A method for late-stage cysteine-selective arylation modification of polypeptides and / or proteins in aqueous phase with palladium catalysis

Abstract

The application discloses a kind of water phase palladium catalysis polypeptide and / or protein's late cysteine selective arylation modification method, with unprotected polypeptide containing cysteine or natural protein containing solvent exposed cysteine as raw material, arylthianthrenium salt is arylating reagent, metal palladium is used as catalytic reagent, buffer is solvent, reaction is carried out under inert gas, and the selective arylation of polypeptide and / or protein is realized.The reaction is carried out in water phase buffer at 37 DEG C, without adding alkali, photo-initiator or organic cosolvent, and the scope of aryl substrate is widely used, shows extremely high functional group tolerance, and can introduce drug fragments, biotin, saccharide, fluorophore and other various groups into unprotected polypeptide and natural protein (such as BSA) with excellent chemical selectivity.In addition, by constructing targeted polypeptide drug conjugate, the application value of the method in the construction of tumor targeted therapy drug is verified.

Description

Technical Field

[0001] This invention belongs to the field of peptide and / or protein modification, specifically relating to an aqueous palladium-catalyzed method for late-stage cysteine ​​selective arylation modification of peptides and proteins. Background Technology

[0002] Peptides and proteins are core functional biomolecules that regulate almost all biological life activities in the body. In recent years, biocompatible chemical reactions have become a core means of functional modification of peptides and proteins, enabling the efficient conjugation of fluorescent groups, functional linkers, bioactive motifs, and drug molecules to peptide and protein molecules. This type of bioconjugation technology not only helps researchers to deeply understand the physiological functions of proteins and the mechanisms of intermolecular interactions, but also greatly promotes the development of targeted therapies such as peptide-drug conjugates (PDCs) and antibody-drug conjugates (ADCs), possessing extremely high application value in the biomedical field. However, peptide-protein bioconjugation reactions themselves have many limitations. For example, the entire reaction must be carried out in an aqueous environment with near-neutral pH and physiological temperature. Furthermore, the presence of multiple competing nucleophilic amino acid residues such as thiol, amino, carboxyl, and hydroxyl groups on the surface of peptides / proteins makes site-specific modification difficult, greatly restricting the clinical application of bioconjugation technology.

[0003] Currently, bioconjugation technology primarily selects cysteine ​​(Cys), a naturally occurring substance with low abundance and strong nucleophilicity, as the site for targeted modification of peptides / proteins. However, existing cysteine ​​bioconjugation methods have significant shortcomings. The current mainstream modification method is the maleimide-thiol reaction. This technology is mature and widely used in the preparation of marketed antibody-drug conjugates, but it generates a mixture of diastereomers, and the product has poor stability, easily undergoing the reverse Michael reaction, causing drug loading loss and leading to severe off-target toxicity, compromising drug safety. To address the product stability issue, researchers have attempted to construct aryl sulfide structures to replace traditional alkyl sulfide structures. Currently, the mainstream methods for constructing aryl sulfides fall into three categories, all of which have significant drawbacks: the first category is aromatic nucleophilic substitution mediated by electron-deficient electrophilic reagents (S... NThe first type involves the Ar (Ar) reaction, which offers mild reaction conditions but has a narrow substrate range. The second type involves coupling methods mediated by transition metals such as nickel, palladium, and gold, effectively broadening the substrate range. However, this method requires the prior preparation of aryl metal reagents and must be operated under an inert gas atmosphere, resulting in complex experimental procedures and high operational barriers. The third type involves the direct catalytic coupling reaction of cysteine ​​with aryl electrophilic reagents by transition metals. Theoretically, this is the simplest process, but existing schemes rely on photo / electrochemical activation, the addition of an organic co-solvent, or a high-temperature reaction environment of 50–65°C, which violates the physiological compatibility requirements. In summary, none of the currently reported cysteine ​​arylation modification techniques can simultaneously achieve the requirements of mild reaction conditions, ease of operation, and broad substrate scope.

[0004] Therefore, it is necessary to develop a novel transition metal-catalyzed selective arylation bioconjugation method for cysteine ​​sites that has reaction conditions suitable for physiological environments, a wide substrate range, and is simple and efficient to operate, so as to provide reliable technical support for the precise modification of peptides and proteins and the development of peptide / protein targeted drugs. Summary of the Invention

[0005] Purpose of the Invention: The purpose of this invention is to provide an aqueous palladium-catalyzed method for late-stage cysteine-selective arylation modification of peptides and proteins. This method features mild reaction conditions, strong biocompatibility, and ease of operation. It belongs to the palladium-catalyzed site-selective arylation reaction method for peptides / proteins. The reaction is carried out in an aqueous buffer at 37°C without the need for the addition of alkali, photoinitiator, or organic co-solvent. Furthermore, it has a wide range of applicable aromatic substrates and exhibits extremely high functional group tolerance. It can introduce various groups such as drug fragments, biotin, sugars, and fluorophores into unprotected peptides and natural proteins (such as BSA) with excellent chemoselectivity.

[0006] This application further demonstrates the practical value of this method in the development of novel targeted therapeutic drugs by constructing receptor-targeted peptide-drug conjugates (PDCs).

[0007] To address the aforementioned technical problems, this invention discloses an aqueous palladium-catalyzed method for selective arylation of cysteine ​​in peptides and / or proteins. The method uses unprotected peptides containing cysteine ​​or natural proteins containing solvent-exposed cysteine ​​as raw materials, arylthianthaneonium salt as the arylation reagent, palladium as the catalyst, and buffer solution as the solvent. The reaction is carried out under an inert gas atmosphere to achieve selective arylation of peptides and / or proteins.

[0008] The amino acids of the polypeptide or protein are either D-type or L-type.

[0009] The general structural formula of arylthianthracene salts is:

[0010] Where X is OTf - CF3CO2 or BF4 - Ar is any one of a benzene ring, a substituted benzene ring, or a heteroaromatic ring, wherein the substituted benzene ring is a group obtained by substituting a hydrogen atom at the ortho, meta, and / or para positions on the benzene ring with a substituent, and the substituent is any one of an alkoxy group, an alkyl group, a cyano group, a nitro group, a halogen group, an ester group, or an acyl group.

[0011] The palladium catalyst is RuPhos-Pd-G3 or (COD)Pd(CH2TMS)2 and SSPhos ligand. Protein BSA modification uses the palladium catalyst (COD)Pd(CH2TMS)2 and SSPhos ligand.

