Fluorescent probe for detecting ONOO <-> as well as preparation method and application of fluorescent probe

Abstract

The invention belongs to the technical field of biomedical probes. The invention provides a fluorescent probe for detecting ONOO <->. The chemical structural formula of the fluorescent probe is shown as a formula 11; in the formula 11, the R substituent is Cl or Br. The fluorescent probe provided by the invention can be used for detecting and imaging ONOO <-> in tendon stem cells (TDSCs) and tendon lesions. The sensitivity of the fluorescent probe can reach 27.9 nM, meanwhile, the fluorescent probe has excellent stability, has high response speed to ONOO <-> in an aqueous solution, and can be used for detecting different stages of tendon lesions in vivo and treating ONOO <-> changes in a rat model. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical probes and particularly relates to a method for detecting ONOO - Fluorescent probe and preparation method and application thereof. Background Art

[0002] Tendinopathy is one of the most common diseases of the musculoskeletal system and is reported to account for a high proportion (approximately 30%) of general musculoskeletal consultations. The incidence of lower limb tendinopathy is as high as 10.52 cases per 1000 person-years, exceeding that of osteoarthritis (8.4 cases per 1000 person-years) in previous cross-sectional studies. The clinical features of tendinopathy include pain, swelling, and limited mobility, which seriously affect the quality of life of patients and bring about a huge socioeconomic burden. Despite great efforts, the exact pathogenic and therapeutic mechanisms of tendinopathy remain undetermined. Studies in recent years have shown that early therapeutic intervention can effectively promote the repair of damaged tendons and prevent heterotopic ossification. However, due to the absence of pain and functional limitations, early tendinopathy is difficult to diagnose. Therefore, it is necessary and important to find effective methods for early diagnosis of tendinopathy.

[0003] Inflammation is increasingly recognized as a key factor in the development and progression of tendinopathy. High levels of reactive species (ROS / RNS / RCS) are produced in large quantities by inflammatory responses and post-injury stress. - ) is a highly reactive substance that can be produced in living systems by non-enzymatic interactions between nitric oxide (NO) and superoxide anions (O2·-), and its excessive formation can cause cellular dysfunction. Recent studies have shown that the neutralization of ROS plays a key role in regulating the differentiation of tendon stem cells (TDSCs). This targeted intervention can effectively reduce the risk of ectopic calcification. Ectopic calcification is a common problem in tendinopathy. In addition, the disruption of the internal microstructure of the tendon stimulates TDSCs to upregulate the expression of cartilage genes by activating endoplasmic reticulum (ER) stress. However, ONOO - The role of ONOO in the ER during tenogenic and osteogenic differentiation of TDSCs and its potential association with ER stress processes remain unclear. - The fluctuation of tendinopathy is of great significance for the diagnosis and prognosis of tendinopathy.

[0004] Currently, in the case of ONOO - Among the existing probes, few can be used for direct in vivo imaging and are not sensitive enough. For example, Chinese patent ZL202011243835.1 provides an ONOO - Fluorescent probe for ONOO -The detection limit was only 85.7 nM, and the in vivo imaging capability was not investigated. Summary of the invention

[0005] In view of the defects of the prior art, the object of the present invention is to provide a method for treating peroxynitrite (ONOO - ) detection with high sensitivity and specificity.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A method for detecting ONOO - A fluorescent probe, the chemical structure of which is shown in Formula 11:

[0008]

[0009] In Formula 11, the R substituent is Cl or Br.

[0010] Preferably, in Formula 11, the R substituent is Br.

[0011] The present invention also provides a method for preparing the fluorescent probe, which comprises the following steps:

[0012] (1) CH3I, compound 1, and K2CO3 were mixed in DMF, stirred and reacted at 50°C in the dark, and ultrapure water was added after cooling, and extracted with ethyl acetate; the organic phase was washed with brine and dried with Na2SO4, and the solvent was removed and purified by silica gel column chromatography to obtain compound 2;

[0013] (2) AlCl3 and compound 5 are dissolved in C2H2Cl4, stirred for reaction, and then compound 3 is added, followed by stirring and reflux reaction; then ice water is added and stirred; the obtained solid phase is filtered and purified by silica gel column chromatography to obtain compound 6;

[0014] Alternatively, AlCl3 and compound 5 are dissolved in C2H2Cl4, stirred for reaction, and then compound 4 is added, followed by stirring and reflux reaction; then ice water is added and stirred; the obtained solid phase is filtered and purified by silica gel column chromatography to obtain compound 7;

[0015] (3) Compound 2 was added to concentrated sulfuric acid, and then cooled to 0°C, and then Compound 6 was added and stirred vigorously, and then the temperature was raised to react, and then cooled, the reaction precipitate was filtered and washed with water, and purified by silica gel column chromatography to obtain Compound 8;

[0016] Alternatively, compound 2 is added to concentrated sulfuric acid, then cooled to 0°C, compound 7 is added and stirred vigorously, the temperature is raised to react, and then cooled, the reaction precipitate is filtered and washed with water, and purified by silica gel column chromatography to obtain compound 9;

[0017] (4) Compound 8 and Compound 10 are dissolved in DMF, reacted at room temperature, and then concentrated under reduced pressure, and then purified by silica gel column chromatography to obtain the compound of formula 11; wherein the R substituent in formula 11 is Cl;

[0018] Alternatively, compound 9 and compound 10 are dissolved in DMF, reacted at room temperature, and then concentrated under reduced pressure, and then purified by silica gel column chromatography to obtain the compound of formula 11; wherein the R substituent in formula 11 is Br;

[0019] Wherein, the chemical structural formulas of compounds 1 to 10 are shown in Formulas 1 to 10 respectively;

[0020]

[0021] Preferably, in step (1), the added amounts of CH3I, compound 1 and K2CO3 are 4.2 mL, 2.0 g and 3.77 g respectively. During the reaction, the mixture is stirred at 50°C in the dark for 24 hours. After cooling to room temperature, 100 mL of ultrapure water is added, and the mixture is extracted with ethyl acetate.

