A dopamine derivative, a conjugate compound, and a preparation method and application thereof

Abstract

The present invention provides a dopamine derivative, a conjugate compound, and a preparation method and application thereof, belonging to the technical field of chemical biology. The dopamine derivative provided by the present invention has the structure shown in Formula I. As a small molecule probe, the dopamine derivative can specifically enrich dopamine-modified protein substrates without relying on modified antibodies. Combining with mass spectrometry methods, it can identify dopamine-modified protein substrates and modification sites, and is particularly suitable for identifying protein substrates and modification sites that can undergo dopamine modification in organisms such as any kind of cells, tissues, and animals, laying a foundation for exploring the biological functions of dopamine modification, and contributing to analyzing the pathogenesis of diseases and developing drugs for regulating the dopamine modification level related to diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical biology, and particularly relates to a dopamine derivative, a coupling compound, and a preparation method and application thereof. Background Art

[0002] Dopmainylation refers to the covalent addition of dopamine molecules as a modifier monomer to the amino acid residue glutamine (Q) of a protein under the action of the catalytic enzyme transglutaminase (TGM). Dopmainylation was discovered as early as 60 years ago, but so far only a limited number of dopmainylated protein substrates have been identified. This is because the level of post-translational modification of proteins is usually not high, so the identification of dopmainylated protein substrates generally relies on the enrichment of dopmainylation antibodies or chemical biology methods. At present, there is no report on a probe for specifically enriching dopmainylated protein substrates in the prior art. Summary of the Invention

[0003] The purpose of the present invention is to provide a dopamine derivative, a coupling compound, and a preparation method and application thereof. The dopamine derivative provided by the present invention contains an alkynyl group, does not rely on dopmainylation antibodies, and through the alkynyl group, it is convenient to introduce a functional compound (such as biotin) that can specifically enrich dopmainylated protein substrates. Then, combined with mass spectrometry methods, dopmainylated protein substrates and modification sites can be identified.

[0004] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a dopamine derivative having the structure shown in Formula I:

[0006]

[0007] The present invention provides a preparation method of the dopamine derivative according to the above technical solution, including the following steps:

[0008] Mix dopamine, Boc 2 O, a first base reagent, a first organic solvent and water, and carry out an amino protection reaction to obtain an amino protection intermediate;

[0009] Mix the amino protection intermediate, propargyl halide, a second base reagent and a second organic solvent, and carry out a substitution reaction to obtain a substitution intermediate;

[0010] Carry out a deprotection reaction on the substitution intermediate to obtain a dopamine derivative having the structure shown in Formula I;

[0011] The structural formulas of the amino-protected intermediate and the substitution intermediate are as follows in sequence:

[0012]

[0013] Preferably, the temperature of the amino-protection reaction is 10 - 20 °C and the time is 10 - 15 h;

[0014] the temperature of the substitution reaction is 15 - 25 °C and the time is 10 - 15 h;

[0015] The deprotection reaction is carried out in the presence of an acid reagent; the temperature of the deprotection reaction is 20 - 30 °C and the time is 30 - 40 h.

[0016] The present invention provides the application of the dopamine derivative as described in the above technical solution in the preparation of a preparation for enriching dopamine-modified protein substrates, a preparation for identifying dopamine-modified protein substrates, or a preparation for identifying the modification sites of dopamine-modified protein substrates, wherein the dopamine-modified protein substrate is obtained by modifying a protein with the dopamine derivative.

[0017] Preferably, the modification is carried out in vivo or in vitro; the modification is carried out in the presence of transglutaminase.

[0018] Preferably, the organism includes cells, tissues or animals.

[0019] The present invention provides a coupling compound, including a matrix substance, a dopamine derivative and a functional substance, wherein the matrix substance, the dopamine derivative and the functional substance are covalently connected in sequence;

[0020] the matrix substance includes a protein or a polypeptide, the dopamine derivative is the dopamine derivative as described in the above technical solution, and the functional substance includes biotin containing an azide group or a fluorescent probe containing an azide group;

[0021] Wherein the matrix substance and the dopamine derivative are connected by an amide bond, and the dopamine derivative and the functional substance are connected by a group formed by a click chemical reaction between an alkyne group and an azide group.

[0022] The present invention provides a preparation method of the coupling compound as described in the above technical solution, including the following steps:

[0023] Mix the matrix substance, the dopamine derivative and a first solvent, and carry out an amidation reaction to obtain an intermediate;

[0024] Mix the intermediate, the functional substance, a copper catalyst, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, tris(2-carboxyethyl)phosphine hydrochloride and a second solvent, and carry out a click chemical reaction to obtain the coupling compound.

[0025] Preferably, the temperature of the amidation reaction is 15 - 25 °C and the time is 1 - 3 h;

[0026] The temperature of the click chemical reaction is 15 - 25 °C and the time is 0.5 - 1.5 h.

