Protein site-specific labeling probes and methods based on reversible michael addition-triggered benzylic participation reactions

Abstract

The present application relates to a kind of protein site-specific labeling probe and labeling method based on reversible michael addition triggered ortho group participation reaction, probe contains two active difference reactive groups, the michael acceptor group of high activity acrylamide part and the group of lower reactivity; It can be by the way of forming covalent bond to carry out site-directed modification to the protein containing double cysteine motif.Probe compound molecular weight is smaller, stability is high, improves the resolution of obtaining protein information, the influence caused to protein is less, maintains protein biological activity;Compared with other thiol chemical methods, significantly reduce the consumption of intracellular GSH, avoid the problem of cell damage caused by excessive consumption of GSH in the process of labeling, provide more selectable tools for high-resolution research of intracellular protein.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry, and in particular relates to a protein site-specific labeling probe and labeling method based on a reversible Michael addition-triggered neighbor-group-involved reaction. Background Technology

[0002] Proteins are fundamental building blocks of life, participating in almost all activities of organisms. The crowded, confined, and multi-component spatial environment within cells differs from dilute solutions, necessitating consideration of the unique characteristics of living cells for in-depth research into protein structure and function. However, techniques for obtaining atomic-resolution images of protein structure or dynamic changes within their native cellular environment are extremely limited. Specific labeling of intracellular proteins, achieving precise labeling and visualization with minimal disturbance to their structure, biological function, and distribution, remains one of the most significant challenges in chemical biology.

[0003] Commonly used protein labeling methods include fusion fluorescent proteins, fusion self-labeled enzyme tags (such as SNAP- and Halo-tags), diarsenic-tetracysteine ​​tags, His Tag / NiII-NTA systems, ligand-directed chemistry, and orthogonal click chemistry of non-natural amino acid binding biomolecules. However, each method has its advantages and disadvantages. Fusion fluorescent proteins are the most widely used protein labeling method, but they also have limitations. Fluorescent proteins have a large molecular weight (approximately 27 kDa), and their fusion with the target protein (POI) can sometimes affect its function and localization. The β-barrel structure is relatively conservative and limited in its ability to support a wider range of functions. Fusion self-labeled enzyme tags offer high substrate specificity, allowing for the binding of substrates to diverse functional dyes. They are highly flexible, versatile, and well-suited for fluorescence imaging. However, besides their large size (>20 kDa), most dyes show no significant difference in fluorescence signal before and after labeling, requiring thorough washing to remove non-specific staining and easily introducing background interference. In bis-arsenic tetracysteine ​​tags, the introduced cysteine ​​polypeptide sequence is relatively short (approximately 0.5 kDa), and its impact on the target protein is smaller than that of protein tags. It is often used to label specific proteins in living cells, but its toxicity and reversibility limit its widespread application. His Tag / NiII-NTA systems have been widely used for the specific labeling of His-tag fusion proteins. Most probes lack cell permeability and require the assistance of cell-penetrating peptides to enter the cell. Furthermore, the tag placement is limited to the N-terminus or C-terminus of the protein, making it impossible to obtain fine conformational changes of the target protein. Ligand-directed chemistry is a proximal protein labeling method driven by ligand and protein recognition, which can be used to label endogenous proteins. However, the selectivity of the labeling site needs improvement. Additionally, target proteins based on affinity tags often lose their inherent activity because the recognition portion permanently occupies its ligand-binding pocket. This may hinder research on protein participation in related cellular processes. Introducing non-natural amino acid-binding bioorthogonal click chemistry is also widely used for the localization and transport of intracellular target proteins. These chemical modifications exhibit high levels of site selectivity; however, the cumbersome synthesis of aminoacylated tRNAs limits their general applicability. Furthermore, these modifications produce long, flexible side chains, making them unsuitable for obtaining high-resolution information about target proteins. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a protein site-specific labeling probe and labeling method based on a reversible Michael addition-triggered neighbor-group-involved reaction.

[0005] The technical solution adopted in this invention is: a compound with the structure shown in Formula 1;

[0006]

[0007] Where n is 0, 1, 2, 3 or 4; R1 is hydrogen or fluorine; R2 is sulfone, p-methylsulfone, methylsulfone or propenyl; R3 is hydrogen, methyl, halogen or nitro; R4 is hydrogen or a fluorescent group; R5 is hydrogen or a fluorescent group; R6 is hydrogen or methyl.

[0008] Preferably, when R4 and R5 are hydrogen, the compound of Formula 1 is a magnetic probe; when R4 or R5 is a fluorescent group, the compound of Formula 1 is a fluorescent probe.

[0009] Preferably, the fluorescent group is BODIPY or Rhodamine fluorescent group.

[0010] Preferably, it is a combination of one or more of formulas T1-T11;

[0011]

[0012]

[0013] Application of compound 1 in probes.

