Synthesis and application of cysteine fluorescent probe based on quinazolinone-benzoindole
By developing a fluorescent probe based on quinazolinone-benzoindole, the problem that fluorescent probes in the prior art are difficult to achieve long-term cysteine imaging in cells, high sensitivity and selectivity near-infrared fluorescence imaging is achieved, and anti-diffusion properties are achieved.
Patent Information
- Application Number
- CN202411919762.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-04-04
AI Technical Summary
Existing fluorescent probes are difficult to achieve long-term imaging of cysteine in cells, and their emission wavelength is short and they are easily disturbed by light scattering and autofluorescence, which limits their application in biomedical imaging.
A fluorescent probe based on quinazolinone-benzoindole was developed to react HPQ-BI with acryloyl chloride and triethylamine through the synthetic route to generate the red solid fluorescent probe HPQ-BI-C, which has long-term fluorescence imaging and diffusion resistance.
The probe shows a significant near-infrared emission peak at 750 nm, with a detection range from 1 μM to 100 μM, and a detection limit of 0.35 μM. It has high sensitivity and selectivity, can achieve long-term cysteine imaging in cells, and has anti-diffusion properties.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to the synthesis and application of a quinazolinone-benzindolecysteine fluorescent probe. Background Art
[0002] As an important biological thiol, cysteine participates in catalysis, transport and protein synthesis in the body, and plays a key role in maintaining cellular redox balance (Wang Y, Li M, Yu H, Chen Y, Cui M, Ji M, Yang F, A near-infrared fluorescent dye with tunable emission wavelength and stokes shift as a high-sensitivity cysteine nanoprobe for monitoring ischemic stroke [J]. ACS Nano, 2024, 18: 15978-15990). Many diseases, such as liver damage, edema, tumors, skin lesions, neurotoxicity, psoriasis and Parkinson's disease, are closely related to abnormal levels of cysteine in biological systems (Qin JC, Li ZW, Fu ZH, Zhang ZH, Development of a NIR fluorescent probe for detection of cysteine and its application in bioimaging [J]. Sensors and Actuators B: Chemical, 2022, 357: 131430). Therefore, developing analytical methods that can sensitively monitor cysteine in vivo in real time is of great significance for biomedical research.
[0003] Traditional methods for detecting cysteine include voltammetry, mass spectrometry, spectrophotometry, high-performance liquid chromatography and capillary electrophoresis. However, these methods cannot be detected inside cells (Fu GQ, Song Q, Wang Z Q, Chao JJ, Zhang H, Mao GJ, Chen DH, Li CY, Long-term imaging of Cys in cells and tumor mice by a solid-state fluorescence probe[J]. Analytical Chemistry, 2023, 95: 17559-17567; Zhou J, Yu CM, Li Z, Peng PP, Zhang DT, Han X, Tang HZ, Wu Q, Li L, Huang W, Arapid and highly selective paper-based device for high-throughput detection of cysteine with red fluorescence emission and a large Stokes shift[J]. Analytical Methods, 2019, 11: 1312). In recent years, small molecule fluorescent probes have been widely used for the specific detection of cysteine due to their advantages such as fast response speed, high sensitivity, strong selectivity, non-invasive imaging capability, and real-time high-resolution spatiotemporal imaging capability (Jiao X, Li Y, Niu J, Xie X, Wang X, Tang B, Small-molecule fluorescent probes for imaging and detection of reactive oxygen, nitrogen, and sulfur species in biological systems[J]. Analytical Chemistry, 2018, 90: 533-555; Yang Y, Wang H, Wei YL, Zhou J, Zhang JF, Zhou Y, A selective coumarin-based “turn-on” fluorescent sensor for the detection of cysteine and its applications for bioimaging[J]. Chinese Chemical Letters, 2017, 28: 2023).At present, a variety of fluorescent probes for detecting cysteine have been developed, but after most probes react with cysteine, the fluorescent signals released cannot be imaged for a long time in cells (Hou X, Li Z, Li Y, Zhou Q, Liu C, Fan D, Wang J, Xu R, Xu Z, ICT-modulated NIR water-soluble fluorescent probe with large Stokes shift for selective detection of cysteine in living cells and zebrafish[J]. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2021, 246: 119030; Zhang S, Wu D, Wu J, Xia Q, Jia X, Song X, Zeng L, Yuan Y, A water-soluble near-infrared fluorescent probe for sensitive and selective detection of cysteine[J]. Talanta, 2019, 204: 747-752). Because after these probes react with cysteine, the released soluble fluorescent dye will quickly diffuse out from its generation site, and the released fluorescent signal will even penetrate into the outside of the cell, resulting in a too large fluorescence imaging range, and it is impossible to achieve long-term imaging of the precise range (Liu HW, Li K, Hu XX, Zhu L, Rong Q, Liu Y, Zhang XB, Hasserodt J, Qu FL, Tan W, In situ localization of enzyme activity in live cells by a molecular probe releasing a precipitating fluorochrome [J]. Angewandte Chemie International Edition, 2017, 56: 11788). Therefore, it is still a challenge to develop a probe that can provide long-term cysteine fluorescence imaging in cells and living bodies.
