Conjugation method of sulfanilic acid and polypeptide

Abstract

The invention discloses a method for conjugating sulfanilic acid and polypeptide, which belongs to the technical field of biological organic chemistry, and comprises the following steps: S1, conjugating sulfanilic acid and polypeptide by using strain-promoted azide-alkynyl cycloaddition reaction: firstly, ensuring that a reactive azide group (-N3) exists on sulfanilic acid, and then, carrying out strain-promoted azide-alkynyl cycloaddition reaction on sulfanilic acid; an alkynyl (-C = CH) is arranged at the tail end or side chain of the polypeptide; s2, introducing an azide group through chemical reaction, and introducing alkynyl through solid phase peptide synthesis (SPPS); the azide-alkynyl click reaction is carried out by using the Cu (I) catalyst, so that the reaction efficiency and the selectivity are relatively high, the conjugation can be quickly completed under relatively mild conditions, and the side reaction is reduced. Alkynyl is introduced to a specific position of the polypeptide, so that high-specificity conjugation is realized, and the biological activity and stability of a polypeptide drug can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of bioorganic chemistry, and in particular to a method for conjugating p-aminobenzenesulfonic acid with a polypeptide. Background Art

[0002] p-Aminobenzenesulfonic acid is an organic compound, a white or off-white crystalline powder with strong acidity, usually used as an intermediate in organic synthesis. Peptides are biological macromolecules composed of two or more amino acids connected by peptide bonds (amide bonds). They are the precursors of proteins and perform various functions in organisms.

[0003] Existing conjugation methods need to be carried out under high temperature, high pressure or the use of harmful solvents, and often lack sufficient site specificity, resulting in reactions at non-target positions on the polypeptide chain, thereby generating a variety of by-products and reducing the purity and efficiency of the product. To this end, a method for conjugating p-aminobenzenesulfonic acid with polypeptides is proposed. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems that the conjugation method in the prior art produces a variety of byproducts, which reduces the purity and efficiency of the product. To this end, one object of the present invention is to provide a method for conjugating p-aminobenzenesulfonic acid with a polypeptide.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for conjugating p-aminobenzenesulfonic acid with a polypeptide comprises the following steps:

[0007] S1. Use strain-promoted azide-alkynyl cycloaddition reaction to conjugate aminobenzenesulfonic acid to peptides. First, ensure that there is a reactive azide group (-N3) on aminobenzenesulfonic acid and an alkynyl group (-C≡CH) on the end or side chain of the peptide.

[0008] S2. Introducing the azide group by chemical reaction and the alkyne group by solid phase peptide synthesis (SPPS);

[0009] S3. Optimizing the reaction conditions;

[0010] S4. Under the optimized reaction conditions, the aminobenzenesulfonic acid containing an azide group and the polypeptide containing an alkynyl group are mixed, an appropriate amount of Cu(I) salt is added, and the mixture is stirred at room temperature for 1 h;

[0011] S5. Remove the copper catalyst using excess sodium thiosulfate and purify the conjugated product by HPLC;

[0012] S6. Use mass spectrometry, nuclear magnetic resonance, and infrared spectroscopy to confirm the structure of the product.

[0013] As a further optimization scheme of the present invention, in S2, the specific steps of introducing the azide group by chemical reaction are:

[0014] S201. aminobenzenesulfonic acid is used as a starting material, sodium azide is used as an azidation reagent, DCC (dicyclohexylcarbodiimide) and HOBt (1-hydroxybenzotriazole) are used as coupling reagents, and DMF (N,N-dimethylformamide) is used as a solvent, and triethylamine is used as an acid-base regulator to adjust the reaction pH;

[0015] S202. Under dry conditions, dissolving aminobenzenesulfonic acid in an anhydrous solvent, adding equimolar amounts of DCC and HOBt to form an active intermediate;

[0016] S203. Under nitrogen protection, the activated aminobenzenesulfonic acid solution is cooled to 0-5°C, and NaN3 is slowly added, and the addition rate is controlled to prevent the temperature from rising. The reaction mixture is stirred at low temperature for 4-6h to complete the azidation reaction;

[0017] S204. Use TLC to monitor the progress of the reaction to ensure that the azidation reaction is complete. After the reaction is completed, remove the insoluble matter by filtration, use solvent extraction to separate and purify the azidaminobenzenesulfonic acid, and remove the solvent from the purified azidaminobenzenesulfonic acid solution by rotary evaporation. The dried solid is placed under vacuum nitrogen protection to prevent the azide from being degraded by water.

