Dopamine derivative, coupling compound and preparation method and application thereof

Abstract

The invention provides a dopamine derivative, a coupling compound and a preparation method and application thereof, and belongs to the technical field of chemical biology. The dopamine derivative provided by the invention has a structure as shown in a formula I. The dopamine derivative as a small molecule probe can specifically enrich a dopamine modified protein substrate, and can identify the dopamine modified protein substrate and a modification site without depending on a modified antibody and combining a mass spectrometry method; the method is particularly suitable for identifying protein substrates and modification sites capable of being subjected to dopamine modification in organisms such as any type of cells, tissues and animals, lays a foundation for exploring biological functions of dopamine modification, and is beneficial to analyzing pathogenesis of diseases and researching and developing drugs for regulating and controlling dopamine modification levels related to the diseases. # imgabs0 #

Description

Technical Field

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

[0002] Dopamine modification (dopmainylation) refers to the covalent addition of dopamine as a modifier to the amino acid glutamine (Q) residue of a protein, catalyzed by the enzyme transglutaminase (TGM). Dopamine modification was discovered as early as 60 years ago, but to date, only a limited number of dopamine-modified protein substrates have been identified. This is because the level of post-translational modification of proteins is generally low. Therefore, identification of dopamine-modified protein substrates generally requires enrichment with dopamine-modifying antibodies or chemical biological methods. Currently, no probes that specifically enrich dopamine-modified protein substrates have been reported. Summary of the Invention

[0003] The object of the present invention is to provide a dopamine derivative, a coupled compound, and a preparation method and application thereof. The dopamine derivative provided by the present invention contains an alkynyl group and does not require reliance on dopamine-modified antibodies. The alkynyl group facilitates the introduction of functional compounds (such as biotin) that can specifically enrich dopamine-modified protein substrates. Subsequently, combined with mass spectrometry methods, dopamine-modified protein substrates and modification sites can be identified.

[0004] In order to achieve the above-mentioned object of the invention, 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 method for preparing the dopamine derivatives described in the above technical solution, comprising the following steps:

[0008] mixing dopamine, Boc2O, a first base reagent, a first organic solvent and water to perform an amino protection reaction to obtain an amino protected intermediate;

[0009] Mixing the amino-protected intermediate, halopropyne, a second base reagent and a second organic solvent to carry out a substitution reaction to obtain a substituted intermediate;

[0010] The substituted intermediate is subjected to a deprotection reaction to obtain a dopamine derivative having a structure shown in Formula I;

[0011] The structural formulas of the amino-protected intermediate and the substituted intermediate are shown below:

[0012]

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

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

[0015] 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.

[0016] The present invention provides the use of the dopamine derivative described in the above technical solution in preparing 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.

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

[0018] Preferably, the organism comprises a cell, a tissue or an animal.

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

[0020] The matrix material 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;

[0021] 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 alkyne group and an azide group.

[0022] The present invention provides a method for preparing the coupling compound described in the above technical solution, comprising the following steps:

[0023] mixing a base substance, a dopamine derivative and a first solvent, and performing an amidation reaction to obtain an intermediate;

[0024] 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 a second solvent are mixed to perform a click chemistry reaction to obtain the coupling compound.

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

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

[0027] The present invention provides the use of the coupling compound described in the above technical solution in preparing 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.

[0028] The present invention provides a dopamine derivative having a 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, eliminating the need to rely on dopamine-modifying antibodies to enrich dopamine-modified protein substrates. Combined with mass spectrometry, the dopamine-modified protein substrates and modification sites can be identified. The dopamine derivative is particularly suitable for identifying protein substrates and modification sites that can undergo dopamine modification in any type of cell or tissue, laying the foundation for preliminary exploration of the biological functions of dopamine modification, and contributing to the analysis of the pathogenesis of diseases related to dopamine modification and the development of drugs that intervene in dopamine modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart for preparing the dopamine modified probe pDA-1 in an embodiment of the present invention;

[0030] Figure 2 This is the LC-MS test result diagram in Test Example 1;

[0031] Figure 3 This is the WB analysis result diagram in test case 1;

[0032] Figure 4 This is the cell imaging test result diagram in Test Example 2;

[0033] Figure 5 Schematic diagram of identification of dopamine-modified protein substrates in Test Example 2;

[0034] Figure 6 This is a diagram showing the verification results of the dopamine-modified substrates DDC, SIRT2, and histone H4 in Test Example 2;

[0035] Figure 7 Schematic diagram of identification of dopamine modification sites in Test Example 3;

[0036] Figure 8 This is the secondary mass spectrum of dopamine modification of histone H427 in vitro in Test Example 4;

[0037] Figure 9This is a graph showing the biological function of dopamine modification (H4Q27dop) on histone H4 in Test Example 4. DETAILED DESCRIPTION

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

[0039]

[0040] The present invention provides a method for preparing the dopamine derivatives described in the above technical solution, comprising the following steps:

[0041] mixing dopamine, Boc2O, a first base reagent, a first organic solvent and water to perform an amino protection reaction to obtain an amino protected intermediate;

[0042] Mixing the amino-protected intermediate, halopropyne, a second base reagent and a second organic solvent to carry out a substitution reaction to obtain a substituted intermediate;

[0043] The substituted intermediate is subjected to a deprotection reaction to obtain a dopamine derivative having a structure shown in Formula I;

[0044] The structural formulas of the amino-protected intermediate and the substituted intermediate are shown below:

[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 using methods well known to those skilled in the art. The preparation method of the dopamine derivatives of the present invention is described in detail below.

[0047] The present invention mixes dopamine, Boc2O, a first base reagent, a first organic solvent and water to carry out an amino protection reaction to obtain an amino protected intermediate. In the present invention, the first base reagent is preferably an alkali metal bicarbonate, and the alkali metal bicarbonate is preferably NaHCO3. In the present invention, the mass ratio of dopamine, Boc2O and the first base reagent is preferably 50:55 to 60:53 to 57, and 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 to 3:1, and more preferably 2.5:1; the ratio of the mass of the dopamine to the total volume of the first organic solvent and water is preferably 50g:300 to 400mL, and more preferably 50g:350mL.

[0048] The present invention preferably dissolves dopamine in a mixture of a first organic solvent and water, and then adds a first alkaline reagent and Boc2O to carry out the amino protection reaction. In the present invention, the temperature of the amino protection reaction is preferably 10 to 20°C, more preferably 15°C; the time is preferably 10 to 15 hours, more preferably 12 hours; the present invention specifically monitors the reaction progress by TLC. After the amino protection reaction is completed, the present invention preferably adds H2O to the resulting product system, then extracts with EtOA, separates the organic phase, and removes the solvent by rotary evaporation to obtain a crude product, which is then separated by column chromatography to obtain the amino protection intermediate. 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 to 10:1.

[0049] After the amino-protected intermediate is obtained, the present invention mixes the amino-protected intermediate, the halopropyne, the second alkaline reagent and the second organic solvent to carry out a substitution reaction to obtain a substituted intermediate. In the present invention, the halopropyne is preferably bromopropyne (i.e., 3-bromo-1-propyne); the second alkaline reagent is preferably an alkali metal carbonate, and the alkali metal carbonate is preferably K2CO3. In the present invention, the mass ratio of the amino-protected intermediate, the halopropyne and the second alkaline reagent is preferably 30:15 to 20:20 to 30, more preferably 30:17.6:24.6. In the present invention, the second organic solvent is preferably N,N-dimethylformamide; the amount ratio of the amino-protected intermediate to the second organic solvent is preferably 30g:250 to 350mL, more preferably 30g:300mL.

