Sulfuryl pyridine derivative-based disulfide bond bridging reagent, conjugate as well as preparation method and application of sulfuryl pyridine derivative-based disulfide bond bridging reagent and conjugate

Abstract

The invention discloses a disulfide bond bridging reagent based on a sulfuryl pyridine derivative, a conjugate as well as a preparation method and application of the disulfide bond bridging reagent. The disulfide bond bridging reagent provided by the invention can be used for re-bridging disulfide bonds in biomolecules (polypeptide and protein) containing disulfide bonds and further coupling small molecules and functional molecules. The invention also provides a preparation method and application of the conjugates, including but not limited to bioimaging and targeted therapy. The bridging reagent is good in reactivity, the generated conjugate is high in stability, successful coupling of small molecules shows that the coupling method has the potential of coupling various functional molecules, and coupling and application of part of functional groups are carried out. And a novel tool integrating water solubility, reactivity, product stability and functional group coupling is provided for biomolecule coupling.

Description

Technical Field

[0001] This invention relates to the field of biomolecular coupling technology, and in particular to a disulfide bond bridging reagent, coupling compound, preparation method and application based on sulfone pyridine derivatives. Background Technology

[0002] Peptide-drug conjugates (PDCs) are a novel class of targeted therapeutic agents composed of peptides and small molecule drugs covalently linked together. Their mechanism of action is similar to that of antibody-drug conjugates (ADCs). Compared to antibodies, peptides have smaller molecular weights and sizes, enabling them to cross membrane transport proteins into cells and thus access a wider range of intracellular target proteins. J. Pharm. Pharmacol. ,2008, 60 ,543-585), in addition, PDC has the advantages of low toxicity and side effects and low difficulty in modification ( J. Med. Chem. 2024 67 ,1641−1661). However, PDC also has certain limitations in its effects, such as the need for repeated administration within a short period of time to ensure efficacy due to its short half-life and rapid renal excretion rate; and poor stability in the digestive system environment, making oral administration difficult ( Chem. Soc. Rev. 2021 50 , 1480-1494. Several strategies have been proposed to improve the chemical stability of PDCs, among which the cyclic peptide construction strategy can stabilize the secondary structure of peptides, thereby increasing their affinity for the target and significantly improving peptide stability. Chem. Eur. J. ,2016, 22 (17112-17129). Due to the low natural abundance of cysteine ​​(Cys) and its strong nucleophilicity, reactions targeting cysteine ​​residues exhibit high specificity and reactivity. Consequently, disulfide bond re-bridging peptide cyclization strategies have been widely applied in PDCs. Developing disulfide bond re-bridging strategies for cyclic peptides is of great significance for stabilizing cyclic peptide structures, enhancing the binding ability of cyclic peptides to targets, and developing PDCs.

[0003] The bridging method for bromomethylbenzene and its derivatives involves introducing methylene bromide at different sites on the benzene ring. The substitution of bromine with thiol groups can achieve the construction of monocyclic to polycyclic peptides. It can also ensure a high reaction rate when bridging monocyclic peptides against a pair of disulfide bonds. However, it is difficult to couple functional groups and faces the problem of difficulty in derivatization. ChemBioChem ,2005, 6, 821-824). Allyl sulfone / bis-sulfone can introduce functional groups on the Michael acceptor, initiate addition-elimination reaction, and can take into account both bridging and coupling functions, but its reaction rate is low, and bis-sulfone also has the disadvantage of poor water solubility, which needs to add 40% acetonitrile to assist the reaction system, which may bring uncertain changes to the structure of polypeptide or protein ( Nat. Protoc. , 2006, 1 , 2241-2252; Chem. Sci. , 2016, 7 , 3234-3239). Maleimide derivatives have the highest reactivity of dibromomaleimide, and can also achieve derivatization by connecting functional groups on N, but its coupling product is poor in stability and is easily exchanged with other sulfhydryl groups in the biological environment, thereby causing the premature dissociation of the functional groups, which brings certain limitations to its practical application ( J. Am. Chem. Soc. , 2010, 132 , 1960-1965), in addition, dibromopyridazinedione also faces similar problems ( Chem. Commun. , 2011, 47 , 8781-8783).

[0004] To ensure that the bridged polypeptide or protein still has its own biological activity, the bridging reaction should be carried out under mild conditions, and the bridged product formed should be more stable than the unmodified biological molecule. At the same time, the reaction of disulfide bond bridging should have high selectivity, that is, the reaction reagent is not affected by other natural nucleophiles, and on this basis, the reaction is efficient. In order to further realize the coupling function, the design of the molecule should add modification or connection site of functional groups, so as to form an ideal bridging reagent. At present, the field still needs a method that can take into account water solubility, reactivity, product stability and coupling functional groups. SUMMARY

[0005] The present application aims to provide a kind of polypeptide or protein disulfide bond bridging and coupling and method. The reagent is designed to have bridging and coupling functions of groups on the skeleton of sulfone pyridine molecule, realizes the disulfide bond rebridging of cyclic peptide and protein containing disulfide bond, further completes the coupling of small molecule and functional groups, and carries out biomedical application. The bridging reagent of the present application has good reactivity, and the coupling product generated has high stability. The successful coupling of small molecules shows that the coupling method has the potential to couple various functional molecules, and some functional groups are coupled and applied, providing a new tool for biomolecule coupling that takes into account water solubility, reactivity, product stability and coupling functional groups.

[0006] The technical scheme adopted to achieve the purpose of the present application is: The first aspect of the present application is a disulfide bridge reagent for polypeptides or proteins based on sulfonyl pyridine derivatives, which has the general structure as shown in formula IA or IB: , IA IB; wherein R in formula IA, IB is the same or different, each independently selected from aryl or C1-C10 alkyl; R1 in formula IA, IB is the same or different, each independently selected from -SO2X 1 , -CH2X 2 or -CHO; wherein X 1 is selected from aryl or C1-C10 alkyl, and X 2 is selected from one of halogen; R2 in formula IA, IB is the same or different, each independently selected from -CN or -CHO.

[0007] The reagent comprises sulfonyl pyridine derivatives with disulfide bridge and coupling functions, which uses the existing sulfonyl group as one of the bridge groups, and undergoes aromatic nucleophilic substitution reaction with thiol during the bridging reaction, and uses R1 as another bridge group to complete the disulfide bridge with the sulfonyl group to form a disulfide covalent bridge modified biomolecule. The functional group R2 serves as a coupling group to couple with small molecules or functional molecules to form a disulfide covalent bridge modified biomolecule-small molecule or functional molecule conjugate.

[0008] Preferably, R in formula IA, IB is each independently selected from aryl or C1-C5 alkyl; R1 in formula IA, IB is each independently selected from -SO2Ph, -CH2Br or -CHO; R2 in formula IA, IB is each independently selected from -CN, -CHO; and the halogen is selected from Cl, Br, I.

[0009] More preferably, the disulfide bridge reagent is selected from at least one of the following compounds: .

[0010] The second aspect of the present application further comprises a preparation method of the disulfide bridge reagent, which is selected from method one, method two, method three or method four: Method one has the following reaction general formula: ; Method two has the following reaction general formula: ; Method three has the following reaction general formula: ; Method four has the following reaction general formula:

[0011] Method one comprises the following steps: IA-1 is used as a starting material, dissolved in an organic solvent, and then reacted with NaSO2R to obtain compound IA-2; wherein the structural formula of compounds IA-1 and IA-2 are as follows: ; wherein R, R2, X 1 are the same as R, R2, X 1 in compound IA, respectively; Method two comprises the following steps: Step (1), compound IA-3 is used as a starting material, dissolved in an organic solvent, and then reacted with oxalyl chloride under ice bath conditions to obtain compound IA-4; wherein the structural formula of compounds IA-3 and IA-4 are as follows: ; Step (2), IA-4 is dissolved in an organic solvent, and then reacted with a reducing agent under ice bath conditions to obtain compound IA-5; wherein the structural formula of compound IA-5 is as follows: ; Step (3), IA-5 is dissolved in an organic solvent, and then reacted with an oxidizing agent one under ice bath conditions and then warmed to obtain compound IA-6; wherein the structural formula of compound IA-6 is as follows: ; Step (4), compound IA-6 is reacted with an oxidizing agent two in an organic solvent to obtain compound IA-7, wherein the compound IA-7 is compound IA-7-1 or IA-7-2; the structural formula of compound IA-7-1 is as follows: , and the structural formula of compound IA-7-2 is as follows: ; Compound IA-7-1 is reacted with hydroxylamine hydrochloride in methanol to obtain compound IA-7-2, which is dissolved in acetonitrile, and then reacted with triethylamine and dimethyl sulfoxide, and then dropwise added with oxalyl chloride under ice bath conditions to obtain compound IA-7-2, Step (5), one of compounds IA-7 is reacted with trifluoroacetic anhydride in an organic solvent to obtain compound IA-8; wherein the compound IA-7 is selected from at least one of compounds IA-7-1 and IA-7-2; the structural formula of compound IA-7 is as follows: ; wherein R2 is the same as R2 in compound IA; the structural formula of compound IA-8 is as follows: ; wherein R2 is the same as R2 in compound IA; Step (6), compound IA-8 is reacted with NaSO2R in an organic solvent to obtain compound IA-9; the structural formula of compound IA-9 is as follows: ; wherein, R, R2 correspond to R, R2 in compound IA respectively; Step (7) is step (7-1) or step (7-2): Step (7-1), compound IA-9, an organic solvent are added into PX 2 3 under ice bath condition, and then the reaction is warmed to obtain compound IA-10; the structural formula of compound IA-10 is as follows ; wherein, R, R2, X 2 correspond to R, R2, X 2 in compound IA respectively; Step (7-2), compound IA-9 is oxidized by adding an oxidant in an organic solvent to obtain compound IA-10-1; the structural formula of compound IA-10-1 is as follows: ; Method three comprises the following steps: Step (1), compound IB-1 is dissolved in an organic solvent, and sodium borohydride is added under ice bath condition to perform reduction reaction to obtain compounds IB-2-1 and IB-2-2; wherein, the structural formulae of compounds IB-1, IB-2-1 and IB-2-2 are as follows: ; Step (2), compound IB-2-1 is reacted with NaSO2R in an organic solvent to obtain compound IB-3-1, and compound IB-2-2 is reacted with NaSO2R in an organic solvent to obtain compound IB-3-2; wherein, the structural formulae of compounds IB-3-1 and IB-3-2 are as follows: ; wherein, R corresponds to R in compound IB; Step (3), IB-3-1 is dissolved in an organic solvent, and PX 2 3 is added, and then the reaction is warmed to obtain compound general formula IB-4-1, and the structural formula of compound IB-4-1 is as follows: ; wherein, R corresponds to R in compound IB, and X 2 corresponds to X 2 in compound IB respectively; Step (4), compound IB-4-1 is added with sodium borohydride in an organic solvent to perform secondary reduction reaction to obtain compound IB-5-1, and the structural formula of compound IB-5-1 is as follows: ; wherein, R corresponds to R in compound IB, and X 2 corresponds to X 2 in compound IB respectively; Step (5), compound IB-5-1 is subjected to one oxidation reaction in an organic solvent by adding an oxidant to obtain compound IB-6-1; compound IB-3-2 is subjected to one oxidation reaction in an organic solvent by adding an oxidant to obtain compound IB-6-2; the structural formulae of compounds IB-6-1 and IB-6-2 are as follows: ; wherein R corresponds to R of compound IB, X 2 corresponds to X of compound IB; 2 Step (6), compound IB-6-1 is subjected to one reaction in a methanol solvent by adding hydroxylamine hydrochloride to obtain a compound, which is dissolved in an acetonitrile solvent, and then triethylamine and dimethyl sulfoxide are added, and oxalyl chloride is added dropwise under ice bath to generate compound IB-7 through two reactions; the structural formula of compound IB-7 is as follows: ; wherein R corresponds to R of compound IB, X 2 corresponds to X of compound IB; 2 Method four comprises the following steps: Compound IB-11 is used as a starting material, dissolved in an organic solvent, and then reacted with NaSO2R to obtain compound IB-12; the structural formulae of compounds IB-11 and IB-12 are as follows: ; wherein R, R2 and X 1 are respectively the same as R, R2 and X of compound IB. 1

[0012] Preferably, in method one, the organic solvent is dimethyl sulfoxide; the molar ratio of compound IA-1 to NaSO2R is 1:4.5-5.5; the usage ratio of compound IA-1 to the organic solvent is 1 g:30-40 mL; the reaction temperature is 45-55°C; and the reaction time is 1.5-3.0 hours.

