Beta-glucuronidase fluorescent probe with large stokes shift based on aie mechanism and preparation method and application thereof

Abstract

Disclosed is a fluorescent probe for glucose oxidase β The application discloses a kind of with big stokes shift The structure of the fluorescent probe QM-GLU is shown as formula I.The QM-GLU has almost no fluorescence at 560 nm when excited by a 450 nm wavelength, but can selectively undergo a fluorescence opening reaction with GLU.The fluorescence intensity at 560 nm gradually increases with the increase of GLU concentration, and other common interfering substances do not significantly interfere with the detection of GLU by QM-GLU and have no effect.Due to the characteristics of its AIE mechanism, and has a large stokes shift (110 nm), which greatly reduces the interference of system background fluorescence and improves the sensitivity of GLU activity detection.Therefore, QM-GLU is suitable for high selectivity and high sensitivity fluorescence detection of GLU.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a fluorescent probe QM-GLU based on the AIE mechanism that can be used for β-glucuronidase, its preparation method, and its application. Background Technology

[0002] β Glucuronidase (GLU) is a lysosomal enzyme widely distributed in various tissues, especially abundant in the intestine. It hydrolyzes the glucuronic acid bonds at the non-reducing ends of glycosaminoglycans, leading to the degradation of proteoglycans and the destruction of the extracellular matrix, ultimately promoting tumor invasion and metastasis. It also plays a crucial role in the enterohepatic circulation of drugs and endogenous substances. Abnormal overexpression of GLU is associated with the development of various cancers, including liver cancer, colon cancer, prostate cancer, kidney cancer, and cervical cancer; therefore, GLU has been considered an important tumor biomarker. Thus, detecting GLU activity in vivo is extremely important for early tumor diagnosis, real-time imaging, and treatment evaluation.

[0003] Currently reported methods for measuring GLU activity include colorimetry, electrochemical methods, and fluorescence sensing. Among these, fluorescence sensing based on fluorescent probes is increasingly being applied to in-situ detection and imaging of GLU due to its advantages such as high spatiotemporal resolution, strong anti-interference ability, simple operation, and non-destructive detection of cells, tissues, and organisms. However, the fluorescent probes developed for GLU detection generally suffer from drawbacks such as small Stokes shift, poor tissue penetration, high excitation light interference, autofluorescence interference, and fluorescence quenching (ACQ). Aggregation-induced emission (AIE) fluorescent dyes have rotatable structures or twisted propeller-shaped conformations. In aqueous solutions (under physiological conditions), they emit strong fluorescence due to the restriction of intramolecular motion, enabling real-time long-term tracking and in-situ imaging of organisms.

[0004] Therefore, developing a GLU fluorescent probe with AIE characteristics and a large Stokes shift is particularly important for realizing real-time, long-term imaging and tracking detection of endogenous GLU activity. Summary of the Invention

[0005] One object of the present invention is to provide a fluorescent probe molecule with AIE properties, large Stokes shift, high sensitivity and high selectivity—QM-GLU.

[0006] The QM-GLU provided by this invention has the structural formula shown in Formula I:

[0007] Another object of the present invention is to provide a method for preparing QM-GLU as shown in Formula I.

[0008] The preparation method of QM-GLU provided by this invention is shown in the flowchart below. Figure 1 Specifically, it includes the following steps: 1) Reaction of quinaldine iodoethane with malononitrile produces compound 1; 2) React compound 1 with p-hydroxybenzaldehyde to generate QM-OH; 3) React p-hydroxybenzaldehyde with acetyl bromide α -D-glucuronide methyl ester reacts to form compound 2; 4) After the reaction of compound 2 with sodium borohydride is complete, phosphorus tribromide is added to generate compound 3; 5) React compound 3 with QM-OH to generate compound 4; 6) React compound 4 with sodium methoxide to obtain compound QM-GLU as shown in Formula I;

[0009] In step 1) of the above method, the specific method for reacting quinaldine iodoethane with malononitrile to generate compound 1 is as follows: quinaldine iodoethane is completely dissolved in ethanol, malononitrile and sodium ethoxide are added in an ice-water bath (0°C) and stirred for 0.5 h, and then the reaction continues to obtain compound 1; wherein, the molar ratio of quinaldine iodoethane, malononitrile and sodium ethoxide is 1:1.5:1.5, the reaction temperature is 25°C and the reaction time is 3.5 h.

[0010] In step 2) of the above method, the specific method for reacting compound 1 with p-hydroxybenzaldehyde to generate QM-OH is as follows: under nitrogen protection, compound 1 and p-hydroxybenzaldehyde are dissolved in anhydrous acetonitrile, piperidine is added dropwise with stirring, and the mixture is heated to reflux to obtain QM-OH; wherein, the molar ratio of compound 1, p-hydroxybenzaldehyde and piperidine is 1:1:5, the reaction temperature is 85℃, and the reaction time is 8 h.

[0011] In step 3) of the above method, p-hydroxybenzaldehyde is reacted with acetyl bromide. α The specific method for reacting -D-glucuronide methyl ester to generate compound 2 is as follows: Under nitrogen protection, p-hydroxybenzaldehyde and acetyl bromide- α -D-glucuronide methyl ester was dissolved in anhydrous acetonitrile, silver oxide was added, and the reaction was carried out in the dark to give compound 2; wherein, the p-hydroxybenzaldehyde and acetyl bromide- α The molar ratio of methyl D-glucuronide to silver oxide is 1.7:1:9.2, the reaction temperature is 25℃, and the reaction time is 8 h.

[0012] In step 4) of the above method, after the reaction of compound 2 with sodium borohydride is complete, phosphorus tribromide is added to generate compound 3. The specific method is as follows: First step: Under ice-water bath, compound 2 is dissolved in dichloromethane / methanol (4:1.2). v / v In the first step, a mixed solution was reacted with sodium borohydride. After the reaction was completed, the reaction was quenched with saturated ammonium chloride solution, extracted with dichloromethane, dried with anhydrous Na2SO4, and the solvent was removed by rotary evaporation to obtain a white solid. In the second step, the obtained white solid was dissolved in dichloromethane solution and reacted with phosphorus tribromide under nitrogen protection and an ice-water bath to obtain compound 3. In the first step, the molar ratio of compound 2 and sodium borohydride was 1:2, the reaction temperature was 0℃, and the reaction time was 1 h. In the second step, the molar ratio of white solid to phosphorus tribromide was 1:2, the reaction temperature was 0℃, and the reaction time was 3 h.

[0013] In step 5) of the above method, the specific method for reacting compound 3 with QM-OH to generate compound 4 is as follows: under a nitrogen atmosphere, compound 3 and QM-OH are dissolved in N,N-dimethylformamide, potassium carbonate is added, and the reaction is carried out at room temperature to obtain compound 4; wherein, the molar ratio of compound 3, QM-OH and K2CO3 is 1:1:2, the reaction temperature is 25℃, and the reaction time is 18 h.

