Synthetic method of sulfonated polypeptide

Abstract

The invention discloses a synthetic method of sulfonated polypeptide. The synthetic method comprises the following steps: S1, synthesizing an intermediate peptide from amino acid raw materials including at least one of an amino acid monomer A with a side chain containing a-NHR1 group and an amino acid monomer B with a side chain containing a-CHR2OR3 group and a tyrosine monomer; a main chain-NH2 of an amino acid monomer serving as a nitrogen terminal in the amino acid raw material is protected as-NHR1; r1 and R3 are protecting groups which can be removed through normal-pressure hydrogenation, and R2 is selected from H and methyl; s2, selectively sulfonating tyrosine phenolic hydroxyl groups in the intermediate peptide to obtain sulfonated intermediate peptide; and S3, carrying out normal-pressure hydrogenation on the sulfonated intermediate peptide to remove R1 or R1 and R3 protecting groups to obtain the sulfonated polypeptide.

Description

Technical Field

[0001] This invention relates to the field of peptide synthesis technology, and more specifically to a synthesis method for selectively achieving peptide sulfonation after orthogonal protection. Background Technology

[0002] Sulfonated peptides are a class of bioactive small molecules modified with sulfonic acid groups, known as sulfopeptides in plant hormone research. These compounds are structural derivatives formed by introducing sulfonic acid groups (-SO3H) at specific sites on the polypeptide chain. Sulfonation modification, as an important post-translational modification, can significantly alter the physicochemical properties of peptide molecules, including but not limited to enhancing water solubility, improving membrane permeability, and regulating biological activity. Studies have shown that this modification can mimic the functional characteristics of naturally occurring sulfonated proteins, playing a crucial role in intercellular communication and molecular recognition.

[0003] In the field of plant physiological regulation, sulfopeptides represent a class of small polypeptide hormones with intercellular signal transduction functions. Four major sulfopeptide families have been identified in the model plant Arabidopsis thaliana: phytosulfonapeptide (PSK), phytosulfonapeptide Y (PSY), root growth factor (RGF), and casparian band integrity factor (CIF). Taking PSK as an example, it participates in the regulation of cell division and organ development through receptor-mediated signal transduction pathways. In recent years, with the development of plant molecular biology, the biosynthetic pathways and mechanisms of action of these sulfonated small peptides are being gradually elucidated.

[0004] From the perspective of chemical modification, sulfonation reactions mainly occur on the side chains of hydroxyl-containing amino acid residues (such as tyrosine (Tyr), serine (Ser), and threonine (Thr). Currently, the main sulfonation methods include: 1) chemical synthesis: using sulfuric acid derivatives (such as chlorosulfonic acid and sulfur trioxide complexes) as sulfonating agents; 2) enzymatic catalysis: utilizing sulfonyltransferases for biocatalysis; and 3) solid-phase synthesis: using solid-phase synthesis technology to gradually construct sulfonated peptides from complexly modified sulfonated monomers.

[0005] The following describes several existing chemical synthesis methods for sulfonated peptides: Scheme 1 (Angew. Chem. Int. Ed. 2009, 48, 2024-2026) is as follows: starting from sulfonyl chloride, a dichlorovinyl (DCV) protected sulfonated tyrosine monomer Fmoc-Tyr(SO3-DCV)-OH is synthesized through 6 steps of conversion. Then, the target sulfonated peptide precursor is synthesized in a solid phase and then hydrogenated to remove the DCV protecting group to obtain the target sulfonated peptide.

[0006] Scheme 2 (Angew. Chem. 2016, 128, 1867-1870) involves the selective sulfonation of tyrosine monomers using sulfonyl fluoride gas to synthesize fluorine-protected monomers Fmoc-Tyr(SO2F)-OH. Subsequently, the target sulfonated peptide precursor is synthesized in a solid phase, followed by hydrolysis with ethylene glycol and cesium carbonate to remove the fluorine-protecting group and obtain the target sulfonated peptide.

