Synthetic methods for two types of dimethylsulfonylphenyloxadiazole

Abstract

This invention discloses two novel methods for synthesizing dithiol-bridged labeled bis(methylsulfonyl)phenyloxadiazole. These methods utilize inexpensive, commercially available raw materials and conventional transformations such as condensation and coupling to prepare two bis(methylsulfonyl)phenyloxadiazoles suitable for dithiol-bridged labeling of antibodies, peptides, and hydrogels. The preparation methods of this invention are simple, use readily available raw materials, and produce high-value-added products, demonstrating significant economic benefits and market potential.

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis, specifically to two novel methods for synthesizing dithiol-bridged labeled methanesulfonylphenyloxadiazoles. These methods utilize inexpensive, commercially available raw materials to prepare two methanesulfonylphenyloxadiazoles suitable for dithiol-bridged labeling of antibodies and peptides. The preparation methods of this invention are simple, use readily available raw materials, and produce high-value-added products, demonstrating significant economic benefits and market potential. Background Technology

[0002] Maleimide is obtained by reacting maleic anhydride with an amine derivative. It readily undergoes a 1,4-addition reaction with a thiol group (-SH) to form a stable thioether bond. Maleimide reacts with thiols at pH 6.5-7.5 at a rate 1000 times faster than amine reactions, thus enabling complete conversion with minimal reagent amounts. Furthermore, the biggest challenge in maleimide alkylation is the reverse addition reaction of the thiol group, whose rate is highly dependent on the pKa of the specific cysteine ​​residue to which it is attached. Figure 1 This reverse addition reaction may lead to premature release of the drug's linker moiety during in vivo circulation, and coupling with other biomolecules containing free thiol groups, such as albumin, resulting in off-target collateral damage. Furthermore, the thiol groups of biomolecules such as proteins are primarily in the form of disulfide bonds, playing a crucial role in stabilizing the spatial structure of peptide chains. Single-thiol labeling schemes require the permanent disruption of protein disulfide bonds, potentially posing unpredictable risks to the stability of protein higher-order structures (Nat. Biotechnol., 2012, 30, 184-189; Nat. Chem., 2019, 11, 310-319).

[0003] Thiol-bridged site-specific conjugation technology is a technique that can effectively solve the above problems, and can develop uniform and stable antibody-drug conjugates (ADCs) without requiring any specific engineering of the antibody. These ADCs have shown high efficacy in both in vitro and in vivo models, with high binding capacity and plasma stability. This method can perform site-specific conjugation without affecting the native structure of the antibody. It mainly consists of the following three steps: (1) reducing the disulfide bond to obtain two active cysteine ​​residues; (2) reacting the relinking reagent in the load with one of the cysteine ​​residues; (3) reacting the remaining cysteine ​​residue with the cysteine-relinking reagent complex generated in the previous step to generate a new link.

[0004] In 2020, Eric and his collaborators first reported a novel bioconjugating reagent, bis(methanesulfonyl)phenyloxadiazole (DiPODS), and successfully applied it to fluorescent dye-protein conjugates. This reagent contains a pair of oxadiazole methyl sulfone moieties, which can irreversibly form covalent bonds with the two thiol groups obtained from the reduction of proteins or peptides, while simultaneously bridging disulfide bonds. Figure 2 (Bioconjugate Chem., 2020, 31(12), 2789–2806). Subsequently, they synthesized a radionuclide antibody conjugate (US2022024904A1) using a similar strategy.

[0005] A literature search revealed only a few reports on the synthesis of di(methanesulfonyl)phenyloxadiazole DiPODS to date (CN103864711A; CN103864711B; US2022024904A1; Bioconjugate Chem., 2020, 31(12), 2789-2806; Bioconjugate Chem., 2023, 34(11), 2123-2132). Given the potential applications of di(methanesulfonyl)phenyloxadiazole DiPODS in mercapto-bridging site-specific coupling and its importance in pharmaceuticals and materials, it is essential to design and synthesize novel di(methanesulfonyl)phenyloxadiazole reagents (DiPODS) to meet the growing market demand. Summary of the Invention

[0006] The purpose of this invention is to provide two novel bis(methanesulfonyl)phenyloxadiazole reagents (DiPODS) and their preparation methods.