[0014] In step 6) of the above method, compound 4 is reacted with sodium methoxide to obtain compound QM-GLU as shown in Formula I. The specific method is as follows: under an ice-water bath, dissolve compound 4 in dichloromethane / methanol (1:2, v / v In a mixed solution, sodium methoxide solution is added dropwise to react and compound QM-GLU is obtained; wherein the molar ratio of compound 4 to sodium methoxide is 1:10, the reaction temperature is 0℃, and the reaction time is 1 h.

[0015] Another object of the present invention is to provide the use of QM-GLU.

[0016] The QM-GLU application provided by this invention is selected from at least one of the following 1)-8): 1) Fluorescent probes made of QM-GLU; 2) Applications of QM-GLU as a fluorescent probe or as a fluorescent probe for detecting GLU; 3) Chemical sensors containing QM-GLU; 4) Application of QM-GLU in the preparation of chemical sensors or chemical sensors for detecting GLU; 5) Application of QM-GLU in GLU detection; 6) Application of the fluorescent probe described in 1) above in the detection of GLU; 7) Application of the chemical sensor described in 3) above in the detection of GLU.

[0017] 8) Application of QM-GLU in the screening and / or evaluation of GLU inhibitors.

[0018] The fluorescent probe and chemical sensor can be used for the detection and / or fluorescence imaging of GLU.

[0019] The GLU is either exogenous GLU or endogenous GLU.

[0020] In some embodiments of the present invention, the fluorescent probe or chemical sensor may be applied to cells (such as cells containing endogenous GLU) or animals (such as tumor-bearing mice).

[0021] In some embodiments of the present invention, the fluorescent probe or chemical sensor can be used for fluorescence imaging of endogenous GLU in cancer cells.

[0022] In a specific embodiment of the present invention, the cancer cells are human liver cancer cells (HepG2 cells) or human colon cancer cells (LoVo cells).

[0023] In some embodiments of the present invention, the animal is a liver cancer-bearing mouse.

[0024] In a specific embodiment of the present invention, the animal is a HepG2 tumor-bearing mouse.

[0025] The inventors of this invention experimentally demonstrated that, using 450 nm as the excitation wavelength, QM-GLU itself does not emit fluorescence at 560 nm. However, when QM-GLU and GLU coexist, the system exhibits a fluorescence emission peak at 560 nm. As the GLU concentration gradually increases (0–52.1 μg / mL), the fluorescence emission intensity at 560 nm gradually increases, and the fluorescence emission intensity F at 560 nm… 560 nm The method exhibits a good linear relationship with GLU concentration, forming an excellent optical system with a large Stokes shift characteristic (110 nm) and good sensitivity and selectivity for GLU. The detection limit is 0.56 ng / mL. Therefore, QM-GLU is suitable for the highly selective and sensitive detection of GLU, which can be performed using fluorescence spectroscopy.

[0026] Meanwhile, QM-GLU exhibits excellent selectivity for the fluorescence response of GLU, and is effective against common ions and interfering substances (Ca). 2+ Zn 2 + Mg2+ Fe 3+ Na + K + Mn 2+ H2O2, NO2 - CO3 2- SO4 2- I - ,Br - S 2- ,Gln,Gly,GSH,Cys,Met,Arg, β -galactosidase, α The presence of γ-glucosidase, lysozyme, elase, tyrosinase, and chymotrypsin (GLU) provides minimal interference with GLU determination, thus eliminating the influence of numerous interfering ions and species on the detection results, resulting in high detection specificity. Furthermore, when using QM-GLU for GLU detection, the high sensitivity necessitates only a small sample volume, broadening the application range of this method. Attached Figure Description

[0027] Figure 1 This is a flowchart of the preparation process for QM-GLU.

[0028] Figure 2 The 1H NMR spectrum of compound 1.

[0029] Figure 3 This is the carbon NMR spectrum of compound 1.

[0030] Figure 4 This is the high-resolution mass spectrum of compound 1.

[0031] Figure 5 The 1H NMR spectrum of QM-OH is shown.

[0032] Figure 6 The carbon NMR spectrum of QM-OH is shown.

[0033] Figure 7 This is a high-resolution mass spectrometry of QM-OH.

[0034] Figure 8 The 1H NMR spectrum of compound 2.

[0035] Figure 9 This is the carbon NMR spectrum of compound 2.

[0036] Figure 10 This is the high-resolution mass spectrum of compound 2.

[0037] Figure 11 The 1H NMR spectrum of compound 3 is shown.

[0038] Figure 12 The image shows the carbon NMR spectrum of compound 3.

[0039] Figure 13 This is the high-resolution mass spectrum of compound 3.

[0040] Figure 14 The 1H NMR spectrum of compound 4.

[0041] Figure 15 This is the carbon NMR spectrum of compound 4.

[0042] Figure 16 This is the high-resolution mass spectrum of compound 4.

[0043] Figure 17 This is the hydrogen NMR spectrum of QM-GLU.

[0044] Figure 18 This is the carbon NMR spectrum of QM-GLU.

[0045] Figure 19 This is a high-resolution mass spectrum of QM-GLU.

[0046] Figure 20 Spectral characteristics of QM-GLU (5 μM) and QM-OH (5 μM) in mixed solvents (water / ethanol) with different water contents are shown. (a) and (b) are the UV spectra of QM-GLU and QM-OH, respectively; (c) is the fluorescence spectrum of QM-GLU; (d) is the relative fluorescence intensity of QM-GLU in the mixed solvent. I / I 0) Relationship between water content and solvent composition, inset: QM-GLU under UV light at different water contents (f w (e) Fluorescence spectrum of QM-OH; (f) Relationship between relative fluorescence intensity (I / I0) of QM-OH in mixed solvents and solvent composition. Inset: QM-OH under UV light at different water contents (f... w The image below. Where: I It is the fluorescence intensity at 560 nm. I λ is the fluorescence intensity of QM-GLU or QM-OH at 560 nm in 0% water. ex / λ em = 450 nm / 560 nm.

[0047] Figure 21 (a) and (b) are the UV absorption and fluorescence spectra of the QM-GLU coexisting system with GLU (52.1 μg / mL) before and after the reaction, respectively; (c) is the fluorescence spectrum of the QM-GLU coexisting system with different concentrations of GLU; (d) is the fluorescence intensity F at 560 nm of the QM-GLU coexisting system with different concentrations of GLU.560 nm Graph showing the trend of GLU concentration, with inset: F 560 nm Linearity graphs between QM-GLU and different concentrations of GLU (0-5.2 μg / mL); (e) Fluorescence intensity F at 560 nm of the coexistence system of QM-GLU and different concentrations of GLU. 560 nm (f) Linear relationship of QM-GLU and GLU (52.1 μg / mL) coexisting systems at different concentrations; (f) Linear relationship of the Michaelis equation for different concentrations of QM-GLU and GLU (52.1 μg / mL).

[0048] Figure 22 The graph shows the fluorescence intensity changes of the coexisting system of QM-GLU and GLU (52.1 μg / mL) and the system of QM-GLU alone at different pH (a) and different temperatures (b).

[0049] Figure 23 (a) Fluorescence spectra of different concentrations of baicalin in coexistence systems with QM-GLU (5 μM) and GLU (52.1 μg / mL); (b) Inhibition rate of different concentrations of baicalin on GLU.

