleavage agent includes one or more of a thiol compound, an acid or a phenolic compound, water, and a silane derivative; and the cleavage conditions are: cleavage at 10 to 45° C. for 1 to 10 hours.

[0017] Preferably, in the solid phase synthesis method provided by the present invention, the mass-volume ratio of the peptide resin to the cleavage agent and / or the cleavage agent is 1g:6ml~15ml; the cleavage agent includes one or more of water, trifluoroacetic acid, 1,2-ethanedithiol and / or triisopropylsilane; and the cleavage conditions are cleavage at 15~30°C for 2-6 hours.

[0018] Preferably, in the solid phase synthesis method provided by the present invention, high performance liquid chromatography separation is performed using a C8, C18 filler chromatographic column and / or an amino acid column, the filler particle size is 5 to 20 μm, the chromatographic column diameter is greater than 100 mm, the length is greater than 250 mm, and the theoretical plate number is greater than 3000;

[0019] When a C18 packed column is used, the mobile phase is a mixed system of acetic acid and / or trifluoroacetic acid, the acetic acid mixed system includes 0.02%~1.0% acetic acid solution, acetonitrile and / or 0.05mol / L~1.0mol / L ammonium acetate solution, and the organic term ratio is 1%~60%; preferably 0.05%~0.5% acetic acid solution, acetonitrile and / or 0.1mol / L~0.5mol / L ammonium acetate solution, and the organic term ratio is 2%~45%.

[0020] Through creative research and repeated experimental verification by the inventors, the reaction system described in the present invention, including the solid phase synthesis process, the preparation of the pre-purified solution, the synthetic resin, the condensation agent, the coupling conditions, the cleavage agent and the HPLC purification conditions, can improve the purity and yield of the peptides in the present invention, reduce the toxic reaction to the chromatographic column, further improve the production efficiency and save the production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 This is the chromatogram of pentapeptide-18 after HPLC purification;

[0023] Figure 2 This is the chromatogram of hexapeptide-9 after HPLC purification;

[0024] Figure 3 This is the chromatogram of nonapeptide-1 after HPLC purification;

[0025] Figure 4 This is the chromatogram of acetyl tetrapeptide-2 after purification by HPLC;

[0026] Figure 5 This is the chromatogram of acetyl tetrapeptide-5 after purification by HPLC;

[0027] Figure 6 This is the chromatogram of acetyl tetrapeptide-9 after HPLC purification;

[0028] Figure 7 This is the chromatogram of pentapeptide-18 after HPLC purification (without pretreatment). DETAILED DESCRIPTION

[0029] In order to further illustrate the present invention, examples are given below. It should be noted that these examples are purely illustrative. The purpose of providing these examples is to fully illustrate the meaning and content of the present invention, but the present invention is not limited to the scope of the embodiments.

[0030] Example 1 Solid Phase Synthesis of Peptides

[0031] The present invention provides a solid phase synthesis method of peptides, wherein the peptides include pentapeptide-18, hexapeptide-9, nonapeptide-1, acetyl tetrapeptide-2, acetyl tetrapeptide-5 and / or acetyl tetrapeptide-9.

[0032] 1) The peptide resin was used for synthesis, the resin used was CTC Resin, and the substitution value range was 1.0 mmol / g to 1.4 mmol / g.

[0033] The peptide resin synthesis includes a coupling step, in which the molar ratio of the Fmoc-protected amino acid to the resin is (1.0-10.0):1; the coupling step is carried out in an organic solution, the coupling temperature is 10-40°C, and the time is more than 100 minutes. In another embodiment, the molar ratio of the Fmoc-protected amino acid to the resin is (2.0-5.0):1, the coupling step is carried out in an N,N-dimethylformamide solution, the coupling temperature is 15-30°C, and the coupling time is 200-800 minutes.

[0034] Starting from the first amino acid at the C-terminus, the peptide chains are sequentially condensed, and the condensing agent is selected from one or more of carbodiimide type, phosphorus cation type, urea cation type, iminium cation type, organic phosphorus condensing agent and / or other types of condensing agents. In one embodiment, the condensing agent is selected from one or more of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), chloro-1-hydroxybenzotriazole (Cl-HOBT) / 1-hydroxybenzotriazole (HOBT), and / or 1,3-diisopropylcarbodiimide (DIC).

[0035] The peptide resin was obtained after washing and drying.