[0007] The bis(methanesulfonyl)phenyloxadiazole reagent (DiPODS) provided by this invention has the structural formula shown in Formula I or II:

[0008]

[0009] In formula I or II, n = 1 to 3, R is selected from substituents such as methyl, ethyl, propyl, and phenyl. 1 Selected from the following groups:

[0010]

[0011] This invention prepares two novel bis(methylsulfonyl)phenyloxadiazole reagents (DiPODS) for dithiol bridging labeling of antibodies, peptides, and hydrogels using inexpensive commercial raw materials through conventional transformations such as condensation and coupling. The side chains of these reagents are predominantly characterized by alkyl hydrophobic chains, which complement the hydrophilic chains of polyethylene glycol currently reported and used. The preparation method of this invention is simple, uses readily available raw materials, and produces high-value-added products, demonstrating significant economic benefits and market potential. Attached Figure Description

[0012] Figure 1 Reverse Michael addition reaction of maleimide with thiol group

[0013] Figure 2 Novel thiol-bridging site-directed coupling reagent DiPODS (dimethylsulfonylphenyloxadiazole) Detailed Implementation

[0014] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0015] Example 1

[0016]

[0017] [Step 1]

[0018] Compound 1-1 (20.0 g, 81.6 mmol) was dissolved in a mixed solvent of tert-butanol and toluene (200 mL, v:v = 1:1) at room temperature. Boc2O (4.0 eq., 71.2 g) and DMAP (4.0 eq., 39.9 g) were added sequentially to the system. The reaction was carried out at room temperature for 16 hours under TLC monitoring. After the reaction was completed, the system was extracted with ethyl acetate. The combined organic phases were washed sequentially with 1 N citric acid solution, saturated sodium bicarbonate solution and saturated brine. The organic phase was dried over sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by rapid column chromatography to give a white solid product 1-2 (11.5 g).

[0019] [Step 2]

[0020] Compound 1-2 (5.8 g, 16.1 mmol) was dissolved in 1,4-dioxane (70 ml) under a nitrogen atmosphere and at room temperature. Methyl 5-hexynate (1.2 eq., 2.4 g), Pd(dppf)Cl2 (0.05 eq., 585 mg), cuprous iodide (0.2 eq., 613 mg), and potassium carbonate (3.0 eq., 6.7 g) were added sequentially. The mixture was heated to 80 °C and reacted for 16 hours under TLC monitoring. After the reaction was completed, the mixture was cooled to room temperature. The reaction solution was diluted with ethyl acetate and filtered through diatomaceous earth. The filtrate was directly concentrated, mixed, and purified by rapid column chromatography to obtain a pale yellow oily product 1-3 (5.7 g).

[0021] [Step 3]

[0022] Compounds 1-3 (5.7 g, 14.2 mmol) were dissolved in dichloromethane (60 mL) under ice-water bath conditions, and trifluoroacetic acid (20 mL) was added dropwise. The mixture was heated to room temperature and reacted for 16 hours under TLC monitoring. After the reaction was completed, the reaction solution was directly concentrated, and the residue was slurried with ethyl acetate / n-hexane to obtain a white solid product 1-4 (3.8 g). No further purification was required, and it was used directly in the next step.

[0023] [Step 4]

[0024] Compounds 1-4 (3.8 g, 13.1 mmol) were dissolved in dichloromethane (50 mL) under ice-water bath conditions. Then, tert-butyl hydrazinoformate (2.5 eq., 4.3 g), DMAP (5.0 eq., 8.0 g), and EDCI (3.0 eq., 7.5 g) were added sequentially. The mixture was heated to room temperature and stirred for 16 hours. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was extracted with dichloromethane. The organic phases were combined, washed with water, 0.5 M hydrochloric acid, and saturated brine. The organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a pale yellow solid product 1-5 (6.4 g). No further purification was required, and it was used directly in the next step.