[0012] The buffer solution is any one of PBS buffer, PB buffer, Tris HCl buffer, TES buffer, HEPES buffer, NaPi buffer, and Sorensen buffer, with a pH range of 6.0-9.0, preferably pH 8.5 Tris HCl buffer.

[0013] The buffer solution contains 2%-20% (v / v) of a co-solvent, wherein the co-solvent is any one of acetonitrile (MeCN), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), dimethyl sulfoxide (DMSO), and trifluoroethanol (TFE). DMSO is preferred.

[0014] The molar ratio of the polypeptide to the arylthiaanthraium salt is 1:1.05-1.25, preferably 1:1.2; the molar ratio of the protein to the arylthiaanthraium salt is 1:1-100, preferably 1:5.

[0015] The molar ratio of the polypeptide to the palladium catalyst is 1:0-1, preferably 1:0.2; the molar ratio of the protein to the palladium catalyst is 1:0-100, preferably 1:5.

[0016] The concentration of the polypeptide is 5 mm-20 mM, preferably 5 mM; the concentration of the protein is 50-600 µM, preferably 200 µM.

[0017] The reaction conditions are as follows: reaction temperature 25-45℃, preferably 37℃; reaction time 6-24 hours, preferably 10 hours.

[0018] Beneficial effects: Compared with the prior art, this application has the following advantages:

[0019] (1) Mild reaction conditions and strong biocompatibility: The new method developed in this invention is carried out at a physiological temperature of 37℃ in a Tris-HCl buffer system. No external alkali, photoinitiator or organic solvent is required for dissolution. The operation is simple and will not destroy the higher-order structure of peptides and natural proteins. It is suitable for the modification of biological macromolecules.

[0020] (2) The method of the present invention has site selection for cysteine: it is compatible with various amino acid residues, the reaction only occurs on thiol cysteine, and no side reactions occur for a large number of competitive nucleophilic groups (lysine, serine, tyrosine, histidine, aspartic acid, etc.) in polypeptides / proteins, and the modification site is single and controllable;

[0021] (3) The arylation substrate range is extremely wide and the functional group tolerance is excellent: This invention has no special requirements for the substituents on the arylation reagent - arylthionium salt. Electron-rich, neutral and electron-deficient aromatic rings can be coupled efficiently. It is compatible with a variety of functional groups such as cyano, nitro, fluorine, carboxyl and alkynyl. Fluorescent dyes, biotin, sugars, alkynyl and small molecule drug fragments can be introduced in the later stage of peptide / protein modification to achieve multifunctional modification of biomolecules in the later stage.

[0022] (4) It can achieve efficient labeling of unprotected peptides and natural proteins: The method of the present invention is not only applicable to various peptide substrates such as long and short chains, cyclic peptides, antimicrobial peptides, and neuropeptides, but also to site-directed modification of natural intact protein BSA. The protein disulfide bonds are stable and do not break during the reaction, and fluorescent and biotinylated protein probes can be constructed quickly.

[0023] (5) The method of this invention constructs stable aryl thioether linkages, overcoming the instability defects of traditional PDCs and ADCs alkylation linkages, and can be directly used for the preparation of PDC-targeted drug conjugates. This method can modularly assemble receptor-targeting PDCs to achieve tumor-targeted killing, providing a universal synthetic platform for the development of next-generation targeted therapies. Attached Figure Description

[0024] Figure 1 The polypeptide structure used in the embodiments of this application;

[0025] Figure 2 This is a schematic diagram of the structure of bovine serum albumin used in the examples;

[0026] Figure 3 The structure is that of the arylthianthaneonium salt or derivative used in the examples;

[0027] Figure 4 The mass spectrum of arylthianthracene onium salt 2m is shown.

[0028] Figure 5 The HPLC chromatogram of purified product 3a is shown. Figure 6 High-resolution mass spectrum of purified product 3a;

[0029] Figure 7 The HPLC chromatogram of purified product 5a is shown. Figure 8 High-resolution mass spectrum of purified product 5a;

[0030] Figure 9 The HPLC chromatogram of the reaction conversion in Example 5 is shown. Figure 10 Here is the MS mass spectrum of Example 5;

[0031] Figure 11 The HPLC chromatogram of the purified product 5c; Figure 12 High-resolution mass spectrum of purified product 5c;

[0032] Figure 13 The HPLC chromatogram of the purified product is shown in Figure 5. Figure 14 High-resolution mass spectrum of the purified product 5 days later;

[0033] Figure 15 The HPLC chromatogram of the purified product 5e; Figure 16 High-resolution mass spectrum of purified product 5e;

[0034] Figure 17 The HPLC chromatogram of purified product 5f; Figure 18 High-resolution mass spectrum of purified product 5f;

[0035] Figure 19 The HPLC chromatogram shows the conversion rate of 5g reaction. Figure 20 This is a high-resolution mass spectrum of 5g.

[0036] Figure 21 The HPLC chromatogram shows the conversion rate after 5 hours of reaction. Figure 22 The high-resolution mass spectrum of the product after 5 hours;

[0037] Figure 23 The HPLC chromatogram shows the conversion rate of the 5i reaction. Figure 24 The high-resolution mass spectrum of product 5i;

[0038] Figure 25 The HPLC chromatogram shows the conversion rate of the 5J reaction. Figure 26 The high-resolution mass spectrum of product 5j is shown.

[0039] Figure 27 The HPLC chromatogram shows the reaction conversion at 5k. Figure 28 The high-resolution mass spectrum of the product at 5k is shown.

[0040] Figure 29 This is a schematic diagram of biotin modification of bovine serum albumin (BSA).

[0041] Figure 30The results show the modification of bovine serum albumin (BSA) with a fluorescein derivative.

[0042] Figure 31 The HPLC chromatogram of purified product 12 (SP-RhB) is shown below.

[0043] Figure 32 High-resolution mass spectrum of purified product 12 (SP-RhB);

[0044] Figure 33 Fluorescence microscopy results of conjugate 12 (SP-RhB) binding to the NK1 receptor;

[0045] Figure 34 The results of in vitro cytotoxicity experiments are shown, including (A) the dose-dependent effects of PDC14 (SP-Exa) and Exatecanmesylate (Exa) on the viability of MDA-MB-231 cells; and (B) the dose-dependent effects of PDC14 (SP-Exa) and Exa on the viability of MCF-7 cells. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer. The embodiments described below are merely some embodiments of this application.