[0007] Scheme 3 (J. Am. Chem. Soc. 2006, 128, 1605-1610) is as follows: the isobutyl-protected sulfonated tyrosine monomer Fmoc-Tyr(SO2F)-OH is synthesized in 3 steps, followed by solid-phase synthesis of the target sulfonated peptide precursor and removal of the fluorine protecting group by sodium iodide to obtain the target sulfonated peptide.

[0008] The above technical approach involves synthesizing sulfonated tyrosine monomers with different protecting groups, followed by solid-phase synthesis to obtain sulfonated peptide precursors, and then selectively deprotecting them to synthesize the target sulfonated peptide. However, solid-phase peptide synthesis requires a significant excess of amino acid monomers to ensure purity and efficiency. Furthermore, the synthesis of sulfonated monomers itself involves multiple steps, complex operations, and expensive or toxic reagents (sulfonyl fluoride is highly toxic and requires strict regulation), further increasing the overall economic, time, and labor costs of sulfonated peptide synthesis. Additionally, under the solid-phase synthesis conditions in Scheme 1, the piperidine solution used in conventional Fmoc-SPPS conditions significantly increases the degradation and shedding of sulfonic acid groups during Fmoc protecting group removal, leading to increased side reactions. Therefore, sterically hindered 2-methylpiperidine is needed as a substitute. Under the solid-phase synthesis conditions in Scheme 2, some sulfonyl fluoride undergoes hydrolysis.

[0009] Existing technologies typically have the following limitations: (1) Direct sulfonation requires harsh reaction conditions (strong acid, high temperature), which can easily lead to polypeptide chain degradation; (2) The synthesis route of pre-sulfonated amino acid monomers is complex and the yield is low; (3) The sulfonic acid group protection / deprotection step in solid-phase synthesis affects the final yield; (4) It is difficult to achieve selective sulfonation at multiple sites.

[0010] Selective sulfonation of sulfonated peptide precursors can significantly reduce synthesis costs. Taking the synthesis of PSK-α as an example, using an unprotected sulfonation strategy, after sulfonating the sulfonated precursor H2N-YIYTQ-COOH with chlorosulfonic acid, sulfur trioxide-N,N-dimethylformamide complex, etc., more than 10 related products can be detected in the system. Theoretically, in a system with an excess of sulfonating agent, the R group... 1 -R 5 All can be sulfonated, and there are up to 32 (2^5=32) related compounds in the system. Although the conversion rate is high, the separation and purification are extremely difficult, and the yield of the target product is also low (<10%). Therefore, there is an urgent need in this field to develop a mild and efficient selective sulfonation method that can achieve precise modification of specific sites while maintaining the structural integrity of peptides. This technology will help promote the application and development of sulfonated peptides in fields such as agricultural biostimulants and novel peptide drugs. Summary of the Invention

[0011] To address the aforementioned technical problems and shortcomings in the field, this invention provides a route for the efficient synthesis of sulfonated peptides by selective sulfonation of peptides based on an orthogonal protecting group strategy. After protecting the amino acid side chains with specific orthogonal protecting groups, solid-phase peptide synthesis (SPPS) and purification are performed, followed by an efficient, low-yield, and selective sulfonation process, and gentle removal of the side chain protecting groups, to rapidly obtain sulfonated peptides with high crude purity.

[0012] The specific technical solution is as follows: A method for synthesizing a sulfonated polypeptide, comprising the following steps: S1, utilizing side chains containing -NHR 1 Amino acid monomer A with a side chain containing -CHR 2 OR 3 An intermediate peptide is synthesized from amino acid raw materials, including at least one of amino acid monomers B and a tyrosine monomer; wherein the main chain -NH2 of the amino acid monomer at the nitrogen terminus is protected as -NHR. 1 ;R 1 R 3 R is a protecting group that can be removed by hydrogenation at atmospheric pressure. 2 Selected from H, methyl; S2, selectively sulfonate the tyrosine phenolic hydroxyl groups in the intermediate peptide to obtain sulfonated intermediate peptide; S3, the sulfonated intermediate peptide undergoes atmospheric pressure hydrogenation and de-R 1 Or R 1 and R 3 The sulfonated polypeptide is obtained by protecting the group.