[0014] Preferably, the compound of Formula 1 specifically labels proteins containing a biscysteine ​​motif.

[0015] Preferably, the specific label for the compound of Formula 1 contains two markers spaced apart by a distance of [missing information]. Proteins with a biscysteine ​​motif.

[0016] Preferably, the labeling is specific to intracellular or extracellular proteins.

[0017] Preferably, when labeling intracellular proteins, the cells are bacterial cells or mammalian cells.

[0018] A protein site-specific labeling probe and labeling method based on a reversible Michael addition-triggered neighbor-group-involved reaction, wherein the compound of Formula 1 is used as a probe to be added to a protein sample containing a biscysteine ​​motif, and the protein is detected by one or more of the following techniques: mass spectrometry, NMR and fluorescence.

[0019] Preferably, when R4 and R5 are hydrogen, the compound of formula 1 serves as a magnetic probe; the labeling effect is detected by mass spectrometry and / or NMR.

[0020] When R4 or R5 is a fluorescent group, the compound of Formula 1 is a fluorescent probe, and the labeling effect is detected by one or more combinations of mass spectrometry, NMR and fluorescence techniques.

[0021] The advantages and positive effects of this invention are: the probe compounds provided have small molecular weights and high stability, which improves the resolution of protein information acquisition, has little impact on proteins, and maintains protein biological activity; compared with other thiol chemistry methods, it significantly reduces the consumption of intracellular GSH, avoids the problem of cell damage caused by excessive GSH consumption during the labeling process, and provides more optional tools for high-resolution study of intracellular proteins.

[0022] The synthesis steps of probe compounds are simple, easy to produce, and convenient to modify; the labeling methods are simple and fast, with high yield, short labeling time, high selectivity, stable labeled products, and the ability to achieve site-specific labeling; in addition, probe compounds have diverse functions, and can be used to study intracellular proteins using both NMR and fluorescence techniques. They are versatile in their application and can be used for various detection methods, enabling the study of the localization and dynamic interaction mechanisms of biomolecules in living cells at a high resolution level. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of site-specific labeling of Ub E24C / A28C by probe T based on neighbor-based assistance.

[0024] Figure 2 This is a schematic diagram of site-specific labeling of Ub E24C / A28C using T1 with neighbor-based assistance.

[0025] Figure 3 As in Example 12 of the present invention 19 Before and after the reaction of F-label T1 with Ub E24C / A28C 19 F-NMR spectrum;

[0026] Figure 4 The LC / Q TOF mass spectrum of Ub E24C / A28C in Example 12;

[0027] Figure 5 This is the LC / Q TOF mass spectrum of Ub E24C / A28C after complete reaction with T1 in Example 12;

[0028] Figure 6 In Example 13 19 F-NMR verification of T1 selectively labeling Ub E24C / A28 in cells; a) Collection of samples after incubation of T1 with cells expressing Ub E24C / A28C. 19 b) F NMR spectrum; b) Supernatant collected from sample a 19 F NMR spectrum; c) Collection of lysate from sample a 19 F NMR spectrum; d) T1 reacts completely with 1 equivalent of Ub E24C / A28C in buffer solution.19 FNMR spectrum; e) Reaction of T1 with 1 equivalent of GSH in buffer solution. 19 F NMR spectrum;

[0029] Figure 7 In Example 13 19 Analysis of the proportion of F-tag T1 in intracellular linker products;

[0030] Figure 8 Example 14: Fluorescent staining of Ub E24C / A28C by fluorescent tag T6 in E. coli cells. Detailed Implementation

[0031] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0032] This invention relates to a protein site-specific labeling probe and labeling method based on a reversible Michael addition-triggered neighbor-group reaction. First, a compound that can be used as a probe is synthesized, with the structure shown in Formula 1.

[0033]

[0034] Wherein, n is 0, 1, 2, 3, or 4; R1 is hydrogen or fluorine; R2 is sulfone, p-methylsulfone, methylsulfone, or propenyl; R3 is hydrogen, methyl, halogen, or nitro; R4 is hydrogen or a fluorescent group; R5 is hydrogen or a fluorescent group; R6 is hydrogen or methyl; the fluorescent group is BODIPY or rhodamine fluorescent group. In Formula 1, R1 and R3 of the probe are inherent groups in the raw material. The leaving group R2 can be introduced onto pyridine by reacting sodium benzenesulfinate or sodium methylsulfinate with a halopyridine derivative. The Michael acceptor portion of the acrylamide moiety is obtained in one step through dehydration condensation of nicotinic acid derivatives and cyanoacetamide derivatives. R4 and R5, being fluorescent groups, can be linked to the probe via amide bonds. When R6 is methyl, it can be introduced by reacting iodomethane with an amino group.