[0047] The polypeptide structures used in the examples are as follows: Figure 1 As shown. The protein used in the examples is bovine serum albumin (BSA), which contains 17 disulfide bonds and a solvent-exposed cysteine ​​residue at position 34, as shown in the diagram. Figure 2 As shown. The structures of the arylthiathaneonium salts or derivatives used in the examples are as follows. Figure 3 As shown.

[0048] Example 1: The synthesis of arylthianthaneonium salt 2m is as follows:

[0049]

[0050] Under an argon atmosphere, trifluoromethanesulfonic acid (TfOH, 98 µL, 1.1 mmol, 1.1 equiv) was added to a MeCN solution of 1-chloro-4-(hepta-6-yn-1-oxy)benzene (1.0 mmol, 1.0 equiv) and TTO (thiaanthracene-S-oxide, 232.1 mg, 1.0 mmol, 1.0 equiv) in 4 mL at 0 °C. Trifluoroacetic anhydride (417 μL, 3.0 mmol, 3.0 equiv) was then added dropwise over approximately 30 seconds, followed by a second addition of TfOH (98 µL, 1.1 mmol, 1.1 equiv). The reaction mixture was stirred at 400 rpm for 1 hour at 0 °C, and then at 25 °C for 16 hours. The solvent was removed by rotary evaporation, and the residue was dissolved in dichloromethane (20 mL). The resulting solution was washed successively with saturated sodium bicarbonate aqueous solution (10 mL) and sodium borofluoride aqueous solution (2 × 20 mL, 10 w / w%). The dichloromethane layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane / methanol as the eluent. The mass spectrum of the arylthiaanthium salt at 2m is shown below. Figure 4 As shown.

[0051] 1 H NMR (600 MHz, DMSO) δ 8.34 (dd, J = 8.0, 1.1 Hz, 2H), 8.10 (dd, J= 7.9, 1.0 Hz, 2H), 7.90 (m, J = 7.8, 1.3 Hz, 2H), 7.83 (m, J = 7.9, 1.2 Hz,2H), 7.77 (dd, J = 8.9, 2.5 Hz, 1H), 7.40 (d, J = 9.0 Hz, 1H), 6.56 (d, J =2.5 Hz, 1H), 4.20 (t, J = 6.5 Hz, 2H), 2.92 (t, J = 2.6 Hz, 1H), 2.29 (m, J =7.0, 2.6 Hz, 2H), 1.83 (m, J = 14.5, 6.5 Hz, 2H), 1.56 (p, J = 7.1 Hz, 2H). 13C NMR (151 MHz, DMSO) δ 156.14, 136.59, 136.09, 135.83, 135.09, 131.01,130.00, 128.70, 125.31, 118.16, 117.07, 111.25, 84.65, 72.28, 70.45, 27.55,24.78, 17.84. 19 F NMR (565 MHz, DMSO) δ -148.15, -148.21. HRMS (ESI) m / z found423.0586; calcd for C 24 H 20 ClOS2 + 423.0639.

[0052] Example 2: Arylization modification of decapeptide 1a

[0053] Decapeptide 1a (0.005 mmol), arylthiazine 2a (1.2 equiv), and RuPhos-Pd-G3 (0.2 equiv) were weighed into a 2 mL reaction flask, and 1 mL of Tris-HCl buffer (1.0 M, pH 8.5) was added. The mixture was then incubated at 37 °C for 10 hours. After the reaction was complete, the reaction solution was purified by semi-preparative chromatography and lyophilized at low temperature to obtain 3.2 mg of the target product 3a, with a separation yield of 58%. The purity was characterized by HPLC, and the target product was identified by high-resolution mass spectrometry. The HPLC chromatogram and high-resolution mass spectrum of the purified product 3a are shown below. Figure 5 and Figure 6 As shown.

[0054] HRMS (ESI) m / z found calcd for C 52 H 76 N 13 O 13 S⁺,1122.5328.

[0055] Example 3: Arylation modification of peptide 4a

[0056] Peptide 4a (0.005 mmol), arylthiazine 2a (1.2 equiv), and RuPhos-Pd-G3 (0.2 equiv) were weighed into a 2 mL reaction flask, and 1 mL of Tris-HCl buffer (1.0 M, pH 8.5) was added. The mixture was then incubated at 37 °C for 10 hours. After the reaction was complete, the reaction solution was purified by semi-preparative chromatography and lyophilized at low temperature to obtain 1.93 mg of the target product 5a, with a separation yield of 62%. The purity was characterized by HPLC, and the target product was identified by high-resolution mass spectrometry. The HPLC and high-resolution mass spectra of the purified product 5a are shown below. Figure 7 and Figure 8 As shown. Target product 5a HRMS (ESI) m / z found 626.0983; calcd for C 26 H 40 N7O7S2 + 626.2352.

[0057]

[0058] Example 4: Arylation modification of cyclic peptide 4b

[0059] Peptide 4b (0.005 mmol), arylthiaphenonium salt 2a (1.2 equiv), and palladium catalyst (0.2 equiv) were weighed into a 2 mL reaction flask, and 1 mL of Tris-HCl buffer (1.0 M, pH 8.5) was added. The flask was then incubated at 37 °C for 10 hours. After the reaction was complete, the conversion rate was characterized by HPLC as 97%. The HPLC chromatogram of the conversion rate is shown below. Figure 9 As shown, the MS mass spectrum is as follows: Figure 10 As shown. MS (ESI) m / z found calcd for C 30 H 39 N8O7S⁺, 655.2584.

[0060]

[0061] Example 5: Arylation modification of peptide 4c

[0062] Peptide 4c (0.005 mmol), arylthianonium salt 2a (1.2 equiv), and RuPhos-Pd-G3 (0.2 equiv) were weighed into a 2 mL reaction flask, and 1 mL of Tris-HCl buffer (1.0 M, pH 8.5) was added. The mixture was then incubated at 37 °C for 10 hours. After the reaction was complete, the reaction solution was purified by semi-preparative chromatography and lyophilized at low temperature to obtain 4.5 mg of the target product 5c, with a separation yield of 55%. The purity was characterized by HPLC, and the target product was identified by high-resolution mass spectrometry. Target product 5c HRMS (ESI) m / z found 626.0983; calcd for C 26 H 40 N7O7S2 + 626.2352. The HPLC chromatogram and resolved mass spectrum of the purified product 5c are shown below. Figure 11 and 12 As shown. Target product 5c HRMS (ESI) m / z found [M+2H] 2⁺ 813.7228 calcd for C 76 H 113 N 20 O 16 S2 + , [M + H] ⁺ 1626.9760; [M + 2H] 2⁺ 813.9880.