[0013] In method two, in step (1), the organic solvent is dichloromethane and N,N-dimethylformamide; the molar ratio of compound IA-3 to oxalyl chloride is 1:2-4; the usage ratio of compound IA-3 to dichloromethane is 1 g:10-15 mL; the usage ratio of compound IA-3 to N,N-dimethylformamide is 1 g:10-15 μL; the one-reaction temperature is 0-4°C; the one-reaction time is 4-6 hours; the usage ratio of compound IA-3 to methanol is 1 g:10-15 mL; the molar ratio of compound IA-3 to triethylamine is 1:1.5-2; the two-reaction temperature is 20-25°C; and the two-reaction time is 4-6 hours; ​​​In Method Two, in step (2), the organic solvent is tetrahydrofuran; the reducing agent is lithium aluminum hydride; the molar ratio of compound IA-4 to the reducing agent is 1 : 1-1.5; the amount ratio of compound IA-4 to the organic solvent is 1 g : 10-15 mL; the temperature of the reduction reaction is 0-4°C; and the time of the reduction reaction is 1-2 hours. In Method Two, in step (3), the organic solvent is dichloromethane; the oxidizing agent I is meta-chloroperoxybenzoic acid; the molar ratio of compound IA-5 to the oxidizing agent I is 1 : 1.2-1.5; the amount ratio of compound IA-5 to the organic solvent is 1 g : 9-12 mL; the temperature of the oxidation reaction is 20-25°C; and the time of the oxidation reaction is 1-1.5 hours. In Method Two, in step (4), the organic solvent is at least one of dichloromethane, chloroform or tetrahydrofuran; the oxidizing agent II is active manganese dioxide, tin dioxide or Dess-Martin oxidizing agent; the molar ratio of compound IA-6 to the oxidizing agent II is 1 : 3-5; the amount ratio of compound IA-6 to the organic solvent is 1 g : 20-30 mL; the temperature of the second oxidation reaction is 60-70°C; and the time of the second oxidation reaction is 8-10 hours. In Method Two, in step (4), the molar ratio of compound IA-7-1 to hydroxylamine hydrochloride is 1 : 10-15; the amount ratio of compound IA-7-1 to methanol is 1 g : 10-50 mL; the temperature of the first reaction is 20°C-25°C; the time of the first reaction is 5-8 hours; the molar ratio of compound IA-7-1 to triethylamine is 1 : 2-3; the molar ratio of compound IA-7-1 to dimethyl sulfoxide is 80-100 : 1; the molar ratio of compound IA-7-1 to oxalyl chloride is 1 : 1.0-1.5; the amount ratio of compound IA-7-1 to acetonitrile is 1 g : 12-18 mL; the temperature of the second reaction is 20°C-25°C; and the time of the second reaction is 9-11 hours. In Method Two, in step (5), the organic solvent is chloroform; the molar ratio of compound IA-7 to trifluoroacetic anhydride is 1 : 2-3; the amount ratio of compound IA-7 to the organic solvent is 1 g : 8-12 mL; the temperature of the reaction is 20°C-25°C; and the time of the reaction is 10-25 hours. In Method Two, in step (6), the organic solvent is dimethyl sulfoxide; the molar ratio of compound IA-8 to NaSO2R is 1 : 2.5-3.5; the amount ratio of compound IA-8 to the organic solvent is 1 g : 15-20 mL; the temperature of the reaction is 45°C-55°C; and the time of the reaction is 2-4 hours. In Method Two, in step (7-1), the organic solvent is chloroform; the molar ratio of compound IA-9 to PX 2molar ratio of compound 3 to compound 2 is 1 : 2.0-4.0; the usage ratio of compound 2 to organic solvent is 1 g : 20-30 mL; the reaction temperature is 20°C-25°C; the reaction time is 2-4 hours; In step (7-2), the organic solvent is at least one of dichloromethane, chloroform or tetrahydrofuran; the oxidant II is active manganese dioxide, tin dioxide or des Martin oxidant; the molar ratio of compound IA-9 to oxidant II is 1 : 3-5; the usage ratio of compound IA-9 to organic solvent is 1 g : 20-30 mL; the oxidation reaction temperature is 60-70°C; the oxidation reaction time is 8-10 hours.

[0014] In method three, in step (1), the organic solvent is dichloromethane and methanol; the molar ratio of compound IB-1 to sodium borohydride is 1 : 1.1-3; the usage ratio of compound IB-1 to dichloromethane is 1 g : 10-15 mL; the usage ratio of compound IB-1 to methanol is 1 g : 3-5 mL; the reduction reaction temperature is 20-30°C; the reduction reaction time is 10-14 hours; In method three, in step (2), the organic solvent is acetonitrile; the molar ratio of compound IB-2-1 to NaSO2R is 1 : 2.0-4.0; the molar ratio of compound IB-2-2 to NaSO2R is 1 : 4.5-5.5; the usage ratio of compound IB-2-1 to organic solvent is 1 g : 10-15 mL; the usage ratio of compound IB-2-2 to organic solvent is 1 g : 30-40 mL; the reaction temperature is 78-80°C; the reaction time is 20-30 hours; In method three, in step (3), the organic solvent is dichloromethane; the molar ratio of compound IB-3-1 to PX3 is 1 : 2.0-4.0; the usage ratio of compound IB-3-1 to organic solvent is 1 g : 20-30 mL; the reaction temperature is 40°C-45°C; the reaction time is 6-8 hours; In method three, in step (4), the organic solvent is dichloromethane and methanol; the molar ratio of compound IB-4-1 to sodium borohydride is 1 : 1.1-3; the usage ratio of compound IB-4-1 to dichloromethane is 1 g : 10-15 mL; the usage ratio of compound IB-4-1 to methanol is 1 g : 3-5 mL; the secondary reduction reaction temperature is 25°C; the secondary reduction reaction time is 10-14 hours; In the third method, in step (5), the organic solvent is at least one of dichloromethane, chloroform or tetrahydrofuran; the oxidant is active manganese dioxide, tin dioxide or des Martin oxidant; the molar ratio of compound IB-5-1 to the oxidant is 1:8-12; the molar ratio of compound IB-3-2 to the oxidant is 1:10-16; the usage ratio of compound IB-5-1 to the organic solvent is 1g:20-30 mL; the usage ratio of compound IB-3-2 to the organic solvent is 1g:50-70 mL; the temperature of the first oxidation reaction is 20-25℃; and the time of the first oxidation reaction is 8-10 hours. In the third method, in step (6), the molar ratio of compound IB-6-1 to hydroxylamine hydrochloride is 1:10-15; the usage ratio of compound IB-6-1 to methanol is 1g:10-50 mL; the temperature of the first reaction is 20-25℃; the time of the first reaction is 5-8 hours; the molar ratio of compound IB-6-1 to triethylamine is 1:2-3; the molar ratio of compound IB-6-1 to dimethyl sulfoxide is 80-100:1; the molar ratio of compound IB-6-1 to oxalyl chloride is 1:1.0-1.5; the usage ratio of compound IB-6-1 to acetonitrile is 1g:12-18 mL; the temperature of the second reaction is 20-25℃; and the time of the second reaction is 9-11 hours. In the fourth method, the organic solvent is dimethyl sulfoxide; the molar ratio of compound IB-11 to NaSO2R is 1:4.5-5.5; the usage ratio of compound IB-11 to the organic solvent is 1g:30-40 mL; the temperature of the reaction is 45-55℃; and the time of the reaction is 1.5-3.0 hours.

[0015] The third aspect of the present application further comprises a disulfide bond bridged modified biomolecule, which has the following general structure formula II A or formula II B or a salt type thereof:

[0016] IIA II B; The disulfide bond bridged modified biomolecule has the following general structure formula II A or formula II B: The corresponding precursor is a biomolecule containing at least one pair of disulfide bonds The R2 is -CN or -CHO.

[0017] As preferred, the biomolecule is a protein The corresponding precursor is a cyclic peptide, an antibody or an antibody fragment containing at least one pair of disulfide bonds; more preferably, the cyclic peptide is a cyclic peptide formed by at least one pair of cysteine thiol disulfide bonds, having a binding or regulating ability with the corresponding receptor, and further preferably at least one of Octreotide, Somatostatin, Lanreotide, Atosiban, Eptifibatide, Terlipressin, Lysipressin, Argiprestocin, Oxytocin, iRGD and its homologues; The antibody or antibody fragment is an antibody or antibody fragment containing at least one pair of disulfide bonds, and the antibody fragment includes one or more parts of a single antibody, which retains the ability to specifically bind to an epitope, and further preferably a whole antibody or antibody fragment of Ablximab, Cetuximab, Trastuzumab, Sacituzumab, Palivizumab.

[0018] The fourth aspect of the present application also includes a preparation method of the disulfide bridge modified biomolecule, which comprises the following steps: A reducing agent and the disulfide bridge reagent are added to a biomolecule solution respectively, the pH is adjusted with an acid or a base, a bridging reaction is carried out, and a disulfide bridge modified biomolecule is obtained; the biomolecule is ; The reaction formula is: .

[0019] As preferred, the molar ratio of the disulfide bridge reagent to the biomolecule is ≥1; The solvent of the biomolecule solution is at least one of a phosphate buffer, an ammonium bicarbonate buffer, a sodium acetate buffer, and a 4-hydroxyethylpiperazine ethanesulfonic acid buffer; the reducing agent is at least one of tris(2-carboxyethyl)phosphine and tris(hydroxypropyl)phosphine; the molar ratio of the reducing agent to the biomolecule is >1; the pH is adjusted with hydrochloric acid or sodium hydroxide; the pH of the bridging reaction is 7.0-8.5; the temperature of the bridging reaction is 25-37°C; and the time of the bridging reaction is 0.5-6 hours.