[0004] HPQ (2-(2′-hydroxyphenyl)-4(3H)-quinazolinone) is a highly insoluble fluorophore that emits strong solid-state fluorescence and is resistant to diffusion, allowing for long-term fluorescence imaging. Currently, HPQ-based fluorescent probes have been successfully applied to fluoride ions, enzyme proteases, hypochlorous acid (ClO -) and the detection of aminopeptidase N (APN), etc. (Zhao Z, Bi X, Mao W, Xu X, A novel HPQ-based turn-on fluorescent probe for detection of fluoride ions in living cells [J]. Tetrahedron Letters, 2017, 58: 4129 - 4132; Li K, Hu XX, Liu HW, Xu S, Huan SY, Li JB, Deng TG, Zhang XB, In situ imaging of furin activity with a highly stable probe by releasing of precipitating fluorochrome [J]. Analytical Chemistry, 2018, 90: 11680 - 11687; Zhou L, Lu DQ, Wang Q, Hu S, Wang H, Sun H, Zhang X, A high-resolution mitochondria-targeting ratiometric fluorescent probe for detection of the endogenous hypochlorous acid [J]. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2016, 166: 129 - 134; Liu Y, Xu C, Liu HW, Teng L, Huan S, Yuan L, Zhang XB, Precipitated fluorophore-based molecular probe for in situ imaging of aminopeptidase N in living cells and tumors [J].Analytical Chemistry, 2021, 93: 6463-6471; Ji Y, Liu S, Zhang J, Qu L, Wu J, Liu H, Cheng Z, Construction of HPQ-based activatable fluorescent probe for peroxynitrite and its application inferroptosis and mice model of LPS-induced inflammation[J]. Bioorganic Chemistry, 2023, 138: 106650. Fu GQ, Song Q, Wang ZQ, Chao JJ, Zhang H, Mao GJ, Chen DH, Li CY, Long-term imaging of Cys in cells and tumor mice by a solid-state fluorescence probe[J]. Analytical Chemistry, 2023, 95: 17559-17567). However, the emission wavelength of these fluorescent probes is short (<700nm) and they are easily interfered by light scattering and autofluorescence in biological tissues, which limits their application in biomedical imaging. Therefore, the development of solid-state fluorescent probes with longer wavelengths is of great significance for biomedical imaging. Summary of the invention
[0005] According to the requirements, the inventors conducted in-depth research on this, and after a lot of creative work, provided a quinazolinone-benzindole-based fluorescent probe that can be used to detect cysteine.
[0006] The technical solution of the present invention is a quinazolinone-benzoindole cysteine fluorescent probe, the structural formula of which is as follows:
[0007]
[0008] A synthesis of a quinazolinone-benzindole cysteine fluorescent probe. The steps are as follows:
[0009] Under nitrogen protection, 1 equivalent of HPQ-BI was dissolved in a two-necked bottle containing 15-20 mL of dichloromethane. Subsequently, 0.5-2 equivalents of triethylamine were added by syringe under ice bath, and after stirring for 10-30 minutes, 18-24 equivalents of acryloyl chloride were slowly added to the reaction solution, and stirred for 30-60 minutes under ice bath, and then the temperature was raised to 25-30°C, and the reaction was continued for 30 minutes. After the reaction stopped, it was extracted 3-5 times with saturated salt water and dichloromethane, the organic phase was dried with anhydrous sodium sulfate, the solvent was removed by reduced pressure distillation, and the crude product was subjected to column chromatography using dichloromethane as eluent, and finally a red solid product was obtained, which was the solid-state fluorescent probe HPQ-BI-C.