[0025] [Step 5]

[0026] Compounds 1-5 (6.4 g, 12.3 mmol) were dissolved in 1,4-dioxane (80 mL) under ice-water bath conditions, and a solution of isopropyl acetate containing hydrogen chloride (6 M, 10 mL) was added dropwise. The mixture was heated to room temperature and stirred for 3 hours. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was directly concentrated, and the residue was slurried with ethyl acetate / n-hexane to give a white solid product 1-6 (4.8 g). No further purification was required, and it was used directly in the next step.

[0027] [Step 6]

[0028] Compounds 1-6 (4.8 g, 15.1 mmol) were dissolved in a mixed solvent of 1,4-dioxane and water (100 mL, v / v = 1:1) at room temperature. An appropriate amount of potassium phosphate solid was added, and the pH of the reaction solution was adjusted to neutral. Potassium phosphate solid (3.0 eq., 9.6 g) and carbon disulfide (2.5 eq., 2.9 g) were then added. The mixture was heated to 60 °C and stirred for 9 hours. The reaction was monitored by HPLC. After the reaction was completed, the mixture was cooled to room temperature. The reaction solution was concentrated under reduced pressure and extracted with ethyl acetate. The aqueous phase was retained, and the pH of the aqueous phase was adjusted to about 3-4 with 1.0 M hydrochloric acid. A light green solid precipitated out. The solid was filtered to obtain product 1-7 (6.0 g).

[0029] [Step 7]

[0030] Compounds 1-7 (4.3 g, 10.8 mmol) were dissolved in tetrahydrofuran (60 mL) under ice-water bath conditions. Triethylamine (4.0 eq., 2.9 mL) and iodomethane (2.5 eq., 1.7 mL) were added dropwise. After the addition was complete, the mixture was naturally heated to room temperature and stirred for 4 hours. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was adjusted to weak acidity with 1 M hydrochloric acid, extracted with ethyl acetate, and the organic phases were combined. The mixture was washed with water and saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a white solid product 1-8. No further purification was required, and it was directly used for the next hydrolysis reaction.

[0031] [Step 8]

[0032] Compounds 1-8 (6.4 g, 14.9 mmol) were dissolved in a mixed solvent of tetrahydrofuran, methanol, and water (90 mL, v / v / v = 1:1:1) under ice-water bath conditions. Lithium hydroxide monohydrate solid (2.5 eq., 1.6 g) was added, and the reaction was stirred at room temperature for 3 hours under HPLC monitoring. After the reaction was completed, the reaction solution was concentrated, the concentrate was adjusted to weak acidity with 1 M hydrochloric acid, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain pale yellow solid product 1-9, which could be used directly in the next oxidation reaction without further purification.

[0033] [Step 9]

[0034] Compounds 1-9 (3.4 g, 8.2 mmol) were dissolved in dichloromethane (60 mL) under ice-water bath conditions, and 85% m-chloroperoxybenzoic acid (m-CPBA, 6.0 eq., 8.5 g) was added. The mixture was heated to room temperature and stirred for 48 hours. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly purified by dry silica gel chromatography to obtain a white solid crude product. The crude product was then purified a second time by acetonitrile / water reversed-phase column chromatography to obtain a white solid final product 1-10 (1.1 g).

[0035] 1 H NMR (400MHz, DMSO-d6, ppm): δ12.21(brs,1H),8.67(s,1H),8.30(s,2H),3.75(s,6H),2.63-2.49(m,2H),2.45-2.34(m,2H),1.89-1.76(m,2H);

[0036] MS(ESI)m / z calculated for C 18 H 15 N4O8S2[MH] - :479.04,found:479.13.

[0037] Example 2

[0038]

[0039] [Step 1]

[0040] Compound 2-1 (10.0 g, 40.8 mmol) was dissolved in DMF (100 mL) at room temperature, followed by the addition of benzyl bromide (2.2 eq., 10.7 mL) and potassium carbonate (2.4 eq., 13.5 g). The mixture was heated to 100 °C and reacted for 2 hours under TLC monitoring. After the reaction was complete, the mixture was cooled to room temperature, extracted with ethyl acetate, and the organic phases were combined, washed multiple times with water and saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a white solid product 2-2. This product was used directly in the next step without further purification.

