Polypeptide or protein modification method for promoting desulfurization of cysteine by light

Abstract

The invention discloses a polypeptide or protein modification method for photopromoting cysteine desulfurization, which comprises the following steps: by taking a naked peptide containing cysteine or a protein containing solvent-exposed cysteine as a raw material, taking a free radical receptor as another raw material, taking pyridinium as an activating reagent, taking a buffer solution as a reaction solvent, and carrying out photolysis reaction to obtain the polypeptide or protein. In the presence of a reducing agent and an additive and under the irradiation of visible light, cysteine desulfurization of polypeptide or protein is realized to form a stereoscopically retained C-X bond, and the free radical receptor is any one of electron-deficient olefin, acrylamide, acrylate, an organic selenium compound, 2, 2, 6, 6-tetramethylpiperidine-N-oxygen free radical or N-heterouracil. The invention relates to a method for constructing a C-X bond, which is promoted by visible light, stereoscopically retains a modification site and is compatible with a disulfide bond under a biocompatible condition, and can be used for site selective accurate modification of polypeptide and protein.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of polypeptide or protein modification, in particular to a polypeptide or protein modification method for promoting cysteine desulfurization by light. BACKGROUND

[0002] Compared with the phage display technology which can insert unnatural amino acids into proteins, site-selective direct chemical modification of polypeptides and proteins under biocompatible conditions is a more easily implemented post-translational modification method targeting specific amino acids.

[0003] Cysteine has become a specific reaction handle for polypeptide and protein modification due to its good nucleophilicity and low abundance on the protein surface (~2%), especially based on the 2e process (a two-electron process converts thiol to S-X, X = C, N, O, S, etc.). However, it is difficult to construct a C-C bond at the beta position of cysteine based on the 2e process. The C-C bond at the beta position can be constructed by Michanel addition or Giese reaction by converting cysteine to dehydroalanine. However, this approach will result in the loss of stereochemistry at the modification site, producing an undesirable mixture of diastereomers.

[0004] Cysteine desulfurization produces configuration-maintained alanine radicals, which is considered to be a method to solve this problem. There are currently two main methods for cysteine desulfurization to generate alanine radicals: one is trivalent phosphorus-mediated cysteine desulfurization, and the other is pentafluoropyridine-mediated cysteine desulfurization. Trivalent phosphorus-mediated cysteine desulfurization has two problems: one is that trivalent phosphorus is a strong reducing agent, which easily destroys disulfide bonds in polypeptides and proteins; the other is that the thiol group of cysteine needs to generate a sulfur radical first, and then add to the trivalent phosphorus to generate a phosphine radical in the reaction process. The generation of sulfur radicals will lead to the generation of thioether or disulfide byproducts, thereby reducing the conversion rate of raw materials. SUMMARY

[0005] The present application aims to solve the technical problems of the prior art and provide a polypeptide or protein modification method for promoting cysteine desulfurization by light, which is a method for constructing C-X bonds under biocompatible conditions with visible light promotion, stereochemical preservation of the modification site, and compatibility with disulfide bonds, and can be used for site-selective precise modification of polypeptides and proteins.

[0006] In order to solve the above technical problems, the application discloses a cysteine desulfurization and stereospecific polypeptide modification method, which takes a naked peptide containing cysteine or a protein containing a soluble solvent exposed cysteine as a raw material, takes a free radical acceptor as another raw material, takes a pyridine salt as an activating agent, takes a buffer as a reaction solvent, and realizes the desulfurization of cysteine in the polypeptide or the protein to form a stereospecific C-X bond under the participation of a reducing agent and an additive and the irradiation of visible light, wherein the free radical acceptor is any one of an electron-deficient olefin, acrylamide, acrylate, an organic selenium compound, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) or N-hetero uracil. After the reaction is completed, the polypeptide can be separated and purified through semi-preparative chromatography, analyzed for purity through high performance liquid chromatography and identified for the product through high resolution mass spectrometry; and the protein can be purified through a protein purification instrument and identified for the product through high resolution mass spectrometry.

[0007] In the above reaction, there is no requirement for the configuration of the amino acid, and both D type and L type are acceptable. There is no requirement for the structural type of the natural amino acid. The reaction container can be a glass tube or a quartz tube, and preferably a quartz tube with better light transmittance.