[0022] Preferably, in step (2), the amount of AlCl3 added is 1.2 g, the amount of compound 5 added is 520 mg, the amount of compound 3 added is 432 mg, and the amount of compound 4 added is 564 mg; when the stirring reflux reaction treatment is carried out, it is treated at 100°C for 10 hours.

[0023] Preferably, in step (3), the amount of compound 2 added is 189 mg, the amount of concentrated sulfuric acid added is 1 mL, the amount of compound 6 added is 146 mg, and the amount of compound 7 added is 168 mg; when the temperature rise reaction is carried out, the reaction is carried out at 90° C. for 1.5 hours.

[0024] Preferably, in step (4), the amount of compound 8 added is 160 mg, the amount of compound 10 added is 130 mg, and the amount of compound 9 added is 176 mg; when reacting at room temperature, the reaction time is 1.5 hours.

[0025] The present invention also provides the above-mentioned one kind of detection ONOO - The fluorescent probe was prepared for detecting ONOO - Application of products in this area.

[0026] Beneficial effects of the present invention:

[0027] The fluorescent probe provided by the present invention can be used to detect and image ONOO in TDSCs and tendinopathy - The sensitivity of the fluorescent probe of the present invention can reach 27.9nM, and it has excellent stability and is sensitive to ONOO in aqueous solution. - It has a fast response rate and can be used to detect ONOO in different stages of tendinopathy and treat ONOO in rat models in vivo - change. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 and Figure 2 Schematic diagram of the synthesis route of the probe compound of the present invention;

[0029] Figure 3 and Figure 4 The probe compound Rod-Cl 1 HNMR and HRMS spectra;

[0030] Figure 5 and Figure 6 The probe compound Rod-Br 1 H NMR and HRMS spectra;

[0031] Figure 7 In the above example, part A and part B are the absence and absence of ONOO - Absorption and fluorescence spectra of Rod-Cl (5 μM) in the presence and absence of ONOO in Parts C and D - (10μM), the absorption and fluorescence spectra of Rod-Br (5μM); λ ex =575 nm, 100 mM PBS containing 0.05% DMSO;

[0032] Figure 8 The probe Rod-Cl and ONOO - Mass spectra before and after the reaction, where part A is the mass spectrum of Rod-Cl and part B is the mass spectrum of Rod-Cl in ONOO - Mass spectrum in positive ion mode in the presence of, part C is Rod-Cl and ONOO - Mass spectrum after the reaction;

[0033] Fig. 9 The probe Rod-Br of the present invention and ONOO - Mass spectra before and after the reaction, where part A is the mass spectrum of Rod-Br and part B is the mass spectrum of Rod-Br in ONOO - Mass spectrum in positive ion mode in the presence of, part C is Rod-Cl and ONOO -Mass spectrum after the reaction;

[0034] Fig.10 The reaction of Rod-Cl and Rod-Br with ONOO under different pH conditions - The fluorescence change results before and after the reaction; the concentrations of Rod-Cl and Rod-Br are both (5μM), λ ex =575 nm, 100 mM PBS containing 0.05% DMSO;

[0035] Fig.11 For the probe of the present invention and ONOO - The reaction time results are shown in Figure 2; the concentrations of Rod-Cl and Rod-Br are both (5 μM), λ ex =575 nm, 100 mM PBS containing 0.05% DMSO;

[0036] Fig.12 The effects of Rod-Cl and Rod-Br on the different concentrations of ONOO - Response results of the graph; Part A shows the different concentrations of ONOO - (0~20μM) fluorescence spectrum of Rod-Cl (5μM), part B is the fluorescence intensity of Rod-Cl (5μM) and ONOO - The linear relationship between the two groups is shown in Figure 2. The response of Rod-Cl (5 μM) to different analytes is shown in Figure 2. The response of Rod-Br (5 μM) to different concentrations of ONOO is shown in Figure 2. - The fluorescence spectrum of (0~20μM), part E is Rod-Br (5μM) and ONOO - (0.1~5μM) linear relationship of fluorescence intensity, part F is the response of Rod-Br (5μM) to different analytes, ONOO - :10μM, HOCl,·OH,NO,·O 2- :25μM, others: 100μM; 100mM PBS, containing 0.05% DMSO, pH 8.0, λ ex =575nm;

[0037] Fig.13 The probe of the present invention is used to detect different concentrations of ONOO - Fluorescence intensity in the range of 0 to 20 μM, where part A is Rod-Cl and part B is Rod-Br; λex = 575 nm, 100 mM PBS containing 0.05% DMSO; Fig.14The fluorescence imaging results of Rod-Br in TDSCs; control group: TDSCs were treated with 5μM of brominated rod (Rod-Br) and 1μg / mL of Hoechst for 10min; SIN-1: TDSCs were first treated with SIN-1 (1mM) for 20min, and then treated with Rod-Br (5μM) and Hoechst (1μg / mL) for 10min; TEMPO: TDSCs were treated with TEMPO (1mM) for 2h, and then treated with Rod-Br (5μM) and Hoechst (1μg / mL) for 10min; AG: TDSCs were treated with AG (1mM) 2h, then treated with Rod-Br (5μM) and Hoechst (1μg / mL) for 10min; Hoechst: λex=405nm, λem=425~480nm; Rod-Br: λex=575nm, λem=590~670nm; Scale bar=10μm; Fig.15 The figure is the time-dependent fluorescence imaging result of Rod-Br in TDSCs; the specific process is: TDSCs cells were treated with 5μM Rod-Br and 1μg / mL Hoechst for 10min, and then treated with 0.1mM SIN-1 for 0-2h, Hoechst: λex=405nm, λem=425~480nm; Rod-Br: λex=575nm, λem=590~670nm; scale bar=10μm;