[0013] In some preferred embodiments, the method for synthesizing the sulfonated polypeptide, R 1 R 3 They are independently selected from benzyl-type protecting groups and alkoxycarbonyl-type protecting groups, respectively.

[0014] In some preferred embodiments, the method for synthesizing the sulfonated polypeptide, R 1 R 3 Each of the following is independently selected from benzyloxycarbonyl protecting group Cbz, benzyl Bn, and p-methoxybenzyl PMB.

[0015] In some preferred embodiments, the method for synthesizing the sulfonated polypeptide, step S1 employs solid-phase polypeptide synthesis, in which the phenolic hydroxyl group in the tyrosine monomer is protected as -O. t Bu, the intermediate peptide was obtained by cleavage, the lysis buffer was a trifluoroacetic acid system, the tyrosine phenolic hydroxyl group of the intermediate peptide was unprotected, and the C-terminal carboxyl group of the intermediate peptide was unprotected.

[0016] In some preferred embodiments, in step S2 of the method for synthesizing the sulfonated polypeptide, the sulfonating agent includes one or more of sulfur trioxide-organic amine, sulfur trioxide-N,N-dimethylformamide, and chlorosulfonic acid.

[0017] In some preferred embodiments, the method for synthesizing the sulfonated polypeptide includes sulfur trioxide-organic amine, which comprises sulfur trioxide-triethylamine (SO3·NEt3).

[0018] In some preferred embodiments, in the method for synthesizing the sulfonated polypeptide, step S2, the sulfonation is carried out in the presence of a base.

[0019] In some preferred embodiments, the base in the method for synthesizing the sulfonated polypeptide includes pyridine.

[0020] In some preferred embodiments, in the method for synthesizing the sulfonated polypeptide, step S3, the atmospheric pressure hydrogenation employs an ammonium formate buffer system.

[0021] In some preferred embodiments, the method for synthesizing the sulfonated polypeptide is wherein the sulfonated polypeptide is PSK-α, PSK-β, PSK-γ, PSK-δ, or PSK-ε.

[0022] This invention proposes an orthogonal protecting group strategy compatible with solid-phase peptide synthesis and subsequent selective deprotection. It utilizes N / O protecting groups that can be removed by atmospheric pressure hydrogenation (including but not limited to alkoxycarbonyl protecting groups, such as benzyloxycarbonyl protecting group Cbz, or benzyl protecting groups, such as benzyl Bn, p-methoxybenzyl PMB, etc.) as side-chain protecting groups for amino acid monomers in solid-phase peptide synthesis (SPPS) (e.g., serine, threonine, glutamic acid, aspartic acid, lysine, ornithine, etc.). These protecting groups are then used as main-chain nitrogen atom protecting groups for the nitrogen-terminated amino acids, allowing for conventional solid-phase peptide synthesis. After cleavage, a chain-like peptide containing tyrosine with specific protecting groups on both the side chain and main-chain N / O atoms is obtained. The phenolic hydroxyl group on the Tyr side chain is unprotected, and the carboxyl group at the carbon terminus is unprotected. Subsequently, selective sulfonation of the phenolic hydroxyl groups of the Tyr side chain of the peptide is achieved under sulfonation conditions (e.g., SO3·NEt3 and pyridine) to obtain a selectively sulfonated precursor (e.g., Cbz-Y(SO3H)IY(SO3H)T(Bn)Q-OH). The crude product after post-treatment is then subjected to atmospheric pressure hydrogenation in an ammonium formate buffer system to remove orthogonal protecting groups, thereby obtaining the final tyrosine sulfonated peptide product (e.g., PSK-α).

