Difunctional near-infrared fluorescent probe as well as synthesis method and application thereof

Abstract

The invention discloses a difunctional near-infrared fluorescent probe as well as a synthesis method and application thereof. The structural formula of the fluorescent probe is shown as a formula (I). The fluorescent probe DTPD provided by the invention can be used for fluorescent quantitative detection of hypochlorous acid and viscosity, and the fluorescent probe can realize detection of hypochlorous acid with high selectivity in an aqueous solution and is not interfered by other coexisting substances. Besides, the near-infrared fluorescent probe provided by the invention has relatively strong organism penetrability in a near-infrared region, can also reduce the interference of autofluorescence of organisms, and has a good cell imaging effect. And # imgabs0 #.

Description

Technical Field

[0001] The invention relates to hypochlorous acid and a viscosity detection reagent, and in particular to a dual-function near-infrared fluorescent probe, a synthesis method thereof, and applications thereof. Background Art

[0002] Intracellular viscosity is a key parameter that influences numerous physiological processes in the microenvironment, such as signal transduction, diffusion, and metabolite transport. Studies have shown that abnormal intracellular viscosity is often associated with a variety of diseases, such as diabetes, atherosclerosis (AS), Alzheimer's disease (AD), and malignant tumors. Therefore, real-time tracking of viscosity fluctuations in living cells is crucial for revealing the relationship between viscosity and clinical diseases and will aid in the pre-diagnosis of viscosity-related diseases.

[0003] In organisms, hypochlorite ions (ClO - ) is one of the important reactive oxygen species (ROS), which can resist the invasion of certain pathogens and regulate cell apoptosis. However, excessive ClO - It can cause oxidative stress and lead to serious diseases, including kidney disease, neurodegeneration, atherosclerosis and cancer. Therefore, maintaining appropriate ClO - Physiological concentration is very important for maintaining normal cell function, so the observation and detection of ClO in living cells, tissues and organisms is very important. - Changes are very necessary for studying the relevant pathological mechanisms.

[0004] Fluorescence emission probes, with their outstanding advantages such as excellent spatiotemporal resolution, non-invasive operation, and real-time visualization, have become ideal tools for detecting various biological analytes in living systems when combined with fluorescence microscopy. In recent years, a series of fluorescent probes with different reaction sites have been developed for the specific detection of hypochlorite.

[0005] Currently, there is no single fluorescent probe for simultaneous detection of viscosity and ClO - In fact, changes in ROS have been shown to be associated with changes in intracellular viscosity. Therefore, simultaneous monitoring of changes in viscosity and ROS can provide more comprehensive information and is of great significance for studying various physiological and pathological mechanisms. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a dual-function near-infrared fluorescent probe and its synthesis method and application, so as to achieve the purpose of simultaneous fluorescence detection of hypochlorous acid and viscosity by a single probe.

[0007] To solve the above technical problems, according to one aspect of the present invention, a dual-function near-infrared fluorescent probe (DTPD) is provided, the structural formula of which is shown in formula (I): . According to another aspect of the present invention, a method for preparing the above-described bifunctional near-infrared fluorescent probe (DTPD) is provided, wherein the compound of formula (III) and N,N-diisopropylethylamine (DIPEA) are added to N,N-dimethylformamide (DMF) containing the compound of formula (II), and the mixture is stirred at room temperature for 8-12 hours; the reaction formula is as follows: .

[0008] Furthermore, after the reaction is completed, the bifunctional near-infrared fluorescent probe is obtained by column chromatography separation, and the developing solvent of the column chromatography separation is dichloromethane:methanol=(15~20):1 (v / v).

[0009] According to another aspect of the present invention, provided is the use of the above-mentioned dual-function near-infrared fluorescent probe (DTPD) in the preparation and detection of hypochlorous acid preparations.

[0010] According to another aspect of the present invention, provided is the use of the above-mentioned dual-function near-infrared fluorescent probe (DTPD) in the preparation of a preparation for detecting viscosity.

[0011] According to another aspect of the present invention, provided is the use of the above-mentioned dual-function near-infrared fluorescent probe (DTPD) in the preparation of an intracellular endogenous hypochlorous acid imaging preparation.