[0020] The fifth aspect of the present application also includes a disulfide bridge modified biomolecule-small molecule conjugate, which has the following general formula IIIA, general formula IIIB or a salt type thereof:

[0021] III A III B; wherein, the R2in formula III A, formula III B is the corresponding precursor is a cyclic peptide, an antibody or an antibody fragment containing at least one pair of disulfide bonds; more preferably, the cyclic peptide is a cyclic peptide formed by at least one pair of cysteine thiol disulfide bonds, having binding or regulatory ability with the corresponding receptor, further preferably at least one of Octreotide, Somatostatin, Lanreotide, Atosiban, Eptifibatide, Terlipressin, Lysipressin, Argiprestocin, Oxytocin, iRGD and its homologues; the antibody or antibody fragment is an antibody or antibody fragment containing at least one pair of disulfide bonds, the antibody fragment including one or more parts of a single antibody, which retains the ability to specifically bind to an epitope, further preferably a whole antibody or antibody fragment of Ablximab, Cetuximab, Trastuzumab, Sacituzumab, Palivizumab; the R3is selected from thiazolidine and its derivatives, thiazoline and its derivatives, oxime and its derivatives; preferably: ; Further preferably, the disulfide bridge modified biomolecule-small molecule conjugate is selected from at least one of the following structural formula: .

[0022] The sixth aspect of the present application also includes a preparation method of the disulfide bridge modified biomolecule-small molecule conjugate, the preparation method comprising the following steps: adding a small molecule compound to the solution of the disulfide bridge modified biomolecule, adjusting the pH with an acid or a base, and performing a coupling reaction to obtain the disulfide bridge modified biomolecule-small molecule conjugate; the structure of the disulfide bridge modified biomolecule is shown in general formula II A or general formula II B; the small molecule compound is a small molecule compound capable of reacting with R2in general formula II A or general formula II B; The reaction formula is: ; Preferably, the small molecule compound is selected from the group consisting of molecules containing 1, 2-aminothiol, hydroxylamine groups, more preferably: .

[0023] As preferred, the molar ratio of the small molecule compound to the disulfide bridge modified biomolecule is ≥1; the solvent of the disulfide bridge modified biomolecule solution is at least one of phosphate buffer, ammonium bicarbonate buffer, sodium acetate buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer; the pH is adjusted by hydrochloric acid or sodium hydroxide; the pH of the coupling reaction is 6.0-8.5; the temperature of the coupling reaction is 25-37°C; the time of the coupling reaction is 1-24 hours.

[0024] The seventh aspect of the present application also includes a disulfide bridge modified biomolecule-function molecule conjugate, the disulfide bridge modified biomolecule-function molecule conjugate has the following general formula IV A, general formula IV B or a salt type thereof:

[0025] IV A IV B; Among them, the R1, R2, R3, R4 in formula IV A, formula IV B The corresponding precursor is a cyclic peptide, an antibody or an antibody fragment containing at least one pair of disulfide bonds; more preferably, the cyclic peptide is a cyclic peptide formed by at least one pair of cysteine thiol disulfide bonds, which has binding or regulating ability with the corresponding receptor, further preferably at least one of Octreotide, Somatostatin, Lanreotide, Atosiban, Eptifibatide, Terlipressin, Lysipressin, Argiprestocin, Oxytocin, iRGD and its homologues; The antibody or antibody fragment is an antibody or antibody fragment containing at least one pair of disulfide bonds, the antibody fragment includes one or more parts of a single antibody, which retains the ability to specifically bind to an epitope, further preferably a whole antibody or antibody fragment of Ablciximab, Cetuximab, Trastuzumab, Sacituzumab, Palivizumab; The R4 is selected from a fluorescent group, a drug molecule; preferably rhodamine, monomethyl australin E, monomethyl australin F, camptothecin, irinotecan, pyrrolobenzodiazepine; Further preferably, the disulfide bridge modified biomolecule-function molecule conjugate is selected from at least one of the following compounds of the following structural formulae: .

[0026] The eighth aspect of the present application further comprises a preparation method of the disulfide bridge modified biomolecule-function molecule conjugate, the preparation method comprising the following steps: adding a function molecule to the disulfide bridge modified biomolecule solution, adjusting pH with an acid or a base, and performing a conjugation reaction to obtain the disulfide bridge modified biomolecule-function molecule conjugate; The reaction formula is: ; The disulfide bridge modified biomolecule is of general formula II A or general formula II B; The function molecule is a function molecule capable of reacting with R2 in the disulfide bridge modified biomolecule; Preferably, the function molecule has the following structural formula: ; The R4 corresponds to R4 in formula IV A or formula IV B.

[0027] More preferably, the molar ratio of the function molecule to the disulfide bridge modified biomolecule is ≥1; the solvent of the disulfide bridge modified biomolecule solution is at least one of phosphate buffer, ammonium bicarbonate buffer, sodium acetate buffer, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer; pH is adjusted with hydrochloric acid or sodium hydroxide; the pH of the conjugation reaction is 4.0-8.5; the temperature of the conjugation reaction is 25-37°C; and the time of the conjugation reaction is 1-24 hours.

[0028] The ninth aspect of the present application further comprises the use of the disulfide bridge linking reagent, the disulfide bridge modified biomolecule, the disulfide bridge modified biomolecule-small molecule conjugate, or the disulfide bridge modified biomolecule-function molecule conjugate in the preparation of a drug.

[0029] Preferably, the disulfide bridge modified biomolecule-function molecule conjugate is used for preparing a polypeptide-drug conjugate or an antibody-drug conjugate; more preferably, the disulfide bridge modified biomolecule-function molecule conjugate is used for preparing a drug for treating cancer, an inflammatory disease, an autoimmune disease, a cardiovascular disease, or a neurological disease.

[0030] Compared with the prior art, the present application has the following beneficial effects: 1. The invention provides a class of disulfide bond bridging reagents based on sulfonyl pyridine derivatives, which can re-bridge the disulfide bond of polypeptides and proteins containing disulfide bonds, and by adjusting the position and type of substituents, high reactivity disulfide bond bridging reagents are obtained. 2. The disulfide bond bridging reagent of the invention is small in size and good in water solubility, and can complete the bridging reaction in a complete buffer solution environment. 3. The disulfide bond bridging reagent provided by the invention has high stability, and no significant reduction is observed after incubation in high-concentration glutathione and plasma for 24 h. 4. The polypeptide-functional molecule conjugate constructed by the disulfide bond bridging reagent provided by the invention can ensure the biological activity of the polypeptide itself, can recognize receptors and enter cells, and can still ensure the killing effect of the drug on tumor cells after conjugation with the drug. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a schematic diagram of the disulfide bond bridging reagent for disulfide bond bridging of biomolecules and conjugation of small molecules or functional groups.

[0032] Figure 2 It is an LC-MS graph of the application of the disulfide bond bridging reagent to re-bridge the disulfide bond of octreotide in Example 6 of the invention.

[0033] Figure 3 It is an LC-MS graph of the application of the disulfide bond bridging reagent to re-bridge the disulfide bond of oxytocin in Example 7 of the invention.

[0034] Figure 4 It is an LC-MS graph of the further conjugation of the disulfide bond bridging modified octreotide with small molecule cysteine in Example 8 of the invention.

[0035] Figure 5 It is an HPLC graph of the continuous sampling of the octreotide-cysteine conjugate in Example 9 of the invention in 10 mM glutathione and 30% human plasma, respectively, in which naphthol is used as an internal standard.

[0036] Figure 6 It is an LC-MS graph of the further conjugation of the disulfide bond bridging modified octreotide with fluorescent molecule rhodamine in Example 10 of the invention.

[0037] Figure 7 It is a fluorescence confocal imaging graph of the internalization of the uncoupled rhodamine and the octreotide-rhodamine conjugate into HepG2 cells in Example 11 of the invention.

[0038] Figure 8 It is an LC-MS graph of the further conjugation of the disulfide bond bridging modified octreotide with drug molecule camptothecin in Example 12 of the invention.

[0039] Figure 9 IC50 plot of the cytotoxicity of unconjugated camptothecin and octreotide-camptothecin conjugate on HepG2 cells in Example 13 of the present application.

[0040] Figure 10 LC-MS plot of disulfide bridge reduction and cysteine conjugation of trastuzumab Fab fragment in Example 14 of the present application. DETAILED DESCRIPTION

[0041] The present application will be further described in conjunction with specific examples. It should be understood, however, that the specific examples, while indicating preferred embodiments of the application, are included merely for purposes of illustration and are not intended to limit the scope of the application.

[0042] Example 1: Synthesis of T1

[0043] Into a 100 mL round bottom flask, compound 1-1 (528 mg), sodium benzene sulfinate (1.64 g) and dimethyl sulfoxide (20 mL) were added in sequence. The reaction was stirred at 50 °C for 2 hours. Diluted with 100 mL ethyl acetate, washed with saturated sodium chloride solution, dried the organic phase with anhydrous sodium sulfate, filtered and concentrated under reduced pressure, the residue was separated by silica column (petroleum ether: ethyl acetate 1:1) to give the target product T1 (658 mg, yield 85%, light yellow solid).1H NMR (400 MHz, CDCl3) δ 10.94 (s, 1H), 9.15 (s, 1H), 8.70 (s, 1H), 8.10 (d, J = 7.2 Hz, 2H), 8.00 (d, J = 7.2 Hz, 2H), 7.82 - 7.74 (m, 1H), 7.74 - 7.65 (m, 3H), 7.61 (t, J = 7.8 Hz, 2H). Example 2: Synthesis of T2

