Quinoxaline-derived Aza-BODIPY AIE fluorescent probe as well as synthesis method and application thereof

Abstract

The invention belongs to the technical field of biomedical detection, and relates to a quinoxaline-derived Aza-BODIPY AIE fluorescent probe, the probe is composed of three parts of fluorophore Aza-BODIPY, rotor benzothiazole and lysosome targeted morpholine, the Aza-BODIPY and benzothiazole are connected through a-C = N-C-group, and the probe is a quinoxaline-derived Aza-BODIPY-AIE fluorescent probe. A precursor intermediate probe 1 is formed by connecting a fluorophore Aza-BODIPY and a rotor benzothiazole through a-C-C = N-group. The invention further discloses a synthesis method of the fluorescent probe and application of the fluorescent probe to lysosome viscosity detection. The probe system disclosed by the invention has significantly increased Stokes shift, and effectively avoids overlapping of excitation / emission spectrums; the fluorescent probe shows typical aggregation-induced emission (AIE) characteristics, and fluorescence enhancement is most remarkable under the condition that the water content is 40-50%; the fluorescent probe has wide-range viscosity response capability (2-610cP), 24 times of fluorescence enhancement can be generated at most, and the detection limit is as low as 0.27 cP; the lysosome co-localization performance is excellent, and the cell compatibility is good; the high-contrast real-time imaging monitoring on the viscosity of the living cell lysosome can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection technology, relates to lysosomal microenvironment detection, and in particular to a quinoxaline-derived Aza-BODIPY AIE fluorescent probe, a synthesis method thereof, and applications thereof. Background Art

[0002] As a key regulator of the cellular microenvironment, viscosity plays a vital role in controlling intracellular diffusion and metabolism. Significant viscosity differences exist between different functional regions within the cell, and each subcellular organelle maintains a different microviscosity profile. Lysosomes are essential organelles in eukaryotic cells, playing a key role in various physiological processes such as protein degradation, substance secretion, plasma membrane repair, and autophagy. Lysosomal viscosity is not only an important indicator of lysosomal function, but also reflects the overall health of the cell. Impaired degradation of lysosomal macromolecules can lead to their abnormal accumulation, resulting in a significant increase in lysosomal viscosity. This change is closely associated with a variety of lysosomal storage diseases and neurodegenerative diseases, making it an important biomarker for diagnosing lysosomal dysfunction.

[0003] Small molecule fluorescent probes have become powerful tools for monitoring microenvironmental changes in biological systems due to their simplicity, non-invasiveness, and excellent spatiotemporal performance. In particular, fluorescent probes based on the molecular rotor mechanism have attracted considerable attention for their potential use in real-time viscosity sensing in biological systems. These probes typically consist of a fluorophore and a molecular rotor, with the fluorophore serving as the signal output unit and the molecular rotor as the viscosity sensing component. Their detection mechanism relies on the degree of restricted intramolecular rotation. Under low viscosity conditions, free intramolecular rotation promotes nonradiative relaxation, leading to fluorescence quenching; whereas high viscosity or steric hindrance restricts molecular rotation, significantly enhancing fluorescence intensity. Among various fluorophores, BODIPY is an ideal platform for constructing high-performance, viscosity-sensitive fluorescent rotors due to its strong absorption and fluorescence properties, high fluorescence quantum yield, and the ability to fine-tune its spectral properties through structural modification.

[0004] However, existing BODIPY-based probes have significant drawbacks, including a small Stokes shift (<30 nm, which can easily lead to spectral crosstalk), a lack of synergistic effects between aggregation-induced emission (AIE) and viscosity response, and insufficient lysosomal targeting, which limit their application in complex biological systems. Therefore, there is an urgent need to develop new fluorescent probes that combine AIE performance, large Stokes shift, precise lysosomal targeting, low toxicity, and the ability to monitor viscosity changes in real time.