[0018] As a further optimization scheme of the present invention, in S2, an alkynyl group is introduced into the polypeptide by the SPPS method, and the specific steps are as follows:

[0019] Alkynyl groups were introduced by solid phase peptide synthesis (SPPS). First, propargyl glycine was used to ensure that the side chain contained an alkynyl group and was compatible with Fmoc (9-fluorenylmethoxycarbonyl) and Boc (tert-butyloxycarbonyl) groups. The Wang resin of the alkynyl amino acid was loaded into a peptide synthesis column. Piperidine was used to remove the Fmoc protecting group to expose the amino group of the amino acid. The DIPEA system of the alkynyl amino acid was reacted with the amino group on the resin to form a peptide bond. The "deprotection-coupling" step was repeated, and other amino acids were added in sequence until the polypeptide chain was synthesized. The polypeptide was cut from the resin using a thioanisole mixture, the polypeptide was purified using HPLC technology, and the structure of the polypeptide was characterized by mass spectrometry.

[0020] As a further optimization scheme of the present invention, in S3, the optimized reaction conditions are specifically: using N,N-dimethylformamide as the reaction solvent, reacting at 25° C., and the reaction time is 1-2 h.

[0021] As a further optimization scheme of the present invention, in S4, the specific steps of mixing the aminobenzenesulfonic acid containing an azide group with the polypeptide containing an alkynyl group are:

[0022] S401. First, ensure that the aminobenzenesulfonic acid containing an azide group and the polypeptide containing an alkynyl group have been purified and are in a dry state, calculate and weigh the appropriate amount of reactants to ensure that the molar ratio of azide to alkynyl is 1:1;

[0023] Place the aminobenzenesulfonic acid solution containing an azide group in a clean round-bottom flask, slowly add the polypeptide solution containing an alkynyl group at a rotation speed of 50-150 rpm, and maintain a stirring speed of 20-50 rpm during mixing to avoid bubbles or local overheating;

[0024] S402. After mixing the reactants, immediately add 0.1-1mmol of Cu(I) salt, the content of which is 0.1-1 molar ratio of the reactants. When adding the catalyst, use ultrasound to help dissolve it to ensure that it is fully dissolved in the solvent. After adding the catalyst, start timing immediately, set the initial reaction time to 1h, take samples regularly to monitor the reaction progress, and use HPLC technology to monitor the reaction progress. If the reaction is not complete within 1h, extend the reaction time appropriately according to the monitoring results until the reaction reaches a satisfactory conversion rate.

[0025] As a further optimization scheme of the present invention, in S5, the specific steps of removing the copper catalyst using excess sodium thiosulfate are:

[0026] After the reaction is completed, the reaction mixture is transferred to a centrifuge tube, 50 mL of sodium thiosulfate aqueous solution is prepared with a concentration of 0.1-0.3 M, the prepared sodium thiosulfate solution is added to the reaction mixture containing the copper catalyst, ensuring that the amount of sodium thiosulfate is at least 10 times the molar amount of the copper catalyst, the mixture is gently stirred, and allowed to react at room temperature for 10-30 minutes, so that the sodium thiosulfate reacts with the copper catalyst to form an insoluble copper sulfide precipitate, and after the reaction is completed, the mixture is placed in a dialysis bag and dialyzed in deionized water to remove small molecule impurities and catalysts.

[0027] As a further optimization scheme of the present invention, in S5, the specific steps of purifying the conjugated product by HPLC are:

[0028] According to the molecular weight and hydrophobicity of the conjugated product, a reversed phase C18 column was selected, and a gradient elution system consisting of an aqueous phase and an organic phase was prepared. The sample after the catalyst removal step was appropriately diluted and filtered through a 0.22 μm filter membrane to remove possible particulate matter. The sample was loaded into the HPLC system, and the flow rate, temperature and detection wavelength were set. The gradient elution program was run, and the target fractions were collected according to the retention time of the product. The fractions containing the conjugated product were collected, and the solvent was removed by freeze-drying to concentrate and purify the product.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The azide-alkyne click reaction using Cu(I) catalyst in the present invention has high reaction efficiency and selectivity, can quickly complete conjugation under relatively mild conditions, and reduce the occurrence of side reactions. By introducing alkyne groups at specific positions of polypeptides, highly specific conjugation can be achieved, which can improve the biological activity and stability of polypeptide drugs.