[0041] [Step 2]

[0042] Compound 2-2 (6.5 g, 15.3 mmol) was dissolved in 1,4-dioxane (80 mL) under a nitrogen atmosphere and at room temperature. Methyl 5-hexynate (1.2 eq., 2.3 g), Pd(dppf)Cl2 (0.05 eq., 556 mg), cuprous iodide (0.2 eq., 583 mg), and potassium carbonate (3.0 eq., 6.4 g) were added sequentially. The mixture was heated to 80 °C and reacted for 16 hours under TLC monitoring. After the reaction was completed, the mixture was cooled to room temperature. The reaction solution was diluted with ethyl acetate and filtered through diatomaceous earth. The filtrate was directly concentrated, mixed, and purified by rapid column chromatography to obtain a pale yellow oily product 2-3 (7.3 g).

[0043] [Step 3]

[0044] Compound 2-3 (7.3 g, 15.5 mmol) was dissolved in tetrahydrofuran (75 mL) under a nitrogen atmosphere and at room temperature. 10% palladium on carbon (1.5 g) was added, and the mixture was evacuated three times with a hydrogen balloon. The reaction was continued for 16 hours under a hydrogen atmosphere (1 atm) with stirring and TLC monitoring. After the reaction was completed, the reaction solution was diluted with ethyl acetate and filtered through diatomaceous earth. The filtrate was directly concentrated to obtain a white solid product 2-4 (3.8 g), which could be used directly in the next step without further purification.

[0045] [Step 4]

[0046] Compound 2-4 (3.8 g, 12.9 mmol) was dissolved in dichloromethane (50 mL) at room temperature. Tert-butyl hydrazinoformate (2.5 eq., 4.3 g), DMAP (5.0 eq., 7.9 g), and EDCI (3.0 eq., 7.4 g) were added sequentially. The mixture was stirred for 12 hours and monitored by HPLC. After the reaction was complete, the reaction solution was extracted with dichloromethane. The organic phases were combined, washed with water, 0.5 M hydrochloric acid, and saturated brine. The organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a pale yellow solid product 2-5 (6.0 g). No further purification was required, and it was used directly in the next step.

[0047] [Step 5]

[0048] At room temperature, compound 2-5 (6.0 g, 11.5 mmol) was dissolved in 1,4-dioxane (90 mL), and a 1,4-dioxane solution of hydrogen chloride (4.0 M, 10 mL) was added dropwise. The reaction was stirred for 4 hours and monitored by HPLC. After the reaction was completed, the reaction solution was directly concentrated under reduced pressure to obtain product 2-6 (3.2 g), which could be used directly in the next step without further purification.

[0049] [Step 6]

[0050] Compound 2-6 (3.2 g, 10.0 mmol) was dissolved in a mixed solvent of 1,4-dioxane and water (60 mL, v / v = 1:1) at room temperature. An appropriate amount of potassium phosphate solid was added, and the pH of the reaction solution was adjusted to neutral. Potassium phosphate solid (3.0 eq., 6.4 g) and carbon disulfide (2.5 eq., 1.9 g) were then added. The mixture was heated to 110 °C and stirred for 6 hours. The reaction was monitored by HPLC. After the reaction was completed, the mixture was cooled to room temperature, and the reaction solution was directly concentrated under reduced pressure. The residue was extracted with ethyl acetate, and the aqueous phase was retained. The pH of the aqueous phase was adjusted to about 3-4 with 0.5 M hydrochloric acid, and a large amount of white solid precipitated. The product 2-7 (4.4 g) was obtained by filtration and drying. No further purification was required, and it was directly used in the next step.

[0051] [Step 7]

[0052] Compound 2-7 (2.1 g, 5.2 mmol) was dissolved in tetrahydrofuran (50 mL) under a nitrogen atmosphere and an ice-water bath. Triethylamine (4.0 eq., 2.9 mL) and iodomethane (2.5 eq., 0.8 mL) were added dropwise. After the addition was complete, the mixture was naturally heated to room temperature and stirred for 2 hours. The reaction was monitored by HPLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure. 80 mL of water was added, and the pH was adjusted to weakly acidic with 1.0 M hydrochloric acid. The mixture was extracted with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain product 2-8 (2.0 g). No further purification was required, and it was directly used in the next hydrolysis reaction.