[0010] The beneficial effect of the present invention is that it provides a quinazolinone-benzindole cysteine fluorescent probe with good spectral response performance. First, the fluorescence spectral properties of the probe are studied. A weak fluorescence signal was observed at 750nm for the probe itself; when cysteine was added to the solution, an obvious near-infrared emission peak appeared at 750nm, and its near-infrared fluorescence intensity continued to increase with the increase in cysteine concentration. The detection range of the probe is from 1μM to 100μM, and the detection limit (3σ / k) is 0.35μM, which shows that the probe can be used for the detection of cysteine with high sensitivity. Next, the ultraviolet absorption spectrum of the probe was studied. In a PBS buffer solution of 30% isopropanol, the probe itself has an absorption peak at 485nm, and after adding cysteine, the absorption peak is red-shifted to 570nm. Then, the selectivity of the probe was studied, and the probe and various amino acids (Lys, Leu, Val, Met, Ile, Thr, Phe), active sulfur species (SO3 2- , H2S), reactive nitrogen species (NO2 - ,ONOO - ), active oxygen (H2O2, HClO), common ions (Na + ,Fe 3+ ,K + ,Cl-,Br -) and biothiols (GSH, Hcy, Cys). The results showed that only by adding cysteine could the fluorescence intensity of the probe be significantly increased. When other analytes were added, the fluorescence intensity of the probe did not change significantly. Subsequently, the change of pH value on the fluorescence intensity of the probe before and after the addition of cysteine was studied. The results showed that the probe was stable at different pH values. After the addition of cysteine, the fluorescence intensity of the probe reached a maximum in the pH range of 7.0 to 8.0. In addition, the fluorescence intensity of the probe at different cysteine concentrations was studied through time-dependent experiments. The fluorescence intensity reached a peak at 20 minutes, indicating that the probe responded quickly to cysteine. In addition, the solid-state luminescence properties of the fluorophore were studied. By comparing the absorption spectrum and fluorescence spectrum of the fluorophore HPQ-BI before and after filtration. In the absorption spectrum, when the fluorophore HPQ-BI was not filtered, the solution had a maximum absorption peak at 570nm. After filtration, the absorption peak at 570nm almost disappeared. A similar phenomenon was observed in the fluorescence spectrum. When HPQ-BI was not filtered, the solution emitted strong fluorescence at 750nm. After filtration, the fluorescence of the solution at 750nm was significantly reduced, indicating that the solubility of the fluorophore HPQ-BI in water was very low, and it was in an aggregated or solid state in water and emitted strong solid-state fluorescence. Finally, the anti-diffusion performance of the probe was studied. By comparing the diffusion behavior of HPQ-BI and the fluorescent dye 4-methylumbelliferone (4-MU) on agar, 4-MU diffused rapidly in all directions within 30min, and the fluorescence signal was gradually diluted, while HTPQ had almost no diffusion within 0-120min, indicating that HPQ-BI has the characteristics of long-term fluorescence imaging.
[0011] An application of a quinazolinone-benzindole cysteine fluorescent probe. Human transitional bladder cancer cells (T24) were selected for the experiment. When the fluorescent probe was added to the cells, obvious fluorescence could be observed. When the thiol inhibitor N-ethylmaleimide (NEM) was added to pre-treat the T24 cells and incubated with the probe, no obvious fluorescence was observed. On this basis, exogenous cysteine Cys was added, and a strong fluorescent signal appeared in the cells. This shows that the probe can detect endogenous and exogenous cysteine Cys, which will provide a reliable means for monitoring cysteine-related lesions in the human body. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 The synthetic route of fluorescent probe.
[0013] Figure 2 The fluorescence spectra of the fluorescent probe in Cys solutions with different concentrations.
[0014] The horizontal axis is wavelength, and the vertical axis is fluorescence intensity. The concentration of the fluorescent probe is 20 μM, the concentration of Cys is 1, 3, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM, the emission wavelength range is 680-850 nm, and the corresponding excitation wavelength is 570 nm.
[0015] Figure 3 This is the relationship between the fluorescence intensity of the fluorescent probe and the Cys concentration.