[0036] 2) The peptide resin is subjected to a cleavage reaction, and then concentrated, precipitated, and dried to obtain a crude peptide. The mass-volume ratio of the peptide resin to the cleavage agent is 1g: 5 ml to 20 ml; the cleavage agent includes one or more of thiol compounds, acids or phenolic compounds, water, and silane derivatives; the cleavage conditions are: cleavage at 10 to 45°C for 1 to 10 hours. In another embodiment, the mass-volume ratio of the peptide resin to the cleavage agent and / or the cleavage agent is 1g: 6ml to 15ml; the cleavage agent includes one or more of water, trifluoroacetic acid, 1,2-ethanedithiol, and / or triisopropylsilane; the cleavage conditions are cleavage at 15 to 30°C for 5 hours.

[0037] 3) The crude peptide is pretreated and purified by high performance liquid chromatography: The pretreatment process includes extracting the crude peptide with a 0.1%-10% alcohol solution, ultrafiltration to prepare a pre-purified solution, and separation and purification of the pre-purified solution by high performance liquid chromatography.

[0038] In one embodiment, the alcohol solution includes one or more of C1~C5 monosubstituted and / or polysubstituted alcohols, and / or sugar alcohols. In another embodiment, the pretreatment process uses 0.2%-5% methanol, ethanol, isopropanol, glycerol and / or sugar alcohol solution for extraction, and the sugar alcohol is selected from xylitol, maltitol, erythritol, lactitol, sorbitol and / or mannitol. The ultrafiltration is selected from cellulose 1~10kD ultrafiltration membrane package, preferably regenerated cellulose 1~5kD ultrafiltration membrane package.

[0039] When separating by high performance liquid chromatography, a C8 or C18 filler chromatographic column and / or an amino acid column is used, the filler particle size is 5 to 20 μm, the chromatographic column diameter is greater than 100 mm, the length is greater than 250 mm, and the theoretical plate number is greater than 3000; when a C18 filler chromatographic column is used, the mobile phase is a mixed system of acetic acid and / or trifluoroacetic acid, and the acetic acid mixed system includes 0.02% to 1.0% acetic acid solution, acetonitrile and / or 0.05 mol / L to 1.0 mol / L ammonium acetate solution, and the organic term ratio is 1% to 60%. In another embodiment, 0.05% to 0.5% acetic acid solution, acetonitrile and / or 0.1 mol / L to 0.5 mol / L ammonium acetate solution are selected, and the organic term ratio is 2% to 45%.

[0040] Example 2 Solid Phase Synthesis of Pentapeptide-18

[0041] Chinese name: Pentapeptide-18

[0042] English name: Pentapeptide-18

[0043] Sequence: H-Tyr-D-Ala-Gly-Phe-Leu-OH

[0044] 1) Synthesis of peptide resin of pentapeptide-18

[0045] Take 3.0 mol of Fmoc-Leu-OH and dissolve it completely with appropriate amount of DMF. Take another 3.0 mol of hydroxyl functional group resin such as 2-CTC Resin (S=1.0 mmol / g~1.4mmol / g), add it to the Fmoc-Leu-OH solution and stir evenly. Add condensation agent Cl-HBTU / DIC, control the reaction temperature at 40℃, couple the reaction for 100 minutes, filter and wash to obtain Fmoc-Leu-2-Cl-TrtResin resin.

[0046] The above resin was deprotected and washed, and the next amino acid activation preparation was carried out in the process. 3.0 mol Fmoc-Phe-OH and 3.0 ml O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) and 1,3-diisopropylcarbodiimide (DIC) were dissolved in an appropriate amount of DMF and activated for use.

[0047] Then, the pretreated Fmoc-Phe-OH, HBTU and DIC solution were added to the deprotected and washed peptide resin for condensation reaction. Starting from the second amino acid at the C-terminus, the condensation was repeated in a cycle (amino acid pretreatment, peptide resin washing, deprotection, washing, adding pretreated amino acid condensation reaction, and then washing the resin as one cycle), and finally washed again to obtain the peptide resin: Tyr(tBu)-D-Ala-Gly-Phe-Leu-2-Cl-Trt Resin.

[0048] 2) Peptide resin cleavage of pentapeptide-18

[0049] Take the prepared peptide resin of pentapeptide-18, the cleavage agents are trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), triisopropylsilane (TIS) and water (H2O), the mass volume ratio of peptide resin to cleavage agent is 1g:5ml, stir evenly, react at 45℃ for 1.0 hour, filter the reaction mixture with a sand core funnel, collect the filtrate, combine the filtrate and concentrate under reduced pressure, add methyl tert-butyl ether (MBTE) for precipitation, wash with MBTE for 3 to 5 times, evaporate MTBE, and dry the crude product under reduced pressure at room temperature to constant weight.