[0044] Step 1: Synthesis of 2-2 Into a 500 mL round bottom flask was added compound 2-1 (9.21 g), N,N- dimethylformamide (100 μί), and anhydrous dichloromethane (100 mL) sequentially. Oxalyl chloride (15.2 g) was added dropwise under ice bath condition, after addition, the reaction was stirred at room temperature for 6 hours. Concentrated under reduced pressure, anhydrous methanol (100 mL) and triethylamine (10.1 g) were added slowly under ice bath condition sequentially, after addition, the reaction was stirred at room temperature, TLC monitored the reaction was completed after 4 hours, concentrated under reduced pressure, the residue was separated by silica gel column (petroleum ether: ethyl acetate 5: 1) to give the target product 2-2 (8.3 g, yield 75%, yellowish solid).1H NMR (400 MHz, CDCl3) δ 8.95 (s, 1H), 7.28 (s, 1H), 3.96 (s, 3H), 2.59 (s, 3H). Step 2: synthesis of 2-3 Into a 500 mL round bottom flask was added compound 2-2 (7.40 g) and anhydrous tetrahydrofuran (100 mL). Lithium aluminum hydride (1.52 g) was added in portions under ice bath condition, the reaction was stirred at this temperature for 1 hour. 1.52 mL water and 15% sodium hydroxide aqueous solution were added sequentially, the stirring was continued for 15 minutes, the diatomite was filtered and the filtrate was collected, the filtrate was dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was separated by silica gel column (petroleum ether: ethyl acetate 1: 1) to give the target product 2-3 (8.3 g, yield 86%, white solid).1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 7.87 (s, 1H), 4.05 (s, 2H), 3.65 (s, 3H), 2.01 (s, 3H). Step 3: synthesis of 2-4 To a 250 mL round bottom flask was added compound 2-3 (5.2 g) and anhydrous dichloromethane (50 mL). M-CPBA (6.9 g) was added portion wise under ice bath conditions and the reaction was stirred at room temperature for 1 h. 100 mL of 10% aqueous potassium carbonate solution was added and stirring was continued for 15 min. The aqueous phase was washed with dichloromethane (3 x 100 mL) and the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica (dichloromethane:methanol 100:1) to give the target product 2-4 (4.9 g, 94% yield, white solid).1H NMR (400 MHz, CDCl3): δ 8.85 (s, 1H), 7.06 (s, 1H), 4.25 (s, 2H), 3.79 (s, 3H), 2.65 (s, 3H).13C NMR (101 MHz, CDCl3) δ 169.23, 163.21, 158.21, 153.29, 150.98, 58.23, 30.27. HRMS (ESI) calcd for [C7H8CINO2+ H] + : 174.0316, found: 174.0319. Step 4: Synthesis of 2-5 To a 250 mL round bottom flask was added compound 2-4 (4.3 g), activated manganese dioxide (8.7 g) and chloroform (100 mL) sequentially. The reaction was stirred at 65 °C for 8 h and the reaction was monitored by TLC. The reaction mixture was filtered through celite and the filtrate was collected, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica (dichloromethane:methanol 100:1) to give the target product 2-5 (3.9 g, 91% yield, white solid).1H NMR (400 MHz, CDCl3) δ 9.87 (s, 1H), 8.02 (s, 1H), 7.61 (s, 1H), 3.67 (s, 3H), 2.03 (s, 3H). Step 5: Synthesis of 2-6 Into a 250 mL round bottom flask was added compound 2-5 (3.4 g) and chloroform (30 mL). Trifluoroacetic anhydride (10 mL) was added dropwise under ice bath condition and the reaction was stirred at room temperature for 10 hours. The reaction mixture was concentrated under reduced pressure to give a brown oily liquid, 30 mL dichloromethane was added and concentrated under reduced pressure, finally 50 mL dichloromethane and 50 mL saturated sodium bicarbonate solution were added successively and stirred at room temperature for 10 hours. The organic phase was washed with dichloromethane (3 x 50 mL), the combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, the residue was purified by silica gel column chromatography (petroleum: ethyl acetate 1:1) to give the target product 2-6 (1.8 g, 10.4 mmol, yield 52%, light yellow solid).1H NMR (400 MHz, CDCl3) δ 9.93 (s, 1H), 8.01 (s, 1H), 7.02 (s, 1H), 4.24 (s, 2H), 3.59 (s, 3H). Step 6: synthesis of 2-7 Into a 100 mL round bottom flask was added compound 2-6 (1.72 g), sodium benzenesulfinate (4.92 g) and dimethyl sulfoxide (30 mL) successively. The reaction was stirred at 50 °C for 2 hours. Diluted with 100 mL ethyl acetate, washed with saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, the residue was purified by silica gel column chromatography (petroleum: ethyl acetate 1:1) to give the target product 2-7 (2.6 g, yield 93%, light yellow solid).1H NMR (400 MHz, CDCl3) δ 9.98 (s, 1H), 8.69 (s, 1H), 8.43 (s, 1H), 8.12 - 8.01 (m, 2H), 7.56 - 7.40 (m, 1H), 7.34 - 7.19 (m, 2H). 4.65 (s, 2H). Step 7: synthesis of T2 Into a 100 mL round bottom flask, compound 2-7 (831 mg) and chloroform (20 mL) were added successively. Phosphorous tribromide (976 mg) was added slowly at 0 °C, and the reaction was continued at room temperature for 2 h. The reaction was monitored by TLC. The reaction mixture was neutralized with saturated sodium bicarbonate solution, diluted with 200 mL of ethyl acetate, washed with saturated sodium chloride solution (3 x 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by column chromatography on silica gel (petroleum ether: ethyl acetate 20: 1) to give the target product T2 (918 mg, 90% yield, white solid).1H NMR (400 MHz, CDCl3) δ 10.88 (s, 1H), 9.15 (s, 1H), 8.09 (s, 1H), 8.03 - 7.94 (m, 1H), 7.78 - 7.69 (m, 2H), 7.68 - 7.61 (m, 2H), 4.66 (s, 2H). Example 3: Synthesis of T3, T4 and T5

[0045] Step 1: Synthesis of 3-2-1 Methyl 4-chloro-2,6-pyridinedicarboxylate (2.912 g, 12.68 mmol, 1 eq.) was dissolved in a mixture of 100 mL of dichloromethane and 30 mL of methanol. Sodium borohydride (528 mg, 13.95 mmol, 1.1 eq.) was added in three portions with an interval of 15 min under an ice-water bath. After the addition, the reaction was stirred at room temperature for 13 h until no gas bubbles were generated. The reaction was monitored by TLC (PE:EA = 1:1). After the reaction was completed, the solvent was removed by rotary evaporation. Then, 30 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated by column chromatography to give 2.400 g of white solid with a yield of 94.2%.1H NMR (400 MHz, CDCl3) δ ppm: 8.42 (s, 1H), 8.28 (s, 1H), 5.39 (s, 1H), 5.12 (s, 2H), 3.90 (s, 3H).13C NMR (101 MHz, CDCl3) δ ppm: 165.4, 156.5, 149.2, 143.1, 126.8, 122.2, 63.4, 51.5. Step 2: Synthesis of 3-2-2 Methyl 4-chloro-2,6-diformylpyridine-2-carboxylate (2.912 g, 12.68 mmol, 1 eq.) was dissolved in a mixture of 100 mL dichloromethane and 30 mL methanol, sodium borohydride (1.440 g, 38.04 mmol, 3 eq.) was added in three portions with 15 min interval under ice water bath, after the addition, the reaction was stirred at room temperature for 13 h until no more bubbles were generated, TLC was used to monitor the reaction (PE:EA = 1:1), after the reaction was completed, the solvent was removed by rotary evaporation, 30 mL saturated sodium bicarbonate solution was added, the mixture was extracted with dichloromethane, the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The product was separated by column chromatography to obtain 2.300 g of white solid with a yield of 96.2%.1H NMR (400 MHz, CDCl3) δ ppm: 8.42 (s, 1H), 8.28 (s, 1H), 5.39 (s, 2H), 5.12 (s, 4H). 13C NMR (101 MHz, CDCl3) δ ppm: 156.5, 149.2, 143.1, 126.8, 122.2, 63.4. Step 3: Synthesis of 3-3-1 Methyl 4-chloro-2,6-diformylpyridine-2-carboxylate (2.912 g, 12.68 mmol, 1 eq.) was dissolved in a mixture of 100 mL dichloromethane and 30 mL methanol, sodium borohydride (1.440 g, 38.04 mmol, 3 eq.) was added in three portions with 15 min interval under ice water bath, after the addition, the reaction was stirred at room temperature for 13 h until no more bubbles were generated, TLC was used to monitor the reaction (PE:EA = 1:1), after the reaction was completed, the solvent was removed by rotary evaporation, 30 mL saturated sodium bicarbonate solution was added, the mixture was extracted with dichloromethane, the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The product was separated by column chromatography to obtain 2.300 g of white solid with a yield of 96.2%.1H NMR (400 MHz, CDCl3) δ ppm: 8.42 (s, 1H), 8.28 (s, 1H), 5.39 (s, 2H), 5.12 (s, 4H). 13C NMR (101 MHz, CDCl3) δ ppm: 156.5, 149.2, 143.1, 126.8, 122.2, 63.4. Step 4: Synthesis of 3-3-2 Dissolve 4-chloro-6-hydroxymethylpyridine (3.038 g, 17.50 mmol, 1 eq.) in 100 mL acetonitrile, add sodium benzenesulfinate (8.61 g, 52.47 mmol, 3 eq.) and glacial acetic acid (1 mL, 17.49 mmol, 1 eq.), heat to reflux at 80 °C for 24 h, monitor the reaction by TLC (PE:EA = 1:1). Add about 1 g potassium carbonate solid, stir, filter, concentrate, and purify by column chromatography to give 2.862 g of white solid with a yield of 58.6%.1H NMR (400 MHz, CDCl3) δ ppm: 9.01 (s, 1H), 8.87 (s, 1H), 7.93 (d, J = 6.3 Hz, 1H), 7.83 (d, J = 7.7 Hz, 2H), 7.74 (t, J = 7.2 Hz, 2H), 5.39 (s, 2H), 5.12 (s, 4H).13C NMR (101 MHz, CDCl3) δ ppm: 156.5, 147.8, 141.4, 133.7, 129.7, 128.3, 124.4, 119.8, 63.9. Step 5: Synthesis of 3-4-1 Dissolve 4-benzenesulfonyl-6-hydroxymethyl-2-pyridinecarboxylic acid methyl ester (2.923 g, 9.511 mmol, 1 eq.) in 150 mL dichloromethane, slowly add phosphorus tribromide (1.79 mL, 19.023 mmol, 2 eq.), heat to reflux at 42 °C for 7 h, monitor the reaction by TLC (PE:EA = 1:1). After the reaction is completed, pour into ice water, adjust the pH to 10 with potassium carbonate solid, extract with dichloromethane three times, wash with saturated brine, separate, dry over anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to give 2.293 g of white solid with a yield of 65.1%.1H NMR (400 MHz, CDCl3) δ ppm: 8.83 (s, 1H), 8.39 (s, 1H), 7.93 (d, J = 6.3 Hz, 1H), 7.83 (d, J = 7.7 Hz, 2H), 7.74 (t, J = 7.2 Hz, 2H), 4.89 (s, 2H), 3.90 (s, 3H).13C NMR (101 MHz, CDCl3) δ ppm: 165.4, 155.9, 148.8, 147.8, 141.4, 133.7, 129.7, 128.3, 124.4, 119.8, 51.5, 33.2. Step 6: Synthesis of 3-5-1 Methyl 6-bromomethyl-4-phenylsulfonyl-2-pyridinecarboxylate (2.38 g, 6.43 mmol, 1 eq.) was dissolved in a mixture of 100 mL dichloromethane and 30 mL methanol, sodium borohydride (0.270 g, 7.07 mmol, 1.1 eq.) was added in three portions with 15 min interval under ice water bath, after the addition, the reaction was stirred at room temperature for 16 h until no more bubbles were generated, TLC was used to monitor the reaction (PE:EA=1:1), after the reaction was completed, the solvent was removed by rotary evaporation, 80 mL saturated sodium bicarbonate solution was added, the mixture was extracted with dichloromethane twice, the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The product was purified by column chromatography to obtain 0.960 g of white solid with a yield of 43.6%.1H NMR (400 MHz, CDCl3) δ ppm: 8.23 (s, 1H), 7.93 (s, 1H), 7.93 (d, J=4.9 Hz, 1H), 7.83 (d, J=6.5 Hz, 2H), 7.74 (t, J=6.1 Hz, 2H), 5.12 (s, 2H), 4.89 (s, 2H).13C NMR (101 MHz, CDCl3) δ ppm: 156.3, 156.1, 146.1, 141.4, 133.7, 129.7, 128.3, 118.0, 64.2, 33.5. Step 7: Synthesis of T3 Manganese (IV) oxide (2.43 g, 28.0 mmol, 10 eq.) was added to a solution of 6-bromomethyl-4-phenylsulfonyl-2-hydroxymethylpyridine (0.960 g, 2.81 mmol, 1 eq.) in chloroform. The reaction was stirred at room temperature for 10 h, and TLC was used to monitor the reaction (PE:EA=1:1). The filter residue was removed by filtration, concentrated, and purified by column chromatography to obtain 0.890 g of white solid with a yield of 94.6%.1H NMR (400 MHz, CDCl3) δ ppm: 9.71 (s, 1H), 8.79 (s, 1H), 8.41 (s, 1H), 7.93 (d, J=4.9 Hz, 1H), 7.83 (d, J=6.5 Hz, 2H), 7.74 (t, J=6.1 Hz, 2H), 4.89 (s, 2H).13C NMR (101 MHz, CDCl3) δ ppm: 192.6, 157.2, 153.1, 147.2, 141.4, 133.7, 129.7, 128.3, 125.1, 116.0, 33.2. Step 8: Synthesis of T5 Dissolve 4-benzenesulfonyl-2-hydroxymethylpyridine (0.700 g, 2.51 mmol, 1 eq.) in chloroform, add manganese dioxide (2.17 g, 25.0 mmol, 10 eq.), stir at room temperature for 10 h, monitor the reaction by TLC (PE:EA = 1:1). Filter off the residue, concentrate, and purify by column chromatography to give 0.646 g of white solid with a yield of 93.6%.1H NMR (400 MHz, CDCl3) δ ppm: 9.71 (s, 2H), 9.45 (s, 2H), 7.93 (d, J=6.3 Hz, 1H), 7.83 (d, J=6.9 Hz, 2H), 7.74 (t, J=7.8 Hz, 2H).13C NMR (101 MHz, CDCl3) δ ppm: 192.6, 154.2, 148.3, 141.4, 133.7, 129.7, 128.3, 123.1. Step 9: Synthesis of T4 Dissolve E-4-benzenesulfonyl-6-bromomethyl-2-pyridinecarboxaldehyde oxime (0.500 g, 1.41 mmol, 1 eq.) in acetonitrile, add triethylamine (0.499 g, 4.93 mmol, 3.5 eq.) and dimethyl sulfoxide (2 mg), and dropwise add oxalyl chloride (0.214 g, 1.69 mmol, 1.2 eq.). Stir at room temperature for about 10 h. After the reaction is complete, spin off the acetonitrile, add NaHCO3solution to remove the acid, extract the aqueous phase with dichloromethane three times, combine the organic layers, dry over anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to give 350 mg of white solid with a yield of 73.7%.1H NMR (400 MHz, CDCl3) δ ppm: 8.74 (s, 1H), 8.46 (s, 1H), 7.93 (d, J=6.8 Hz, 1H), 7.83 (d, J=7.6 Hz, 2H), 7.74 (t, J=7.9 Hz, 2H), 4.89 (s, 2H).13C NMR (101 MHz, CDCl3) δ ppm: 157.9, 147.5, 141.4, 134.2, 133.7, 129.7, 128.3, 124.6, 123.6, 117.1, 32.7. Example 4: Preparation and characterization of polypeptide-small molecule or functional molecule conjugate

