Application of 4-alkylidenemalononitrile-benzopyran derivative fluorescent dye in bio-fluorescent imaging

Abstract

This invention discloses the application of a 4-methylenemalonium-benzopyran derivative fluorescent dye in biofluorescence imaging. The 4-methylenemalonium-benzopyran derivative fluorescent dye, as shown in formula (I), is obtained by a Knoevenagel condensation reaction of 9-aldehyde julonidine with 2-(2-methyl-4H-benzopyran-4-methylene)malonium under piperidine catalysis. The preparation method of this compound is simple, the reaction conditions are mild, and the yield is high. The 4-methylenemalonium-benzopyran derivative fluorescent dye exhibits excellent two-photon absorption properties and can be applied to confocal fluorescence imaging of HeLa cells and two-photon excitation microscopy of HEK cells. Furthermore, this compound has good biocompatibility and can achieve mitochondrial localization, showing great promise for applications in molecular biology and medicine.

Description

Technical Field

[0001] This invention belongs to the field of two-photon fluorescent materials and biofluorescence imaging, and relates to the application of a 4-methylenemalonium-benzopyran derivative fluorescent dye in biofluorescence imaging. Background Technology

[0002] Fluorescence imaging, as a non-invasive imaging technique, boasts advantages such as speed, convenience, real-time non-destructive processing, high sensitivity, and low cost, making it widely used in the biomedical field. A crucial aspect of this is the development and application of imaging agents. Small-molecule fluorescent chromophores, in particular, are not only easy to use but also offer advantages such as low cost and high sensitivity, enabling visualization of living systems at the subcellular level. Therefore, many researchers have explored various high-performance chromophores for this purpose, including classic chromophores such as coumarin, fluorescein, indole, bodily oil, and rhodamine. However, these dye molecules typically suffer from severe light scattering and high energy consumption for single-photon excitation in traditional single-photon confocal microscopy (1PM), resulting in poor penetration and limiting their application in the life sciences. Therefore, in the past two decades, two-photon confocal microscopy (2PM), based on the nonlinear optical interaction between light and matter, has become an important tool for imaging biological species. Compared to lasers that excite single-photon absorption, the wavelength of the radiation source for two-photon absorption lasers is generally located in the visible-near-infrared region. Two-photon excitation has a longer wavelength, resulting in lower phototoxicity to biomolecules and the ability to penetrate deeper into specimens. