Methyl cyclopropene benzoic acid probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of probe molecules and protein post-translational modification, in particular to a methylcyclopropene benzoic acid probe as well as a preparation method and application thereof. The methylcyclopropene benzoic acid probe is any one of a compound 4, a compound 5 and a compound 6; wherein R1 is-COOCH3 which is subjected to ortho-position substitution, meta-position substitution or para-position substitution; r < 2 > is meta-substituted or para-substituted-COOH; and R3 is-COOCH2OCOCH3 which is subjected to meta-position substitution or para-position substitution. According to the invention, methylcyclopropene in the methylcyclopropene benzoic acid probe is used as a dienophile part and can form a unique probe pair with the S-tetrazine coupled diene probe, so that efficient and accurate labeling of benzoylation protein post-translational modification is realized; the problems of long time consumption, radioactive hazard, low enrichment efficiency, poor specificity and background fluorescence interference in PTMs detection are solved. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the technical field of probe molecules and protein post-translational modification, and in particular to a methylcyclopropenebenzoic acid probe and a preparation method and application thereof. Background Art

[0002] Histone post-translational modifications, or PTMs for short, have received extensive research attention in recent years as key epigenetic mechanisms regulating gene transcription and chromatin structure. These modifications not only control the basic functions of cells, but are also closely related to the occurrence and development of a variety of diseases, such as tumorigenesis, inflammation, autoimmune diseases, neurodegeneration, and viral infections. PTMs are considered "targetable" therapeutic targets. Among them, histone benzoylation, as a unique type of acylation modification containing an aromatic ring, has significant biological properties due to its aromatic ring structure and has become one of the research hotspots.

[0003] The uniqueness of benzoylation lies in the large volume and strong hydrophobicity of its benzene ring structure, which distinguishes it from other aliphatic chain acylation modifications in biological function. Benzoyl-CoA is the substrate of benzoylation, and its source and metabolic pathway suggest that benzoylation may respond to different cellular metabolic pathways and thus participate in a variety of biological processes.

[0004] However, despite the increasing importance of benzoylation, the methods for its detection and labeling are relatively limited. Traditional biological methods, such as radioisotope labeling, antibody recognition, immunoprecipitation, and non-radioactive spectroscopy, have many limitations in the detection of new PTMs, such as time-consuming, radioactive hazards, low enrichment efficiency, poor specificity, and background fluorescence interference, which restrict the in-depth study and understanding of benzoylation. Summary of the invention

[0005] In order to solve the problems of long time consumption, radioactive hazards, low enrichment efficiency, poor specificity and background fluorescence interference in PTMs detection, the purpose of the present invention is to provide a methylcyclopropenebenzoic acid probe and its preparation method and application.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows.

[0007] The first aspect of the present invention provides a methylcyclopropene benzoic acid probe, wherein the methylcyclopropene benzoic acid probe is any one of compound 4, compound 5 and compound 6:

[0008] Among them, R1 is -COOCH3 substituted at the ortho position, meta position or para position; R2 is -COOH substituted at the meta position or para position; R3 is -COOCH2OCOCH3 substituted at the meta position or para position.

[0009] The methylcyclopropene benzoic acid probe molecule of the present invention utilizes the methylcyclopropene in the probe molecule as a dienophile part, and can form a unique probe pair with an S-tetrazine-coupled diene probe, thereby achieving efficient and accurate labeling of post-translational modifications of benzoylated proteins, and solving the problems of long time consumption, radioactive hazards, low enrichment efficiency, poor specificity and background fluorescence interference in PTMs detection.

[0010] The second aspect of the present invention provides a method for preparing the methylcyclopropenebenzoic acid probe according to the first aspect, comprising the following steps:

[0011] Compound 1 reacts with vinyltrimethylsilane under the catalysis of metal palladium to construct a precursor compound 2 of cyclopropane; the precursor compound 2 of cyclopropane reacts with tribromoform and hexadecyltrimethylammonium bromide through a carbene addition reaction with a double bond to obtain compound 3; compound 3 reacts with iodomethane under the action of n-butyl lithium to obtain an intermediate 3-1; intermediate 3-1 is eliminated by tetrabutylammonium fluoride to obtain compound 4; compound 4 is hydrolyzed to remove the methyl ester group under the action of lithium hydroxide to obtain compound 5; compound 5 undergoes a substitution reaction with bromomethyl acetate to cover the carboxyl group with an acetoxy group to obtain compound 6; the specific synthesis route is as follows:

[0012]

[0013] Preferably, the preparation method of the cyclopropane precursor compound 2 comprises the following steps:

[0014] Compound 1 is stirred and mixed with a metal palladium catalyst and silver nitrate in a solvent one, triethylamine and vinyltrimethylsilane are added, and the reaction is carried out at 70°C±5°C under a protective atmosphere to obtain a cyclopropane precursor compound 2; the molar ratio of compound 1 to the metal palladium catalyst, silver nitrate, triethylamine and vinyltrimethylsilane is 1:0.01-0.05:1-1.5:1.5:1.8-2.5; and the solvent one is acetonitrile.

[0015] Preferably, the preparation method of compound 3 comprises the following steps:

[0016] The precursor compound 2 of cyclopropane is stirred and mixed with bromoform and hexadecyltrimethylammonium bromide in solvent 2, and then an alkali solution is added to carry out a carbene addition reaction with a double bond at 25°C±5°C to obtain compound 3; the molar ratio of compound 2 to bromoform and hexadecyltrimethylammonium bromide is 1:4-5:0.1-0.3; the molar ratio of compound 2 to the alkali is 1:10-12; the concentration of the alkali solution is 50wt%±5wt%; the solvent 2 is dichloromethane; and the alkali is sodium hydroxide or potassium hydroxide.

[0017] Preferably, the preparation method of compound 4 comprises the following steps:

[0018] The compound 3 is mixed evenly in the solvent three, cooled to -75 to -80°C under a protective atmosphere, and then iodomethane and n-butyllithium are added under stirring to react to obtain an intermediate 3-1; the intermediate 3-1 and tetrabutylammonium fluoride are subjected to an elimination reaction in the solvent three to obtain a compound 4; the molar ratio of the compound 3 to iodomethane and n-butyllithium is 1:1.8 to 2:1.5 to 2; the molar ratio of the intermediate 3-1 to tetrabutylammonium fluoride is 1:1.5 to 2.0; the solvent three is tetrahydrofuran.

[0019] Preferably, the preparation method of compound 5 comprises the following steps:

[0020] Compound 4 and lithium hydroxide are stirred and reacted in solvent four to hydrolyze and remove the methyl ester group to obtain compound 5; the molar ratio of compound 4 to lithium hydroxide is 1:4.5-5; solvent four is a mixed solution of tetrahydrofuran and water.

[0021] Preferably, the preparation method of compound 6 comprises the following steps:

[0022] Compound 5 is subjected to a substitution reaction with bromomethyl acetate and N,N-diisopropylethylamine in solvent five to mask the carboxyl group with an acetoxy group to obtain compound 6; the molar ratio of compound 5 to bromomethyl acetate and N,N-diisopropylethylamine is 1:2-2.2:2-2.5; solvent five is acetonitrile.

[0023] The third aspect of the present invention provides a fluorescent probe, which is generated by reacting a methylcyclopropene benzoic acid derivative with S-tetrazine-BODIPY through an IEDDA cycloaddition reaction to generate a fluorescent probe for releasing a fluorescent signal; the synthetic reaction equation of the fluorescent probe is as follows:

[0024] Among them, the compound shown in formula I is a methylcyclopropene benzoic acid derivative; the compound shown in formula II is a fluorescent probe; the methylcyclopropene benzoic acid derivative is the methylcyclopropene benzoic acid probe described in claim 1, and R is selected from R1, R2 or R3.

[0025] The invention designs and synthesizes methylcyclopropene benzoic acid probe molecules based on IEDDA reaction. These probe molecules use methylcyclopropene as a dienophile part to form a unique probe pair with an S-tetrazine coupled diene probe for labeling research on post-translational modification of benzoylated proteins.

[0026] The fourth aspect of the present invention provides a use of the methylcyclopropenebenzoic acid probe described in the first aspect or the fluorescent probe described in the third aspect in the preparation of a benzoylated protein post-translational modification detection reagent.

[0027] Beneficial effects of the present invention:

[0028] 1. The methylcyclopropene benzoic acid probe molecule of the present invention utilizes the methylcyclopropene in the probe molecule as the dienophile part, which can form a unique probe pair with the S-tetrazine coupled diene probe, thereby achieving efficient and accurate labeling of post-translational modifications of benzoylated proteins, and solving the problems of long time consumption, radioactive hazards, low enrichment efficiency, poor specificity and background fluorescence interference in PTMs detection.

[0029] 2. The present invention uses the IEDDA reaction, i.e., the inverse electron demand Diels-Alder reaction of olefins / S-tetrazine, to generate a stable six-membered cyclic compound through a rapid and synergistic addition reaction between a dienophile and a diene, thereby achieving labeling of the target protein.

[0030] 3. The present invention aims to screen out a better probe pair by investigating the bio-orthogonal correlation performance of benzoic acid probes in vitro, and realize efficient and accurate labeling of post-translational modification of benzoylated proteins. The present invention will not only provide a new method for detecting post-translational modification of benzoylated proteins, but also provide an effective way to quickly locate post-translational modification of benzoylated proteins, which will help promote the research on the role and function of post-translational modification of new proteins in life activities. At the same time, the present invention provides a theoretical basis and experimental basis for the development of targeted drugs and therapies for benzoylation modification, which has important scientific significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the synthetic route of the methylcyclopropenebenzoic acid probe.

[0032] Figure 2 It is the product of the reaction of compound 4b with S-tetrazine-BODIPY 1 H NMR spectrum.

[0033] Figure 3 The results of the selectivity evaluation of the reaction of compound 4b with S-tetrazine-BODIPY are shown in Figure 1. (a) is the fluorescence spectrum at 500nm to 650nm; (b) is the fluorescence intensity change at the maximum emission wavelength of 514nm after treatment with different interfering substances.

[0034] Figure 4 The figure is a comparison of the effects of different concentrations of compound 4b on the activity of RAW264.7 and HeLa cells. A is the activity of RAW264.7 cells; B is the activity of HeLa cells.

[0035] Figure 5 Confocal live cell imaging of methylcyclopropenebenzoic acid probe 4b and S-tetrazine-BODIPY. Among them, A1~A3 are the images without DAPI addition, with DAPI addition, and merged images of the -Probe+BDP FL Tz group, respectively; B1~B3 are the images without DAPI addition, with DAPI addition, and merged images of the +4a+BDP FL Tz group, respectively; C1~C3 are the images without DAPI addition, with DAPI addition, and merged images of the +4b+BDP FL Tz group, respectively; D1~D3 are the images without DAPI addition, with DAPI addition, and merged images of the +4c+BDP FL Tz group, respectively; E1~E3 are the images without DAPI addition, with DAPI addition, and merged images of the +5b+BDP FL Tz group, respectively; F1~F3 are the images without DAPI addition, with DAPI addition, and merged images of the +5c+BDP FL Tz group, respectively; G1~G3 are the images without DAPI addition, with DAPI addition, and merged images of the +6b+BDP FL Tz group, respectively; H1~H3 are the images without DAPI addition, with DAPI addition, and merged images of the +6c+BDP FL Tz group, respectively.

[0036] Figure 6 These are the fluorescence statistics of methylcyclopropenebenzoic acid probe 4b and S-tetrazine-BODIPY.

[0037] Figure 7 It is a dynamic detection of post-translational modification images of benzoylated proteins in cells. Among them, A1~A3 are the images of the control group without DAPI, with DAPI, and merged images; B1~B3 are the images of the first group without DAPI, with DAPI, and merged images; C1~C3 are the images of the second group without DAPI, with DAPI, and merged images; D1~D3 are the images of the third group without DAPI, with DAPI, and merged images.

[0038] Figure 8 is the fluorescence statistics for dynamic detection of benzoylation in cells. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0041] One of the keys of the present invention is to efficiently prepare the methylcyclopropene benzoic acid probe. The ideal cyclopropene-substituted acylated derivative should be to directly connect the cyclopropene to the lysine benzoyl compound. Since the difficulty of cyclopropene synthesis lies in the diversification of cyclopropene chemical reactivity, high temperature, strong oxidant, strong reducing agent, expensive metal rhodium and explosive diazo compound should be avoided in the synthesis process. The present invention designs and synthesizes a novel protein lysine post-translationally modified methylcyclopropene benzoic acid probe.

[0042] The synthetic routes of methylcyclopropenebenzoic acid probes 4a, 4b-6b, 4c-6c are as follows: Figure 1 As shown. The specific synthesis method is as follows: commercial iodobenzoic acid methyl ester 1a~1c is used as the starting material to obtain the precursor compound 2a~2c for constructing cyclopropane under the catalysis of metal palladium; 2a~2c is subjected to carbene addition to the double bond to obtain dibromocyclopropane 3a~3c, and further eliminated by tetrabutylamine fluoride under the action of n-butyl lithium to obtain candidate benzoic acid probes 4a~4c. Benzoic acid probes 5b~5c are obtained by hydrolyzing the candidate benzoic acid probes 4b~4c under the action of lithium hydroxide to remove the methyl ester group to generate acid; benzoic acid probes 5b~5c are substituted with bromomethyl acetate to cover the carboxyl group with an acetoxy group to obtain benzoic acid probes 6b~6c. It is expected that this uncharged probe can easily penetrate the cell membrane. Among them, o- represents the ortho position, m- represents the meta position, and p- represents the para position.

[0043] The technical solution of the present invention is further described below through specific embodiments.

[0044] In the following examples, the methods described are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0045] TBAF represents tetrabutylammonium fluoride. THF represents tetrahydrofuran; DIPEA represents N,N-diisopropylethylamine. The room temperature is 25°C ± 5°C.

[0046] Example 1

[0047] like Figure 1 , a method for preparing a methylcyclopropenebenzoic acid probe 4a, comprising the following steps:

[0048] Synthesis of compound 2a: Palladium acetate and silver nitrate were dissolved in acetonitrile, and 4.0 g of compound 1a, triethylamine, and vinyltrimethylsilane were added in sequence by syringe, wherein the molar ratio of compound 1a, palladium acetate, silver nitrate, triethylamine, and vinyltrimethylsilane was 1:0.03:1:1.5:2; magnetic stirring was performed under nitrogen protection, and the reaction was heated at 70°C±5°C for 17 hours. When compound 1a was consumed, the mixture was cooled and filtered, extracted with ethyl acetate, and the combined organic phase was washed with water, dried with Na2SO4, and spun to obtain compound 2a with a yield of 95.4%.

[0049] The NMR data of compound 2a are as follows: 1 H NMR (400MHz, CDCl3): δ7.85 (dd, J=8.0, 1.4Hz, 1H), 7.68~7.58 (m, 1H), 7.47 (td, J=7.7, 1 .4Hz, 1H), 7.30 (td, J=7.6, 1.3Hz, 1H), 6.40 (d, J=18.9Hz, 1H), 3.91 (s, 1H), 0.18 (s, 3H). 13 C NMR (100MHz, CDCl3): δ169.46, 143.78, 141.80, 134.56, 133.40, 131.65, 129.84, 128.76, 128.54, 53.49, 0.16.

[0050] Synthesis of compound 3a: 2.65g of compound 2a was dissolved in dichloromethane, bromoform and hexadecyltrimethylammonium bromide were added, and an alkali solution was added to the mixture and stirred at room temperature for 50h; wherein the molar ratio of compound 2a, bromoform and hexadecyltrimethylammonium bromide was 1:4.76:0.1; the alkali solution was a 50wt% sodium hydroxide solution, and the molar ratio of compound 2a to sodium hydroxide was 1:12. After the reaction was completed, water was added to quench, extracted with dichloromethane, dried with Na2SO4 and spun to dryness, to obtain compound 3a with a yield of 85.4%.

[0051] The NMR data of compound 3a are as follows: 1 H NMR (400MHz, CDCl3): δ8.10~8.04 (m, 1H), 7.54~7.46 (m, 1H), 7.40 (td, J=7.6, 1.3Hz, 1H), 7.0 5 (d, J=7.7Hz, 1H), 4.04 (s, 3H), 3.25 (d, J=11.0Hz, 1H), 1.35 (d, J=10.9Hz, 1H), 0.28 (s, 9H). 13C NMR (100MHz, CDCl3): δ169.76, 141.09, 134.45, 133.40, 132.95, 131.86, 129.82, 54.94, 41.01, 37.19, 30.88, 0.81.

[0052] Synthesis of compound 4a: Compound 3a was dissolved in tetrahydrofuran, placed in a low-temperature thermostatic reactor, cooled to -78°C under nitrogen protection and stirred, methyl iodide was added to the reaction system, and then n-butyl lithium was added, and stirred and reacted at -78°C for 2h. The molar ratio of compound 3a, methyl iodide and n-butyl lithium was 1:2:1.5; after the reaction was completed, it was quenched with saturated ammonium chloride solution. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layer was washed with brine, dried with MgSO4 and spin-dried to obtain intermediate 3a-1.

[0053] The intermediate 3a-1 was dissolved in tetrahydrofuran, and tetrabutylammonium fluoride was added, wherein the molar ratio of the intermediate 3a-1 to tetrabutylammonium fluoride was 1:1.5, and stirred at room temperature for 2 hours. After the reaction was completed, the mixture was quenched with water and extracted with ethyl acetate. The combined organic phase was washed with water, dried with Na2SO4, filtered and dried by spin drying to obtain compound 4a with a yield of 77.2%.

[0054] The NMR data of compound 4a are as follows: 1 H NMR (400MHz, CDCl3): δ7.74 (dd, J=7.8, 1.5Hz, 1H), 7.37 (td, J=7.7, 1.5Hz, 1H), 7.16 (td, J=7.6, 1. 4Hz, 1H), 6.96 (dd, J=7.8, 1.3Hz, 1H), 6.62 (s, 1H), 3.92 (s, 3H), 3.10 (d, J=1.8Hz, 1H), 2.15 (s, 3H). 13 C NMR (100MHz, CDCl3): δ169.09, 148.37, 131.43, 129.85, 126.01, 124.79, 118.12, 99.22, 52.02, 20.51, 10.64.