[0046] The following are examples of the preparation and analytical characterization of polypeptide-functional molecule conjugates. It should be noted that the following examples are only partial embodiments and are not limited to the embodiments described herein. For example, the polypeptides are not limited to the polypeptides shown herein, but can also be any disulfide polypeptide; the functional molecules are not limited to the fluorescent and drug molecules shown herein, but can also be any functional molecule that can be conjugated.

[0047] General procedure for disulfide bridge modification of polypeptides with disulfide bridge reagents As an example, some commercially available cyclic peptides are used, including but not limited to octreotide, somatostatin, oxytocin, terlipressin, lanreotide, atosiban, lysyl vasopressin, arginine vasopressin, eptifibatide, iRGD, and the like, and the reaction of these cyclic peptides with disulfide bridge reagents T1-T5 is used as an example. In a 1.5 mL centrifuge tube, a polypeptide solution with a concentration of 0.1 mM (solvent: 20 mM phosphate buffer, pH 7.5) is added, a reducing agent TCEP (0.15 mM, 1.5 eq.) is added, and after mixing, a reduction reaction is carried out for 30-60 min, followed by the addition of a disulfide bridge reagent (0.11 mM, 1.1 eq.), adjustment to the desired pH value with hydrochloric acid or sodium hydroxide, and mixing, after which the mixture is placed in a metal heating module at 30°C for 1-3 h, and the disulfide bridge modification is completed.

[0048] General procedure for the preparation of disulfide bridge modified polypeptide-small molecule conjugates As an example, small molecules containing 1,2-aminothiol or hydroxylamine are used, including but not limited to cysteine, hydroxylamine hydrochloride, benzyl hydroxylamine, and the like, and the reaction of these small molecules with disulfide bridge modified polypeptides is used as an example. In a 1.5 mL centrifuge tube, a disulfide bridge modified polypeptide solution with a concentration of 0.1 mM (solvent: 20 mM phosphate buffer, pH 7.5) is added, a small molecule that reacts with it (0.6 mM 6 eq.) is added, and the pH is adjusted to the desired value with hydrochloric acid or sodium hydroxide, and after mixing, the mixture is placed in a metal heating module at 30°C for 3-20 h, and the conjugation of the small molecule to the polypeptide is completed.

[0049] General procedure for the preparation of disulfide bridge modified polypeptide-functional molecule conjugates Take the functional molecule containing 1,2-aminothiol as an example, the functional groups include but are not limited to rhodamine, monomethyl auristatin E, monomethyl auristatin F, camptothecin, irinotecan, pyrrolobenzodiazepine, and the reaction of these functional molecules with disulfide bridge modified polypeptides as an example. In a 1.5 mL centrifuge tube, add a disulfide bridge modified polypeptide solution with a concentration of 0.1 mM (solvent: 20 mM phosphate buffer, pH 7.5), add the functional molecule to be reacted (0.15 mM 1.5 eq.), adjust to the desired pH value with hydrochloric acid or sodium hydroxide, mix well and place on a metal heating module at 30°C for 3-20h to complete the coupling of small molecules and polypeptides.

[0050] General characterization method of disulfide bridge modified polypeptides and their conjugates (a) RP-HPLC analysis High performance liquid chromatograph: high performance liquid chromatography system.

[0051] Chromatographic column: BioResolve RP C8 (4.6x150 mm, 3.2 μm) (manufacturer: Phenomenex).

[0052] Mobile phase: mobile phase A: 0.1% H2O (containing 0.1% TFA); mobile phase B: ACN (containing 0.1% TFA); eluted according to the following elution program: 0-2 min mobile phase A volume 90%, mobile phase B volume 10%; 2-12 min mobile phase A volume 90%-40%, mobile phase B volume 10%-60%, linear change with time; 12-15 min mobile phase A volume 40%-10%, mobile phase B volume 60%-90% linear change with time; set the flow rate of mobile phase to 1 ml / min, column temperature 30°C.

[0053] Detection conditions: detection wavelength 280 nm.

[0054] Experimental procedure: take 5 μg sample (converted to mass and volume according to concentration), add storage buffer to a final volume of 25 μL, vortex mix. Centrifuge at 12000 rpm for 5 min, take 20 μL supernatant and inject into high performance liquid chromatograph, elute using the above elution program, and record the chromatogram.

[0055] (b) LC-MS (ESI-Q-TOF) analysis All samples were analyzed using Agilent 1290 series high performance liquid chromatograph (HPLC) system equipped with 6545 series liquid chromatograph-electrospray ionization-quadrupole time-of-flight (LC-ESI-Q-TOF) mass spectrometer (Agilent).

[0056] Chromatographic column: Agilent ZORBAX 300SB-C8 (2.1 x 150 mm) Mobile phase: Mobile phase A: H2O (containing 0.1% formic acid), mobile phase B: ACN (containing 0.1% formic acid). Elution was performed according to the following elution program: 0-2 min mobile phase A volume 95%, mobile phase B volume 5%; 2-12 min mobile phase A volume 90%-40%, mobile phase B volume 10%-60%, linear change over time; 12-14 min mobile phase A volume 40%-5%, mobile phase B volume 60%-95%, linear change over time; set the flow rate of mobile phase to 0.5 ml / min, column temperature 70 °C.

[0057] Mass spectrometry: The column effluent was continuously analyzed by a capillary electrospray source connected to an Agilent 6545 Q-TOF mass spectrometer. Mass spectra were obtained in positive electrospray ionization (ESI) mode, with an acquisition range set to 100 to 2500 Da, using profile mode, acquiring total ion chromatograms. The retention peak of the target protein in the total ion chromatogram was selected using Bioconfirm software (Agilent Technologies Inc., V10.0), and raw mass spectral data with different charges were obtained.

[0058] Example 5: Preparation and characterization of antibody fragment-small molecule conjugates The following is an example of the preparation and analytical characterization of antibody fragment-small molecule conjugates. It should be noted that the following example is only a partial embodiment and is not limited to the presently described embodiment. For example, the antibody fragment is not limited to the presently demonstrated antibody, but can also be any other disulfide bond-containing antibody fragment.

[0059] General procedure for disulfide bond bridging modification of antibody fragments by disulfide bond bridging reagents With some monoclonal antibody fragments that retain the ability to specifically bind to epitopes as examples, including but not limited to cetuximab, certuximab, mitumumab, patitumumab, sacituzumab, trastuzumab, etc. antibody fragments, and taking the reaction of these antibody fragments with disulfide bridge linking reagent T1-T5 as an example. In a 1.5 mL centrifuge tube, add an antibody fragment solution with a concentration of 0.1 mM (solvent is 20 mM phosphate buffer, pH 7.5), add reducing agent THP (0.5 mM, 5 eq.), mix well and perform reduction reaction for 30-60 min, remove THP using Zeba™ desalting centrifuge column, 7K MWCO (Zeba™ Spin Desalting Columns, 7K MWCO), then add disulfide bridge linking reagent (0.12 mM, 1.2 eq.), adjust to the desired pH value with hydrochloric acid or sodium hydroxide, mix well and place on a metal heating module at 30°C for 1-5 h to complete the disulfide bridge linking modification.

[0060] General preparation process of disulfide bridge linking modified antibody fragment-small molecule conjugates With small molecules containing 1,2-aminothiol or hydroxylamine as examples, including but not limited to cysteine, hydroxylamine hydrochloride, benzyl hydroxylamine, etc. small molecules, and taking the reaction of these small molecules with disulfide bridge linking modified antibody fragments as an example. In a 1.5 mL centrifuge tube, add a disulfide bridge linking modified antibody fragment solution with a concentration of 0.1 mM (solvent is 20 mM phosphate buffer, pH 7.5), add the small molecule to be reacted (0.6 mM 6 eq.), adjust to the desired pH value with hydrochloric acid or sodium hydroxide, mix well and place on a metal heating module at 30°C for 3-20 h to complete the coupling of the small molecule with the antibody or antibody fragment.