[0038] Fig.16 The co-localization results of Rod-Br after TDSCs were treated with 10μM Rod-Br and different tracking agents for 20 minutes; Part A is ER-tracker Green (1μM), Part B is Mito-tracker Green (200nM), and Part C is Lyso-tracker Green (100nM); Rod-Br:λ ex =575nm,λ em =590-670nm; ER-tracker Green:λ ex =504nm,λ em =510-580nm, Mito-tracker Green:λ ex =490nm,λ em =500-560nm, Lyso-tracker Green:λ ex =490nm,λ em =500-560nm; scale bar = 10μm;

[0039] Fig.17The results of different probes detecting ROS levels in TDSCs treated with different concentrations of IL-1β (0, 5, 10, 20 ng / mL); Part A is the ROS fluorescence imaging of TDSCs after treatment with DCFH-DA and different concentrations of IL-1β, scale bar = 200 μm, DCFH-DA: FITC channel; Part B is ONOO in TDSCs treated with 5 μM Rod-Br and different concentrations of IL-1β - Fluorescence imaging, scale bar = 10 μm, Rod-Br: λ ex =575nm,λ em =590~670nm; Part C is the fluorescence intensity statistics of DCFH-DA; Part D is the fluorescence intensity statistics of Rod-Br;

[0040] Fig.18 ONOO in TDSCs treated with 5 μM br and different concentrations of IL-1β - Fluorescence imaging results, scale bar = 200 μm, Rod-Br:TRITC;

[0041] Fig.19 This is the H&E staining result of each organ after 24h of Rod-Br (100μM, 50μL); scale bar = 100μM;

[0042] Fig. 20 Exogenous ONOO in living rats - Time-dependent fluorescence imaging results; the left leg was treated with 100 μM Rod-Br, and the right leg was treated with 100 μM Rod-Br and 200 μM ONOO - After treatment, fluorescence was recorded for 1 min; ex =570nm,λ em =640nm; N = 3;

[0043] Fig.21 In the A part, at 15min and 30min, λ ex =570nm,λ em =640nm, the in vivo imaging ONOO-fluctuation results of 100μM Rod-Br treated rat model and tendinopathy on different days; Part B is the immunofluorescence and corresponding H&E staining results of IL-1β in tendinopathy on different days and treated rat model tissues, IL-1β: FITC channel, immunofluorescence and H&E scales are 200μm and 100μm, respectively; Part C is the average fluorescence result of in vivo imaging; Part D is the average fluorescence result of IL-1β immunofluorescence;

[0044] Fig. 22Part A shows the RT-PCR analysis results of the expression of inflammatory factors TNF-α and IL-6; Part B shows the WB analysis results of the expression of endoplasmic reticulum stress markers CHOP and GPR78; Part C shows the RT-PCR analysis results of tendon differentiation markers SCX, TNMD, and COL 1; Part D shows the RT-PCR analysis results of osteogenic differentiation markers BMP-2, ALP, and RUNX2. DETAILED DESCRIPTION

[0045] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technical personnel in this field based on the above invention content still fall within the scope of protection of the present invention.

[0046] Example 1

[0047] 1. Basic experimental materials and experimental methods:

[0048] 1.1 Raw materials and experimental equipment:

[0049] Unless otherwise stated, materials were obtained from commercial suppliers and used without further purification. 1H NMR and 13C NMR spectra were measured using a Bruker AM400 NMR instrument. Proton chemical shifts in NMR spectra are given in ppm relative to the internal reference TMS (1H, 0.00 ppm). HRMS spectral data were recorded on a Bruker Daltonics BioTOF mass spectrometer. Absorption and photoluminescence spectra were performed on Agilent Cary 3500 UV-Vis and Horiba Fluoromax+ fluorescence spectrophotometers, respectively. Cells and tissues were imaged using a white light laser under a Leica TCS SP8 confocal microscope. Animal experiments were performed using PerkinElmer Spectroscopy.

[0050] 1.2 Preparation of cationic and anionic solutions

[0051] Dissolve 10 mmol of inorganic salts {NaCl, KCl, CaCl2·2H2O, CuCl2, ZnCl2, MgCl2·6H2O, FeCl3·6H2O, Na2S·9H2O, glutathione (GSH), cysteine ​​(Cys)} in distilled water (50 mL) to form the corresponding aqueous solution. When necessary, dilute the stock solution with water to the required concentration.

[0052] 1.3 Preparation of reactive oxygen species (ROS) and reactive nitrogen species (RNS)

[0053] Prepare HOCl by diluting commercial NaClO solution with deionized water, using ε292nm=350M-1 cm -1 The concentration of peroxynitrite (ONOO) was determined by spectrophotometry. Hydrogen peroxide was prepared by direct dilution of H2O2 solution. Nitric oxide (NO) was prepared using NOC-9 as raw material. - ), using a spectrophotometer ε302nm=1670M -1 cm -1 The concentration of peroxynitrite was determined. Fe 2+ The solution was added to an excess of H2O2 solution to prepare hydroxyl radicals. Superoxide was generated from KO2 using saturated dimethyl sulfoxide (DMSO) as solvent.

[0054] 1.4 Fluorescence analysis

[0055] 10 mM stock solutions of Rod-Cl and Rod-Br were prepared in dimethyl sulfoxide (DMSO). The 10 mM stock solutions were diluted to make 10 μM final test solutions of Rod-Cl and Rod-Br.