[0008] Specifically, the pyridine salt is an N-alkyl pyridine salt, and the structural general formula is as follows:

[0009] wherein X 1 is F, Cl, Br, I or SO2Me, preferably F; X 2 is Cl, Br, I, BF4, ClO4 or OTf, preferably OTf; R 1 is Me or Et, preferably Me; R 2 is H, Me, Et, OMe, CF3 or CH3CO, preferably Me; R 2 The substitution site can be located at the 3, 4 or 5 position of the pyridine ring, and preferably the 3 position.

[0010] The additive is 2,6-dimethylpyridine, triethylamine, diisopropylethylamine or none, and preferably 2,6-dimethylpyridine; the reducing agent is Hans ester, reduced nicotinamide adenine dinucleotide (NADH), tertiary amine or none. Different free radical acceptors select different types of reducing agents. In the Giese reaction (i.e., the type of free radical acceptor is an electron-deficient olefin, acrylamide, acrylate, an organic selenium compound or TEMPO), the reducing agent is Hans ester or NADH; in the Minisci reaction (i.e., the type of free radical acceptor is N-hetero uracil), no reducing agent is required

[0011] The reaction solvent is a buffer solution with pH 6-9, and the buffer solution is any one of PBS buffer, PB buffer, Tris HCl buffer, TES buffer, HEPES buffer, NaPi buffer or Sorensen buffer. Preferably, the buffer solution is PBS buffer or Tris HCl buffer.

[0012] In some embodiments, 2%-50% of a cosolvent is added to the buffer solution, and the cosolvent is acetonitrile (MeCN), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), dimethyl sulfoxide (DMSO), trifluoroethanol (TFE), preferably acetonitrile and trifluoroethanol.

[0013] The molar ratio of the polypeptide to the N-alkyl pyridine salt is 1:1.05-1.25, preferably 1:1.1; the molar ratio of the protein to the N-alkyl pyridine salt is 1:1.05-1.5, preferably 1:1.25; the molar ratio of the polypeptide to the radical acceptor is 1:2-5, preferably 1:5; the molar ratio of the protein to the radical acceptor is 1:50-100, preferably 1:100.

[0014] The molar ratio of the polypeptide to the additive is 1:0-5, preferably 1:3; the molar ratio of the protein to the additive is 1:0-5, preferably 1:0, i.e. when the reactant is a protein, no additive can be added; the molar ratio of the polypeptide to the reducing agent is 1:2-5, preferably 1:5; the molar ratio of the protein to the reducing agent is 1:50-100, preferably 1:100; wherein when the radical acceptor is an N-heterocyclic uracil, no reducing agent is added to the reaction.

[0015] Preferably, the concentration of the polypeptide is 10mM-80mM, preferably 40mM and 20mM; the concentration of the protein is 50-600μM, preferably 200μM.

[0016] Preferably, the reaction temperature is 20-30℃, the reaction time is 1-10 hours, and the reaction is carried out in an inert gas or air.

[0017] The visible light is visible light with a wavelength of 390-430nm, preferably 400nm, or 420-430nm.

[0018] The present application provides a method for constructing a C-X bond in a polypeptide and a protein. The thiol group of the cysteine side chain undergoes a rapid aromatic nucleophilic substitution (SN NThe cysteine-pyridine salt adduct is irradiated by visible light to break the C(sp3)-S bond and generate a stereospecific alanine radical. The alanine radical can react with various types of radical acceptors to construct the desired C-X bond in a polypeptide or a protein. The method uses visible light irradiation to avoid the use of ultraviolet light; the reaction of the stereospecific alanine radical with various radical acceptors does not lose its own stereochemistry, and a single configuration product can be obtained; and the use of N-alkyl pyridine salt does not damage the disulfide bond. Therefore, the present application provides a more mild, efficient and widely applicable method for site-selective and stereospecific modification of polypeptides and proteins.

[0019] Advantages: Compared with the prior art, the present application has the following advantages:

[0020] (1) The present application uses N-methyl pyridine salt as a pre-activation reagent for cysteine desulfurization, avoids the use of trivalent phosphine, and is compatible with disulfide bonds in polypeptides and proteins.

[0021] (2) The light source used in the present application is visible light of 390-430 nm, which is lower in energy than ultraviolet light and is relatively mild, and can protect the reactive groups (such as aromatic amino acids and nucleotide bases) in the biological system which are easily activated by ultraviolet light.