[0016] The horizontal axis is the concentration of Cys, and the vertical axis is the fluorescence intensity. The concentration of the fluorescent probe is 20 μM.
[0017] Figure 4 This is the UV absorption spectrum of the fluorescent probe before and after the reaction with Cys.
[0018] Figure 5 Selectivity diagram of the fluorescent probe.
[0019] The concentration of the fluorescent probe was 20 μM, and the concentration of other analytes was 100 μM.
[0020] Figure 6 This is a diagram showing the effect of pH on the fluorescent probe.
[0021] Figure 7 This is a curve diagram showing the relationship between the fluorescence intensity and time after the fluorescent probe interacts with Cys.
[0022] Figure 8 Absorption spectra of the fluorophore HPQ-BI in 30% isopropanol in PBS buffer before and after filtration.
[0023] Fig. 9 Fluorescence spectra of the fluorophore HPQ-BI in 30% isopropanol in PBS buffer before and after filtration.
[0024] Fig.10 The figure is a graph of the agar diffusion resistance of the fluorescent probe. The agar diffusion resistance of 4-methylumbelliferone and the probe under 365nm ultraviolet excitation.
[0025] Fig.11 This is a diagram of a cytotoxicity experiment. The horizontal axis is the concentration of the fluorescent probe, and the vertical axis is the cell survival rate.
[0026] Fig.12 This is the fluorescence imaging diagram of the fluorescent probe responding to intracellular Cys.
[0027] Fig.13 The relative fluorescence intensity graph is shown. DETAILED DESCRIPTION
[0028] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but is not limited thereto.
[0029] Embodiment 1:
[0030] Synthesis of fluorescent probes
[0031] Synthesis of probe HTPQC: Synthesis route as follows Figure 1 . Under nitrogen protection, 0.120 g of HPQ-BI (0.2 mmol) was added to a two-necked bottle containing 15.0 mL of dichloromethane. Subsequently, 0.014 mL of triethylamine (0.1 mmol) was added with a syringe at 0°C. After stirring for 10 min, 0.362 g of acryloyl chloride (4 mmol) was slowly added to the reaction solution. After stirring for 2 h in an ice bath, the mixture was extracted with saturated saline and dichloromethane. The product was dissolved in the organic phase, which was dried over anhydrous sodium sulfate. The solvent was removed by reduced pressure distillation. The crude product was subjected to column chromatography using dichloromethane as an eluent, and 0.095 g of red solid product HPQ-BI-C (yield: 72%) was obtained. 1 HNMR (400MHz, Acetone-d6) δ9.03(d,J=15.9Hz,1H),8.51(d,J=8.4Hz,1H),8.40(d,J=2.3Hz,1H),8.34(d,J=8.9Hz, 1H),8.29(d,J=4.0Hz,1H),8.27–8.23(m,1H),8.20–8.16(m,2H),8.15(s,1H),8.13(d,J=2.4Hz,1H),7.95(d,J=4.0 Hz,1H),7.87(dd,J=8.7,2.6Hz,2H),7.77(d,J=7.7Hz,1H),7.74(d,J=8.7Hz,1H),7.60(d,J=8.5Hz,1H),6.33(d,J= 1.8Hz,1H),6.14(d,J=4.8Hz,1H),5.91(d,J=1.8Hz,1H),5.02(q,J=7.4Hz,2H),2.22(s,6H),1.73(t,J=7.2Hz,3H). 13C NMR(400MHz,DMSO-d6)δ181.0,173.5,162.8,161.1,154.3,145.5,140.3,1 38.9,138.7,138.4,135.4,133.4,131.8,130.5,130.1,128.5,128.8,128. 6,128.4,128.0,127.4,127.3,126.1,125.8,125.5,123.5,122.6,120.0,1 19.8,119.5,115.0,114.0,108.8,44.1,42.3,29.5,14.3.MS(TOF):657.2.
[0032] Embodiment 2:
[0033] Preparation of fluorescent probe and Cys solutions of different concentrations
[0034] Preparation of probe solution: Weigh a certain amount of probe and dissolve it in dimethyl sulfoxide to make 2×10 -4 A reserve solution of M.