[0056] 1.5 Cell isolation and culture

[0057] Primary tendon stem cells (TDSCs) were isolated from male Sprague Dawley rats, 4–5 weeks old and weighing 100–120 g, with approval from the Animal Care and Utilization Committee of Sichuan University.

[0058] 1.6 Cytotoxicity assay

[0059] Cytotoxicity was determined by CCK-8 assay. TDSCs of passage 3 were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum for 24 hours in a 96-well microplate. Fresh medium containing different concentrations of brominated rods was then added for 24 hours. After the medium was removed, CCK-8 reagent was diluted with fresh DMEM and added to each well. The absorbance was then measured at 450 nm.

[0060] 1.7 Data Analysis

[0061] GraphPad Prism software (version 9; GraphPad Prism, San Diego, CA, USA) was used. Data are presented as mean ± standard deviation (SD). Differences between groups were analyzed using one-way ANOVA, one-way ANOVA, and t-test. For all experiments, *P<0.05 was considered significant.

[0062] 2. Preparation of probe

[0063] The preparation process of the probe of the present invention is as follows Figure 1 and Figure 2 As shown, the specific structure of the corresponding reaction raw material (such as compound 1) in the following specific synthesis scheme is as follows Figure 1 and Figure 2 shown.

[0064] 2.1 Synthesis of Compound 2:

[0065] A mixture of CH3I (4.2 mL, 68.2 mmol), compound 1 (2.0 g, 12.4 mmol) and K2CO3 (3.77 g, 27.3 mmol) was stirred in DMF (100 mL) at 50 °C in a dark room for 24 h. After cooling to room temperature, ultrapure water (100 mL) was added and extracted with ethyl acetate. The organic phase was washed with brine and dried over Na2SO4. The solvent was removed and purified by silica gel column chromatography to obtain compound 1 (1.13 g, 48% yield). 1 H NMR (400MHz, DMSO-d6) δ (ppm): 7.70 (1H, d, J = 8.8Hz), 6.63 (1H, dd, J1 = 2.8Hz, J2 = 9.2Hz ), 6.48 (1H, d, J = 2.4Hz), 3.00 (6H, s), 2.83 (2H, t, J = 6.0Hz), 2.43 (2H, m), 1.95 (2H, m). 13 C NMR (100MHz, DMSO-d6) δ (ppm): 195.18, 153.28, 146.23, 128.33, 120.95, 110.08, 109.40, 38.37, 29.76, 23.17. HRMS m / z: calcd 190.1226, found 190.1228[M+H] + .

[0066] 2.2 Synthesis of compound 6:

[0067] A mixture of AlCl3 (1.2 g, 9.1 mmol) and compound 5 (520 mg, 3.5 mmol) was dissolved in C2H2Cl4, stirred at 0°C for 1 h, and then compound 3 (432 mg, 3.0 mmol) was added. The resulting solution was stirred and refluxed at 100°C for 10 h. After the reaction was completed, the solvent was poured into ice water and stirred continuously. A yellow solid was obtained by filtration and purified by silica gel column chromatography. (0.54 g, yield 62%). 1HNMR (400MHz, DMSO-d6) δ (ppm): 13.20 (1H, s), 11.89 (2H, s), 8.00 (1H, d, J = 7.6Hz), 7.72 (1H,t,J=6.8Hz),7.65(1H,t,J=7.6Hz),7.44(1H,d,J=7.2Hz),6.99(1H,s),6.55(1H,s). 13 C NMR(100MHz,DMSO-d6)δ(ppm):199.35,166.72,162.12,160.04,139.86,132 .86,132.44,130.00,129.94,129.42,127.29,114.13,111.31,103.83.HRMS m / z:calcd291.0066,found 291.0070[M+H] + .

[0068] 2.3 Synthesis of compound 7:

[0069] A mixture of AlCl3 (1.2 g, 9.1 mmol) and compound 5 (520 mg, 3.5 mmol) was dissolved in C2H2Cl4, stirred at 0°C for 1 h, and then compound 4 (564 mg, 3.0 mmol) was added. The resulting solution was stirred and refluxed at 100°C for 10 h. After the reaction was completed, the solvent was poured into ice water and stirred continuously. A yellow solid was obtained by filtration and purified by silica gel column chromatography. (0.66 g, yield 65%). 1 H NMR (400MHz, DMSO-d6) δ (ppm): 13.20 (1H, s), 11.86 (2H, s), 8.00 (1H, d, J = 7.6Hz), 7.73 ( 1H,t,J=6.8Hz),7.65(1H,t,J=6.8Hz),7.45(1H,d,J=7.2Hz),7.14(1H,s),6.54(1H,s). 13 C NMR(100MHz,DMSO-d6)δ(ppm):199.21,166.72,163.06,162.65,160.94,13 9.84,136.00,132.44,129.98,129.44,127.29,114.91,103.59,99.90.HRMS m / z:calcd 334.9561,found 334.9567[MH] - .

[0070] 2.4 Synthesis of Compound 8:

[0071] Compound 2 (189 mg, 1 mmol) was slowly added to concentrated H2SO4 (1 mL) and cooled to 0°C. Compound 6 (146 mg, 0.5 mmol) was then added in portions and stirred vigorously. The reaction mixture was heated at 90°C for 1.5 h, cooled to room temperature, and poured onto ice. The resulting precipitate was filtered and washed with cold water. The solid was purified by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ (ppm): 13.20 (1H, s), 11.86 (2H, s), 8.00 (1H, d, J = 7.6Hz), 7.73 ( 1H,t,J=6.8Hz),7.65(1H,t,J=6.8Hz),7.45(1H,d,J=7.2Hz),7.14(1H,s),6.54(1H,s). 13 C NMR(100MHz,DMSO-d6)δ(ppm):199.21,166.72,163.06,162.65,160.94,13 9.84,136.00,132.44,129.98,129.44,127.29,114.91,103.59,99.90.HRMS m / z:calcd 446.1154,found446.1143[M] + .