[0050] Figure 24 This demonstrates the selectivity of QM-GLU for GLU fluorescence detection.

[0051] Figure 25 The cell survival rate was determined by co-incubating HepG2 cells or LoVo cells with different concentrations of QM-GLU (0~100 μM).

[0052] Figure 26 Confocal fluorescence imaging of endogenous GLU in HepG2 cells. (a) Fluorescence imaging of HepG2 cells co-incubated with QM-GLU (25 μM) for different time periods; (b) Fluorescence imaging of HepG2 cells pretreated with baicalin (650 μM) and incubated with QM-GLU (25 μM) for different time periods; (c) and (d) are the average fluorescence intensities corresponding to Figure (a) and Figure (b), respectively.

[0053] Figure 27 shows confocal fluorescence imaging of endogenous GLU in LoVo cells. (a) Fluorescence imaging of LoVo cells co-incubated with QM-GLU (25 μM) for different time periods; (b) Fluorescence imaging of LoVo cells pretreated with baicalin (650 μM) incubated with QM-GLU (25 μM) for different time periods; (c) and (d) are the average fluorescence intensities corresponding to Figure (a) and Figure (b), respectively.

[0054] Figure 28 Laser confocal imaging of QM-GLU for long-term tracking of endogenous GLU in HepG2 and LoVo cells.

[0055] Figure 29 Laser confocal imaging of QM-GLU tracing endogenous GLU in tumor-bearing mice. (a) Fluorescence imaging after QM-GLU (100 μL, 50 μM) was injected into the tumor of HepG2 tumor-bearing mice; (b) Fluorescence imaging after the GLU inhibitor Baicalin (50 μL, 650 μM) was injected into the tumor of HepG2 tumor-bearing mice, incubated for 40 min, and then QM-GLU (100 μL, 50 μM) was injected into the peritoneal cavity of the tumor-bearing mice.

[0056] Figure 30 Laser confocal imaging of the whole body and individual organs of tumor-bearing mice after oral administration of QM-GLU. (a) Fluorescence imaging of HepG2 tumor mice after oral administration of 200 μL QM-GLU (100 μM); (b) Fluorescence imaging of different organs (including heart, liver, spleen, lung, kidney and small intestine) of tumor-bearing mice. Detailed Implementation

[0057] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0059] Example 1: Preparation of the chemical sensor molecule QM-GLU The reaction process is as follows Figure 1 As shown, the specific method is as follows: Quinaldinyl iodoethane (300 mg, 1 mmol) was completely dissolved in 20 mL of ethanol. Malononitrile (99.1 mg, 1.5 mmol) and sodium ethoxide (102 mg, 1.5 mmol) were added under ice-water bath conditions (0 °C), and the mixture was stirred for 0.5 h. The reaction was then carried out at room temperature (25 °C) for 3.5 h. After the reaction was complete, a yellow solid precipitated. The solid was filtered, and the filter cake was washed with ice-cold ethanol to give compound 1 in 45% yield.

[0060] Under nitrogen protection, compound 1 (117.6 mg, 0.5 mmol) and p-hydroxybenzaldehyde (61.1 mg, 0.5 mmol) were dissolved in 5 mL of anhydrous acetonitrile. Piperidine (250 μL, 2.5 mmol) was added dropwise with stirring, and the mixture was refluxed at 85 °C for 8 h. After the reaction was complete, the mixture was cooled to room temperature and filtered to give an orange-yellow solid compound QM-OH in 57% yield.

[0061] Under nitrogen protection, p-hydroxybenzaldehyde (103.7 mg, 0.85 mmol) and methyl acetyl bromide-α-D-glucuronide (198 mg, 0.5 mmol) were dissolved in 5 mL of anhydrous acetonitrile, and silver oxide (534 mg, 4.6 mmol) was added. The reaction was carried out at room temperature (25 °C) in the dark for 8 h. After the reaction was completed, the mixture was filtered, rotary evaporated under reduced pressure, redissolved in ethyl acetate, washed with saturated sodium chloride solution, extracted with ethyl acetate, dried over anhydrous Na₂SO₄, and the solvent was removed by rotary evaporation to give compound 2 in 47% yield.

[0062] Compound 2 (110 mg, 0.25 mmol) was dissolved in DCM / MeOH (4:1.2). v / v In a mixed solution, sodium borohydride (18.9 mg, 0.5 mmol) was slowly added to the reaction system under ice-water bath (0 °C). After reacting for 1 h, the reaction was quenched with saturated ammonium chloride solution. The mixture was extracted with dichloromethane, dried over anhydrous Na₂SO₄, and the solvent was removed by rotary evaporation to obtain a white solid. Under nitrogen protection, the white solid (110 mg, 0.25 mmol) was dissolved in anhydrous dichloromethane. Phosphorus tribromide (47 μL, 0.5 mmol) was added dropwise under ice-water bath (0 °C). After reacting for 3 h, the reaction was quenched with saturated ammonium chloride solution. The mixture was extracted with ethyl acetate, dried over anhydrous Na₂SO₄, and the solvent was removed by rotary evaporation to obtain compound 3, with a yield of 74%.

[0063] Under nitrogen protection, compound 3 (68 mg, 0.14 mmol) and QM-OH (47.5 mg, 0.14 mmol) were dissolved in DMF, and K2CO3 (40 mg, 0.28 mmol) was added. The reaction was carried out at room temperature for 18 h. After the reaction was complete, the mixture was extracted with ethyl acetate, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The mixture was then purified by column chromatography, with dichloromethane / ethyl acetate as the eluent (15:1). v / v Compound 4 was obtained with a yield of 26%.

[0064] Compound 4 (120 mg, 0.16 mmol) was dissolved in 9 mL of DCM / MeOH (1:2). v / vIn a mixed solution, sodium methoxide (54 mg, 1.6 mmol) was added at 0 °C and the mixture was stirred for 1 h. Then, Amberlite IR-120 plus (H) was added. + Adjust the pH to 5, filter, remove the solvent by rotary evaporation, and purify by column chromatography. The eluent is ethyl acetate / methanol (10:1). v / v The yellow compound QM-GLU was obtained in 47% yield.

[0065] NMR and high-resolution mass spectrometry identification results of compound 1: 1 H NMR (DMSO- d 6,500 MHz) = 8.89 (d, J = 8.5 Hz, 1H); 8.05 (d, J = 8.9 Hz, 1H); 7.90 (t, J = 8.5 Hz, 1H); 7.59 (d, J =7.8 Hz, 1H); 6.80 (s, 1H); 4.48-4.43 (m, 2H); 2.66 (s, 3H); 1.35 (t, J = 6.2Hz, 3H). 13 C NMR (DMSO- d 6, 125 MHz) = 153.4, 151.5, 138.9, 134.4, 126.1, 125.7, 121.6, 121.5, 120.2, 118.9, 110.1, 46.8, 44.1, 22.1, 14.3. The 1H and 1C NMR spectra are shown below. Figure 2 and Figure 3 Instrument model: Bruker Avance. HR-MS (ESI, m / z) calibrated for C 15 H 13 N3 + [M+Na] + : 258.1007, found: 258.1008. See results. Figure 4 Instrument model: UPLC-Q / TOF Xevo G2-XS. The above results confirm that the obtained compound is indeed the target compound 1.

