Synthesis method of tyrosine sulfonated polypeptide growth factor

Abstract

The invention provides a process route for industrially producing a plant sulfonated peptide growth factor, which comprises the following steps: synthesizing a molecular block by using a liquid phase method, constructing the molecular block into polypeptide by using a solid phase method to obtain polypeptide containing Tyr (SO2F), converting the Tyr (SO2F) of the polypeptide into Tyr (SO3H) under an alkaline condition, and purifying the product to obtain tyrosine sulfonated polypeptide. According to the process route, a common structure of a series of sulfonated peptide growth factors is comprehensively considered, intermediate polypeptide is constructed by using molecular building blocks and then is converted into sulfonated polypeptide, the advantages of a solid phase and a liquid phase are comprehensively considered, meanwhile, exposure of sulfonated growth factor products to high-concentration TFA is avoided, the whole reaction condition is mild, repeatability is high, raw materials are cheap and easy to obtain, and the method is suitable for industrial production. The development of large-scale industrial production is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide synthesis, and specifically relates to a new process route for sulfonating pure natural plant growth factors. Background Art

[0002] In 1991, scientists discovered a class of plant polypeptide growth factors through research on injured Solanaceae plants. After nearly 30 years of continuous efforts, nearly 43 types of polypeptide plant growth factors have been discovered. Among them, polypeptide growth factors in which some amino acid side chains have been modified are called post-translationally modified polypeptide growth factors.

[0003] In 1996, Japanese scientists used asparagus leaf flesh cell suspension culture to find that the conditioned medium after high-density culture could stimulate the low-density culture cells that did not divide to undergo mitosis, and this effect is unmatched by known plant hormones. After chromatography and HPLC purification, the purified product was identified by amino acid sequence analysis and rapid atom bombardment mass spectrometry to determine its molecular structure. It is mainly a tyrosine sulfonated polypeptide PSK series growth factor, which is also the first sulfonated modified plant polypeptide growth factor discovered. Its structure is: Tyr(SO 3 H)-Ile-Tyr(SO 3 H)-Thr-Gln-OH (PSK-α, Phytosulfokine-α), Tyr(SO 3 H)-Ile-Tyr(SO 3 H)-Thr-OH (PSK-β, Phytosulfokine‐β).

[0004] Scientists have found that plants synthesize sulfonated polypeptide growth factors by transferring the sulfonate group on PAPs (3'-phosphoadenosine transfer sulfate 5'-phosphosulfate) to the side chain hydroxyl group of tyrosine in proteins under the action of sulfotransferase (TPST) to form sulfonated modification of tyrosine. The more eye-catching plant growth factors in nature also include the RGFs / GLVs / PSYs / CIF series of growth factors. The common feature of these growth factors is that the side chain of tyrosine in the polypeptide sequence has undergone sulfonation modification. Taking RGF1 and GLV8 as examples, their structures can be expressed as follows: RGF1: Asp-Tyr(SO 3 H)-Ser-Asn-Pro-Gly-His-His-Pro-Pro-Arg-His-Asn-OH GLV8: Asp-Tyr(SO 3 H)-Gln-Gly-Pro-Lys-Pro-Arg-Ser-Lys-Pro-Leu-Lys-Asn-OH

[0005] After the tyrosine in the polypeptide structure is sulfonated on the side chain, the negatively charged sulfonate plays a very critical role in the extracellular biomolecular interaction of the organism, such as cell adhesion, leukocyte transport, hormone activity and immune response. These natural polypeptide growth factors after sulfonation are involved in controlling the development, growth regulation and reproductive process of the root and stem apical meristem, can effectively promote the division and growth of plant roots, stems, leaves and fruits, can control the ripening of fruits, can increase the fiber length of cotton, can play a synergistic role in improving the synthesis of paclitaxel, and tyrosine sulfonated polypeptides can also stimulate the plant immune system to prevent diseases and insect pests. Sulfonated polypeptide plant growth factors have attracted widespread attention from industry insiders because of their excellent performance. The call for the industrialization of such polypeptides has become increasingly high. So far, the main reported synthesis method is biological recombination fermentation, which is not conducive to large-scale production and has brought great difficulties to the promotion and use of products. In addition, since sulfonated peptides are easily decomposed under acidic conditions, if conventional peptide solid-phase synthesis methods are used, the strong acid used in the process of removing the protecting group will simultaneously remove the sulfonic acid group. Therefore, this method will greatly reduce the yield and make post-processing very difficult, making large-scale production impossible.

[0006] The present invention provides a novel method for synthesizing a tyrosine sulfonated polypeptide growth factor. The method has low cost, high yield and simple operation, combines the advantages of a solid phase and a liquid phase, synthesizes molecular building blocks first and then constructs a polypeptide structure, can avoid the problem that the sulfonated polypeptide is unstable when exposed to acid, and efficiently produces the sulfonated growth factor. Summary of the invention

[0007] One purpose of the present invention is to solve the limitations of the existing process and provide a process route for the industrialization of tyrosine sulfonated polypeptide growth factors.

[0008] The product solution is mainly achieved through the following technical solutions. Its characteristics are: the growth factors are all with Tyr (SO 3 H) structure, synthesizing a universal molecular building block P-Tyr(SO 2 F)-OH, and then the universal molecular building blocks are constructed into different polypeptide structures, and then converted into sulfonated growth factor peptides under alkaline conditions.

[0009] Step 1, synthesis of P-Tyr(SO 2 F) -OH as a molecular building block.

[0010] Step 2, the molecular building blocks obtained in step 1 are constructed into different polypeptide structures to form key intermediates.

[0011] Step 3, the Tyr(SO2 F) The polypeptide is hydrolyzed into sulfonated polypeptide under alkaline conditions.

[0012] After the above steps are completed, the product can be purified and freeze-dried to obtain the final product. The obtained crude product is purified by high performance liquid preparative chromatography, the chromatographic column uses C4, C8 or C18 as the column filler, and the mobile phase uses one or a combination of acetic acid system, ammonium acetate system, TEAP system, and ammonia system.