[0050] The crude product was dissolved with an appropriate amount of aqueous solution. During the dissolution process, the pH was controlled at about 8.5 with 5% dilute ammonia water while adding the sample. After the solution was completely dissolved, it was filtered and then concentrated for 30 minutes to obtain a crude solution of pentapeptide-18.

[0051] 3) Purification of crude pentapeptide-18

[0052] The crude product of pentapeptide-18 was extracted with 0.1% methanol solution, and the pre-purified solution was prepared by ultrafiltration using a regenerated cellulose 1kD ultrafiltration membrane. The pre-purified solution was separated and purified by high-performance liquid chromatography. The purification was carried out by high-performance liquid chromatography. The chromatographic filler used for purification was a C18 filler column with a filler particle size of 5 μm, a chromatographic column diameter of 100 mm, a length of 250 mm, and a theoretical plate number of more than 3000. After acetic acid mixed system (i.e., mobile phase: 0.02% HAC.H 2 O, acetonitrile, 0.05 mol / L NH 4 AC.H 2 O), the organic phase ratio is 1%~60%, and the qualified components are collected for purification. After the main peak is collected and concentrated, it is a colorless clear liquid. After freeze-drying, pentapeptide-18 powder (acetate) is obtained with a purity of 99.0% and a yield of 65.3%.

[0053] Example 3 Solid Phase Synthesis of Hexapeptide-9

[0054] Chinese name: Hexapeptide-9

[0055] English name: Hexapeptide-9

[0056] Sequence: H-Gly-Pro-Gln-Gly-Pro-Gln-OH

[0057] 1) Synthesis of peptide resin of hexapeptide-9

[0058] Take 30.0 mol of Fmoc-Gln-OH and dissolve it completely with an appropriate amount of DMF; take another 3.0 mol of hydroxyl functional group resin such as 2-CTC Resin (S=1.0 mmol / g~1.4mmol / g), add it to the Fmoc-Gln-OH solution and stir evenly, add condensation agent N,N-diisopropylethylamine (DIEA), control the reaction temperature at 10℃, couple the reaction for 800 minutes, filter and wash to obtain Fmoc-Gln-2-Cl-Trt Resin resin.

[0059] The above resin was deprotected and washed, and the next amino acid activation preparation was carried out simultaneously. 30.0 mol Fmoc-Pro-OH and 30.0 ml chloro-1-hydroxybenzotriazole (Cl-HOBT) and 1,3-diisopropylcarbodiimide (DIC) were slowly added into the DMF solution under stirring and activated for use.

[0060] Then, the pretreated Fmoc-Pro-OH / Cl-HOBT / DIC / DMF solution was added to the deprotected and washed peptide resin for condensation reaction. Starting from the second amino acid at the C-terminus, the condensation was repeated in a cycle (amino acid pretreatment, peptide resin washing, deprotection, washing, adding pretreated amino acid for condensation reaction, and then washing the resin as one cycle), and finally washed again to obtain the peptide resin: Gly-Pro-Gln(Trt)-Gly-Pro-Gln(Trt)-2-Cl-Trt Resin.

[0061] 2) Peptide resin cleavage of hexapeptide-9

[0062] Take the obtained peptide resin of hexapeptide-9, the cleavage agent is trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) or triisopropylsilane (TIS) and water (H2O), the mass volume ratio of peptide resin to cleavage agent is 1g:20ml, stir evenly, react at 10℃ for 10.0 hours, filter the reaction mixture with a sand core funnel, collect the filtrate, combine the filtrate and concentrate under reduced pressure, add methyl tert-butyl ether (MBTE) for precipitation, wash with MBTE for 3 to 5 times, evaporate MTBE, and dry the crude product under reduced pressure at room temperature to constant weight.

[0063] The crude product was dissolved with an appropriate amount of aqueous solution. During the dissolution process, the pH was controlled at about 8.5 with 5% dilute ammonia water while adding the sample. After the solution was completely dissolved, it was filtered and then concentrated for 30 minutes to obtain the crude solution of hexapeptide-9.