[0050] The present invention preferably dissolves the amino-protected intermediate in a second organic solvent, and then adds a second base reagent and a halopropyne 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 hours, more preferably 12 hours; the present invention specifically monitors the reaction progress by TLC; the substitution reaction is preferably carried out under stirring. After the substitution reaction is completed, the present invention preferably adds H2O to the resulting product system to adjust the pH value of the resulting mixture to acidic (preferably pH 6.8), then extracts with EtOA, separates the organic phase, and removes the solvent by rotary evaporation to obtain a crude product, which is first separated by column chromatography and then separated by preparative HPLC to obtain the substituted intermediate. In the present invention, the reagent used to adjust the pH value of the mixture 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 preparative HPLC secondary separation include: the chromatographic column used is preferably Xtimate C18, the size is preferably 250mm×80mm, and the filler particle size is preferably 10μm; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is preferably an NH4HCO3 aqueous solution with a concentration of 10mM, and the mobile phase B is preferably acetonitrile; gradient elution, from 0 to 25min, the volume fraction of mobile phase B is uniformly increased from 15% to 45%.

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

[0052] The present invention preferably dissolves the substituted intermediate in an acid reagent to carry out a deprotection reaction. In the present invention, the temperature of the deprotection reaction is preferably 20 to 30°C, more preferably 25°C; the time is preferably 30 to 40h, more preferably 36h; the present invention specifically monitors the reaction progress by TLC; the deprotection reaction is preferably carried out under stirring. After the deprotection reaction is completed, the present invention preferably adds H2O to the obtained product system, then extracts with EtOA, separates the organic phase, and removes the solvent by rotary evaporation to obtain a crude product, which is then separated by column chromatography to obtain a dopamine derivative having a 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 to 10:1.

[0053] The present invention provides the use of the dopamine derivatives described in the above technical solution in the preparation of a preparation for enriching a dopamine-modified protein substrate, identifying a preparation for a dopamine-modified protein substrate, or identifying a preparation for 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. The present invention is not particularly limited to the protein, and any protein well known to those skilled in the art can be used; in an embodiment of the present invention, histone H3, histone H4, aromatic amino acid decarboxylase (DDC), acylation demodification enzyme (SIRT2) and heat shock protein (HSPA8) are specifically used as examples to verify the enrichment effect of the dopamine derivatives of the present invention on dopamine-modified protein substrates, the identification effect on dopamine-modified protein substrates, and the identification effect on 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 restrictions on the specific operating conditions of the modification, and methods well known to those skilled in the art can be used.

[0054] The present invention provides a coupling compound, comprising a matrix substance, a dopamine derivative and a functional substance, wherein the matrix substance, the dopamine derivative and the functional substance are covalently linked in sequence;

[0055] The matrix material 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] 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 alkyne group and an azide group.

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

[0058]

[0059] R in Formula III 1 Specifically, it can be provided by a protein or a polypeptide. 2 Specifically, it can be provided by biotin or a fluorescent probe (the alkyne group of the dopamine derivative and the azide group of the functional substance undergo a knock-in chemical reaction to form a nitrogen heterocycle, thereby achieving 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, biotin containing an azide group (azide-biotin) and TAMRA fluorescent dye are specifically used as examples for description.

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

[0062] mixing a base substance, a dopamine derivative and a first solvent, and performing an amidation reaction to obtain an intermediate;

[0063] 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 a second solvent are mixed to perform a click chemistry reaction to obtain the coupling compound.

[0064] In the present invention, a substrate, a dopamine derivative, and a first solvent are mixed to perform an amidation reaction to obtain an intermediate. The present invention does not particularly limit the type of the first solvent; any solvent familiar to those skilled in the art can be used as long as it ensures a smooth amidation reaction. In the present invention, during the amidation reaction, the working concentration of the dopamine derivative is preferably 150 to 250 μM, more preferably 200 μM. In the present invention, the amidation reaction temperature is preferably 15 to 25°C, more preferably 20 to 25°C, and the reaction time is preferably 1 to 3 hours, more preferably 2 hours.

[0065] After obtaining the intermediate, the present invention mixes the intermediate, the functional substance, the copper catalyst, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) with a second solvent to perform a click chemistry reaction to obtain the coupling compound. The present invention does not specifically limit the type of the second solvent. A solvent of a type familiar to those skilled in the art can be used as long as the click chemistry reaction can proceed smoothly. In the present invention, the copper catalyst preferably includes CuSO4. In the present invention, during the click chemistry reaction, the working concentration of the functional substance is preferably 50 to 150 μM, more preferably 100 μM; the working concentration of the copper catalyst is preferably 0.5 to 1.5 mM, more preferably 1 mM; the working concentration of TBTA is preferably 50 to 150 μM, more preferably 100 μM; the working concentration of TCEP is preferably 0.5 to 1.5 mM, more preferably 1 mM. In the present invention, the temperature of the click chemistry reaction is preferably 15 to 25° C., more preferably 20 to 25° C.; the time is preferably 0.5 to 1.5 h, more preferably 0.5 to 1 h.

