A dual-responsive near-infrared fluorescent probe NIR-CE and its application in HSO3 - and CE detection

Abstract

The application discloses a dual-response near-infrared fluorescent probe NIR-CE and application of the dual-response near-infrared fluorescent probe NIR-CE in HSO3 ‑ and CE detection, wherein the structure of the dual-response near-infrared fluorescent probe NIR-CE is shown in the following formula: The dual-response near-infrared fluorescent probe NIR-CE of the application can show good selectivity and sensitive dual-response fluorescent signals, the red channel emission wavelength responding to carboxylesterase is 650 nm, the green channel emission wavelength responding to HSO3 ‑ is 560 nm, and the two channels do not interfere with each other. ‑ Confocal fluorescence microscopic imaging experiments show that the probe has good light stability for HepG2 cells, can effectively detect carboxylesterase and HSO3 ‑ , and is suitable for carrying out dual-channel confocal fluorescence imaging on extracellular and endogenous carboxylesterase and HSO3 ‑ .

Description

TECHNICAL FIELD

[0001] The present application relates to a dual-responsive near-infrared fluorescent probe NIR-CE and its application in HSO3 - and CE detection, to achieve carboxylesterase and HSO3 - dual-responsive near-infrared fluorescence imaging with the advantages of selective specificity, efficient detection, and good biocompatibility. BACKGROUND

[0002] Carboxylesterases (CEs) belong to the α, β-hydrolase fold family and are commonly found in various life systems. CEs play a key role in catalyzing the hydrolysis of endogenous esters, including short- and long-chain acyl-glycerols, long-chain acyl-nitriles, long-chain acyl-coenzyme A esters, and the like. In addition, these enzymes are involved in the hydrolysis of xenobiotics, including various drugs, pesticides, environmental toxins, and carcinogens. In particular, it is well known that CEs are essential in the disposition of various ester prodrugs. In addition to metabolizing endogenous and exogenous compounds, CEs are closely related to metabolic diseases; in particular, they can be considered as unique biomarkers of hepatocellular carcinoma (HCC). Therefore, it is highly desirable to develop an effective and efficient method to evaluate CE levels.

[0003] In nature, SO2 is one of the most widely distributed atmospheric pollutants, mainly produced by coal combustion and industrial production. SO2 is easily hydrated to exist in equilibrium with SO3 2- and HSO3 - . Sulfur dioxide derivatives are widely used as adhesion agents and antioxidants in food and beverages. However, excessive SO2 derivatives can cause respiratory diseases such as asthma, chronic bronchitis, emphysema, and nervous system diseases.

[0004] Optical imaging technology based on small organic molecule probes has inherent advantages such as high spatiotemporal resolution, real-time detection, and non-invasive biological imaging, and has become a powerful tool for specific and selective imaging of enzyme activity in complex life systems. Although various optical probes for CE imaging and detection have been developed, they have many limitations. For example, existing CE probes are based on fluorescence imaging methods, which are detrimental to in vivo biological imaging due to notorious autofluorescence. Many methods have been developed to detect RSS, such as chromatography, chemiluminescence, flow injection analysis, and electrochemical analysis. However, these methods usually require expensive equipment, complex operation, and tedious sample pretreatment, which hinders their application in practical analysis. In summary, it is imperative to develop a rapid, sensitive, and specific method for monitoring SO2 derivatives and carboxylesterases in living cells and the environment. SUMMARY

[0005] The present application aims at the deficiencies of the prior art, and provides a dual-response near-infrared fluorescent probe NIR-CE and application thereof in HSO3 - and CE detection. The technical problem to be solved is to obtain a dual-channel fluorescence imaging probe which can identify extracellular and intracellular HSO3 - and CE activities, has the advantages of specific selectivity, good light stability, dual-channel imaging and near-infrared imaging.