[0035] Preparation of Cys solution: Dissolve a certain amount of Cys in ultrapure water, transfer to a 500 mL volumetric flask, and add water to the scale to obtain a concentration of 1.0 × 10 -3 M Cys solution. 1.0×10 -3 The Cys solution was gradually diluted to obtain 1.0×10 -4 -1.0×10 -3 mol·L -1 1.0 mL of probe solution and 1.0 mL of Cys solution were added to a 10 mL volumetric flask and the volume was fixed with buffer solution to obtain a concentration of 2.0 × 10 -5 M fluorescent probe and 1.0×10 -5 -1.0×10 -4 M of Cys mixed with the test solution.
[0036] Embodiment 3:
[0037] Determination of fluorescence spectra of fluorescent probes in Cys solutions with different concentrations
[0038] Figure 2The fluorescence spectra of the solid-state fluorescent probe in response to Cys solutions of different concentrations. The concentration of the fluorescent probe is 20 μM, and the concentrations of cysteine Cys are 1, 3, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM, respectively. The excitation wavelength used in the experiment is 570 nm, the emission wavelength range is 680-850 nm, the slit width is 10.0 nm / 10.0 nm, and the fluorescence measurement instrument used is Hitachi F4600 fluorescence spectrophotometer. Figure 2 It can be seen that due to the quenching effect of acrylate, the probe itself only emits weak fluorescence. After adding Cys, an obvious emission peak appears at 750nm. This is because the recognition group acrylate group in the probe undergoes conjugate addition with Cys to form a thioether intermediate, followed by intramolecular cyclization, which leads to the cleavage of the allyl formate, releasing the fluorophore and generating a fluorescent signal. As the Cys concentration increases, the fluorescence intensity of the probe molecule gradually increases. Figure 3 The linear response of the probe to different Cys concentrations. The fluorescence intensity is linearly related to the Cys concentration, and the linear range is 1.0×10 -5 ~1.0×10 -4 M, and the detection limit (3σ / k) was 0.35 μM. This indicates that the probe can be used for the detection of Cys with high sensitivity.
[0039] Embodiment 4:
[0040] Determination of absorption spectra of fluorescent probes before and after reaction with Cys
[0041] Figure 4 The figure shows the UV-visible absorption spectra before and after the reaction of the fluorescent probe and Cys. The concentration of the fluorescent probe is 20 μM and the concentration of Cys is 100 μM. The instrument used for the UV-visible absorption spectrum measurement is Agilent Cary60 UV-visible spectrophotometer. Figure 4 It can be seen that the probe itself has no absorption peak at 570nm. After adding Cys, the probe has an obvious absorption peak at 570nm.
[0042] Embodiment 5:
[0043] Selectivity of fluorescent probes for Cys determination
[0044] Figure 5 To investigate the selectivity of the fluorescent probe for Cys, the fluorescent probe at a concentration of 20 μM was tested for its interaction with various amino acids (Lys, Leu, Val, Met, Ile, Thr, Phe), active sulfur species (SO3 2- , H2S), reactive nitrogen species (NO2 - ,ONOO - ), active oxygen (H2O2, HClO), common ions (Na+ ,Fe 3+ ,K + ,Cl-,Br - ) and biothiols (GSH, Hcy, Cys). Figure 5 It can be seen that only Cys can cause significant changes in the fluorescence spectrum, and other interferents have no obvious changes in the fluorescence intensity of the probe, indicating that the fluorescent probe has good selectivity in response to Cys and can specifically detect cysteine.
[0045] Embodiment 6:
[0046] Effect of Solution pH on Fluorescence Properties of Solid-State Fluorescent Probes for Cys Determination
[0047] The effect of pH on the fluorescence spectrum of Cys measured by fluorescent probe was investigated. Figure 6 . The pH range of the study was 4.0-11.0, the concentration of the fluorescent probe was 20 μM, and the concentration of cysteine was 100 μM. As can be seen from the figure, the fluorescence intensity of the fluorescent probe remained basically stable at different pH values, indicating that the effect of pH on the probe was small. However, after the addition of Cys, the fluorescence intensity of the probe was significantly enhanced when the pH value was in the range of 7.0 to 8.0. In summary, when the pH value is between 7.0 and 8.0, it will not affect the determination of Cys by the fluorescent probe, and it is a relatively suitable pH range, which is conducive to the use of the probe for the detection of Cys in actual samples.
[0048] Embodiment 7:
[0049] Study on the response time of fluorescent probe to Cys
[0050] The response time of the probe to different concentrations of Cys was studied through time-dependent experiments. When different concentrations of Cys (0, 50, 100 μM) were added, the fluorescence intensity of the probe increased with time and reached a peak at around 20 minutes, indicating that the probe responded relatively quickly to Cys and could meet the requirements for monitoring in actual samples. Figure 7 It can also be observed that after the fluorescence intensity reaches the maximum value, it remains stable for a subsequent period of time, indicating that the fluorescent probe has good photostability.
[0051] Embodiment 8:
[0052] Filter experiments with fluorophores
[0053] Through the filtration experiment of the fluorophore, the solid-state luminescence performance of the fluorophore HPQ-BI in 30% isopropanol PBS buffer solution was explored. The absorption and fluorescence spectra of the fluorophore HPQ-BI before and after filtration were compared. The results are shown in Figure 8 and Fig. 9As shown. Figure 8 It can be observed that when the fluorophore HPQ-BI is not filtered, the solution has a maximum absorption peak at 570nm; after filtering, the absorption peak at 570nm almost disappears. Fig. 9 It can be observed that the fluorescence spectrum shows a similar phenomenon. When HPQ-BI is not filtered, the solution emits strong fluorescence at 750nm. After filtering, the fluorescence of the solution at 750nm is significantly weakened. This shows that the fluorophore HPQ-BI has good solid-state luminescence properties.
[0054] Embodiment 9:
[0055] Anti-diffusion experiment of fluorescent probe
[0056] The anti-diffusion ability of the fluorescent probe was investigated by agar experiment. Under 365nm ultraviolet excitation, the diffusion behavior of HPQ-BI and the fluorescent dye 4-methylumbelliferone (4-MU) on agar was compared. The results are as follows: Fig.10 As shown in the figure, it can be observed that 4-MU diffuses rapidly in all directions within 30 minutes, and the fluorescence signal is gradually diluted, while HPQ-BI hardly diffuses within 120 minutes, indicating that HPQ-BI has the characteristics of long-term fluorescence imaging.
[0057] Embodiment 10:
[0058] Application of fluorescent probes in living cells
[0059] First, the toxicity of the probe to cells was investigated, e.g. Fig.11 As shown. When 0-30 μM of the fluorescent probe was added, the survival rate of human bladder transitional cell carcinoma cells (T24) was above 90%. This indicates that the fluorescent probe has low toxicity and is suitable for detecting cysteine in living cells. Subsequently, the application of the fluorescent probe in living cells was studied, and human bladder transitional cell carcinoma cells (T24) were selected for confocal microscopy imaging. The results are shown in Fig.12 After adding the fluorescent probe to the cells, an obvious fluorescence signal can be observed; after adding the thiol inhibitor N-ethylmaleimide (NEM) to pre-treat T24 cells and incubating with the probe, no obvious fluorescence was observed; subsequently, exogenous Cys was added to the NEM-pretreated cells, and a strong fluorescence signal appeared in the cells, indicating that the probe can detect endogenous and exogenous cysteine Cys, providing a reliable means for the detection of Cys in biological samples.
Claims
1. A quinazolinone-benzoindole cysteine fluorescent probe, HPQ-BI-C, characterized in that: The structure is as follows:
2. The synthesis of a quinazolinone-benzoindole cysteine fluorescent probe according to claim 1, characterized in that: The reaction steps are as follows: Under nitrogen protection, 1 equivalent of HPQ-BI was dissolved in a two-necked bottle containing 15-20 mL of dichloromethane. Subsequently, 0.5-2 equivalents of triethylamine were added with a syringe under ice bath. After stirring for 10-30 minutes, 18-24 equivalents of acryloyl chloride were slowly added to the reaction solution. Stirring was continued for 30-60 minutes under ice bath. Then the temperature was raised to 25-30°C and the reaction was continued for 30 minutes. After the reaction stopped, it was extracted with saturated brine and dichloromethane for 3-5 times. The organic phase was dried with anhydrous sodium sulfate, and the solvent was removed by reduced pressure distillation. The crude product was subjected to column chromatography using dichloromethane as eluent to obtain a red solid product, which was the solid-state fluorescent probe HPQ-BI-C.
3. The use of a quinazolinone-benzoindole cysteine fluorescent probe according to claim 1, characterized in that: The fluorescent probe can be applied to the detection of cysteine in living cells.