[0053] [Step 8]

[0054] Compound 2-8 (3.5 g, 8.1 mmol) was dissolved in a mixed solvent of tetrahydrofuran, methanol, and water (45 mL, v / v / v = 1:1:1) at room temperature. Lithium hydroxide monohydrate solid (1.5 eq., 511 mg) was added, and the mixture was stirred for 3 hours under HPLC monitoring. After the reaction was complete, the reaction solution was extracted with ethyl acetate, and the aqueous phase was retained. The pH of the aqueous phase was adjusted to weakly acidic with 0.5 M HCl, and extracted again with ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a white solid product 2-9 (2.1 g). No further purification was required, and it was directly used in the next reaction.

[0055] [Step 9]

[0056] Compound 2-9 (2.0 g, 4.5 mmol) was dissolved in dichloromethane (75 mL) at room temperature, and 85% m-chloroperoxybenzoic acid (m-CPBA, 6.0 eq., 5.4 g) was added. The mixture was stirred for 48 hours and monitored by HPLC. After the reaction was completed, the reaction solution was directly purified by dry silica gel chromatography to obtain a white solid crude product. The crude product was then purified a second time by acetonitrile / water reversed-phase column chromatography to obtain a white solid product 2-10 (520 mg).

[0057] [Step 10]

[0058] Compound 2-10 (200 mg, 0.4 mmol) was dissolved in DMF (2 mL) at room temperature, and EDCI (2.0 eq., 153 mg) and N-hydroxysuccinimide (1.1 eq., 44 mg) were added sequentially. The mixture was stirred for 12 hours and monitored by TLC. After the reaction was completed, the reaction solution was extracted with ethyl acetate, the organic phases were combined, washed with water, saturated citric acid solution, and saturated brine, the organic phase was dried over sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by rapid column chromatography to obtain a white solid final product 2-11 (150 mg).

[0059] 1 H NMR (400MHz, DMSO-d6, ppm): δ8.70(s,1H),8.22(s,2H),3.56(s,6H),2.88-2.82(m,6H),2.64-2.60(m,2H),1.85-1.75(m,4H),1.55-1.25(m,2H);

[0060] MS(ESI)m / z calculated for C 22 H 24 N5O 10 S2[M+H] + :582.09,found:582.29.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Two bis(methanesulfonyl)phenyloxadiazoles for dithiol-bridging labeling of antibodies, peptides, and hydrogels, with structural formulas as shown in Formula I or II: In formula I or II, n = 1, 2, or 3, and R is selected from substituents such as methyl, ethyl, propyl, and phenyl. 1 Selected from the following groups:

2. A method for synthesizing bis(methylsulfonyl)phenyloxadiazole for dithiol-bridged labeling of antibodies, peptides, and hydrogels, characterized in that, As described in Formula I of claim 1, when R is methyl, R 1 When the hydrogen content is 3, the synthesis steps are as follows:

3. The method for synthesizing bis(methylsulfonyl)phenyloxadiazole for dithiol-bridged labeling of antibodies, peptides, and hydrogels according to claim 2, characterized in that: The specific method for synthesizing compound 1-2 is as follows: compound 1-1 is dissolved in a mixed solvent of toluene and tert-butanol, and ditert-butyl dicarbonate and DMAP are added sequentially. The reaction is carried out at room temperature. After the reaction is completed, the temperature is lowered to room temperature. The reaction solution is extracted, desolventized, and purified to obtain compound 1-2. The specific method for synthesizing compounds 1-3 is as follows: under a nitrogen atmosphere and at room temperature, compounds 1-2 are dissolved in 1,4-dioxane, and methyl 5-hexynate, Pd(dppf)Cl2, cuprous iodide and potassium carbonate are added sequentially. The temperature is raised to react. After the reaction is completed, the temperature is lowered to room temperature. The reaction solution is diluted with ethyl acetate and filtered through diatomaceous earth. The filtrate is desolventized and purified to obtain compounds 1-3. The specific method for synthesizing compounds 1-4 is as follows: under room temperature conditions, compounds 1-3 are dissolved in dichloromethane, trifluoroacetic acid is added dropwise, the mixture is stirred and reacted, and after the reaction is completed, the reaction solution is directly concentrated and slurried to obtain compounds 1-4 without further purification, and can be directly used for the next reaction. The specific method for synthesizing compounds 1-5 is as follows: under room temperature conditions, compounds 1-4 are dissolved in dichloromethane, and tert-butyl hydrazinoformate, DMAP and EDCI are added sequentially. The mixture is stirred and reacted. After the reaction is completed, the reaction solution is extracted and the solvent is removed to obtain compounds 1-5. No further purification is required, and they can be directly used in the next reaction. The specific method for synthesizing compounds 1-6 is as follows: under room temperature conditions, compounds 1-5 are dissolved in 1,4-dioxane, and a solution of 1,4-dioxane containing hydrogen chloride is added dropwise. The reaction is continued with stirring. After the reaction is completed, the reaction solution is purified by desolventizing and slurrying to obtain products 1-6. No further purification is required, and they can be directly used in the next step of the reaction. The specific method for synthesizing compounds 1-7 is as follows: under room temperature conditions, compounds 1-6 are dissolved in a mixed solvent of 1,4-dioxane and water, an appropriate amount of potassium phosphate solid is added, the pH of the reaction solution is adjusted to neutral, potassium phosphate solid and carbon disulfide are added, the temperature is raised and the reaction is carried out. After the reaction is completed, the temperature is lowered to room temperature, the reaction solution is directly concentrated under reduced pressure, the pH of the residue is adjusted to about 3-4 with hydrochloric acid, a solid is precipitated, and the solid is filtered to obtain compounds 1-7. The specific method for synthesizing compounds 1-8 is as follows: under ice-water bath conditions, compounds 1-7 are dissolved in tetrahydrofuran, triethylamine and iodomethane are added dropwise, the mixture is naturally heated to room temperature and stirred to react, and after the reaction is completed, the reaction solution is extracted and the solvent is removed to obtain compounds 1-8, which do not require further purification and can be directly used for the next hydrolysis reaction. The specific method for synthesizing compounds 1-9 is as follows: under room temperature conditions, compounds 1-8 are dissolved in a mixed solvent of methanol, tetrahydrofuran and water, solid lithium hydroxide is added, the reaction is stirred, and after the reaction is completed, the reaction solution is concentrated. The pH of the aqueous phase of the residue is adjusted to about 3-4 with dilute HCl. After extraction and solvent removal, compounds 1-9 are obtained. No further purification is required, and they can be directly used for the next oxidation reaction. The specific method for synthesizing compounds 1-10 is as follows: under room temperature conditions, compounds 1-9 are dissolved in dichloromethane, m-chloroperoxybenzoic acid is added, the mixture is stirred and reacted, and after the reaction is completed, the reaction solution is directly purified by dry silica gel mixing and rapid column chromatography to obtain white solid product 1-10.

4. Another method for synthesizing bis(methylsulfonyl)phenyloxadiazole active esters for dithiol-bridged labeling of antibodies, peptides, and hydrogels, characterized in that, The bis(methanesulfonyl)phenyloxadiazole active ester according to formula II of claim 1, wherein R is methyl, R 1 When the sample is succinimide and n=3, the synthesis steps are as follows:

5. The method for synthesizing bis(methanesulfonyl)phenyloxadiazole active esters for dithiol-bridged labeling of antibodies, peptides, and hydrogels according to claim 4, characterized in that: The specific method for synthesizing compound 2-2 is as follows: compound 2-1 is dissolved in DMF solution, potassium carbonate and benzyl bromide are added sequentially, the temperature is raised and the reaction is carried out. After the reaction is completed, the temperature is lowered to room temperature, and the reaction solution is extracted and the solvent is removed to obtain compound 2-2. No further purification is required, and it can be directly used for the next reaction. The specific method for synthesizing compound 2-3 is as follows: under a nitrogen atmosphere and at room temperature, compound 2-2 is dissolved in 1,4-dioxane, and methyl 5-hexynate, Pd(dppf)Cl2, cuprous iodide and potassium carbonate are added in sequence. The temperature is raised to react. After the reaction is completed, the temperature is lowered to room temperature. The reaction solution is filtered through diatomaceous earth. The filtrate is desolventized and purified to obtain compound 2-3. The specific method for synthesizing compound 2-4 is as follows: under a nitrogen atmosphere and at room temperature, compound 2-3 is dissolved in tetrahydrofuran, 10% palladium on carbon is added, the gas is evacuated three times by a hydrogen balloon, and the reaction is stirred under a hydrogen atmosphere. After the reaction is completed, the reaction solution is diluted with ethyl acetate and filtered through diatomaceous earth. The filtrate is desolventized to obtain compound 2-4, which does not require further purification and can be directly used in the next step of the reaction. The specific method for synthesizing compound 2-5 is as follows: under room temperature conditions, compound 2-4 is dissolved in dichloromethane, and tert-butyl hydrazide formate, DMAP and EDCI are added sequentially. The mixture is stirred and reacted. After the reaction is completed, the reaction solution is extracted and the solvent is removed to obtain compound 2-5. No further purification is required, and it can be directly used in the next reaction. The specific method for synthesizing compounds 2-6 is as follows: under room temperature conditions, compounds 2-5 are dissolved in 1,4-dioxane, and a solution of 1,4-dioxane containing hydrogen chloride is added dropwise. The reaction is continued with stirring. After the reaction is completed, the reaction solution is desolventized to obtain product 2-6, which does not require further purification and can be directly used in the next step of the reaction. The specific method for synthesizing compounds 2-7 is as follows: under room temperature conditions, compounds 2-6 are dissolved in a mixed solvent of 1,4-dioxane and water, an appropriate amount of potassium phosphate solid is added, the pH of the reaction solution is adjusted to neutral, potassium phosphate solid and carbon disulfide are added, the temperature is raised and the reaction is carried out. After the reaction is completed, the temperature is lowered to room temperature, the reaction solution is directly concentrated under reduced pressure, and the pH of the residue is adjusted to about 3-4 with hydrochloric acid, a solid is precipitated, the solid is filtered to obtain the crude product; the crude product is extracted, solvent removed and purified to obtain compounds 2-7. The specific method for synthesizing compounds 2-8 is as follows: under a nitrogen atmosphere and an ice-water bath, compounds 2-7 are dissolved in tetrahydrofuran, and triethylamine and iodomethane are added dropwise. The mixture is then naturally heated to room temperature and stirred. After the reaction is completed, the reaction solution is extracted and the solvent is removed to obtain compounds 2-8. No further purification is required, and the mixture can be directly used for the next hydrolysis reaction. The specific method for synthesizing compounds 2-9 is as follows: under room temperature conditions, compounds 2-8 are dissolved in a mixed solvent of acetonitrile and water, lithium hydroxide solid is added, the reaction is stirred, and after the reaction is completed, the reaction solution is extracted with ethyl acetate, the aqueous phase is retained, the pH of the aqueous phase is adjusted to about 3-4 with dilute HCl, and compounds 2-9 are obtained by extraction and solvent removal. No further purification is required, and they can be directly used for the next oxidation reaction. The specific method for synthesizing compound 2-10 is as follows: under room temperature conditions, compound 2-9 is dissolved in dichloromethane, m-chloroperoxybenzoic acid is added, the mixture is stirred and reacted, and after the reaction is completed, the reaction solution is directly purified by dry silica gel mixing and rapid column chromatography to obtain white solid product 2-10. The specific method for synthesizing compound 2-11 is as follows: under room temperature conditions, compound 2-10 is dissolved in DMF, and EDCI and N-hydroxysuccinimide are added sequentially. The mixture is stirred and reacted. After the reaction is completed, the reaction solution is extracted, desolventized, and purified to obtain compound 2-11.

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