[0022] (3) Compared with the construction of Cβ-X bond based on dehydroalanine, the present application uses N-methyl pyridine salt to treat cysteine, and generates alanine radical after desulfurization under light irradiation. The alanine radical can effectively maintain the original configuration without changing during the subsequent reaction with the radical acceptor, so that the product generated is a stereospecific single configuration.

[0023] (4) The reaction conditions of the present application are carried out in a buffer, and the reaction temperature is 20-30℃, which is biocompatible.

[0024] (5) The alanine radical can react with various types of radical acceptors, so the present application has a wide range of applications and can be used to construct Cβ-X bond and perform site-selective modification of polypeptides and proteins.

[0025] (6) The present application does not use reducing agents or uses biologically derived reducing agents (Hans ester and NADH), so the method is more green and mild, and is more suitable for biological systems. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figures 1-2 are the H NMR and C NMR results of GSH-1 adduct, respectively. 1 H NMR and 13 C NMR results.

[0027] Figures 3-5 GSH-2 respectively 1 H NMR, 13 C NMR and 31 P NMR characterization diagram;

[0028] Figures 6-7 GSH-3 respectively 1 H NMR and 13 C10 NMR characterization diagram;

[0029] Figures 8-9 GSH-4 respectively 1 H NMR and 13 C NMR characterization diagram;

[0030] Figures 10-11 GSH-5 respectively 1 H NMR and 13 C NMR characterization diagram;

[0031] Figures 12-13 GSH-6 respectively 1 H NMR and 13 C10 NMR characterization diagram;

[0032] Figures 14-15 GSH-7 respectively 1 H NMR and 13 C10 NMR characterization diagram;

[0033] Figure 16 The diagram shows the structural characterization of the CG-β-1 adduct, where a is the HPLC result of CG-β-1; b is the MS spectrum of CG-β-1 (calculated mass [M+2H]2+: 703.7801; measured mass [M+2H]2+). 2+ (703.8427). Purity analysis was performed at UNIMICRO. Performed on the -3030 instrument, using C18-AP column (pore size) Particle size 5 μm (4.6 mm × 250 mm), using linear gradient elution, changed from 10% A / 90% B to 75% A / 25% B within 20 minutes;

[0034] Figure 17 In the image, a is the analytical high-performance liquid chromatography (HPLC) chromatogram of CG-β-4, and b is the mass spectrum of CG-β-4 (calculated mass [M+2H]). 2+ : 639.7835; Measured mass [M+2H] 2+ Purity analysis was performed using UNIMICRO (639.8427). - on a -3030 liquid chromatograph using C18-AP column (pore size 5 μm, dimensions 4.6 mm x 250 mm) with linear gradient elution, adjusting the mobile phase ratio from 10% A / 90% B to 75% A / 25% B in 20 minutes; C18-AP column (pore size 5 μm, dimensions 4.6 mm x 250 mm) with linear gradient elution, adjusting the mobile phase ratio from 10% A / 90% B to 75% A / 25% B in 20 minutes;

[0035] Figure 18 a is the analytical high performance liquid chromatography (HPLC) profile of CG-β-5; b is the mass spectrum profile of CG-β-5 (calculated mass: [M+H] + 1392.4731 ; [M+2H] 2+ 696.7422; found mass: [M+H] + 1392.3788; [M+2H] 2+ 696.6906); purity analysis on UNIMICRO - on a -3030 liquid chromatograph using C18-AP column (pore size 5 μm, dimensions 4.6 mm x 250 mm) with linear gradient elution, adjusting the mobile phase ratio from 10% A / 90% B to 75% A / 25% B in 20 minutes; C18-AP column (pore size 5 μm, dimensions 4.6 mm x 250 mm) with linear gradient elution, adjusting the mobile phase ratio from 10% A / 90% B to 75% A / 25% B in 20 minutes;

[0036] Figure 19 a is the analytical high performance liquid chromatography (HPLC) profile of CG-β(Cys-Ala); b is the mass spectrum profile of CG-β(Cys-Ala) (calculated mass: [M+H] + 1238.5284; [M+2H] 2+ 619.7679; found mass: [M+H] + 1238.4505; [M+2H] 2+ 619.7281); purity analysis on UNIMICRO - on a -3030 liquid chromatograph using C18-AP column (pore size 5 μm, dimensions 4.6 mm x 250 mm) with linear gradient elution, adjusting the mobile phase ratio from 10% A / 90% B to 75% A / 25% B in 20 minutes; C18-AP column (pore size 5 μm, dimensions 4.6 mm x 250 mm) with linear gradient elution, adjusting the mobile phase ratio from 10% A / 90% B to 75% A / 25% B in 20 minutes;

