Synthesis and application of bifunctional fluorescent probe for simultaneously and differentially detecting hypochlorous acid and viscosity

Abstract

The invention discloses a difunctional fluorescent probe for simultaneously distinguishing and detecting hypochlorous acid and viscosity. The chemical structural formula of the difunctional probe is as follows: # imgabs0 #. According to the bifunctional fluorescent probe, a 1, 8-naphthalimide derivative and an indole derivative are combined through a simple condensation reaction, hypochlorous acid is recognized through a sulfenyl oxidation strategy, and the viscosity is detected through a molecular rotor strategy. When the probe reacts with hypochlorous acid, strong green fluorescence of 540 nm is emitted under the excitation wavelength of 430 nm; when acting with viscosity, the fluorescent powder emits near-infrared fluorescence of 724 nm under the excitation wavelength of 650 nm. The probe for detecting hypochlorous acid and viscosity has the advantages of good selectivity, quick response and the like, and has a huge application prospect in the technical fields of analytical chemistry, life science, biomedical treatment and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and specifically relates to the synthesis and application of a dual-function fluorescent probe for simultaneously distinguishing and detecting hypochlorous acid and viscosity. The probe combines two fluorophores through a simple condensation reaction and can rapidly and selectively detect hypochlorous acid and viscosity from various biologically active substances. The green fluorescent channel selectively detects hypochlorous acid, and the near-infrared fluorescent channel selectively detects viscosity. It has the advantages of large Stokes shift, high detection sensitivity, and visual detection. Background Art

[0002] Hypochlorous acid (HOCl) is considered one of the most important biological reactive oxygen species and plays an important role in the immune system, including defense and eradication of pathogens (J. Am. Chem. Soc., 2013, 135:9944–9; J. Lumin., 2020, 226:117460). Endogenous hypochlorous acid is produced by white blood cells through myeloperoxidase (MPO)-mediated chloride peroxidation, which can kill invading pathogens and bacteria in the body (J. Am. Chem. Soc., 2011, 133:5680–5682). However, uncontrolled production of HOCl may lead to severe tissue damage and various diseases, such as liver damage, atherosclerosis, rheumatoid arthritis, cardiovascular disease, arthritis and cancer (Am. J. Physiol Gastrointest Liver Physiol., 2005, 289:760–7; J. Orthop. Res., 2007, 25:1128–35). Cell viscosity is an important microenvironmental parameter in biological systems. It can show the orderly function of cells and can also dominate signal transduction, electron transfer, diffusion of reactive metabolites and biomolecular interactions in living cells (Biochem. Pharmacol., 1995, 49, 1589). However, abnormal changes in cell viscosity are closely related to a variety of diseases and dysfunctions (Anal. Chem., 2020, 92, 21, 14667–14675).

[0003] At present, a variety of methods have been developed to detect hypochlorous acid and viscosity, such as liquid chromatography-mass spectrometry (LCMS), high-performance liquid chromatography, gas chromatography-mass spectrometry (GCMS) and fluorescence spectroscopy. Among these detection methods, fluorescent probe analysis is generally favored due to its rapid response and high sensitivity, as well as spatial resolution and satisfactory biocompatibility (Angew. Chem. Int. Ed., 2017, 56, 16611–16615; Anal. Chem., 2016, 76,166–181). Many fluorescent probes for detecting hypochlorous acid and viscosity have been reported, and most of them detect one of them alone (RSC Adv., 2024,14, 11151–11156; Chem. Commun., 2018, 54, 8522-8525). Therefore, the simultaneous detection of hypochlorous acid and viscosity is still a huge challenge at present. Summary of the invention

[0004] In view of the above situation, in order to overcome some of the deficiencies of the prior art, the present invention aims to provide a dual-function fluorescent probe for simultaneously distinguishing and detecting hypochlorous acid and viscosity. The probe can rapidly and selectively detect hypochlorous acid and viscosity from various bioactive substances under specific detection conditions.

[0005] The present invention also aims to provide a method for synthesizing and applying the above-mentioned bifunctional fluorescent probe with simple preparation method, high sensitivity, low detection limit and low cost.

[0006] The specific technical solution adopted by the present invention to solve the problem is to synthesize a dual-function fluorescent probe for simultaneously distinguishing and detecting hypochlorous acid and viscosity, and to prepare a device for quantitatively analyzing hypochlorous acid and viscosity in the environment, and simultaneously distinguishing and imaging hypochlorous acid and viscosity in living cells. The chemical structure of the dual-function probe is as follows: .

[0007] A synthesis of a dual-function fluorescent probe for simultaneously distinguishing and detecting hypochlorous acid and viscosity, characterized in that the preparation method of the dual-function fluorescent probe comprises the following steps: Step 1. Synthesis of tert-butyl (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H-yl)ethyl)carbamate Add tert-butyl (2-6-bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl)carbamate N,N-dimethylformamide (DMF), then add thiomorpholine, react at 60 degrees Celsius overnight, after the reaction is complete, add the reaction solution into deionized water, filter, and spin dry the solid to obtain tert-butyl (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H-yl)ethyl)carbamate; Step 2. Synthesis of 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H)-yl)ethan-1-amine-2,2,2-trifluoroacetate Add tert-butyl (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H-yl)ethyl)carbamate to anhydrous dichloromethane, then add trifluoroacetic acid, stir at room temperature overnight, after the reaction is complete, spin dry the reaction system, separate and purify by column chromatography to obtain 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate; Step 3. Synthesis of the bifunctional fluorescent probe 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodide is added to anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP) is added, and the reaction is carried out at room temperature for 5 minutes. Subsequently, 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate is added and stirred for 5 minutes, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added, and the reaction is stirred at room temperature overnight. After the reaction is complete, the reaction system is spin-dried, and the bifunctional fluorescent probe is purified and separated by column chromatography.

[0008] The present invention discloses a method for using a dual-function fluorescent probe for simultaneously distinguishing and detecting hypochlorous acid and viscosity: unless otherwise specified, the dual-function probe is usually dissolved in dimethyl sulfoxide (DMSO) at room temperature, and used for analysis and detection in an environment where the volume ratio of the organic phase to the aqueous phase is 1:99, the organic phase is dimethyl sulfoxide, and the aqueous phase is a phosphate buffer solution (PBS) with a pH of 7.4. The specific characteristics of the dual-function fluorescent probe for simultaneously distinguishing and detecting hypochlorous acid and viscosity are as follows: the dual-function fluorescent probe is dissolved in DMSO, the dual-function probe is dissolved in an organic phase and an aqueous phase (1:99, v / v) solution, and after 30 minutes with hypochlorous acid, it emits 540 nm green fluorescence at an excitation wavelength of 430 nm; the probe is tested for response viscosity in a glycerol-methanol system with different ratios, and emits 724 nm near-infrared fluorescence at an excitation wavelength of 650 nm. Therefore, the detection of a specific analyte with a specific excitation and fluorescence emission signal is achieved, and when both substances are present, the two can be well distinguished by using different excitation and fluorescence emission signals. The above-mentioned bifunctional fluorescent probe can simultaneously distinguish and detect hypochlorous acid and viscosity under different detection conditions, and has no obvious response to other active oxygen, active sulfur, common amino acids, metal ions and active nitrogen. The detection limits of hypochlorous acid and viscosity are as low as 3.2 nM and 0.12 cP, respectively. Therefore, the bifunctional fluorescent probe disclosed in the present invention can achieve highly sensitive distinguishing quantitative detection of the two. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the bifunctional fluorescent probe of the present invention.

[0010] Figure 2 The dual-function fluorescent probe of the present invention responds to ultraviolet and fluorescence spectra of hypochlorous acid and viscosity.