[0061] General characterization method of disulfide bridge linking modified antibody fragments and their conjugates (a) SEC-HPLC analysis High performance liquid chromatograph: Agilent 1260 liquid chromatograph.

[0062] Chromatographic column: Waters Xbridge BEH200 SEC (7.8 x 300 mm, 3.5 μm) Mobile phase: 50 mM PB, 100 mM NaCl, pH 7.2, eluted according to the following elution program, 0-30 min mobile phase A volume is 100%; set the flow rate of mobile phase to 1 ml / min, column temperature is 30°C.

[0063] Detection condition: detection wavelength is 280 nm.

[0064] Experimental procedure: 20 μg of sample (converted to mass and volume according to concentration) was taken, supplemented with storage buffer to a final volume of 25 μL, vortexed and mixed. 12000 rpm centrifugation for 5 min, 20 μL of supernatant was injected into the high performance liquid chromatograph, eluted using the above elution program, and the chromatogram was recorded.

[0065] (b) LC-MS (ESI-Q-TOF) analysis All samples were analyzed using an Agilent 1290 series high performance liquid chromatograph (HPLC) system equipped with a 6545 series liquid chromatograph-electrospray ionization-quadrupole time-of-flight (LC-ESI-Q-TOF) mass spectrometer (Agilent).

[0066] Chromatographic column: Agilent ZORBAX 300SB-C8 (2.1 x 150 mm) Mobile phase: Mobile phase A: H2O (containing 0.1% formic acid), mobile phase B: ACN (containing 0.1% formic acid). Eluted according to the following elution program: 0-2 min mobile phase A volume 95%, mobile phase B volume 5%; 2-12 min mobile phase A volume 90%-40%, mobile phase B volume 10%-60%, linearly changing with time; 12-14 min mobile phase A volume 40%-5%, mobile phase B volume 60%-95%, linearly changing with time; 14-15 min mobile phase A volume 5%-95%, mobile phase B volume 95%-5%, linearly changing with time; set the flow rate of mobile phase to 0.5 ml / min, column temperature 70°C.

[0067] Mass spectrometry: The column effluent was continuously analyzed by a capillary electrospray source connected to an Agilent 6545 Q-TOF mass spectrometer. The mass spectrum was obtained in positive electrospray ionization (ESI) mode, the acquisition range was set to 600 to 3200 Da, the profile mode was used, and the total ion chromatogram was obtained. The Bioconfirm software (Agilent Technologies Inc., V10.0) was used to select the retention peak of the target protein in the total ion chromatogram, and the original mass spectrum data was converted to zero charge mass spectrum using the maximum entropy deconvolution algorithm.

[0068] The deconvolution settings are configured as follows: mass range: 600.0-3200.0 daltons, mass step: 1.0000 daltons, baseline factor: 7.00, adduct: proton, isotope width: automatic. This setting provides a comprehensive analysis of the protein sample, allowing its mass and composition to be accurately characterized.

[0069] Example 6: Preparation of octreotide modified by disulfide bridge

[0070] According to the general process of disulfide bridge modification of polypeptides by disulfide bridge reagents, in a 1.5 mL centrifuge tube, octreotide solution with a concentration of 0.1 mM (solvent: 20 mM phosphate buffer, pH 7.5) was added, and a reducing agent TCEP (0.15 mM, 1.5 eq.) was added. After mixing, the reduction reaction was carried out for 60 min, and then in three parallel experiments, disulfide bridge reagents T1, T2, and T4 (0.11 mM, 1.1 eq.) were added, respectively. The pH was adjusted to 7.5 with sodium hydroxide, and after mixing, it was placed on a metal heating module at 30°C for 2 h to complete the disulfide bridge modification. From Figure 2 It can be seen that the reduced octreotide has completed the bridging reaction with T1, T2, and T4, respectively, and the bridging ratio is nearly 100%.

[0071] Example 7: Preparation of oxytocin modified by disulfide bridge

[0072] According to the general process of disulfide bridge modification of polypeptides by disulfide bridge reagents, in a 1.5 mL centrifuge tube, oxytocin solution with a concentration of 0.1 mM (solvent: 20 mM phosphate buffer, pH 7.5) was added, and a reducing agent TCEP (0.15 mM, 1.5 eq.) was added. After mixing, the reduction reaction was carried out for 60 min, and then in three parallel experiments, disulfide bridge reagents T1, T2, and T4 (0.11 mM, 1.1 eq.) were added, respectively. The pH was adjusted to 7.5 with sodium hydroxide, and after mixing, it was placed on a metal heating module at 30°C for 2 h to complete the disulfide bridge modification. From Figure 3 It can be seen that the reduced oxytocin has completed the bridging reaction with T1, T2, and T4, respectively, and the bridging ratio is nearly 100%.

[0073] Example 8: Preparation of octreotide-cysteine conjugate

[0074] According to the general preparation process of disulfide bond bridging modified polypeptide-small molecule conjugates, in a 1.5 mL centrifuge tube, a T4 bridging modified octreotide solution with a concentration of 0.1 mM (solvent: 20 mM phosphate buffer, pH 7.5) was added, cysteine (0.6 mM 6 eq.) was added, hydrochloric acid was added to adjust the pH to 6.1, and after mixing, it was placed on a metal heating module at 30°C for 20 h to complete the coupling of cysteine and octreotide. Figure 4 It can be seen that T4 bridging modified octreotide almost completely reacts with cysteine, and the cyan group on T4 forms a thiazoline with cysteine.

[0075] Example 9: Stability analysis of octreotide-cysteine conjugate In a 1.5 mL centrifuge tube, 0.1 mM octreotide-cysteine was added to a GSH solution with a concentration of 10 mM and human plasma with a volume fraction of 30%, and 0.05 mM naphthol was added as an internal reference. After mixing, it was placed on a metal heating module at 30°C for no less than 36 h, and samples were taken at 0, 1, 3, 5, 8, 12, 24, and 36 h. HPLC was used for monitoring. Figure 5 It can be seen that the octreotide-cysteine conjugate can stably exist in high-concentration GSH and human plasma within 36 h.

[0076] Example 10: Preparation of octreotide-fluorescent molecule rhodamine conjugate

[0077] According to the general preparation process of disulfide bond bridging modified polypeptide-functional molecule conjugates, in a 1.5 mL centrifuge tube, a T1 bridging modified octreotide solution with a concentration of 0.1 mM (solvent: 20 mM phosphate buffer, pH 7.5) was added, and a rhodamine molecule modified with a 1, 2-aminothiol group (0.3 mM 3 eq.) was added. Hydrochloric acid was added to adjust the pH to 7.0, and after mixing, it was placed on a metal heating module at 30°C for 20 h to complete the coupling of rhodamine and octreotide. Figure 6 It can be seen that T1 bridging modified octreotide almost completely couples with rhodamine, and the aldehyde group on T1 forms a thiazolidine with 1, 2-aminothiol.

[0078] Example 11: Application of octreotide-rhodamine fluorescent molecule conjugate in cell fluorescence imaging To verify the function of the conjugated octreotide to bind to the receptor and be endocytosed into the cell, HepG2 cells (somatostatin receptor positive) were used as an example, and 20000 cells / well were seeded on poly-D-lysine coated coverslips in a 24-well culture plate. The cells were attached for 24 h at 37°C in a 5% CO2 environment. The control group was treated with 25 μM Rhodamin B, and the experimental group was treated with 25 μM octreotide-Rhodamin B conjugate. Each was treated for 10 min at 37°C in the dark, followed by washing, fixation, and staining of the cell nucleus using Hoechst 33342, and finally the coverslips with the fixed cells were loaded onto a glass slide.

[0079] Imaging of the cells was performed using a confocal laser scanning microscope. Confocal imaging was performed using an Olympus IX83 inverted microscope equipped with a FluoView 3000 scanning system (Olympus, Center Valley, PA). DM405 / 488 / 561 standard emission filters were used. Kaede-red 561 nm and Hoechst 33342 405 nm were chosen as dyes and detectors. All images were acquired using a UPLSAPO x 40 numerical aperture 0.95 dry objective lens (Olympus).

[0080] As Figure 7 It can be seen that the octreotide-Rhodamin conjugate is effectively internalized into the cell, while the Rhodamin molecule without conjugation does not show red fluorescence in the cell, indicating that the octreotide modified by disulfide bridge connection retains the ability to recognize the receptor and be internalized into the cell.

[0081] Example 12: Preparation of octreotide-drug molecule camptothecin conjugate

[0082] According to the general preparation process of disulfide bridge modified polypeptide-function molecule conjugate, in a 1.5 mL centrifuge tube, a T1 bridge modified octreotide solution with a concentration of 0.1 mM (solvent: 20 mM phosphate buffer, pH 7.5) was added, 1, 2-amino mercapto group modified camptothecin molecules (0.3 mM 3 eq.) were added, hydrochloric acid was added to adjust the pH to 7.0, and after mixing, the mixture was placed on a metal heating module for reaction at 30°C for 20 h to complete the conjugation of camptothecin and octreotide. After HPLC separation and lyophilization, pure octreotide-camptothecin conjugate was obtained. From Figure 8 It can be seen that the T1 bridge modified octreotide is almost completely conjugated with camptothecin, and the aldehyde group on T1 forms a thiazolidine with 1, 2-amino mercapto.

[0083] Example 13: Application of octreotide-drug molecule camptothecin conjugate in cytotoxicity To verify the ability of octreotide to carry drug molecules and exert drug toxicity, HepG2 cells (somatostatin receptor positive) were used as an example, and the cells were seeded in a 96-well culture plate at a density of 3000 cells / well. The cells were attached for 24 h at 37°C in a 5% CO2 environment. The experimental group was octreotide-camptothecin conjugate (Oct-CPT), and the control group was uncoupled camptothecin (CPT). The cells were treated with gradient concentrations, and after 72 h, the drug was removed and CCK8 was used for cell activity detection. The treated cells were measured for absorbance at 450 nm using a microplate reader, and IC50 curves were plotted using Graphpad Prism 9. From Figure 9 It can be seen that the conjugate exhibits cytotoxicity close to that of the uncoupled drug, indicating that the bridging reagent does not weaken the toxicity of the cytotoxic drug, and the conjugated polypeptide can develop targeting under the premise of ensuring toxicity.

[0084] Example 14: Preparation of trastuzumab antibody fragment-cysteine conjugate

[0085] According to the general process of disulfide bond bridging modification of antibody fragments by disulfide bond bridging reagents, in a 1.5 mL centrifuge tube, a trastuzumab Fab fragment solution with a concentration of 0.1 mM (solvent: 20 mM phosphate buffer, pH 7.5) was added, a reducing agent THP (0.5 mM, 5 eq.) was added, and after mixing, a reduction reaction was performed for 60 min. THP was removed using Zeba™ desalting centrifuge columns, 7K MWCO (Zeba™ Spin Desalting Columns, 7K MWCO), and then the bridging reagent T4 (0.12 mM, 1.2 eq.) was added. The pH was adjusted to 7.5 with sodium hydroxide, and after mixing, it was placed in a metal heating module for 1 h at 30°C for disulfide bond bridging modification.