[0027] The present invention provides the use of the coupling compound according to the above technical solution in the preparation of a preparation for enriching dopamine-modified protein substrates, a preparation for identifying dopamine-modified protein substrates, or a preparation for identifying the modification sites of dopamine-modified protein substrates.

[0028] The present invention provides a dopamine derivative having the structure shown in Formula I. The dopamine derivative provided by the present invention can be used as a small molecule probe for specifically enriching dopamine-modified protein substrates. It does not rely on dopamine-modified antibodies to enrich dopamine-modified protein substrates. Combining with mass spectrometry methods, it can identify dopamine-modified protein substrates and modification sites, especially suitable for identifying protein substrates and modification sites that can undergo dopamine modification in organisms such as any type of cell or tissue, laying a foundation for preliminarily exploring the biological functions of dopamine modification, and contributing to analyzing the pathogenesis of diseases related to dopamine modification and developing drugs for intervening in dopamine modification. Brief Description of the Drawings

[0029] Figure 1 It is a flow chart for preparing the dopamine modification probe pDA-1 in the examples of the present invention;

[0030] Figure 2 It is a LC-MS detection result graph in Test Example 1;

[0031] Figure 3 It is a WB analysis result graph in Test Example 1;

[0032] Figure 4 It is a cell imaging detection result graph in Test Example 2;

[0033] Figure 5 It is a schematic diagram for identifying dopamine-modified protein substrates in Test Example 2;

[0034] Figure 6 It is a verification result graph for dopamine-modified substrates DDC, SIRT2, and histone H4 in Test Example 2;

[0035] Figure 7 It is a schematic diagram for identifying dopamine modification sites in Test Example 3;

[0036] Figure 8 It is a secondary mass spectrometry graph of dopamine modification occurring at histone H427 in vitro in Test Example 4;

[0037] Figure 9Results graph for testing the biological function of dopamine modification (H4Q27dop) on histone H4 in Test Example 4. Detailed implementation mode

[0038] The present invention provides a dopamine derivative having the structure shown in Formula I:

[0039]

[0040] The present invention provides a preparation method of the dopamine derivative described in the above technical solution, including the following steps:

[0041] Mix dopamine, Boc 2 O, a first base reagent, a first organic solvent and water, and carry out an amino protection reaction to obtain an amino protection intermediate;

[0042] Mix the amino protection intermediate, propargyl halide, a second base reagent and a second organic solvent, and carry out a substitution reaction to obtain a substitution intermediate;

[0043] Carry out a deprotection reaction on the substitution intermediate to obtain a dopamine derivative having the structure shown in Formula I;

[0044] The structural formulas of the amino protection intermediate and the substitution intermediate are shown as follows in sequence:

[0045]

[0046] In the present invention, unless otherwise specified, the raw materials and reagents used are commercially available products well-known to those skilled in the art or are prepared by methods well-known to those skilled in the art. The preparation method of the dopamine derivative in the present invention is described in detail below.

[0047] The present invention mixes dopamine, Boc 2 O, a first base reagent, a first organic solvent and water, and carries out an amino protection reaction to obtain an amino protection intermediate. In the present invention, the first base reagent is preferably an alkali metal bicarbonate, and the alkali metal bicarbonate is preferably NaHCO 3 . In the present invention, the mass ratio of dopamine, Boc 2 O to the first base reagent is preferably 50:55-60:53-57, more preferably 50:57.5:55.4. In the present invention, the first organic solvent is preferably an organic solvent miscible with water, and the first organic solvent is more preferably tetrahydrofuran or 1,4-dioxane; the volume ratio of the first organic solvent to water is preferably 2-3:1, more preferably 2.5:1; the mass of dopamine to the total volume of the first organic solvent and water is preferably 50 g:300-400 mL, more preferably 50 g:350 mL.

[0048] In the present invention, dopamine is preferably dissolved in a mixture of a first organic solvent and water, and then a first base reagent and Boc 2 O are added to carry out the amino protection reaction. In the present invention, the temperature of the amino protection reaction is preferably 10-20 °C, more preferably 15 °C; the time is preferably 10-15 h, more preferably 12 h; the progress of the reaction is specifically monitored by TLC in the present invention. After the amino protection reaction is completed, the present invention preferably adds H 2 O to the obtained product system, then extracts with EtOA, separates the organic phase, removes the solvent by rotary evaporation to obtain a crude product, and then separates by column chromatography to obtain the amino protection intermediate. In the present invention, the eluent used for column chromatography separation is preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 20:1-10:1.

[0049] After the amino protection intermediate, the present invention mixes the amino protection intermediate, propargyl halide, a second base reagent and a second organic solvent to carry out a substitution reaction to obtain a substitution intermediate. In the present invention, the propargyl halide is preferably propargyl bromide (i.e., 3-bromo-1-propyne); the second base reagent is preferably an alkali metal carbonate, and the alkali metal carbonate is preferably K 2 CO 3 . In the present invention, the mass ratio of the amino protection intermediate, propargyl halide and the second base reagent is preferably 30:15-20:20-30, more preferably 30:17.6:24.6. In the present invention, the second organic solvent is preferably N,N-dimethylformamide; the dosage ratio of the amino protection intermediate to the second organic solvent is preferably 30 g:250-350 mL, more preferably 30 g:300 mL.

[0050] In the present invention, the amino protection intermediate is preferably dissolved in a second organic solvent, and then a second base reagent and propargyl halide are added to carry out the substitution reaction. In the present invention, the temperature of the substitution reaction is preferably 15-25 °C, more preferably 20 °C; the time is preferably 10-15 h, more preferably 12 h; the progress of the reaction is specifically monitored by TLC in the present invention; the substitution reaction is preferably carried out under stirring conditions. After the substitution reaction is completed, the present invention preferably adds H 2O, adjust the pH value of the obtained mixed material to be acidic (the pH value is preferably 6.8), then extract with EtOA, separate the organic phase, and remove the solvent by rotary evaporation to obtain a crude product. The crude product is first separated by column chromatography and then further separated by preparative HPLC to obtain the substituted intermediate. In the present invention, the reagent used to adjust the pH value of the mixed material is preferably hydrochloric acid, and the concentration of the hydrochloric acid is preferably 0.5 - 1.5 mol / L, more preferably 1 mol / L. In the present invention, the eluent used for the column chromatography separation is preferably petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is preferably 5:1 - 0:1. In the present invention, the conditions for the further separation by preparative HPLC include: the chromatographic column used is preferably Xtimate C18, the size is preferably 250 mm × 80 mm, and the filler particle size is preferably 10 μm; the mobile phase includes mobile phase A and mobile phase B, and mobile phase A is preferably an aqueous solution of NH 4 HCO 3 with a concentration of 10 mM, and mobile phase B is preferably acetonitrile; gradient elution is carried out, and from 0 to 25 min, the volume fraction of mobile phase B is uniformly increased from 15% to 45%.

[0051] After obtaining the substituted intermediate, the present invention conducts a deprotection reaction on the substituted intermediate to obtain a dopamine derivative having the structure shown in Formula I. In the present invention, the deprotection reaction is preferably carried out in the presence of an acid reagent, and the acid reagent is preferably hydrochloric acid, and the concentration of the hydrochloric acid is preferably 0.5 - 1.5 mol / L, more preferably 1 mol / L; the dosage ratio of the substituted intermediate to the acid reagent is preferably 3.5 g:200 - 300 mL, more preferably 3.5 g:240 mL.

[0052] The present invention preferably dissolves the substituted intermediate in the acid reagent for the deprotection reaction. In the present invention, the temperature of the deprotection reaction is preferably 20 - 30 °C, more preferably 25 °C; the time is preferably 30 - 40 h, more preferably 36 h; the present invention specifically monitors the reaction process by TLC; the deprotection reaction is preferably carried out under stirring conditions. After the deprotection reaction is completed, the present invention preferably adds H 2 O to the obtained product system, then extracts with EtOA, separates the organic phase, removes the solvent by rotary evaporation to obtain a crude product, and then separates by column chromatography to obtain a dopamine derivative having the structure shown in Formula I. In the present invention, the eluent used for the column chromatography separation is preferably dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is preferably 20:1 - 10:1.

[0053] The present invention provides the use of the dopamine derivative described in the above technical solution in the preparation of a preparation for enriching dopamine-modified protein substrates, a preparation for identifying dopamine-modified protein substrates, or a preparation for identifying the modification sites of dopamine-modified protein substrates, wherein the dopamine-modified protein substrate is obtained by modifying a protein with the dopamine derivative. The present invention has no special limitation on the protein, and any protein well-known to those skilled in the art can be used; in the examples of the present invention, histone H3, histone H4, aromatic amino acid decarboxylase (DDC), acyl-modifying enzyme (SIRT2), and heat shock protein (HSPA8) are specifically used as examples to verify the enrichment effect of the dopamine derivative described in the present invention on dopamine-modified protein substrates, the identification effect on dopamine-modified protein substrates, and the identification effect on the modification sites of dopamine-modified protein substrates. In the present invention, the modification is preferably carried out in vivo or in vitro; the organism preferably includes cells, tissues, or animals. In the present invention, the modification is preferably carried out in the presence of transglutaminase (specifically transglutaminase 2, TGM2); the present invention has no special limitation on the specific operating conditions of the modification, and any method well-known to those skilled in the art can be used.

[0054] The present invention provides a conjugate compound, including a matrix substance, a dopamine derivative, and a functional substance, wherein the matrix substance, the dopamine derivative, and the functional substance are covalently connected in sequence;

[0055] The matrix substance includes a protein or a polypeptide, the dopamine derivative is the dopamine derivative described in the above technical solution, and the functional substance includes biotin containing an azide group or a fluorescent probe containing an azide group;

[0056] Among them, the matrix substance and the dopamine derivative are connected by an amide bond, and the dopamine derivative and the functional substance are connected by a group formed by the click chemical reaction of an alkyne group and an azide group.