[0037] Figure 20 a is the analytical high performance liquid chromatography (HPLC) profile of CG-β-5; b is the mass spectrum profile of CG-β-5 (calculated mass: [M+H]

[0038] Figure 21 fluorescence imaging map after fluorescent molecule labeling. DETAILED DESCRIPTION

[0039] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings in which:

[0040] The polypeptide used in the examples is glutathione (GSH) and bioactive peptide Chorionic Gonadotropin-β (CG-β), the structure of which is as follows:

[0041]

[0042] The protein used in the examples is bovine serum albumin (BSA), which contains 17 pairs of disulfide bonds and one free, solvent-exposed cysteine (position 34) in its structure.

[0043] The free radical acceptor used in the following examples has the following structure:

[0044]

[0045] The N-methylpyridinium salt used in the examples has the following structure:

[0046] Example 1: Synthesis of GSH-1 adduct.

[0047] In a glove box, glutathione (GSH, 0.06 mmol), compound 1 (5 equiv), pyridine salt PyS1 (1.1 equiv), and Hans ester (4 equiv) were weighed into a 10 ml quartz photoreactor tube, 1.5 mL Tris-HCl buffer (1 M, pH 7.2, 20% v / v TFE (trifluoroethanol)) was added, followed by 2,6-dimethylpyridine (4 equiv). After sealing the photoreactor tube, it was removed from the glove box and placed under a 100 W, 400 nm direct light for 3 hours. After the reaction was completed, dichloromethane was added for extraction, and the aqueous solution was purified by semi-preparative chromatography and low-temperature lyophilization to obtain the target product GSH-1 adduct. The characterization results are shown in Figures 1-2 The structural characterization parameters are as follows:

[0048]

[0049] White flocculent solid, yield: 75% (20 mg).

[0050] 1H NMR (400 MHz, D20) δ 7.80-7.78 (m, 2H), 7.68-7.65 (m, 1H), 7.58-7.54 (m, 2H), 4.15-4.12 (m, 1H), 3.89-3.76 (m, 3H), 3.30-3.27 (m, 2H), 2.35 (t, J = 8.0 Hz, 2H), 2.05-1.99 (m, 2H), 1.75-1.72 (m, 1H), 1.65-1.58 (m, 3H).

[0051] 13 C NMR (101 MHz, D20) δ 174.18, 173.84, 172.77, 171.77, 136.42, 134.74, 129.62, 127.74, 54.14, 52.83, 52.35, 40.87, 30.74, 29.10, 25.44, 18.66.

[0052] HRMS (ESI) C 18 H 26 N3O8S [M+H] + calcd: 444.1456, found: 444.1435.

[0053] Example 2: Synthesis of GSH-2 adduct.

[0054] GSH (0.06 mmol), compound 2 (5 equiv), pyridine salt PyS1 (1.1 equiv) and Hans ester (4 equiv) were weighed in a 10 ml quartz photoreaction tube in a glove box, 1.5 mL Tris-HCl buffer (1 M, pH 7.2, 20% v / v TFE (trifluoroethanol)) was added, followed by 2,6-dimethylpyridine (4 equiv). After sealing the photoreaction tube, it was removed from the glove box and placed under a 100 W, 400 nm direct light for 3 hours. After the reaction was completed, dichloromethane was added for extraction, and the aqueous solution was purified by semi-preparative chromatography and low-temperature lyophilization to obtain the target product GSH-2 adduct. Its characterization chart is shown in Figures 3-5 The structure characterization parameters are as follows:

[0055]

[0056] White flocculent solid, yield: 52% (13.7 mg).

[0057] 1H NMR (400 MHz, D20) δ 4.23 - 4.19 (m, 1H), 4.04 - 3.86 (m, 7H), 2.47 - 2.40 (m, 2H), 2.14 - 2.06 (m, 2H), 1.85 - 1.79 (m, 3H), 1.77 - 1.67 (m, 1H), 1.59 - 1.50 (m, 2H), 1.18 (t, J = 4.0 Hz, 6H).

[0058] 31 P NMR (162 MHz, D20) δ 35.40.

[0059] 13 C NMR (101 MHz, D20) δ 174.41, 174.33, 172.94, 172.03, 63.27 (t, J = 7.0 Hz), 53.27, 52.59, 40.98, 31.45, 31.28, 30.86, 25.56, 23.81, 22.43, 18.02 (d, J = 4.0 Hz), 15.52 (d, J = 6.0 Hz).

[0060] HRMS (ESI) C 16 H 31 N3O9P [M + H] + calcd: 440.1798, found: 440.1789.

[0061] Example 3: Synthesis of GSH-3 adduct.

[0062] GSH (0.06 mmol), compound 3 (5 equiv), pyridine salt PyS1 (1.1 equiv) and Hans ester (4 equiv) were weighed in a 10 ml quartz photoreaction tube in a glove box, 1.5 mL Tris-HCl buffer (1 M, pH 7.2, 20% v / v TFE (trifluoroethanol)) was added, followed by 2,6-dimethylpyridine (4 equiv). After sealing the photoreaction tube, it was removed from the glove box and placed under a 100 W, 400 nm direct light for 3 hours. After the reaction was completed, dichloromethane was added for extraction, and the aqueous solution was purified by semi-preparative chromatography and low-temperature lyophilization to obtain the target product GSH-3 adduct. The standard results are shown in Figures 6-7 The structure characterization parameters are as follows:

[0063]

[0064] White flocculent solid, yield: 35% (10.4 mg).

[0065] 1H NMR (600 MHz, D20) δ 8.48 (d, J = 6.6 Hz, 2H), 7.66 (d, J = 6.6 Hz, 2H), 4.21 - 4.19 (m, 1H), 3.88 - 3.79 (m, 3H), 2.84 (t, J = 6.6 Hz, 2H), 2.43 - 2.35 (m, 2H), 2.03 (q, J = 7.2 Hz, 2H), 1.75 - 1.64 (m, 4H).

[0066] 13 C NMR (150 MHz, D20) δ 174.43, 174.37, 172.99, 172.27, 164.32, 163.12, 162.77, 140.29, 127.11, 53.54, 52.73, 40.96, 34.74, 30.86, 30.23, 25.58, 24.97.

[0067] HRMS (ESI) C 17 H 25 N4O6[M]+calcd: 381.1781, found: 381.1769.

[0068] Example 4: Synthesis of GSH-4 adduct

[0069] GSH (0.06 mmol), compound 4 (5 equiv), pyridine salt PyS1 (1.1 equiv) and Hans ester (4 equiv) were weighed in a 10 ml quartz photoreactor tube inside the glove box, 1.5 mL Tris-HCl buffer (1 M, pH 7.2, 20% v / v TFE (trifluoroethanol)) was added, followed by 2,6-dimethylpyridine (4 equiv). After sealing the photoreactor tube, it was removed from the glove box and placed under a 100 W, 400 nm direct light for 3 hours. After the reaction was completed, dichloromethane was added for extraction, and the aqueous solution was purified by semi-preparative chromatography and low-temperature lyophilization to obtain the target product GSH-4 adduct. Its characterization chart is shown in Figures 8-9 The structure characterization parameters are as follows:

[0070]

[0071] White flocculent solid, yield: 26% (6.8 mg).

[0072] 1H NMR (400 MHz, D20) δ 5.78 - 5.68 (m, 1H), 4.98 - 4.70 (m, 2H), 4.21 - 4.17 (m, 1H), 3.93 - 3.82 (m, 3H), 2.51 - 2.38 (m, 2H), 2.14 - 1.94 (m, 4H), 1.85 - 1.65 (m, 2H).

[0073] 13 C NMR (101 MHz, D20) δ 174.84, 174.33, 172.97, 171.83, 163.12, 162.77, 137.28, 115.72, 53.22, 52.40, 40.94, 30.82, 30.01, 29.19, 25.48.

[0074] HRMS (ESI) C 13 H 22 N3O6[M]+calcd: 316.1504, found: 316.1503.

[0075] Example 5: Synthesis of GSH-5 adduct

[0076] GSH (0.06 mmol), compound 5 (5 equiv), pyridine salt PyS1 (1.1 equiv) and Hans ester (4 equiv) were weighed in a 10 ml quartz photoreactor tube inside the glove box, 1.5 mL Tris-HCl buffer (1 M, pH 7.2, 20% v / v TFE (trifluoroethanol)) was added, followed by 2,6-dimethylpyridine (4 equiv). After sealing the photoreactor tube, it was removed from the glove box and placed under a 100 W, 400 nm direct light for 3 hours. After the reaction was completed, dichloromethane was added for extraction, and the aqueous solution was purified by semi-preparative chromatography and low-temperature lyophilization to obtain the target product GSH-5 adduct. The characterization results are shown in Figures 10-11 The structure characterization parameters are as follows:

[0077]

[0078] White flocculent solid, yield: 56% (14.5 mg).