[0013] In the process of synthesizing the molecular building block in step 1, P is Boc or Fmoc, and Boc-Tyr-OH or Fmoc-Tyr-OH is used as the raw material. The obtained molecular building blocks are Boc-Tyr(SO 2 F)-OH or Fmoc-Tyr(SO 2 F)-OH, SO 2 F 2 Gas is used as an acyl fluorination reaction reagent, boric acid, PBS, sodium bicarbonate, sodium carbonate is used as a base, dichloromethane, ethyl acetate, acetonitrile, tetrahydrofuran, cyclohexane, dioxane is used as a solvent, and the reaction temperature is 15-40°C.

[0014] In step 2, Wang resin or CTC resin is selected as a carrier for constructing a polypeptide sequence, and the degree of substitution of the resin is 0.4-1.5 mmol / g. It mainly includes de-Fmoc protection and coupling reaction. Piperidine, DBU or methyl piperidine are selected for de-Fmoc protection. The condensation reagents selected for the condensation reaction include DIC, DCC, HBTU, HATU, PyBOP, PyAOP, TBTU. NMM, DIEA, TEA are selected as bases. The condensation reagents and bases are used in combination.

[0015] In the process of constructing the polypeptide sequence, except for Tyr which is a molecular building block, other protected amino acids are Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Boc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, and Fmoc-Val-OH.

[0016] In step 2, after the peptide is constructed on the resin, the cleavage reagent is a mixed component of concentrated TFA, the TFA content is 85-95%, and the others include one or a mixture of TIS, water, EDT, phenol, TES, and DODT.

[0017] The product obtained in step 2 is: PSK-α(SO 2 F): Tyr(SO 2 F)-Ile-Tyr(SO 2 F)-Thr-Gln-OH CIF1(SO 2 F): Asp-Tyr(SO 2 F)-Gly-Asn-Asn-Ser-Pro-Ser-Pro-Arg-Leu-Glu-Arg-Pro-Pro-Phe-Lys-Leu-Ile-Pro-Asn-OH RGF1(SO 2 F): Asp-Tyr(SO 2 F)-Ser-Asn-Pro-Gly-His-His-Pro-Pro-Arg-His-Asn-OH GLV8(SO 2 F): Asp-Tyr(SO 2 F)-Gln-Gly-Pro-Lys-Pro-Arg-Ser-Lys-Pro-Leu-Lys-Asn-OH PSY1(SO 2 F): Asp-Tyr(SO 2 F)-Gly-Asp-Pro-Ser-Ala-Asn-Pro-Lys-His-Asp-Pro-Gly-Val-Pro-Pro-Ser-OH

[0018] In step 3, preferably, the solvent is one of ethylene glycol, methanol, ethanol, acetonitrile, tetrahydrofuran, dioxane, water, dichloromethane, ethyl acetate, DMF, or a mixture of several thereof.

[0019] In step 3, preferably, pyridine, cesium carbonate, DBU, DMAP, piperidine, potassium carbonate, lithium carbonate, sodium carbonate are used as the base, and the amount of the base used is 2-20 times that of the polypeptide.

[0020] In step 3, the structural formula of the product is: PSK-α: Tyr(SO 3 H)-Ile-Tyr(SO3 H)-Thr-Gln-OH CIF1: Asp-Tyr(SO 3 H)-Gly-Asn-Asn-Ser-Pro-Ser-Pro-Arg-Leu-Glu-Arg-Pro-Pro-Phe-Lys-Leu-Ile-Pro-Asn-OH RGF1: Asp-Tyr(SO 3 H)-Ser-Asn-Pro-Gly-His-His-Pro-Pro-Arg-His-Asn-OH GLV8: Asp-Tyr(SO 3 H)-Gln-Gly-Pro-Lys-Pro-Arg-Ser-Lys-Pro-Leu-Lys-Asn-OH PSY1: Asp-Tyr(SO 3 H)-Gly-Asp-Pro-Ser-Ala-Asn-Pro-Lys-His-Asp-Pro-Gly-Val-Pro-Pro-Ser-OH

[0021] The advantages of this process are as follows: 1. This type of growth factor has Tyr (SO 3 H) This feature, the liquid phase method is used to first synthesize molecular building blocks, and the solid phase method is used to assemble and construct different growth factor precursors, and then the precursors are converted into growth factors in solution, combining the advantages of solid phase and liquid phase, increasing the production scale and shortening the production cycle. 2. The hydrolysis process and the subsequent treatment conditions are all very warm alkaline conditions, with a high conversion rate, which can effectively avoid the problem of instability of sulfonated polypeptide growth factor series products under acidic conditions. DETAILED DESCRIPTION

[0022] In order to explain the process technology scheme and its advantages in the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] Since the patent protected in this article involves multiple series of plant sulfonated peptides, we use a representative synthesis process to describe each series of plant sulfonated peptides. The process of the representative compound is also fully applicable to other sulfonated growth factor peptides in the series.

[0024] The abbreviations used in the present invention are compared as follows:

[0025] Some typical examples are given below to illustrate our inventive ideas. The description of the sulfonated peptides in the examples is carried out according to the step description in the invention content.

[0026] Example 1: Molecular building block Boc-Tyr(SO 2 Synthesis of F)-OH

[0027] Boc-Tyr-OH (10 g, 35.5 mmol) was added to dichloromethane (500 mL), and after stirring to dissolve, saturated boric acid solution (500 mL) was added. The heterogeneous solution was vigorously stirred for 10 minutes, and the air in the solution was replaced by gas replacement. SO 2 F 2 After 3 hours of reaction, a large amount of white solid precipitated. After removing dichloromethane by rotary evaporation at 35°C, the obtained white solid suspension solution was filtered. The filter cake was washed with water 4 times, and the white solid was placed in a blast drying oven at 60°C and dried overnight. The white powder solid Boc-Tyr(SO 2 F) -OH 10.7 g, yield 82.9%.

[0028] Example 2: Molecular building block Fmoc-Tyr(SO 2 Synthesis of F)-OH

[0029] Fmoc-Tyr-OH (9.8 g, 24.3 mmol) was added to dichloromethane (200 mL), stirred to dissolve, and then saturated boric acid solution (700 mL) was added. The obtained heterogeneous solution was vigorously stirred for 10 minutes, and the air in the solution was replaced by gas replacement. SO 2 F 2 After 3 hours of reaction, a large amount of white solid precipitated. 1M hydrochloric acid solution (100 mL) was added to the reaction solution. After removing dichloromethane by rotary evaporation at 35°C, the obtained white solid suspension solution was filtered. The filter cake was washed with water 4 times, and the white solid was placed in a blast drying oven at 60°C and dried overnight. Then, it was taken out to obtain a white powder solid Fmoc-Tyr(SO 2 F) -OH 9.2g, yield 78%.