[0064] 3) Purification of crude hexapeptide-9

[0065] The crude hexapeptide-9 was extracted with 10% ethanol solution, and the pre-purified solution was prepared by ultrafiltration using a regenerated cellulose 3kD ultrafiltration membrane. The pre-purified solution was separated and purified by high performance liquid chromatography. The purification was carried out by high performance liquid chromatography. The chromatographic filler used for purification was a C8 filler column with a filler particle size of 10 μm, a chromatographic column diameter of 150 mm, a length of 250 mm, and a theoretical plate number of more than 3000. After acetic acid mixed system (i.e., mobile phase: 0.05% HAC.H 2 O, acetonitrile, 0.1 mol / L NH 4 AC.H 2 O), the organic phase ratio is 1%~60%, and the qualified components are collected for purification. After the main peak is collected and concentrated, it is a colorless clear liquid. After freeze-drying, hexapeptide-9 powder (acetate) is obtained with a purity of 99.2% and a yield of 66.2%.

[0066] Example 4 Solid Phase Synthesis Method of Nonapeptide-1

[0067] Chinese name: Nonapeptide-1

[0068] English name: Nonapeptide-1

[0069] Sequence: H-Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val-NH2

[0070] 1) Synthesis of peptide resin of nonapeptide-1

[0071] Take 6.0 mol of Fmoc-Val-NH2 and dissolve it completely with appropriate amount of DMF; take another 3.0 mol of resin such as CTC Resin (S=1.0 mmol / g~1.4mmol / g), add it to the Fmoc-Val-NH2 solution and stir evenly, add condensation agent HOBt / DIC, control the reaction temperature at 15℃, couple the reaction for 600 minutes, filter and wash to obtain Fmoc-Val-Cl-Trt Resin resin.

[0072] The above resin was deprotected and washed, and the next amino acid activation preparation was carried out in the process. 6.0 mol Fmoc-Pro-OH and 6.0 mol HOBT were dissolved in an appropriate amount of DMF; 6.0 mol DIC was also taken and slowly added to the DMF solution under stirring, and it was activated and set aside.

[0073] Then, the pretreated Fmoc-Pro-OH / HOBt / DIC / DMF solution was added to the deprotected and washed peptide resin for condensation reaction. Starting from the second amino acid at the C-terminus, the condensation was repeated in a cycle (amino acid pretreatment, peptide resin washing, deprotection, washing, adding pretreated amino acid for condensation reaction, and then washing the resin as one cycle), and finally washed again to obtain the peptide resin: Met-Pro-D-Phe-Arg(Pbf)-D-Trp-Phe-Lys(Boc)-Pro-Val-AM Resin.

[0074] 2) Peptide resin cleavage of nonapeptide-1

[0075] Take the obtained peptide resin of nonapeptide-1, the cleavage agent is trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) or triisopropylsilane (TIS) and water (H2O), the mass volume ratio of peptide resin to cleavage agent is 1g:6ml, stir evenly, react at 15℃ for 2.0 hours, filter the reaction mixture with a sand core funnel, collect the filtrate, combine the filtrate and concentrate under reduced pressure, add methyl tert-butyl ether (MBTE) for precipitation, wash with MBTE for 3 to 5 times, evaporate MTBE, and dry the crude product under reduced pressure at room temperature to constant weight.

[0076] The crude product was dissolved with an appropriate amount of aqueous solution. During the dissolution process, the pH was controlled at about 8.5 with 5% dilute ammonia water while adding the sample. After the solution was completely dissolved, it was filtered and then concentrated for 30 minutes to obtain the crude solution of nonapeptide-1.

[0077] 3) Purification of crude nonapeptide-1

[0078] The crude product of nonapeptide-1 was extracted with 0.2% isopropanol solution, and the pre-purified solution was prepared by ultrafiltration using a regenerated cellulose 5kD ultrafiltration membrane. The pre-purified solution was separated and purified by high performance liquid chromatography. The purification was carried out by high performance liquid chromatography. The chromatographic filler used for purification was a C18 filler column with a filler particle size of 10 μm, a chromatographic column diameter of 200 mm, a length of 250 mm, and a theoretical plate number of more than 3000. After acetic acid mixed system (i.e., mobile phase: 1.0% HAC.H 2 O, acetonitrile, 1.0 mol / L NH 4 AC.H 2 O), the organic phase ratio was 2%~45%, and the qualified components were collected for purification. After the main peak was collected and concentrated, it was a colorless clear liquid. After freeze-drying, nonapeptide-1 powder (acetate) was obtained with a purity of 99.3% and a yield of 66.4%.

