A method for selectively modifying lysine by visible light catalysis and its application

Abstract

The present invention provides a method for selectively modifying lysine by visible light catalysis. Using a thiocarboxylic acid compound, a riboflavin-based catalyst is added under visible light irradiation for modifying lysine; the chemical reaction formula for the reaction of the thiocarboxylic acid compound with lysine is: wherein, R is selected from one or more of methyl, phenyl, azide substituent, alkyne substituent, and biotin. The present invention also provides the application of the above method in protein modification. The present invention uses a thiocarboxylic acid and adds a riboflavin-based catalyst, which can specifically modify lysine under visible light irradiation without modifying cysteine, greatly eliminating the interference of cysteine and improving the chemoselectivity of lysine modification.

Description

Technical Field

[0001] The present invention belongs to the field of chemical biology, and relates to a selective modification of lysine, in particular to a method for selectively modifying lysine by visible light catalysis and its application. Background Art

[0002] Lysine has a high proportion on the protein surface, with an abundance of about 5.9% in natural protein residues. At the same time, lysine residues also have certain nucleophilic reaction activities. Therefore, lysine has gradually become one of the ideal targets for targeted drugs.

[0003] Currently, strategies for using lysine as a modification site include methylation, acetylation, succinylation, crotonylation, etc. The modification methods mainly involve reactions with nucleophiles. However, the presence of nucleophilic groups such as cysteine on the protein surface will cause competitive interference to the above reactions, thereby affecting the chemoselectivity and modification efficiency of the reactions. Therefore, it is particularly important to develop new lysine modification strategies with stronger chemoselectivity for the development of new targeted drugs.

[0004] Photocatalytic reactions, due to their unique medium selectivity and low reactivity, have been widely studied in chemoselective coupling. At the same time, photocatalytic reactions have also been developed for bioconjugation applications under mild conditions. Because they have higher chemoselectivity, the reactions are initiated under light irradiation with a suitable catalyst. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for selectively modifying lysine by visible light catalysis and its application, which improves the chemoselectivity of the lysine modification strategy through the controllable action of a photocatalyst, and avoids the problems of strong interference from nucleophilic amino acids such as cysteine in the existing lysine modification technology and poor chemoselectivity.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A method for selectively modifying lysine by visible light catalysis, which uses a thiocarboxylic acid compound and adds a riboflavin-based catalyst under visible light irradiation for modifying lysine;

[0008] The chemical structural formula of the thiocarboxylic acid compound is:

[0009]

[0010] The chemical reaction formula for the reaction of the thiocarboxylic acid compound with lysine is as follows:

[0011]

[0012] Among them, R is selected from one or more of methyl, phenyl, azide substituent, alkynyl substituent, and biotin.

[0013] As one of the preferred embodiments of the present invention, the synthesis method of the thio-carboxylic acid compound is as follows:

[0014]

[0015] As one of the preferred embodiments of the present invention, when R is selected from methyl, phenyl, azide substituent, alkynyl substituent, or biotin, the corresponding thio-carboxylic acid compound is thioacetic acid, potassium thioacetate, thiobenzoic acid, 5-azidopentanoic acid, 4-alkynylpentanoic acid, or thio-carboxybiotin.

[0016] As one of the preferred embodiments of the present invention, the lysine to be modified is independent lysine, lysine on a polypeptide, or lysine on a protein; and among them, the protein includes one of pure white protein, antibody, protein system, and living cell.

[0017] As one of the preferred embodiments of the present invention, the riboflavin-based catalyst is selected from one of riboflavin, tetrabutyl riboflavin, tetraethyl riboflavin, and riboflavin phosphate.

[0018] As one of the preferred embodiments of the present invention, the visible light is selected from one of 450 nm blue light and 440 nm blue light.

[0019] An application of the above visible light-catalyzed selective modification method of lysine in protein modification.

[0020] As one of the preferred embodiments of the present invention, add the protein to be modified and the thio-carboxylic acid compound, and add the riboflavin-based catalyst under visible light irradiation for reaction; the modification site is lysine on the protein, and a photocatalytic reaction occurs between the thio-carboxylic acid compound and lysine;

[0021] Among them, the feeding amount of the thio-carboxylic acid compound is 0.1 to 20 equivalents of the protein, and the feeding amount of the riboflavin-based catalyst is 0.01 equivalent of the thio-carboxylic acid compound.

[0022] As one of the preferred embodiments of the present invention, the reaction solvent used in the photocatalytic reaction is selected from water or phosphate solution.