[0030] Example 3: Tyr(SO 3 H)-Ile-Tyr(SO 3 Synthesis of H)-Thr-Gln-OH (PSK-α)

[0031] 3.1 Tyr(SO 2 F)-Ile-Tyr(SO 2 Synthesis of F)-Thr-Gln-OH

[0032] 3.1.1 Synthesis of amino acid resin: Fmoc-Gln(Trt)-OH (6.7 g, 11 mmol) was added to dichloromethane (100 mL), and the mixture was stirred to dissolve. CTC resin (1.10 mmol / g, 5 g, 5.5 mmol) was added to the above reaction solution, and DIEA (3.6 g, 27.5 mmol) was added. The reaction solution was stirred at room temperature for 3 hours, and then 5 mL of methanol was added. After the reaction was continued for 15 minutes, the reaction solution was drained, washed with dichloromethane (100 mL x 3), and then washed with MTBE (100 mL x 2), and dried to obtain 8.1 g of Fmoc-Gln(Trt)-CTC resin. The degree of substitution was measured by absorptiometry to be 0.64 mmol / g.

[0033] 3.1.2 Deprotection of the first amino acid: Fmoc-Gln(Trt)-CTC resin (8.1 g, 5.2 mmol) was suspended in DMF (50 mL). After swelling for 1 hour, the Fmoc deprotection step was started. A 20% piperidine solution in DMF (80 mL) was added to the resin. After reacting for 30 minutes under stirring, the reaction solution was drained and washed with DMF (60 mL x 5) before the second amino acid coupling reaction was carried out.

[0034] 3.1.3 Second amino acid coupling: Fmoc-Thr(tBu)-OH (4.1 g, 10.4 mmol), HOBt (1.4 g, 10.4 mmol) and NMM (1.05 g, 10.4 mmol) were added to the resin and stirred. HBTU (3.7 g, 9.9 mmol) was added and stirred for 1 hour. The reaction was detected by the ninhydrin method. The reaction was continued for 10 minutes and then the reaction solution was drained and washed with DMF (100 mL x 4).

[0035] 3.1.4 Deprotection of the second amino acid: Add 20% piperidine in DMF solution (60 mL) to the resin, react for 30 minutes under stirring, drain the reaction solution, and wash with DMF (60 mL x 5).

[0036] 3.1.5 The third amino acid building block Fmoc-Tyr(SO 2 F)-OH coupling: Add Fmoc-Tyr(SO 2 F)-OH (5.1 g, 10.4 mmol), HOBt (1.4 g, 10.4 mmol) and NMM (1.05 g, 10.4 mmol) were added and stirred, and HBTU (3.7 g, 9.9 mmol) was added and stirred for 1 hour. The reaction was detected to be complete by the ninhydrin method. The reaction was continued for 10 minutes, and then the reaction solution was drained and washed with DMF (100 mL x 4).

[0037] 3.1.6 Deprotection of the third amino acid molecular building block: Add 20% piperidine DMF solution (80 mL) to the resin, react for 30 minutes under stirring, drain the reaction solution, and wash with DMF (60 mL x 5).

[0038] 3.1.7 The fourth amino acid coupling: Fmoc-Ile-OH (3.7 g, 10.4 mmol), HOBt (1.4 g, 10.4 mmol) and NMM (1.05 g, 10.4 mmol) were added to the resin and stirred. HBTU (3.7 g, 9.9 mmol) was added and stirred for 1 hour. The reaction was detected to be complete using the ninhydrin method. The reaction was continued for 10 minutes and then the reaction solution was drained and washed with DMF (100 mL x 4).

[0039] 3.1.8 Deprotection of the fourth amino acid: Add 20% piperidine DMF solution (80 mL) to the resin, react for 30 minutes under stirring, drain the reaction solution, and wash with DMF (60 mL x 5).

[0040] 3.1.9 The fifth amino acid building block Boc-Tyr(SO 2 F)-OH coupling: Add Boc-Tyr(SO 2 F)-OH (3.78 g, 10.4 mmol), HOBt (1.4 g, 10.4 mmol) and NMM (1.05 g, 10.4 mmol) were stirred, and DIC (1.3 g, 10.4 mmol) was added and stirred for 1 hour. The reaction was detected to be complete by the ninhydrin method. After the reaction was continued for 10 minutes, the reaction solution was drained and washed with DMF (100 mL x 4). After washing with DCM (60 mL x 3) and MTBE (60 mL x 2), it was dried to obtain Boc-Tyr (SO 2 F)-Ile-Tyr(SO 2 F) -Thr(tBu)-Gln(Trt)-CTC resin.

[0041] 3.1.10 Cleavage of peptide resin: Add cleavage solution (TFA:TIS:H 2 O=95:2.5:2.5, 200 mL), stirred at room temperature for 2 hours and filtered, precipitated with ether (200 mL) and washed to obtain the PSK intermediate Tyr(SO 2 F)-Ile-Tyr(SO 2 F) -Thr-Gln-OH 4.2 g, yield 95%, purity 95.04%.

[0042] 3.2Tyr(SO 2F)-Ile-Tyr(SO 2 F)-Thr-Gln-OH hydrolysis: Tyr(SO 2 F)-Ile-Tyr(SO 2 F)-Thr-Gln-OH (4.2 g, 4.95 mmol) was dissolved in 45 mL of ethylene glycol and stirred at room temperature until dissolved (about 10 minutes). Cesium carbonate (8 g, 24.8 mmol) was added to the reaction solution in batches. The solution gradually became clear and transparent. After all the cesium carbonate solid was added, the reaction solution was stirred at room temperature. The reaction was monitored by HPLC and samples were taken every 30 minutes until the raw material content in the reaction solution was less than 1%, and the reaction was terminated.