[0012] According to another aspect of the present invention, provided is the use of the above-mentioned dual-function near-infrared fluorescent probe (DTPD) in the preparation of an intracellular viscosity imaging preparation.

[0013] According to another aspect of the present invention, a method for fluorescent quantitative detection of hypochlorous acid is provided, comprising the following steps: Step 1: Prepare the DTPD stock solution of the above-mentioned dual-function near-infrared fluorescent probe, prepare it with hypochlorous acid to prepare a series of standard solutions, and measure the fluorescence intensity at 650 nm on a fluorescence photometer. - The concentration is the horizontal axis, and the fluorescence intensity I 650nm Draw labeled curves for the ordinate and obtain linear equations; Step 2: Take the test solution containing hypochlorous acid, add it to the DTPD stock solution described in step 1 and dilute to volume with solvent, measure the fluorescence intensity at 650 nm on a fluorescence photometer, and obtain ClO according to the linear equation obtained in step 1. - concentration.

[0014] Furthermore, the fluorescence intensity I 650nm and ClO - The concentration range was 200 mM-760 mM, and the R 2=0.97595.

[0015] According to another aspect of the present invention, a method for fluorescence quantitative detection of viscosity is provided, comprising the following steps: Step 1: Prepare the DTPD stock solution of the above-mentioned dual-function near-infrared fluorescent probe, take the DTPD stock solution and hypochlorous acid solution, mix them, add methanol and glycerol solution systems respectively, and as the proportion of glycerol increases, the fluorescence intensity at 650 nm measured on a fluorescence photometer gradually increases. The fluorescence intensity at 650 nm is I 650nm The viscosity value (logη) shows a good linear relationship in the range of 0-6, R 2 =0.9751, log(η) is taken as the horizontal axis, and the fluorescence intensity log(I 650nm ) is plotted as the ordinate to obtain a linear equation between viscosity and fluorescence intensity; Step 2: Mix the DTPD stock solution and hypochlorous acid solution described in step 1, add the sample solution to be tested, and then adjust the volume with the solvent. Detect the fluorescence intensity on a fluorescence spectrophotometer, and calculate the viscosity of the solution to be tested according to the linear equation in step 1.

[0016] The dual-function near-infrared fluorescent probe (DTPD) developed by this invention can rapidly, selectively, and sensitively simultaneously detect hypochlorous acid and viscosity in aqueous solutions using spectroscopy. The process is simple, convenient, and quick, and the results are clear and easily discernible. More importantly, the fluorescent probe can also be used for cellular imaging of endogenous hypochlorous acid and viscosity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the H-NMR spectrum of the near-infrared fluorescent probe DTPD obtained in Example 1; Figure 2 is the fluorescence spectrum of Example 2; Figure 3 This is the working curve diagram of Example 2; Figure 4 This is a comparison diagram of the effects of coexisting ions on the detection of hypochlorous acid by near-infrared fluorescent probe in Example 2; Figure 5 is the fluorescence spectrum of Example 3; Figure 6 This is the working curve diagram of Example 3; Figure 7 This is a fluorescence imaging diagram of endogenous hypochlorous acid and viscosity in living cells using DTPD in Example 4. DETAILED DESCRIPTION

[0018] A typical embodiment of the present invention provides a dual-function near-infrared fluorescent probe (DTPD) having a structural formula as shown in formula (I), . The compound of formula (I) can realize fluorescence detection of multiple target molecules (hypochlorous acid and viscosity) by a single probe.

[0019] Add the compound of formula (III) and N,N-diisopropylethylamine (DIPEA) to N,N-dimethylformamide (DMF) containing the compound of formula (II) and stir at room temperature for 8-12 hours; the reaction formula is as follows: .

[0020] The compound of formula (II) is an existing product and can be synthesized with reference to the following literature: Yanli Chen, Wenhui Shi, Yanqi Xua and Peng Wang, Real-time visualization of sulfatase inliving cells and in vivo with a ratiometric AIE fluorescent probe. Chem. Commun., 2023, 59, 9754. The compound of formula (III) is N,N-dimethylthiocarbonyl chloride.