[0005] Compared with the traditional BODIPY fluorophore, its heteroatom-substituted derivative Aza-BODIPY exhibits significantly enhanced photophysical properties, including a higher molar extinction coefficient, stronger fluorescence emission, and excellent photostability. Although viscosity probes based on the BODIPY backbone have been widely explored, there is still a gap in the development of viscosity-responsive probes based on the Aza-BODIPY system. Given the excellent optical properties and stability of Aza-BODIPY, the development of viscosity probes based on this structure has great research potential. However, to date, there have been no reports of probes based on the quinoxaline-Aza-BODIPY backbone that simultaneously possess the dual functions of AIE effect and viscosity response, and can achieve highly specific lysosomal targeting and a large Stokes shift. Summary of the Invention

[0006] In response to the technical problems in the prior art such as poor probe targeting, small Stokes shift, lack of aggregation-induced emission (AIE) properties and viscosity-sensitive dual functionality, the first object of the present invention is to disclose a quinoxaline-derived Aza-BODIPY AIE fluorescent probe.

[0007] Technical Solution

[0008] A quinoxaline-derived Aza-BODIPY AIE fluorescent probe, comprising a fluorophore Aza-BODIPY, a rotor benzothiazole, and a lysosome-targeting morpholine. Aza-BOIDPY and benzothiazole are linked by a -C=NC- group and have the following molecular structure:

[0009] .

[0010] In a preferred embodiment of the present invention, the precursor intermediate probe 1 of the quinoxaline-derived Aza-BODIPY AIE fluorescent probe is composed of the fluorophore Aza-BODIPY and the rotor benzothiazole connected by a -CC=N- group, and its molecular structure is shown below:

[0011] .

[0012] In a preferred disclosed example of the present invention, the quinoxaline-derived Aza-BODIPY AIE fluorescent probe has a Stokes shift ≥43 nm (43 nm for probe 1 and 51 nm for probe 2), the AIE effect is strongest at a water content of 40-50% (the AIE effect is strongest when probe 1 is at 50% water content and probe 2 is at 40% water content), and the cell viability is >80% at a concentration of 0-40 μM (the cell viability is >80% at a concentration of probe 1 at 0-40 μM and probe 2 at 0-40 μM).

[0013] The second object of the present invention is to disclose a method for synthesizing the above-mentioned quinoxaline-derived Aza-BODIPY AIE fluorescent probe.

[0014] A method for synthesizing a quinoxaline-derived Aza-BODIPY AIE fluorescent probe comprises the following steps:

[0015] S1. Synthesis of Probe 1: 6,7-Dimethyl-1,4-dihydroquinoxaline-2,3-dione and 2-aminobenzothiazole were dissolved in anhydrous o-dichlorobenzene solvent, titanium tetrachloride (TiCl4) and triethylamine (NEt3) were added sequentially, and the mixture was heated to reflux. After the condensation reaction was complete, boron trifluoride diethyl ether complex (BF3·OEt2) was added to the system and the reflux complexation reaction was continued. After the reaction was completed, the product was separated and purified by silica gel column chromatography to obtain Probe 1.

[0016] S2. Synthesis of Probe 2: Probe 1 and 4-(2-chloroethyl)morpholine were dissolved in anhydrous DMF. Nucleophilic substitution occurred at the benzothiazole nitrogen atom under alkaline conditions and nitrogen protection. After sufficient reaction, the reaction mixture was washed, dried, purified by column chromatography, and concentrated under reduced pressure to obtain probe 2, a quinoxaline-derived Aza-BODIPY AIE fluorescent probe. The reaction equation is:

[0017] .

[0018] In a preferred embodiment of the present invention, in step S1, the molar ratio of the reaction materials 6,7-dimethyl-1,4-dihydroquinoxaline-2,3-dione, 2-aminobenzothiazole, TiCl4, NEt3, and BF3·OEt2 is 0.10-4.00 mmol: 0.40-24.00 mmol: 0.40-28.00 mmol: 1.00-68.00 mmol: 14.00-80 mmol, preferably 0.25 mmol: 1.13 mmol: 1.38 mmol: 3.63 mmol: 3.98 mmol.

[0019] According to a preferred embodiment of the present invention, in step S1, the heating reflux reaction is carried out at 110-180°C for 3-12 hours, preferably at 180°C for 3 hours; and the reflux complexation reaction is carried out at 110-180°C for 3-24 hours, preferably at 180°C for 15 hours.

[0020] In a preferred embodiment of the present invention, in step S1, the sufficient reaction is confirmed by thin layer chromatography (TLC) analysis to confirm the formation of imine.

[0021] In a preferred disclosed embodiment of the present invention, in step S1, the column chromatography purification uses dichloromethane / petroleum ether (DCM / PE) = 2:1 (volume ratio) and pure ethyl acetate (EA) as eluents, and the crude product is purified by silica gel column chromatography.

[0022] According to a preferred embodiment of the present invention, in step S2, the alkaline condition is prepared by placing probe 1, 4-(2-chloroethyl)morpholine and anhydrous potassium carbonate in a container, adding DMF as a reaction solvent, purging with nitrogen and then mixing.

[0023] According to a preferred embodiment of the present invention, in step S2, the molar ratio of the reaction materials probe 1, 4-(2-chloroethyl)morpholine, and anhydrous potassium carbonate is 0.10-2.00 mmol: 0.12-3.40 mmol: 0.12-3.40 mmol, preferably 0.155 mmol: 0.224 mmol: 0.224 mmol.

[0024] In a preferred embodiment of the present invention, in step S2, the sufficient reaction is monitored and confirmed by thin layer chromatography (TLC) to confirm the formation of new compounds.

[0025] According to a preferred embodiment of the present invention, in step S2, the washing and drying is performed by extracting the reaction mixture with dichloromethane (DCM), washing the organic layer three times with water, and drying the combined organic layers with anhydrous sodium sulfate, filtering, and concentrating under reduced pressure to obtain a crude product.

[0026] In a preferred disclosed embodiment of the present invention, in step S2, the column chromatography purification adopts silica gel column chromatography with an elution gradient of "100% dichloromethane → 100% ethyl acetate".

[0027] The third object of the present invention is to disclose the application of the quinoxaline-derived Aza-BODIPY AIE fluorescent probes 1 and 2 in lysosomal viscosity detection.

[0028] In a preferred embodiment of the present invention, the colocalization Pearson coefficient of the probe with the lysosomal dye is ≥0.83 (0.83 for probe 1 and 0.86 for probe 2), the probe exhibits a linear "on" fluorescence response in the viscosity range of 2-610 cP, and the detection limit is as low as ≤0.26 cP (the detection limit is as low as 0.27 cP (probe 1) and 0.26 cP (probe 2) respectively).

[0029] In a preferred embodiment of the present invention, the detection includes real-time dynamic imaging of lysosome viscosity in living cells, and is particularly suitable for detecting abnormal lysosome viscosity induced by dexamethasone.

[0030] A preferred disclosed example of the present invention is used for real-time monitoring of dexamethasone-induced abnormal lysosomal viscosity in living cells, with an excitation wavelength of 488 nm and an emission wavelength of 505-550 nm.

[0031] A lysosomal viscosity detection kit comprises the above-mentioned quinoxaline-derived Aza-BODIPY AIE fluorescent probe and instructions for use.

[0032] The present invention synthesizes probe 1 by one-pot Schiff base condensation. Probe 1 is an AIE-type viscosity probe with quinoxaline-Aza-BODIPY as the fluorescent core and benzothiazole as the rotor. Then, a morpholine group is selectively introduced into the nitrogen atom of the benzothiazole rotor of probe 1 to construct a lysosomal viscosity probe (probe 2) with a benzothiazole / morpholine dual-targeting unit and a base-catalyzed nucleophilic substitution synthesis method thereof. Probe 2 is synthesized by incorporating the lysosomal-targeting morpholine group into the rotatable benzothiazole group of probe 1.

[0033] Beneficial effects

[0034] The present invention synthesizes an intermediate probe 1 through a one-pot Schiff base condensation reaction, and then introduces a morpholine targeting group through a nucleophilic substitution reaction to obtain a probe 2. Its molecular structure uses quinoxaline-Aza-BODIPY as a fluorescent chromophore, a benzothiazole group as a molecular rotor unit, and the benzothiazole and morpholine groups simultaneously as lysosomal targeting modules. This probe system exhibits excellent performance metrics: a significantly increased Stokes shift (Probe 1: 43 nm; Probe 2: 51 nm), effectively avoiding excitation / emission spectral overlap; typical aggregation-induced emission (AIE) properties, with peak fluorescence enhancement at 40-50% water content; a wide viscosity response range (2-610 cP), with up to 24-fold fluorescence enhancement, and detection limits as low as 0.27 cP (Probe 1) and 0.26 cP (Probe 2), respectively; excellent lysosomal colocalization (Pearson coefficient: 0.83 for Probe 1 and 0.86 for Probe 2); and excellent cytocompatibility (HK-2 cell viability >80% at 40 μM). These properties enable high-contrast, real-time imaging of lysosomal viscosity in living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 X-ray single crystal diffraction patterns of probe 1 and probe 2, front view (a and b) and side view (c and d);