[0066] NMR and high-resolution mass spectrometry identification results of QM-OH: 1 H NMR (DMSO- d 6, 500 MHz): = 9.96 (s, 1H), 8.85 (dd, J 1 = 8.5 Hz J 2 = 1.3 Hz, 1H), 8.01 (d, J = 8.9 Hz, 1H), 7.86 (td, J 1 = 7.3 Hz J 2 = 1.4 Hz, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.53 (t, J = 8.0 Hz, 1H), 7.30(d, J = 15.7 Hz, 1H), 7.24 (d, J = 15.7 Hz, 1H), 6.94 (s, 1H), 6.81(d, J = 8.7 Hz,2H), 4.53-4.48 (m, 2H), 1.37 (t, J = 7.1 Hz, 3H). 13 C NMR (DMSO- d 6, 125 MHz) =159.3, 152.1, 149.5, 139.9, 137.8, 133.5, 129.9 (2C), 126.3, 125.1, 124.7, 120.6, 118.0, 116.8, 115.7 (2C), 106.4, 48.6, 46.5, 43.7, 13.6. The 1H and 1C NMR spectra are shown below. Figure 5 and Figure 6 Instrument model: Bruker Avance. HR-MS (ESI, m / z) cacld for C 22 H 17 N3O + [M+H] + 340.1450, found: 340.1444. See results below. Figure 7 Instrument model: UPLC-Q / TOF XevoG2-XS. The above results confirm that the obtained compound is indeed the target compound QM-OH.

[0067] NMR and high-resolution mass spectrometry identification results of compound 2: 1 H NMR (CDCl3, 500 MHz) = 9.92 (s, 1H), 7.85 (d, J= 8.6 Hz, 2H), 7.11 (d, J = 8.6 Hz, 2H), 5.36 (d, J = 7.9 Hz, 2H), 5.30 (d, J = 5.0 Hz, 2H), 4.25 (d, J = 6.0 Hz, 1H), 3.71 (s, 3H), 2.05 (s, 9H). 13 CNMR (CDCl3, 125 MHz) =190.8, 170.2, 169.4, 169.3, 166.8, 132.1, 132.0 (2C), 117.0 (2C), 98.2, 72.9, 71.7, 71.1, 68.9, 53.2, 20.7 (2C), 20.6. The 1H and 1C NMR spectra are shown below. Figure 8 and Figure 9 Instrument model: Bruker Avance. HR-MS (ESI, m / z) cacld for C 20 H 22 O 11 + [M+Na] + : 461.1056, found: 461.1060. See results. Figure 10 Instrument model: UPLC-Q / TOFXevo G2-XS. The above results confirm that the obtained compound is indeed the target compound 2.

[0068] NMR and high-resolution mass spectrometry identification results of compound 3: 1 H NMR (CDCl3, 400 MHz) = 7.32 (d, J =5.8 Hz, 2H), 6.95 (d, J = 5.8 Hz, 2H), 5.32-5.36 (m, 2H), 5.26-5.28 (m, 1H),5.15 (d, J = 4.9 Hz, 1H), 4.47 (s, 2H), 4.18-4.19 (m, 1H), 3.72 (s, 3H), 2.05(s, 3H), 2.04 (s, 3H), 2.03 (s, 3H). 13 C NMR (CDCl3, 100 MHz) = 170.2, 169.5, 169.3, 166.9, 156.7, 133.1, 130.6 (2C), 117.4 (2C), 99.1, 72.8, 71.9, 71.1, 69.2, 53.1, 33.2, 20.7, 20.6. The 1H and 1C NMR spectra are shown below. Figure 11 Figure 12 Instrument model: Bruker Avance. HR-MS (ESI, m / z) calibrated for C 22 H 23 BrO 10 + [M+Na] + : 525.0372, found: 525.0370. See results. Figure 13 Instrument model: UPLC-Q / TOF Xevo G2-XS. The above results confirm that the obtained compound is indeed the target compound 3.

[0069] NMR and high-resolution mass spectrometry identification results of compound 4: 1 H NMR (CD2Cl2, 600 MHz) = 9.08 (d, J = 7.8 Hz, 1H), 7.78 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.56 (d, J = 7.2 Hz, 2H),7.47 (s, 1H), 7.40 (d, J = 7.2 Hz, 2H), 7.31 (d, J = 15.0 Hz, 1H), 7.12 (s, 1H), 7.08-6.92 (m, 5H), 5.39-5.33 (m, 1H), 5.30-5.22 (m, 2H), 5.21 (s, 1H), 5.07(s, 2H), 4.39 (d, J = 6.0 Hz, 2H), 4.22 (d, J = 9.0 Hz, 1H), 3.71 (s, 3H), 2.05 (s, 3H), 2.03 (s, 6H), 1.54 (s, 3H). 13 C NMR (CD2Cl2, 150 MHz) = 170.22,169.76, 169.57, 167.31, 160.62, 156.92, 153.73, 148.90, 140.08, 138.63,133.55, 132.18, 129.69, 129.60, 128.50, 127.00, 124.85, 121.87, 120.76,119.53, 117.72, 117.49, 116.82, 115.66, 107.80, 99.39, 72.88, 71.99, 71.28,69.94, 69.51, 53.25, 50.63, 44.47, 20.79, 20.69, 14.10. The 1H and 1C NMR spectra are shown below. Figure 14 and Figure 15 Instrument model: Bruker Avance. HR-MS (ESI, m / z) calibrated for C 42 H 39 N3O 11 + [M+Na] + : 784.2482, found: 784.2480. See results below. Figure 16 Instrument model: UPLC-Q / TOF Xevo G2-XS. The above results confirm that the obtained compound is indeed the target compound 4.

[0070] NMR and high-resolution mass spectrometry identification results of QM-GLU: 1 H NMR (DMSO- d 6,400 MHz) = 8.90 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 8.8 Hz, 1H), 7.91 (t, J = 7.6 Hz, 1H), 7.76 (d, J = 8.8Hz, 2H), 7.59 (t, J = 7.6 Hz, 1H), 7.39 (s, 1H), 7.37 (s, 3H), 7.08 (d, J = 8.8Hz, 2H), 7.05 (d, J = 8.4 Hz, 3H), 7.00 (s, 1H), 5.31 (s, 1H), 5.10 (s, 2H),5.09(s, 1H), 4.85 (d, J= 7.2 Hz, 1H), 4.55 (q, J = 7.0 Hz, 2H), 3.43 (d, J = 10.0Hz, 1H), 3.30-3.20 (m, 2H), 3.16 (q, J = 9.6 Hz, 1H), 1.39 (t, J = 7.2 Hz, 3H). 13 CNMR (DMSO- d 6, 100 MHz,) = 172.2, 159.8, 157.4, 152.2, 149.5, 139.5, 137.9, 133.7, 129.8, 129.8, 129.2, 127.9, 125.1, 124.9, 120.6, 118.1, 116.3, 115.3, 106.6, 100.3, 76.7, 73.6, 73.2, 72.1, 69.1, 46.6, 43.8, 13.7. The 1H and 1C NMR spectra are shown below. Figure 17 Figure 18 Instrument model: Bruker Avance. HR-MS (ESI, m / z) calibrated for C 35 H 31 N3O8 + [M+Na] + : 644.2009, found: 644.2016. See results. Figure 19 Instrument model: UPLC-Q / TOF Xevo G2-XS. The above results confirm that the obtained compound is indeed the target compound QM-GLU.