[0006] The dual-response near-infrared fluorescent probe NIR-CE of the present application is based on a coumarin derivative as a parent structure, and the structure is shown as follows:

[0007]

[0008] The preparation method of the dual-response near-infrared fluorescent probe NIR-CE of the present application comprises the following steps:

[0009] Step 1: 1.38 g of compound 1 (10 mmol) is mixed with 2.22 g of methyl acetoacetate (19 mmol) as a solvent, refluxed at 75 DEG C, and after the reaction is completed as monitored by TLC, the reaction solution is cooled, dried by spinning, then slurried, filtered, and the filter cake is intermediate 2;

[0010] Step 2: Compound 2 (313 mg, 1 mmol) and compound 3 (204 mg, 1 mmol) are dissolved in methanesulfonic acid (3 ml), and stirred at 90 DEG C overnight; after the reaction is completed, the reaction solution is cooled to room temperature, then the reaction solution is dropped into 25 ml of saturated brine, a large amount of solid is precipitated, and the solid is purified by column chromatography (DCM:MeOH = 20:1, v / v) after being washed with 100 ml of ice water, filtered and washed three times, to obtain purple-black solid NIR-OH;

[0011] Step 3: Compound NIR-OH (154.3 mg, 0.32 mmol) is dissolved in appropriate DMF, and appropriate Cs2CO3 is added, dissolved in an ice bath, and N,N-dimethylaminomethyl chloride is dropped into the solution (added completely after about 20 minutes), and after dropping, the ice bath solution is removed, stirred at 40 DEG C, and the reaction is completed as monitored by TLC; the solvent is evaporated under reduced pressure to obtain purple crude product, which is purified by column chromatography (DCM:MeOH = 30:1, v / v) to obtain dark yellow solid NIR-CE.

[0012] The synthesis route is shown as follows:

[0013]

[0014] The dual-response near-infrared fluorescent probe NIR-CE of the present application is applied in HSO3 - and CE detection.

[0015] The detection reagent was prepared using the fluorescent probe NIR-CE, and the detection reagent is capable of dual-channel imaging to detect endogenous and exogenous HSO3 in cells. - And CE testing.

[0016] The detection method is as follows:

[0017] The NIR-CE of this invention was dissolved in DMSO (5 mL) to prepare a 2 mM stock solution. 15 μL of this stock solution was then added to 3 mL of HEPES solvent containing different concentrations of carboxylesterase (the carboxylesterase used in the detection process is CAS: 9016-18-6) to obtain a final test solution with a concentration of 10 μM. The spectral properties of NIR-CE were detected using UV-vis absorption and fluorescence emission spectra obtained in 0.2 M HEPES buffer (pH = 7.4). The maximum UV absorption was observed at 580 nm. With 580 nm as the excitation wavelength, the fluorescence was strongest at 650 nm. The fluorescence intensity at 650 nm showed a good linear relationship with CE concentration (R = 0.9943), with a linear equation of y = 127.11x + 66.18 and a detection limit (LOD) of 4.4 U / L. To further investigate the response of NIR-CE to CE, the time course of the fluorescence response of NIR-CE to CE was determined by fluorescence spectroscopy. In the presence of CE (6 U / mL), the fluorescence intensity at 650 nm reached a stable state after 90 minutes. Furthermore, the fluorescence of NIR-CE showed only slight changes across pH values ​​from 6 to 10. In addition, we investigated the resistance of NIR-CE to interference from other common species in physiological environments. The response of NIR-CE to CE was specific, and the influence of other species was negligible.

[0018] Take 15 μL of NIR-CE stock solution and add it to 3 mL of HSO3 with different concentrations. - The probe NIR-CE was obtained in HEPES solvent (pH = 7.4) under different HSO3 conditions. - UV-vis absorption and fluorescence emission spectra at various concentrations were observed. In the reaction system, the absorption peak of NIR-CE decreased at 560 nm, and a new peak appeared at 500 nm. Using 470 nm as the excitation wavelength, the fluorescence was strongest at 560 nm. The fluorescence intensity at 560 nm and HSO3... - The concentration-relativity curve shows a good linear relationship R. 2 =0.9948, the linear equation is y = 80.94x + 6.26, and the detection limit is LOD = 1.8 nM. To further investigate the effect of NIR-CE on HSO3... - To assess the response of NIR-CE to HSO3, we used fluorescence spectroscopy to determine its effect. - The time process of the fluorescence response in HSO3.- The fluorescence intensity at 560 nm reached a steady state after 100 s in the presence of 22 μM NIR-CE. The fluorescence of NIR-CE was not affected by pH values from 6 to 10. In addition, we also studied the anti-interference ability of NIR-CE in the physiological environment. NIR-CE has specificity for the reaction of CE, and the influence of other species can be basically ignored.

