Synthesis and Application of a Bifunctional Fluorescent Probe for Simultaneously Differentiating and Detecting Hypochlorous Acid and Viscosity

Abstract

This invention discloses a bifunctional fluorescent probe that simultaneously distinguishes between hypochlorous acid and viscosity. The chemical structure of the bifunctional probe is as follows: This bifunctional fluorescent probe combines a 1,8-naphthalenedimide derivative and an indole derivative through a simple condensation reaction. It recognizes hypochlorous acid through a sulfhydryl oxidation strategy and detects viscosity through a molecular rotor strategy. When the probe reacts with hypochlorous acid, it emits strong green fluorescence at 540 nm under an excitation wavelength of 430 nm; when it reacts with viscosity, it emits near-infrared fluorescence at 724 nm under an excitation wavelength of 650 nm. This probe exhibits good selectivity and rapid response in detecting hypochlorous acid and viscosity, and has great application potential in analytical chemistry, life sciences, and biomedicine.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to the synthesis and application of a bifunctional fluorescent probe that simultaneously distinguishes between hypochlorous acid and viscosity. This probe combines two fluorophores through a simple condensation reaction and can rapidly and selectively detect hypochlorous acid and viscosity from various bioactive substances. The green fluorescence channel selectively detects hypochlorous acid, while the near-infrared fluorescence channel selectively detects viscosity. It possesses advantages such as a large Stokes shift, high detection sensitivity, and visual detection capabilities. Background Technology

[0002] Hypochlorous acid (HOCl) is considered one of the most important biological reactive oxygen species, playing a crucial role in the immune system, including the 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 chloride ion peroxidation mediated by myeloperoxidase (MPO), which can kill invading pathogens and bacteria in the body (J. Am. Chem. Soc., 2011,133:5680–5682). However, uncontrolled production of HOCl can lead to severe tissue damage and various diseases, such as liver injury, atherosclerosis, rheumatoid arthritis, cardiovascular disease, arthritis, and cancer (Am. J. Physiol GastrointestLiver Physiol., 2005, 289:760–7; J. Orthop. Res., 2007, 25:1128–35). Cell viscosity is an important microenvironmental parameter in biological systems, revealing the orderly functioning of cells and governing 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 associated with a variety of diseases and functional disorders (Anal. Chem., 2020, 92, 21, 14667–14675).

[0003] Currently, various methods have been developed for the detection of hypochlorous acid and viscosity, such as liquid chromatography-mass spectrometry (LCMS), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GCMS), and fluorescence spectroscopy. Among these methods, fluorescent probe analysis is generally favored due to its rapid response, high sensitivity, 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 viscosity have been reported, most of which detect only one of the two (RSC Adv., 2024, 14, 11151–11156; Chem. Commun., 2018, 54, 8522-8525). Therefore, simultaneously distinguishing between the detection of hypochlorous acid and viscosity remains a significant challenge. Summary of the Invention

[0004] In view of the above, and to overcome some shortcomings of existing technologies, the present invention aims to provide a bifunctional fluorescent probe that can simultaneously distinguish and detect hypochlorous acid and viscosity. This 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, which is simple to prepare, highly sensitive, has a low detection limit, and is low in cost.

[0006] The specific technical solution adopted by this invention to solve the problem is the synthesis and preparation of a bifunctional fluorescent probe that can simultaneously distinguish and detect hypochlorous acid and viscosity, and the application of a device for quantitative analysis of hypochlorous acid and viscosity in the environment and simultaneous imaging of hypochlorous acid and viscosity in living cells. The chemical structural formula of the bifunctional probe is as follows:

[0007] .

[0008] Synthesis of a bifunctional fluorescent probe capable of simultaneously distinguishing between hypochlorous acid and viscosity, characterized in that the preparation method of the bifunctional fluorescent probe includes the following steps:

[0009] Step 1. Synthesis of tert-butyl (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinoline-2(3H-yl)ethyl)carbamate

[0010] Add tert-butyl 2-6-bromo-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)carbamate to N,NAdd thiomorpholine to dimethylformamide (DMF) and react overnight at 60°C. After the reaction is complete, add the reaction solution to deionized water, filter, and evaporate the solid to dryness to obtain tert-butyl (2-(1,3-dioxo-6-thiomorpholine-1H-benzo[de]isoquinoline-2(3H-yl)ethyl)carbamate.

[0011] Step 2. Synthesis of 2-(1,3-dioxo-6-thiomorpholine-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate

[0012] (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinoline-2(3H-yl)ethyl)carbamate tert-butyl ester was added to anhydrous dichloromethane, followed by the addition of trifluoroacetic acid. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction system was dried by rotary evaporation, and the product was purified by column chromatography to obtain 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate.

[0013] Step 3. Synthesize the bifunctional fluorescent probe.

[0014] 5-Carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodide was added to anhydrous dichloromethane, followed by the addition of 4-dimethylaminopyridine (DMAP). The reaction was carried out at room temperature for 5 min, and then 2-(1,3-dioxo-6-thiomorpholine-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate was added and stirred for 5 min. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and stirred at room temperature overnight. After the reaction was complete, the reaction system was evaporated to dryness, and the bifunctional fluorescent probe was purified by column chromatography.