[0071] Example 2: QM-GLU as an analytical reagent for fluorescence detection of GLU. 1. Sensitivity of QM-GLU for fluorescence detection of GLU Weigh an appropriate amount of QM-GLU and dissolve it in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 1.0 mM, labeled as QM-GLU stock solution. Dissolve GLU in a certain amount of phosphate buffer solution (PBS, 10 mM, pH = 6.0) to prepare a stock solution with a concentration of 1 mg / mL, labeled as GLU stock solution.

[0072] Add 20 μL of reagent-QM-GLU stock solution and an appropriate volume of GLU stock solution to a 5 mL plastic EP tube, then add an appropriate amount of PBS / ethanol mixture (8:2). v / v The concentrations of GLU in each test system were 0, 1, 2.6, 4.2, 5.2, 10.4, 15.6, 20.9, 26.1, 31.3, 36.5, 41.7, 47, and 52.1 μg / mL, respectively, using PBS (10.0 mM, pH = 6.0). The concentration of QM-GLU was 5 μM. After incubation at 37 °C for 30 min, the system was transferred to a 1 cm quartz cell, and the UV absorption and fluorescence spectra of the reaction system were measured.

[0073] Figure 20 The spectral characteristics of QM-GLU (5 μM) and QM-OH (5 μM) in mixed solvents (water / ethanol) with different water contents are shown. Figure 20 (a) and Figure 20 (b) The UV spectra of QM-GLU and QM-OH are shown in the figure. It can be seen from the figure that both QM-GLU and QM-OH have two peaks in the UV-Vis absorption range (300-500 nm), located at about 345 nm and 437 nm, respectively. Figure 20 (c) shows the fluorescence spectrum of QM-GLU; Figure 20 (d) represents the relative fluorescence intensity of QM-GLU in the mixed solvent. I / I 0) Relationship between solvent composition and solvent composition. From Figure 20 (c) and Figure 20 (d) It can be seen that QM-GLU hardly emits fluorescence under different water content systems. Figure 20 (e) is the fluorescence spectrum of QM-OH; Figure 20 (f) is a graph showing the relationship between the relative fluorescence intensity (I / I0) of QM-OH in a mixed solvent and the solvent composition. From... Figure 20 (e) and Figure 20 (f) It can be seen that when the water content is ≤70%, QM-OH exhibits almost no fluorescence at 560 nm. However, when the water content exceeds 70%, the fluorescence intensity of the system rapidly increases, reaching its maximum value when the water content reaches 80%, producing a bright yellow fluorescence. Compared with the pure ethanol system, the fluorescence intensity of QM-OH is enhanced by 42 times, indicating that QM-OH is an organic fluorescent molecule exhibiting the AIE phenomenon.

[0074] Figure 21 (a) and (b) are the UV absorption and fluorescence spectra of the QM-GLU and GLU (52.1 μg / mL) coexisting system before and after the reaction, respectively. The excitation wavelength of the system was 450 nm, and the emission wavelength was 560 nm. Figure 21As can be seen from (a) and (b), the maximum absorption wavelength of QM-GLU is at 437 nm. When GLU is added, the ultraviolet absorption wavelength of the system redshifts to 450 nm. QM-GLU itself has no obvious fluorescence at 560 nm, but when GLU is added to the system, the fluorescence intensity at 560 nm of the system increases significantly. Figure 21 In the middle (c) and (d), respectively, the fluorescence spectra of QM-GLU coexisting with different concentrations of GLU and the fluorescence intensity F at 560 nm of the QM-GLU coexisting with different concentrations of GLU coexisting system are shown. 560 nm The graph shows the trend of GLU concentration. When the GLU concentration gradually increases from 0 μg / mL to 26.1 μg / mL, the fluorescence intensity of the probe QM-GLU at 560 nm gradually increases, and the fluorescence intensity remains stable in the range of 26.1-52.1 μg / mL. Figure 21 (d) Illustration is F 560 nm Linearity graphs showing the relationship between the fluorescence intensity F at 560 nm and different concentrations of GLU (0–5.2 μg / mL) within the range of 0–5.2 μg / mL. 560 nm The fluorescence intensity was linearly related to the GLU concentration, with the linear equation being Y = 75.123X + 62.553. The limit of detection was calculated as 0.56 ng / mL by dividing the standard deviation of the blank signal by three times. Figure 21(e) shows the fluorescence intensity F at 560 nm of the coexistence system of QM-GLU and different concentrations of GLU. 560 nm A graph showing how the relationship changes over time. Figure 21 (e) It can be seen that at different GLU concentrations (0 μg / mL, 2.6 μg / mL, 13.1 μg / mL and 52.1 μg / mL), the fluorescence intensity of the QM-GLU and GLU coexistence system gradually increased with time. When the GLU concentration was 52.1 μg / mL, the fluorescence intensity of the system basically stabilized at 30 min. Figure 21 (f) shows the linear relationship of the Michaelis-Menten equation for different concentrations of QM-GLU and GLU (52.1 μg / mL). The maximum enzymatic reaction rate V was obtained from the Michaelis-Menten equation. max It is 2.967 μM / min, and the Michaelis constant (k m The concentration was 63.237 μM, indicating that GLU has a strong binding and catalytic ability for QM-GLU.

[0075] Figure 22(a) The fluorescence intensity changes of the coexisting system of QM-GLU and GLU (52.1 μg / mL) and the system containing QM-GLU alone at different pH values ​​are shown in the figure. It can be seen from the figure that when the pH range is 4.0–10.0, QM-GLU shows no obvious fluorescence at 560 nm, and its fluorescence intensity hardly changes with pH. When GLU is added to the system, the fluorescence intensity reaches its highest value at pH = 6.0, and there is still a relatively high fluorescence intensity in the system at pH = 6.5, indicating that QM-GLU is suitable for fluorescence recognition of GLU under weakly acidic conditions. Figure 22 (b) shows the fluorescence intensity changes of the coexisting system of QM-GLU and GLU (52.1 μg / mL) and the system of QM-GLU alone at different temperatures. The figure shows that at four different temperatures (25℃, 30℃, 37℃, and 45℃), QM-GLU alone did not show significant fluorescence; when QM-GLU and GLU coexisted, the system emitted the strongest fluorescence at 37℃, indicating that 37℃ is the optimal reaction temperature for QM-GLU and GLU.