[0036] Figure 2 a) Fluorescence spectra of probe 1 and c) probe 2 (both at 44 μM) in a glycerol / DMSO mixture with the glycerol content increasing from 0 to 90%; (λ ex = 386 nm, λ of probe 2ex = 420 nm); b) Linear fitting of fluorescence intensity and viscosity of probe 1 and d) probe 2;

[0037] Figure 3 a) Fluorescence spectra of probe 1 and c) probe 2 (both at 44 μM concentration) in H2O / DMSO mixed solvents, with H2O content increasing from 0 to 90% or 95% (λ ex = 386 nm, λ of probe 2 ex = 420 nm); b) Linear fitting of the fluorescence intensity of probe 1 (wavelength 501 nm) and d) probe 2 (wavelength 521 nm) with the H2O content;

[0038] Figure 4 HK-2 cells were incubated with different concentrations of a) Probe 1 and b) Probe 2 at 37°C for 4 h, and cell viability was measured. Data are presented as mean ± SD (n = 5).

[0039] Figure 5 Laser confocal imaging and Pearson coefficient of colocalization of probes 1 and 2 with LysoTracker Deep Red.

[0040] Figure 6 a) Cells were pretreated with different concentrations of dexamethasone for 40 min and then incubated with probe 1 (30 μM) for another 30 min; b) The intensity of probe 1 (λ) after pretreatment with different concentrations of dexamethasone ex = 488nm, λ em = 505-535 nm), data represent mean ± SD (n = 6), *P < 0.05, **P < 0.01;

[0041] Figure 7 a) Cells were pretreated with different concentrations of dexamethasone for 40 min and then incubated with probe 2 (30 μM) for another 30 min; b) The intensity of probe 2 (λ) after pretreatment with different concentrations of dexamethasone ex = 488nm, λ em = 505-535 nm), data are mean ± SD (n = 6), *P < 0.05. DETAILED DESCRIPTION

[0042] The present invention will be described in detail below with reference to the following examples so that those skilled in the art can better understand the present invention. However, the present invention is not limited to the following examples.

[0043] Example 1

[0044] Synthesis of probe 1

[0045] 6,7-Dimethyl-1,4-dihydroquinoxaline-2,3-dione (48 mg, 0.25 mmol) and 2-aminobenzothiazole (169 mg, 1.13 mmol) were dissolved in dry o-dichlorobenzene (6 mL) and refluxed at 180°C for 10 min. TiCl₄ (0.15 mL, 1.38 mmol) and triethylamine (0.5 mL, 3.63 mmol) were added dropwise, and reflux was continued for 3 h. BF₃·OEt₂ (0.48 mL, 3.98 mmol) was then added, and refluxed for 15 h. The reaction mixture was cooled and purified by silica gel column chromatography (DCM / PE 2:1→EA 100%) to afford yellow solid probe 1 in 46% yield. HR-MS (ESI) [M + ] (m / z): 503.1100 (theoretical value is 502.1017). 1 H NMR (400MHz, CDCl3) δ [ppm] = 10.50 (br, 1H), 8.19 (s, 1H), 8.07 (s, 1H), 8.11–8.13(d, 1H), 7.90–7.91 (d, 1H), 7.83–7.85 (d, 1H), 7.75–7.77 (d, 1H), 7.59–7.63 (dt, 1H), 7.45–7.52 (m, 2H), 7.33–7.37 (t, 1H), 2.46–2.51 (d, 6H).

[0046] Example 2

[0047] Synthesis of probe 2

[0048] Probe 1 (57.8 mg, 0.155 mmol), 4-(2-chloroethyl)morpholine (33.5 mg, 0.224 mmol), and anhydrous K2CO3 (31.0 mg, 0.224 mmol) were dissolved in anhydrous DMF (4 mL) and reacted at 50°C under nitrogen for 48 h. After completion of the reaction, the product was extracted with dichloromethane, washed with saturated NaCl, dried over anhydrous Na2SO4, concentrated, and purified by silica gel column chromatography (DCM→EA 100%) to afford probe 2 as a yellow solid in a 38% yield. HR-MS (ESI) m / z [M + ] 616.1941 (theoretical value 615.1858). 1H NMR (400MHz, CDCl3) δ [ppm] = 8.21 (s, 1H), 8.06 (d, 1H), 7.77–7.66 (m, 3H), 7.56–7.52 (m, 1H), 7.49–7.45 (m, 1H), 7.43–7.35 (m, 2H), 7.30–7.28 (d, 1H), 4.79–4.75 (t, 2H), 3.78 (s, 4H), 2.94–2.85 (m, 6H), 2.49 (s, 3H), 2.43 (s, 3H).