[0063]

[0064] Example 6: Arylization modification of peptide 4d

[0065] Peptide 4d (0.005 mmol), arylthiazine 2q (1.2 equiv), and RuPhos-Pd-G3 (0.2 equiv) were weighed into a 2 mL reaction flask, and 1 mL of Tris-HCl buffer (1.0 M, pH 8.5) was added. The mixture was then incubated at 37 °C for 10 hours. After the reaction was complete, the reaction solution was purified by semi-preparative chromatography and lyophilized at low temperature to obtain the target product 5d 3.7 mg, with a separation yield of 58%. The purity was characterized by HPLC, and the target product was identified by high-resolution mass spectrometry. The HPLC chromatogram and high-resolution mass spectrum of the purified product 5d are shown below. Figure 13 and 14 As shown. 5d HRMS (ESI) m / z found 1279.5038 ;calcd for C 55 H80 ClN 12 O 19 S⁺, 1279.4994.

[0066]

[0067] Example 7: Arylation modification of peptide 4e

[0068] Peptide 4e (0.005 mmol), arylthiazine 2q (1.2 equiv), and RuPhos-Pd-G3 (0.2 equiv) were weighed into a 2 mL reaction flask, and 1 mL of Tris-HCl buffer (1.0 M, pH 8.5) was added. The mixture was then incubated at 37 °C for 10 hours. After the reaction was complete, the reaction solution was purified by semi-preparative chromatography and lyophilized at low temperature to obtain 4.3 mg of the target product 5e, with a separation yield of 52%. The purity was characterized by HPLC, and the target product was identified by high-resolution mass spectrometry. The HPLC chromatogram and high-resolution mass spectrum of the purified product 5e are shown below. Figure 15 and 16 As shown. 5e HRMS (ESI) m / z found ;calcd for C 78 H 100 ClN 18 O 18 S⁺, 1643.6794.

[0069]

[0070] Example 8: Arylation modification of peptide 4f

[0071] Peptide 4f (0.005 mmol), arylthiazine 2q (1.2 equiv), and RuPhos-Pd-G3 (0.2 equiv) were weighed into a 2 mL reaction flask, and 1 mL of Tris-HCl buffer (1.0 M, pH 8.5) was added. The mixture was then incubated at 37 °C for 10 hours. After the reaction was complete, the reaction solution was purified by semi-preparative chromatography and lyophilized at low temperature to obtain 4.1 mg of the target product 5f, with a separation yield of 47%. The purity was characterized by HPLC, and the target product was identified by high-resolution mass spectrometry. The HPLC chromatogram and high-resolution mass spectrum of the purified product 5f are shown below. Figure 17 and 18 As shown. HRMS (ESI) m / z found ; calcdfor C 80 H 114 ClN 18 O 23S⁺, 1761.7635.

[0072]

[0073] Example 9: Arylization modification of polypeptide 4g

[0074] 4 g (0.005 mmol) of peptide, 2 t (1.2 equiv) of arylthiaphenonium salt, and 0.2 equiv of RuPhos-Pd-G3 were weighed into a 2 mL reaction flask. 1 mL of Tris-HCl buffer (1.0 M, pH 8.5) was added, and the mixture was incubated at 37 °C for 10 hours. After the reaction was complete, the conversion rate was characterized by HPLC (89%), and the target product (5 g) was identified by high-resolution mass spectrometry. The HPLC chromatogram and high-resolution mass spectrometry chromatogram of the 5 g reaction conversion are shown below. Figure 19 and Figure 20 As shown.

[0075] HRMS: calculated m / z for [M + 3H]³⁺ 710.4796, [M + 4H] 4 ⁺ 533.1098, [M+ 5H] 5 ⁺ 426.6878; found m / z [M + 3H]³⁺ 710.6885, [M + 4H] 4 ⁺ 533.2681, [M + 5H] 5 ⁺ 426.8162.

[0076]

[0077] Example 10: Arylation modification of peptide 4h

[0078] The peptide 4h (0.001 mmol), arylthiazine 7 (1.2 equiv), and RuPhos-Pd-G3 (0.2 equiv) were weighed into a 2 mL reaction flask, and 1 mL of Tris-HCl buffer (1.0 M, pH 8.5, 5% v / v DMSO) was added. The mixture was then incubated at 37°C for 10 hours. After the reaction was complete, the conversion rate was characterized by HPLC (48%), and the target product was identified by high-resolution mass spectrometry. The HPLC chromatogram and high-resolution mass spectrometry chromatogram of the 5-hour reaction conversion are shown below. Figure 21 and 22 shown. HRMS: calculated for [M+ 4H] 4 ⁺ 798.9782; found m / z [M + 4H] 4 ⁺ 798.6896.

[0079]

[0080] The synthesis of arylthianthaneonium salt 7 is as follows:

[0081]