[0076] Figure 23 (a) and (b) show the fluorescence spectra of different concentrations of baicalin in coexisting systems with QM-GLU (5 μM) and GLU (52.1 μg / mL), respectively, and the inhibition rate of different concentrations of baicalin on GLU. Figure 23 As shown in (a) and (b), the fluorescence intensity of the reaction system at 560 nm gradually decreased with increasing baicalin concentration (0-1000 μM), and the relative activity of GLU decreased with increasing baicalin concentration. The degree of inhibition was determined according to (F0-F1) / (F0-F2), where F0 represents the fluorescence intensity in the absence of baicalin, F1 represents the fluorescence intensity in the presence of baicalin, and F2 represents the fluorescence intensity in the absence of both baicalin and GLU. Based on the correlation between inhibition efficiency and baicalin concentration, the IC50 was determined. 50 The value was 650.964 μM. Therefore, the probe QM-GLU shows great promise in the evaluation and screening of GLU inhibitors.

[0077] 2. Specificity of QM-GLU for fluorescence detection of GLU Simultaneously, take several 5 mL EP tubes and perform similar operations as above, except that instead of adding GLU, add various common interfering ions or substances. Samples 1-27 correspond to: Ca 2+ Zn 2+ Mg 2+ Fe 3+ Na+ K + Mn 2+ H2O2, NO2 - CO3 2- SO4 2- I - ,Br - S 2- Gln, Gly, GSH, Cys, Met, Arg (interfering ion or substance concentration of 100 μM), β -galactosidase, α -glucosidase, lysozyme, elase, tyrosinase, chymotrypsin (all at 20 equivalents) and no interfering substances (blank). Results are shown in [link to results]. Figure 24 .from Figure 24 As can be seen, before the addition of GLU, only the probe QM-GLU (5 μM) and various interfering ions or substances were present in the system, and the fluorescence intensity of the system did not change significantly, indicating that the aforementioned interfering ions or substances did not have a significant fluorescence response to the probe QM-GLU. Subsequently, GLU (52.1 μg / mL) was added to the systems 1-27 above, and the fluorescence intensity of the system at 560 nm increased significantly, indicating that the probe QM-GLU has a good response to GLU. This experimental phenomenon shows that the presence of the aforementioned interfering ions and substances will not significantly interfere with or affect the detection of GLU by QM-GLU as an analytical reagent. As a detection reagent, QM-GLU has high selectivity for the fluorescence detection of GLU.

[0078] 3. Performance comparison of QM-GLU with other GLU fluorescent probes The fluorescence detection performance of QM-GLU on GLU was summarized and compared with that of other GLU fluorescent probes in the literature, and the results are shown in Table 1. Table 1 shows that most reported probes exhibit fluorescence quenching (ACQ) caused by aggregation, which may result in low photostability. QM-GLU, however, exhibits AIE characteristics, a large Stokes shift (110 nm), and good detection sensitivity (0.56 ng / mL), and its overall performance is superior to that of similar probes reported in the literature.

[0079] Table 1. Performance comparison of QM-GLU and GLU fluorescent probes in the literature.

[0080] ND indicates no detection limit.

[0081] References [1]Wang J. M., Zhang L., Su Y. L., Qu Y., Cao Y. P., Qin W. W., LiuY., A novel fluorescent probe strategy activated by β -glucuronidase forassisting surgical resection of liver cancer. Anal. Chem., 2022, 94, 7012-7020.[2]Yu Z. X., Zhang J. X., Chen J. X., Zhao L. Y., Yu D. B., Liu L., DongS. J., A new fluorescent probe tool: ERNathG. Anal. Chem., 2023, 95, 4261-4265. [3]Jin Y. Z., Tian X. G., Jin L. L., Cui Y. L., Liu T., Yu Z. L., HuoX. K., Cui J. N., Sun C. P., Wang C., Ning J., Zhang B. J., Feng L., Ma X.C., Highly specific near-infrared fluorescent probe for the real-timedetection of β -glucuronidase in various living cells and animals. Anal. Chem.,2018, 90, 3276-3283. [4]Briciu-Burghina C., Heery B., Regan F., Continuous fluorometricmethod for measuring β -glucuronidase activity: comparative analysis of threefluorogenic substrates.Analyst, 2015,140, 5953-5964. [5]Feng L., Yang Y. L., Huo X. K., Tian X. G., Feng Y. J., Yuan H.W., Zhao L. J., Wang C., Chu P., Long F. D., Wang W., Ma X. C., Highlyselective NIR probe for intestinal β -glucuronidase and high-throughputscreening inhibitors to therapy intestinal damage. ACS Sens., 2018, 3, 1727-1734. [6]Huo X. K., Tian X. G., Li Y. N., Feng L., Cui Y. L., Wang C., CuiJ. N., Sun C. P., Liu K. X., Ma X. C., A highly selective ratiometricfluorescent probe for real-time imaging of β -glucuronidase in living cells andzebrafish. Sens. Actuators B: Chem., 2018, 262, 508-515. [7]Cheng T. C., Roffler S. R., Tzou S. C., Chuang K. H., Su Y. C.,Chuang C. H., Kao C. H., Chen C. S., Harn I. H., Liu K. Y., Cheng T. L., LeuY. L., An activity-based near-infrared glucuronide trapping probe for imaging β -glucuronidase expression in deep tissues.J. Am. Chem. Soc., 2012, 134,3103-3110. [8]Lu S. M., Li G. L., Lv Z. X., Qiu N. N., Kong W. H., Gong P.W.,Chen G., Xia L., Guo X. X., You J. M., Wu Y. N., Facile and ultrasensitivefluorescence sensor platform for tumor invasive biomaker β -glucuronidasedetection and inhibitor evaluation with carbon quantum dots based on inner-filter effect.Biosens. Bioelectron., 2016, 85, 358-362. [9]Chen M., Cheng K. W., Chen Y. J., Wang C. H., Cheng T. C., ChangK. C., Kao A. P., Chuang K. H., Real-time imaging of intestinal bacterial β -glucuronidase activity by hydrolysis of a fluorescent probe.Sci. Rep.,2017,7, 3142.

[10] Magro G., Bain E. S. R., Woodall A. C., Matthews L. R., Gundry W.S., Davis P. A., Synthesis and application of resorufin β -D-glucuronide, alow-cost chromogenic substrate for detecting escherichia coli in drinkingwater. Environ. Sci. Technol., 2014, 48, 9624-9631.

[11] Heery B., Briciu-Burghina C., Zhang D., Duffy G., Brabazon D., O’Connor N., Regan F., ColiSense, today's sample today: a rapid on-sitedetection of β -D-glucuronidase activity in surface water as a surrogate for E.coli. Talanta, 2016, 148, 75-83.

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[13] Gong P.W., Sun L., Wang F., Liu X. C., Yan Z. Q., Wang M. Z.,Zhang L., Tian Z. Z., Liu Z., You J. M., Highly fluorescent N-doped carbondots with two-photon emission for ultrasensitive detection of tumor markerand visual monitor anticancer drug loading and delivery. Chem. Eng. J., 2019,356, 994-1002.