[0066] In an embodiment of the present invention, taking neuroblastoma cells SK-N-SH as an example, when the functional substance is biotin containing an azide group, the neuroblastoma cells SK-N-SH are treated with a dopamine derivative at a working concentration of 200 μM at room temperature for 2 hours, and then the cells are digested with trypsin, washed with PBS buffer solution, and the cell pellet is collected. The cell pellet is lysed with PBS buffer solution containing 0.1% (volume fraction) Triton, and the resulting lysate is centrifuged at 14,000 × g for 30 minutes. The protein concentration of the obtained supernatant is determined by the BCA method and diluted to 2 mg / mL; 2 mL of the resulting total protein lysate is mixed with CuSO4, TBTA, an azide-biotin compound (azide-biotin) and TCEP, wherein the concentration of CuSO4 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, and then incubated at room temperature for 1 hour to obtain the coupling compound.

[0067] In an embodiment of the present invention, taking neuroblastoma cells SK-N-SH as an example, when the functional substance is TAMRA fluorescent dye, specifically, the neuroblastoma cells SK-N-SH are plated on a cell culture sheet and incubated at 37°C overnight; treated with a dopamine derivative at a working concentration of 200 μM at room temperature for 2 hours, and then added with freshly prepared 4wt% formaldehyde for fixation for 15 minutes, and then blocked with 1wt% BSA solution for 1 hour, and the fixed cells are mixed with CuSO4, TBTA, TAMRA fluorescent dye and TCEP, wherein the concentration of CuSO4 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, and then incubated at room temperature for 0.5 hours to obtain the coupling compound.

[0068] The present invention provides the use of the coupling compound described in the above technical solution 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 modification sites on a dopamine-modified protein substrate. The dopamine derivative described in the present invention contains an alkynyl group, which facilitates the introduction of a functional compound (such as biotin or a fluorescent probe) that can specifically enrich a dopamine-modified protein substrate through a click reaction. Subsequently, combined with mass spectrometry, the dopamine-modified protein substrate and modification site can be identified.

[0069] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0070] Example 1

[0071] according to Figure 1 The dopamine modified probe pDA-1 was prepared as shown in the flowchart. The steps are as follows:

[0072] (1) Synthesis of Compound 2

[0073] 50 g of compound 1 (dopamine) was dissolved in 350 mL of a mixed solvent of tetrahydrofuran (THF) and H2O (the volume ratio of THF to H2O was 2.5:1), followed by the addition of 55.4 g of NaHCO3 and 57.5 g of Boc2O. The mixture was reacted at 15°C for 12 h, and TLC showed that the reaction was complete. 100 mL of H2O was added to the resulting product system, followed by extraction with EtOA, the organic phase was separated, and the solvent was removed by rotary evaporation to obtain a crude product, which was then separated by column chromatography (the eluents used were dichloromethane and methanol, wherein the volume ratios of dichloromethane to methanol were 20:1, 15:1, and 10:1, respectively), to obtain 62 g of compound 2.

[0074] H NMR spectrum data of compound 2: 1 H NMR (400MHz, DMSO-d6) δ8.53-8.80 (2H, m) 6.82 (1H, brt, J = 5.30Hz) 6.62 (1H, d, J = 7.87Hz) 6.5 5(1H,d,J=1.91Hz)6.41(1H,dd,J=7.99,1.79Hz)2.92-3.09(2H,m)2.47(2H,brs)1.37(9H,s).