[0049] Example 3

[0050] Lysosomal viscosity imaging

[0051] MTT assay: The cytotoxicity of probes 1 and 2 was evaluated in HK-2 cells using the standard MTT assay. Cells in logarithmic growth phase were plated at 8 × 10 3 Cells were seeded at a density of 100 μL in a 96-well plate, and 100 μL of complete medium was added to each well. After incubation for 24 hours to allow cells to adhere, the medium was aspirated and the cells were gently washed with PBS. Subsequently, 100 μL of medium containing a gradient of concentrations of probe 1 or probe 2 (0, 0.125, 2.5, 5, 10, 20, and 40 μM) was added to the wells and incubated for 4 hours. After treatment, 20 μL of MTT solution (5 mg / mL in PBS) was added to each well and incubated for an additional 4 hours under standard culture conditions. After incubation, the supernatant was carefully removed to remove unreacted MTT, and 150 μL of DMSO was added to each well to dissolve the resulting formazan crystals. The plate was gently shaken for 10 minutes to ensure complete dissolution. Absorbance was measured at 490 nm using a microplate reader. Cell viability was calculated as follows: Cell viability (%) = 100 × (OD1 – OD2) / (OD3 – OD2), where OD1, OD2, and OD3 represent the absorbance values ​​of the experimental group, blank group, and control group, respectively. All experiments were repeated at least five times.

[0052] Lysosomal localization: HK-2 cells in logarithmic growth were plated at uniform density and cultured under standard conditions for 24 hours. After incubation, the medium was removed and the cells were incubated with probe 1 or 2 (30 μM) and the organelle-specific dye, LysoTracker Deep Red (100 nM), for 30 minutes at 37°C in a 5% CO2 incubator. After staining, the supernatant was discarded and the cells were rinsed three times with PBS. The cells were then incubated in phenol red-free medium until imaging. Confocal fluorescence imaging was performed using an LSM 800 laser scanning confocal microscope. Excitation parameters were set as follows: LysoTracker Deep Red was excited at 633 nm and emission was collected in the 650-670 nm range; probes 1 and 2 were excited at 488 nm and emission was collected in the 505-535 nm range.

[0053] Viscosity response: HK-2 cells were plated at 5×10 4 Cells were seeded at a density of cells / well in confocal culture dishes and incubated in a 37°C, 5% CO2 incubator for 24 hours. The original culture medium was discarded, and fresh culture medium containing 10, 20, or 40 μM dexamethasone was added, followed by an additional 40 minutes of incubation. The drug solution was removed, and culture medium containing 30 μM probe 2 (or probe 1 control) was added, followed by incubation for 30 minutes in the dark. The cells were washed three times with PBS, and images were acquired using a confocal microscope (excitation wavelength 488 nm, emission channel 505–535 nm).

[0054] from Figure 1 X-ray single crystal diffraction confirmed that probe 1 has a highly coplanar structure. In probe 2, the morpholine group is selectively attached to the nitrogen atom of the rotor benzothiazole. The morpholine group is perpendicular to the plane formed by the aza-BODIPY and rotor benzothiazole, and the morpholine group enhances π electron delocalization, leading to a compact head-to-tail stacking arrangement in probe 2.