[0031] In the present invention, after the conjugation reaction, the HPLC technology is used for purification, which can efficiently separate and purify the target conjugated product, and the purification efficiency and the purity of the product can be further improved by finely adjusting the parameters of HPLC. At the same time, the reagents and catalysts used are more environmentally friendly, and the operation steps are simple and easy to scale production. It is not only suitable for the conjugation of p-aminobenzenesulfonic acid with polypeptides, but can also be extended to the conjugation of other small molecule drugs with polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The flowchart of the conjugation method of p-aminobenzenesulfonic acid and polypeptide of the present invention;

[0033] Figure 2 The flowchart of the invention is a process for introducing an azide group through a chemical reaction;

[0034] Figure 3 The figure is a flowchart of the process of introducing an alkyne group by solid phase peptide synthesis in the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Embodiment 1

[0037] See also Figure 1-Figure 3 , the present invention provides a technical solution:

[0038] A method for conjugating p-aminobenzenesulfonic acid with a polypeptide comprises the following steps:

[0039] S1. Use strain-promoted azide-alkynyl cycloaddition reaction to conjugate aminobenzenesulfonic acid to peptides. First, ensure that there is a reactive azide group (-N3) on aminobenzenesulfonic acid and an alkynyl group (-C≡CH) on the end or side chain of the peptide.

[0040] S2. Introducing the azide group by chemical reaction and the alkyne group by solid phase peptide synthesis (SPPS);

[0041] S3. Optimizing the reaction conditions;

[0042] S4. Under the optimized reaction conditions, the aminobenzenesulfonic acid containing an azide group and the polypeptide containing an alkynyl group are mixed, an appropriate amount of Cu(I) salt is added, and the mixture is stirred at room temperature for 1 h;

[0043] S5. Remove the copper catalyst using excess sodium thiosulfate and purify the conjugated product by HPLC;

[0044] S6. Use mass spectrometry, nuclear magnetic resonance, and infrared spectroscopy to confirm the structure of the product.

[0045] In S2, the specific steps of introducing the azide group by chemical reaction are:

[0046] S201. aminobenzenesulfonic acid is used as a starting material, sodium azide is used as an azidation reagent, DCC (dicyclohexylcarbodiimide) and HOBt (1-hydroxybenzotriazole) are used as coupling reagents, and DMF (N,N-dimethylformamide) is used as a solvent, and triethylamine is used as an acid-base regulator to adjust the reaction pH;

[0047] S202. Under dry conditions, dissolving aminobenzenesulfonic acid in an anhydrous solvent, adding equimolar amounts of DCC and HOBt to form an active intermediate;

[0048] Specifically, DMSO was used and stirred overnight by adding anhydrous sodium sulfate, and then filtered to remove the drying agent.

[0049] S203. Under nitrogen protection, the activated aminobenzenesulfonic acid solution is cooled to 0-5°C, and NaN3 is slowly added, and the addition rate is controlled to prevent the temperature from rising. The reaction mixture is stirred at low temperature for 4-6h to complete the azidation reaction;

[0050] S204. Use TLC to monitor the progress of the reaction to ensure that the azidation reaction is complete. After the reaction is completed, remove the insoluble matter by filtration, use solvent extraction to separate and purify the azidaminobenzenesulfonic acid, and remove the solvent from the purified azidaminobenzenesulfonic acid solution by rotary evaporation. The dried solid is placed under vacuum nitrogen protection to prevent the azide from being degraded by water.

[0051] In S2, an alkynyl group is introduced into a polypeptide by the SPPS method, and the specific steps are as follows:

[0052] Alkynyl groups were introduced by solid phase peptide synthesis (SPPS). First, propargyl glycine was used to ensure that the side chain contained an alkynyl group and was compatible with Fmoc (9-fluorenylmethoxycarbonyl) and Boc (tert-butyloxycarbonyl) groups. The Wang resin of the alkynyl amino acid was loaded into a peptide synthesis column. Piperidine was used to remove the Fmoc protecting group to expose the amino group of the amino acid. The DIPEA system of the alkynyl amino acid was reacted with the amino group on the resin to form a peptide bond. The "deprotection-coupling" step was repeated, and other amino acids were added in sequence until the polypeptide chain was synthesized. The polypeptide was cut from the resin using a thioanisole mixture, the polypeptide was purified using HPLC technology, and the structure of the polypeptide was characterized by mass spectrometry.