[0086] According to the general preparation process of disulfide bond modified antibody fragment-small molecule conjugate, in a 1.5 mL centrifuge tube, a T4 modified trastuzumab Fab fragment solution with a concentration of 0.1 mM (solvent: 20 mM phosphate buffer, pH 7.5) was added, cysteine (0.6 mM 6 eq.) was added, and the pH was adjusted to 8.0 with sodium hydroxide. After mixing, it was placed in a metal heating module for 24 h at 30°C for coupling of cysteine with the antibody fragment. From Figure 10 It can be seen that the trastuzumab Fab has almost completely completed the disulfide bond bridging modification and further coupled with cysteine within 24 h, with a conversion rate of about 40%, proving that the conjugate also has the potential to couple functional groups to antibody fragments.

[0087] In summary, the present application provides a disulfide bond bridging reagent based on a sulfonyl pyridine derivative, which can re-bridge the disulfide bond of a polypeptide or protein containing a disulfide bond and couple a small molecule or a functional molecule. The disulfide bond bridging reagent of the present application is small in size, good in water solubility, and can complete the bridging reaction in a complete buffer solution environment, and the constructed biomolecule-small molecule conjugate is high in stability. The present application also provides a preparation method of a biomolecule-functional molecule conjugate and its application at a cell level, and exhibits application potential in cancer diagnosis and treatment.

[0088] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A disulfide bond bridging reagent for peptides or proteins based on sulfonylurimidine derivatives, characterized in that, Having a general structural formula as shown in formula IA or IB: ， IA IB; in, In formulas IA and IB, R may be the same or different, and each is independently selected from aryl or C1-C10 alkyl groups, preferably aryl or C1-C5 alkyl groups; Whether R1 in equations IA and IB is the same or different, each is independently selected from -SO2X. 1 -CH2X 2 Or -CHO, where X 1 Selected from aryl or C1-C10 alkyl groups, preferably aryl; X 2 Selected from one of the halogens, preferably Cl, Br, or I, and more preferably Br; The R2 values ​​in formulas IA and IB may be the same or different, and each is independently selected from -CN or -CHO.

2. The disulfide bond bridging reagent as described in claim 1, characterized in that, The disulfide bridging agent is selected from at least one of the following compounds: 。 3. The method for preparing the disulfide bond bridging reagent as described in claim 1, characterized in that, Choose from Method 1, Method 2, Method 3, or Method 4: Method 1 has the following general reaction formula: ； Method 2 has the following general reaction formula: ； Method 3 has the following general reaction formula: ； Method 4 has the following general reaction formula: ； Method 1 includes the following steps: Using IA-1 as the starting material, it was dissolved in an organic solvent, and then NaSO2R was added to react and give compound IA-2; the structural formulas of compounds IA-1 and IA-2 are as follows: ;where R, R2, X 1 Relative to R, R2, and X in compound IA 1 The same applies; preferably, the organic solvent is dimethyl sulfoxide; the molar ratio of compound IA-1 to NaSO2R is 1:4.5-5.5; the molar ratio of compound IA-1 to organic solvent is 1g:30-40mL; the reaction temperature is 45-55℃; and the reaction time is 1.5-3.0 hours. Method 2 includes the following steps: Step (1): Compound IA-3 was dissolved in an organic solvent, and oxalyl chloride was added under ice bath conditions for a primary reaction. After concentration under reduced pressure, methanol and triethylamine were added for a secondary reaction to obtain compound IA-4. The structural formulas of compounds IA-3 and IA-4 are as follows: Preferably, the organic solvents are dichloromethane and N,N-dimethylformamide; the molar ratio of compound IA-3 to oxaloyl chloride is 1:2-4; the molar ratio of compound IA-3 to dichloromethane is 1 g:10-15 mL; the molar ratio of compound IA-3 to N,N-dimethylformamide is 1 g:10-15 μL; the primary reaction temperature is 0-4°C; the primary reaction time is 4-6 hours; the molar ratio of compound IA-3 to methanol is 1 g:10-15 mL; the molar ratio of compound IA-3 to triethylamine is 1:1.5-2; the secondary reaction temperature is 20-25°C; the secondary reaction time is 4-6 hours. Step (2): Dissolve IA-4 in an organic solvent, add a reducing agent under ice bath conditions to carry out a reduction reaction to obtain compound IA-5; wherein, the structural formula of compound IA-5 is as follows: Preferably, the organic solvent is tetrahydrofuran; the reducing agent is lithium aluminum hydride; the molar ratio of compound IA-4 to the reducing agent is 1:1-1.5; the volume ratio of compound IA-4 to the organic solvent is 1 g:10-15 mL; the temperature of the reduction reaction is 0-4°C; and the time of the reduction reaction is 1-2 hours. Step (3): Dissolve IA-5 in an organic solvent, add oxidant one under ice bath conditions, and then heat to carry out the oxidation reaction to obtain compound IA-6; the structural formula of compound IA-6 is as follows: Preferably, the organic solvent is dichloromethane; the oxidant is m-chloroperoxybenzoic acid; the molar ratio of compound IA-5 to oxidant is 1:1.2-1.5; the volume ratio of compound IA-5 to organic solvent is 1 g:9-12 mL; the oxidation reaction temperature is 20-25°C; and the oxidation reaction time is 1-1.5 hours. Step (4): Compound IA-6 undergoes a secondary oxidation reaction in an organic solvent with the addition of oxidant II to obtain compound IA-7, wherein compound IA-7 is either compound IA-7-1 or IA-7-2; the structural formula of compound IA-7-1 is as follows: The structural formula of compound IA-7-2 is as follows: ; Compound IA-7-1 is reacted in methanol with the addition of hydroxylamine hydrochloride in a primary reaction, yielding a compound dissolved in acetonitrile. Triethylamine and dimethyl sulfoxide are then added, followed by dropwise addition of oxaloyl chloride under ice bath conditions, resulting in a secondary reaction that generates compound IA-7-2. Preferably, the organic solvent is at least one of dichloromethane, chloroform, or tetrahydrofuran; the oxidant is active manganese dioxide, tin dioxide, or Desmartin oxidant; the molar ratio of compound IA-6 to oxidant is 1:3-5; the molar ratio of compound IA-6 to organic solvent is 1 g:20-30 mL; the temperature of the secondary oxidation reaction is 60-70°C; and the reaction time is 8-10 hours. The molar ratio of compound IA-7-1 to hydroxylamine hydrochloride was 1:10-15; the molar ratio of compound IA-7-1 to methanol was 1 g:10-50 mL; the temperature of the first reaction was 20°C-25°C; the reaction time was 5-8 hours; the molar ratio of compound IA-7-1 to triethylamine was 1:2-3; the molar ratio of compound IA-7-1 to dimethyl sulfoxide was 80-100:1; the molar ratio of compound IA-7-1 to oxaloyl chloride was 1:1.0-1.5; the molar ratio of compound IA-7-1 to acetonitrile was 1 g:12-18 mL; the temperature of the second reaction was 20°C-25°C; the reaction time was 9-11 hours. Step (5): One of the compounds IA-7 is reacted with trifluoroacetic anhydride in an organic solvent to obtain compound IA-8; compound IA-7 is selected from at least one of compounds IA-7-1 and IA-7-2; the structural formula of compound IA-7 is as follows: R2 corresponds to the same R2 in compound IA; the structural formula of compound IA-8 is as follows: Wherein, R2 corresponds to the same R2 in compound IA; preferably, the organic solvent is chloroform; the molar ratio of compound IA-7 to trifluoroacetic anhydride is 1:2-3; the molar ratio of compound IA-7 to organic solvent is 1g:8-12mL; the reaction temperature is 20°C-25°C; the reaction time is 10-25 hours; Step (6): Compound IA-8 reacts with NaSO2R in an organic solvent to obtain compound IA-9; the structural formula of compound IA-9 is as follows: Wherein, R and R2 correspond to the same R and R2 in compound IA; preferably, the organic solvent is dimethyl sulfoxide; the molar ratio of compound IA-8 to NaSO2R is 1:2.5-3.5; the molar ratio of compound IA-8 to organic solvent is 1g:15-20mL; the reaction temperature is 45°C-55°C; and the reaction time is 2-4 hours. Step (7) is either step (7-1) or step (7-2): Step (7-1): Compound IA-9 and organic solvent are added to PX under ice bath conditions. 2 After step 3, the reaction was heated to obtain compound IA-10; the structural formula of compound IA-10 is as follows: Among them, R, R2, X 2 With the R, R2, and X of compound IA 2 The corresponding compounds are the same; preferably, the organic solvent is chloroform; compound IA-9 and PX 2 The molar ratio of compound IA-9 to organic solvent is 1:2.0-4.0; the molar ratio of compound IA-9 to organic solvent is 1g:20-30mL; the reaction temperature is 20°C-25°C; and the reaction time is 2-4 hours. Step (7-2): Compound IA-9 is oxidized in an organic solvent with the addition of oxidant II to obtain compound IA-10-1; the structural formula of compound IA-10-1 is as follows: Preferably, the organic solvent is at least one of dichloromethane, chloroform, or tetrahydrofuran; the oxidant is active manganese dioxide, tin dioxide, or Desmartin oxidant; the molar ratio of compound IA-9 to oxidant is 1:3-5; the volume ratio of compound IA-9 to organic solvent is 1g:20-30mL; the oxidation reaction temperature is 60-70°C; and the oxidation reaction time is 8-10 hours. Method 3 includes the following steps: Step (1): Compound IB-1 is dissolved in an organic solvent, and sodium borohydride is added under ice bath conditions to carry out a reduction reaction to obtain compounds IB-2-1 and IB-2-2; wherein, the structural formulas of compounds IB-1, IB-2-1, and IB-2-2 are as follows: Preferably, the organic solvents are dichloromethane and methanol; the molar ratio of compound IB-1 to sodium borohydride is 1:1.1-3; the molar ratio of compound IB-1 to dichloromethane is 1 g:10-15 mL; the molar ratio of compound IB-1 to methanol is 1 g:3-5 mL; the reduction reaction temperature is 20-30°C; and the reduction reaction time is 10-14 hours. Step (2): Compound IB-2-1 is reacted with NaSO2R in an organic solvent to obtain compound IB-3-1, and compound IB-2-2 is reacted with NaSO2R in an organic solvent to obtain compound IB-3-2; wherein, the structural formulas of compounds IB-3-1 and IB-3-2 are as follows: Wherein, R corresponds to the same R in compound IB; preferably, the organic solvent is acetonitrile; the molar ratio of compound IB-2-1 to NaSO2R is 1:2.0-4.0; the molar ratio of compound IB-2-2 to NaSO2R is 1:4.5-5.5; the volume ratio of compound IB-2-1 to organic solvent is 1 g: 10-15 mL; the volume ratio of compound IB-2-2 to organic solvent is 1 g: 30-40 mL; the reaction temperature is 78-80℃; the reaction time is 20-30 hours; Step (3): Dissolve IB-3-1 in an organic solvent and add PX. 2 After step 3, the reaction was heated to obtain the compound with the general formula IB-4-1. The structural formula of compound IB-4-1 is as follows: Where R corresponds to the same R in compound IB, X 2 X of compound IB 2 The corresponding values ​​are the same; preferably, the organic solvent is dichloromethane; the molar ratio of compound IB-3-1 to PX3 is 1:2.0-4.0; the volume ratio of compound IB-3-1 to organic solvent is 1 g:20-30 mL; the reaction temperature is 40℃-45℃; and the reaction time is 6-8 hours. In step (4), sodium borohydride was added to an organic solvent to carry out a secondary reduction reaction of compound IB-4-1 to obtain compound IB-5-1. The structural formula of compound IB-5-1 is as follows: Where R corresponds to the same R in compound IB, X 2 With X in compound IB 2 The corresponding values ​​are the same; preferably, the organic solvents are dichloromethane and methanol; the molar ratio of compound IB-4-1 to sodium borohydride is 1:1.1-3; the molar ratio of compound IB-4-1 to dichloromethane is 1 g:10-15 mL; the molar ratio of compound IB-4-1 to methanol is 1 g:3-5 mL; the secondary reduction reaction temperature is 25°C; the secondary reduction reaction time is 10-14 hours; Step (5): Compound IB-5-1 is subjected to a single oxidation reaction in an organic solvent with the addition of an oxidizing agent to obtain compound IB-6-1; compound IB-3-2 is subjected to a single oxidation reaction in an organic solvent with the addition of an oxidizing agent to obtain compound IB-6-2; the structural formulas of compounds IB-6-1 and IB-6-2 are as follows: Where R corresponds to the same R in compound IB, X 2 X of compound IB 2 The corresponding parameters are the same; preferably, the organic solvent is at least one of dichloromethane, chloroform, or tetrahydrofuran; the oxidant is active manganese dioxide, tin dioxide, or Dysmartin oxidant; the molar ratio of compound IB-5-1 to oxidant is 1:8-12; the molar ratio of compound IB-3-2 to oxidant is 1:10-16; the volume ratio of compound IB-5-1 to organic solvent is 1g:20-30 mL; the volume ratio of compound IB-3-2 to organic solvent is 1g:50-70 mL; the temperature of the first oxidation reaction is 20-25℃; the time of the first oxidation reaction is 8-10 hours; Step (6): Compound IB-6-1 was dissolved in methanol solvent, and hydroxylamine hydrochloride was added. After a single reaction, the compound was dissolved in acetonitrile solvent, and triethylamine and dimethyl sulfoxide were added. Oxaloyl chloride was added dropwise under ice bath. After a second reaction, compound IB-7 was generated. The structural formula of compound IB-7 is as follows: Where R corresponds to the same R in compound IB, X 2 With X in compound IB 2 The corresponding values ​​are the same; preferably, the molar ratio of compound IB-6-1 to hydroxylamine hydrochloride is 1:10-15; the molar ratio of compound IB-6-1 to methanol is 1g:10-50 mL; the temperature of the first reaction is 20℃-25℃; the reaction time is 5-8 hours; the molar ratio of compound IB-6-1 to triethylamine is 1:2-3; the molar ratio of compound IB-6-1 to dimethyl sulfoxide is 80-100:1; the molar ratio of compound IB-6-1 to oxalyl chloride is 1:1.0-1.5; the molar ratio of compound IB-6-1 to acetonitrile is 1g:12-18 mL; the temperature of the second reaction is 20℃-25℃; the reaction time is 9-11 hours. Method four includes the following steps: Using compound IB-11 as a starting material, it was dissolved in an organic solvent, and then reacted with NaSO₂R to obtain compound IB-12; the structural formulas of compounds IB-11 and IB-12 are as follows: ;where R, R2, X 1 Relative to R, R2, and X in compound IB 1 The same applies; preferably, the organic solvent is dimethyl sulfoxide; the molar ratio of compound IB-11 to NaSO2R is 1:4.5-5.5; the volume ratio of compound IB-11 to organic solvent is 1g:30-40mL; the reaction temperature is 45-55℃; and the reaction time is 1.5-3.0 hours.