[0082] Et3N (1.1 mL, 7.8 mmol, 1.5 equiv) was added to a 26 mL solution of 5-(5-(2-carboxyethyl)-2-methoxyphenyl)-5H-thiaanthracene-5-onium tetrafluoroborate 2i (2.5 g, 5.2 mmol, 1.0 equiv), HOBT (843.1 mg, 6.2 mmol, 1.2 equiv), and EDCI (1.2 g, 6.2 mmol, 1.2 equiv) in DMF at room temperature. After stirring for 20 minutes, tert-butyl-(2-(2-(2-aminoethoxy)ethyl)-carbamate (6.2 mmol, 1.2 equiv) was added, and the resulting mixture was stirred at room temperature for 12 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to remove most of the DMF. The residue was diluted with CH2Cl2 and washed successively with saturated sodium bicarbonate aqueous solution, citric acid aqueous solution, and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2.7 g (3.5 mmol), a yellow solid product, in 67% yield. The resulting yellow solid was added to TFA (8 mL) and stirred at room temperature for 2 hours to remove the Boc protecting group. The reaction mixture was concentrated under reduced pressure, and the residue was treated with DIPEA to neutralize the residual acid. Additionally, a solution of biotin (855.1 mg, 3.5 mmol, 1.0 equiv) and HATU (1.6 g, 4.2 mmol, 1.2 equiv) in DMF (17.5 mL) was treated with DIPEA (2.5 mL, 14.0 equiv). The mixture was treated with 4.0 mmol (equiv) and stirred at room temperature for 10 minutes. The solution obtained in the deprotection step was then added, and the resulting mixture was stirred at room temperature for 8 hours. After the reaction was complete, the crude reaction mixture was purified directly by reversed-phase C18 chromatography (mobile phase: water / acetonitrile containing 0.1% TFA, acetonitrile ratio 5–65%) to give a white solid product, arylthiaanthracite salt 7 (112.4 mg, 130.0 μmol, yield 4%). 1H NMR (600 MHz, DMSO) δ 8.40 (dd, J = 8.0, 1.2 Hz, 2H),8.05 (dd, J = 8.0, 1.1 Hz, 2H), 7.91 – 7.85 (m, 4H), 7.80 (dd, J = 7.9, 1.3Hz, 2H), 7.49 (dd, J = 8.5, 2.0 Hz, 1H), 7.26 (d, J = 8.6 Hz, 1H), 6.48 (d, J= 2.0 Hz, 1H), 4.30 (dd, J = 7.6, 4.7 Hz, 1H), 4.11 (dd, J = 7.7, 4.4 Hz,1H), 3.91 (s, 3H), 3.47 (h, J = 2.8 Hz, 4H), 3.38 (t, J = 6.0 Hz, 2H), 3.34(t, J = 5.8 Hz, 2H), 3.16 (dt, J = 17.3, 5.8 Hz, 4H), 3.09 – 3.04 (m, 1H),2.82 – 2.76 (m, 1H), 2.64 (t, J = 7.4 Hz, 2H), 2.57 (d, J = 12.4 Hz, 1H),2.21 (t, J = 7.5 Hz, 2H), 2.05 (t, J = 7.4 Hz, 2H), 1.62 – 1.57 (m, 1H), 1.51– 1.42 (m, 3H), 1.32 – 1.24 (m, 2H). 13 C NMR (151 MHz, DMSO) δ 172.64, 171.19,163.20, 156.02, 136.82, 135.95, 135.38, 134.90, 130.81, 129.89, 128.71,118.05, 117.49, 114.72, 109.75, 69.98, 69.95, 69.60, 61.49, 59.64, 57.41,55.91, 55.40, 40.46, 38.98, 38.86, 36.77, 35.55, 29.97, 28.67, 28.50, 25.74. 19 F NMR (565 MHz, DMSO) δ -74.47. HRMS (ESI) m / z found [M+H] + 751.2603; calcdfor C38 H 47 N4O6S3 + 751.2652.

[0083] Example 11: Arylation modification of peptide 4i

[0084] Peptide 4i (0.001 mmol), arylthiazine 7 (1.2 equiv), and RuPhos-Pd-G3 (0.2 equiv) were weighed into a 2 mL reaction flask, and 1 mL of Tris-HCl buffer (1.0 M, pH 8.5, 5% v / v DMSO) was added. The mixture was then incubated at 37 °C for 10 hours. After the reaction was complete, the conversion rate was characterized by HPLC (59%), and the target product was identified by high-resolution mass spectrometry. The HPLC chromatogram and high-resolution mass spectrometry chromatogram of the 5i reaction conversion are shown below. Figure 23 and 24 As shown.

[0085] HRMS: calculated m / z for [M + 4H] 4 ⁺ 967.1410, [M + 5H] 5 ⁺ 773.9128, [M+ 6H] 6 ⁺ 645.0940; found m / z [M + 4H] 4 ⁺ 967.0345, [M + 5H] 5 ⁺ 773.8293, [M + 6H] 6 ⁺ 645.0254.

[0086]

[0087] Example 12: Arylation modification of peptide 4j

[0088] Peptide 4j (0.001 mmol), arylthiamium salt 7 (1.2 equiv), and RuPhos-Pd-G3 (0.2 equiv) were weighed into a 2 mL reaction flask, and 1 mL of Tris-HCl buffer (1.0 M, pH 8.5, 5% v / v DMSO) was added. The mixture was then incubated at 37 °C for 10 hours. After the reaction was complete, the conversion rate was characterized by HPLC (70%), and the target product was identified by high-resolution mass spectrometry. The HPLC chromatogram and high-resolution mass spectrometry chromatogram of the 5j reaction conversion are shown below. Figure 25 and 26 As shown.

[0089] HRMS: calculated m / z for [M + 4H]4 ⁺ 831.459, [M + 5H] 5 ⁺ 665.3672;found m / z [M + 4H] 4 ⁺ 831.4204, [M + 5H] 5 ⁺ 665.3377.

[0090]

[0091] Example 13: Arylation modification of polypeptide 4k containing 33 amino acids

[0092] Peptide 4k (0.001 mmol), arylthiazine 7 (1.2 equiv), and RuPhos-Pd-G3 (0.2 equiv) were weighed into 2 mL reaction flasks, and 1 mL of Tris-HCl buffer (1.0 M, pH 8.5, 5% v / v DMSO) was added. The mixture was then incubated at 37 °C for 10 hours. After the reaction was complete, the conversion rate was characterized by HPLC (61%), and the target product was identified by high-resolution mass spectrometry. The HPLC chromatogram and high-resolution mass spectrometry chromatogram of the 5k reaction conversion are shown below. Figure 27 and Figure 28 As shown.

[0093] HRMS: calculated m / z for [M + 4H] 4 ⁺ 949.3865, [M + 5H] 5 ⁺ 759.7092, [M+ 6H] 6 ⁺ 633.2576; found m / z [M + 4H] 4 ⁺ 949.2833, [M + 5H] 5 ⁺ 759.6283, [M + 6H] 6 ⁺ 633.1916.

[0094]

[0095] Example 14: Biotin modification of bovine serum albumin (BSA).

[0096] like Figure 29As shown, in a 2 mL vial, 50 μL of a mixed solution of arylthianonium salt 7, (COD)Pd(CH2TMS)2, and s-SPhos (all at a concentration of 20 μmol / mL) was added to a Tris-HCl buffer solution (pH 8.5, 950 μL) containing BSA (13.2 mg, 0.2 μmol, 1.0 equiv). The reaction was carried out on a shaker at 37°C for 10 hours. The reaction mixture was then diluted with water to a concentration of 4 × 10⁻⁻⁻⁻⁻⁶. 6 The reaction system was analyzed by UPLC-HRMS at a concentration of mol / L, and the biotin-labeled target protein was detected with a conversion rate of 77% (HRMS: 66962.984).