[0079] Example 5 Solid Phase Synthesis of Acetyl Tetrapeptide-2

[0080] Chinese name: Acetyl tetrapeptide-2

[0081] English Name: Acetyl Tetrapeptide-2

[0082] Sequence: Ac-Lys-Asp-Val-Tyr-OH

[0083] 1) Synthesis of peptide resin of acetyl tetrapeptide-2

[0084] Take 15.0 mol of Fmoc-Tyr-OH and dissolve it completely with an appropriate amount of DMF; take another 3.0 mol of hydroxyl functional group resin such as 2-CTC Resin (S=1.0 mmol / g~1.4mmol / g), add it to the Fmoc-Tyr-OH solution and stir evenly, add condensation agent HOBt / DIC, control the reaction temperature at 30℃, couple the reaction for 200 minutes, filter and wash to obtain Fmoc-Tyr-2-Cl-TrtResin resin.

[0085] The above resin was deprotected and washed, and the next amino acid activation preparation was carried out in the process. 15.0 mol Fmoc-Val-OH and 15.0 mol HOBT were dissolved in an appropriate amount of DMF; 15.0 mol DIC was also taken and slowly added to the DMF solution under stirring, and it was activated and set aside.

[0086] Then add the pretreated Fmoc-Val-OH / HOBt / DIC / DMF solution to the deprotected and washed peptide resin for condensation reaction. Repeat the condensation cycle starting from the second amino acid at the C-terminus (amino acid pretreatment, peptide resin washing, deprotection, washing, adding pretreated amino acid condensation reaction, and then washing the resin as one cycle), and finally wash again to obtain the peptide resin: Ac-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)-2-Cl-Trt Resin.

[0087] 2) Peptide resin cleavage of acetyl tetrapeptide-2

[0088] Take the prepared peptide resin of acetyl tetrapeptide-2, the cleavage agent is trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) or triisopropylsilane (TIS) and water (H2O), the mass volume ratio of peptide resin to cleavage agent is 1g:15ml, stir evenly, react at 30℃ for 6.0 hours, filter the reaction mixture with a sand core funnel, collect the filtrate, combine the filtrate and concentrate under reduced pressure, add methyl tert-butyl ether (MBTE) for precipitation, wash with MBTE for 3 to 5 times, evaporate MTBE, and dry the crude product under reduced pressure at room temperature to constant weight.

[0089] The crude product was dissolved with an appropriate amount of aqueous solution. During the dissolution process, the pH was controlled at about 8.5 with 5% dilute ammonia water while adding the sample. After the solution was completely dissolved, it was filtered and then concentrated for 30 minutes to obtain the crude solution of acetyl tetrapeptide-2.

[0090] 3) Purification of crude acetyl tetrapeptide-2

[0091] The crude product of acetyl tetrapeptide-2 was extracted with 5% glycerol solution, and the pre-purified solution was prepared by ultrafiltration using a regenerated cellulose 1kD ultrafiltration membrane. The pre-purified solution was separated and purified by high performance liquid chromatography. The purification was carried out by high performance liquid chromatography. The chromatographic filler used for purification was an amino acid column with a filler particle size of 10 μm, a chromatographic column diameter of 300 mm, a length of 250 mm, and a theoretical plate number of more than 3000. After acetic acid mixed system (i.e., mobile phase: 0.5% HAC.H 2 O, acetonitrile, 0.5 mol / L NH 4 AC.H 2 O), the organic phase ratio is 2%~45%, and the qualified components are collected for purification. After the main peak is collected and concentrated, it is a colorless clear liquid. After freeze-drying, acetyl tetrapeptide-2 powder (acetate) is obtained with a purity of 99.5% and a yield of 67.3%.

[0092] Example 6 Solid Phase Synthesis of Acetyl Tetrapeptide-5

[0093] Chinese name: Acetyl tetrapeptide-5

[0094] English Name: Acetyl Tetrapeptide-5

[0095] Sequence: Ac-β-Ala-His-Ser-His-OH

[0096] 1) Synthesis of peptide resin of acetyl tetrapeptide-5

[0097] Take 9.0 mol of Fmoc-His-OH and dissolve it completely with an appropriate amount of DMF; take another 3.0 mol of hydroxyl functional group resin such as 2-CTC Resin (S=1.0 mmol / g~1.4mmol / g), add it to the Fmoc-His-OH solution and stir evenly, add condensation agent HOBt / DIC, control the reaction temperature at 20℃, couple the reaction for 400 minutes, filter and wash to obtain Fmoc-His-2-Cl-TrtResin resin.