[0023] As one of the preferred embodiments of the present invention, the reaction time of the photocatalytic reaction is 10 s to 2 min according to the type of protein participating in the reaction.

[0024] As one of the preferred embodiments of the present invention, the reaction temperature of the photocatalytic reaction is room temperature or 37 °C according to the substrate participating in the reaction.

[0025] Modification principle:

[0026] In the present invention, thioacetic acid and lysine form an amide bond under the action of a riboflavin-based catalyst through visible light irradiation to complete the specific modification of lysine.

[0027] The advantages of the present invention compared with the prior art are as follows: The present invention utilizes a thioacetic acid compound and adds a riboflavin-based catalyst, which can specifically modify lysine under visible light irradiation and does not modify cysteine, greatly eliminating the interference of the presence of cysteine, improving the chemoselectivity of lysine modification in the present invention, making the lysine modification strategy of the present invention have greater value, and further providing a strong theoretical basis for the development of antibody-drug conjugates. Description of the Drawings

[0028] Figure 1 It is a diagram showing the blue light catalytic reaction process and product conversion rate results of different thioacetic acid compounds for different polypeptide lysines in Example 3;

[0029] Figure 2 It is the attempt result of the blue light catalytic reaction on proteins, nanobodies, and antibodies in Example 4 (in the figure, Figure A is a schematic diagram of the reaction process, Figures B - E are mass spectrometry result diagrams of the blue light catalytic reaction on lysine pure protein, lysine nanobody, and lysine antibody, Figures F - G are circular dichroism result diagrams of the protein after modification, Figure H is a schematic diagram of the protein fluorescence labeling experiment, and Figure I is a fluorescence protein gel diagram of the protein after modification);

[0030] Figure 3 It is the attempt result of the blue light catalytic reaction in proteomics at the cell lysate level in Example 5 (in the figure, Figure A is a schematic diagram of the reaction assembly process, Figures B - C are experimental result diagrams of the chemoselectivity of the modification strategy of the present invention for lysine, and Figures D - E are experimental result diagrams of the present invention at the proteomics level). Detailed Embodiments

[0031] The following details the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments. At the same time, the reagent products and experimental methods used in the present invention, unless otherwise specified, are all conventional reagents or methods in the art and will not be elaborated further.

[0032] Example 1: Preparation of 5-azidothiopentanoic acid (a kind of thioacetic acid):

[0033]

[0034] Add 1.5 g of benzotriazole to a 100 mL round-bottom flask, add 15 mL of dichloromethane, start magnetic stirring, and stir for 10 min; dissolve 500 mg of 5-azidopentanoic acid in 5 mL of dichloromethane, drop the solution into the reaction solution, and stir the mixture overnight at room temperature; after the reaction is complete, filter the precipitate to obtain the product - a white solid. The NMR data of this compound are as follows: 1 H NMR(500MHz,Chloroform-d).δ8.26(dt,J=8.2,1.0Hz,1H),8.10(dt,J=8.2,1.0Hz,1H),7.64(ddd,J=8.3,7.1,1.0Hz,1H),7.49(ddd,J=8.2,7.1,1.0Hz,1H),3.46(t,J=7.3Hz,2H),3.38(t,J=6.7Hz,2H),2.11-1.90(m,2H),1.87-1.69(m,2H). 13 C NMR(101MHz,Chloroform-d)δ172.0,146.2,131.1,130.6,126.3,120.3,114.5,51.1,35.0,28.4,21.6。

[0035] Add 1 g of the white solid to a 100 mL round-bottom flask, add 10 mL of water, add NaSH, start magnetic stirring, and stir the reaction overnight; add ethyl acetate for extraction, combine the aqueous phases, and lyophilize to obtain the product "5-azidothiopentanoic acid", with a yield of 97%. The NMR data are as follows: 1 H NMR(400MHz,Chloroform-d)δ3.12(ddd,J=6.4,5.0,1.0Hz,1H),2.75-2.45(m,1H),2.11-1.87(m,2H). 13 CNMR(101MHz,Chloroform-d)δ201.5,53.5,41.3,30.5,23.0,22.8。

[0036] Example 2: Preparation of thiocarboxybiotin (a kind of thiocarboxylic acid):

[0037]

[0038] Add 2.5 g of biotin, 2.7 g of triphenylmethanethiol, and 100 mg of 4-dimethylaminopyridine into a 100 mL round-bottom flask; add 25 mL of dimethylformamide and start magnetic stirring; place the round-bottom flask in an ice-water bath and slowly add 1.9 g of 1-ethyl-3-(3-dimethylpropylamine)carbodiimide hydrochloride; after the addition is complete, transfer the round-bottom flask to room temperature and stir the mixture overnight at room temperature; after the reaction is complete, add 50 mL of water until precipitation occurs; filter the precipitate and dry it under vacuum to obtain a white solid, which is the intermediate product.