[0079] 1H NMR (400 MHz, D20) δ 7.46 - 7.44 (m, 2H), 7.22 - 7.21 (m, 3H), 7.44 (q, J = 4.0 Hz, 1H), 3.77 (t, J = 8.0 Hz, 1H), 3.66 (s, 2H), 3.27 - 3.22 (m, 1H), 3.13 - 3.08 (m, 1H), 2.29 - 2.17 (m, 2H), 1.98 - 1.91 (m, 2H).

[0080] 13 C NMR (101 MHz, D20) δ 174.12, 172.89, 172.45, 172.26, 133.33, 129.43, 127.97, 127.92, 53.69, 52.77, 40.98, 30.87, 27.71, 25.53.

[0081] HRMS (ESI) C 16 H 22 N3O6Se [M+H]+ calcd: 432.0671, found: 432.0669.

[0082] Example 6: Synthesis of GSH-6 adduct.

[0083] GSH (0.06 mmol), compound 6 (5 equiv), pyridine salt PyS1 (1.1 equiv) and Hans ester (4 equiv) were weighed in a 10 ml quartz photoreactor tube inside the glove box, 1.5 mL Tris-HCl buffer (1 M, pH 7.2, 20% v / v TFE (trifluoroethanol)) was added, followed by 2,6-dimethylpyridine (4 equiv). After sealing the photoreactor tube, it was removed from the glove box and placed under a 100 W, 400 nm direct light for 3 hours. After the reaction was completed, dichloromethane was added for extraction, and the aqueous solution was purified by semi-preparative chromatography and low-temperature lyophilization to obtain the target product GSH-6 adduct. The characterization results are shown in Figures 12-13 The characterization results are shown in the structure characterization parameters as follows:

[0084]

[0085] White flocculent solid, yield: 39% (10 mg).

[0086] 1H NMR (400 MHz, D20) δ 4.86 (t, J = 6.0 Hz, 1H), 4.54-4.51 (m, 1H), 4.47-4.44 (m, 1H), 3.92 (s, 2H), 3.88-3.86 (m, 1H), 2.49 (t, J = 6.0 Hz, 2H), 2.12-2.06 (m, 2H), 1.78-1.67 (m, 5H), 1.53-1.47 (m, 1H), 1.36 (s, 3H), 1.34 (s, 3H), 1.31 (s, 3H), 1.27 (s, 3H).

[0087] 13 C NMR (101 MHz, D20) δ 174.25, 174.23, 172.86, 169.72, 76.64, 71.13, 71.09, 52.68, 51.52, 41.22, 36.54, 36.49, 30.98, 27.44, 27.40, 25.51, 19.41, 14.72.

[0088] HRMS (ESI) C 19 H 35 N4O7[M+H] + calcd: 431.2513, found: 431.2500.

[0089] Example 7: Synthesis of GSH-7 adduct

[0090] GSH (0.06 mmol), compound 7 (5 equiv), pyridine salt PyS1 (1.1 equiv) and Hans ester (4 equiv) were weighed in a 10 ml quartz photoreaction tube inside the glove box, 1.5 mL Tris-HCl buffer (1 M, pH 7.2, 20% v / v TFE (trifluoroethanol)) was added, followed by 2,6-dimethylpyridine (4 equiv). After sealing the photoreaction tube, it was removed from the glove box and placed under a 100 W, 400 nm direct light for 3 hours. After the reaction was completed, dichloromethane was added for extraction, and the aqueous solution was purified by semi-preparative chromatography and low-temperature lyophilization to obtain the target product GSH-7 adduct. The characterization results are shown in Figures 14-15 The structure characterization parameters are as follows:

[0091]

[0092] White flocculent solid, yield: 49% (14.4 mg).

[0093] 1H NMR (600 MHz, D20) δ 4.17-4.15 (m, 1H), 3.92-3.83 (m, 3H), 3.03 (t, J = 7.2 Hz, 2H), 2.47-2.38 (m, 2H), 2.12-2.04 (m, 2H), 1.84 (s, 3H), 1.73-1.66 (m, 1H), 1.63-1.57 (m, 1H), 1.41-1.37 (m, 2H), 1.31-1.22 (m, 2H).