[0098] The above resin was deprotected and washed, and the next amino acid activation preparation was carried out in the process. 9.0 mol Fmoc-Ser-OH and 9.0 mol HOBT were dissolved in an appropriate amount of DMF; 9.0 mol DIC was also taken and slowly added to the DMF solution under stirring, and it was activated and set aside.

[0099] Then add the pretreated Fmoc-Ser-OH / HOBt / DIC / DMF solution to the deprotected and washed peptide resin for condensation reaction. Starting from the second amino acid at the C-terminus, the condensation is repeated in a cycle (amino acid pretreatment, peptide resin washing, deprotection, washing, adding pretreated amino acid condensation reaction, and then washing the resin as one cycle), and finally washing again to obtain the peptide resin: Ac-β-Ala-His(Trt)-Ser(tBu)-His(Trt)-2-Cl-Trt Resin.

[0100] 2) Peptide resin cleavage of acetyl tetrapeptide-5

[0101] Take the prepared peptide resin of acetyl tetrapeptide-5, the cleavage agent is trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) or triisopropylsilane (TIS) and water (H2O), the mass volume ratio of peptide resin to cleavage agent is 1g:12ml, stir evenly, react at 20℃ with stirring for 5.0 hours, filter the reaction mixture with a sand core funnel, collect the filtrate, combine the filtrate and concentrate under reduced pressure, add methyl tert-butyl ether (MBTE) for precipitation, wash with MBTE for 3 to 5 times, evaporate MTBE, and dry the crude product under reduced pressure at room temperature to constant weight.

[0102] The crude product was dissolved with an appropriate amount of aqueous solution. During the dissolution process, the pH was controlled at about 8.5 with 5% dilute ammonia water while adding the sample. After the solution was completely dissolved, it was filtered and then concentrated for 30 minutes to obtain the crude solution of acetyl tetrapeptide-5.

[0103] 3) Purification of crude acetyl tetrapeptide-5

[0104] The crude product of acetyl tetrapeptide-5 was extracted with 0.5% mannitol solution, and the pre-purified solution was prepared by ultrafiltration using a regenerated cellulose 3kD ultrafiltration membrane package. The pre-purified solution was separated and purified by high performance liquid chromatography. The purification was carried out by high performance liquid chromatography. The chromatographic filler used for purification was a C18 filler column with a filler particle size of 20 μm, a chromatographic column diameter of 200 mm, a length of 250 mm, and a theoretical plate number of more than 3000. After acetic acid mixed system (i.e., mobile phase: 0.15% HAC.H 2 O, acetonitrile, 0.3 mol / L NH 4 AC.H 2O), the organic phase ratio is 2%~45%, and the qualified components are collected for purification. After the main peak is collected and concentrated, it is a colorless clear liquid. After freeze-drying, acetyl tetrapeptide-5 powder (acetate) is obtained with a purity of 99.6% and a yield of 68.8%.

[0105] Example 7 Solid Phase Synthesis of Acetyl Tetrapeptide-9

[0106] Chinese name: Acetyl tetrapeptide-9

[0107] English Name: Acetyl Tetrapeptide-9

[0108] Sequence: Ac-Gln-Asp-Val-His-OH

[0109] 1) Synthesis of peptide resin of acetyl tetrapeptide-9

[0110] Take 12.0 mol of Fmoc-His-OH and dissolve it completely with an appropriate amount of DMF; take another 3.0 mol of hydroxyl functional group resin such as 2-CTC Resin (S=1.0 mmol / g~1.4mmol / g), add it to the Fmoc-His-OH solution and stir evenly, add condensation agent N,N-diisopropylethylamine (DIEA), control the reaction temperature at 20℃, couple the reaction for 400 minutes, filter and wash to obtain Fmoc-His-2-Cl-Trt Resin resin.

[0111] The above resin was deprotected and washed, and the next amino acid activation preparation was carried out in the process. 12.0 mol Fmoc-Val-OH and 12.0 mol HOBT were dissolved in an appropriate amount of DMF; 12.0 mol DIC was also taken and slowly added to the DMF solution under stirring, and it was activated and set aside.