[0039] Add 1 g of the intermediate into a 100 mL round-bottom flask, add 10 mL of dichloromethane, and start magnetic stirring; add 2 mL of trifluoroacetic acid dropwise to the reaction solution and stir the reaction solution at room temperature for 4 h; remove the solvent using a rotary evaporator, add ether to the round-bottom flask, place the reaction flask in an ice-water bath, start stirring and wait for solid precipitation, and dry it under vacuum to obtain the product "thiocarboxy biotin". The NMR data is as follows: 1 H NMR(400MHz,DMSO-d 6 )δ6.45(s,1H),4.30(dd,J=7.7,4.9Hz,1H),4.13(ddd,J=7.6,4.4,2.8Hz,1H),3.09(ddd,J=8.4,6.2,4.3Hz,1H),2.95-2.76(m,2H),2.74-2.53(m,2H),1.73-1.18(m,6H). 13 C NMR(101MHz,DMSO-d 6 )δ193.4,162.8,61.1,61.0,59.2,55.4,55.3,46.1,42.0,28.0,27.9,27.8,27.7,26.2,25.0。

[0040] Example 3: Photocatalytic modification of polypeptide lysine:

[0041] Add 1 mM of a polypeptide phosphate solution containing lysine, 0.5 mM of riboflavin, and 10 mM of a thiocarboxylic acid compound into a 1.5 mL centrifuge tube. At room temperature, start a blue light reactor and irradiate with blue light (wavelength 450 nm) for 10 s to 1 min. After stopping the irradiation, purify the reaction solution by HPLC to obtain the product and determine the conversion rate.

[0042] Refer to Figure 1, in this embodiment, the selection of the polypeptide Peptides can specifically be one of Peptides2, Peptides3, Thymopentin 4, Oxytocin 5 (pH 6.0), Somatostain 6, Melanotan 7, Type I collagenfragment 8, EGFR fragmen 9, Peptides10; the selection of the thio-carboxylic acid compound can be one of potassium thioacetate (Formula a), thioacetic acid (Formula b), thiobenzoic acid (Formula c), 4-alkynylthiopentanoic acid (Formula d), 5-azidothiopentanoic acid (Formula e, refer to Example 1), thio-carboxybiotin (Formula f, refer to Example 2).

[0043] Correspondingly, under the combination of different polypeptides and different thio-carboxylic acids, the conversion rate results of the obtained product Products are respectively as Figure 1 shown. For example, for Peptides2, its combination with potassium thioacetate (Formula a) is 2a, and its combination with thioacetic acid is 2b, and so on.

[0044] From Figure 1 the results, it can be seen that different thio-carboxylic acids have high efficiency in modifying lysine of different polypeptides.

[0045] Example 4: Modification of protein lysine:

[0046] Add 50 μM protein phosphate solution containing lysine, 10 μM riboflavin, and 1 mM thio-carboxylic acid compound into a 1.5 mL centrifuge tube. At room temperature, start the blue light reactor and irradiate with blue light (wavelength 450 nm) for 1 min. After stopping the irradiation, through purification means such as ultrafiltration, the modified protein conjugate is obtained, and experimental operations such as mass spectrometry detection, circular dichroism detection, and protein gel are completed.

[0047] In this embodiment, the protein types tried are pure proteins containing lysine, nanobodies, and antibodies; the thio-carboxylic acid compound is selected as 5-azidothiopentanoic acid or thio-carboxybiotin; the reaction process is as Figure 2 shown in A ( Figure 2 in A, 5-azidothiopentanoic acid is Formula e, and thio-carboxybiotin is Formula f); the mass spectrometry results of the modification of lysine pure protein, lysine nanobody, and lysine antibody by blue light catalytic reaction are as Figure 2 shown in B to 2E; the circular dichroism results of the protein after modification are as Figure 2 shown in F to 2G; the protein fluorescence labeling experiment is as Figure 2 shown in H; the fluorescence protein gel diagram results of the protein after modification are as Figure 2 shown in I.