[0021] Preferably, the molar ratio of the compound of formula (III), N,N-diisopropylethylamine (DIPEA), and the compound of formula (II) is 12:4:1. The reaction time can be 8 hours, 9 hours, 11 hours, 12 hours, and preferably 10 hours.

[0022] After the reaction is completed, the bifunctional near-infrared fluorescent probe is obtained by column chromatography separation. The developing solvent for the column chromatography separation is dichloromethane:methanol = (15-20):1 (v / v), for example: dichloromethane:methanol = 15:1 (v / v), dichloromethane:methanol = 20:1 (v / v), dichloromethane:methanol = 18:1 (v / v), dichloromethane:methanol = 16:1 (v / v). More preferably, dichloromethane:methanol = 17:1 (v / v).

[0023] The compound of formula (I) is a near-infrared fluorophore that can be excited by near-infrared light and emit fluorescence at a longer wavelength. N,N-dimethylthioformyl chloride, a strong electron-withdrawing group, quenches the fluorescence of the fluorophore due to the weakened intramolecular charge transfer effect when attached to the near-infrared fluorophore. However, when N,N-dimethylthioformyl chloride is attacked by hypochlorous acid, the resulting electron-donating hydroxyl group further enhances the intramolecular charge transfer process, leading to the restoration of the fluorophore's fluorescence. By observing the fluorescence changes before and after the probe reacts with hypochlorous acid, hypochlorous acid can be monitored in real time. Furthermore, in low-viscosity media, the intramolecular rotation of DTPD forms a TICT effect, resulting in weak fluorescence emission. However, in high-viscosity environments, the intramolecular rotation of DTPD is hindered, resulting in an increase in fluorescence intensity.

[0024] Based on this, the fluorescent probe DTPD provided by the present invention can be used for the quantitative fluorescent detection of hypochlorous acid and viscosity. This fluorescent probe can highly selectively detect hypochlorous acid in aqueous solutions without interference from other coexisting substances. Furthermore, the near-infrared fluorescent probe provided by the present invention has strong bio-penetration in the near-infrared region and can also reduce interference from autofluorescence, resulting in excellent cell imaging.

[0025] Illustratively, a method for fluorescent quantitative detection of hypochlorous acid using the fluorescent probe DTPD comprises the following steps: (1) Configuration 10 -3 M DTPD DMSO stock solution, prepared 10 -2 M Aqueous solution of hypochlorous acid; (2) Take 20 μL of DTPD stock solution and add it to a clean colorimetric tube. With the addition of hypochlorous acid, the solution gradually changes from orange to light yellow. With the increase of hypochlorous acid concentration, the fluorescence intensity at 650 nm measured on the fluorescence photometer gradually increases. The fluorescence intensity of the system at 650 nm I 650nm and [ClO - ] showed a good linear relationship in the concentration range of 200 mM-760 mM (R 2 =0.97595), with ClO - The concentration is the horizontal axis, and the fluorescence intensity I 650nm Plotting as the vertical axis, we get ClO - The linear equation of concentration and fluorescence intensity: F=5.55219[ClO - ]-852.94073; (3) Take 20 mL of DTPD stock solution and add it to a clean colorimetric tube. Take x mL of the sample solution to be tested and add it to the colorimetric tube. Use a mixed solution of secondary water and DMSO (v / v = 1:1) to make the volume 5 mL. Detect it on a fluorescence spectrophotometer. Substitute the measured fluorescence intensity into the linear equation of step (2) to obtain [ClO- ], the sample to be tested [ClO - ] 待测 =5000mL´[ClO - ] ´10 -6 / x mL, the concentration of hypochlorite ions can be calculated.