[14] Wei XH, Wu QP, Feng Y., Chen ML, Zhang SH, Chen MT, Zhang JM, Yang GZ, Ding YD, Yang XJ, Ye QH, Zhang YX, Gu QH, Wang J., Wu S., Pang R., Li Y., Off-on fluorogenic substrate harnessing ESIPTand AIE features for in situ and long-term tracking of β -glucuronidase inEscherichia coli, Sens. Actuators B: Chem., 2020, 304, 127242.

[15] Tong CY, Cai GH, Wei QS, Cao YX, Chen YX, Shi S Y.,Highly specific β glucuronidase light-up natural fluorescent probe with aggregation-induced emission and excited-state intramolecular proton transfer for inhibitors screening and in situ imaging in Escherichia coli, Microchem.J., 2022, 174, 107104. Example 3: QM-GLU as an analytical reagent for in situ imaging and long-term tracking imaging of GLU in cells. Given the excellent performance of QM-GLU in in vitro experiments, we further investigated its ability to track GLU in living cells. First, using human hepatocellular carcinoma cells (HepG2 cells) and human colon cancer cells (LoVo cells) as cell models, we evaluated the cytotoxicity of QM-GLU using the CCK-8 assay. The results are shown below. Figure 25As can be seen from the figure, after treatment with different concentrations of QM-GLU (0~100 μM), the survival rates of HepG2 cells and LoVo cells were both greater than 85%, indicating that QM-GLU has low toxicity to HepG2 cells and LoVo cells and good biocompatibility, which supports the further application of QM-GLU.

[0082] HepG2 cells and LoVo cells overexpressing GLU were used as model cells to further investigate the imaging ability of QM-GLU on endogenous GLU. The culture and imaging procedures for HepG2 and LoVo cells were as follows: HepG2 cells and LoVo cells (5 × 10⁻⁶ cells) were cultured at 37℃. 5 Cells (cells / mL) were placed in sterile culture dishes and cultured in DMEM medium containing 10% embryonic serum for 12 h, maintaining a 5% CO2 atmosphere. The cells were then transferred to 96-well plates and incubated overnight before being divided into three groups. Group 1: HepG2 and LoVo cells were each incubated with 25 μM QM-GLU at 37°C, and timed, with confocal imaging performed every 10 min. Group 2: HepG2 and LoVo cells were incubated with 650 μM baicalin at 37°C for 1 h, followed by incubation with 25 μM QM-GLU at 37°C, and timed, with confocal imaging performed every 10 min. Group 3: HepG2 and LoVo cells were each incubated with 25 μM QM-GLU at 37°C for 30 min, and timed, with confocal imaging performed every 3 h.

[0083] Figure 26 (a) and (b) are fluorescence imaging images of HepG2 cells co-incubated with QM-GLU (25 μM) for different times and fluorescence imaging images of HepG2 cells pretreated with baicalin (650 μM) incubated with QM-GLU (25 μM) for different times, respectively. Figure 26 (c) and (d) are respectively Figure 26 (a) and (b) show the average fluorescence intensity. Figure 27 (a) and (b) are fluorescence images of LoVo cells co-incubated with QM-GLU (25 μM) for different times and fluorescence images of LoVo cells pretreated with baicalin (650 μM) incubated with QM-GLU (25 μM) for different times, respectively. Figure 27 (c) and (d) are respectively Figure 27 The average fluorescence intensity corresponding to (a) and (b). From Figure 26 and Figure 27It can be seen that the fluorescence intensity of QM-GLU (25 μM) co-incubated with two types of cancer cells (HepG2 cells and LoVo cells) gradually increased with time. This is because GLU promotes the formation of nanoaggregates of QM-OH released from deglycosylation within the cells, resulting in the AIE effect. In contrast, HepG2 cells showed a greater increase in fluorescence intensity than LoVo cells. Figure 26 c and Figure 27 c) indicates that HepG2 cells have more endogenously expressed GLU than LoVo cells. However, after HepG2 or LoVo cells pretreated with the GLU inhibitor baicalin (650 μM) and then co-incubated with QM-GLU (25 μM), the intracellular fluorescence signal significantly decreased and eventually disappeared, indicating that GLU activity was completely inhibited by baicalin. These results show that QM-GLU can be activated by overexpressed GLU in tumor cells, enabling in situ monitoring of GLU activity. Furthermore, QM-GLU can be used for visual screening of GLU inhibitors. Based on the good results of in situ imaging of endogenous GLU activity in HepG2 and LoVo cells, we further investigated the ability of QM-GLU to long-term trace GLU activity in HepG2 and LoVo cells; the results are shown in [Figure number missing]. Figure 28 .from Figure 28 As can be seen, significant fluorescence signals appeared in HepG2 and LoVo cells after 3 h of incubation with QM-GLU (25 μM). Even after 6 h of incubation, the fluorescence intensity of the probe remained stable. This is because QM-GLU interacts with endogenous GLU in the cells to generate QM-OH, which forms nanoaggregates in situ, making it difficult for these aggregates to diffuse out of the cells. When the incubation time was increased to 9 h, although the intracellular fluorescence intensity decreased slightly, the signal obtained by QM-GLU was confined to a localized region with low background fluorescence, still demonstrating the ability to perform high-resolution long-term tracking of GLU overexpression activity in tumor cells. These results clearly indicate that the QM-GLU probe can be used for long-term tracking imaging of endogenous GLU.

[0084] Example 4: In situ imaging of GLU in tumor-bearing mice using QM-GLU as an analytical reagent. We further investigated the ability of QM-GLU to perform in situ imaging of GLU in tumor-bearing mice. First, HepG2 tumor-bearing mice were constructed as follows: HepG2 cells in logarithmic growth phase were selected, and trypsin was added to digest the cells. Complete culture medium was added to terminate the digestion. The cell suspension was transferred to 15 mL centrifuge tubes, centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were resuspended in 3 mL of pre-chilled PBS. The cells were centrifuged and washed twice to remove residual serum. Finally, PBS was added to resuspend the cells, and the cell count was adjusted to 4 × 10⁶ cells / mL.6 200 μL of cell suspension was injected subcutaneously into nude mice and observed once a day until the tumor grew to a diameter of about 7-9 mm, thus successfully constructing HepG2 tumor-bearing mice.

[0085] The following experiments were conducted on the HepG2 tumor-bearing mice: 1) QM-GLU (100 μL, 50 μM) was injected into the tumors of the HepG2 tumor-bearing mice, and images were taken every 10 min; 2) The GLU inhibitor Baicalin (50 μL, 650 μM) was injected into the tumors of the HepG2 tumor-bearing mice, incubated for 40 min, and then QM-GLU (100 μL, 50 μM) was injected into the peritoneal cavity of the tumor-bearing mice. Images were taken every 10 min; 3) 200 μL of QM-GLU (100 μM) was orally administered to the HepG2 tumor-bearing mice, and images were taken every 10 min. Finally, the tumor-bearing mice were euthanized, and in vitro fluorescence imaging was performed on different organs (including heart, liver, spleen, lung, kidney, and small intestine). The excitation wavelength for the above fluorescence imaging was 480 nm, and the emission wavelength was 560 nm.