[0097] Example 15

[0098] In a 2 mL vial, 50 μL of a mixed solution of arylthianonium salt 9, (COD)Pd(CH2TMS)2, and s-SPhos (all at a concentration of 20 μmol / mL) was added to a Tris-HCl buffer solution (pH 8.5, 950 μL) containing BSA (13.2 mg, 0.2 μmol, 1.0 equiv). The reaction was carried out on a shaker at 37°C for 10 hours. The reaction system was analyzed by SDS-PAGE. Figure 30 As shown, the SDS-PAGE results are as follows: lane 1 is the protein marker, lane 2 is unmodified BSA, and lane 3 is BSA-6-carboxyfluorescein derivative conjugate 10. Coomassie brilliant blue staining showed that each sample had a protein band at approximately 66 kDa. Fluorescence imaging showed that only lane 3 had a significant fluorescence signal at the corresponding position, proving that BSA was successfully labeled with fluorescein.

[0099] The synthesis of arylthianthaneonium salt 9 is as follows:

[0100]

[0101] Add Et3N (1.1 mL, 7.8 mmol, 1.5 equiv) to a DMF (26 mL) solution of 5-(5-(2-carboxyethyl)-2-methoxyphenyl)-5H-thiaanthracene-5-onium tetrafluoroborate 2i (2.5 g, 5.2 mmol, 1.0 equiv), HOBT (843.1 mg, 6.2 mmol, 1.2 equiv), and EDCI (1.2 g, 6.2 mmol, 1.2 equiv) at room temperature. After stirring for 20 minutes, tert-butyl(2-(2-(2-aminoethoxy)ethyl)carbamate (6.2 mmol, 1.2 equiv) was added, and the resulting mixture was stirred at room temperature for 12 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to remove most of the DMF. The residue was diluted with CH2Cl2 and washed successively with saturated aqueous sodium bicarbonate solution, aqueous citric acid solution, and saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2.7 g (3.5 mmol), a yellow solid product, in 67% yield. The product was then reacted with tert-butyl(2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazaheptadecane-17-yl)-2-methoxyphenyl)-5H-thiaanthracene-5-onium (3.9 g, 5.0 mmol, 1.0 equiv) in TFA (11.5 mL, 30.0 mmol). The Boc protecting group was removed by stirring the solution at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the residue was treated with DIPEA. Separately, 6-carboxyfluorescein (2.3 g, 6.0 mmol, 1.2 equiv) and HATU (2.3 g, 6.0 mmol, 1.2 equiv) were dissolved in DMF (25 mL) and stirred at room temperature for 10 minutes. The solution obtained from the deprotection step was added dropwise to this mixture, followed by the addition of DIPEA (2.6 mL, 15.0 mmol, 3.0 equiv). The resulting reaction mixture was stirred at room temperature for 8 hours. After the reaction was complete, the crude reaction mixture was purified directly by reversed-phase C18 chromatography (mobile phase: water / acetonitrile system containing 0.1% TFA, acetonitrile ratio 5–65%) to give a yellow solid product 9 (49.8 mg, 0.05 mmol, yield 1%). 1H NMR(600 MHz, DMSO) δ 10.17 (s, 2H), 8.76 (t, J = 5.7 Hz, 1H), 8.38 (dd, J = 8.0,1.2 Hz, 2H), 8.16 (dd, J = 8.1, 1.4 Hz, 1H), 8.07 (d, J = 8.2 Hz, 1H), 8.03(dd, J = 8.0, 1.1 Hz, 2H), 7.87 (td, J = 7.8, 1.4 Hz, 2H), 7.80 – 7.72 (m,3H), 7.68 (s, 1H), 7.48 (dd, J = 8.5, 2.0 Hz, 1H), 7.24 (d, J = 8.6 Hz, 1H),6.69 (d, J = 2.3 Hz, 2H), 6.59 – 6.54 (m, 3H), 6.48 (d, J = 2.0 Hz, 1H), 3.90(s, 3H), 3.46 – 3.45 (m, 3H), 3.42 – 3.39 (m, 3H), 3.35 (q, J = 5.8 Hz, 2H),3.28 (t, J = 5.8 Hz, 2H), 3.09 (q, J = 5.7 Hz, 2H), 2.64 – 2.60 (m, 2H), 2.18(t, J = 7.5 Hz, 2H). 13 C NMR (151 MHz, DMSO) δ 171.14, 168.51, 165.06, 160.08,156.03, 153.21, 152.29, 140.97, 136.81, 135.94, 135.91, 135.40, 134.90,130.80, 129.89, 129.85, 129.73, 128.72, 128.69, 125.38, 122.74, 118.06,114.74, 113.21, 109.78, 109.60, 102.71, 83.74, 69.92, 69.90, 69.57, 69.07,57.42, 40.52, 38.94, 36.74, 29.97. 19 F NMR (565 MHz, DMSO) δ -74.18. HRMS(ESI) m / z found 883.2213; calcd for C 49 H 43 N2O10 S2 + , 883.2354.

[0102] Example 16: Fluorescent labeling of neuropeptide SP (4c)

[0103]

[0104] In a 3 mL vial equipped with a magnetic stir bar, peptide 4c (10.0 μmol, 15.5 mg, 1.0 equiv), thiaanthraquinone 11 (12.0 μmol, 12.7 mg, 1.2 equiv), and RuPhos–Pd–G3 (2.0 μmol, 1.67 mg, 20 mol%) were added. DMSO / Tris–HCl buffer (1.0 M, pH 8.5, 2.0 mL, v / v = 5:95) was added. The resulting reaction mixture was stirred at 37°C for 10 hours. The crude product was directly purified by reversed-phase C18 chromatography (5–65% acetonitrile / water + 0.1% TFA) to obtain a red solid product 12 (SP-RhB, 4.1 mg, 1.0 μmol, 18% yield). The HPLC and high-resolution mass spectra of the purified product 12 (SP-RhB) are shown below. Figure 31 and 32 As shown. HRMS(ESI) m / z found [M+4H] 4⁺ 571.5449 calcd for C 114 H 161 N 24 O 22 S2 + [M + 3H] 3⁺ 761.7218; [M + 4H] 4⁺ 571.5413.