[0019] The fluorescent probe NIR-CE of the present application takes the coumarin ring as the reaction site of HSO3 - . After the reaction with HSO3 - , the fluorescence of NIR-CE in the green channel is obviously enhanced. After the reaction with CE, the N,N-dimethylaminocarbonyl in the probe molecule can be removed (carboxylesterase CAS: 9016-18-6 is used in detection), and an enhanced fluorescence signal in the near-infrared (NIR) channel is generated. NIR-CE has high selectivity and sensitivity for HSO3 - and CE, fast response speed, low detection limit, large Stokes shift, and good water solubility. In addition, NIR-CE can detect HSO3 - and CE in living cells through double-channel non-interference fluorescence imaging.

[0020] The double-response near-infrared fluorescent probe of the present application has good response ability for CE and HSO3 - in solution and cells. The present application realizes fluorescence imaging of HSO3 - and CE in cells through near-infrared double-response fluorescence detection. It has stable fluorescence imaging ability, and the confocal fluorescence microscopic imaging experiment shows that NIR-CE can perform fluorescence imaging of HSO3 - and CE through double channels. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The schematic diagram of the mechanism of the double-response fluorescent probe NIR-CE for detecting HSO3 - and CE.

[0022] Figure 2UV-Vis (a) absorption and (b) fluorescence spectra of NIR-CE (10 mM) after addition of CE (0-6.3 U / mL) in HEPES (pH = 7.4); (c) Change in fluorescence intensity (650 nm) of NIR-CE (10 mM) with concentration of CE (0-6.3 U / mL) in HEPES (pH = 7.4); (d) Time variation of fluorescence spectra of NIR-CE (10 mM) in the presence of 6 U / mL CE; (e) Fluorescence intensity (650 nm) of NIR-CE (10 mM) at different pH values in the presence of CE (6 U / mL); (f) Fluorescence variation (650 nm) of NIR-CE (10 mM) in HEPES (pH = 7.4) for other biological species: 1 : none, 2: Fe 3+ , 3: Mg 2+ , 4: K + , 5: Cu 2+ , 6: Zn 2+ , 7: Na + , 8: Ca 2+ , 9: ClO - , 10: H2PO4 - , 11: NO2 - , 12: HCO3 - , 13: CO3 2- , 14: Cl - , 15: I - , 16: SO3 2- , 17: SO4 2- , 18: H2O2, 19: Cys, 20: Hcy, 21: GGT, 22: LAP, 23: tyrosinase, 24: Ache, 25: HSO3 - , 26: CE. (Carboxylesterase CAS: 9016-18-6 used for the assay).

[0023] Figure 3 UV-Vis (a) absorption and (b) fluorescence spectra of NIR-CE (10 mM) after addition of HSO3 - (0-26 mM) in HEPES (pH = 7.4); (c) Change in fluorescence intensity (560 nm) of NIR-CE (10 mM) with concentration of HSO3 - (0-26 mM) in HEPES (pH = 7.4); (d) Time variation of fluorescence spectra of NIR-CE (10 mM) in the presence of HSO3 - (22 mM); (e) Fluorescence intensity (560 nm) of NIR-CE (10 mM) at different pH values in the presence of HSO3 -(f) Fluorescence changes (560 nm) of NIR-CE (10 mM) in HEPES (pH = 7.4) in the presence of other biological species: 1: none, 2: Fe 3+ , 3: Mg 2+ , 4: K + , 5: Cu 2+ , 6: Zn 2+ , 7: Na + , 8: Ca 2+ , 9: ClO - , 10: H2PO4 - , 11: NO2 - , 12: HCO3 - , 13: CO3 2- , 14: Cl - , 15: I - , 16: SO3 2- , 17: SO4 2- , 18: H2O2, 19: Cys, 20: Hcy, 21: GGT, 22: LAP, 23: tyrosinase, 24: Ache, 25: HSO3 - .