[0035] The specific steps for preparing the compound of formula 1 are as follows:

[0036] Step 1: A sulfone group can be introduced at the 4-position of pyridine by reacting a 4-halogenated nicotinic aldehyde compound with a sodium sulfinate derivative;

[0037]

[0038] Wherein, R can be one of the following groups:

[0039]

[0040] Alternatively, a vinyl group can be introduced at the 4-position of pyridine by reacting a 4-halogenated nicotinic aldehyde compound with a vinyl borate pinacol ester, and the catalyst can be tetrakis(triphenylphosphine)palladium.

[0041]

[0042] Step 2: React the reaction product of Step 1 with a cyanoacetamide compound under the action of a base (such as triethylamine);

[0043]

[0044] R5 can be hydrogen or the following groups:

[0045]

[0046] R6 can be hydrogen or methyl;

[0047] When R4 is a fluorescent group, it can be synthesized in the following way.

[0048]

[0049] It can also be synthesized via acid-amine condensation, with condensing agents such as EDCI, DCC, and HATU.

[0050]

[0051] It can also be synthesized by forming a Schiff base from an amino group and an aldehyde group.

[0052] R7 can be one of the following groups:

[0053]

[0054] These probes contain two reactive sites with different activities: a Michael acceptor group in the highly reactive acrylamide moiety and a less reactive R2 group. They can perform site-specific modification of proteins containing biscysteine ​​motifs through covalent bonding. For example... Figure 1 As shown, the free thiol group first undergoes a rapid and reversible covalent binding with the Michael acceptor group of the acrylamide moiety. Then, another free thiol group of the protein undergoes an SN2 reaction with the less active R2 group in the probe, thereby achieving Michael addition and neighboring group participation effects, thus realizing the site-directed irreversible introduction of functional small molecule probes.

[0055] Depending on the selection of R4 and R5, magnetic or fluorescent probes can be constructed. Based on the probe properties, detection can be achieved through one or more combinations of mass spectrometry, NMR, and fluorescence techniques, thereby enabling the detection or localization of labeled proteins. When R4 and R5 are hydrogen, the compound of Formula 1 serves as a magnetic probe, and the labeling effect is detected by mass spectrometry and / or NMR; when R4 or R5 is a fluorescent group, the compound of Formula 1 serves as a fluorescent probe, and the labeling effect is detected by one or more combinations of mass spectrometry, NMR, and fluorescence techniques.

[0056] When used as a probe, the compound shown in Formula 1 can detect or label samples containing proteins with a biscysteine ​​motif. Due to its small molecular weight, it can also be used to label proteins containing a biscysteine ​​motif within living cells. The probe enters cells directly through incubation, selectively contacting and ligating with proteins expressing a biscysteine ​​motif. In use, the probe is added to the sample or cell sample to be labeled, and after incubation, the probe ligates to the protein. The incubation time is 0.1-2 hours, and the reaction can be carried out at room temperature. The operation is convenient and requires no additional chemical modification of the protein to be labeled. The protein ligation efficiency of the probe is 70%-100%. The specific labeling steps are as follows:

[0057] Step 1: Weigh the probe and add it to DMF to prepare the probe solution;

[0058] Step 2: Add 1-5 equivalents of probe solution to the protein sample to be labeled and react at room temperature; wherein the concentration of the protein containing cysteine ​​is 0.01-0.2 mM;

[0059] Step 3: Use 19 F-NMR, 1 H- 15 The reaction between the probe and the protein is monitored using N HSQC or LC / Q-TOF mass spectrometry until the reaction is complete and the modification is finished; the reaction time is 0.1-3 hours.

[0060] Step 4: Journey to the West 19 F-NMR and 1 H- 15 After the reaction was completed, the reaction solution was diluted with ultrapure water and then directly analyzed by LC / Q-TOF mass spectrometry to verify the ligation products.

[0061] In some embodiments of the present invention, the probe can also be used for labeling cell samples. 0.02-0.5 mM of the probe is added and incubated with cells for 1-2 hours. After the reaction, the labeling is performed using… 19 F-NMR or fluorescent gel imaging can be used to verify the labeling of target proteins within cells. Because the probe compound molecules are small, their impact on protein function is minimal after binding to the protein. Furthermore, the use of this probe results in low GSH consumption within the cell, avoiding excessive GSH depletion and cell damage, thus providing a foundation for the application of probe compounds in labeling living cells.

[0062] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0063] Example 1: Synthesis of probe T1 (2-cyano-3-(2-fluoro-4-(benzenesulfonyl)pyridin-3-yl)acrylamide)

[0064]

[0065] 2-Fluoro-4-iodopyridine-3-carboxaldehyde (2 mmol, 504 mg) and sodium benzenesulfinate (5 mmol, 805 mg) were dissolved in 20 mL of dimethyl sulfoxide and reacted at room temperature for 2 h. The mixture was then diluted with 50 mL of ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain intermediate 2.