[0026] For example, the fluorescence quantitative analysis method for detecting viscosity using the fluorescent probe DTPD comprises the following steps: (1) Take 20 μL of DTPD stock solution and 120 mL of ClO - The solution was added to a clean colorimetric tube, shaken, and 5 mL of methanol and glycerol solution systems with different volume ratios (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10) were added respectively. As the glycerol ratio increased, the fluorescence intensity at 650 nm measured on a fluorescence photometer gradually increased. The fluorescence intensity of the system at 650 nm I 650nm and viscosity value (logη) show a good linear relationship in the range of 0-6 (R 2 =0.9751), with log(η) as the horizontal axis and fluorescence intensity log(I 650nm ) as the ordinate, and the linear equation of viscosity value and fluorescence intensity was obtained: Y=0.26975x+6.25806; (2) Take 20 mL of DTPD stock solution and 120 mL of ClO - The solution is added to a clean colorimetric tube, x mL of the sample solution to be tested is added, and the volume is made up to 5 mL with methanol. The fluorescence intensity is measured on a fluorescence spectrophotometer. Substitute the measured fluorescence intensity into the linear equation of step (1) to obtain log(η). The log(η) of the sample to be tested is 待测 =5000mL´log(η) ´10 -6 / x, the viscosity value can be obtained.

[0027] The technical solutions claimed in the present invention are further illustrated below by means of some examples. However, the examples are intended to illustrate the embodiments of the present invention and do not exceed the scope of the subject matter of the present invention. The scope of protection of the present invention is not limited by these examples. Unless otherwise specified, the materials and reagents used in the present invention can be obtained from commercial products in the art.

[0028] Example 1 Synthesis and Characterization of Near-Infrared Fluorescent Probe (DTPD)

[0029] First, 0.5 mmol of the compound of formula (II) was dissolved in 3 mL of N,N-dimethylformamide. Then, 6 mmol of N,N-dimethylthiocarbonyl chloride and 2 mmol of N,N-diisopropylethylamine were added to the above solution and stirred at room temperature for 10 hours. After the reaction, the solvent was removed in vacuo to obtain a deep red solid. The crude product was then chromatographed on a silica gel column. The developing solvent for column chromatography was dichloromethane:methanol = 17:1 (v / v), and a bifunctional near-infrared fluorescent probe was finally obtained.

[0030] 1 H NMR (DMSO-d6)δ: 8.17 (m, 2H), 7.90 (d, 1H), 7.82 (dd, 2H), 7.63 (d,1H), 7.48 (d, 1H), 7.47(d, 1H), 7.40(dd, 2H),7.32 (d, 1H), 7.28 (d, 1H), 5.76(s, 1H), 4.09 (m, 2H), 3.17(m, 6H), 3.14 (t, 3H). H NMR spectrum of fluorescent probe DTPD is shown in Figure 1 .

[0031] Example 2 Fluorescence Quantitative Detection of Hypochlorous Acid (1) Fluorescence spectrometric determination of hypochlorous acid Take the dual-function near-infrared fluorescent probe obtained in Example 1, prepare 1mM DTPD stock solution with DMSO, prepare 0.01M hypochlorous acid solution with distilled water, and prepare HEPES buffer solution with pH=7.4 and concentration of 0.025M. Take 20mL of DTPD stock solution and add it to a clean colorimetric tube, add different volumes of hypochlorous acid (10 mL, 20 mL, 30 mL, 40 mL, 50mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 110 mL, 120 mL), 0.5mL HEPES buffer, and dilute to 5mL with a mixed solution of secondary water and DMSO (v / v=1:1). After shaking, take 2.5mL and add it to a clean colorimetric dish. Detect on a fluorescence spectrophotometer (excitation wavelength is 436 nm). With the addition of hypochlorous acid, the solution gradually changes from orange to light yellow, and the fluorescence intensity at 650 nm gradually increases. The fluorescence spectrum is shown in Figure 2 .

[0032] (2) Determination of the linear relationship of hypochlorous acid According to the fluorescence spectrum titration experiment, with the addition of hypochlorous acid, the fluorescence intensity at 650 nm gradually increased, and the fluorescence intensity of the system at 650 nm I 650nm and [ClO -] showed a good linear relationship in the concentration range of 200 mM-760 mM (R 2 =0.97595), with [ClO - ] is the horizontal axis, and the fluorescence intensity I 650 nm As the vertical axis, the linear equation of hypochlorous acid concentration and fluorescence intensity is obtained: F=5.55219[ClO - ]-852.94073, [ClO - ] is in units of 10 -6 mol / L; working linear diagram see Figure 3 .