[0053] Specifically, propargylglycine (Gly) is used as the alkynylating amino acid, and the target polypeptide sequence is: Arg-Gly-Asp-Ser-Gly (RGDSG), wherein propargylglycine will replace the second glycine residue.

[0054] Amino acids added in order:

[0055] Step 1: Load Arg (Fmoc-Arg-Wang resin) into a peptide synthesis column.

[0056] Step 2: Use Piperidine to remove the Fmoc protecting group on Arg to expose the amino group of Arg.

[0057] Step 3: The activated ester of propargylglycine reacts with Arg on the resin to form a peptide bond.

[0058] Step 4: Repeat the “deprotection-coupling” step, adding Asp, Ser and finally Gly in sequence, and removing the Fmoc protecting group of the newly added amino acid.

[0059] The activated ester of the next amino acid is coupled to the amino group on the resin until all amino acids have been added in sequence.

[0060] In S3, the optimized reaction conditions are as follows: using N,N-dimethylformamide as the reaction solvent, reacting at 25° C., and reacting for 1-2 h.

[0061] In S4, the specific steps of mixing the aminobenzenesulfonic acid containing an azide group with the polypeptide containing an alkynyl group are:

[0062] S401. First, ensure that the aminobenzenesulfonic acid containing an azide group and the polypeptide containing an alkynyl group have been purified and are in a dry state, calculate and weigh the appropriate amount of reactants to ensure that the molar ratio of azide to alkynyl is 1:1;

[0063] Place the aminobenzenesulfonic acid solution containing an azide group in a clean round-bottom flask, slowly add the polypeptide solution containing an alkynyl group at a rotation speed of 50-150 rpm, and maintain a stirring speed of 20-50 rpm during mixing to avoid bubbles or local overheating;

[0064] Specifically, the purification of aminobenzenesulfonic acid: aminobenzenesulfonic acid is purified by recrystallization in methanol, and aminobenzenesulfonic acid is separated and purified using silica gel or gel filtration column chromatography technology. Further purification by high performance liquid chromatography ensures that the aminobenzenesulfonic acid of the azide group reaches the required purity.

[0065] Purification of alkynylated peptides: Gel filtration chromatography was used to remove unreacted reagents and impurities, and a C18 column was used to purify the peptides, which were separated based on their hydrophobicity. Mass spectrometry was used to confirm the molecular weight and structure of the peptides.

[0066] When calculating and weighing the appropriate amount of reactants, first determine the molar mass, calculate the molar mass of aminobenzenesulfonic acid and alkynylated peptide, and determine that the molar ratio of azide to alkyne is 1: 1. When 1 mmol of conjugated product is required, then 1 mmol of aminobenzenesulfonic acid and 1 mmol of alkynylated peptide are required.

[0067] Use a precision balance to weigh the masses of aminobenzenesulfonic acid and alkynylated polypeptide corresponding to the calculated molar number. When the molar mass of aminobenzenesulfonic acid is 200 g / mol, the mass of 1 mmol is 200 mg.

[0068] S402. After mixing the reactants, immediately add an appropriate amount of Cu(I) salt, the content of which is 0.1-1 molar ratio of the reactants. When adding the catalyst, use ultrasound to help dissolve it to ensure that it is fully dissolved in the solvent. After adding the catalyst, start timing immediately, set the initial reaction time to 1 hour, take samples regularly during the period to monitor the reaction progress, and use HPLC technology to monitor the reaction progress. If the reaction is not complete within 1 hour, appropriately extend the reaction time according to the monitoring results until the reaction reaches a satisfactory conversion rate.

[0069] Specifically, calculate the amount of Cu(I) salt:

[0070] Minimum amount (0.1 molar ratio):

[0071] The number of moles of Cu(I) salt = the number of moles of reactants × 0.1

[0072] The number of moles of Cu(I) salt = 1 mmol x 0.1 = 0.1 mmol

[0073] Maximum amount (1 molar ratio):

[0074] The number of moles of Cu(I) salt = the number of moles of reactants × 1

[0075] The number of moles of Cu(I) salt = 1 mmol x 1 = 1 mmol

[0076] Cu(I) iodide (CuI) was used as a catalyst, and its molar mass was 190.45 g / mol.