[0011] Figure 3 Fluorescence quantitative analysis graph of the dual-function fluorescent probe of the present invention in response to hypochlorous acid and viscosity.

[0012] Figure 4 The dual-function fluorescent probe of the present invention simultaneously distinguishes and images intracellular hypochlorous acid and viscosity cell maps. DETAILED DESCRIPTION

[0013] The present invention is further described in conjunction with the following formula.

[0014] The synthesis route of the bifunctional fluorescent probe of the present invention is as follows:

[0015] Example 1. Synthesis of tert-butyl (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H-yl)ethyl)carbamate Add 3.00 g (7.16 mmol) of tert-butyl (2-6-bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl)carbamate to 30 mL N,N -dimethylformamide (DMF), and then add 1.48 g (14.31 mmol) of thiomorpholine, react at 60 degrees Celsius overnight, after the reaction is complete, add the reaction solution into 200 mL of deionized water, filter, and spin-dry the solid to obtain 2.60 g of tert-butyl (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinoline-2(3H-yl)ethyl)carbamate, with a yield of 82.30%.

[0016] Example 2. Synthesis of 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H)-yl)ethan-1-amine-2,2,2-trifluoroacetate 2.00 g (4.53 mmol) of tert-butyl (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H-yl)ethyl)carbamate was added to 20 mL of anhydrous dichloromethane, and then 3 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature overnight. After the reaction was complete, the reaction system was dried by spin drying and separated and purified by column chromatography to obtain 1.80 g of 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate with a yield of 87.25%.

[0017] Example 3. Synthesis of the bifunctional fluorescent probe 100.0 mg (193.64 µmol) of 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodide was added to 8 mL of anhydrous dichloromethane, and then 3 mg of 4-dimethylaminopyridine (DMAP) was added. The mixture was reacted at room temperature for 5 min. Subsequently, 88.2 mg (193.64 µmol) of 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H)-yl)ethan-1-amine-2,2,2-trifluoroacetate was added and stirred for 5 min. Then, 55.7 mg (290.46 The mixture was stirred at room temperature overnight. After the reaction was complete, the reaction system was dried by rotary evaporation and purified by column chromatography to obtain 120.0 mg of the bifunctional fluorescent probe with a yield of 78.48%.

[0018] Example 4. Dual-functional fluorescent probe for simultaneous detection of hypochlorous acid and viscosity in vitro The spectral property experiment of the bifunctional fluorescent probe described in the present invention: the bifunctional probe is dissolved in dimethyl sulfoxide (DMSO) to prepare a probe solution with a concentration of 1 mM, and a solution with a concentration of 10 mM hypochlorous acid and different viscosities (different ratios of methanol: glycerol) is prepared. The specific test method is: take 20 μL of 1 mM probe solution, add 20 μL of 10 mM hypochlorous acid solution, and finally add 1960 μL PBS. All tests maintain the volume ratio of organic phase to aqueous phase at 1:99 (the total volume of each test sample is 2 mL). For example, when the fluorescence intensity of hypochlorous acid with a concentration of 100 μM is required to be tested, the sample preparation is: take 20 μL of 1 mM probe solution, 20 μL of 10 mM hypochlorous acid aqueous solution, and then add 1960 μL of PBS buffer solution in a 2 mL sample tube. After shaking at room temperature for 30 minutes, the fluorescence emission intensity can be measured with an excitation wavelength of 430 nm. When detecting viscosity, the specific test method is: take 20 μL of 1 mM probe solution, add 1980 μL of prepared solutions of different viscosities, shake at room temperature for 30 minutes, and measure its fluorescence emission intensity with an excitation wavelength of 650 nm. This dual-function probe can distinguish and detect two biologically active substances, hypochlorous acid and viscosity, using different excitation wavelengths and fluorescence emission signals. It has high sensitivity, with detection limits as low as 3.2 nM and 0.12 cP, respectively, and is very suitable for imaging / quantitative analysis of endogenous hypochlorous acid and viscosity in living cells.

