A hemicyanine dye derivative bs-1 and a synthesis method and application thereof

Abstract

The application provides a semi-florin dye derivative BS-1 and a synthesis method and application thereof. The specific name of the semi-florin dye derivative is (E)-3-(2-(2-(6-(4-(dimethylamino)phenyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3,3-dimethyl-3H-indol-1-ium-1-yl)propane-1-sulfonate. The application also provides a synthesis method of the derivative BS-1 and a mitochondrial microenvironment polarity level fluorescence imaging experiment of the derivative BS-1 in LX-2 (human liver stellate cells). The derivative BS-1 has high sensitivity response to the polarity level in vitro test and cells and has relatively significant fluorescence imaging capability through fluorescence spectrum and laser confocal microscope verification. Therefore, the semi-florin dye derivative BS-1 can be used as a fluorescence probe for dynamic detection of the mitochondrial microenvironment polarity level in vitro and cells.

Description

Technical Field

[0001] This invention relates to hemicyanine dye derivatives and polar-responsive fluorescent probes, specifically to a biological method for synthesizing a hemicyanine dye derivative and its application as a polar-responsive fluorescent probe. Background Technology

[0002] Polarity is a crucial physicochemical parameter in the cellular microenvironment, playing a key role in maintaining cellular homeostasis, regulating biomolecular interactions, and mediating various physiological activities. Processes such as immune responses, cell activation, and differentiation can all induce changes in cell polarity, and abnormalities in cell polarity are closely related to cellular dysfunction and even the development of various diseases—abnormal polarity alterations can lead to tumors, diabetes, Alzheimer's disease, cancer, and many other diseases. In recent years, increasing research has shown that changes in the polarity of the physiological microenvironment have been considered effective biomarkers for diseases such as tumors. Therefore, real-time, in-situ, and precise monitoring of the dynamic changes in intracellular polarity is of great significance for a deeper understanding of the molecular mechanisms of related physiological and pathological processes and for promoting the early diagnosis and treatment of diseases.

[0003] Fluorescence imaging technology has become an important tool for studying changes in the intracellular microenvironment due to its advantages such as ease of operation, high sensitivity, non-invasiveness, low toxicity, and excellent spatiotemporal resolution. Among them, small molecule fluorescent probes have attracted much attention for detecting intracellular polarity changes due to their tunable structure, good biocompatibility, and ease of functionalization. Polarity-responsive fluorescent probes typically consist of three parts: an intramolecular electron donor (such as an amino group), a conjugated linker region, and an electron acceptor. Under photoexcitation, they undergo intramolecular charge transfer (ICT), thereby producing a sensitive spectral response to changes in the polarity of the surrounding environment.

[0004] Hemicyanine dyes belong to an important branch of cyanine dyes. They possess a typical D-π-A (donor-π-conjugated-acceptor) structure, where electron-withdrawing and electron-donating groups are linked by conjugated chains containing an odd number of carbon atoms. Hemicyanine dyes exhibit significant advantages such as high molar absorptivity, long absorption and emission wavelengths (extending to the near-infrared region), high fluorescence quantum yield, and large Stokes shift, making them highly favored in the design and synthesis of fluorescent probes. More importantly, the unique D-π-A structure of hemicyanine dyes endows them with a significant solvation-induced color change effect—their absorption and emission spectra shift markedly with changes in solvent polarity. This characteristic makes hemicyanine derivatives an ideal molecular platform for constructing polar-responsive fluorescent probes.

[0005] Based on this, the present invention uses hemicyanine dye as the probe backbone and covalently links a triphenylamine derivative to the end of the hemicyanine dye to enhance electron-donating ability. This significantly improves the optical properties of the hemicyanine dye derivative BS-1 of the present invention, such as sensitivity, selectivity, and signal-to-background ratio. Summary of the Invention

[0006] The purpose of this invention is to provide a hemicyanine dye derivative BS-1, its synthesis method, and the application of this derivative BS-1 as a fluorescent probe for detecting mitochondrial microenvironment polarity in vitro and in cells.