[0043] 3.3Tyr(SO 3 H)-Ile-Tyr(SO 3 Purification of H)-Thr-Gln-OH: After diluting the reaction solution with 5 times the volume of water, purify it by preparative high performance liquid chromatography, collect the components with a purity of more than 95%, re-inject it into the preparative column, wash 2 column volumes with acetic acid system, rinse the chromatographic column with 30% acetonitrile / acetic acid solution to elute the product, and collect the eluate by freeze drying to obtain the product Tyr(SO 3 H)-Ile-Tyr(SO 3 H)-Thr-Gln-OH(PSK-α) 3.6 g, yield 85.7%, purity 98.4%.

[0044] Example 4: Asp-Tyr(SO 3 Synthesis of H-Gly-Asn-Asn-Ser-Pro-Ser-Pro-Arg-Leu-Glu-Arg-Pro-Pro-Phe-Lys-Leu-Ile-Pro-Asn-OH (CIF1)

[0045] 4.1Asp-Tyr(SO 2 F) Synthesis of -Gly-Asn-Asn-Ser-Pro-Ser-Pro-Arg-Leu-Glu-Arg-Pro-Pro-Phe-Lys-Leu-Ile-Pro-Asn-OH.

[0046] 4.1.1 Synthesis of amino acid resin: Fmoc-Asn(Trt)-OH (1.2 g, 2 mmol) was added to dichloromethane (15 mL), and stirred to dissolve. CTC resin (1.00 mmol / g, 1 g, 1 mmol) was added to the above reaction solution, and DIEA (0.65 g, 5 mmol) was added. The reaction solution was stirred at room temperature for 2.5 hours, and 1 mL of methanol was added. After continuing the reaction for 15 minutes, the reaction solution was drained, washed with dichloromethane (20 mL x 3), and then washed with MTBE (20 mL x 2). After drying, 1.5 g of Fmoc-Asn(Trt)-CTC resin was obtained. The degree of substitution was measured by absorptiometry to be 0.61 mmol / g.

[0047] 4.1.2 Deprotection of the first amino acid: Fmoc-Asn(Trt)-CTC resin (1.5 g, 0.91 mmol) was suspended in DMF (10 mL). After swelling for 1 hour, the Fmoc deprotection step was started. A 20% piperidine solution in DMF (20 mL) was added to the resin. After reacting for 30 minutes under stirring, the reaction solution was drained and washed with DMF (20 mL x 5) before proceeding to the next step.

[0048] 4.1.3 Construction of the peptide chain of the second to nineteenth amino acids: Fmoc-Pro-OH (0.61 g, 1.82 mmol), HOBt (0.25 g, 1.82 mmol) and NMM (0.24 g, 1.82 mmol) were added to the resin and stirred. HBTU (0.66 g, 1.73 mmol) was added and stirred for 1 hour. The reaction was detected to be complete using the ninhydrin method. The reaction was continued for 10 minutes and then the reaction solution was drained and washed with DMF (100 mL x 4). Repeat the amino acid coupling and deprotection steps, and then add Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-O H, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, and Fmoc-Gly-OH were constructed on the resin to obtain Gly-Asn(Trt)-Asn(Trt)-Ser(tBu)-Pro-Ser(tBu)-Pro-Arg(Pbf)-Leu-Glu(OtBu)-Arg(Pbf)-Pro-Pro-Phe-Lys(Boc)-Leu-Ile-Pro-Asn(Trt)-CTC resin.

[0049] 4.1.4 The twentieth amino acid building block Fmoc-Tyr(SO 2 F)-OH coupling: Add Fmoc-Tyr(SO 2 F)-OH (0.88 g, 1.82 mmol), HOBt (0.25 g, 1.82 mmol) and DIC (0.23 g, 1.82 mmol) were added and stirred for 2 hours. The reaction was detected to be complete by the ninhydrin method. The reaction was continued for 10 minutes and then the reaction liquid was drained and washed with DMF before the next step of reaction.

[0050] 4.1.5 The twentieth amino acid Fmoc-Tyr(SO 2 F) Deprotection of -OH: Add 20% piperidine DMF solution (20 mL) to the resin, react for 30 minutes under stirring, drain the reaction solution, and wash with DMF (60 mL x 5).

[0051] 4.1.6 Coupling of the twenty-first amino acid Boc-Asp(OtBu)-OH: Boc-Asp(OtBu)-OH (0.53 g, 1.82 mmol), HOBt (0.25 g, 1.82 mmol) and NMM (0.24 g, 1.82 mmol) were added to the resin and stirred. HBTU (0.66 g, 1.73 mmol) was added and stirred for 1 hour. The reaction was detected by the ninhydrin method. After the reaction was continued for 10 minutes, the reaction solution was drained, washed with DMF (50 mL x 4) and then dried with MTBE (50 mL x 3) to obtain Boc-Asp(OtBu)-Tyr(SO 2 F) -Gly-Asn(Trt)-Asn(Trt)-Ser(tBu)-Pro-Ser(tBu)-Pro-Arg(Pbf)-Leu-Glu(OtBu)-Arg(Pbf)-Pro-Pro-Phe-Lys(Boc)-Leu-Ile-Pro-Asn(Trt)-CTC resin.

[0052] 4.1.7 Peptide resin cleavage: Peptide resin is added to the cleavage solution (TFA:TIS:EDT:H 2 O=90:4:4:2, 10mL), stirred at room temperature for 2 hours, filtered and washed with TFA (2mLx2), the peptide in the filtrate was precipitated with MTBE (40mL), centrifuged and washed twice, and then dried to obtain Asp-Tyr(SO 2 F)-Gly-Asn-Asn-Ser-Pro-Ser-Pro-Arg-Leu-Glu-Arg-Pro-Pro-Phe-Lys-Leu-Ile-Pro-Asn-OH 2.3g.

[0053] 4.2Asp-Tyr(SO 2 F) Hydrolysis of -Gly-Asn-Asn-Ser-Pro-Ser-Pro-Arg-Leu-Glu-Arg -Pro-Pro-Phe-Lys-Leu-Ile-Pro-Asn-OH: Dissolve 2.3 g of the product obtained in step 4.1.7 in 20 mL of ethylene glycol and stir at room temperature until dissolved (approximately 10 minutes). Then, add cesium carbonate (2.4 g, 7.2 mmol) to the reaction solution in batches. The solution gradually becomes clear and transparent. After all the cesium carbonate solid is added, the reaction solution is stirred at room temperature and the reaction is monitored by HPLC. Samples are taken every 30 minutes until the raw material content in the reaction solution is less than 1%, then the reaction is terminated.