[0066] Intermediate 2 (2.0 mmol, 530 mg), cyanoacetamide (2.5 mmol, 210 mg), and triethylamine (3 mmol, 303 mg) were dissolved in 10 mL of ethanol and reacted at room temperature for 6 h. The mixture was concentrated under reduced pressure and separated by column chromatography (petroleum ether: ethyl acetate 1:1) to obtain T1566 mg. The two-step yield was 85%. 1 H NMR (400MHz, MeOD) δppm: 8.66 (d, J = 5.1Hz, 1H), 8.40 (s, 1H), 8.16 (d, J = 5.1Hz, 1H), 7. 92(d,J=7.7Hz,2H),7.78(t,J=7.5Hz,1H),7.64(t,J=7.8Hz,2H).HRMS(ESI)m / z[M+H] + calcd for C 15 H 11 FN3O3S + :332.0500,found:332.0502.

[0067] Example 2: Synthesis of probe T2

[0068]

[0069] 4-Chloro-3-pyridinecarboxaldehyde 3 (5.0 mmol, 708 mg) and sodium benzenesulfinate (3.28 g, 20 mmol) were dissolved in dimethyl sulfoxide and reacted at room temperature for 2 hours. TLC showed complete reaction. The mixture was concentrated under reduced pressure, and ethanol, cyanoacetamide (3.0 mmol, 252 mg), and 1-methylpiperazine (2.0 mmol, 222 μL) were added. The reaction was continued at room temperature for 1 hour. The mixture was concentrated under reduced pressure and separated by column chromatography to give a white solid compound T2 477 mg, in 76% yield. 1H NMR(400MHz,MeOD)δ9.03(d,J=5.1Hz,1H),8.97(s,1H),8.72(s,1H),8.23(d,J=5.1Hz,1 H),7.96–7.91(m,2H),7.76(t,J=7.5Hz,1H),7.63(t,J=7.8Hz,2H).HRMS(ESI)m / z[M+H] + calcd for C 15 H 11 FN3O3S + :313.0500,found:313.0501.

[0070] Example 3: Synthesis of probe T3

[0071]

[0072] 2-Fluoro-4-iodopyridin-3-carboxaldehyde 1 (2 mmol, 0.5 g) and sodium 4-methylbenzenesulfinate (5 mmol, 1.34 g) were dissolved in dimethyl sulfoxide and reacted at room temperature for 2 h. The mixture was then concentrated under reduced pressure, and the resulting intermediate 4 could be used directly in the next step without purification. Intermediate 5, cyanoacetamide (2.5 mmol, 210 mg), and triethylamine (3 mmol, 303 mg) were added to ethanol and reacted at room temperature for 6 h. The mixture was then concentrated under reduced pressure, and column chromatography was used to separate T3 621 mg. The two-step yield was 90%. 1 H NMR(400MHz,Chloroform-d)δ8.58(d,J=5.1Hz,1H),8.51(s,1H),8.01(d,J=5.1Hz,1H),7.76(dd,J =8.4,2.1Hz,2H),7.37(d,J=8.0Hz,2H),6.21(s,1H),5.87(s,1H),2.45(s,3H).HRMS(ESI)m / z[M+H] + calcd forC 16 H 13 FN3O3S + :346.0656,found:346.0655.

[0073] Example 4: Synthesis of probe T4

[0074]

[0075] 2-Fluoro-4-iodopyridine-3-carboxaldehyde (2 mmol, 0.5 g) and sodium methanesulfonate (2 mmol, 204 mg) were dissolved in dimethyl sulfoxide and reacted at room temperature for 2 h. The mixture was then concentrated under reduced pressure, and intermediate 5 was obtained directly for the next step without purification. Intermediate 6, cyanoacetamide (2.5 mmol, 210 mg), and triethylamine (3 mmol, 303 mg) were dissolved in ethanol and reacted at room temperature for 6 h. The mixture was then concentrated under reduced pressure, and column chromatography was used to separate T4 323 mg. The two-step yield was 60%. 1 H NMR(400MHz, Methanol-d4)δ8.67(d,J=5.1Hz,1H),8.53(s,1H),8.02(d,J=5.1Hz,1H),3.28(s,3H).HRMS(ESI)m / z[M+H] + calcd forC 10 H 10 FN3O3S + :270.0343,found:270.0340.