[0023] Compared with the prior art, the beneficial effects of this invention are as follows: 1. Economic Efficiency and Availability of Raw Materials: The sulfonated amino acid precursor selected in this invention is a monomer protected by a conventional protecting group (such as Fmoc). Its price is comparable to that of the amino acid monomers used in the common Fmoc-SPPS technology, and it has been commercialized and is readily available at low cost. Compared with the sulfonated monomers that require multiple synthesis steps in existing technical solutions 1-3, this invention significantly reduces raw material costs and reduces synthesis steps, thereby significantly saving economic, time, and labor costs.

[0024] 2. Protecting Group Compatibility and Synthetic Stability: This invention uses N,O- protecting groups such as benzyl and alkoxycarbonyl groups that can be hydrogenated and removed, which are well compatible with the Fmoc-SPPS solid-phase synthesis conditions. This protecting group strategy eliminates the need for the addition of 2-methylpiperidine to suppress side reactions and avoids the risk of hydrolysis removal of traditional protecting groups under acidic conditions, thereby significantly improving the yield of solid-phase synthesis.

[0025] 3. Efficient deprotection and high-purity product: The protecting group selected in this invention is highly compatible with the hydrogenation deprotection conditions (Pd / C, H2) of the sulfonated peptide precursor, ensuring product stability. After hydrogenation deprotection, the purity of the crude sulfonated peptide is >90%, significantly reducing the difficulty of separation and purification.

[0026] 4. Suppression of Over-sulfonation and High Yield: Compared to unprotected sulfonation strategies, this invention effectively suppresses the formation of over-sulfonation byproducts by blocking the N,O- sites with protecting groups, while ensuring that the terminal carboxyl group is retained under optimal conditions. After post-processing, high-purity sulfonated peptide precursors can be obtained, and the overall yield after hydrogenation deprotection is increased from <10% in traditional methods to 59%-71% (as shown in Examples 1-5).

[0027] 5. The method of the present invention represents a significant technological advancement in the field of sulfonated peptide synthesis, is applicable to drug development and structure-activity relationship studies, and has promising prospects for industrial application. Detailed Implementation

[0028] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0029] Example 1: Synthesis of PSK-α: Cbz-YIYT(Bn)Q-OH Synthesis: 2.0 g (1.2 mmol, 0.6 mmol / g loading) of CTC (2-chlorotriphenylmethyl chloride) resin was weighed and placed in a polypeptide solid-phase synthesis apparatus, and then...N , N Dimethylformamide (DMF) and dichloromethane (DCM) were used to swell the resin in 10 mL for 10 minutes, followed by washing three times with DCM. Fmoc-Gln(Trt)-OH (6.0 mmol, 5 equiv) and diisopropylethylamine (DIEA, 12.0 mmol, 10 equiv) were dissolved in 12 mL of DCM to obtain a clear solution, which was then added to the aforementioned resin. The mixture was shaken on a shaker at room temperature for 2 hours. 5 mL of methanol was added, and the reaction was continued for 15 minutes. The reaction solution was then dried under vacuum, washed with dichloromethane (10 mL × 3), and then washed with DMF (10 mL × 2). Subsequently, 10 mL of 20% piperidine / DMF solution was added, and the reaction was continued for 10 minutes. The reaction solution was then dried under vacuum, and the mixture was washed with dichloromethane (10 mL × 3), and then washed with DMF (10 mL × 2). The second amino acid coupling was then carried out. Fmoc-Thr(Bn)-OH (6.0 mmol, 5 equiv) and O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU, 6.0 mmol, 5 equiv) were dissolved in 12 mL of DMF. Then, DIEA (12.0 mmol, 10 equiv) was added, and the mixture was shaken to activate for 30 seconds. This was then mixed with the resin and reacted on a shaker at room temperature for 1 hour. The reaction solution was dried under vacuum, washed with dichloromethane (10 mL × 3), and then washed with DMF (10 mL × 2). Subsequently, 10 mL of 20% piperidine / DMF solution was added, and the reaction was allowed to proceed for 10 minutes. The reaction solution was then removed, and another 10 mL of 20% piperidine / DMF solution was added, and the reaction was allowed to proceed for 10 minutes. The reaction solution was then removed, and the mixture was washed with dichloromethane (10 mL × 3), and then washed with DMF (10 mL × 2). Subsequently, Fmoc-Thr(Bn)-OH was added sequentially. t Bu)-OH, Fmoc-Ile-OH, Cbz-Tyr( t Bu)-OH acts as an amino acid monomer for peptide chain elongation, with the last monomer (N-terminus of the peptide chain) being Cbz-Tyr( tAfter the coupling of Bu-OH was completed, the reaction solution was dried under vacuum, washed with dichloromethane (10 mL × 3), and then washed with DMF (10 mL × 2). Then, 10 mL of 20% piperidine / DMF solution was added, and the reaction was carried out for 10 minutes. The reaction solution was then removed, and another 10 mL of 20% piperidine / DMF solution was added, and the reaction was carried out for 10 minutes. The reaction solution was then removed, washed with DMF (10 mL × 2), and then washed with dichloromethane (10 mL × 3). Peptide resin lysis: lysis buffer (TFA:triisopropylsilane TIS:H2O = 95:2.5:2.5, 20 mL) was added to the resin. After stirring at room temperature for 2 hours, the mixture was filtered, washed with dichloromethane (10 mL × 3), and the filtrates were combined. The solvent was dried using an air pump, precipitated with diethyl ether (50 mL), and washed to obtain the PSK-α precursor Cbz-YIYT(Bn)Q-OH, with a crude product yield of 94%. This was directly added to the next reaction step.