[0072] 2.5 Synthesis of compound 9:

[0073] Compound 2 (189 mg, 1 mmol) was slowly added to concentrated H2SO4 (1 mL) and cooled to 0°C. Then, compound 7 (168 mg, 0.5 mmol) was added in portions and stirred vigorously. The reaction mixture was heated at 90°C for 1.5 h, cooled to room temperature, and poured onto ice. The resulting precipitate was filtered and washed with cold water. The solid was purified by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ (ppm): 13.20 (1H, s), 11.86 (2H, s), 8.00 (1H, d, J = 7.6Hz), 7.73 ( 1H,t,J=6.8Hz),7.65(1H,t,J=6.8Hz),7.45(1H,d,J=7.2Hz),7.14(1H,s),6.54(1H,s). 13C NMR(100MHz,DMSO-d6)δ(ppm):199.21,166.72,163.06,162.65,160.94,13 9.84,136.00,132.44,129.98,129.44,127.29,114.91,103.59,99.90.HRMS m / z:calcd 490.0648,found490.0646[M] + .

[0074] 2.6 Synthesis of probe compound Rod-Cl:

[0075] Compound 8 (160 mg, 0.36 mmol) and compound 10 (130 mg, 0.44 mmol) were dissolved in dry DMF and stirred at room temperature for 1.5 h. After the reaction was completed, the mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (56 mg, yield 27%). 1 H NMR (400MHz, DMSO-d6) δ (ppm): 8.19 (1H, d, J = 4.0Hz), 7.84 (1H, t, J = 4.0Hz), 7.75(2H,m),7.56(2H,d,J=4.0Hz),7.41(1H,d,J=3.6Hz),7.00(2H,d,J=4.0 Hz),6.74(2H,m),6.55(1H,s),6.47(1H,s),5.06(2H,dd,J1=10.4Hz,J2=16. 4Hz),3.08(6H,s),2.69(1H,m),2.53(1H,m),2.24(1H,m),2.13(1H,m).HRMS m / z:calcd 580.1693,found 580.1699[M+H] + .

[0076] 2.7 Synthesis of probe compound Rod-Br:

[0077] Compound 9 (176 mg, 0.36 mmol) and compound 10 (130 mg, 0.44 mmol) were dissolved in dry DMF and stirred at room temperature for 1.5 h. After the reaction was completed, the mixture was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (56 mg, yield 23%). 1HNMR (400MHz, DMSO-d6) δ (ppm): 8.19 (1H, d, J = 8.0Hz), 7.85 (1H, t, J = 7.6Hz), 7.76 (2H, dd, J1=8.8Hz, J2=15.6Hz), 7.56(2H,d,J=8.0Hz), 7.42(1H,d,J=7.6Hz), 7.01(2H,d,J=7.6Hz), 6.96(1H,s),6.75(1H,dd,J1=2.4Hz,J2=8.8Hz),6.56(1H,s),6.46(1H,s),5.07(2H,dd,J1= 12.4Hz, J2=19.6Hz),3.09(6H,s),2.67(1H,m),2.55(1H,m),2.23(1H,m),2.13(1H,m).HRMS m / z:calcd 624.1188,found 624.1196[M+H] + .

[0078] 3. Probe detection effect experiment

[0079] 3.1 Cell imaging:

[0080] To detect exogenous ONOO - , TDSCs cultured in the confocal dish at passage 3 were washed with PBS. Subsequently, the cells were exposed to 100 μM SIN-1 for 0.5, 1, 2, and 4 hours. Following this treatment, 5 μM Rod-Br was given for 10 minutes. Finally, the cells were detected and imaged.

[0081] To detect endogenous ONOO - The tendon stem cells (TDSCs) cultured in the confocal culture dish were washed with PBS. The cells were treated with IL-1β0, 5, 10, 20 ng / mL for 48 h or IL-1β20 ng / mL for 1, 3, 7 days, and then 5 μM Rod-Br was added for 10 min. Finally, the cells were detected and imaged.

[0082] To detect the colocalization of Rod-Br at the subcellular level, TDSCs cultured in confocal dishes were washed with PBS. Then, cells were treated with 5 μM Rod-Br and ER Tracker Green / Lyso Tracker Green / Mito Tracker Green for 10 min. Finally, cells were subjected to detection and imaging procedures.

[0083] 3.2 Tendinopathy rat model:

[0084] The rats used in the experiment were approved by the Animal Ethics Committee of West China Hospital, Sichuan University (No.: 20240226307). Eight-week-old male Sprague-Dawley– (SD) rats were randomly divided into four groups (control group, 7-day tendinopathy group, 14-day tendinopathy group, and treatment group), with three rats in each group. First, the rats were anesthetized by intraperitoneal injection of ketamine (80-100 mg / kg). During anesthesia, 50 μL (40 mg / mL) of type I collagenase was injected into the Achilles tendon tissue. Treatment method: One week after modeling, 300 mg / kg of N-acetylcysteine ​​was injected intraperitoneally every 3 days.

[0085] The rats were killed on the 7th and 14th days after modeling. Then the main organs were collected, rinsed with saline and subjected to H&E staining. Tendon tissues from each group were taken for H&E staining and immunofluorescence staining.