[0075] (2) Synthesis of compound 3

[0076] 30 g of compound 2 was dissolved in 300 mL of N, N-dimethylformamide (DMF), and then 24.6 g of K2CO3 and 17.6 g of propargyl bromide were added. The reaction was stirred at 20 ° C for 12 h. TLC showed that the reaction was complete. 50 mL of H2O was added to the resulting product system, and then the pH value was adjusted to 6.8 with 1 M hydrochloric acid. After that, it was extracted with EtOA, the organic phase was separated, and the solvent was removed by rotary evaporation to obtain a crude product. The crude product was first separated by column chromatography (the eluents used were petroleum ether and ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate was 5:1, 2:1, and 0:1, respectively), and then separated by preparative HPLC for secondary separation (the chromatographic column used was Xtimate C18, size 250mm×80mm, filler particle size 10μm; mobile phase includes mobile phase A and mobile phase B, mobile phase A is 10mM NH4HCO3 aqueous solution, and mobile phase B is acetonitrile; gradient elution, from 0 to 25min, the volume fraction of mobile phase B increases uniformly from 15% to 45%), to obtain compound 3.

[0077] H-NMR spectrum data of compound 3: 1HNMR(400MHz,DMSO-d6)δ9.00(s,1H),6.75-6.90(m,2H),6.58-6.66(m,1H),6.46-6.57(m,1H) ,4.68(d,J=2.20Hz,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] 3.5 g of compound 3 was dissolved in 240 mL of 1 M hydrochloric acid and stirred at 25°C for 36 h. TLC indicated the reaction was complete. 50 mL of H2O was added to the resulting product system, followed by extraction with EtOA. The organic phase was separated and the solvent was removed by rotary evaporation to obtain a crude product, which was then separated by column chromatography (eluents: dichloromethane and methanol, with the volume ratios of dichloromethane to methanol being 20:1, 15:1, and 10:1, respectively) to obtain the dopamine-modified probe pDA-1 (yield 44.5%).

[0080] Dopamine modified probe pDA-1 H NMR spectrum data: 1 HNMR(400MHz,MeOH-d4)δ7.01(d,J=8.16Hz,1H),6.76(d,J=2.20Hz,1H),6.70(dd,J=8.16,2.21H z, 1H), 4.75 (d, J = 2.43Hz, 2H), 3.08-3.16 (m, 2H), 2.93 (t, J = 2.43Hz, 1H), 2.84 (t, J = 7.72Hz, 2H).

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

[0082] 1. In vitro enzyme activity test

[0083] Chemically synthesized histone H3 polypeptide fragment H3 1-14 , probe pDA-1, 9 μg of TGM2 were dissolved in 25 μL of enzymatic hydrolysis buffer (25 mM Tris-Cl, pH = 7.5, 5 mM CaCl2, 1 mM PMSF and 1 mM DTT), where the concentration of probe pDA-1 was 5 mM, and incubated in the dark at room temperature for 5 h; the resulting product system was centrifuged at 4°C for 20 min to remove the precipitate, and the supernatant was detected by LC-MS.

[0084] Figure 2 The LC-MS test results are shown in Figure 1. Figure 2It was found that the probe pDA-1 can be covalently added to the known modification substrate histone H3.

[0085] 2. Cell experiments

[0086] Neuroblastoma cells SK-N-SH were treated with 200 μM probes pDA-1 and DA for 2 h, respectively. The cells were digested with trypsin and the cell pellets were collected after washing with PBS buffer solution. The cell pellets were lysed with PBS buffer solution containing 0.1% (volume fraction) Triton. The resulting lysate was centrifuged at 14,000 × g for 30 min. The protein concentration of the obtained supernatant was determined by BCA method and diluted to 2 mg / mL. 1 mL of the obtained total protein lysate was mixed with CuSO4 , TBTA, an azide-biotin compound (azide-biotin) and TCEP were mixed, wherein the concentration of CuSO4 was 1mM, the concentration of TBTA was 100μM, the concentration of the azide-biotin compound was 100μM, and the concentration of TCEP was 1mM. The mixture was then incubated at room temperature for 1h. After the incubation, the resulting product system was precipitated with pre-cooled methanol and centrifuged at 14000×g for 30min. After removing the supernatant, the precipitate was dissolved with 1mL PBS buffer solution containing 1.2wt% SDS, and the protein labeled with probe pDA-1 was enriched with magnetic beads containing streptavidin, and then eluted with 2× loading buffer. The eluate was subjected to WB analysis.