[0094] 13 C NMR (150 MHz, D20) δ 174.79, 174.33, 173.94, 172.95, 171.78, 163.12, 162.77, 53.79, 52.39, 40.92, 39.02, 30.80, 30.51, 27.72, 25.48, 22.22, 21.76.

[0095] HRMS (ESI) C 15 H 27 N4O7[M]+calcd: 375.1875, found: 375.1874.

[0096] Example 8: Synthesis of CG-β-1 adduct.

[0097] Chorionic Gonadotropin-β (CG-β, 5 μmol), compound 1 (5 equiv), pyridine salt PyS1 (1.1 equiv) and NADH (5 equiv) were weighed in a 10 ml quartz photoreaction tube in the glove box, 125 μL PBS buffer (0.1 M, pH 6.5, 20% v / v TFE) was added, followed by 2,6-dimethylpyridine (4 equiv). After sealing the photoreaction tube, it was removed from the glove box and placed under a 24 W, 420-430 nm direct light for 5 hours. After the reaction was completed, the reaction solution was purified by semi-preparative chromatography, and then freeze-dried at low temperature to obtain the target product CG-β-1 adduct. The structure characterization results are shown in Figure 16 .

[0098] Example 9: Synthesis of CG-β-4 adduct

[0099] Chorionic Gonadotropin-β (CG-β, 5 μmol), compound 4 (5 equiv), pyridine salt PyS1 (1.1 equiv) and NADH (5 equiv) were weighed in a 10 ml quartz photoreactor tube in a glove box, 125 μL PBS buffer (0.1 M, pH 6.5, 20% v / v TFE) was added, followed by 2,6-dimethylpyridine (4 equiv). After sealing the photoreactor tube, it was removed from the glove box and placed under a 24 W, 420-430 nm direct light for 5 hours. After the reaction was completed, the reaction solution was purified by semi-preparative chromatography, and then freeze-dried at low temperature to obtain the target product CG-β-4 adduct. The structural characterization results thereof are shown in Figure 17 .

[0100] Example 10: Synthesis of CG-β-5 adduct

[0101] Chorionic Gonadotropin-β (CG-β, 5 μmol), compound 5 (5 equiv), pyridine salt PyS1 (1.1 equiv) and NADH (5 equiv) were weighed in a 10 ml quartz photoreactor tube in a glove box, 125 μL PBS buffer (0.1 M, pH 6.5, 20% v / v TFE) was added, followed by 2,6-dimethylpyridine (4 equiv). After sealing the photoreactor tube, it was removed from the glove box and placed under a 24 W, 420-430 nm direct light for 5 hours. After the reaction was completed, the reaction solution was purified by semi-preparative chromatography, and then freeze-dried at low temperature to obtain the target product CG-β-5 adduct. The structural characterization results thereof are shown in Figure 18 .

[0102] Example 11: Synthesis of CG-β (Cys-Ala)

[0103] Chorionic Gonadotropin-β (CG-β, 5 μmol), pyridine salt PyS1 (1.1 equiv) and NADH (5 equiv) were weighed in a 10 ml quartz photoreactor tube in a glove box, 125 μL PBS buffer (0.1 M, pH 6.5, 20% v / v TFE) was added, followed by 2,6-dimethylpyridine (4 equiv). After sealing the photoreactor tube, it was removed from the glove box and placed under a 24 W, 420-430 nm direct light for 5 hours. After the reaction was completed, the reaction solution was purified by semi-preparative chromatography, and then freeze-dried at low temperature to obtain the target product CG-β (Cys-Ala). The structural characterization results thereof are shown in Figure 19 .

[0104] Example 12: Site-selective modification and fluorescent molecule labeling of bovine serum albumin BSA.