[0055] Figure 2 : Probe 1 exhibits weak emission in DMSO, which is attributed to the free rotation of the benzothiazole part in the soluble state, thereby promoting the non-radiative decay pathway and quenching the fluorescence. As the glycerol volume fraction increases from 10% to 80%, the viscosity gradually increases, and the fluorescence intensity in the 450-650nm region gradually increases. The fluorescence intensity of probe 1 reaches a maximum when the glycerol volume fraction is 80%. Probe 2 also exhibits similar behavior. As the glycerol volume fraction increases from 10% to 90%, the fluorescence emission of probe 2 at 505nm increases significantly. By plotting the fluorescence intensity at 488nm (log I 488 ) and viscosity (logη) ( Figure 2 b), it is calculated that probe 1 has a good linear relationship, R 2The value was 0.94. The limit of detection (LOD) was calculated using the equation LOD = 3σ / S, where S is the slope of the calibration curve and σ is the standard deviation of the blank, and the obtained LOD was 0.27 cP. As shown in Figure 2d, the fluorescence intensity of probe 2 at 505 nm (log I 505 ) shows a two-stage linear relationship, with good R in the low viscosity range of 2-24 cP (glycerol volume fraction is 0% to 20%) and the high viscosity range of 49 to 610 cP (glycerol volume fraction is 30% to 90%). 2 The detection limit of probe 2 was further calculated to be 0.26 cP using the first-stage viscosity range. In addition, both probes 1 and 2 exhibited approximately 24-fold emission enhancement over a wide viscosity range of 2 to 610 cP.

[0056] Figure 3 : When the water fraction in the DMSO / water mixture gradually increased from 10% to 50%, the fluorescence intensity of probe 1 significantly increased to a maximum value, indicating the onset of molecular aggregation. As the water volume further increased (60% to 90%), the fluorescence intensity also decreased slightly, which may be due to excessive aggregation or partial precipitation of probe 1 at high water content ( Figure 3 a). Similar to probe 1, probe 2 also exhibits obvious AIE characteristics ( Figure 3 b). When the water content increases to 40%, the fluorescence intensity increases significantly, and the corresponding emission maximum shifts from 507 nm to 522 nm, reaching a peak intensity, indicating optimal aggregate formation. As the water content continues to increase from 50% to 90%, the fluorescence intensity gradually decreases, and the emission maximum further red-shifts from 522 nm to 536 nm. Both probes exhibit excellent AIE properties.

[0057] Figure 4 :The cytotoxicity test was performed using the MTT method. Figure 4 As shown, probes 1 and 2 exhibited negligible cytotoxicity after 4 h of incubation at concentrations ranging from 0 to 40 μM, with cell viability consistently exceeding 80%.

[0058] Figure 5 Both probes successfully entered HK-2 cells, emitting green fluorescence under 488 nm excitation. LysoTracker Red is excited at 633 nm. Colocalization analysis revealed good overlap between the two probes and LysoTracker Red, with Pearson correlation coefficients (R) of 0.83 and 0.86, respectively, indicating significant lysosomal targeting by the benzothiazole moiety. Probe 2 exhibited a higher Pearson correlation coefficient than probe 1, attributable to the dual targeting ability of the benzothiazole and morpholine moieties.

[0059] Figure 6 Confocal fluorescence imaging revealed that probe 1 exhibited weak fluorescence in untreated cells. In contrast, cells pretreated with dexamethasone exhibited significantly enhanced fluorescence emission in the 505-535 nm range under 488 nm excitation, indicating a viscosity-induced "on" fluorescence response within the lysosomal environment. The fluorescence intensity of probe 1 increased significantly with increasing dexamethasone concentration, demonstrating its effectiveness in monitoring changes in lysosomal viscosity.

[0060] Figure 7 Probe 2 showed a more pronounced sensitivity to changes in viscosity within lysosomes. With increasing dexamethasone concentration, the fluorescence emission of Probe 2 in the 508-550nm range increased significantly, especially at 40μM, with clear changes in fluorescence intensity visible in the cell images. Importantly, the fluorescence intensity of Probe 2 consistently exceeded that of Probe 1, demonstrating its superior sensitivity and detection performance. These results confirm that both Probe 1 and Probe 2 can precisely locate lysosomes and sensitively respond to viscosity fluctuations.