[0057] In the present invention, the structural formula of the conjugate compound is as shown in Formula III:

[0058]

[0059] R in Formula III 1 can specifically be provided by a protein or a polypeptide, and the R 2 can specifically be provided by biotin or a fluorescent probe (the alkyne group of the dopamine derivative and the azide group of the functional substance form a nitrogen heterocycle through a click chemical reaction, thereby realizing the covalent connection between the dopamine derivative and the functional substance).

[0060] In the present invention, the functional substance includes biotin containing an azide group or a fluorescent probe containing an azide group; in the embodiments of the present invention, specifically, biotin containing an azide group (azide-biotin) and TAMRA fluorescent dye are taken as examples for illustration.

[0061] The present invention provides a method for preparing the coupling compound described in the above technical solution, comprising the following steps:

[0062] Mix a matrix substance, a dopamine derivative and a first solvent, and carry out an amidation reaction to obtain an intermediate;

[0063] Mix the intermediate, the functional substance, a copper catalyst, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, tris(2-carboxyethyl)phosphine hydrochloride and a second solvent, and carry out a click chemical reaction to obtain the coupling compound.

[0064] In the present invention, a matrix substance, a dopamine derivative and a first solvent are mixed, and an amidation reaction is carried out to obtain an intermediate. The present invention has no special limitation on the type of the first solvent, and a solvent well-known to those skilled in the art can be used as long as it can ensure the smooth progress of the amidation reaction. In the present invention, during the process of carrying out the amidation reaction, the working concentration of the dopamine derivative is preferably 150-250 μM, more preferably 200 μM. In the present invention, the temperature of the amidation reaction is preferably 15-25 °C, more preferably 20-25 °C; the time is preferably 1-3 h, more preferably 2 h.

[0065] After obtaining the intermediate, in the present invention, the intermediate, the functional substance, a copper catalyst, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and a second solvent are mixed, and a click chemical reaction is carried out to obtain the coupling compound. The present invention has no special limitation on the type of the second solvent, and a solvent well-known to those skilled in the art can be used as long as it can ensure the smooth progress of the click chemical reaction. In the present invention, the copper catalyst preferably includes CuSO 4 . In the present invention, during the process of carrying out the click chemical reaction, the working concentration of the functional substance is preferably 50-150 μM, more preferably 100 μM; the working concentration of the copper catalyst is preferably 0.5-1.5 mM, more preferably 1 mM; the working concentration of TBTA is preferably 50-150 μM, more preferably 100 μM; the working concentration of TCEP is preferably 0.5-1.5 mM, more preferably 1 mM. In the present invention, the temperature of the click chemical reaction is preferably 15-25 °C, more preferably 20-25 °C; the time is preferably 0.5-1.5 h, more preferably 0.5-1 h.

[0066] In an embodiment of the present invention, taking the neuroblastoma cell SK-N-SH as an example, when the functional substance is biotin containing an azide group, specifically, the neuroblastoma cell SK-N-SH is treated with a dopamine derivative at a working concentration of 200 μM at room temperature for 2 h, and then the cells are digested with trypsin. After washing with PBS buffer solution, the cell precipitate is collected. The cell precipitate is lysed with PBS buffer solution containing 0.1% (volume fraction) Triton, and the obtained lysate is centrifuged at 14,000×g for 30 min. The protein concentration of the obtained supernatant is measured by the BCA method and diluted to 2 mg / mL; 2 mL of the obtained total protein lysate is mixed with CuSO 4 , TBTA, azide-biotin compound, and TCEP, wherein the concentration of CuSO 4 is 1 mM, the concentration of TBTA is 100 μM, the concentration of the azide-biotin compound is 100 μM, and the concentration of TCEP is 1 mM. Then, it is incubated at room temperature for 1 h to obtain the conjugate compound.

[0067] In an embodiment of the present invention, taking the neuroblastoma cell SK-N-SH as an example, when the functional substance is TAMRA fluorescent dye, specifically, the neuroblastoma cell SK-N-SH is plated on a cell culture slide and incubated overnight at 37 °C; it is treated with a dopamine derivative at a working concentration of 200 μM at room temperature for 2 h, and then freshly prepared 4 wt% formaldehyde is added for fixation for 15 min. Then, it is blocked with 1 wt% BSA solution for 1 h. The fixed cells are mixed with CuSO 4 , TBTA, TAMRA fluorescent dye, and TCEP, wherein the concentration of CuSO 4 is 1 mM, the concentration of TBTA is 100 μM, the concentration of TAMRA fluorescent dye is 100 μM, and the concentration of TCEP is 1 mM. Then, it is incubated at room temperature for 0.5 h to obtain the conjugate compound.