[0030] Weigh out the crude PSK-α precursor Cbz-YIYT(Bn)Q-OH (0.1 mmol, 1 equiv) and pyridine (pyr, 100 μL), dissolve them in tetrahydrofuran / acetonitrile (THF / ACN = 1:1, 2 mL), and add the sulfur trioxide-triethylamine complex (SO3·NEt3, 90 mg, 0.5 mmol, 5 equiv) in portions under ice bath conditions. Stir the reaction mixture at room temperature for 3 hours, remove tetrahydrofuran and acetonitrile under reduced pressure at room temperature, and then add 1 mmol of the solution under ice bath conditions. Excess sulfur trioxide-triethylamine complex was quenched with sodium bicarbonate solution, followed by extraction three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure at room temperature to obtain crude Cbz-Y(SO3H)IY(SO3H)T(Bn)Q-OH. This was then dissolved in 5 mL of methanol, and Pd / C (10 mg, 10 wt% Pd, 55 wt% water) and ammonium formate (63 mg, 1 mmol, 10 equiv) were added. Hydrogenation was carried out at atmospheric pressure for 3–6 hours, with HPLC monitoring and sampling every 30 minutes. After the reaction, the mixture was filtered through diatomaceous earth, washed with methanol, concentrated, and then purified by reversed-phase chromatography. The corresponding fractions were collected and lyophilized to obtain PSK-α (55 mg, 65% yield). Example 2: Synthesis of PSK-β: Cbz-YIYT(Bn)-OH synthesis: 2.0 g (1.2 mmol, 0.6 mmol / g loading) of CTC (2-chlorotriphenylmethylchloro) resin was weighed and placed in a polypeptide solid-phase synthesis apparatus, and then used sequentially... N , N10 mL of dimethylformamide (DMF) and dichloromethane (DCM) were used to swell the resin for 10 minutes, followed by washing three times with DCM. Fmoc-Thr(Bn)-OH (6.0 mmol, 5 equiv) and diisopropylethylamine (DIEA, 12.0 mmol, 10 equiv) were dissolved in 12 mL of DCM to obtain a clear solution, which was then added to the aforementioned resin. The mixture was shaken on a shaker at room temperature for 2 hours. 5 mL of methanol was added, and the reaction was continued for 15 minutes. The reaction solution was then dried under vacuum, washed with dichloromethane (10 mL × 3), and then washed with DMF (10 mL × 2). 10 mL of 20% piperidine / DMF solution was then added, and the reaction was continued for 10 minutes. The reaction solution was then dried under vacuum, washed with dichloromethane (10 mL × 3), and then washed with DMF (10 mL × 2). Then, the coupling of the second amino acid was carried out, and Fmoc-Tyr was weighed. t Bu)-OH (6.0 mmol, 5 equiv), O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU, 6.0 mmol, 5 equiv) was dissolved in 12 mL of DMF, followed by the addition of DIEA (12.0 mmol, 10 equiv), shaken to activate for 30 seconds, then mixed with the resin, and reacted on a shaker at room temperature for 1 hour. The reaction solution was then dried, washed with dichloromethane (10 mL × 3), and then washed with DMF (10 mL × 2). Subsequently, 10 mL of 20% piperidine / DMF solution was added, reacted for 10 minutes, the reaction solution was removed, and then 10 mL of 20% piperidine / DMF solution was added again, reacted for 10 minutes, the reaction solution was removed, washed with dichloromethane (10 mL × 3), and then washed with DMF (10 mL × 2). Subsequently, Fmoc-Ile-OH, Cbz-Tyr ( t Bu)-OH acts as an amino acid monomer for peptide chain elongation, with the last monomer (N-terminus of the peptide chain) being Cbz-Tyr( tAfter the coupling of Bu-OH was completed, the reaction solution was dried under vacuum, washed with dichloromethane (10 mL × 3), and then washed with DMF (10 mL × 2). Then, 10 mL of 20% piperidine / DMF solution was added, and the reaction was allowed to proceed for 10 minutes. The reaction solution was then removed, and another 10 mL of 20% piperidine / DMF solution was added, and the reaction was allowed to proceed for 10 minutes. The reaction solution was then removed, and the solution was washed with DMF (10 mL × 2), and then washed with dichloromethane (10 mL × 3). Peptide resin lysis: Lysis buffer (TFA:TIS:H2O = 95:2.5:2.5, 20 mL) was added to the resin. After stirring at room temperature for 2 hours, the mixture was filtered, washed with dichloromethane (10 mL × 3), and the filtrates were combined. The solvent was dried using an air pump, and the product was precipitated with diethyl ether (50 mL) and washed to obtain the PSK-β precursor Cbz-YIYT(Bn)-OH, with a crude product yield of 92%. This crude product was directly added to the next reaction step.