[0015] A method for using a bifunctional fluorescent probe for simultaneously distinguishing and detecting hypochlorous acid and viscosity according to the present invention: Unless otherwise specified, the bifunctional probe is typically dissolved in dimethyl sulfoxide (DMSO) at room temperature, and analyzed in an environment where the organic phase and aqueous phase volume ratio is 1:99. The organic phase is DMSO, and the aqueous phase is phosphate buffered saline (PBS) at pH = 7.4. The specific characteristics of the bifunctional fluorescent probe for simultaneously distinguishing and detecting hypochlorous acid and viscosity are as follows: The bifunctional fluorescent probe is dissolved in DMSO in an organic phase and aqueous phase (1:99, v / v) solution. After reacting with hypochlorous acid for 30 minutes, it emits 540 nm green fluorescence at an excitation wavelength of 430 nm. The probe is tested for viscosity response in glycerol-methanol systems with different ratios, emitting 724 nm near-infrared fluorescence at an excitation wavelength of 650 nm. Therefore, it enables the detection of specific analytes using specific excitation and fluorescence emission signals, and when both substances are present, they can be well distinguished using different excitation and fluorescence emission signals. The aforementioned bifunctional fluorescent probe enables the simultaneous detection of hypochlorous acid and viscosity under different detection conditions. It shows no significant response to other reactive oxygen species, reactive sulfur, common amino acids, metal ions, and reactive nitrogen. The detection limits for hypochlorous acid and viscosity are as low as 3.2 nM and 0.12 cP, respectively. Therefore, the bifunctional fluorescent probe disclosed in this invention can achieve highly sensitive and quantitative detection of both. Attached Figure Description

[0016] Figure 1 The proton NMR spectrum of the bifunctional fluorescent probe described in this invention.

[0017] Figure 2 The UV and fluorescence spectra of the bifunctional fluorescent probe described in this invention in response to hypochlorous acid and viscosity.

[0018] Figure 3 The fluorescence quantitative analysis diagram of the bifunctional fluorescent probe of the present invention in response to hypochlorous acid and viscosity.

[0019] Figure 4 The bifunctional fluorescent probe described in this invention can simultaneously distinguish between intracellular hypochlorous acid and viscous cell images. Detailed Implementation

[0020] The present invention will be further explained in conjunction with the following formula.

[0021] The synthetic route of the bifunctional fluorescent probe described in this invention is as follows:

[0022]

[0023] Example 1. Synthesis of tert-butyl (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinoline-2(3H-yl)ethyl)carbamate

[0024] Add 3.00 g (7.16 mmol) of tert-butyl 2-6-bromo-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)carbamate to 30 mL N,N 1.48 g (14.31 mmol) of thiomorpholine was added to dimethylformamide (DMF), and the mixture was reacted overnight at 60°C. After the reaction was complete, the reaction solution was added to 200 mL of deionized water, filtered, and the solid was evaporated to dryness to obtain 2.60 g of tert-butyl (2-(1,3-dioxo-6-thiomorpholine-1H-benzo[de]isoquinoline-2(3H-yl)ethyl)carbamate, with a yield of 82.30%.

[0025] Example 2. Synthesis of 2-(1,3-dioxo-6-thiomorpholine-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate

[0026] 2.00 g (4.53 mmol) of tert-butyl 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinoline-2(3H-yl)ethyl)carbamate was added to 20 mL of anhydrous dichloromethane, followed by 3 mL of trifluoroacetic acid. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was evaporated to dryness, and the solution was purified by column chromatography to obtain 1.80 g of 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate, with a yield of 87.25%.

[0027] Example 3. Synthesis of the bifunctional fluorescent probe.

[0028] 100.0 mg (193.64 µmol) of 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodide was added to 8 mL of anhydrous dichloromethane, followed by 3 mg of 4-dimethylaminopyridine (DMAP). The mixture was reacted at room temperature for 5 min. Subsequently, 88.2 mg (193.64 µmol) of 2-(1,3-dioxo-6-thiomorpholine-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate was added and stirred for 5 min. Then, 55.7 mg (290.46 µmol) of 2-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)-2-(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate was added. The bifunctional fluorescent probe was prepared by stirring 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride at room temperature overnight. After the reaction was completed, the reaction system was evaporated to dryness and purified by column chromatography to obtain 120.0 mg of the bifunctional fluorescent probe, with a yield of 78.48%.

[0029] Example 4. Bifunctional fluorescent probes simultaneously distinguish between hypochlorous acid and viscosity in an in vitro environment.