[0024] Figure 4 is the confocal fluorescence imaging of NIR-CE (10 mM) to investigate the optical stability of NIR-CE responding to CE and HSO3 - .

[0025] Figure 5 is the confocal fluorescence imaging of NIR-CE probe (10 mM) responding to exogenous HSO3 - in HeLa cells.

[0026] Figure 6 is the confocal fluorescence imaging of NIR-CE probe (10 mM) responding to endogenous CE in HeLa cells. DETAILED DESCRIPTION

[0027] The application is further illustrated by the following examples.

[0028] Example 1: Synthesis of NIR-CE

[0029] The compound NIR-OH (154.3 mg, 0.32 mmol) was dissolved in an appropriate amount of DMF, and an appropriate amount of Cs₂CO₃ was added. The solution was dissolved in an ice bath. N,N-dimethylcarbamoyl chloride was added dropwise to the solution (completely added after about 20 minutes). After the addition was complete, the ice bath was removed, and the mixture was stirred at 40 °C. After the reaction was monitored by TLC, the solvent was evaporated under reduced pressure to obtain a purple crude product. The crude product was purified by column chromatography (DCM:MeOH = 30:1, v / v) to obtain a deep yellow solid NIR-CE.

[0030] 1 H NMR (CDCl3, 400MHz, ppm): 1.22 (t, 6H, J = 7.2Hz), 3.41 (q, 4H, J = 7.2Hz), 6.10 (d, 1H, J = 0.8Hz), 6.36 ( m,2H),6.51(d,2H,J=8.8Hz),6.67(m,1H),6.75(s,1H),7.20(t,2H,J=8.4Hz),7.31(d,2H,J=9.2Hz), 13 C NMR (CDCl3, 100MHz, ppm): δ12.57,44.46,97.38,103.74,109.26,109.75,116.65,118.26,118.72,123.98,125.06,126.59,127.28 ,128.53,129.47,129.78,133.81,134.54,139.35,145.86,149.44,152.59,153.45,143.64,153.93,157.57,163.69,169.69.FT-MS m / z:[NIR-CE] + :calcd.,553.1969; found,553.1981.

[0031] Example 2: NIR-CE response to CE

[0032] The NIR-CE of this invention was dissolved in DMSO (5 mL) to prepare a 2 mM stock solution. 15 μL of the NIR-CE stock solution was added to 3 mL of HEPES solvent (pH = 7.4). Different concentrations of CE were added to obtain the UV and fluorescence spectra of the probe NIR-CE at different CE concentrations. Figure 2 a, b). The fluorescence intensity at 650 nm versus CE concentration curves show a good linear relationship. 2 =0.9943, the linear equation is y = 127.11x + 66.18, and the detection limit (LOD) is 4.4 U / L. Figure 2c) In the presence of 6 U / mL of CE, the fluorescence intensity at 650 nm reached a steady state after 110 min (2d). To test the response of NIR-CE to the environmental pH of CE, the fluorescence intensity of the probe NIR-CE in different pH solvents was obtained in the presence of 6 U / mL of CE. When the pH value of the environment changed from 6 to 10, the fluorescence intensity of NIR-CE was not substantially affected by the environmental pH Figure 2 f).

[0033] Example 3: Response of NIR-CE to HSO3 - Example 3: Response of NIR-CE to HSO3

[0034] A 2 mM stock solution of NIR-CE was prepared by dissolving NIR-CE in DMSO (5 mL). 15 μL of the NIR-CE stock solution was added to 3 mL of HEPES solvent (pH = 7.4) with different concentrations of HSO3 - , and the UV spectrum and fluorescence spectrum of the probe NIR-CE at different concentrations of HSO3 - were obtained Figure 3 a, b). The curve of the fluorescence intensity at 560 nm versus the concentration of HSO3 - showed a good linear relationship R 2 = 0.9948, the linear equation y = 80.94x + 6.26, and the detection limit was LOD = 1.8 nM Figure 3 c). In the presence of HSO3 - (22 μM), the fluorescence intensity at 560 nm reached a steady state after 100 s Figure 3 d). To test the response of NIR-CE to the environmental pH, the fluorescence intensity of the probe NIR-CE in different pH solvents was obtained in the presence of HSO3 - (22 μM). When the pH value of the environment changed from 6 to 10, the fluorescence intensity of NIR-CE was not substantially affected by the environmental pH Figure 3 f).