[0019] Example 5. Dual-channel fluorescence imaging analysis of endogenous hypochlorous acid and viscosity in HepG2 (liver cancer cell) cells HepG2 cells were passaged into a confocal dish cell culture medium, and after culturing for 24 hours under standard growth conditions, an appropriate amount of probe (5 μM) was added and continued to be cultured under standard growth conditions for 30 minutes, and then photographed under a confocal fluorescence microscope, and endogenous hypochlorous acid and viscosity of HepG2 cells were imaged using a 500-600 nm green channel and a 700-750 nm near-infrared fluorescence channel, respectively. The dual-functional fluorescent probe of the present invention can emit two different fluorescences in cells to simultaneously distinguish and detect hypochlorous acid and viscosity in cells, and successfully realizes dual-channel fluorescence imaging analysis of endogenous hypochlorous acid and viscosity in cells.

[0020] The present invention provides a dual-function fluorescent probe for simultaneously distinguishing and detecting hypochlorous acid and viscosity. The 1,8-naphthalimide derivative and the indole derivative are connected by a simple condensation reaction, and hypochlorous acid is detected by a sulfur oxidation strategy, and viscosity is detected by a molecular rotor strategy. When reacting with hypochlorous acid, a strong green fluorescence of 540 nm is emitted at an excitation wavelength of 430 nm; when acting with viscosity, a near-infrared fluorescence of 724 nm is emitted at an excitation wavelength of 650 nm, and the fluorescence phenomenon is obvious. And the product after the reaction has good water solubility, fast response speed, large Stokes shift and the like. It has great practical application value in the fields of biochemistry, analysis and detection. Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be regarded as a limitation of the present invention. After reading the above content, it will be obvious to those skilled in the art for various modifications and substitutions of the present invention. Therefore, fluorescent probes with similar technical features as described herein all fall within the scope of protection of this patent.

Claims

1. A dual-function fluorescent probe for simultaneously distinguishing and detecting hypochlorous acid and viscosity, characterized in that: The chemical structure of the bifunctional fluorescent probe is as follows: 。 2. The synthesis of the bifunctional fluorescent probe according to claim 1, characterized in that: The synthesis method of the bifunctional fluorescent probe comprises the following steps: Step 1. Synthesis of tert-butyl (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H-yl)ethyl)carbamate Add tert-butyl (2-6-bromo-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl)carbamate N, N -dimethylformamide (DMF), then add thiomorpholine, react at 60 degrees Celsius overnight, after the reaction is complete, add the reaction solution into deionized water, filter, and spin dry the solid to obtain tert-butyl (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H-yl)ethyl)carbamate; Step 2. Synthesis of 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H)-yl)ethan-1-amine-2,2,2-trifluoroacetate Add tert-butyl (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H-yl)ethyl)carbamate to anhydrous dichloromethane, then add trifluoroacetic acid, stir at room temperature overnight, after the reaction is complete, spin dry the reaction system, separate and purify by column chromatography to obtain 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate; Step 3. Synthesis of the bifunctional fluorescent probe 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodide is added to anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP) is added, and the reaction is carried out at room temperature for 5 minutes. Subsequently, 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate is added and stirred for 5 minutes, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added, and the reaction is stirred at room temperature overnight. After the reaction is complete, the reaction system is spin-dried, and the bifunctional fluorescent probe is separated and purified by column chromatography.

3. The method for synthesizing a bifunctional probe according to claim 2, wherein: The molar ratio of 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodide and 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinolin-2(3H)-yl)ethan-1-amine-2,2,2-trifluoroacetate in step 3 is 1:

1.

4. The use of the bifunctional fluorescent probe according to claim 1 for preparing a device, characterized in that: The fabricated device is capable of quantitatively analyzing hypochlorous acid and viscosity in the environment, as well as simultaneously differentiating and imaging hypochlorous acid and viscosity in cells, tissues, and living bodies.

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