[0068] The present invention provides the use of the conjugate compound described in the above technical solution in the preparation of a preparation for enriching dopamine-modified protein substrates, a preparation for identifying dopamine-modified protein substrates, or a preparation for identifying the modification sites of dopamine-modified protein substrates. The dopamine derivative of the present invention contains an alkynyl group, and through the alkynyl group, it is convenient to introduce a functional compound (such as biotin or a fluorescent probe) that can specifically enrich dopamine-modified protein substrates through a click reaction. Then, combined with a mass spectrometry method, dopamine-modified protein substrates and modification sites can be identified.

[0069] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0070] Example 1

[0071] According to Figure 1 the shown flow chart, the dopamine-modified probe pDA-1 was prepared as follows:

[0072] (1) Synthesis of Compound 2

[0073] Dissolve 50 g of Compound 1 (dopamine) in a mixed solvent of 350 mL of tetrahydrofuran (THF) and H 2 O (the volume ratio of THF to H 2 O is 2.5:1), then add 55.4 g of NaHCO 3 and 57.5 g of Boc 2 O, react at 15 °C for 12 h, and TLC shows that the reaction is complete; add 100 mL of H 2 O to the obtained product system, then extract with EtOA, separate the organic phase, rotary evaporate to remove the solvent to obtain the crude product, and then separate by column chromatography (the eluent used is dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 20:1, 15:1, 10:1 in sequence) to obtain 62 g of Compound 2.

[0074] 1H NMR data of Compound 2: 1 H NMR (400 MHz, DMSO-d 6 ) δ 8.53 - 8.80 (2H, m) 6.82 (1H, brt, J = 5.30 Hz) 6.62 (1H, d, J = 7.87 Hz) 6.55 (1H, d, J = 1.91 Hz) 6.41 (1H, dd, J = 7.99, 1.79 Hz) 2.92 - 3.09 (2H, m) 2.47 (2H, brs) 1.37 (9H, s).

[0075] (2) Synthesis of Compound 3

[0076] Dissolve 30 g of Compound 2 in 300 mL of N,N-dimethylformamide (DMF), then add 24.6 g of K 2 CO 3 and 17.6 g of propargyl bromide, stir and react at 20 °C for 12 h, and TLC shows that the reaction is complete; add 50 mL of H 2O, then adjust the pH value to 6.8 with 1 M hydrochloric acid, and then extract with EtOA. Separate the organic phase and rotary evaporate to remove the solvent to obtain the crude product. The crude product is first separated by column chromatography (the eluent used is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 5:1, 2:1, 0:1 in turn), and then separated by preparative HPLC for the second time (the chromatographic column used is Xtimate C18, with a size of 250 mm × 80 mm and a packing particle size of 10 μm; the mobile phase includes mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of 10 mM NH 4 HCO 3 , and mobile phase B is acetonitrile; gradient elution, from 0 to 25 min, the volume fraction of mobile phase B is increased from 15% to 45% at a constant speed), to obtain Compound 3.

[0077] 1H NMR data of Compound 3: 1 1H NMR (400 MHz, DMSO-d 6 ) δ 9.00 (s, 1H), 6.75 - 6.90 (m, 2H), 6.58 - 6.66 (m, 1H), 6.46 - 6.57 (m, 1H), 4.68 (d, J = 2.20 Hz, 2H), 3.43 - 3.53 (m, 1H), 2.94 - 3.10 (m, 2H), 2.49 - 2.55 (m, 2H), 1.35 (s, 9H).

[0078] (3) Synthesis of dopamine-modified probe pDA-1

[0079] Dissolve 3.5 g of Compound 3 in 240 mL of 1 M hydrochloric acid, stir and react at 25 °C for 36 h. TLC shows that the reaction is complete; add 50 mL of H 2 O to the resulting product system, then extract with EtOA, separate the organic phase, rotary evaporate to remove the solvent to obtain the crude product, and then separate by column chromatography (the eluent used is dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 20:1, 15:1, 10:1 in turn) to obtain dopamine-modified probe pDA-1 (yield: 44.5%).

[0080] 1H NMR data of dopamine-modified probe pDA-1: 1 1H NMR (400 MHz, MeOH-d 4 ) δ 7.01 (d, J = 8.16 Hz, 1H), 6.76 (d, J = 2.20 Hz, 1H), 6.70 (dd, J = 8.16, 2.21 Hz, 1H), 4.75 (d, J = 2.43 Hz, 2H), 3.08 - 3.16 (m, 2H), 2.93 (t, J = 2.43 Hz, 1H), 2.84 (t, J = 7.72 Hz, 2H).

[0081] Verification of the Activity of Probe pDA-1 in Test Example 1

[0082] 1. In vitro enzyme activity experiment

[0083] Mix the chemically synthesized polypeptide fragment H3 of histone H3 1-14 , probe pDA-1, and 9 μg of TGM2 with 25 μL of enzymatic digestion buffer (25 mM Tris-Cl, pH = 7.5, 5 mM CaCl 2 , 1 mM PMSF and 1 mM DTT), where the concentration of probe pDA-1 is 5 mM, and incubate in the dark at room temperature for 5 h; centrifuge the obtained product system at 4 °C for 20 min to remove the precipitate, and detect the supernatant by LC-MS.