[0030] The bifunctional fluorescent probe of this invention is used for spectral property experiments. The bifunctional probe is dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM probe solution. Solutions of 10 mM hypochlorous acid and different viscosities (different ratios of methanol:glycerol) are also prepared. The specific testing method is as follows: Take 20 μL of the 1 mM probe solution, add 20 μL of the 10 mM hypochlorous acid solution, and finally add 1960 μL of PBS. For all tests, the volume ratio of the organic phase to the aqueous phase is maintained at 1:99 (the total volume of each test sample is 2 mL). For example, when testing the fluorescence intensity of 100 μM hypochlorous acid, the sample preparation is as follows: Take 20 μL of the 1 mM probe solution, 20 μL of the 10 mM hypochlorous acid aqueous solution, and add 1960 μL of PBS buffer solution to a 2 mL sample tube. After shaking and mixing at room temperature for 30 minutes, the fluorescence emission intensity can be measured using an excitation wavelength of 430 nm. When detecting viscosity, the specific testing method is as follows: Take 20 μL of 1 mM probe solution, add 1980 μL of prepared solutions of different viscosities, shake well at room temperature for 30 minutes, and then measure the fluorescence emission intensity using an excitation wavelength of 650 nm. This bifunctional probe enables the differentiation and detection of 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, making it ideal for imaging / quantitative analysis of endogenous hypochlorous acid and viscosity in live cells.

[0031] Example 5. Dual-channel fluorescence imaging analysis of endogenous hypochlorous acid and viscosity in HepG2 (liver cancer cells) cells.

[0032] HepG2 cells were passaged into confocal cell culture medium and cultured under standard growth conditions for 24 hours. Then, an appropriate amount of probe (5 μM) was added and cultured under standard growth conditions for another 30 minutes. The cells were then photographed under a confocal fluorescence microscope, and the endogenous hypochlorous acid and viscosity of HepG2 cells were imaged using the 500-600 nm green channel and the 700-750 nm near-infrared fluorescence channel, respectively. The bifunctional fluorescent probe of this invention can emit two different fluorescences in the cells to simultaneously distinguish and detect hypochlorous acid and viscosity in the cells, successfully realizing the bichannel fluorescence imaging analysis of endogenous hypochlorous acid and viscosity in cells.

[0033] This invention provides a bifunctional fluorescent probe that simultaneously distinguishes between hypochlorous acid and viscosity. It links a 1,8-naphthalenedimide derivative and an indole derivative through a simple condensation reaction, detecting hypochlorous acid via a sulfhydryl oxidation strategy and viscosity via a molecular rotor strategy. When reacting with hypochlorous acid, it emits strong green fluorescence at 540 nm under an excitation wavelength of 430 nm; when reacting with viscosity, it emits near-infrared fluorescence at 724 nm under an excitation wavelength of 650 nm, exhibiting significant fluorescence. Furthermore, the reaction product has good water solubility, fast response speed, and a large Stokes shift. It has significant practical application value in biochemistry, analytical detection, and other fields. Although the invention has been described in detail through the above preferred embodiments, it should be understood that the above description should not be considered as limiting the invention. Various modifications and substitutions to the invention will be obvious to those skilled in the art after reading the above content. Therefore, fluorescent probes with similar technical features as described herein fall within the protection scope of this patent.

Claims

1. A bifunctional fluorescent probe for simultaneously distinguishing detection of hypochlorous acid and viscosity, characterized in that, The chemical structure of the bifunctional fluorescent probe is shown below: 。 2. The synthesis of bifunctional fluorescent probe as claimed in claim 1 wherein, The method for synthesizing the bifunctional fluorescent probe includes the following steps: Step 1. Synthesis of tert-butyl (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinoline-2(3H-yl)ethyl)carbamate Add tert-butyl 2-6-bromo-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl)carbamate to N, N Add thiomorpholine to dimethylformamide (DMF) and react overnight at 60°C. After the reaction is complete, add the reaction solution to deionized water, filter, and evaporate the solid to dryness to obtain tert-butyl (2-(1,3-dioxo-6-thiomorpholine-1H-benzo[de]isoquinoline-2(3H-yl)ethyl)carbamate. Step 2. Synthesis of 2-(1,3-dioxo-6-thiomorpholine-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate (2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinoline-2(3H-yl)ethyl)carbamate tert-butyl ester was added to anhydrous dichloromethane, followed by the addition of trifluoroacetic acid. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction system was dried by rotary evaporation, and the product was purified by column chromatography to obtain 2-(1,3-dioxo-6-thiomorpholino-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate. Step 3. Synthesize 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 was added to anhydrous dichloromethane, followed by the addition of 4-dimethylaminopyridine (DMAP). The reaction was carried out at room temperature for 5 min, and then 2-(1,3-dioxo-6-thiomorpholine-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate was added and stirred for 5 min. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and stirred at room temperature overnight. After the reaction was complete, the reaction system was evaporated to dryness, and the bifunctional fluorescent probe was purified by column chromatography.

3. The method for synthesizing a bifunctional probe as described in claim 2, characterized in that, The molar ratio of 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodide and 2-(1,3-dioxo-6-thiomorpholine-1H-benzo[de]isoquinoline-2(3H)-yl)ethyl-1-amine-2,2,2-trifluoroacetate in step 3 is 1:

1.

4. The application of the bifunctional fluorescent probe as described in claim 1 in device fabrication, characterized in that, The fabricated device can quantitatively analyze hypochlorous acid and viscosity in the environment, and simultaneously distinguish and image hypochlorous acid and viscosity in cells, tissues and living organisms.

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