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

[0088] Test Example 2

[0089] 1. Cell or tissue imaging

[0090] Neuroblastoma cells SK-N-SH (2×10 5 ) were spread on cell culture slides and incubated at 37°C overnight; treated with 200μM probe pDA-1 for 2h, then added with freshly prepared 4wt% formaldehyde for fixation for 15min, and then blocked with 1wt% BSA solution for 1h. The fixed cells were mixed with CuSO4, TBTA, TAMRA fluorescent dye and TCEP, wherein the concentration of CuSO4 was 1mM, the concentration of TBTA was 100μM, the concentration of TAMRA fluorescent dye was 100μM, and the concentration of TCEP was 1mM. The cells were then incubated at room temperature for 0.5h. After the incubation, the reaction solution was removed, the cell nuclei were stained with DAPI, and then the slides were sealed and the cells were imaged using confocal microscopy.

[0091] Figure 4 Figure 1 shows the results of a cell imaging assay (scale bar, 100μm). Cells were treated with dopamine (DA) and the probe pDA-1, respectively. TAMRA fluorescent dye was introduced via a click reaction, and the dopamine-modified protein substrate was excited to emit red fluorescence. Cell nuclei were stained with DAPI. The results show that dopamine modification is present throughout SK-N-SH cells, demonstrating that the probe pDA-1 can penetrate the cell and nuclear membranes and act on the entire cell. It also confirms that dopamine modification is distributed throughout the cell.

[0092] 2. Identification of potential dopamine modification substrates

[0093] Neuroblastoma cells SK-N-SH were treated with 200 μM probe pDA-1 and cell culture medium for 2 h, respectively. The cells were digested with trypsin, washed with PBS buffer solution, and the cell pellets were collected. The cell pellets were lysed with PBS buffer solution containing 0.1% (volume fraction) Triton. 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, an azide-biotin compound (azide-biotin) and TCEP were mixed, wherein the concentration of CuSO4 was 1mM, the concentration of TBTA was 100μM, the concentration of the azide-biotin compound was 100μM, and the concentration of TCEP was 1mM. The mixture was then incubated at room temperature for 1h. After the incubation, the resulting product system was precipitated with pre-cooled methanol and centrifuged at 14000×g for 30min. After removing the supernatant, the precipitate was dissolved with 1mL PBS buffer solution containing 1.2wt% SDS. The protein labeled with probe pDA-1 was enriched with magnetic beads containing streptavidin, and then alkylated and cleaved by trypsin based on on-beads for proteomic analysis and compared with the Uniprot protein library.

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

[0095] Table 1 Histone enrichment effect by probe pDA-1

[0096]

[0097] 3. Verification of modified substrates

[0098] Using the above-mentioned enrichment strategy, the present invention identified a total of 4133 possible dopamine-modified protein substrates, including histone H4, aromatic amino acid decarboxylase (DDC) and acylation demodification enzyme (SIRT2). Since the expression levels of DDC and SIRT2 in neuroblastoma cells SK-N-SH are relatively low, the present invention constructed cell lines overexpressing DDC and SIRT2, respectively. Subsequently, the wild-type, DDC and SIRT2 overexpressing cell lines were treated with probes pDA-1 and DA, respectively, and the strategy in the existing literature (Fulton, SL et al. Histone H3 dopaminylation in ventral tegmental area underlies heroin-induced transcriptional and behavioral plasticity in malerats. Neuropsychopharmacology 47, 1776-783, 2022) was used to verify whether histone H4, DDC and SIRT2 are modified substrates of dopamine.