[0031] Weigh out the crude compound PSK-β precursor Cbz-YIYT(Bn)-OH (0.1 mmol, 1 equiv) and pyridine (pyr, 100 μL), dissolve them in tetrahydrofuran / acetonitrile (THF / ACN = 1:1, 2 mL), and add the sulfur trioxide-triethylamine complex (SO3·NEt3, 90 mg, 0.5 mmol, 5 equiv) in portions under ice bath conditions. Stir the reaction at room temperature for 3 hours, remove tetrahydrofuran and acetonitrile under reduced pressure at room temperature, and then add 1 mmol of the solution under ice bath conditions. Excess sulfur trioxide-triethylamine complex was quenched with sodium bicarbonate solution, followed by three extractions with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure at room temperature to obtain crude Cbz-Y(SO3H)IY(SO3H)T(Bn)-OH. This was then dissolved in 5 mL of methanol, and Pd / C (10 mg, 10 wt% Pd, 55 wt% water) and ammonium formate (63 mg, 1 mmol, 10 equiv) were added. Hydrogenation was carried out at atmospheric pressure for 3–6 hours, with HPLC monitoring and sampling every 30 minutes. After the reaction, the mixture was filtered through diatomaceous earth, washed with methanol, concentrated, and then purified by reversed-phase chromatography. The corresponding fractions were collected and lyophilized to obtain PSK-β (44 mg, 62% yield). Example 3: Synthesis of PSK-γ: Synthesis of Cbz-YVYT(Bn)Q-OH: Refer to Example 1, except that Fmoc-Val-OH is used instead of Fmoc-Ile-OH. All other aspects are the same. The PSK-γ precursor Cbz-YVYT(Bn)Q-OH was obtained with a crude product yield of 94%, which was directly fed into the next reaction.