[0076] Example 5: Synthesis of probe T5

[0077]

[0078] 2-Fluoro-4-iodopyridin-3-carboxaldehyde (10 mmol, 2.5 g), vinylboronic acid pinacol ester (15 mmol, 2.3 g), and tetrakis(triphenylphosphine)palladium (0.1 mmol, 115 mg) were dissolved in 20 mL of N,N-dimethylformamide, followed by the addition of 4 mL of 2 M sodium carbonate solution. The reaction was carried out at 120 °C for 1 h, cooled to room temperature, concentrated under reduced pressure, and separated by column chromatography to give intermediate 7 0.5 g (30% yield). Intermediate 7 (3 mmol, 0.5 g), cyanoacetamide (3.6 mmol, 303 mg), and triethylamine (4.5 mmol, 455 mg) were dissolved in ethanol, stirred at room temperature for 6 h, concentrated under reduced pressure, and separated by column chromatography to give target compound T5 292 mg (45% yield). 1 H NMR (400MHz, CDCl3-d) δ8.41(d,J=1.9Hz,1H),8.30(d,J=5.4Hz,1H),7.42(d,J=5.4Hz,1H),6.76(ddd,J=17.5,11.0, 1.9Hz,1H),6.38(s,1H),6.00(dd,J=17.3,2.0Hz,1H),5.85(s,1H),5.77(dd,J=11.0,1.9Hz,1H).HRMS(ESI)m / z[M+H] + calcd for C 11 H9FN3O+ :218.0724,found:218.0722.

[0079] Example 6: Synthesis of probe T6

[0080]

[0081] ABP (1 mmol, 283 mg), cyanoacetic acid (1.2 mmol), HATU (1.2 mmol, 456 mg), and N,N-diisopropylethylamine (1.5 mmol, 194 mg) were dissolved in 10 mL of anhydrous dichloromethane. The mixture was stirred at room temperature for 12 h, concentrated under reduced pressure, and separated by column chromatography (eluent: dichloromethane) to give intermediate 8 175 mg, yield 51%. Intermediate 8 (0.5 mmol, 175 mg), 2-fluoro-4-benzenesulfonylpyridine-3-carboxaldehyde (0.6 mmol, 159 mg), and triethylamine (0.75 mmol, 76 mg) were dissolved in 10 mL of anhydrous ethanol. The mixture was stirred at room temperature for 6 h, concentrated under reduced pressure, and separated by column chromatography (dichloromethane:methanol 20:1) to give target compound T6 119 mg, yield 40%. 1 H NMR (800MHz, DMSO-d6) δ10.97(s,1H),8.80(d,J=5.2Hz,1H),8.48(s,1H),8.20(t,J=4.8Hz,1H),8.16(s,2H),8.00(d,J=8.0Hz,2H),7.97(d,J= 7.8Hz,2H),7.85(t,J=7.6Hz,1H),7.78(d,J=8.2Hz,2H),7.70(t,J=7.7Hz,2H),7.13(d,J=4.2Hz,2H),6.74(t,J=6.0Hz,2H).HRMS(ESI)m / z[MH] - calcd for C 30 H 18 BF3N5O3S - :596.1181,found:596.1180.

[0082] Example 7: Synthesis of Probe T7

[0083]

[0084] Compound 9 (2 mmol, 399 mg) and sodium benzenesulfinate (5 mmol, 821 mg) were dissolved in dimethyl sulfoxide and reacted at room temperature for 2 h. The mixture was then concentrated under reduced pressure to give intermediate 10 (580 mg, yield: 95%). Intermediate 10 (1.9 mmol, 580 mg) was dissolved in 20 mL of THF / H₂O (1:1), and 3.8 mmol of lithium hydroxide was added. The mixture was stirred at room temperature for 6 h, and the pH was adjusted with hydrochloric acid until a white solid precipitated. The solid was collected by filtration to give intermediate 11 (553 mg, 92%). Intermediate 11 (1.9 mmol, 553 mg), AMP (0.2 mmol, 566 mg), EDCI (3 mmol, 575 mg), and DMAP (0.2 mmol, 24 mg) were dissolved in dichloromethane and stirred at room temperature for 20 h. The solvent was removed under reduced pressure, and the mixture was separated by column chromatography to give intermediate 12. Compound 12, cyanoacetamide (2.5 mmol, 210 mg), and triethylamine (3 mmol, 303 mg) were dissolved in ethanol and reacted at room temperature for 6 h. The mixture was concentrated under reduced pressure and separated by column chromatography to obtain T7 614 mg, with a yield of 52%. 1 H NMR (400MHz, DMSO-d6) δ9.12 (s, 1H), 8.88 (s, 1H), 8.67 (d, J = 8.0Hz, 2H), 8. 56(s,1H),8.20(s,1H),8.00(d,J=8.0Hz,2H),7.87(d,J=7.8Hz,2H),7.84(t ,J=7.6Hz,1H),7.76(d,J=8.2Hz,2H),7.77(d,J=8.2Hz,2H),7.70(s,1H),7 .55(t,J=7.7Hz,2H),7.14(s,1H),6.37(d,J=8.2Hz,2H)HRMS(ESI)m / z[M+H] + calcd for C 31 H 21 BF2N6O4S - :621.1531,found:621.1535.