[0105] The pyridine salt (0.7 mg) was dissolved in 0.25 mL of ultrapure water. The BSA (2.7 mg) was dissolved in 175 mL of PBS buffer (0.1 M, pH 8.0) followed by the addition of 5 μL of the pyridine salt solution (10 mM). After the mixture was shaken on a shaker for 10 min, 20 μL of a PBS buffer solution (0.1 M, pH 8.0, 20% v / v MeCN) of the radical acceptor 8 was added in a glove box and transferred to a 10 mL quartz photoreaction tube. After the photoreaction tube was sealed, it was removed from the glove box and placed under a 100 W, 400 nm direct light for 8 h. After the reaction was completed, the reaction system was subjected to HRMS detection, and the results are shown in Figure 20

[0106] After the above reaction was completed, FAM-PEG3-N3 (100 equiv), tris(3- hydroxypropyltriazole methyl) amine (THPTA, 200 equiv), CuSO4·5H2O (100 equiv), and a DMF (10 μL) solution of sodium vitamin C (200 equiv) were added to the reaction system, and the reaction mixture was placed on a shaker and shaken for 1 h. 10 μL of the reaction solution was taken and diluted with 10 μL of ultrapure water. Each sample was added to a 12% SDS-PAGE gel in a 12-well format, and electrophoresis was performed at room temperature and a voltage of 160 V for 150 min. Subsequently, fluorescence imaging was performed at 460 nm on a Typhoon FLA 9500 (GE). The fluorescence imaging results are shown in Figure 21

[0107] The present application provides a polypeptide and protein construction C-X bond ideas and methods, the method and approach to realize the technical scheme is many, the above described is only the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled person in the art, without departing from the principle of the present application, can make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application. The components not explicitly described in the present embodiment can be realized by existing technology.​​

Claims

1. A method for modifying polypeptides or proteins by photo-promoting cysteine ​​desulfurization, characterized in that, Using naked peptides containing cysteine ​​or proteins containing solvent-exposed cysteine ​​as raw materials, and free radical acceptors as another raw material, pyridinium salts as activating reagents, and buffer solutions as reaction solvents, under the participation of reducing agents and additives and irradiation with visible light, cysteine ​​desulfurization of peptides or proteins is achieved to form stereoretained CX bonds. The free radical acceptor is any one of electron-deficient olefins, acrylamide, acrylates, organoselenic compounds, 2,2,6,6-tetramethylpiperidine-N-oxygen free radicals, or N-heterouracil.

2. The method according to claim 1, characterized in that, The pyridinium salt is an N-alkylpyridinium salt with the following general structural formula: , Among them, X 1 For F, Cl, Br, I or SO2Me; X 2 For Cl, Br, I, BF4, ClO4, or OTf; R 1 For Me or Et; R 2 For H, Me, Et, OMe, CF3 or CH3CO; R 2 The substitution site can be located at position 3, 4 or 5 of the pyridine ring.

3. The method according to claim 1, characterized in that, The additive is 2,6-dimethylpyridine, triethylamine, diisopropylethylamine or none, preferably 2,6-dimethylpyridine; the reducing agent is hans ester, reduced nicotinamide adenine dinucleotide, tertiary amine or none.

4. The method according to claim 1, characterized in that, The reaction solvent is a buffer solution with a pH of 6-9, and the buffer solution is any one of PBS buffer, PB buffer, Tris HCl buffer, TES buffer, HEPES buffer, NaPi buffer, or Sorensen buffer.

5. The method according to claim 4, characterized in that, The buffer solution is supplemented with a co-solvent at a volume ratio of 2%-50%, wherein the co-solvent is acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, or trifluoroethanol.

6. The method according to claim 1, characterized in that, The molar ratio of polypeptide to N-alkylpyridinium salt is 1:1.05-1.25; the molar ratio of protein to N-alkylpyridinium salt is 1:1.05-1.5; the molar ratio of polypeptide to free radical acceptor is 1:2-5; and the molar ratio of protein to free radical acceptor is 1:50-100.

7. The method according to claim 1, characterized in that, The molar ratio of polypeptide to additive is 1:0-5; the molar ratio of protein to additive is 1:0-5; the molar ratio of polypeptide to reducing agent is 1:2-5; the molar ratio of protein to reducing agent is 1:50-100; wherein, when the free radical acceptor is N-heterocyclic uracil, no reducing agent is added to the reaction.

8. The method according to claim 1, characterized in that, The concentration of peptides is 10 mM to 80 mM, preferably 40 mM and 20 mM; the concentration of proteins is 50 to 600 µM, preferably 200 µM.

9. The method according to claim 1, characterized in that, The reaction temperature is 20-30℃, the reaction time is 1-10 hours, and the reaction is carried out in an inert gas or air.

10. The method according to claim 1, characterized in that, The visible light refers to visible light with a wavelength of 390-430 nm.

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