[0054] 4.3 Asp-Tyr(SO 3Purification of H)-Gly-Asn-Asn-Ser-Pro-Ser-Pro-Arg-Leu-Glu-Arg -Pro-Pro-Phe-Lys-Leu-Ile-Pro-Asn-OH: The reaction solution was diluted with 5 volumes of water and purified by preparative high performance liquid chromatography. The components with a purity of more than 95% were collected and re-injected into the preparative column. After washing with acetic acid system for 2 column volumes, the chromatographic column was rinsed with 30% acetonitrile / acetic acid solution to elute the product. The eluate was lyophilized and collected to obtain 1.45 g of CIF1, with a yield of 64.4% and a purity of 99.5%.

[0055] Example 5: Asp-Tyr(SO 3 H) Synthesis of -Ser-Asn-Pro-Gly-His-His-Pro-Pro-Arg-His-Asn-OH (RGF1).

[0056] 5.1 Asp-Tyr(SO 2 F) Synthesis of -Ser-Asn-Pro-Gly-His-His-Pro-Pro-Arg-His-Asn-OH.

[0057] 5.1.1 Synthesis of amino acid resin: Fmoc-Asn(Trt)-OH (3.6 g, 6 mmol) was added to dichloromethane (40 mL), and stirred to dissolve. CTC resin (1.00 mmol / g, 3 g, 3 mmol) was added to the above reaction solution, and DIEA (1.9 g, 15 mmol) was added. The reaction solution was stirred at room temperature for 2.5 hours, and 3 mL of methanol was added. After continuing the reaction for 15 minutes, the reaction solution was drained, washed with dichloromethane (50 mL x 3), and then washed with MTBE (50 mL x 2), and dried to obtain 4.6 g of Fmoc-Asn(Trt)-CTC resin. The degree of substitution was measured by absorptiometry to be 0.6 mmol / g.

[0058] 5.1.2 Deprotection of the first amino acid: Fmoc-Asn(Trt)-CTC resin (4.6 g, 2.76 mmol) was suspended in DMF (40 mL). After swelling for 1 hour, the Fmoc deprotection step was started. A 20% piperidine DMF solution (80 mL) was added to the resin. After reacting for 30 minutes under stirring, the reaction solution was drained and washed with DMF (60 mL x 5) before the amino acid coupling reaction was carried out.

[0059] 5.1.3 Construction of the peptide chain of the second to eleventh amino acids: Fmoc-His(Trt)-OH (3.4 g, 5.52 mmol), HOBt (0.75 g, 5.52 mmol) and NMM (0.71 g, 5.52 mmol) were added to the resin and stirred. HBTU (1.99 g, 5.2 mmol) was added and stirred for 1 hour. The reaction was detected by the ninhydrin method. After continuing the reaction for 10 minutes, the reaction solution was drained and washed with DMF (100 mL x 4). Repeat the common amino acid coupling and deprotection steps, and construct Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, and Fmoc-Ser(tBu)-OH onto the resin to obtain Ser(tBu)-Asn(Trt)-Pro-Gly-His(Trt)-His(Trt)-Pro-Arg(Pbf)-His(Trt)-Asn(Trt)-CTC resin.

[0060] 5.1.4 The twelfth amino acid building block Fmoc-Tyr(SO 2 F)-OH coupling: Add Fmoc-Tyr(SO 2 F)-OH (2.7 g, 5.52 mmol), HOBt (0.75 g, 5.52 mmol) and DIC (0.7 g, 5.52 mmol) were stirred for 2 hours, and the reaction was detected to be complete by the ninhydrin method. After continuing the reaction for 10 minutes, the reaction solution was drained and washed with DMF before the next step of reaction.

[0061] 5.1.5 The twelfth amino acid Fmoc-Tyr(SO 2 F) Deprotection of -OH: Add 20% piperidine DMF solution (80 mL) to the resin, react for 30 minutes under stirring, drain the reaction solution, and wash with DMF (60 mL x 5).

[0062] 5.1.6 Coupling of the thirteenth amino acid Boc-Asp(OtBu)-OH: Add Boc-Asp(OtBu)-OH (1.5 g, 5.52 mmol), HOBt (0.75 g, 5.52 mmol) and NMM (0.71 g, 5.52 mmol) to the resin and stir. Add HBTU (1.99 g, 5.2 mmol) and stir for 1 hour. Use the ninhydrin method to detect that the reaction is complete. Continue the reaction for 10 minutes and then drain the reaction solution. Wash with DMF (100 mL x 4) and then dry the resin with MTBE (100 mL x 3) to obtain Boc-Asp(OtBu)-Tyr(SO 2 F)-Ser(tBu)-Asn(Trt)-Pro-Gly-His(Trt)-His(Trt)-Pro-Pro-Arg(Pbf)-His(Trt)-Asn(Trt)-CTC resin.

[0063] 5.1.7 Peptide resin cleavage: Add peptide resin to cleavage solution (TFA:TIS:EDT:H 2 O=90:4:4:2, 10 mL), stirred at room temperature for 2 hours, filtered and washed with TFA (10 mL x 2), the peptide in the filtrate was precipitated with MTBE (100 mL), centrifuged and washed twice, and then dried to obtain Asp-Tyr (SO 2 F)-Ser-Asn-Pro-Gly-His-His-Pro-Pro-Arg-His-Asn-OH 4.3g.

[0064] 5.2Asp-Tyr(SO 2 F) Hydrolysis of Ser-Asn-Pro-Gly-His-His-Pro-Pro-Arg-His-Asn-OH: 4.3 g of Asp-Tyr(SO 2 F)-Ser-Asn-Pro-Gly-His-His-Pro-Pro-Arg-His-Asn-OH was dissolved in ethylene glycol (50 mL). After stirring at room temperature until dissolved, cesium carbonate (8.1 g, 24.84 mmol) was added to the reaction solution in batches, and the solution gradually became clear and transparent. After all the cesium carbonate solid was added, the reaction solution was stirred at room temperature and the reaction was monitored by HPLC. Samples were taken every 30 minutes until the raw material content in the reaction solution was less than 1%, and the reaction was terminated.