[0085] Example 8: Synthesis of probe T8

[0086]

[0087] Compound 13 (2 mmol, 432 mg) and sodium benzenesulfinate (5 mmol, 821 mg) were dissolved in dimethyl sulfoxide and reacted at room temperature for 2 h. The mixture was then concentrated under reduced pressure to give intermediate 14 (526 mg, yield: 90%). Intermediate 14 (1.9 mmol, 580 mg), cyanoacetamide (2.5 mmol, 210 mg), and triethylamine (3 mmol, 303 mg) were dissolved in ethanol and reacted at room temperature for 6 h. The mixture was then concentrated under reduced pressure and separated by column chromatography to give intermediate 15 (524 mg, yield: 85%). Compound 15 (1.5 mmol, 524 mg) and activated manganese dioxide (15 mmol, 1.3 g) were dissolved in dichloromethane (20 mL) and stirred at room temperature for 24 h. The manganese dioxide was filtered off, the solvent was removed under reduced pressure, and the mixture was separated by column chromatography to give intermediate 16501 mg, yield: 98%. Compound 15 (1.47 mmol, 501 mg) and ABP (1.5 mmol, 424 mg) were dissolved in methanol, stirred at room temperature for 6 h, methanol was removed under reduced pressure, and T8, 267 mg, was obtained by column chromatography, with a yield of 30%. 1 H NMR (800MHz, DMSO-d6) δ8.81 (s, 1H), 8.67 (s, 1H), 8.53 (d, J = 8.0Hz, 2H), 8. 47(s,1H),8.18(s,1H),8.00(d,J=8.0Hz,2H),7.89(d,J=7.8Hz,2H),7.85(t ,J=7.6Hz,1H),7.76(d,J=8.2Hz,2H),7.78(d,J=8.2Hz,2H),7.70(s,1H),7 .66(t,J=7.7Hz,2H),7.13(s,1H),6.32(d,J=8.2Hz,2H)HRMS(ESI)m / z[M+H] + calcd for C 31 H 21 BF2N6O3S - :606.1457,found:606.1459.

[0088] Example 9: Synthesis of Probe T9

[0089]

[0090] Compounds 7 (1 mmol, 151 mg), 8 (1.2 mmol, 420 mg), and triethylamine (1.5 mmol, 152 mg) were dissolved in ethanol, stirred at room temperature for 6 h, concentrated under reduced pressure, and separated by column chromatography to obtain 217 mg of the target compound T9, with a yield of 45%. 1H NMR(400MHz, CDCl3-d)δ8.98(s,1H),8.52(d,J=1.9Hz,1H),8.46(d,J=8.0Hz,2H), 8.11(d,J=5.4Hz,1H),7.98(d,J=7.8Hz,2H),7.85(d,J=8.2Hz,2H),7.77(t,J=7.7H z,2H),7.45(d,J=5.4Hz,1H),6.80(d,J=8.2Hz,2H)6.54(ddd,J=17.5,11.0,1.9Hz, 1H),6.13(dd,J=17.3,2.0Hz,1H),5.48(dd,J=11.0,1.9Hz,1H)HRMS(ESI)m / z[M+H] + calcd for C 26 H 17 BF3N5O + :483.1478,found:483.1480.

[0091] Example 10: Synthesis of probe T10

[0092]

[0093] Compound 17 (1 mmol, 191 mg) was dissolved in 10 mL of THF / H₂O (1:1), and 1.2 mmol of lithium hydroxide was added. The mixture was stirred at room temperature for 6 h, and the pH was adjusted with hydrochloric acid until a white solid precipitated. The solid was collected by filtration to give intermediate 18 (94 mg, 53%). Intermediate 18 (0.53 mmol, 94 mg), AMP (0.6 mmol, 170 mg), EDCI (0.9 mmol, 172 mg), and DMAP (0.06 mmol, 8 mg) were dissolved in dichloromethane, stirred at room temperature for 20 h, concentrated under reduced pressure, and separated by column chromatography to give intermediate 19 (152 mg, 65% yield). Compound 19 (0.34 mmol, 152 mg), cyanoacetamide (0.4 mmol, 34 mg), and triethylamine (0.5 mmol, 50 mg) were dissolved in ethanol, reacted at room temperature for 6 h, concentrated under reduced pressure, and separated by column chromatography to give T10 (77 mg, 45% yield). 1H NMR(400MHz, CDCl3-d)δ8.56(s,1H),8.49(s,J=1.9Hz,1H),8.46(d,J=8.0Hz,2H) ,7.86(d,J=7.8Hz,2H),7.74(d,J=8.2Hz,2H),7.69(t,J=7.7Hz,2H),7.46(s,1H), 6.80(ddd,J=17.5,11.0,1.9Hz,1H),6.78(d,J=8.2Hz,2H)6.43(s,1H),6.37(dd,J =17.3,2.0Hz,1H),5.78(s,1H),5.66(dd,J=11.0,1.9Hz,1H).HRMS(ESI)m / z[M+H] + calcd for C 27 H 19 BF2N6O2 + :508.1631,found:508.1625.