[0105] The synthesis of arylthianthaneonium salt 11 is as follows:

[0106]

[0107] Add Et3N (1.1 mL, 7.8 mmol, 1.5 equiv) to a DMF (26 mL) solution of 5-(5-(2-carboxyethyl)-2-methoxyphenyl)-5H-thiaanthracene-5-onium tetrafluoroborate 2i (2.5 g, 5.2 mmol, 1.0 equiv), HOBT (843.1 mg, 6.2 mmol, 1.2 equiv), and EDCI (1.2 g, 6.2 mmol, 1.2 equiv) at room temperature. After stirring for 20 minutes, tert-butyl(2-(2-(2-aminoethoxy)ethyl)carbamate (6.2 mmol, 1.2 equiv) was added, and the resulting mixture was stirred at room temperature for 12 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to remove most of the DMF. The residue was diluted with CH2Cl2 and washed successively with saturated aqueous sodium bicarbonate solution, aqueous citric acid solution, and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2.7 g (3.5 mmol), a yellow solid product, in 67% yield. 5-(5-(2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14-diazaheptadecane-17-yl)-2-methoxyphenyl)-5H-thiaanthracene-5-onium (3.9 g, 5.0 mmol, 1.0 equiv) in TFA (11.5 mL, 30.0 mmol) was added. The solution was stirred at room temperature for 2 hours to remove the Boc protecting group. The reaction mixture was concentrated under reduced pressure, and the residue was treated with DIPEA. Separately, Rhodamine B (2.9 g, 6.0 mmol, 1.2 equiv) and HATU (2.3 g, 6.0 mmol, 1.2 equiv) were dissolved in DMF (25 mL) and stirred at room temperature for 10 minutes. The solution obtained from the deprotection step was added dropwise to this mixture, followed by the addition of DIPEA (2.6 mL, 15 mmol, 3.0 equiv). The resulting reaction mixture was stirred at room temperature for 8 hours. After the reaction was complete, the crude product was purified directly by reversed-phase C18 chromatography (mobile phase: water / acetonitrile containing 0.1% TFA, acetonitrile ratio 5–65%) to give a pink solid product 11 (255.4 mg, 217.0 μmol, yield 4.3%). 1H NMR (600 MHz, DMSO)δ 8.39 (dd, J = 8.0, 1.2 Hz, 2H), 8.03 (dd, J = 7.9, 1.1 Hz, 2H), 7.87 (dd, J = 7.8, 1.5 Hz, H.75 (2H), 7.7. – 7.47 (m, 3H), 7.24 (d, J =8.6 Hz, 1H), 7.03 (dd, J = 6.4, 1.3 Hz, 1H), 6.48 (d, J = 1.9 Hz, 1H), 6.43 –6.88 (J,6 = 3H), 3.90 (s, 3H), 3.32 (q, J = 6.9 Hz,8H), 3.28 – 3.23 (m, 4H), 3.15 (dd, J = 17.8, 6.0 Hz, 4H), 3.08 (q, J = 5.72 Hz, = 2H), 2.62 (t, J = 7.3 Hz, 2H), 2.18 (t, J = 7.4Hz, 2H), 1.06 (t, J = 7.0 Hz, 12H). 13 C NMR (151 MHz, DMSO) δ 171.09, 167.45,156.03, 153.77, 153.04, 148.53, 136.82, 135.95, 139.91, 18134.1, 130.78, 130.73, 129.89, 129.59, 128.91, 128.84, 128.74, 128.78, 119.29, 11.118.06.4. 114.72, 114.08, 113.47, 109.77, 108.99, 69.76, 69.69, 69.60, 67.43, 64.39, 57.41, 44.46, 39.715, 26.71,3 19 F NMR (565 MHz, DMSO) δ −74.59. HRMS (ESI) m / z found950.3960; calcd for C 56 H 62 N4O6S2 2+, 950.4100.

[0108] Example 17: Synthesis of a peptide-drug conjugate (PDC) 14 (SP-Exa) targeting the NK1 receptor:

[0109]

[0110] In a 20 mL vial equipped with a magnetic stir bar, peptide 4c (0.05 mmol, 77.5 mg, 1.0 equiv), thiaanthraquinone 13 (0.06 mmol, 66.2 mg, 1.2 equiv), and RuPhos–Pd–G3 (0.01 mmol, 8.4 mg, 20 mol%) were added. DMSO / Tris–HCl buffer (1.0 M, pH 8.5, 15.0 mL, v / v = 5:95) was added. The resulting reaction mixture was stirred at 37 °C for 10 hours. The crude product was purified directly by reversed-phase C18 chromatography (5–65% acetonitrile / water + 0.1% TFA) to give a yellow solid product 14 (SP-Exa) (20.6 mg, 0.009 mmol, 18% yield). HRMS (ESI) m / z: calcd for [M+2H] 2⁺ 1144.0277; found m / z [M+2H] 2⁺ 1144.5329.

[0111] The synthesis of arylthianthaneonium salt 13 is as follows:

[0112]

[0113] Under an argon atmosphere, (((9H-fluorene-9-yl)methoxy)carbonyl)alanylalanine (191.2 mg, 0.5 mmol, 1.0 equiv) was dissolved in anhydrous DMF (2.5 mL, 0.2 M). DIPEA (261 μL, 1.5 mmol, 3.0 equiv) and piperidine (10 μL, 0.1 mmol, 0.2 equiv) were added sequentially. The resulting reaction mixture was stirred at 40 °C for 1 hour, followed by stirring at 50 °C for another hour. The reaction progress was monitored by LC-MS. After the reaction was complete, the mixture was concentrated under reduced pressure, and the crude product was used directly in the next step without further purification. In another vial, 289.4 mg (289.4 mg, 0.6 mmol, 1.2 equiv) of 5-(5-(2-carboxyethyl)-2-methoxyphenyl)-5H-thiaanthracene-5-onium tetrafluoroborate 2i, HATU (228.1 mg, 0.6 mmol, 1.2 equiv), and DIPEA (174 μL, 1.0 mmol, 2.0 equiv) were dissolved in DMF (2.5 mL) and stirred at room temperature for 15 minutes to prepare an activation solution. This solution was added to the deprotected peptide obtained in the previous step, and the resulting mixture was stirred at room temperature for 4 hours. The reaction was quenched by TFA, and the crude product mixture was directly purified by reversed-phase C18 column chromatography (elution conditions: acetonitrile / water solution containing 0.1% TFA, acetonitrile ratio 5–65%) to give 195.8 mg, 0.3 mmol, white solid intermediate in 60% yield. 5-(5-(3-((1-((1-carboxyethyl)amino)-1-oxopropyl-2-yl)amino)-3-oxopropyl)-2-methoxyphenyl)-5H-thiaanthracene-5-onium trifluoromethanesulfonate (326.3 mg, 0.5 mmol, 1.0 equiv) and HATU (228.1 mg, 0.6 mmol, 1.2 equiv) were dissolved in DMF (2.5 mL, 0.2 M), and DIPEA (261 μL, 1.5 mmol, 3.0 equiv) was added. The mixture was stirred at room temperature for 10 minutes to generate an activated intermediate. Subsequently, a DMF (2.0 mL) suspension of Exatecan methanesulfonate (318.9 mg, 0.6 mmol, 1.2 equiv) was added, and the resulting reaction mixture was stirred at room temperature for 4 hours. After the reaction was confirmed by LC-MS analysis, the crude reaction mixture was directly purified by reverse-phase C18 column chromatography (elution conditions: MeCN / H2O system containing 0.1% TFA, MeCN concentration 5–65%) to obtain product 13 (pale yellow solid, 32.1 mg, 0.03 mmol, yield 6%).