[0032] Weigh out the crude PSK-γ precursor Cbz-YVYT(Bn)Q-OH (0.1 mmol, 1 equiv) and pyridine (pyr, 100 μL), dissolve them in tetrahydrofuran / acetonitrile (THF / ACN = 1:1, 2 mL), and add the sulfur trioxide-triethylamine complex (SO3·NEt3, 90 mg, 0.5 mmol, 5 equiv) in portions under ice bath conditions. Stir the reaction mixture at room temperature for 3 hours, remove tetrahydrofuran and acetonitrile under reduced pressure at room temperature, and then add 1 mmol of the solution under ice bath conditions. Excess sulfur trioxide-triethylamine complex was quenched with sodium bicarbonate solution, followed by extraction three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure at room temperature to obtain crude Cbz-Y(SO3H)VY(SO3H)T(Bn)Q-OH. This was then dissolved in 5 mL of methanol, and Pd / C (10 mg, 10 wt% Pd, 55 wt% water) and ammonium formate (63 mg, 1 mmol, 10 equiv) were added. Hydrogenation was carried out at atmospheric pressure for 3–6 hours, with HPLC monitoring and sampling every 30 minutes. After the reaction, the mixture was filtered through diatomaceous earth, washed with methanol, concentrated, and then purified by reversed-phase chromatography. The corresponding fractions were collected and lyophilized to obtain PSK-γ (59 mg, 71% yield). Example 4: Synthesis of PSK-δ: Synthesis of Cbz-YIYT(Bn)N-OH: Refer to Example 1, except that Fmoc-Gln(Trt)-OH is replaced with an equimolar amount of Fmoc-Asn(Trt)-OH. All other aspects are the same, and the PSK-δ precursor Cbz-YIYT(Bn)N-OH is obtained with a crude product yield of 95%, which is directly fed into the next reaction.

[0033] Weigh out the crude PSK-δ precursor Cbz-YIYT(Bn)N-OH (0.1 mmol, 1 equiv) and pyridine (pyr, 100 μL), dissolve them in tetrahydrofuran / acetonitrile (THF / ACN = 1:1, 2 mL), and add the sulfur trioxide-triethylamine complex (SO3·NEt3, 90 mg, 0.5 mmol, 5 equiv) in portions under ice bath conditions. Stir the reaction mixture at room temperature for 3 hours, remove tetrahydrofuran and acetonitrile under reduced pressure at room temperature, and then add 1 mmol of the solution under ice bath conditions. Excess sulfur trioxide-triethylamine complex was quenched with sodium bicarbonate solution, followed by extraction three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure at room temperature to obtain crude Cbz-Y(SO3H)IY(SO3H)T(Bn)N-OH. This was then dissolved in 5 mL of methanol, and Pd / C (10 mg, 10 wt% Pd, 55 wt% water) and ammonium formate (63 mg, 1 mmol, 10 equiv) were added. Hydrogenation was carried out at atmospheric pressure for 3–6 hours, with HPLC monitoring and sampling every 30 minutes. After the reaction, the mixture was filtered through diatomaceous earth, washed with methanol, concentrated, and then purified by reversed-phase chromatography. The corresponding fractions were collected and lyophilized to obtain PSK-δ (55 mg, 66% yield). Example 5: Synthesis of PSK-ε: Synthesis of Cbz-YVYT(Bn)N-OH: Refer to Example 4, except that Fmoc-Val-OH is used instead of Fmoc-Ile-OH, and everything else is the same. The PSK-ε precursor Cbz-YVYT(Bn)N-OH was obtained with a crude product yield of 94%, which was directly fed into the next reaction.