[0065] 5.3 Asp-Tyr(SO 3Purification of H)-Ser-Asn-Pro-Gly-His-His-Pro-Pro-Arg-His-Asn-OH: After diluting the reaction solution with 5 volumes of water, purify it by preparative high performance liquid chromatography, collect the components with a purity of more than 95%, re-inject them into the preparative column, wash 2 column volumes with acetic acid system, rinse the chromatographic column with 30% acetonitrile / acetic acid solution to elute the product, and collect the eluate by freeze drying to obtain the product Asp-Tyr(SO 3 H)-Ser-Asn-Pro-Gly-His-His-Pro-Pro-Arg-His-Asn-OH (RGF1) 2.4 g, yield 54.5%.

[0066] Example 6: Asp-Tyr(SO 3 H) Synthesis of -Gln-Gly-Pro-Lys-Pro-Arg-Ser-Lys-Pro-Leu-Lys-Asn-OH (GLV8).

[0067] 6.1 Asp-Tyr(SO 2 F) Synthesis of -Gln-Gly-Pro-Lys-Pro-Arg-Ser-Lys-Pro-Leu-Lys-Asn-OH.

[0068] 6.1.1 Synthesis of amino acid resin: Fmoc-Asn(Trt)-OH (6 g, 10 mmol) was added to dichloromethane (50 mL), and stirred to dissolve. CTC resin (1.00 mmol / g, 5 g, 5 mmol) was added to the above reaction solution, and DIEA (3.2 g, 25 mmol) was added. The reaction solution was stirred at room temperature for 2.5 hours, and 5 mL of methanol was added. After continuing the reaction for 15 minutes, the reaction solution was drained, washed with dichloromethane (70 mL x 3), and then washed with MTBE (70 mL x 2), and dried to obtain 7.7 g of Fmoc-Asn(Trt)-CTC resin. The degree of substitution was measured by absorptiometry to be 0.61 mmol / g.

[0069] 6.1.2 Deprotection of the first amino acid: Fmoc-Asn(Trt)-CTC resin (7.7 g, 4.7 mmol) was suspended in DMF (60 mL). After swelling for 1 hour, the Fmoc deprotection step was started. A 20% piperidine solution in DMF (100 mL) was added to the resin. After reacting for 30 minutes under stirring, the reaction solution was drained and washed with DMF (80 mL x 5) before proceeding to the next step.

[0070] 6.1.3 Construction of the peptide chain of the second to twelfth amino acids: Add Fmoc-Lys(Boc)-OH (6.6 g, 14.1 mmol), HOBt (1.9 g, 14.1 mmol) and NMM (1.4 g, 14.1 mmol) to the resin and stir. Add HBTU (5 g, 13.4 mmol) and stir for 1 hour. Use the ninhydrin method to detect that the reaction is complete. Continue the reaction for 10 minutes, then drain the reaction solution and wash with DMF (120 mL x 4). Repeat the amino acid coupling and deprotection steps, and then construct Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, and Fmoc-Gln(Trt)-OH onto the resin to obtain Gln(Trt)-Gly-Pro-Lys(Boc)-Pro-Arg(Pbf)-Ser(tBu)-Lys(Boc)-Pro-Leu-Lys(Boc)-Asn(Trt)-CTC resin.

[0071] 6.1.4 Thirteenth amino acid building block Fmoc-Tyr(SO 2 F)-OH coupling: Add Fmoc-Tyr(SO 2 F) -OH (6.8 g, 14.1 mmol), HOBt (1.9 g, 14.1 mmol) and DIC (1.8 g, 14.1 mmol) were added and stirred for 2 hours. The reaction was detected to be complete by the ninhydrin method. The reaction was continued for 10 minutes and then the reaction liquid was drained and washed with DMF before the next step of reaction.

[0072] 6.1.5 Thirteenth amino acid Fmoc-Tyr(SO 2 F) Deprotection of -OH: Add 20% piperidine DMF solution (120 mL) to the resin, react for 30 minutes under stirring, drain the reaction solution, and wash with DMF (100 mL x 5).

[0073] 6.1.6 Coupling of the fourteenth amino acid Boc-Asp(OtBu)-OH: Add Boc-Asp(OtBu)-OH (4.1 g, 14.1 mmol), HOBt (1.9 g, 14.1 mmol) and NMM (1.4 g, 14.1 mmol) to the resin and stir. Add HBTU (5.1 g, 13.4 mmol) and stir for 1 hour. Use the ninhydrin method to detect that the reaction is complete. Continue the reaction for 10 minutes and then drain the reaction solution. Wash with DMF (120 mL x 4) and then dry the resin with MTBE (120 mL x 3) to obtain Boc-Asp(OtBu)-Tyr(SO 2 F) -Gln(Trt)-Gly-Pro-Lys(Boc)-Pro-Arg(Pbf)-Ser(tBu)-Lys(Boc)-Pro-Leu-Lys(Boc)-Asn(Trt)-CTC resin.

[0074] 6.1.7 Peptide resin cleavage: Add peptide resin to cleavage solution (TFA:TIS:EDT:H 2 O=90:4:4:2, 10 mL), stirred at room temperature for 2 hours, filtered and washed with TFA (15 mL x 2), the peptide in the filtrate was precipitated with MTBE (180 mL), centrifuged and washed twice, and then dried to obtain Asp-Tyr(SO 2 F)-Gln-Gly-Pro-Lys-Pro-Arg-Ser-Lys-Pro-Leu-Lys-Asn-OH 8g.

[0075] 6.2 Asp-Tyr(SO 2 F) Hydrolysis of -Gln-Gly-Pro-Lys-Pro-Arg-Ser-Lys-Pro-Leu-Lys-Asn-OH: 8 g of Asp-Tyr(SO 2 F)-Gln-Gly-Pro-Lys-Pro-Arg-Ser-Lys-Pro-Leu-Lys-Asn-OH was dissolved in ethylene glycol (50 mL) and stirred at room temperature until dissolved (approximately 10 minutes). Cesium carbonate (7.6 g, 23.4 mmol) was added to the reaction solution in batches, and the solution gradually became clear and transparent. After all the cesium carbonate solid was added, the reaction solution was stirred at room temperature and the reaction was monitored by HPLC. Samples were taken every 30 minutes until the raw material content in the reaction solution was less than 1%, and the reaction was terminated.