[0084] Figure 2 is the LC-MS detection result graph. It can be seen from Figure 2 that probe pDA-1 can be covalently added to the known modified substrate histone H3.

[0085] 2. Cell experiment

[0086] Treat neuroblastoma cells SK-N-SH with 200 μM of probe pDA-1 and DA for 2 h respectively. Digest the cells with trypsin, collect the cell precipitate after washing with PBS buffer solution, lyse the cell precipitate with PBS buffer solution containing 0.1% (volume fraction) Triton, then centrifuge the obtained lysate at 14000×g for 30 min. Determine the protein concentration of the obtained supernatant protein by the BCA method and dilute it to 2 mg / mL; mix 1 mL of the obtained total protein lysate with CuSO 4 , TBTA, azide-biotin compound, and TCEP, where the concentration of CuSO 4 is 1 mM, the concentration of TBTA is 100 μM, the concentration of azide-biotin compound is 100 μM, and the concentration of TCEP is 1 mM. Then incubate at room temperature for 1 h. After the incubation, precipitate the obtained product system with pre-cooled methanol, centrifuge at 14000×g for 30 min, remove the supernatant, dissolve the precipitate with 1 mL of PBS buffer solution containing 1.2 wt% SDS, enrich the protein labeled with probe pDA-1 with magnetic beads containing streptavidin, and then elute with 2× loading buffer, and analyze the eluate by WB.

[0087] Figure 3 is the WB analysis result graph. It can be seen from Figure 3It can be seen that the endogenous histone H3, a known protein modification substrate in cells, can be significantly enriched by the probe pDA-1.

[0088] Test Example 2

[0089] 1. Cell or tissue imaging

[0090] Neuroblastoma cells SK-N-SH (2×10 5 ) were plated on cell culture slides and incubated overnight at 37 °C; treated with 200 μM of the probe pDA-1 for 2 h, then fixed with freshly prepared 4 wt% formaldehyde for 15 min, and then blocked with 1 wt% BSA solution for 1 h. The fixed cells were mixed with CuSO 4 , TBTA, TAMRA fluorescent dye, and TCEP, where the concentration of CuSO 4 was 1 mM, the concentration of TBTA was 100 μM, the concentration of TAMRA fluorescent dye was 100 μM, and the concentration of TCEP was 1 mM. After incubation at room temperature for 0.5 h, the reaction solution was removed, the cell nuclei were stained with DAPI, and then the slides were sealed. Confocal microscopy was used to capture cell imaging.

[0091] Figure 4 Figure for cell imaging detection results (scale bar is 100 μm). Specifically, after treating cells with dopamine (DA) and the probe pDA-1 respectively, the TAMRA fluorescent dye was introduced by click reaction. The dopamine-modified protein substrate emitted red fluorescence after being excited, and the cell nuclei were stained with DAPI. The results showed that dopamine modification existed in the whole SK-N-SH cells, indicating that the probe pDA-1 could penetrate the cell membrane and nuclear membrane and act on the whole cell, and at the same time proving that dopamine modification was distributed in all cells.

[0092] 2. Identification of potential dopamine-modified substrates

[0093] Neuroblastoma cells SK-N-SH were treated with 200 μM of the probe pDA-1 and cell culture medium for 2 h respectively. The cells were digested with trypsin, and the cell precipitate was collected after washing with PBS buffer solution. The cell precipitate was lysed with PBS buffer solution containing 0.1% (volume fraction) Triton, and the resulting lysate was centrifuged at 14,000×g for 30 min. The protein concentration of the obtained supernatant was determined by the BCA method and diluted to 2 mg / mL; 2 mL of the obtained total protein lysate was mixed with CuSO 4 , TBTA, azide-biotin, and TCEP, where the concentration of CuSO 4The concentration of [substance] is 1 mM, the concentration of TBTA is 100 μM, the concentration of the biotin compound containing azide is 100 μM, and the concentration of TCEP is 1 mM. Then, it is incubated at room temperature for 1 h. After the incubation, the obtained product system is precipitated with pre-cooled methanol, centrifuged at 14,000×g for 30 min. After removing the supernatant, the precipitate is dissolved in 1 mL of PBS buffer solution containing 1.2 wt% SDS. The protein labeled with probe pDA-1 is enriched with magnetic beads containing streptavidin. Subsequently, alkylation and trypsin digestion are performed based on on-beads, and then proteomic analysis is carried out and compared with the Uniprot protein library.