[0086] Figure 29 This is a laser confocal imaging image of QM-GLU tracing endogenous GLU in tumor-bearing mice. (Comparison) Figure 29 (a) and Figure 29 (b) We found that when QM-GLU (100 μL, 50 μM) was injected into the tumor of HepG2 tumor-bearing mice, the fluorescence in the flank of the mice gradually increased over time. When the GLU inhibitor Baicalin (50 μL, 650 μM) was injected into the tumor of HepG2 tumor-bearing mice and incubated for 40 min, and then QM-GLU (100 μL, 50 μM) was injected into the peritoneal cavity of the tumor-bearing mice, no obvious fluorescence was produced in the flank of the mice. This was because Baicalin significantly inhibited the activity of GLU in the tumor-bearing mice.

[0087] Figure 30 Laser confocal imaging of the whole body and individual organs in tumor-bearing mice after oral administration of QM-GLU. Figure 30(a) It can be seen that after oral administration of 200 μL QM-GLU (100 μM) to HepG2 tumor mice, a large area of ​​fluorescence appeared in the abdomen of the mice. Through dissection and in vitro fluorescence imaging of different organs (including heart, liver, spleen, lung, kidney, and small intestine), we found that the heart, liver, spleen, lung, and kidney showed almost no fluorescence, while the small intestine showed obvious fluorescence. This is because after QM-GLU enters the body, it is activated by GLU in the liver, spleen, kidney, intestine, and tumor cells to emit light, but the fluorescent parent product QM-OH is quickly excreted into the intestine and accumulates in large quantities.

[0088] The above results indicate that the analytical reagent—QM-GLU—has excellent fluorescence properties and can achieve highly sensitive fluorescence detection of GLU.

[0089] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The compound QM-GLU represented by Formula I: 。 2. The method for preparing the compound QM-GLU according to claim 1, comprising the following steps: 1) Reaction of quinaldine iodoethane with malononitrile produces compound 1; 2) React compound 1 with p-hydroxybenzaldehyde to generate QM-OH; 3) React p-hydroxybenzaldehyde with acetyl bromide α -D-glucuronide methyl ester reacts to form compound 2; 4) After the reaction of compound 2 with sodium borohydride is complete, phosphorus tribromide is added to generate compound 3; 5) React compound 3 with QM-OH to generate compound 4; 6) React compound 4 with sodium methoxide to obtain compound QM-GLU as shown in Formula I; 。 3. The preparation method according to claim 2, characterized in that: In step 1), the specific method for reacting quinaldine iodoethane with malononitrile to generate compound 1 is as follows: quinaldine iodoethane is completely dissolved in ethanol, malononitrile and sodium ethoxide are added and stirred for 0.5 h in an ice-water bath (0°C), and then the reaction continues to obtain compound 1; wherein, the molar ratio of quinaldine iodoethane, malononitrile, and sodium ethoxide is 1:1.5:1.5, the reaction temperature is 25°C, and the reaction time is 3.5 h; And / or, in step 2), the specific method for reacting compound 1 with p-hydroxybenzaldehyde to generate QM-OH is as follows: under nitrogen protection, compound 1 and p-hydroxybenzaldehyde are dissolved in anhydrous acetonitrile, piperidine is added dropwise with stirring, and the mixture is heated to reflux to obtain QM-OH; wherein, the molar ratio of compound 1, p-hydroxybenzaldehyde, and piperidine is 1:1:5, the reaction temperature is 85℃, and the reaction time is 8 h; And / or, in step 3), p-hydroxybenzaldehyde is reacted with acetyl bromide. α The specific method for reacting -D-glucuronide methyl ester to generate compound 2 is as follows: Under nitrogen protection, p-hydroxybenzaldehyde and acetyl bromide- α -D-glucuronide methyl ester was dissolved in anhydrous acetonitrile, silver oxide was added, and the reaction was carried out in the dark to give compound 2; wherein, the p-hydroxybenzaldehyde and acetyl bromide- α The molar ratio of methyl D-glucuronide to silver oxide is 1.7:1:9.2, the reaction temperature is 25℃, and the reaction time is 8 h. And / or, in step 4), after the reaction of compound 2 with sodium borohydride is completed, phosphorus tribromide is added to generate compound 3. The specific method is as follows: First step: under ice-water bath, compound 2 is dissolved in a dichloromethane / methanol mixed solution, sodium borohydride is added to react, the reaction is quenched by saturated ammonium chloride solution after the reaction is completed, dichloromethane is extracted, anhydrous Na2SO4 is dried, and the solvent is removed by rotary evaporation to obtain a white solid; Second step: the obtained white solid is dissolved in dichloromethane solution, and under nitrogen protection and ice-water bath, it is reacted with phosphorus tribromide to obtain compound 3; wherein, in the first step, the molar ratio of compound 2 to sodium borohydride is 1:2, the reaction temperature in the first step is 0℃, and the reaction time is 1 h; in the second step, the molar ratio of white solid to phosphorus tribromide is 1:2, the reaction temperature in the second step is 0℃, and the reaction time is 3 h; And / or, in step 5), the specific method for reacting compound 3 with QM-OH to generate compound 4 is as follows: under a nitrogen atmosphere, compound 3 and QM-OH are dissolved in N,N-dimethylformamide, potassium carbonate is added, and the reaction is carried out at room temperature to obtain compound 4; wherein, the molar ratio of compound 3, QM-OH and K2CO3 is 1:1:2, the reaction temperature is 25°C, and the reaction time is 18 h; And / or, in step 6), the specific method for reacting compound 4 with sodium methoxide to obtain compound QM-GLU of formula I is as follows: under an ice-water bath, compound 4 is dissolved in a dichloromethane / methanol mixed solution, and sodium methoxide solution is added dropwise to react and obtain compound QM-GLU; wherein, the molar ratio of compound 4 to sodium methoxide is 1:10, the reaction temperature is 0°C, and the reaction time is 1 h.

4. A fluorescent probe, characterized in that: The fluorescent probe is the compound QM-GLU as described in claim 1.

5. A chemical sensor, characterized in that: The chemical sensor contains the compound QM-GLU as described in claim 1.

6. The use of the compound QM-GLU of claim 1, the fluorescent probe of claim 4, or the chemical sensor of claim 5 in the detection of β-glucuronidase (GLU) or in GLU fluorescence imaging.

7. The use of the compound QM-GLU according to claim 1, wherein the use is selected from at least one of the following: a) Application of QM-GLU as a fluorescent probe or as a fluorescent probe for detecting GLU; b) Application of QM-GLU in the preparation of chemical sensors or in the preparation of chemical sensors for detecting GLU; c) Application of QM-GLU in the screening and / or evaluation of GLU inhibitors.

8. The application according to claim 7, characterized in that: The fluorescent probe or chemical sensor is used for the detection and / or fluorescence imaging of GLU.

9. The application according to claim 7 or 8, characterized in that: The GLU is either exogenous GLU or endogenous GLU.

10. The application according to any one of claims 7-9, characterized in that: The fluorescent probes or chemical sensors are applied to cells or animal bodies; Furthermore, the cells are cancer cells, including but not limited to HepG2 cells and LoVo cells; Furthermore, the animal is a tumor-bearing mouse, including but not limited to liver cancer tumor-bearing mice.

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