[0112] Then add the pretreated Fmoc-Val-OH / HOBt / DIC / DMF solution to the deprotected and washed peptide resin for condensation reaction. Repeat the condensation cycle starting from the second amino acid at the C-terminus (amino acid pretreatment, peptide resin washing, deprotection, washing, adding pretreated amino acid condensation reaction, and then washing the resin as one cycle), and finally wash again to obtain the peptide resin: Ac-Gln(Trt)-Asp(OtBu)-Val-His(Trt)-2-Cl-Trt Resin.

[0113] 2) Peptide resin cleavage of acetyl tetrapeptide-9

[0114] Take the prepared peptide resin of acetyl tetrapeptide-9, the cleavage agent is trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) or triisopropylsilane (TIS) and water (H2O), the mass volume ratio of peptide resin to cleavage agent is 1g:10ml, stir evenly, react at 22℃ with stirring for 5.0 hours, filter the reaction mixture with a sand core funnel, collect the filtrate, combine the filtrate and concentrate under reduced pressure, add methyl tert-butyl ether (MBTE) for precipitation, wash with MBTE for 3 to 5 times, evaporate MTBE, and dry the crude product under reduced pressure at room temperature to constant weight.

[0115] The crude product was dissolved with an appropriate amount of aqueous solution. During the dissolution process, the pH was controlled at about 8.5 with 5% dilute ammonia water while adding the sample. After the solution was completely dissolved, it was filtered and then concentrated for 30 minutes to obtain the crude solution of acetyl tetrapeptide-9.

[0116] 3) Purification of crude acetyl tetrapeptide-9

[0117] The crude product of acetyl tetrapeptide-9 was extracted with 1.0% lactitol solution, and the pre-purified solution was prepared by ultrafiltration using a regenerated cellulose 3kD ultrafiltration membrane package. The pre-purified solution was separated and purified by high-performance liquid chromatography. The purification was carried out by high-performance liquid chromatography. The chromatographic filler used for purification was a C18 filler column with a filler particle size of 10 μm, a chromatographic column diameter of 150 mm, a length of 250 mm, and a theoretical plate number of more than 3000. After acetic acid mixed system (i.e., mobile phase: 0.3% HAC.H 2 O, acetonitrile, 0.3 mol / L NH 4 AC.H 2 O), the organic phase ratio is 2%~45%, and the qualified components are collected for purification. After the main peak is collected and concentrated, it is a colorless clear liquid. After freeze-drying, acetyl tetrapeptide-9 powder (acetate) is obtained with a purity of 99.7% and a yield of 68.5%.

[0118] Comparative Example 1 Solid Phase Synthesis Method of Pentapeptide-18

[0119] Chinese name: Pentapeptide-18

[0120] English name: Pentapeptide-18

[0121] Sequence: H-Tyr-D-Ala-Gly-Phe-Leu-OH

[0122] 1) Synthesis of peptide resin of pentapeptide-18

[0123] Take 3.0 mol of Fmoc-Leu-OH and dissolve it completely with an appropriate amount of DMF. Take another 3.0 mol of hydroxyl functional group resin such as 2-CTC Resin (S=1.0 mmol / g~1.4mmol / g), add it to the Fmoc-Leu-OH solution and stir evenly. Add condensation agent N,N-diisopropylethylamine (DIEA), control the reaction temperature at 40℃, and couple the reaction for 70 minutes. Filter and wash to obtain Fmoc-Leu-2-Cl-Trt Resin resin.

[0124] The above resin was deprotected and washed, and the next amino acid activation preparation was carried out in the process. 3.0 mol Fmoc-Phe-OH and 3.0 ml O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) and 1,3-diisopropylcarbodiimide (DIC) were dissolved in an appropriate amount of DMF and activated for use.

[0125] Then add the pretreated Fmoc-Phe-OH / HBTU / DIC / DMF solution to the deprotected and washed peptide resin for condensation reaction. Repeat the condensation cycle starting from the second amino acid at the C-terminus (amino acid pretreatment, peptide resin washing, deprotection, washing, adding pretreated amino acid for condensation reaction, and then washing the resin as one cycle), and finally wash again to obtain the peptide resin: Tyr(tBu)-D-Ala-Gly-Phe-Leu-2-Cl-Trt Resin.

[0126] 2) Peptide resin cleavage of pentapeptide-18

[0127] Take the prepared peptide resin of pentapeptide-18, the cleavage agent is trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) or triisopropylsilane (TIS) and water (H2O), the mass volume ratio of peptide resin to cleavage agent is 1g:3ml, stir evenly, react at 45℃ for 1.0 hour, filter the reaction mixture with a sand core funnel, collect the filtrate, combine the filtrate and concentrate under reduced pressure, add methyl tert-butyl ether (MBTE) for precipitation, wash with MBTE for 3 to 5 times, evaporate MTBE, and dry the crude product under reduced pressure at room temperature to constant weight.