[0035] Example 4: Time stability of NIR-CE response to CE and HSO3 - Example 4: Time stability of NIR-CE response to CE and HSO3

[0036] The long-term imaging of the probe NIR-CE in cells was tested. As Figure 4 , with the extension of the irradiation time, the fluorescence of both the green channel and the red channel of the probe NIR-CE remained stable. This indicates that the fluorescence of the probe NIR-CE is very stable, and it is suitable for long-term imaging in cells.

[0037] Example 5: Exogenous imaging of HSO3 - in HeLa cells by NIR-CE

[0038] HepG2 cells were first cultured with NIR-CE for 0.5 hours, followed by confocal fluorescence microscopy analysis. Figure 5 As shown, the corresponding images of NIR-CE exhibit weak green fluorescence, confirming the response of NIR-CE to intracellular CE. In exogenous HSO3... - In the detection experiment, a certain concentration of HSO3 was used. - Cells were incubated with the solution for 5 min, followed by incubation with the probe for 0.5 h. The fluorescence intensity of the green channel in the confocal image was significantly enhanced. This confirms that with HSO3… - As the concentration increases, the NIR-CE response also increases.

[0039] Example 6: NIR-CE Imaging of Endogenous CE in HeLa Cells

[0040] HepG2 cells were first cultured with NIR-CE for 2 hours, followed by confocal fluorescence microscopy analysis. Figure 6 As shown, the corresponding NIR-CE images exhibit stronger red fluorescence, confirming the response of NIR-CE to intracellular CE. In the endogenous CE detection experiment, cells were treated with AEBSF for 3 hours to inhibit intracellular CE activity. Figure 5 After incubating with the probe for another 2 hours, the fluorescence intensity of the red channel in the confocal image significantly decreased. This confirms that as CE activity is inhibited, the NIR-CE response also weakens.

Claims

1. A dual-response near-infrared fluorescent probe (NIR-CE) for the preparation of HSO3 - Its application in CE testing reagents is characterized by: The structure of the dual-response near-infrared fluorescent probe NIR-CE is shown below: 。 2. The application according to claim 1, characterized in that: The detection reagent is capable of dual-channel imaging to detect endogenous and exogenous HSO3 in cells. - And CE testing.

3. The application according to claim 1, characterized in that... The dual-response near-infrared fluorescent probe NIR-CE was prepared by a method comprising the following steps: Step 1: Compound 1 and ethyl acetoacetate were mixed with ethanol as solvent and refluxed at 75°C. After the reaction was completed by TLC monitoring, the reaction solution was cooled, evaporated to dryness, then slurryed, filtered, and the filter cake was intermediate 2. Step 2: Compounds 2 and 3 were dissolved in methanesulfonic acid and stirred at 90°C. After the reaction was completed, the reaction solution was cooled to room temperature and then added dropwise to saturated salt water. A large amount of solid was precipitated. The solid was rinsed, filtered, and washed three times with ice water. The obtained solid was purified by column chromatography to obtain a purple-black solid NIR-OH. Step 3: Dissolve compound NIR-OH in DMF, add an appropriate amount of Cs2CO3, dissolve in an ice bath, add N,N-dimethylcarbamoyl chloride dropwise to the solution, remove the ice bath after the addition is complete, stir at 40℃, monitor the reaction by TLC until it ends, evaporate the solvent under reduced pressure to obtain a purple crude product, purify by column chromatography to obtain a deep yellow solid NIR-CE; The synthesis route is shown below: 。

Images