[0076] 6.3 Asp-Tyr(SO 3Purification of H)-Gln-Gly-Pro-Lys-Pro-Arg-Ser-Lys-Pro-Leu-Lys-Asn-OH: After diluting the reaction solution with 5 times the volume of water, purify it by preparative high performance liquid chromatography, collect the components with a purity of more than 95%, re-inject them into the preparative column, wash 2 column volumes with acetic acid system, rinse the chromatographic column with 30% acetonitrile / acetic acid solution to elute the product, and collect the eluate by freeze drying to obtain the product Asp-Tyr(SO 3 H)-Gln-Gly-Pro-Lys-Pro-Arg-Ser-Lys-Pro-Leu-Lys-Asn-OH (GLV8) 6.3 g, yield 79%.

[0077] Example 7: Asp-Tyr(SO 3 H) Synthesis of -Gly-Asp-Pro-Ser-Ala-Asn-Pro-Lys-His-Asp-Pro-Gly-Val-Pro-Pro-Ser-OH (PSY1).

[0078] 7.1 Asp-Tyr(SO 2 F) Synthesis of -Gly-Asp-Pro-Ser-Ala-Asn-Pro-Lys-His-Asp-Pro-Gly-Val-Pro-Pro-Ser-OH.

[0079] 7.1.1 Synthesis of amino acid resin: Add Fmoc-Ser(tBu)-OH (2.3 g, 6 mmol) to dichloromethane (40 mL), stir to dissolve, add CTC resin (1.00 mmol / g, 3 g, 3 mmol) to the above reaction solution, add DIEA (1.9 g, 15 mmol), leave the reaction solution to react at room temperature with stirring for 2.5 hours, add 3 mL of methanol, continue to react for 15 minutes, drain the reaction solution, wash with dichloromethane (50 mL x 3), then wash with MTBE (50 mL x 2), and dry to obtain 4.1 g of Fmoc-Ser(tBu)-CTC resin. The degree of substitution measured by absorptiometry is 0.71 mmol / g.

[0080] 7.1.2 Deprotection of the first amino acid: Fmoc-Ser(tBu)-CTC resin (4.1 g, 2.9 mmol) was suspended in DMF (40 mL). After swelling for 1 hour, the Fmoc deprotection step was started. A 20% piperidine solution in DMF (80 mL) was added to the resin. After reacting for 30 minutes under stirring, the reaction solution was drained and washed with DMF (60 mL x 5) before the amino acid coupling reaction was carried out.

[0081] 7.1.3 Construction of the peptide chain of the second to sixteenth amino acids: Add Fmoc-Pro-OH (2.9 g, 8.7 mmol), HOBt (1.2 g, 8.7 mmol) and NMM (0.8 g, 8.7 mmol) to the resin and stir. Add HBTU (3.1 g, 8.3 mmol) and stir for 1 hour. Use the ninhydrin method to detect that the reaction is complete. Continue the reaction for 10 minutes, then drain the reaction solution and wash with DMF (100 mL x 4). Repeat the common amino acid coupling and deprotection steps, and continue to construct Fmoc-Pro-OH, Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Asp(OtBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Gly-OH onto the resin to obtain Gly-Asp(OtBu)-Pro-Ser(tBu)-Ala-Asn(Trt)-Pro-Lys(Boc)-His(Trt)-Asp(OtBu)-Pro-Gly-Val-Pro-Pro-Ser(tBu)-CTC resin.

[0082] 7.1.4 The seventeenth amino acid building block Fmoc-Tyr(SO 2 F)-OH coupling: Add Fmoc-Tyr(SO 2 F) -OH (4.2 g, 8.7 mmol), HOBt (1.2 g, 8.7 mmol) and DIC (1.1 g, 8.7 mmol) were stirred for 2 hours, and the reaction was detected to be complete by the ninhydrin method. The reaction was continued for 10 minutes, and then the reaction solution was drained and washed with DMF before the next step of reaction.

[0083] 7.1.5 The seventeenth amino acid Fmoc-Tyr(SO 2 F) Deprotection of -OH: Add 20% piperidine DMF solution (80 mL) to the resin, react for 30 minutes under stirring, drain the reaction solution, and wash with DMF (60 mL x 5).

[0084] 7.1.6 Coupling of the eighteenth amino acid Boc-Asp(OtBu)-OH: Add Boc-Asp(OtBu)-OH (2.5 g, 8.7 mmol), HOBt (1.2 g, 8.7 mmol) and NMM (0.71 g, 5.52 mmol) to the resin and stir. Add HBTU (3.1 g, 8.3 mmol) and stir for 1 hour. Use the ninhydrin method to detect that the reaction is complete. Continue the reaction for 10 minutes and then drain the reaction solution. Wash with DMF (100 mL x 4) and then dry the resin with MTBE (100 mL x 3) to obtain Boc-Asp(OtBu)-Tyr(SO 2 F)-Gly-Asp(OtBu)-Pro-Ser(tBu)-Ala-Asn(Trt)-Pro-Lys(Boc)-His(Trt)-Asp(OtBu)-Pro-Gly-Val-Pro-Pro-Ser(tBu)-CTC resin.

[0085] 7.1.7 Peptide resin cleavage: Add peptide resin to cleavage solution (TFA:TIS:EDT:H 2 O=90:4:4:2, 10 mL), stirred at room temperature for 2 hours, filtered and washed with TFA (10 mL x 2), the peptide in the filtrate was precipitated with MTBE (100 mL), centrifuged and washed twice, and then dried to obtain Asp-Tyr(SO 2 F)-Gly-Asp-Pro-Ser-Ala-Asn-Pro-Lys-His-Asp-Pro-Gly-Val-Pro-Pro-Ser-OH 5.5g.

[0086] 7.2Asp-Tyr(SO 2 Hydrolysis of F)-Gly-Asp-Pro-Ser-Ala-Asn-Pro-Lys-His-Asp-Pro-Gly-Val-Pro -Pro-Ser-OH: 5.5 g of Asp-Tyr (SO 2 F)-Gly-Asp-Pro-Ser-Ala-Asn-Pro-Lys-His-Asp-Pro-Gly-Val-Pro-Pro-Ser-OH was dissolved in ethylene glycol (50 mL) and stirred at room temperature until dissolved (approximately 10 minutes). Cesium carbonate (7.6 g, 23.3 mmol) was added to the reaction solution in batches, and the solution gradually became clear and transparent. After all the cesium carbonate solid was added, the reaction solution was stirred at room temperature and the reaction was monitored by HPLC. Samples were taken every 30 minutes until the raw material content in the reaction solution was less than 1%, and the reaction was terminated.