[0128] The crude product was dissolved with an appropriate amount of aqueous solution. During the dissolution process, the pH was controlled at about 8.5 with 5% dilute ammonia water while adding the sample. After the solution was completely dissolved, it was filtered and then concentrated for 30 minutes to obtain a crude solution of pentapeptide-18.

[0129] 3) Purification of crude pentapeptide-18

[0130] The purification was carried out by high performance liquid chromatography, and the chromatographic filler used for purification was a C18 filler column, the filler particle size was 10 μm, the chromatographic column diameter was 100 mm, the length was 250 mm, and the theoretical plate number was greater than 3000. 2 O, acetonitrile, 0.05 mol / L NH 4 AC.H 2 O), the organic phase ratio is 1%~60%, and the qualified components are collected for purification. After the main peak is collected and concentrated, it is a colorless clear liquid. After freeze-drying, pentapeptide-18 powder (acetate) is obtained with a purity of 97.2% and a yield of 45.1%.

[0131] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A solid phase synthesis method for peptides, characterized in that: 1) Synthesizing by peptide resin, condensing peptide chains sequentially from the first amino acid at the C-terminus, washing and drying to obtain peptide resin; 2) The peptide resin is subjected to a cleavage reaction, concentrated, precipitated and dried to obtain a crude peptide; 3) The crude peptide is pretreated and purified by high performance liquid chromatography; The pretreatment process comprises extracting the crude peptide with a 0.5%-1.0% sugar alcohol solution, ultrafiltration to prepare a pre-purified solution, and separating and purifying the pre-purified solution by high performance liquid phase method; the peptide is selected from acetyl tetrapeptide-5 or acetyl tetrapeptide-9; The sugar alcohol is selected from lactitol and / or mannitol; and the ultrafiltration is selected from regenerated cellulose 1-5kD ultrafiltration membrane package.

2. The solid phase synthesis method of peptides according to claim 1, characterized in that: The resin is CTC Resin, and the substitution value range is 1.0mmol / g to 1.4mmol / g; in the synthesis of the peptide resin, the condensing agent is selected from one or more of carbodiimide type, phosphorus cation type, urea cation type, imine cation type, and organic phosphorus condensing agent.

3. The solid phase synthesis method of peptides according to claim 2, characterized in that: The condensing agent is selected from one or more of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), chloro-1-hydroxybenzotriazole (Cl-HOBt), 1-hydroxybenzotriazole (HOBT), and / or 1,3-diisopropylcarbodiimide (DIC).

4. The solid phase synthesis method of peptides according to claim 1, characterized in that: The peptide resin synthesis includes a coupling step, in which the molar ratio of Fmoc-protected amino acid to resin is (2.0-5.0):1, and the coupling step is carried out in N,N-dimethylformamide solution, the coupling temperature is 15-30°C, and the coupling time is 200-800min.

5. The solid phase synthesis method of peptides according to claim 1, characterized in that: The mass-volume ratio of the peptide resin to the cleavage agent is 1g:5ml-20ml; the cleavage agent includes a thiol compound, an acid or a phenolic compound, water, and a silane derivative; and the cleavage condition is 10-45°C for 1-10 hours.

6. The solid phase synthesis method of peptides according to claim 5, characterized in that: The mass-volume ratio of the peptide resin to the cleavage agent is 1 g: 6 ml to 15 ml; the cleavage agent includes water, trifluoroacetic acid, 1,2-ethanedithiol and triisopropylsilane; The lysis conditions are 15-30°C for 2-6 hours.

7. The solid phase synthesis method of peptides according to claim 1, characterized in that: The high performance liquid chromatography separation adopts C8, C18 filler chromatographic column and / or amino acid column, the filler particle size is 5-20 μm, the chromatographic column diameter is greater than 100 mm, the length is greater than 250 mm, and the theoretical plate number is greater than 3000; When a C18 filler chromatographic column is used, the mobile phase adopts an acetic acid mixed system, which includes 0.02% to 1.0% acetic acid solution, acetonitrile and 0.05mol / L to 1.0mol / L ammonium acetate solution, and the organic phase ratio is 1% to 60%.

Images