[0007] This invention provides a hemicyanine dye derivative BS-1, its synthesis method, and its application. The specific name of the hemicyanine dye derivative is (E)-3-(2-(2-(6-(4-(dimethylamino)phenyl)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-3H-indol-1-ium-1-yl)propane-1-sulfonate. Its English name is ((E)-3-(2-(2-(6-(4-(dimethylamino)phenyl)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-3H-indol-1-ium-1-yl)propane-1-sulfonate. Its structural formula is: .

[0008] The method for synthesizing BS-1 provided by this invention includes the following steps: 1) Add 10 equivalents of cyclohexanone and an equal amount of phosphorus oxychloride to a round-bottom flask, then add 15 mL of DMF and stir at room temperature for 2 hours. After the reaction is complete, pour the reaction solution into 100 mL of ice water and adjust the pH to 7.0 using sodium bicarbonate solid. Finally, extract with dichloromethane and evaporate the extract under vacuum to obtain compound B1. 2) Add 5 equivalents of compound B1 and 4-bromo-2-hydroxybenzaldehyde to a round-bottom flask, add 5 equivalents of cesium carbonate to the flask, and use 20 mL of anhydrous DMF as the reaction solvent; after stirring at room temperature for 16 hours, evaporate the reaction liquid under vacuum to obtain the crude product; purify the crude product by silica gel column chromatography using CH3OH and DCM as eluents to obtain yellow compound B2; 3) Add 3 equivalents of compound B2 and an equal equivalent of (4-dimethylaminophenyl)boric acid to a 100 mL round-bottom flask, then add 16 mL of toluene, 6 mL of ethanol and 2 mL of water as reaction solvents; under nitrogen protection, heat to 80 °C and reflux overnight; evaporate the solution to dryness under reduced pressure, then add 10 mL of dichloromethane and extract three times with saturated brine to obtain the crude product; separate the crude product by PE / EA column chromatography to obtain white powder compound B3; 4) Add 2 equivalents of compound B3 and an equal equivalent of 3-(2,3,3-trimethyl-3H-indol-1-onthiol-1-yl)propane-1-sulfonate to a 100 mL round-bottom flask; add a small amount of piperidine as a catalyst and use 10 mL of ethanol as the reaction solvent; heat to 85 °C and reflux overnight; vacuum evaporate to obtain the crude product; use DCM / CH3OH as the eluent for column chromatography to separate the dark blue solid product, i.e., BS-1.

[0009] The structural formulas of compounds B1, B2, and B3 are as follows: .

[0010] The above-mentioned synthesis is based on a hemicyanine dye derivative BS-1 as a fluorescent probe for rapid and specific qualitative detection of polarity changes in vitro, and the application of this derivative BS-1 in LX-2 cells (human hepatic stellate cells) for efficient uptake and detection of cellular oxidative stress.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention is based on hemicyanine dyes, and the detection method is simple. It represents a significant breakthrough in fluorescence wavelength and selectivity, enabling in vitro qualitative and quantitative detection of polarity using fluorescence spectroscopy. The excellent fluorescence imaging capability for polarity has been verified by spectroscopy and confocal microscopy.

[0012] 2. BS-1 was used to achieve a rapid response to polarity and a significant fluorescence imaging capability for polarity in LX-2 cells. Attached Figure Description

[0013] Figure 1 1H NMR spectrum of BS-1 Figure 2 BS-1 carbon NMR spectrum Figure 3 Mass spectrum of BS-1 Figure 4 UV and fluorescence response diagrams of BS-1 Figure 5 Selectivity test chart of BS-1 Figure 6Laser confocal microscopy test diagram of BS-1 on the intrinsic and extrinsic polarities of LX-2 cells. Detailed Implementation

[0014] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the following embodiments.