[0086] 4. Experimental results

[0087] Rhodamine and its derivatives have attracted extensive attention due to their excellent photophysical properties, such as high photostability and quantum yield. - We synthesized two rhodamine derivatives (Rod-Cl and Rod-Br) using rhodamine derivatives as fluorophores and boric acid as response groups. Chlorine and bromine atoms exhibit different electron-withdrawing effects, thus affecting the pKa of rhodamine derivatives. Since phenylboronic acid is attached to the phenol group of rhodamine derivatives, the fluorescence of Cl and Br is effectively quenched. - After removing phenylboronic acid, a strong fluorescence signal of the probe can be observed.

[0088] 4.1 Rod-Cl and Rod-Br for ONOO - The corresponding:

[0089] In order to study the effects of Rod-Cl and Rod-Br on ONOO - We preliminarily detected their absorption and fluorescence spectra before and after the reaction. Figure 7 As shown in Part A and Part C, the maximum absorption wavelengths of Rod-Cl and Rod-Br in aqueous solution (100mM PBS containing 0.05% DMSO) are 579nm and 583nm, respectively. After reacting with ONOO-, the absorption wavelengths are blue-shifted to 573nm and 574nm, respectively. In addition, after adding ONOO-, the fluorescence of Rod-Cl and Rod-Br at 628nm is significantly enhanced ( Figure 7In the Figures B and D). To confirm that the fluorescence enhancement is due to the release of the phenol structure after the reaction of boric acid with ONOO-, we performed mass spectrometry analysis. As shown in Figures S15 and S16, the mass spectrum peak of Rod-Cl is mainly at 579.9 m / z. After reacting with ONOO-, the main mass spectrum peak shifted to 445.9 m / z, indicating that the reaction product was hydrolyzed into a phenol structure (compound 8). Similar results can also be observed in Rod-Br.

[0090] Typically, the probe and ONOO - The pH value of the reaction between Rod-Cl and Rod-Br and ONOO is crucial for cell imaging. - Fluorescence changes before and after the reaction. Fig.10 As shown in the figure, in the pH range of 5.0-9.0, the fluorescence of Rod-Cl and Rod-Br remained relatively stable. Under physiological pH conditions, both probes showed strong responses to ONOO-. Because reaction time is another key factor affecting the detection of ONOO- in cells. Therefore, the reaction time of the two probes with ONOO- was detected. Fig.11 As shown in Figure 2, within 360 s, the fluorescence of Rod-Cl and Rod-Br was weak, and Rod-Br was more stable than Rod-Cl. - The probe also reacted almost completely within 3 minutes, indicating that ONOO - Quick response.

[0091] Subsequently, the effects of Rod-Cl and Rod-Br on the degradation of ONOO at different concentrations were tested. - Response. Fig.12 Parts A and D of Fig.13 As shown, when ONOO - When the concentration is in the range of 0-20 μM, the fluorescence intensity of Rod-Cl and Rod-Br increases continuously. In addition, in the range of 0.1-5 μM, the fluorescence intensity of Rod-Cl and Rod-Br (R2=0.999) shows a good linear relationship with the concentration of ONOO-, and the detection limits are 53.6 nM and 27.9 nM, respectively. Fig.12 To eliminate potential interferences from various molecular ions and other biomolecules in biological samples, we performed selective experiments. Fig.12 As shown in parts C and F of the figure, other active substances that may interfere (25 μM HOCl, NO, ·OH, ·O2-), metal ions (100 μM Fe 3+ Mg 2+ 、Zn 2+ , Cu 2+ , Ca 2+ , K+ 、Na + ) and biomolecules (100 μM S 2- , GSH, Cys), and especially H2O2 (100 μM), hardly triggered fluorescence. However, we were pleased to observe that even 10 μM ONOO - It can also cause significant fluorescence enhancement in Rod-Cl and Rod-Br. These experimental results show that under physiological conditions, Rod-Cl and Rod-Br can be used for ONOO - Due to its higher sensitivity and faster response time, it will be used for further biological research.

[0092] We then further investigated the detection and imaging capabilities of Rod-Br in TDSCs using confocal microscopy. To evaluate whether Rod-Br could be used to image basal levels of ONOO- in TDSCs, we pretreated cells with AG (a ROS scavenger, 1 mM) or TEMPO (a ·O2 scavenger, 1 mM) for 2 h and then incubated them with Rod-Br. Fig.14 As shown, cells treated with Rod-Br alone showed weak fluorescence. In contrast, cells pretreated with AG or TEMPO showed even weaker fluorescence. - When cells were stimulated with Rod-Br, a significant increase in fluorescence was observed. - Time-dependent fluorescence imaging of SIN-1 at different incubation periods. TDSCs were pretreated with Rod-Br and then incubated with SIN-1 (1 mM) for 0, 0.5, 1, and 2 h. Fig.15 As shown in Figure 3, the increase in SIN-1 pretreatment time corresponds to an increase in fluorescence intensity. It is worth noting that the fluorescence intensity enhancement in the 2-hour group was minimal compared to the 1-hour group. These findings suggest that Rod-Br is a promising target for detecting ONOO in living cells. - sensitive probe.

[0093] 4.2 Colocalization of Rod-Br at the subcellular level

[0094] To determine the organelle-specific targeting ability of Rod-Br, we performed a series of colocalization experiments. Fig.16 TDSCs were incubated with Rod-Br together with various organelle-specific trackers (ER-Tracker Green, Mito-Tracker Green, and Lyso-Tracker Green) as indicated. Fig.16As shown in part A, TDSCs co-stained with ER-tracker Green and Rod-Br showed both green and red fluorescence, with a large overlap between the two fluorescence signals. In addition, the intensity profiles along the blue line across the cells were almost identical, with a Pearson correlation coefficient of 0.90, indicating high targeting specificity for ER. However, minimal fluorescence overlap was observed when TDSCs were co-stained with Rod-Br and other commercial trackers such as Mito-Tracker Green and Lyso-TrackerGreen, with relatively low Pearson correlation coefficients ( Fig.16 These results suggest that Rod-Br accumulates preferentially in the ER and may serve as a marker for detecting ONOO in the ER. - potential probes.