[0077] For 0.1 mmol of CuI, mass = 0.1 mmol x 190.45 g / mol = 19.045 mg For 1 mmol of CuI, mass = 1 mmol x 190.45 g / mol = 190.45 mg

[0078] In S5, the specific steps of removing the copper catalyst using excess sodium thiosulfate are:

[0079] After the reaction is completed, the reaction mixture is transferred to a centrifuge tube, 50 mL of sodium thiosulfate aqueous solution is prepared with a concentration of 0.1-0.3 M, the prepared sodium thiosulfate solution is added to the reaction mixture containing the copper catalyst, ensuring that the amount of sodium thiosulfate is at least 10 times the molar amount of the copper catalyst, the mixture is gently stirred, and allowed to react at room temperature for 10-30 minutes, so that the sodium thiosulfate reacts with the copper catalyst to form an insoluble copper sulfide precipitate, and after the reaction is completed, the mixture is placed in a dialysis bag and dialyzed in deionized water to remove small molecule impurities and catalysts.

[0080] Specifically, using 1 mmol of Cu(I) catalyst, the molar amount of sodium thiosulfate is at least 10 mmol. When a concentration of 0.2 M is selected, for 10 mmol of sodium thiosulfate, the required volume is:

[0081] Volume = 10mmol / 0.2M = 0.05L = 50mL

[0082] The specific steps for purifying the conjugated product by HPLC are:

[0083] According to the molecular weight and hydrophobicity of the conjugated product, a reverse phase C18 column was selected, and a gradient elution system consisting of water containing 0.1% TFA and acetonitrile was prepared. The sample after the catalyst removal step was appropriately diluted and filtered through a 0.22 μm filter membrane to remove possible particulate matter. The sample was loaded into the HPLC system and the flow rate was set to 1.0 mL / min, the temperature to 20-25°C, and the detection wavelength to 280 nm. The gradient elution program was run, and the target fractions were collected according to the retention time of the product. The fractions containing the conjugated product were collected, and the solvent was removed by freeze drying to concentrate and purify the product.

[0084] Steps for structural confirmation of aminobenzenesulfonic acid-peptide conjugate products using mass spectrometry, nuclear magnetic resonance, and infrared spectroscopy:

[0085] Sample preparation for mass spectrometry: Ensure the conjugated product is dry or dissolved in a water / acetonitrile mixture and prepare the sample using an autosampler.

[0086] Instrument settings: Select electrospray ionization (ESI), set the spray voltage, cone voltage and source temperature.

[0087] Data acquisition: Run the mass spectrometer to obtain the molecular ion peak ([M]+) and fragment ion peaks of the conjugated product. For high-resolution mass spectra, record the accurate molecular weight.

[0088] Data analysis: Confirm the molecular structure by comparing the experimentally obtained molecular weight with the theoretically calculated value, analyze the fragment ion pattern, and infer the connection points and molecular structure.

[0089] Sample preparation for NMR: Dissolve the conjugated product in heavy water (D2O) or deuterated solvent (such as CD3OD) and adjust the sample concentration. Select the probe and magnetic field strength, pulse sequence and spectral width.

[0090] Data acquisition: 1H NMR and 13C NMR spectra were collected, and two-dimensional NMR experiments were performed.

[0091] Data Analysis: Assignment of atomic environments by chemical shifts, integrals, coupling constants, and peak shapes.

[0092] Sample preparation for IR spectroscopy: For solid samples, use the KBr pellet method, for liquids or solutions, use the ATR accessory. Calibrate the instrument and set the scan range and resolution.

[0093] Data collection: The infrared spectrum of the conjugated product was collected to record the specific functional groups of the radical (-NH2), carbonyl (C=O) and sulfonic acid group (-SO3H).

[0094] Data analysis: Confirm the presence of functional groups by comparing experimental spectra with standard spectra, analyze the intensity and shape of absorption peaks, and infer the molecular structure.

[0095] Embodiment 2

[0096] Introduction of alkyne groups by chemical modification:

[0097] After peptide synthesis, alkyne groups are introduced into specific positions of the peptide using alkynylated succinimidyl esters with the thiol groups of the peptide, followed by an alkyne-azide click reaction.