[0094] Example 11: Synthesis of probe T11

[0095]

[0096] Compound 20 (1 mmol, 163 mg), cyanoacetamide (1.2 mmol, 1.5 mg), and triethylamine (1.5 mmol, 152 mg) were dissolved in ethanol and reacted at room temperature for 6 h. The mixture was concentrated under reduced pressure and separated by column chromatography to give intermediate 21, 149 mg, in 65% yield. Compound 21 (0.65 mmol, 149 mg) and activated manganese dioxide (6.5 mmol, 650 mg) were dissolved in dichloromethane and stirred at room temperature for 24 h. The manganese dioxide was filtered off, the solvent was removed under reduced pressure, and intermediate 22, 148 mg, in 98% yield was obtained by column chromatography. Compound 22 (0.65 mmol, 148 mg) and ABP (1.5 mmol, 424 mg) were dissolved in methanol and stirred at room temperature for 6 h. The methanol was removed under reduced pressure, and intermediate 11, 105 mg, in 33% yield was obtained by column chromatography. 1H NMR(400MHz, CDCl3-d)δ8.98(s,1H),8.89(s,J=1.9Hz,1H),8.43(d,J=8.0Hz,2H) ,7.79(d,J=7.8Hz,2H),7.73(d,J=8.2Hz,2H),7.67(t,J=7.7Hz,2H),7.54(s,1H), 6.78(d,J=8.2Hz,2H)6.69(ddd,J=17.5,11.0,1.9Hz,1H),6.33(s,1H),6.01(dd,J =17.3,2.0Hz,1H),5.80(s,1H),5.56(dd,J=11.0,1.9Hz,1H).HRMS(ESI)m / z[M+H] + calcd for C 27 H 19 BF2N6O + :492.1681,found:492.1680.

[0097] Example 12: Site-specific labeling of Ub E24C / A28C by T1 based on neighbor-based participation

[0098] Probe T1 can specifically label the Ub E24C / A28C site, and the labeling method is as follows: Figure 2 As shown.

[0099] Weigh out the T1 prepared in Example 1 and dissolve it in DMF to prepare a 100 mM T1 solution; take 0.2 mM 500 μL UbE24C / A28C in 20 mM phosphate buffer (pH 7.5), add 0.5 times the amount of TCEP, and let it stand at room temperature for 20 min; slowly add 1 times the amount of T1 solution to the above protein solution, always maintaining the pH of the solution at 7.5, and react at room temperature for 20 min, then collect the results. 19 F-NMR, the results are as follows Figure 3 As shown, from Figure 3 The results indicate that T1 reacted completely with Ub E24C / A28C.

[0100] Samples were collected, diluted with H2O, and the binding of T1 to the Ub E24C / A28C protein was analyzed by LC / Q TOF mass spectrometry. The mass spectrum of the Ub E24C / A28C protein was used as a blank control. The results are as follows: Figure 4-5As shown, the peak of protein Ub E24C / A28C is located at m / z = 8571.16. The mass spectrum of the crude product mixture of T1 and Ub E24C / A28C reaction only shows a peak at m / z = 8760.36, which corresponds to the peak of T1-Ub E24C / A28C, the neighboring product formed by Michael addition and nucleophilic substitution of sulfone group during the reaction of Ub E24C / A28C with T1.

[0101] Example 13: Selective labeling of Ub E24C / A28C by T1 in E. coli cells based on neighbor-based assistance

[0102] The Ub E24C / A28C plasmid was transformed into BL21(DE3)Codon plus competent cells, seeded in LB liquid medium containing 0.1% (v / v) ampicillin, and cultured overnight, followed by subculture with a 1:100 dilution. When OD... 600 When the protein expression reaches 0.6, isopropyl β-D-thiogalactopyranoside (IPTG) (1 mM) is added to induce protein expression at 37°C for 2–3 h.

[0103] Ub E24C / A28C overexpressing E. coli cells were collected by centrifugation, the supernatant was removed, and the bacterial pellet was resuspended in 50 mM PBS buffer (pH 7.3) at room temperature to prepare 500 uL of 0.1-0.15 g / ml E. coli resuspension (containing 10% D2O); probe T1 was added to a final concentration of 0.1 mM and mixed with the cell sample.