[0055] Example 2

[0056] A method for preparing methylcyclopropenebenzoic acid probes 4b to 6b comprises the following steps:

[0057] Synthesis of Compound 2b: The synthesis of Compound 2a in Example 1 was followed, except that Compound 1a was replaced by Compound 1b, to obtain Compound 2b with a yield of 95.4%.

[0058] The NMR data of compound 2b are as follows: 1 H NMR (400MHz, CDCl3): δ8.11 (d, J=2.1Hz, 1H), 7.92 (dd, J=7.6, 1.4Hz, 1H), 7.62 (dd, J=7.4, 2.0Hz, 1H), 7.40 (td, J=7.7, 2.3Hz, 1H), 6.97~6.84 (m, 1H), 6.58 (dd, J=19.2, 2.3Hz, 1H), 3.93 (s, 3H), 0.17 (s, 9H). 13 C NMR (100MHz, CDCl3): δ169.20, 144.50, 140.72, 133.37, 132.81, 132.49, 130.97, 130.69, 129.53, 54.29, 0.79.

[0059] Synthesis of compound 3b: The synthesis method of compound 3a in Example 1 was followed, except that compound 2a was replaced by compound 2b, to obtain compound 3b with a yield of 63.8%.

[0060] The NMR data of compound 3b are as follows: 1 H NMR (400MHz, CDCl3): δ7.99 (d, J=1.1Hz, 1H), 7.91 (t, J=1.0Hz, 1H), 7.45~7.41 (m, 2 H), 3.93 (s, 3H), 2.82 (dd, J=10.7, 0.7Hz, 1H), 1.38 (d, J=10.6Hz, 1H), 0.25 (s, 9H). 13 C NMR (100MHz, CDCl3): δ168.97, 139.68, 135.34, 132.26, 131.93, 130.83, 130.43, 54.38, 40.73, 35.96, 29.61, 0.76.

[0061] Synthesis of compound 4b: The synthesis method of compound 4a in Example 1 was followed, except that compound 3a was replaced by compound 3b to obtain intermediate 3b-1. Intermediate 3a-1 was replaced by intermediate 3b-1 to obtain compound 4b with a yield of 83.5%.

[0062] The NMR data of compound 4b are as follows: 1H NMR (400MHz, CDCl3): δ7.80 (d, J=7.0Hz, 1H), 7.73 (s, 1H), 7.29 (d, J=1.8Hz, 2H), 6.59 (d, J=1.5Hz, 1H), 3.90 (s, 3H), 2.58 (d, J=1.7Hz, 1H), 2.14 (s, 3H). 13 C NMR (100MHz, CDCl3): δ147.87, 130.21, 129.90, 128.07, 126.50, 99.09, 52.15, 22.74, 10.31.

[0063] Synthesis of compound 5b: Compound 4b was dissolved in a mixed solution of tetrahydrofuran and water in a volume ratio of 3:1, stirred at room temperature for 5 min, LiOH was added, and stirred at room temperature overnight; wherein the molar ratio of compound 4b to LiOH was 1:5. After the reaction, tetrahydrofuran was dried by spin-drying, pH was adjusted to acidic pH <2 with 0.5M dilute hydrochloric acid, extracted with ethyl acetate, dried with anhydrous MgSO4 and spin-dried to obtain compound 5b with a yield of 68.6%.

[0064] The NMR data of compound 5b are as follows: 1 H NMR (400MHz, CDCl3): δ7.88 (d, J=4.1Hz, 1H), 7.81 (s, 1H), 7.35 (s, 2H), 6.60 (s, 1H), 2.60 (s, 1H), 2.16 (s, 3H). 13 C NMR (100MHz, CDCl3): δ172.53, 148.06, 131.08, 129.08, 128.19, 127.15, 127.12, 116.33, 99.10, 22.72, 10.29.

[0065] Synthesis of compound 6b: Compound 5b was dissolved in acetonitrile, and N,N-diisopropylethylamine was added, wherein the molar ratio of compound 5b to N,N-diisopropylethylamine was 1:2.5; the mixture was stirred at room temperature for 2 hours, and then bromomethyl acetate was added, wherein the molar ratio of compound 5b to bromomethyl acetate was 1:2.2. After the reaction was completed, acetonitrile was removed under reduced pressure to obtain compound 6b with a yield of 60.9%.

[0066] The NMR data of compound 6b are as follows: 1H NMR (400MHz, CDCl3): δ7.87~7.84(m, 1H), 7.79(s, 1H), 7.34~7.29(m, 2H), 6.58 (s, 1H), 4.85 (s, 2H), 3.79 (s, 3H), 2.58 (s, 1H), 2.14 (d, J=2.1Hz, 3H). 13 C NMR (100MHz, CDCl3): δ168.71, 165.98, 131.60, 130.73, 128.86, 128.43, 128.17, 128.10, 126.93, 93.39, 61.20, 52.46, 29.73, 22.73.

[0067] Example 3

[0068] A method for preparing methylcyclopropenebenzoic acid probes 4c to 6c comprises the following steps:

[0069] Synthesis of Compound 2c: The synthesis of Compound 2a in Example 1 was followed, except that Compound 1a was replaced by Compound 1c, to obtain Compound 2c with a yield of 91.7%.

[0070] The NMR data of compound 2c are as follows: 1 H NMR (400MHz, CDCl3): δ7.99 (dd, J=8.4, 1.6Hz, 1H), 7.48 (dd, J=8.4, 1.6Hz, 1H), 6.90 (d d, J=19.2, 1.4Hz, 1H), 6.62 (dd, J=19.1, 1.5Hz, 1H), 3.91 (d, J=1.4Hz, 3H), 0.17 (s, 9H). 13 C NMR (100MHz, CDCl3): δ169.07, 144.66, 144.56, 135.31, 132.00, 131.31, 128.34, 54.20, 0.99, 0.74.

[0071] Synthesis of compound 3c: The synthesis method of compound 3a in Example 1 was followed, except that compound 2a was replaced by compound 2c, to obtain compound 3c with a yield of 68.5%.