[0048] From Figure 2As can be seen from the results, the method of the present invention has a high modification efficiency for pure proteins, nanobodies, and antibodies containing lysine ( Figure 2 B to 2E); at the same time, combining the results of circular dichroism spectroscopy and fluorescence experiments, the conformation of the modified protein is stable ( Figure 2 F to 2G), and the modification strategy is highly efficient ( Figure 2 I).

[0049] Example 5: Lysine modification at the proteomics level:

[0050] Add 10 μg of cell lysate containing lysine, 10 μM riboflavin, and 1 mM biotin thioacid to a 1.5 mL round-bottom flask, start the blue light reactor, and irradiate for 1 min; after the labeling is completed, enrich the protein with biotin resin, and complete the protein secondary mass spectrometry; perform data analysis.

[0051] In this example, the reaction assembly process is as shown in Figure 3 A; the experimental results of the chemoselectivity of the modification strategy of the present invention for lysine are as shown in Figure 3 B to 3C; the experimental results of the present invention at the proteomics level are as shown in Figure 3 D to 3E.

[0052] From the Figure 3 results, it can be seen that the modification strategy of the present invention has high chemoselectivity for lysine, mainly modifies lysine, and has an advantage in modifying lysine at the corresponding positions in Figure 3 D to 3E.

[0053] In addition, it should also be noted that:

[0054] For the riboflavin-based catalyst in the photocatalytic modification process of the present invention, in addition to riboflavin in the above examples, one of tetrabutyl riboflavin, tetraethyl riboflavin, and riboflavin phosphate can also be used.

[0055] For the visible light irradiation in the photocatalytic modification process, in addition to the 450 nm blue light in the above examples, 440 nm blue light can also be used.

[0056] For the reaction temperature in the photocatalytic modification process, in addition to room temperature (20 - 25 °C) in the above examples, it can also be 37 °C.

[0057] For the reaction solvent used in the photocatalytic modification process, in addition to the phosphate solution in the above examples, water can also be used.

[0058] In the process of photocatalytic modification of protein lysine, the feeding amount of the thioacid compound is 0.1 - 20 equivalents of the protein, that is, in addition to 20 equivalents in the above examples, it can also be 0.1 equivalent, 1 equivalent, or 10 equivalents.

[0059] During the process of photocatalytic modification of protein lysine, the reaction time of the photocatalytic reaction, depending on the type of protein involved in the reaction, in addition to being 1 min in the above embodiments, can also be 10 s, 20 s, etc.

[0060] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for selective modification of lysine by visible light catalysis, characterized in that, a thio-carboxylic acid compound is used, and a riboflavin-based catalyst is added under visible light irradiation for modifying lysine; the chemical structural formula of the thio-carboxylic acid compound is: the chemical reaction formula for the reaction of the thio-carboxylic acid compound with lysine is as follows: Among them, the thio carboxylic acid compound is specifically a structural compound, and R is the corresponding group of the above compound; The lysine is independent lysine, lysine on a polypeptide or lysine on a protein; the riboflavin-based catalyst is selected from one of riboflavin, tetrabutyl riboflavin, tetraethyl riboflavin, and riboflavin phosphate.

2. The method for selective modification of lysine by visible light catalysis according to claim 1, characterized in that, the visible light is selected from one of 450 nm blue light and 440 nm blue light.

3. An application of the method for selective modification of lysine by visible light catalysis according to any one of claims 1 to 2 in protein modification.

4. The application of the method for selective modification of lysine by visible light catalysis according to claim 3 in protein modification, characterized in that, a protein to be modified and a thio-carboxylic acid compound are added, and a riboflavin-based catalyst is added under visible light irradiation for reaction; the modification site is lysine on the protein, and a photocatalytic reaction occurs between the thio-carboxylic acid compound and lysine; wherein, the feeding amount of the thio-carboxylic acid compound is 0.1 to 20 equivalents of the protein, and the feeding amount of the riboflavin-based catalyst is 0.01 equivalent of the thio-carboxylic acid compound.

5. The application of the method for selective modification of lysine by visible light catalysis according to claim 4 in protein modification, characterized in that, the reaction solvent used in the photocatalytic reaction is selected from water or phosphate solution.

6. The method for selective modification of lysine by visible light catalysis according to claim 4, characterized in that, the reaction time of the photocatalytic reaction is 10 s to 2 min.

7. The method for selective modification of lysine by visible light catalysis according to claim 4, characterized in that, the reaction temperature is room temperature or 37 °C.

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