[0034] Weigh out the crude PSK-ε precursor Cbz-YVYT(Bn)N-OH (0.1 mmol, 1 equiv) and pyridine (pyr, 100 μL), dissolve them in tetrahydrofuran / acetonitrile (THF / ACN = 1:1, 2 mL), and add the sulfur trioxide-triethylamine complex (SO3·NEt3, 90 mg, 0.5 mmol, 5 equiv) in portions under ice bath conditions. Stir the reaction at room temperature for 3 hours, remove tetrahydrofuran and acetonitrile under reduced pressure at room temperature, and then add 1 mmol of the solution under ice bath conditions. Excess sulfur trioxide-triethylamine complex was quenched with sodium bicarbonate solution, followed by extraction three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure at room temperature to obtain crude Cbz-Y(SO3H)VY(SO3H)T(Bn)N-OH. This was then dissolved in 5 mL of methanol, and Pd / C (10 mg, 10 wt% Pd, 55 wt% water) and ammonium formate (63 mg, 1 mmol, 10 equiv) were added. Hydrogenation was carried out at atmospheric pressure for 3–6 hours, with HPLC monitoring and sampling every 30 minutes. After the reaction, the mixture was filtered through diatomaceous earth, washed with methanol, concentrated, and then purified by reversed-phase chromatography. The corresponding fractions were collected and lyophilized to obtain PSK-ε (48 mg, 59% yield). Furthermore, it should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method of synthesizing a sulfonated polypeptide, characterized in that, The method comprises the steps of: S1, at least one of an amino acid monomer A including a side chain containing -NHR 1 group and an amino acid monomer B including a side chain containing -CHR 2 OR 3 group, and a tyrosine monomer are used to synthesize an intermediate peptide in an amino acid raw material; a main chain -NH2 of the amino acid monomer at the nitrogen end in the amino acid raw material is protected as -NHR 1 ; R 1 , R 3 is a protecting group that can be removed by normal pressure hydrogenation, and R 2 is selected from H and methyl; S2, selectively phenolic hydroxyl sulfonating tyrosine in the intermediate peptide to obtain a sulfonated intermediate peptide; S3, said sulfonated intermediate peptide is hydrogenated at atmospheric pressure to remove R 1 or R 1 and R 3 to obtain said sulfonated polypeptide.

2. The method of synthesizing a sulfonated polypeptide of claim 1, wherein, R 1 , R 3 are independently selected from each other and are independently selected from the group consisting of benzyl-type protecting groups, alkoxycarbonyl-type protecting groups.

3. The method of synthesizing a sulfonated polypeptide of claim 1, wherein, R 1 , R 3 are each independently selected from the group consisting of a benzyloxycarbonyl protecting group Cbz, a benzyl group Bn, a p-methoxybenzyl group PMB.

4. The method of synthesizing a sulfonated polypeptide of claim 1, wherein, Step S1 employs solid-phase polypeptide synthesis, the phenolic hydroxyl group in the tyrosine monomer is protected as -O t Bu, the cleavage solution employs trifluoroacetic acid system, the tyrosine phenolic hydroxyl group in the intermediate peptide is unprotected, and the carboxyl group at the carbon end of the intermediate peptide is unprotected.

5. The method of claim 1, wherein the sulfated polypeptide is selected from the group consisting of SEQ ID NOs: 1- 12. 5 In step S2, the sulfonating reagent comprises one or more of sulfur trioxide-organic amine, sulfur trioxide-N,N-dimethylformamide, chlorosulfonic acid.

6. The method of synthesizing a sulfonated polypeptide of claim 5, wherein, The sulfur trioxide-organic amine comprises sulfur trioxide-triethylamine.

7. The method for synthesizing sulfonated polypeptides according to claim 1, characterized in that, In step S2, the sulfonating is performed in the presence of a base.

8. The method of synthesizing a sulfonated polypeptide of claim 7, wherein, The base comprises pyridine.

9. The method for synthesizing sulfonated polypeptides according to claim 1, characterized in that, In step S3, the normal-pressure hydrogenation adopts a formic acid ammonium buffer system.

10. The method of claim 1, wherein the sulfated polypeptide is selected from the group consisting of SEQ ID NOs: 1- 10. The sulfonated polypeptide is PSK-α, PSK-β, PSK-γ, PSK-δ or PSK-ε.

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