[0072] The NMR data of compound 3c are as follows: 1 H NMR (400MHz, CDCl3): δ8.03 (d, J=8.4Hz, 2H), 7.36~7.26 (m, 2H), 3.91 (s, 3H), 2.82 (d, J=10.7Hz, 1H), 1.37 (d, J=10.7Hz, 1H), 0.25 (s, 9H).13 C NMR (100MHz, CDCl3): δ168.93, 144.34, 131.61, 131.37, 130.90, 54.29, 40.92, 35.57, 29.92, 0.75.

[0073] Synthesis of compound 4c: The synthesis method of compound 4a in Example 1 was followed, except that compound 3a was replaced by compound 3c to obtain intermediate 3c-1. Intermediate 3a-1 was replaced by intermediate 3c-1 to obtain compound 4c with a yield of 77.4%.

[0074] The NMR data of compound 4c are as follows: 1 H NMR (400MHz, CDCl3): δ7.92 (d, J=8.3Hz, 2H), 7.13 (d, J=8.3Hz, 2H), 6.56 (d, J=1.4Hz, 1H), 3.89 (s, 3H), 2.58 (d, J=1.8Hz, 1H), 2.14 (s, 3H). 13 C NMR (100MHz, CDCl3): δ167.48, 153.45, 129.46, 127.04, 125.40, 115.80, 98.81, 52.00, 23.20, 10.25.

[0075] Synthesis of compound 5c: The synthesis method of compound 5b in Example 2 was followed, except that compound 4b was replaced by compound 4c, to obtain compound 5c with a yield of 56.3%.

[0076] The NMR data of compound 5c are as follows: 1 H NMR (400MHz, CDCl3): δ 8.00 (d, J=8.0Hz, 2H), 7.17 (d, J=8.0Hz, 2H), 6.57 (s, 1H), 2.60 (s, 1H), 2.16 (s, 3H). 13 C NMR (100MHz, CDCl3): δ172.09, 130.61, 130.55, 130.39, 130.32, 129.95, 129.10, 128.94, 127.20, 32.17, 24.52.

[0077] Synthesis of compound 6c: The synthesis method of compound 6b in Example 2 was followed, except that compound 5b was replaced by compound 5c, to obtain compound 6c with a yield of 44.6%.

[0078] The NMR data of compound 6c are as follows: 1H NMR (400MHz, CDCl3): δ7.97 (d, J=8.2Hz, 2H), 7.15 (d, J=8.2Hz, 2H), 6.56 (s, 1H), 4.85 (s, 2H), 3.78 (s, 3H), 2.58 (s, 1H), 2.14 (s, 3H). 13 C NMR (100MHz, CDCl3): δ168.47, 165.93, 130.33, 129.86, 128.91, 127.15, 125.50, 98.73, 61.09, 52.40, 32.13, 23.27.

[0079] The following examples mainly screen the in vitro bioorthogonal performance of the methylcyclopropenebenzoic acid probes 4a, 4b-6b, 4c-6c prepared in Examples 1-3 to find methylcyclopropenebenzoic acid probes with fast reaction speed and good selectivity.

[0080] Example 4

[0081] Kinetic test of methylcyclopropene benzoic acid probe: The methylcyclopropene benzoic acid derivative was connected with S-tetrazine-BODIPY by IEDDA cycloaddition reaction to generate the pyridazine compound 4b-BODIPY, thereby lighting up the BODIPY fluorescent group quenched by tetrazine and releasing a fluorescent signal. By observing the fluorescence enhancement rate of the BODIPY fluorescent group, the reaction rate of the methylcyclopropene benzoic acid probe can be obtained.

[0082] The Chinese name of S-tetrazine-BODIPY is boron fluoride dipyrrol tetrazine, and the English name is BODIPY FLtetrazine, referred to as BDP FL Tz, which was purchased from Xi'an Kangfuno Biological Co., Ltd. The reaction equation of methylcyclopropene benzoic acid probe 4b and S-tetrazine-BODIPY is as follows:

[0083]

[0084] The specific preparation method is as follows:

[0085] The methylcyclopropene benzoic acid probe 4b was dissolved in DMF to obtain a methylcyclopropene benzoic acid 4b stock solution with a concentration of 8 mM. S-tetrazine-BODIPY was dissolved in DMF to obtain a S-tetrazine-BODIPY stock solution with a concentration of 4 mM. 50 μL of methylcyclopropene benzoic acid 4b stock solution and 10 μL of S-tetrazine-BODIPY stock solution were mixed in a mixed solution of DMF and water so that the volume ratio of DMF to water in the reaction system was 9:1, the concentration of methylcyclopropene benzoic acid 4b was 4 mM, and the concentration of S-tetrazine-BODIPY was 400 μM. The reaction was stirred at room temperature, and the corresponding cycloaddition product was monitored by HPLC at different time points. The results showed that S-tetrazine-BODIPY was almost completely consumed within 180 min and converted into the cycloaddition product, with a conversion rate of 93.4%. The cycloaddition product 4b-BODIPY 1 H NMR spectrum Figure 2 shown.

[0086] According to the above method, the second-order rate constant of the reaction of methylcyclopropene benzoic acid derivatives 4a, 4b~6b, 4c~6c with S-tetrazine-BODIPY in 50% DMF / phosphate buffer was determined by fluorescence determination. The specific method is: in a four-way light cuvette, 1.6mM~2.4mM concentration of methylcyclopropene benzoic acid probe and 4μM concentration of S-tetrazine-BODIPY reaction solution were reacted at room temperature, and the reaction solution of 0min~40min was taken, and the fluorescence value at 514nm was measured every 2 minutes, and then the reaction solution with different reaction time was studied by fluorescence spectrum to explore Ex max The relationship between the fluorescence intensity at and the reaction time. The results show that when the reaction time is about 40 minutes, the fluorescence intensity gradually stabilizes, indicating that the methylcyclopropene benzoic acid probe can react completely with S-tetrazine-BODIPY in about 40 minutes.

[0087] The data were analyzed using GraphPad Prism 9.0. The dynamic changes of fluorescence intensity during the reaction of different methylcyclopropene benzoic acid probes with S-tetrazine-BODIPY were measured using an FLS1000 steady-state transient fluorescence spectrometer, and the reaction rate of the pseudo-first-order reaction was obtained by fitting the curves of the exponential growth of these fluorescence intensities over time.