[0033] (3) Determine the concentration of hypochlorous acid in the test solution Take 20 mL of DTPD stock solution and add it to a clean colorimetric tube. Take x mL of the sample solution to be tested and add it to the colorimetric tube. Use a mixed solution of secondary water and DMSO (v / v=1:1) to make it up to 5 mL. Detect it on a fluorescence spectrophotometer. Substitute the measured fluorescence intensity into the linear equation of step (2) to obtain [ClO - ], the sample to be tested [ClO - ] 待测 =5000mL´[ClO - ] ´10 -6 / x mL, the concentration of hypochlorite ions can be calculated.

[0034] (4) Fluorescence determination of the interference of coexisting analytes on the detection of hypochlorous acid by the probe Take 20 mL of DTPD stock solution and add it to a clean colorimetric tube, and add 120 mL of ClO - , 120 mL of various other analytes (CO3 2- , I - , Cl - , Br - , H2PO3 - , HPO3 2- , S2O3 2- , HSO3 - , NO3 - , SO4 2- , GSH, Cys, Hcy), 0.5 mL HEPES buffer, dilute to 5 mL with secondary water and DMSO mixed solution (v / v=1:1), shake well and add 2.5 mL to a clean cuvette, and detect on a fluorescence spectrophotometer. The interference of coexisting analytes on the fluorescence intensity of the probe detecting hypochlorous acid can be seen in Figure 4 The experiment showed that other common analytes did not interfere with the determination of hypochlorous acid by the system.

[0035] Example 3 Fluorescence Quantitative Detection of Viscosity (1) Fluorescence spectroscopy of viscosity Take 20 mL of DTPD stock solution and add it to a clean colorimetric tube, add 120 mL of ClO - The solution was shaken and 5 mL of methanol and glycerol solution with different volume ratios were added respectively. Each sample was ultrasonicated for 10 min to remove bubbles. After standing at room temperature for 30 min, 2.5 mL was added to a clean cuvette and detected on a fluorescence spectrophotometer (excitation wavelength of 436 nm). As the glycerol ratio increased, the fluorescence intensity at 650 nm gradually increased. The fluorescence spectrum is shown in Figure 5 .

[0036] (2) Determination of linear relationship of viscosity According to the fluorescence spectrum titration experiment, as the proportion of glycerol increases, the fluorescence intensity at 650 nm gradually increases. 650nm and viscosity value (logη) show a good linear relationship in the range of 0-6 (R 2 =0.9751), with log(η) as the horizontal axis and fluorescence intensity log(I 650nm ) as the ordinate, and the linear equation of viscosity and fluorescence intensity is obtained: Y=0.26975x+6.25806. The working linear diagram is shown in Figure 6 .

[0037] (3) Determine the viscosity of the solution to be tested Take 20 mL of DTPD stock solution and 120 mL of ClO - The solution is added to a clean colorimetric tube, x mL of the sample solution to be tested is added, and the volume is made up to 5 mL with methanol. The fluorescence intensity is measured on a fluorescence spectrophotometer. Substitute the measured fluorescence intensity into the linear equation in step (2) to obtain log(η). The log(η) of the sample to be tested is 待测 =5000mL´log(η) ´10 -6 / x, the viscosity value can be obtained.

[0038] Example 4 Fluorescence Imaging of Endogenous Hypochlorous Acid and Viscosity in Living Cells Plate an appropriate concentration of RAW 264.7 cells in a cell culture dish and culture until the cells are in good condition. Prepare 1 mM fluorescent probe DTPD stock solution, 1 mM lipopolysaccharide (LPS) solution, 1 mM N-acetyl-l-cysteine (NAC) solution, and 1 mM ABAH solution.

[0039] LPS treatment group: First, 100 μL of LPS solution was added to the cell culture dish and incubated for 3 h. Then, 10 μL of the fluorescent probe DTPD stock solution was added to the culture medium and mixed thoroughly. After incubation for another 10 minutes, the culture medium was discarded and the cells were rinsed three times with PBS buffer (pH = 7.4).

[0040] LPS & NAC treatment group: First, 100 μL of LPS and NAC solution were added to the cell culture dish respectively and incubated for 3 h. Then, 10 μL of probe stock solution was added to the culture medium and mixed thoroughly. After incubation for another 10 minutes, the culture medium was discarded and the cells were rinsed three times with PBS buffer.