[0015] Example 1

[0016] 1) Add 10 equivalents of cyclohexanone and an equal amount of phosphorus oxychloride to a round-bottom flask, then add 15 mL of DMF and stir at room temperature for 2 hours. After the reaction is complete, pour the reaction solution into 100 mL of ice water and adjust the pH to 7.0 using sodium bicarbonate solid. Finally, extract with dichloromethane and evaporate the extract under vacuum to obtain compound B1. 2) Add 5 equivalents of compound 1 and 4-bromo-2-hydroxybenzaldehyde to a round-bottom flask, add 5 equivalents of cesium carbonate to the flask, and use 20 mL of anhydrous DMF as the reaction solvent; place the round-bottom flask at room temperature and stir for 16 hours; after the reaction is completed, evaporate the reaction liquid under vacuum to obtain crude B2; purify the crude product by silica gel column chromatography using CH3OH and DCM as eluents to obtain yellow compound B2; 3) Add 3 equivalents of B2 and an equal equivalent of (4-dimethylaminophenyl)boric acid to a 100 mL round-bottom flask, and add 16 mL of toluene, 6 mL of ethanol, and 2 mL of water as reaction solvents. Under nitrogen protection, reflux at 80 °C overnight. After the reaction is complete, evaporate the solution to dryness under reduced pressure, then add 10 mL of dichloromethane and extract three times with saturated brine to obtain the crude product. Finally, separate by PE / EA column chromatography to obtain a white powdery compound B3.

[0017] 4) Two equivalents of B3 and an equal equivalent of B4 (3-(2,3,3-trimethyl-3H-indol-1-onthium-1-yl)propane-1-sulfonate) were added to a 100 mL round-bottom flask. A small amount of piperidine was added as a catalyst, and 10 mL of ethanol was used as the reaction solvent. The mixture was heated to 85 °C and refluxed overnight. After the reaction was completed, the product was evaporated under vacuum to obtain the crude product. Finally, column chromatography was performed using DCM / CH3OH as the eluent to separate the dark blue solid product, BS-1. 1H NMR (400 MHz, DMSO-d6) δ 8.61 (d, J = 12.0 Hz, 1H), 7.81 – 7.73 (m, 2H), 7.68 (s, 2H), 7.60(s, 2H), 7.54 (t, J = 7.5 Hz, 2H), 7.49 – 7.43 (m, 2H), 6.82 (s, 3H), 4.59(t, J = 8.0 Hz, 2H), 2.98 (s, 6H), 2.71 (s, 4H), 2.61 (t, J = 6.5 Hz, 2H), 2.09 (t, J = 7.5 Hz, 2H), 1.82 (s, 2H), 1.78 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 160.45, 153.56, 151.09, 145.70, 144.20, 142.81, 141.86, 132.78, 129.41,128.46, 128.15, 127.65, 125.54, 123.23, 123.07, 120.13, 115.18, 113.86,112.92, 111.91, 105.89, 50.96, 48.03, 44.43, 29.20, 27.98, 24.50, 24.15,20.49. ESI-MS: [M] + Calcd. For595.2625, Found595.26471. ( Figure 1 , Figure 2 , Figure 3 ).

[0018] Example 2 (1) Prepare a 2 mM BS-1 stock solution; (2) Take 10 μL of BS-1 and add it to 2 mL of the test system in a cuvette. Then add water / 1,4-dioxane in different volume ratios. Detect the fluorescence intensity at 730 nm on a fluorescence spectrophotometer with 640 nm UV-Vis absorption as the excitation source to detect the BS-1 response effect. Figure 4 ).

[0019] (3) Preparation of 1. Blank 2. Asp 3. Pro 4. Glu 5. Gly 6. GABA 7. Thr 8. His 9. Arg 10. Lys 11. Ser 12. Leu 13. Val 14. Trp 15. Phe 16. KCl 17. CaCl2 18. CuSO4 19. MgCl2 20. NaCl 21. Na2CO3 22. NaHCO3 23. Ca(ClO)2 24. H2O2 25. Na2S 26. NaHSO3 27. NaHS 28. Hcy 29. GSH 30. Cys 31. O2 •⁻ Amino acids 500 μM, salt ions 500 μM, reactive oxygen species 200 μM, reactive sulfur 100 μM, and physiologically appropriate concentrations of Cys, Hcy, and GSH 200 μM, 15 μM, and 2 mM, respectively. 10 μL of BS-1 stock solution, 2.0 mL of the test system, and a cuvette were added, along with a blank group and other interfering substances (amino acids 500 μM, salt ions 500 μM, reactive oxygen species 200 μM, reactive sulfur 100 μM, and physiologically appropriate concentrations of Cys, Hcy, and GSH 200 μM, 15 μM, and 2 mM, respectively). The fluorescence intensity at 730 nm was detected using a fluorescence spectrophotometer with 640 nm UV-Vis absorption as the excitation source to determine the selectivity of BS-1 for polarity changes. Figure 5 ).