[0098] Specifically, before the alkyne reaction, a protecting group is used to protect non-target functional groups in the polypeptide to avoid side reactions. When the pH is 7.0-7.5 and the temperature is 25°C-37°C, the alkyne reagent is added to the polypeptide solution, the timing is started, and the reaction progress is monitored by sampling using HPLC. After the alkyne reaction is completed, the protecting group is removed, and the conjugated polypeptide is purified by gel filtration. The purified polypeptide is structurally characterized by mass spectrometry to confirm the introduction of the alkyne group and the structure of the polypeptide.

[0099] Embodiment 3

[0100] Optimization process of alkyne introduction and thiol-alkyne click reaction:

[0101] Step 1: During peptide synthesis, mercaptopropionic anhydride or thioacetic anhydride is used as an introduction reagent to introduce thiol groups onto the predetermined amino acid side chains.

[0102] Step 2: Use alkynylated succinimide ester to react with thiol. Through experiments, the reaction conditions are optimized to be 37°C, pH 7.4, 2h, and a molar ratio of 1:1.5, which can achieve high efficiency and high yield of alkyne introduction.

[0103] Step 3: Under optimized conditions, the alkyne reagent is mixed with the peptide with a thiol group to carry out a thiol-alkyne click reaction in the absence of copper to ensure the purity of the product.

[0104] Step 4: Monitor the reaction progress by UV-visible spectroscopy and conduct qualitative and quantitative analysis of the reaction mixture by high performance liquid chromatography to ensure that the acetylation reaction is complete and no by-products are formed.

[0105] Step 5: After the reaction is completed, unreacted reagents and by-products are removed by dialysis. Subsequently, the conjugated peptide is finely purified using size exclusion chromatography and reversed-phase HPLC to obtain a high-purity target product.

[0106] Step 6: Use high-resolution mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy to confirm the structure of the purified polypeptide and analyze in detail the position and conformation of the introduction of the alkynyl group.

[0107] Parts not involved in the present invention are the same as the prior art or can be implemented by the prior art. Although the embodiments of the present invention have been shown and described, it is understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for conjugating p-aminobenzenesulfonic acid with a polypeptide, characterized in that: The steps include: S1. Use strain-promoted azide-alkynyl cycloaddition reaction to conjugate aminobenzenesulfonic acid to peptides. First, ensure that there is a reactive azide group (-N3) on aminobenzenesulfonic acid and an alkynyl group (-C≡CH) on the end or side chain of the peptide. S2. Introducing the azide group by chemical reaction and the alkyne group by solid phase peptide synthesis (SPPS); S3. Optimizing the reaction conditions; S4. Under the optimized reaction conditions, the aminobenzenesulfonic acid containing an azide group and the polypeptide containing an alkynyl group are mixed, an appropriate amount of Cu(I) salt is added, and the mixture is stirred at room temperature for 1 h; S5. Remove the copper catalyst using excess sodium thiosulfate and purify the conjugated product by HPLC; S6. Use mass spectrometry, nuclear magnetic resonance, and infrared spectroscopy to confirm the structure of the product.

2. The method for conjugating p-aminobenzenesulfonic acid with a polypeptide according to claim 1, characterized in that: In S2, the specific steps of introducing the azide group by chemical reaction are: S201. aminobenzenesulfonic acid is used as a starting material, sodium azide is used as an azidation reagent, DCC (dicyclohexylcarbodiimide) and HOBt (1-hydroxybenzotriazole) are used as coupling reagents, and DMF (N,N-dimethylformamide) is used as a solvent, and triethylamine is used as an acid-base regulator to adjust the reaction pH; S202. Under dry conditions, dissolving aminobenzenesulfonic acid in an anhydrous solvent, adding equimolar amounts of DCC and HOBt to form an active intermediate; S203. Under nitrogen protection, the activated aminobenzenesulfonic acid solution is cooled to 0-5°C, and NaN3 is slowly added, and the addition rate is controlled to prevent the temperature from rising. The reaction mixture is stirred at low temperature for 4-6h to complete the azidation reaction; S204. Use TLC to monitor the progress of the reaction to ensure that the azidation reaction is complete. After the reaction is completed, remove the insoluble matter by filtration, use solvent extraction to separate and purify the azidaminobenzenesulfonic acid, and remove the solvent from the purified azidaminobenzenesulfonic acid solution by rotary evaporation. The dried solid is placed under vacuum nitrogen protection to prevent the azide from being degraded by water.