[0088] According to the time point when the fluorescence intensity reaches half of the maximum value in the reaction progress curve, the half-life t 1 / 2 , combined with formula K obs =1 / [compd.]×t 1 / 2 , where [compd.] represents the concentration of different methylcyclopropene benzoic acid probes, K obsrepresents the apparent reaction rate, which is used to quantify the reaction rate under different concentrations of methylcyclopropene benzoic acid probe. obs The relationship curve between the concentration of methylcyclopropene benzoic acid probe and the reaction rate difference between different methylcyclopropene benzoic acid probes and S-tetrazine-BODIPY is reflected by the slope. Finally, the secondary rate constant k2 is determined according to the slope, and the results are shown in Table 1.

[0089] Table 1 Kinetic test table of the reaction between methylcyclopropene benzoic acid probe and S-tetrazine-BODIPY

[0090]

[0091]

[0092] As shown in Table 1, the secondary rate constant of the candidate probe is 0.7175M -1 s -1 ~2.365M -1 s -1 The range of changes indicates that different substitution positions or different substituents will affect the IEDDA reaction rate of the probe molecule with S-tetrazine-BODIPY. By comparing the IEDDA reaction rates of methylcyclopropene benzoic acid probes 4a~4c with S-tetrazine-BODIPY, it can be obtained that the reaction rate of meta-benzoic acid methyl ester probe 4b is slightly greater than that of ortho-benzoic acid methyl ester probe 4a, and both are greater than para-benzoic acid methyl ester probe 4c; by comparing the IEDDA reaction rates of methylcyclopropene benzoic acid probes 4b~6b with S-tetrazine-BODIPY, it can be obtained that the reaction rate of benzoic acid methyl ester probe 4c is greater than that of acetoxymethyl benzoate probe 6c, and both are greater than benzoic acid probe 5c. By comparing the secondary rate constants k2 of candidate probes 4a, 4b-6b, and 4c-6c, as shown in Table 1, the secondary rate constant k2 of probe molecule 4b has the maximum value, and k2=2.365M -1 s -1 ±0.096M -1 s -1 .

[0093] Example 5

[0094] Selective investigation of the reaction of methylcyclopropene benzoic acid probe 4b with S-tetrazine-BODIPY: homocysteine ​​Hcy, cysteine ​​Cys, dithiothreitol DTT, glucose, vitamin C, nicotinamide adenine dinucleotide NAD + , all purchased from Bidex Pharmaceuticals.

[0095] Various potential interfering substances in the biological environment, including homocysteine ​​Hcy, cysteine ​​Cys, dithiothreitol DTT, glutathione GSH, glucose, sodium ascorbate VitC, nicotinamide adenine dinucleotide phosphate NADPH, hydrogen peroxide, inorganic salts CaCl2 and MgCl2, etc., were monitored in the PBS buffer at pH 7.4 to further study the selectivity of methylcyclopropenebenzoic acid probe 4b and its bioorthogonal properties.

[0096] The specific method is: prepare Hcy, Cys, DTT in DMF and glucose, vitamin C, NAD + , H2O2, CaCl2 or MgCl2 in deionized water, all at a concentration of 10 mM; the above stock solutions were diluted to a concentration of 50 μM with PBS buffer at pH = 7.4 to obtain the PBS buffer of the interferor. The prepared methyl cyclopropene benzoate 4b was prepared into a stock solution with a concentration of 10 mM with DMSO, and then diluted to a final concentration of 50 μM with PBS buffer. The 10 mM S-tetrazine-BODIPY-DMSO stock solution was diluted to a final concentration of 10 μM with the PBS buffer of the interferor. After mixing at 37°C for 30 min, the fluorescence spectra of these solutions were recorded in the range of 500 nm to 650 nm with an excitation wavelength of 495 nm and a slit width of 1 nm. The selectivity was evaluated by comparing the fluorescence intensity.

[0097] The fluorescence spectrum at 500nm to 650nm was detected by a fluorescence instrument, and the fluorescence intensity at the maximum emission point was quantified. Figure 3 As shown in the figure, after treatment with all potential interfering substances, the change in fluorescence intensity at the maximum emission wavelength of 514 nm was negligible. After treatment with methylcyclopropene benzoic acid probe 4b alone, the fluorescence intensity increased significantly, which was 16 times the increase in fluorescence intensity after treatment with interfering substances. This shows that these interfering substances have negligible effects on the IEDDA reaction of methylcyclopropene benzoic acid probe with S-tetrazine-BODIPY, and the IEDDA reaction of methylcyclopropene benzoic acid probe with S-tetrazine-BODIPY is highly specific.

[0098] In the embodiment of the present invention, the photophysical properties of the IEDDA product of methylcyclopropenebenzoic acid probe 4b and S-tetrazine-BODIPY were investigated. The results showed that after the IEDDA reaction, the fluorescence intensity was significantly enhanced compared to the original value. It can be seen that the IEDDA reaction can be optically monitored in real time by using the changes in the measured fluorescence emission intensity.

[0099] Example 6

[0100] The cytotoxicity of the probe molecules was determined by CCK-8 method: Human cervical cancer cells HeLa and mouse mononuclear macrophage leukemia cells RAW264.7 were purchased from Nest. 0.25% trypsin without phenol red was purchased from Gibco.

[0101] Using the recognized cytotoxicity test CCK-8 method, the grown HeLa cells and RAW264.7 cells were taken out of the incubator and placed in a clean bench for operation. First, the cells were digested with trypsin and counted. The concentration of the cell suspension was prepared to 4×10 4 / mL~5×10 4 / mL, and then the cells in the 96-well plate were evenly cultured with the cell suspension in an incubator for 24 hours. The methylcyclopropene benzoic acid probe 4b was prepared in a culture medium with five different concentrations of 25μM, 50μM, 100μM, 200μM and 400μM. The original culture medium in the 96-well plate was removed, and the newly prepared culture medium containing the compound was added to the cells for incubation for 24 hours. After that, 10μL CCK-8 was added to each well for staining. After culturing in the incubator for 2 hours, the cells were imaged with a fluorescence microscope, and the cell viability was evaluated by the CCK-8 method.