[0094] Figure 5 It is a schematic diagram for the identification of dopamine-modified protein substrates. The enrichment effect of histones by probe pDA-1 is shown in Table 1 in detail. As can be seen from Table 1, compared with the negative control group, after treatment with probe pDA-1, in two out of three independent experiments, the proteins with an enrichment ratio ≥1.5-fold are potential dopamine-modified substrates. This indicates that in addition to the known modified substrate histone H3 that can be enriched by probe pDA-1, histone H4, H1.4, DDC, SIRT2, and HSPA8 are also potential dopamine-modified protein substrates.

[0095] Table 1 Enrichment effect of histones by probe pDA-1

[0096]

[0097] 3. Verification of modified substrates

[0098] Using the above enrichment strategy, the present invention identified a total of 4,133 possible dopamine-modified protein substrates, including histone H4, aromatic amino acid decarboxylase (DDC), and acylation demodifying enzyme (SIRT2). Since the expression levels of DDC and SIRT2 in neuroblastoma cell line SK-N-SH are relatively low, the present invention respectively constructed cell lines overexpressing DDC and SIRT2. Subsequently, the wild-type, DDC-overexpressing, and SIRT2-overexpressing cell lines were treated with probe pDA-1 and DA respectively, and the strategies in the existing literature (Fulton, S.L. et al. Histone H3 dopaminylation in ventral tegmental area underlies heroin-induced transcriptional and behavioral plasticity in male rats. Neuropsychopharmacology 47, 1776 - 783, 2022) were used to verify whether histone H4, DDC, and SIRT2 are dopamine-modified substrates.

[0099] Figure 6 Verification results of dopamine-modified substrates DDC, SIRT2, and histone H4. As can be seen from Figure 6 it, histone H4, DDC, and SIRT2 are protein substrates modified by dopamine.

[0100] Test Example 3 Identification of potential novel dopamine modification sites

[0101] Neuroblastoma cells SK-N-SH were treated with 200 μM of probe pDA-1 and cell culture medium for 2 h, the cells were digested with trypsin, and the cell precipitate was collected after washing with PBS buffer solution. The cell precipitate was lysed with PBS buffer solution containing 0.1% (volume fraction) Triton, and the resulting lysate was centrifuged at 14,000×g for 30 min. The protein concentration of the resulting supernatant was determined by the BCA method and diluted to 2 mg / mL; 2 mL of the resulting total protein lysate was mixed with CuSO 4 , TBTA, azide-biotin, and TCEP, where the concentration of CuSO 4 was 1 mM, the concentration of TBTA was 100 μM, the concentration of azide-biotin was 100 μM, and the concentration of TCEP was 1 mM. After incubation at room temperature for 1 h, the resulting product system was precipitated with pre-cooled methanol and centrifuged at 14,000×g for 30 min. After removing the supernatant, the precipitate was dissolved in 1 mL of PBS buffer solution containing 1.2 wt% SDS. The protein labeled with probe pDA-1 was enriched with magnetic beads containing streptavidin, followed by on-beads enzymatic digestion, washing with PBS buffer solution, removing the peptide segments not modified by the probe, and the peptide segments modified by the probe could be released with acetic acid.

[0102] Figure 7 Schematic diagram for the identification of dopamine modification sites. Table 2 shows the corresponding data of the secondary mass spectrometry of the N363 modification of heat shock protein HSAP8. The results show that in addition to the known modification site Q, there is a new modification site asparagine at position 363 of protein HSPA8, that is, N363 (new modification site) on protein HSPA8 can undergo dopamine modification.

[0103] Table 2 Corresponding data of the secondary mass spectrometry of the N363 modification of heat shock protein HSAP8

[0104] Ion type Measured molecular weight Ion type Measured molecular weight <![CDATA[b 2+ > 201.12512 <![CDATA[y 8+ > 774.44348 <![CDATA[b 5+ > 826.39734 <![CDATA[y 9+ -H 2 O]]> 884.48102 <![CDATA[b 5+ -H 2 O]]> 808.40021 <![CDATA[y 9+ -NH 3 > 885.48541 <![CDATA[y 3+ -H 2 O]]> 301.14093 <![CDATA[y 9+ > 902.50110 <![CDATA[y 3+ > 319.16364 <![CDATA[y 10+ > 1001.56671 <![CDATA[y 4+ > 406.19626 <![CDATA[y 11+ > 1072.60547 <![CDATA[y 5+ > 519.28113 <![CDATA[y 13+ > 1200.66516 <![CDATA[y 6+ > 632.36725 <![CDATA[y 14 > 1363.73193 <![CDATA[y 7+ > 703.40204 <![CDATA[y 15+ > 1434.74487

[0105] Test Example 4

[0106] 1. Identification of histone H4 modification sites

[0107] Dissolve the chemically synthesized histone H4 peptide containing Q27, 5 mM of the probe pDA-1, and 9 μg of TGM2 in 25 μL of enzymatic digestion buffer (25 mM Tris-Cl, pH = 7.5, 5 mM CaCl 2 , 1 mM PMSF and 1 mM DTT), and incubate in the dark at room temperature for 5 h; centrifuge the resulting product system at 4 °C for 20 min to remove the precipitate, and detect the supernatant by LC-MS.