[0104] use 19 F NMR confirmed the intracellular selectivity of T1 for Ub E24C / A28C; results are as follows Figure 6 As shown. Figure 6 a is the intracellular data collected after mixing T1 cells with cells expressing Ub E24C / A28C. 19 F NMR spectrum;

[0105] Figure 6 b is the supernatant collected after centrifugation of sample 6a. 19 F NMR spectrum; Figure 6 c represents the cell pellet from sample 6a after centrifugation, resuspended in 50 mM PBS buffer (pH 7.3, containing 10% D2O), and collected as lysate after sonication. 19 F NMR spectrum; Figure 6 d is the reaction of T1 with 1 equivalent of Ub E24C / A28C in 50mM PBS buffer (pH 7.3, containing 10% D2O) for 10 minutes. 19 F NMR spectrum (reaction complete); Figure 6e represents the reaction of T1 with 1 equivalent of GSH in 50 mM PBS buffer (pH 7.3, containing 10% D2O) for 56 minutes. 19 F NMR spectra. Reactions of T1 in buffer with Ub E24C / A28C and GSH, respectively. Figure 6 de as a reference, in Figure 6 In in-cell experiments, the neighboring group-particulate product T1-UbE24C / A28C was observed in the reaction of T1 with Ub E24C / A28C, indicating that T1 can specifically label Ub E24C / A28C within the cell. Although the T1-GSH conjugate product labeled with black spheres was also observed, the NMR signal peaks of the GSH / target protein and T1 conjugate products showed a significant shift, which largely avoids interference from GSH in intracellular protein conformation studies. Figure 6 T1 and its products linked to intracellular small molecules were observed in the cell supernatant of cell b. No protein leakage was observed, proving that the probe can be used to label living cells without killing them. Figure 6 Only the T1-Ub E24C / A28C signal peak was observed in the cell lysate of b.

[0106] In addition, through Figure 6 The integral area of ​​each product in step a was used to calculate and analyze the proportion of the reaction between probe T1 and various intracellular substances. The results are as follows: Figure 7 As shown, the main ligation product of T1 in the cell was T1-Ub E24C / A28C, accounting for 86% of the total product; GSH consumed a small amount of the probe, with the T1-GSH ligation product accounting for 12% of the total product; the remaining 2% of unknown reaction products were negligible. These results demonstrate that T1 can achieve highly selective labeling of Ub E24C / A28C in cells.

[0107] Example 14: Achieving T6 fluorescent selective labeling of Ub E24C / A28C in E. coli cells based on neighbor-based assistance;

[0108] E. coli cells overexpressing Ub E24C / A28C as described in Example 7 were taken, and fluorescent probe T6 was added to a final concentration of 10 μM. The mixture was then incubated. The labeled cells were lysed, proteins were extracted, and the cell lysates were analyzed by SDS-PAGE.

[0109] The results are as follows Figure 8As shown, 1 represents the protein after cell lysis, and 2 represents the control sample with T6-Ub E24C / A28C added to the buffer. By comparison, it can be seen that only one protein band with a molecular weight of about 8kDa in the cell lysate shows yellow fluorescence, proving that Ub E24C / A28C is indeed the only labeled protein, demonstrating the feasibility of using the probe to fluorescently label target proteins in live cells.

[0110] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A compound, characterized in that: The structure is shown in Equation 1; Formula 1; Wherein, n is 0, 1, 2, 3 or 4; R1 is hydrogen or fluorine; R2 is sulfone, p-methylsulfone or methylsulfone; R3 is hydrogen, methyl, halogen or nitro; R4 is hydrogen or a fluorescent group; R5 is hydrogen or a fluorescent group; the fluorescent group is BODIPY; R6 is hydrogen or methyl; when R4 and R5 are hydrogen, the compound of formula 1 is a magnetic probe; when R4 or R5 is a fluorescent group, the compound of formula 1 is a fluorescent probe.

2. A compound, characterized in that: Specifically, it is one of the following formulas: T1, T2, T3, T4, T6, T7, and T8; 。 3. The use of the compound of claim 1 or 2 in a probe, wherein the use is not for disease diagnosis or treatment purposes.

4. The application according to claim 3, characterized in that: Compound 1 specifically labels proteins with biscysteine ​​motifs.

5. The application according to claim 4, characterized in that: Formula 1 compounds specifically label proteins containing two biscysteine ​​motifs spaced 5-9 Å apart.

6. The application according to claim 4, characterized in that: Specific labeling of intracellular or extracellular proteins.

7. A labeling method for protein site-specific labeled probes based on reversible Michael addition-triggered neighbor-group reactions, wherein the labeling method is for non-disease diagnosis and treatment purposes, characterized in that: The compound of claim 1 or 2 is used as a probe to be added to a protein sample containing a biscysteine ​​motif, and detected by one or more of the following techniques: mass spectrometry, NMR, and fluorescence.

8. The marking method according to claim 7, characterized in that: When R4 and R5 are hydrogen, the compound of Formula 1 serves as a magnetic probe; the labeling effect is detected by NMR and / or mass spectrometry. When R4 or R5 is a fluorescent group, the compound of Formula 1 serves as a fluorescent probe; the labeling effect is detected by fluorescence technology and / or mass spectrometry.

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