[0102] Results and analysis: The effect of methylcyclopropene benzoic acid probe 4b on mouse mononuclear macrophage leukemia cells RAW264.7, such as Figure 4 As shown in Figure A, when the concentration of the cyclopropene benzoic acid probe 4b is 25 μM, the cell viability is close to 100%. As the concentration of the cyclopropene benzoic acid probe 4b increases to 400 μM, the cell viability does not change significantly and remains close to 100%, indicating that the cyclopropene benzoic acid probe 4b has no significant effect on the normal growth and survival of RAW264.7 cells and has almost no cytotoxicity.

[0103] Effects of methylcyclopropenebenzoic acid probe 4b on human cervical cancer cells HeLa, such as Figure 4 As shown in Figure B, when the concentration of the cyclopropene benzoic acid probe 4b is 25 μM, the cell viability is 87%. As the concentration of the probe 4b increases to 400 μM, the cell viability does not change significantly and remains in the range of 84% to 100%, indicating that the cyclopropene benzoic acid probe 4b has no significant effect on the normal growth and survival of HeLa cells and has excellent biocompatibility.

[0104] Example 7

[0105] Methylcyclopropenebenzoic acid probe molecule 4b labels post-translational modification of intracellular benzoylated proteins.

[0106] S-tetrazine-BODIPY has good biocompatibility and cytotoxicity, so it is expected to image the post-translational modification of benzoylated proteins in cells through the IEDDA reaction between the methylcyclopropene benzoic acid probe and S-tetrazine-BODIPY. After HepG2 cells were incubated with the methylcyclopropene benzoic acid probe for 6 hours at 37°C, the specific lysine residues of the protein were modified through the biological metabolic pathway in the cell, fixed on the membrane, and then the fluorescent group S-tetrazine-BODIPY was added, and incubated for another hour at 37°C, and the images were collected by laser confocal scanning microscopy.

[0107] Probe molecule labeling and IEDDA reaction in living cells: Under a microscope, 2×10 5 HepG2 cells were seeded on a laser confocal dish and attached for 14 hours. Methylcyclopropene benzoic acid probes 4a, 4b-6b, 4c-6c were diluted with DMEM to a final concentration of 100 μM, and the culture medium with probes or the culture medium without probes as a negative control was incubated with HepG2 cells for 6 hours, respectively, so that the probe molecules could fully label the intracellular histone lysine. The cells were washed three times with fresh PBS buffer solution, and then 1 mL of 4% paraformaldehyde was added to the laser confocal dish to fix the cells. After 10 minutes, the cells were washed three times with PBS buffer solution; then 500 μL of cell staining permeabilizer was added, the active ingredient of which was 0.4% Triton X-100. After incubation for 10 minutes, PBS buffer solution was added to wash the cells three times. Then S-tetrazine-BODIPY was dissolved in PBS buffer solution to prepare a solution with a final concentration of 20 μM, and the solution was incubated with the cells for 1 hour without washing, and the images excited at 488 nm were collected by confocal laser scanning microscopy. DAPI was added as a nuclear localization dye for co-staining to confirm the labeling position of the probe on the post-translational modification of benzoylated proteins.

[0108] The negative control without probe was denoted as -Probe+BDP FL Tz.

[0109] The experimental groups with probes were recorded as +4a+BDP FL Tz, +4b+BDP FL Tz, +4c+BDP FL Tz, +5b+BDP FL Tz, +5c+BDP FL Tz, +6b+BDP FL Tz, and +6c+BDP FL Tz.

[0110] DAPI stands for 4',6-diamidino-2-phenylindole, a fluorescent dye.

[0111] Confocal imaging of living cells: All confocal dishes were imaged on a confocal laser scanning microscope, equipped with a Ti2-E inverted microscope, LU-NV series laser unit. The fluorescent probe molecule S-tetrazine-BODIPY was excited using the 488 nm laser line, and images were acquired using a resonant scanner and DU4 detector 560LP 517 / 50. The built-in Zeiss denoising function immediately removed Poisson noise from the images. Z-stacks were acquired in 0.20 μm steps from fixed cells and triple-labeled samples, and all were presented in maximum intensity projections. The same brightness and contrast adjustments were made for samples that were directly compared to each other to ensure that the relative intensity between samples remained unchanged. Five areas were selected for each confocal dish imaged, and the fluorescence intensity was statistically analyzed by Image J to further compare the intensity of the fluorescence signal.

[0112] Results and Analysis:

[0113] Confocal live cell imaging of the 1 h click reaction of methylcyclopropenebenzoic acid probes 4a, 4b-6b, 4c-6c with S-tetrazine-BODIPY, as shown in Figure 5 Images of 460 nm emitting DAPI and 518 nm emitting probes are presented together with merged images, and imaging was performed in at least three technical replicates. Figure 5 In the above figure, Merge means merging images.

[0114] The experimental results show that the methylcyclopropene benzoic acid probes 4a, 4b~6b, 4c~6c can react with S-tetrazine-BODIPY in cells by IEDDA, ​​and co-localization with DAPI can show that there are labeling signals for histone lysine. The fluorescence intensity is quantified by taking the average fluorescence intensity of five regions in the green fluorescence channel as follows.

[0115] By comparing the fluorescence intensity, we can get Figure 6 The average fluorescence intensity of methylcyclopropene benzoic acid probe 4b has a maximum value of 95.46AU±2.6AU, and the imaging effect is the best. This shows that the methylcyclopropene benzoic acid probe can be used for fixed cell nucleolus staining, and the preliminary imaging results lay the foundation for further exploration of experimental conditions.

[0116] Based on the successful realization of the IEDDA reaction of methylcyclopropene benzoic acid derivatives, it was applied to the experiment of dynamically detecting the post-translational modification of benzoylated proteins in living cells.

[0117] HepG2 cell line was used, and the group without adding methylcyclopropenebenzoic acid probe was first used as the control group, which was marked as +BDPFL Tz.

[0118] In the first group, nicotinamide was added before the probe was added to incubate the cells to inhibit the debenzoylation of SIRT2, and an intracellular fluorescence labeling experiment was performed, which was recorded as +Nicotinamide+4b+BDP FL Tz.