[0087] 7.3Asp-Tyr(SO 3 Purification of H)-Gly-Asp-Pro-Ser-Ala-Asn-Pro-Lys-His-Asp-Pro-Gly-Val-Pro -Pro-Ser-OH (PSY1): After the reaction solution was diluted with 5 volumes of water, it was purified by preparative high performance liquid chromatography, and the components with a purity of more than 95% were collected and re-injected into the preparative column. After washing with acetic acid system for 2 column volumes, the column was rinsed with 30% acetonitrile / acetic acid solution to elute the product, and the eluate was lyophilized and collected to obtain the product Asp-Tyr (SO 3 H)-Gly-Asp-Pro-Ser-Ala-Asn-Pro-Lys-His-Asp-Pro-Gly-Val-Pro-Pro-Ser-OH (PSY1) 3.8 g, yield 69%.

[0088] The process disclosed in this patent can obtain a product with a purity of more than 95% after relatively convenient purification, and the yield is 50%-90%.

Claims

1. The present invention provides a production process for producing natural plant sulfonated peptide growth factor. The following steps are involved: Step 1, synthesis of P-Tyr(SO 2 Step 2: The building blocks obtained in step 1 are constructed into different peptide structures to form key Tyr(SO 2 F) polypeptide intermediate. Step 3, the Tyr(SO 2 F) The polypeptide is dissociated into sulfonated polypeptide under alkaline conditions.

2. According to the synthesis scheme of claim 1, Its characteristics are: In the preparation process of the molecular building block, P in step 1 is an amino protecting group, preferably Boc and Fmoc, and Boc-Tyr-OH or Fmoc-Tyr-OH is used as a raw material, and SO 2 F 2 The gas was used as the reaction reagent, boric acid, PBS, sodium bicarbonate, sodium carbonate were used as the base, dichloromethane, ethyl acetate, acetonitrile, tetrahydrofuran, cyclohexane, dioxane were used as the solvent, the reaction temperature was 15-40°C, and the corresponding molecular building blocks were Boc-Tyr(SO 2 F)-OH or Fmoc-Tyr(SO 2 F)-OH.

3. According to the synthesis scheme of claim 1, Its characteristics are: In step 2, a Tyr(SO 2 F) The peptide sequence is selected to use Wang resin or CTC resin as a carrier, and the resin substitution degree is 0.4-1.5mmol / g. It mainly includes de-Fmoc protection and coupling reaction. Piperidine, DBU or methyl piperidine are used for de-Fmoc protection. The condensation reagents selected for the condensation reaction include DIC, DCC, HBTU, HATU, PyBOP, PyAOP, TBTU. NMM, DIEA, TEA are selected as bases. The condensation reagents and bases can be used in any combination.

4. According to the synthesis scheme of claims 1 and 3, Its characteristics are: Other protected amino acids are selected from Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-His(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Boc-Asp(OtBu)-OH, and Fmoc-Val-OH. After the polypeptide is constructed on the resin, the cleavage reagent is a mixed component containing TFA, and the content of TFA is 85-95%. The scavenger used includes one or a mixture of TIS, water, EDT, phenol, TES, and DODT.

5. According to the synthesis scheme of claims 1-4, the polypeptide structure constructed is characterized by the following sequence: PSK-α(SO 2 F):Tyr(SO 2 F)-Ile-Tyr(SO 2 F)-Thr-Gln-OH CIF1(SO 2 F): Asp-Tyr(SO 2 F)-Gly-Asn-Asn-Ser-Pro-Ser-Pro-Arg-Leu-Glu-Arg-Pro-Pro-Phe-Lys-Leu-Ile-Pro-Asn-OH RGF1(SO 2 F):Asp-Tyr(SO 2 F)-Ser-Asn-Pro-Gly-His-His-Pro-Pro-Arg-His-Asn-OH GLV8(SO 2 F):Asp-Tyr(SO 2 F)-Gln-Gly-Pro-Lys-Pro-Arg-Ser-Lys-Pro-Leu-Lys-Asn-OH PSY1(SO 2 F): Asp-Tyr(SO 2 F)-Gly-Asp-Pro-Ser-Ala-Asn-Pro-Lys-His-Asp-Pro-Gly-Val-Pro-Pro-Ser-OH 6. According to the synthesis scheme of claim 1, Its characteristics are: In step 3, the solvent is one or a mixture of ethylene glycol, methanol, ethanol, acetonitrile, tetrahydrofuran, dioxane, water, dichloromethane, ethyl acetate, DMF. Pyridine, cesium carbonate, DBU, DMAP, piperidine, potassium carbonate, lithium carbonate, sodium carbonate are used as the base, and the amount of the base is 2-20 times that of the polypeptide.

7. According to the synthesis scheme of claims 1-6, it is characterized in that the following polypeptide structure is obtained: PSK-α: Tyr(SO 3 H)-Ile-Tyr(SO 3 H)-Thr-Gln-OH CIF1: Asp-Tyr(SO 3 H)-Gly-Asn-Asn-Ser-Pro-Ser-Pro-Arg-Leu-Glu-Arg-Pro-Pro-Phe-Lys-Leu-Ile-Pro-Asn-OH RGF1:Asp-Tyr(SO 3 H)-Ser-Asn-Pro-Gly-His-His-Pro-Pro-Arg-His-Asn-OH GLV8: Asp-Tyr(SO 3 H)-Gln-Gly-Pro-Lys-Pro-Arg-Ser-Lys-Pro-Leu-Lys-Asn-OH PSY1: Asp-Tyr(SO 3 H)-Gly-Asp-Pro-Ser-Ala-Asn-Pro-Lys-His-Asp-Pro-Gly-Val-Pro-Pro-Ser-OH 8. According to the synthesis scheme of claims 1-7, Its characteristics are: The obtained crude product is purified by high performance liquid chromatography, the chromatographic column uses C4, C8 or C18 as filler, and one or a combination of TFA system, acetic acid system, ammonium acetate system, TEAP system, ammonia system, and perchloric acid system is used as the purification mobile phase.