[0114] Example 18: Fluorescent labeling of MDA-MB-231 and MCF-7 cells by targeting peptide 12

[0115] Neuropeptide 4c has been reported as a targeting peptide with high affinity for the neuropeptide receptor (NK1). In this embodiment, cell-specific labeling experiments were used to verify whether neuropeptide 4c can specifically bind to tumor cells that highly express the NK1 receptor.

[0116] MDA-MB-231 (NK1 receptor highly expressed) and MCF-7 (NK1 receptor low expressed) cells were cultured at 3 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per confocal culture dish and cultured for 24 hours to allow cell adhesion. Subsequently, DAPI, SP-RhB, and an NK1 receptor inhibitor were added to the culture medium, and incubated for 1 hour. After washing three times with PBS, SP-RhB was added to the inhibitor group, and incubation continued for 1 hour, followed by three more washes with PBS. After incubation, the cells were rinsed three times with PBS, and imaging was performed using a laser confocal microscope. All experiments were performed in triplicate.

[0117] Fluorescence microscopy observation results as follows Figure 33 The results showed that MDA-MB-231 cells exhibited a strong fluorescent signal, while MCF-7 cells showed a weaker signal, and the fluorescence intensity was positively correlated with the receptor expression level. After pretreatment with the NK1 receptor antagonist aprepitant, the signal in MDA-MB-231 cells was significantly reduced. These experimental results confirm that conjugate 12 (SP-RhB) can bind to the NK1 receptor with high affinity, indicating that peptide 4c can serve as a targeting agent in targeted peptide drug conjugates.

[0118] Example 19: Detection of cytotoxicity of peptide drug conjugate PDC14 on MDA-MB-231 and MCF-7 cells

[0119] MDA-MB-231 and MCF-7 cells in logarithmic growth phase, treated with trypsin, were collected. After centrifugation at 800 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in fresh culture medium. The cell suspension was transferred to 96-well plates at a density of 8,000 cells per well (100 μL per well) and cultured at 37°C, 5% CO2 for 24 hours. After culture, the cells were exposed to different concentrations of PDC (14, SP-Exa) and Exatecan mesylate for 48 hours. Then, 10 μL of MTT solution was added to each well, and the cells were incubated at 37°C, 5% CO2 for another 4 hours. Finally, 100 μL of DMSO was added to each well, and the absorbance was measured at 490 nm. All experiments were performed in triplicate. All results and statistical analyses are expressed as mean ± standard deviation, and data were analyzed using IBM SPSS 26.0 statistical software.

[0120] In vitro cytotoxicity assays showed that PDC 14 was cytotoxic to MDA-MB-231 cells with high NK1 expression (IC50). 50 = 94.5 ± 2.6 μM) showed stronger killing activity against MCF-7 cells with low NK1 expression (IC50, 94.5 ± 2.6 μM) than against MCF-7 cells with low NK1 expression (IC50, 94.5 ± 2.6 μM). 50 = 232.6 ± 3.1 μM). This embodiment illustrates the practicality of the method of the invention in constructing targeted tumor therapeutic drugs.

[0121] This invention provides a method and approach for the selective arylation of cysteine ​​residues in the later stages of peptides and proteins using aqueous palladium catalysis. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for selective arylation of cysteine ​​residues in the late stage of peptides and / or proteins using aqueous palladium catalysis, characterized in that, Using unprotected peptides containing cysteine ​​or natural proteins containing solvent-exposed cysteine ​​as raw materials, arylthianthaneonium salt as the arylation reagent, palladium as the catalyst, and buffer solution as the solvent, the reaction is carried out under an inert gas atmosphere to achieve selective arylation of peptides and / or proteins.

2. The method according to claim 1, characterized in that, The amino acids of the polypeptide or protein are of the D or L type.

3. The method according to claim 1, characterized in that, The general structural formula of arylthianthaneonium salts is: Where X is OTf - CF3CO2 or BF4 - Ar is any one of a benzene ring, a substituted benzene ring, or a heteroaromatic ring, wherein the substituted benzene ring is a group obtained by substituting a hydrogen atom at the ortho, meta, and / or para positions on the benzene ring with a substituent, and the substituent is any one of an alkoxy group, an alkyl group, a cyano group, a nitro group, a halogen group, an ester group, or an acyl group.

4. The method according to claim 1, characterized in that, The palladium catalyst is RuPhos-Pd-G3 or (COD)Pd(CH2TMS)2 and SSPhos ligand. For protein BSA modification, the palladium catalyst (COD)Pd(CH2TMS)2 and SSPhos ligand are used.

5. The method according to claim 1, characterized in that, The buffer solution is any one of PBS buffer, PB buffer, Tris HCl buffer, TES buffer, HEPES buffer, NaPi buffer, and Sorensen buffer, with a pH range of 6.0-9.0, preferably pH 8.5 Tris HCl buffer.

6. The method according to claim 5, characterized in that, The buffer solution contains 2%-20% (v / v) of a co-solvent, wherein the co-solvent is any one of acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, and trifluoroethanol.

7. The method according to claim 1, characterized in that, The molar ratio of the polypeptide to the arylthiaanthraium salt is 1:1.05-1.25, preferably 1:1.2; the molar ratio of the protein to the arylthiaanthraium salt is 1:1-100, preferably 1:

5.

8. The method according to claim 1, characterized in that, The molar ratio of the polypeptide to the palladium catalyst is 1:0-1, preferably 1:0.2; the molar ratio of the protein to the palladium catalyst is 1:0-100, preferably 1:

5.

9. The method according to claim 1, characterized in that, The concentration of the polypeptide is 5 mm-20 mM, preferably 5 mM; the concentration of the protein is 50-600 µM, preferably 200 µM.

10. The method according to claim 1, characterized in that, The reaction conditions are as follows: reaction temperature 25-45℃, preferably 37℃; reaction time 6-24 hours, preferably 10 hours.

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