3. The method for conjugating p-aminobenzenesulfonic acid with a polypeptide according to claim 1, characterized in that: In S2, an alkynyl group is introduced into a polypeptide by the SPPS method, and the specific steps are as follows: Alkyne groups were introduced by solid phase peptide synthesis (SPPS). First, propargyl glycine was used to ensure that the side chain contained an alkynyl group and was compatible with Fmoc (9-fluorenylmethoxycarbonyl) and Boc (tert-butyloxycarbonyl) groups. The Wang resin of the alkynyl amino acid was loaded into a peptide synthesis column. Piperidine was used to remove the Fmoc protecting group to expose the amino group of the amino acid. The DIPEA system of the alkynyl amino acid was reacted with the amino group on the resin to form a peptide bond. The "deprotection-coupling" step was repeated, and other amino acids were added in sequence until the polypeptide chain was synthesized. The polypeptide was cut from the resin using a thioanisole mixture, and the polypeptide was purified using HPLC technology. The structure of the polypeptide was characterized by mass spectrometry.

4. The method for conjugating p-aminobenzenesulfonic acid with a polypeptide according to claim 1, characterized in that: In S3, the optimized reaction conditions are as follows: using N,N-dimethylformamide as the reaction solvent, reacting at 25° C., and reacting for 1-2 h.

5. The method for conjugating p-aminobenzenesulfonic acid with a polypeptide according to claim 1, characterized in that: In S4, the specific steps of mixing the aminobenzenesulfonic acid containing an azide group with the polypeptide containing an alkynyl group are: S401. First, ensure that the aminobenzenesulfonic acid containing an azide group and the polypeptide containing an alkynyl group have been purified and are in a dry state, calculate and weigh the appropriate amount of reactants to ensure that the molar ratio of azide to alkynyl is 1:1; Place the aminobenzenesulfonic acid solution containing an azide group in a clean round-bottom flask, slowly add the polypeptide solution containing an alkynyl group at a rotation speed of 50-150 rpm, and maintain a stirring speed of 20-50 rpm during mixing to avoid bubbles or local overheating; S402. After mixing the reactants, immediately add 0.1-1mmol of Cu(I) salt, the content of which is 0.1-1 molar ratio of the reactants. When adding the catalyst, use ultrasound to help dissolve it to ensure that it is fully dissolved in the solvent. After adding the catalyst, start timing immediately, set the initial reaction time to 1h, take samples regularly to monitor the reaction progress, and use HPLC technology to monitor the reaction progress. If the reaction is not complete within 1h, extend the reaction time appropriately according to the monitoring results until the reaction reaches a satisfactory conversion rate.

6. The method for conjugating p-aminobenzenesulfonic acid with a polypeptide according to claim 1, characterized in that: In S5, the specific steps of removing the copper catalyst using excess sodium thiosulfate are: After the reaction is completed, the reaction mixture is transferred to a centrifuge tube, 50 mL of sodium thiosulfate aqueous solution is prepared with a concentration of 0.1-0.3 M, the prepared sodium thiosulfate solution is added to the reaction mixture containing the copper catalyst, ensuring that the amount of sodium thiosulfate is at least 10 times the molar amount of the copper catalyst, the mixture is gently stirred, and allowed to react at room temperature for 10-30 minutes, so that the sodium thiosulfate reacts with the copper catalyst to form an insoluble copper sulfide precipitate, and after the reaction is completed, the mixture is placed in a dialysis bag and dialyzed in deionized water to remove small molecule impurities and catalysts.

7. The method for conjugating p-aminobenzenesulfonic acid with a polypeptide according to claim 1, characterized in that: In S5, the specific steps of purifying the conjugated product by HPLC are: According to the molecular weight and hydrophobicity of the conjugated product, a reversed phase C18 column was selected, and a gradient elution system consisting of an aqueous phase and an organic phase was prepared. The sample after the catalyst removal step was appropriately diluted and filtered through a 0.22 μm filter membrane to remove possible particulate matter. The sample was loaded into the HPLC system, and the flow rate, temperature and detection wavelength were set. The gradient elution program was run, and the target fractions were collected according to the retention time of the product. The fractions containing the conjugated product were collected, and the solvent was removed by freeze-drying to concentrate and purify the product.

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