[0095] 4.3ONOO - Imaging studies in the IL-1β-induced TDSC tendinopathy model

[0096] To demonstrate the success of the IL-1β-induced TDSCs tendinopathy model, we first selected the commercially available fluorescent probe DCFH-DA to detect the ROS level in TDSCs treated with different concentrations of IL-1β (0, 5, 10, 20 ng / mL) and found that the fluorescence intensity gradually increased with the increase of IL-1β concentration ( Fig.17 To further test the effect of Rod-Br on endogenous ONOO - To investigate the sensitivity of TDSCs, we also incubated TDSCs with different concentrations of IL-1β (0, 5, 10, 20 ng / mL) for 48 h and then treated them with Rod-Br (5 μM) and Hoechst (1 μg / mL) for 10 min. Fig.17 The B and 3D parts and Fig.18 As shown, compared with the weak fluorescence observed in cells treated with Rod-Br alone, the fluorescence intensity gradually increased with the increase of IL-1β concentration, indicating that ONOO - Taken together, these results indicate that ONOO - ONOO is elevated in the microenvironment of IL-1β-induced tendinopathy, while Rod-Br is able to detect ONOO in injured TDSCs - level.

[0097] 4.4 Observation of ONOO in rat tendinopathy model - Fluctuations and possible mechanisms

[0098] ONOO was successfully detected in IL-1β-induced TDSCs - We then further investigated ONOO -Potential role in tendinopathy in rats. This study is the first to study the effects of Rod-Br on major organs in rats. Rats in the Rod-Br group were treated with 100 μM (50 μL) of Rod-Br for 24 hours, and rats in the control group were treated with the same amount of solvent. The results of H&E staining showed no significant difference compared with the control group, including the heart, liver, spleen, lung, kidney and other organs ( Fig.19 ). We then investigated the effect of exogenous ONOO - Feasibility of in vivo imaging in rats.

[0099] The encouraging result is that Rod-Br can detect ONOO in the Achilles tendon. - , the fluorescence intensity gradually increases with time ( Fig. 20 ). Recent studies have shown that tendon calcification and degeneration caused by tendinopathy, coupled with the resulting pain, are the main causes of movement disorders. Furthermore, during tendinopathy, activation of TDSCs has been identified as an important source of ROS, along with immune cells. Given that Rod-Br detects ONOO in TDSCs in the inflammatory microenvironment, - We are motivated to study ONOO - Potential role in tendinopathy. A rat tendinopathy model was established using type I collagenase. In vivo imaging showed minimal fluorescence in the Achilles tendon of the control group 15 and 30 minutes after the Rod-Br probe was injected into the rat Achilles tendon ( Fig.21 In contrast, the fluorescence of the Achilles tendon was significantly enhanced in the 7th day, 14th day and treatment groups of Achilles tendinopathy, and the fluorescence enhancement was most obvious in the 14th day group. At the same time, the treatment group using the anti-inflammatory drug N-acetylcysteine ​​(NAC) had a significant therapeutic effect on rats with tendinopathy. In addition, we further detected the expression of IL-1β in the Achilles tendon, which is one of the key inflammatory factors in tendinopathy. Fig.21 As shown in Figures B and D, the expression of IL-1β was consistent with the in vivo imaging and H&E staining results. This finding supports the notion that the rat tendinopathy model has been successfully established. Overall, these findings suggest that Rod-Br is a promising candidate for detecting ONOO in tendinopathy. - is effective and indicates that ONOO - Levels can serve as a direct indicator of the extent of inflammation.

[0100] 4.5ONOO - Fluctuations in tendinopathy lead to abnormal differentiation of TDSCs by activating inflammation and endoplasmic reticulum stress

[0101] Encouraged by the above results, we further investigated the possible mechanisms. Excessive oxidative stress can increase the levels of inflammatory factors and ER stress. In addition, ER stress is considered to be a downstream of excessive oxidative stress, and ONOO -However, in IL-1β-induced tendinopathy, ONOO - The relationship between inflammatory factors and ER stress is still unclear. Therefore, we first used RT-PCR to detect the levels of inflammatory factors and then used WB to detect the levels of ER stress at different time points. Fig. 22 As shown in Part A of Figure 3, the expression level of TNF-α gradually increased with the extension of IL-1β exposure time. Interestingly, the expression of IL-6 peaked on day 7 and then decreased on day 14 ( Fig. 22 This phenomenon may be attributed to the dual role of IL-6 as an interleukin, acting both as a pro-inflammatory cytokine and an anti-inflammatory factor. More interestingly, at all observed time points, the prolonged effect of IL-1β was associated with an increase in CHOP and GRP78 protein levels, indicating the onset of ER stress ( Fig. 22 (Part B). It has been reported that abnormal differentiation of TDSCs is one of the causes of tendinopathy. Studies have shown that the accumulation of ROS affects the self-renewal ability of TDSCs and inhibits the expression of tendon-related genes in TDSCs. Therefore, in order to verify the ONOO - To investigate the effects of IL-1β on TDSC differentiation, we examined the gene levels of tenogenic and osteogenic markers at different time points after IL-1β induction. Fig. 22 As shown in the middle C part, the prolonged IL-1β induction time led to a significant inhibition of tenogenic differentiation of TDSCs, including a significant decrease in the expression of tenogenic differentiation markers such as SCX, TNMD and COL I, especially on day 14. In contrast, the expression of osteogenic differentiation markers such as BMP-2, ALP and RUNX2 was enhanced at the same time, especially on day 14 ( Fig. 22 (D in the figure). It has also been reported that the pathological microenvironment of tendon injury can induce endoplasmic reticulum stress, thereby affecting the differentiation of TDSCs. - The intrinsic mechanism of fluctuation may lead to abnormal differentiation of TDSCs by activating inflammation and inducing endoplasmic reticulum stress.