[0020] (4) In the laser confocal microscopy experiment, the intrinsic and extrinsic polarity of LX-2 cells were tested. First, the intrinsic polarity was tested by adding 10 μL of BS-1 to 2 mL of DMEM, shaking well, and then pouring it into a culture dish containing LX-2 cells. The cells were incubated for 10 minutes, then washed three times with PBS solution and placed on a fluorescence confocal microscope. The fluorescence intensity of the red channel in the 680-750 nm wavelength range was detected using a 640 nm excitation source. The cells showed weak red fluorescence. Then, LX-2 cells were incubated with 50 mM LPS and PMA for 30 minutes, then washed three times with PBS solution and placed on a fluorescence confocal microscope. The fluorescence intensity of the red channel in the 680-750 nm wavelength range was detected using a 640 nm excitation source. The red channel fluorescence was enhanced (…). Figure 6 ).

[0021] The above experimental results indicate that BS-1 is a good tool for detecting polarity changes in vitro and intracellularly.

Claims

1. A hemicyanine dye derivative BS-1, characterized in that... The structural formula is: .

2. The method for synthesizing a hemicyanine dye derivative BS-1 as described in claim 1, characterized in that, Includes the following steps: 1) Add 10 equivalents of cyclohexanone and an equal amount of phosphorus oxychloride to a round-bottom flask, then add 15 mL of DMF and stir at room temperature for 2 hours. After the reaction is complete, pour the reaction solution into 100 mL of ice water and adjust the pH to 7.0 using sodium bicarbonate solid. Finally, extract with dichloromethane and evaporate the extract under vacuum to obtain compound B1. 2) Add 5 equivalents of compound B1 and 4-bromo-2-hydroxybenzaldehyde to a round-bottom flask, add 5 equivalents of cesium carbonate to the flask, and use 20 mL of anhydrous DMF as the reaction solvent; after stirring at room temperature for 16 hours, evaporate the reaction liquid under vacuum to obtain the crude product; purify the crude product by silica gel column chromatography using CH3OH and DCM as eluents to obtain yellow compound B2; 3) Add 3 equivalents of compound B2 and an equal equivalent of (4-dimethylaminophenyl)boric acid to a 100 mL round-bottom flask, then add 16 mL of toluene, 6 mL of ethanol and 2 mL of water as reaction solvents; under nitrogen protection, heat to 80 °C and reflux overnight; evaporate the solution to dryness under reduced pressure, then add 10 mL of dichloromethane and extract three times with saturated brine to obtain the crude product; separate the crude product by PE / EA column chromatography to obtain white powder compound B3; 4) Two equivalents of compound B3 and an equal equivalent of 3-(2,3,3-trimethyl-3H-indol-1-onthium-1-yl)propane-1-sulfonate were added to a 100 mL round-bottom flask; a small amount of piperidine was added as a catalyst, and 10 mL of ethanol was used as the reaction solvent; the mixture was heated to 85 °C and refluxed overnight; the crude product was obtained by vacuum evaporation; the crude product was subjected to column chromatography using DCM / CH3OH as the eluent, and the dark blue solid product, BS-1, was separated. The structural formulas of compounds B1, B2, and B3 are as follows: .

3. The hemicyanine dye derivative BS-1 as described in claim 1 is used as a fluorescent probe for fluorescence spectral detection of polarity changes.

4. The application of the hemicyanine dye derivative BS-1 as described in claim 1 in the preparation of a fluorescent probe for detecting the polarity of the mitochondrial microenvironment in cells.

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