[0119] The second group added the same concentration of methyl benzoate at the same time as the methylcyclopropene benzoic acid probe 4b and performed an intracellular fluorescence labeling experiment, which was recorded as +4b+Methyl benzoate+BDP FLTz.

[0120] The third group added methylcyclopropenebenzoic acid probe 4b for the experiment, which was recorded as +4b+BDP FL Tz.

[0121] The images were collected by confocal laser scanning microscopy. Figure 7 .

[0122] Results and analysis: The fluorescence intensity of the green fluorescence channel was quantified as follows.

[0123] like Figure 8 Compared with the fluorescence signal of the third group after adding methylcyclopropene benzoic acid probe 4b, the SIRT2 inhibitor nicotinamide treatment group, i.e. the first group, showed an increase in fluorescence intensity due to benzoylation enrichment; the fluorescence signal of the second group, i.e. after adding methyl benzoate for competitive inhibition, weakened, indicating that the methylcyclopropene benzoic acid probe molecule 4b has the potential for dynamic detection of post-translational modification of benzoylated proteins in living cells.

[0124] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A methylcyclopropenebenzoic acid probe, characterized in that: The methylcyclopropene benzoic acid probe is any one of compound 4, compound 5 and compound 6: Among them, R1 is -COOCH3 substituted at the ortho position, meta position or para position; R2 is -COOH substituted at the meta position or para position; R3 is -COOCH2OCOCH3 substituted at the meta position or para position.

2. A method for preparing the methylcyclopropenebenzoic acid probe according to claim 1, characterized in that: The following steps are involved: Compound 1 reacts with vinyltrimethylsilane under the catalysis of metal palladium to obtain a precursor compound 2 of cyclopropane; The cyclopropane precursor compound 2 reacts with bromoform and hexadecyltrimethylammonium bromide to obtain compound 3 through a carbene addition reaction with the double bond; Compound 3 reacts with iodomethane under the action of n-butyl lithium to obtain intermediate 3-1; intermediate 3-1 is eliminated by tetrabutylammonium fluoride to obtain compound 4; Compound 4 is hydrolyzed under the action of lithium hydroxide to remove the methyl ester group to obtain compound 5; Compound 5 undergoes a substitution reaction with bromomethyl acetate to mask the carboxyl group with an acetoxy group to obtain compound 6; The specific synthetic route is as follows:

3. The method for preparing the methylcyclopropenebenzoic acid probe according to claim 2, characterized in that: The preparation method of the cyclopropane precursor compound 2 comprises the following steps: Compound 1 is mixed with a metal palladium catalyst and silver nitrate in a solvent 1, triethylamine and vinyltrimethylsilane are added, and the mixture is reacted at 70°C±5°C under a protective atmosphere to obtain a cyclopropane precursor compound 2; The molar ratio of compound 1 to metal palladium catalyst, silver nitrate, triethylamine and vinyltrimethylsilane is 1: 0.01-0.05: 1-1.5: 1.5: 1.8-2.5; the first solvent is acetonitrile.

4. The method for preparing the methylcyclopropenebenzoic acid probe according to claim 2, characterized in that: The preparation method of compound 3 comprises the following steps: The precursor compound 2 of cyclopropane is mixed with bromoform and hexadecyltrimethylammonium bromide in solvent 2, and then an alkali solution is added to perform a carbene addition reaction with a double bond at room temperature to obtain compound 3; The molar ratio of compound 2 to bromoform and hexadecyltrimethylammonium bromide is 1:4-5:0.1-0.3; the molar ratio of compound 2 to alkali is 1:10-12; the concentration of the alkali solution is 50wt%±5wt%; the second solvent is dichloromethane; and the alkali is sodium hydroxide or potassium hydroxide.

5. The method for preparing the methylcyclopropenebenzoic acid probe according to claim 2, characterized in that: The preparation method of compound 4 comprises the following steps: Compound 3 is mixed evenly in solvent three, cooled to -75 to -80°C under a protective atmosphere, and then iodomethane and n-butyl lithium are added under stirring to react to obtain intermediate 3-1; intermediate 3-1 and tetrabutylammonium fluoride are subjected to elimination reaction in solvent three to obtain compound 4; The molar ratio of compound 3 to methyl iodide and n-butyl lithium is 1:1.8-2:1.5-2; The molar ratio of the intermediate 3-1 to tetrabutylammonium fluoride is 1:1.5-2; the solvent three is tetrahydrofuran.

6. The method for preparing the methylcyclopropenebenzoic acid probe according to claim 2, characterized in that: The preparation method of compound 5 comprises the following steps: Compound 4 is reacted with lithium hydroxide in solvent 4 by stirring to hydrolyze and remove the methyl ester group to obtain compound 5; The molar ratio of compound 4 to lithium hydroxide is 1:4.5-5; solvent 4 is a mixed solution of tetrahydrofuran and water.

7. The method for preparing the methylcyclopropenebenzoic acid probe according to claim 2, characterized in that: The preparation method of compound 6 comprises the following steps: Compound 5 is subjected to a substitution reaction with bromomethyl acetate and N,N-diisopropylethylamine in solvent 5 to mask the carboxyl group with an acetoxy group to obtain compound 6; The molar ratio of compound 5 to bromomethyl acetate and N,N-diisopropylethylamine is 1:2-2.2:2-2.5; and solvent five is acetonitrile.

8. A fluorescent probe, characterized in that The fluorescent probe is generated by reacting a methylcyclopropenebenzoic acid derivative with S-tetrazine-BODIPY through an IEDDA cycloaddition reaction to generate a fluorescent probe for releasing a fluorescent signal; The synthetic reaction equation of the fluorescent probe is as follows: Among them, the compound shown in formula I is a methylcyclopropene benzoic acid derivative; the compound shown in formula II is a fluorescent probe; the methylcyclopropene benzoic acid derivative is the methylcyclopropene benzoic acid probe described in claim 1, and R is selected from R1, R2 or R3.

9. Use of the methylcyclopropenebenzoic acid probe according to claim 1 or the fluorescent probe according to claim 8 in preparing a reagent for detecting post-translational modification of benzoylated proteins.

Images