[0061] The above-described embodiments are only specific implementation methods of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the description of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A quinoxaline-derived Aza-BODIPY AIE fluorescent probe, characterized in that: The probe consists of three parts: the fluorophore Aza-BODIPY, the rotor benzothiazole, and the lysosome-targeting morpholine. Aza-BOIDPY and benzothiazole are connected by a -C=NC- group and have the following molecular structure: 。 2. The quinoxaline-derived Aza-BODIPY AIE fluorescent probe according to claim 1, characterized in that The precursor intermediate probe 1 of the quinoxaline-derived Aza-BODIPY AIE fluorescent probe is composed of the fluorophore Aza-BODIPY and the rotor benzothiazole connected by a -CC=N- group, and its molecular structure is shown below: 。 3. A method for synthesizing the quinoxaline-derived Aza-BODIPY AIE fluorescent probe according to claim 1, characterized in that: The steps include: S1. Synthesis of Probe 1: 6,7-dimethyl-1,4-dihydroquinoxaline-2,3-dione and 2-aminobenzothiazole were dissolved in anhydrous o-dichlorobenzene solvent, titanium tetrachloride TiCl4 and triethylamine NEt3 were added sequentially, and the mixture was heated to reflux. After the condensation reaction was complete, boron trifluoride etherate complex BF3·OEt2 was added to the system to continue the reflux complexation reaction. After the reaction was completed, the product was separated and purified by silica gel column chromatography to obtain Probe 1. S2. Synthesis of Probe 2: Probe 1 and 4-(2-chloroethyl)morpholine were dissolved in anhydrous DMF. Nucleophilic substitution occurred at the benzothiazole nitrogen atom under alkaline conditions and nitrogen protection. After sufficient reaction, the reaction mixture was washed, dried, purified by column chromatography, and concentrated under reduced pressure to obtain probe 2, a quinoxaline-derived Aza-BODIPY AIE fluorescent probe. The reaction equation is: 。 4. The method for synthesizing the quinoxaline-derived Aza-BODIPY AIE fluorescent probe according to claim 3, wherein: In step S1, the molar ratio of the reaction materials 6,7-dimethyl-1,4-dihydroquinoxaline-2,3-dione, 2-aminobenzothiazole, TiCl4, NEt3, and BF3·OEt2 is 0.10-4.00 mmol: 0.40-24.00 mmol: 0.40-28.00 mmol: 1.00-68.00 mmol: 14.00-80 mmol, preferably 0.25 mmol: 1.13 mmol: 1.38 mmol: 3.63 mmol: 3.98 mmol.

5. The method for synthesizing the quinoxaline-derived Aza-BODIPY AIE fluorescent probe according to claim 3, wherein: In step S1, the heating reflux reaction is carried out at 110-180°C for 3-12 hours, preferably at 180°C for 3 hours; the reflux complexation reaction is carried out at 110-180°C for 3-24 hours, preferably at 180°C for 15 hours.

6. The method for synthesizing the quinoxaline-derived Aza-BODIPY AIE fluorescent probe according to claim 3, wherein: In step S1, the sufficient reaction is confirmed by thin layer chromatography (TLC) analysis to confirm the formation of imine; the column chromatography purification is carried out by sequentially using dichloromethane / petroleum ether (DCM / PE) and pure ethyl acetate (EA) in a volume ratio of 2:1 as eluents, and the crude product is purified by silica gel column chromatography.

7. The method for synthesizing the quinoxaline-derived Aza-BODIPY AIE fluorescent probe according to claim 3, wherein: In step S2, the alkaline condition is to place probe 1, 4-(2-chloroethyl)morpholine and anhydrous potassium carbonate in a container, add DMF as a reaction solvent, purge with nitrogen and mix; the molar ratio of the reaction materials probe 1, 4-(2-chloroethyl)morpholine and anhydrous potassium carbonate is 0.10-2.00mmol: 0.12-3.40mmol: 0.12-3.40mmol, preferably 0.155mmol: 0.224mmol: 0.224mmol; the sufficient reaction is monitored by thin layer chromatography TLC and the formation of a new compound is confirmed; the washing and drying is to extract the reaction mixture with dichloromethane DCM, wash the organic layer three times with water, dry the combined organic layer with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain a crude product; the column chromatography purification is performed using silica gel column chromatography with an elution gradient of "100% dichloromethane → 100% ethyl acetate".

8. Use of the quinoxaline-derived Aza-BODIPY AIE fluorescent probe according to any one of claims 1 or 2, characterized in that: The probe was applied to lysosomal viscosity detection.

9. Use of the quinoxaline-derived Aza-BODIPY AIE fluorescent probe according to claim 8, characterized in that: The detection includes real-time dynamic imaging of lysosome viscosity in living cells, and is suitable for detecting abnormal lysosome viscosity induced by dexamethasone; further, it is used for real-time monitoring of abnormal lysosome viscosity induced by dexamethasone in living cells, with an excitation wavelength of 488 nm and an emission wavelength of 505-550 nm.

10. A lysosomal viscosity detection kit comprising the quinoxaline-derived Aza-BODIPYAIE fluorescent probe according to claim 1 or 2 and instructions for use.

Images