[0108] Figure 8 This is the second-order mass spectrometry diagram of dopamine modification of histone H4 at position 27 in vitro. The results show that the probe pDA-1 can be covalently added to histone H4, that is, glutamine (Q27) at position 27 of histone H4 can undergo dopamine modification.

[0109] 2. Functional study of histone H4 Q27 dop modification

[0110] In the present invention, cells overexpressing histone H4 and with Q27 mutated to alanine A were used, and then these two cell lines were treated with dopamine respectively.

[0111] Figure 9 This is the result diagram of dopamine modification (H4Q27dop) on histone H4 inhibiting cell proliferation. Figure 9 In a, it is the result diagram of SK-N-SH cells treated with different concentrations of dopamine for 48 h. It can be seen that dopamine can inhibit cell proliferation and shows a dose dependence with the concentration; the data presentation method is mean ± standard deviation (n = 3), * indicates p < 0.05, *** indicates p < 0.001, **** indicates p < 0.0001, ns = not significant, t-test. Figure 9 In b, it is the result diagram after dopamine treatment of H4WT and H4Q27A mutant cell lines respectively. It can be seen that the cell proliferation phenotype caused by dopamine is inhibited; the data presentation method is mean ± standard deviation (n = 3), * indicates p < 0.05, *** indicates p < 0.001. Figure 9 In c, it is the specific verification diagram of the H4Q27dop antibody. The dotplot shows that the H4Q27dop modification antibody can recognize the peptide containing H4Q27dop modification, but does not recognize the H3Q5dop dopamine modification and unmodified peptides. Figure 9 In d, it is the diagram of the change of H4Q27dop modification level with dopamine concentration. It can be seen that the mutation of Q27A causes a significant decrease in the H4Q27dop modification level. The data presentation method is mean ± standard deviation (n = 3), ** indicates p < 0.05, ns = not significant, t-test.

[0112] The above cell proliferation experiments confirmed that the cell proliferation induced by dopamine was inhibited by the mutation of Q27A. This indicates that the dopamine modification at histone site 27 can inhibit cell proliferation.

[0113] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A use of a dopamine derivative or a coupled compound in the preparation of a preparation for enriching a dopamine-modified protein substrate, a preparation for identifying a dopamine-modified protein substrate, or a preparation for identifying a modification site of a dopamine-modified protein substrate, wherein the dopamine-modified protein substrate is obtained by modifying a protein with the dopamine derivative, and the protein is histone H4, aromatic amino acid decarboxylase, acylation demodification enzyme, or heat shock protein; The dopamine derivative has a structure shown in Formula I: The coupling compound comprises a matrix substance, a dopamine derivative and a functional substance, wherein the matrix substance, the dopamine derivative and the functional substance are covalently connected in sequence; the matrix substance comprises a protein or a polypeptide, the dopamine derivative is a dopamine derivative having a structure shown in Formula I, and the functional substance comprises biotin containing an azide group or a fluorescent probe containing an azide group; The matrix substance is connected to the dopamine derivative via an amide bond, and the dopamine derivative is connected to the functional substance via a group formed by a click chemical reaction between an alkynyl group and an azide group.

2. The use according to claim 1, characterized in that: The preparation method of the dopamine derivative comprises the following steps: dopamine, Boc2O, a first base reagent, a first organic solvent and water are mixed to perform an amino protection reaction to obtain an amino protected intermediate; The amino-protected intermediate, the halogenated propyne, the second base reagent and the second organic solvent are mixed to carry out a substitution reaction to obtain a substituted intermediate; The substituted intermediate is subjected to a deprotection reaction to obtain a dopamine derivative having a structure shown in Formula I; The structural formulas of the amino-protected intermediate and the substituted intermediate are shown below:

3. The use according to claim 2, characterized in that: The temperature of the amino protection reaction is 10-20°C and the time is 10-15h; The temperature of the substitution reaction is 15-25°C and the time is 10-15h; The deprotection reaction is carried out in the presence of an acid reagent; the temperature of the deprotection reaction is 20 to 30° C. and the time is 30 to 40 hours.

4. The use according to claim 1, characterized in that: The modification is carried out in vivo or in vitro; the modification is carried out in the presence of transglutaminase.

5. The use according to claim 4, characterized in that: The organism includes cells, tissues or animals.

6. The use according to claim 1, characterized in that: The preparation method of the coupling compound comprises the following steps: The base material, the dopamine derivative and the first solvent are mixed to carry out an amidation reaction to obtain an intermediate; The intermediate, the functional substance, the copper catalyst, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, tris(2-carboxyethyl)phosphine hydrochloride and the second solvent are mixed to perform a click chemistry reaction to obtain the coupling compound.

7. The use according to claim 6, characterized in that: The amidation reaction temperature is 15-25°C and the time is 1-3h; The temperature of the click chemistry reaction is 15-25° C. and the reaction time is 0.5-1.5 h.

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