[0041] LPS & ABAH treatment group: First, 100 μL of LPS and ABAH solution were added to the cell culture dish respectively and incubated for 3 h. Then, 10 μL of fluorescent probe DTPD stock solution was added to the culture medium and mixed thoroughly. After incubation for another 10 minutes, the culture medium was discarded and the cells were rinsed three times with PBS buffer.

[0042] The experimental results showed that when cells were pre-incubated with LPS and then treated with the fluorescent probe DTPD, a clear NIR fluorescence signal could be observed. Then, when LPS-induced inflammatory cells were treated with N-acetyl-l-cysteine and ABAH (HClO scavenger), weaker NIR fluorescence signals were observed, indicating that the probe can have a good cell imaging effect in cells (see Figure 7 ).

[0043] The scope of protection claimed by the present invention is not limited to the above specific embodiments. For those skilled in the art, the present invention may have various variations and modifications. Any modifications, improvements and equivalent substitutions made within the concept and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. A dual-function near-infrared fluorescent probe, characterized in that: Its structural formula is shown in formula (I): 。 2. The method for preparing the bifunctional near-infrared fluorescent probe according to claim 1, characterized in that: Add the compound of formula (III) and N,N-diisopropylethylamine (DIPEA) to N,N-dimethylformamide (DMF) containing the compound of formula (II) and stir at room temperature for 8-12 hours; the reaction formula is as follows: 。 3. The method for preparing a bifunctional near-infrared fluorescent probe according to claim 2, wherein: After the reaction is completed, the bifunctional near-infrared fluorescent probe is obtained by column chromatography separation, and the developing solvent of the column chromatography separation is dichloromethane:methanol=(15-20):1 (v / v).

4. Use of the bifunctional near-infrared fluorescent probe according to claim 1 in the preparation and detection of hypochlorous acid preparations.

5. Use of the bifunctional near-infrared fluorescent probe according to claim 1 in preparing a preparation for detecting viscosity.

6. Use of the dual-function near-infrared fluorescent probe according to claim 1 in preparing an intracellular endogenous hypochlorous acid imaging preparation.

7. Use of the dual-function near-infrared fluorescent probe according to claim 1 in preparing an intracellular viscosity imaging preparation.

8. A method for quantitative fluorescence detection of hypochlorous acid, characterized in that: The following steps are involved: Step 1: prepare the DTPD stock solution of the dual-function near-infrared fluorescent probe according to claim 1, prepare a series of standard solutions with hypochlorous acid, and measure the fluorescence intensity at 650 nm on a fluorescence photometer. - The concentration is the horizontal axis, and the fluorescence intensity I 650nm Draw labeled curves for the ordinate and obtain linear equations; Step 2: Take the test solution containing hypochlorous acid, add it to the DTPD stock solution described in step 1 and dilute to volume with solvent, measure the fluorescence intensity at 650 nm on a fluorescence photometer, and obtain ClO according to the linear equation obtained in step 1. - concentration.

9. The method for fluorescent quantitative detection of hypochlorous acid according to claim 8, wherein: Fluorescence intensity I 650nm and ClO - The concentration range was 200 mM-760 mM, and the R 2 =0.97595.

10. A fluorescence quantitative detection method for viscosity, characterized in that: The following steps are involved: Step 1, prepare the DTPD stock solution of the dual-function near-infrared fluorescent probe according to claim 1, take the DTPD stock solution and the hypochlorous acid solution, add methanol and glycerol solution systems respectively, and as the glycerol ratio increases, the fluorescence intensity at 650 nm measured on the fluorescence photometer gradually increases. The fluorescence intensity at 650 nm is 650nm The viscosity value (logη) shows a good linear relationship in the range of 0-6, R 2 =0.9751, log(η) is taken as the horizontal axis, and the fluorescence intensity log(I 650nm ) is plotted as the ordinate to obtain a linear equation between viscosity and fluorescence intensity; Step 2: Mix the DTPD stock solution and hypochlorous acid solution described in step 1, add the sample solution to be tested, and then adjust the volume with the solvent. Detect the fluorescence intensity on a fluorescence spectrophotometer, and calculate the viscosity of the solution to be tested according to the linear equation in step 1.

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