[0102] In summary, we developed two novel fluorescent probes (Rod-Cl and Rod-Br) for the detection and imaging of ONOO in primary TDSCs and tendinopathy. - Among them, Rod-Br has higher sensitivity (27.9nM), stability and kinetics to ONOO in aqueous solution (0.05% DMSO). - In addition, Rod-Br was able to assess the basal level of ONOO- in the ER of TDSCs and measure ONOO- in IL-1β-stimulated TDSCs. -In addition, Rod-Br was able to detect ONOO in different stages of tendinopathy and treatment rat models in vivo. - In addition, ONOO - The intrinsic mechanism of fluctuation in tendinopathy may lead to abnormal differentiation of TDSCs by activating inflammation and inducing endoplasmic reticulum stress. Therefore, the present invention introduces a reliable method to detect ONOO in TDSCs during tendinogenic and osteogenic differentiation. - Dynamic, bringing new perspectives to the understanding and treatment of tendinopathy.

Claims

1. A method for detecting ONOO - A fluorescent probe characterized in that The chemical structure of the fluorescent probe is shown in Formula 11: In Formula 11, the R substituent is Cl or Br.

2. A method for detecting ONOO according to claim 1. - A fluorescent probe characterized in that In Formula 11, the R substituent is Br.

3. A method for detecting ONOO - A method for preparing a fluorescent probe, characterized in that: The fluorescent probe is a detection probe of ONOO according to claim 1 or 2. - The fluorescent probe is prepared by the following steps: (1) CH3I, compound 1, and K2CO3 were mixed in DMF, stirred and reacted at 50°C in the dark, and ultrapure water was added after cooling, and extracted with ethyl acetate; the organic phase was washed with brine and dried with Na2SO4, and the solvent was removed and purified by silica gel column chromatography to obtain compound 2; (2) AlCl3 and compound 5 are dissolved in C2H2Cl4, stirred for reaction, and then compound 3 is added, followed by stirring and reflux reaction; then ice water is added and stirred; the obtained solid phase is filtered and purified by silica gel column chromatography to obtain compound 6; Alternatively, AlCl3 and compound 5 are dissolved in C2H2Cl4, stirred for reaction, and then compound 4 is added, followed by stirring and reflux reaction; then ice water is added and stirred; the obtained solid phase is filtered and purified by silica gel column chromatography to obtain compound 7; (3) Compound 2 was added to concentrated sulfuric acid, and then cooled to 0°C, and then Compound 6 was added and stirred vigorously, and then the temperature was raised to react, and then cooled, the reaction precipitate was filtered and washed with water, and purified by silica gel column chromatography to obtain Compound 8; Alternatively, compound 2 is added to concentrated sulfuric acid, then cooled to 0°C, compound 7 is added and stirred vigorously, the temperature is raised to react, and then cooled, the reaction precipitate is filtered and washed with water, and purified by silica gel column chromatography to obtain compound 9; (4) Compound 8 and Compound 10 are dissolved in DMF, reacted at room temperature, and then concentrated under reduced pressure, and then purified by silica gel column chromatography to obtain the compound of formula 11; wherein the R substituent in formula 11 is Cl; Alternatively, compound 9 and compound 10 are dissolved in DMF and reacted at room temperature, then concentrated under reduced pressure, and then purified by silica gel column chromatography to obtain the compound of formula 11; wherein the R substituent in formula 11 is Br; Wherein, the chemical structural formulas of compounds 1 to 10 are shown in Formulas 1 to 10 respectively; 4. A method for detecting ONOO according to claim 3. - A method for preparing a fluorescent probe, characterized in that: In step (1), the added amounts of CH3I, compound 1, and K2CO3 are 4.2 mL, 2.0 g, and 3.77 g, respectively. During the reaction, the mixture is stirred at 50°C in the dark for 24 hours. After cooling to room temperature, 100 mL of ultrapure water is added, and the mixture is extracted with ethyl acetate.

5. A method for detecting ONOO according to claim 4. - A method for preparing a fluorescent probe, characterized in that: In step (2), the amount of AlCl3 added is 1.2 g, the amount of compound 5 added is 520 mg, the amount of compound 3 added is 432 mg, and the amount of compound 4 added is 564 mg; when the stirring reflux reaction treatment is carried out, it is treated at 100°C for 10 hours.

6. A method for detecting ONOO according to claim 5. - A method for preparing a fluorescent probe, characterized in that: In step (3), the amount of compound 2 added is 189 mg, the amount of concentrated sulfuric acid added is 1 mL, the amount of compound 6 added is 146 mg, and the amount of compound 7 added is 168 mg; when the temperature rise reaction is carried out, the reaction is carried out at 90° C. for 1.5 hours.

7. The method for preparing a fluorescent probe for detecting ONOO- according to claim 6, characterized in that: In step (4), the amount of compound 8 added is 160 mg, the amount of compound 10 added is 130 mg, and the amount of compound 9 added is 176 mg; when reacting at room temperature, the reaction time is 1.5 hours.

8. A method for detecting ONOO according to claim 1 or 2. - The fluorescent probe was prepared for detecting ONOO - Application of products in this area.

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