4. A biomolecule modified with disulfide bonds, characterized in that, The disulfide bond-bridged modified biomolecules have the following general structural formula IIA or general formula IIB or their salt forms: ， IIA IIB; Among them, the The corresponding precursor is a biomolecule containing at least one pair of disulfide bonds. ; R2 is either -CN or -CHO; Preferably, the The corresponding precursor is a cyclic peptide, antibody, or antibody fragment containing at least one pair of disulfide bonds; More preferably, the cyclic peptide is a cyclic peptide that forms a disulfide bond through at least one pair of cysteine ​​sulfhydryl groups, and even more preferably, at least one of octreotide, somatostatin, lanreotide, atorvastatin, epitubatide, terlipressin, lysine vasopressin, arginine vasopressin, oxytocin, iRGD and its homologs. More preferably, the antibody or antibody fragment is an antibody or antibody fragment comprising at least one pair of disulfide bonds, the antibody fragment comprising one or more portions of a single antibody, and more preferably, a whole antibody or antibody fragment of abciximab, cetuximab, trastuzumab, sacetuzumab, or pravitizine.

5. The method for preparing the disulfide bond-bridged modified biomolecule as described in claim 4, characterized in that, Includes the following steps: A reducing agent and the disulfide bond bridging reagent are added to a biomolecule solution, and the pH is adjusted with acid or base to carry out a bridging reaction, thereby obtaining a disulfide bond-bridged biomolecule. The biomolecule is... ; Preferably, the molar ratio of the disulfide bridging reagent to the biomolecule is ≥1; the solvent of the biomolecule solution is at least one selected from phosphate buffer, ammonium bicarbonate buffer, sodium acetate buffer, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer; the reducing agent is at least one selected from tris(2-carboxyethyl)phosphine and tris(hydroxypropyl)phosphine; the molar ratio of the reducing agent to the biomolecule is >1; the pH is adjusted with hydrochloric acid or sodium hydroxide; the pH of the bridging reaction is 7.0-8.5; the temperature of the bridging reaction is 25-37°C; and the time of the bridging reaction is 0.5-6 hours.

6. A disulfide bond-bridged biomolecule-small molecule conjugate, characterized in that, The disulfide bond-bridged biomolecule-small molecule conjugates have the following general structural formulas IIIA, IIIB, or their salts: ， IIIA IIIB; Among them, in formula IIIA and formula IIIB The corresponding precursor is a cyclic peptide, antibody, or antibody fragment containing at least one pair of disulfide bonds; More preferably, the cyclic peptide is a cyclic peptide that forms a disulfide bond through at least one pair of cysteine ​​sulfhydryl groups, and even more preferably, at least one of octreotide, somatostatin, lanreotide, atorvastatin, epitubatide, terlipressin, lysine vasopressin, arginine vasopressin, oxytocin, iRGD and its homologs. More preferably, the antibody or antibody fragment is an antibody or antibody fragment comprising at least one pair of disulfide bonds, the antibody fragment comprising one or more portions of a single antibody, and more preferably, a whole antibody or antibody fragment of abciximab, cetuximab, trastuzumab, saxituzumab, or pravitizumab. R3 is selected from thiazoline and its derivatives, thiazoline and its derivatives, and oxime and its derivatives; preferably: ; More preferably, the disulfide bond-bridged biomolecule-small molecule conjugate is selected from at least one compound with the following structural formula: 。 7. The method for preparing the disulfide bond-bridged biomolecule-small molecule conjugate as described in claim 6, characterized in that, Includes the following steps: Add a small molecule compound to a solution of biomolecules that are bridged by disulfide bonds, adjust the pH with acid or base, and carry out a coupling reaction to obtain biomolecule-small molecule conjugates that are bridged by disulfide bonds. The biomolecule structure modified by disulfide bond bridging is shown in general formula IIA or general formula IIB: ， IIA IIB; The small molecule compound is a small molecule compound that can react with R2 in general formula IIA or general formula IIB; Preferably, the small molecule compound is selected from molecules containing 1,2-aminothiol and hydroxylamine groups; more preferably, ; Further preferably, the molar ratio of the small molecule compound to the disulfide-bridged biomolecule is ≥1; the solvent of the disulfide-bridged biomolecule solution is at least one of phosphate buffer, ammonium bicarbonate buffer, sodium acetate buffer, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer; the pH is adjusted with hydrochloric acid or sodium hydroxide; the pH of the coupling reaction is 6.0-8.5; the temperature of the coupling reaction is 25-37°C; and the time of the coupling reaction is 1-24 hours.

8. A biomolecule-functional molecule conjugate modified with disulfide bonds, characterized in that, The disulfide bond-bridged biomolecule-functional molecule conjugates have the following general structural formulas IVA, IVB, or their salt forms: ， IVA IVB; Among them, in formula IVA and formula IVB The corresponding precursor is a cyclic peptide, antibody, or antibody fragment containing at least one pair of disulfide bonds; More preferably, the cyclic peptide is a cyclic peptide that forms a disulfide bond through at least one pair of cysteine ​​sulfhydryl groups, and even more preferably, at least one of octreotide, somatostatin, lanreotide, atorvastatin, epitubatide, terlipressin, lysine vasopressin, arginine vasopressin, oxytocin, iRGD and its homologs. More preferably, the antibody or antibody fragment is an antibody or antibody fragment comprising at least one pair of disulfide bonds, the antibody fragment comprising one or more portions of a single antibody, and more preferably, a whole antibody or antibody fragment of abciximab, cetuximab, trastuzumab, saxituzumab, or pravitizumab. R4 is selected from fluorescent groups and drug molecules; preferably, rhodamine, methylaurestatin E, methylaurestatin F, camptothecin, irinotecan, and pyrrolobenzodiazepine. More preferably, the disulfide bond-bridged biomolecule-functional molecule conjugate is selected from at least one compound with the following structural formula: 。 9. The method for preparing the disulfide bond-bridged biomolecule-functional molecule conjugate as described in claim 8, characterized in that, Includes the following steps: Add functional molecules to a solution of biomolecules that are bridged by disulfide bonds, adjust the pH with acid or base, and carry out a coupling reaction to obtain biomolecule-functional molecule conjugates that are bridged by disulfide bonds. The biomolecule structure modified by disulfide bond bridging is shown in general formula IIA or general formula IIB: ， IIA IIB; The functional molecule is a functional molecule capable of reacting with R2 in a biomolecule bridged by a disulfide bond; preferably, the functional molecule has the following structural formula: The R4 mentioned is the same as the R4 in formula IVA or formula IVB; More preferably, the molar ratio of the functional molecule to the disulfide-bridged biomolecule is ≥1; the solvent of the disulfide-bridged biomolecule solution is at least one of phosphate buffer, ammonium bicarbonate buffer, sodium acetate buffer, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer; the pH is adjusted with hydrochloric acid or sodium hydroxide; the pH of the coupling reaction is 4.0-8.5; the temperature of the coupling reaction is 25-37°C; and the time of the coupling reaction is 1-24 hours.

10. The application of the disulfide bond bridging reagent as described in claim 1, or the disulfide bond bridging modified biomolecule as described in claim 4, or the disulfide bond bridging modified biomolecule-small molecule conjugate as described in claim 6, or the disulfide bond bridging modified biomolecule-functional molecule conjugate as described in claim 8, in drug preparation; preferably for preparing polypeptide-drug conjugates and antibody-drug conjugates; more preferably, for preparing drugs for treating cancer, inflammatory diseases, autoimmune diseases, cardiovascular diseases, or neurological diseases.

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