[0099] Figure 6 This is the verification result diagram of dopamine-modified substrates DDC, SIRT2 and histone H4. Figure 6 It can be seen that 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 probe pDA-1 and cell culture medium for 2 h, respectively. The cells were digested with trypsin, washed with PBS buffer solution, and the cell pellets were collected. The cell pellets were lysed with PBS buffer solution containing 0.1% (volume fraction) Triton. 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, an azide-biotin compound and TCEP were mixed, wherein the concentration of CuSO4 was 1 mM, the concentration of TBTA was 100 μM, the concentration of the azide-biotin compound was 100 μM, and the concentration of TCEP was 1 mM. The mixture was then incubated at room temperature for 1 hour. After the incubation, the resulting product system was precipitated with pre-cooled methanol and centrifuged at 14,000 × g for 30 minutes. After removing the supernatant, the precipitate was dissolved with 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 hydrolysis and washing with PBS buffer solution. After removing the peptides not modified by the probe, the peptides modified by the probe could be released with acetic acid.

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

[0103] Table 2 Corresponding data of the secondary mass spectrum of 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+ -H2O]]> 884.48102 <![CDATA[b 5+ -H2O]]> 808.40021 <![CDATA[y 9+ -NH3]]> 885.48541 <![CDATA[y 3+ -H2O]]> 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] The chemically synthesized histone H4 peptide containing Q27, 5mM probe pDA-1, and 9μg of TGM2 were dissolved in 25μL of enzymatic hydrolysis buffer (25mM Tris-Cl, pH=7.5, 5mM CaCl2, 1mM PMSF and 1mM DTT) and incubated in the dark at room temperature for 5h; the resulting product system was centrifuged at 4°C for 20min to remove the precipitate, and the supernatant was detected by LC-MS.

[0108] Figure 8 This is the secondary mass spectrometry diagram of dopamine modification of histone H427 in vitro. The results show that probe pDA-1 can be covalently added to histone H4, that is, glutamine at position 27 (Q27) of histone H4 can be modified by dopamine.

[0109] 2. Functional study of histone H4Q27dop modification

[0110] The present invention overexpresses cells in which histone H4 and Q27 are mutated to alanine A, and then the two cell lines are treated with dopamine respectively.

[0111] Figure 9 This figure shows the results of dopamine modification on histone H4 (H4Q27dop) inhibiting cell proliferation. Figure 9 Figure a shows the results of SK-N-SH cells treated with different concentrations of dopamine for 48 h. It can be seen that dopamine can inhibit cell proliferation in a dose-dependent manner. The data are expressed as mean ± standard deviation (n = 3), * indicates p < 0.05, *** indicates p < 0.001, **** indicates p < 0.0001, ns = not significant, t-test. Figure 9 Panel b shows the results after dopamine treatment of H4WT and H4Q27A mutant cell lines, respectively. It can be seen that the cell proliferation phenotype induced by dopamine is inhibited; the data are expressed as mean ± standard deviation (n = 3), * indicates p < 0.05, *** indicates p < 0.001. Figure 9 Figure c is a specificity validation diagram of the H4Q27dop antibody. The dotplot shows that the H4Q27dop modified antibody can recognize peptides containing H4Q27dop modification, but does not recognize H3Q5dop dopamine modified and non-modified peptides. Figure 9 The d in the figure is a graph showing the change of H4Q27dop modification level with dopamine concentration. It can be seen that the H4Q27dop modification level caused by the Q27A mutation is significantly reduced. The data are expressed as mean ± standard deviation (n = 3), ** indicates p < 0.05, ns = not significant, t-test.

[0112] The above cell proliferation experiments confirmed that dopamine-induced cell proliferation was inhibited by the Q27A mutation, indicating that dopamine modification at histone 27 can inhibit cell proliferation.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A dopamine derivative, characterized in that: It has the structure shown in formula I:

2. The method for preparing the dopamine derivative according to claim 1, characterized in that: The following steps are involved: 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 preparation method 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. Use of the dopamine derivative according to claim 1 in the preparation of a preparation enriched in 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.

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

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

7. A coupling compound, comprising a matrix substance, a dopamine derivative and a functional substance, wherein the matrix substance, the dopamine derivative and the functional substance are covalently linked in sequence; The matrix substance includes a protein or a polypeptide, the dopamine derivative is the dopamine derivative according to claim 1, and the functional substance includes 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.

8. The method for preparing the coupling compound according to claim 7, comprising 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.

9. The preparation method according to claim 8, 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.

10. Use of the coupling compound according to claim 7 in preparing 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.

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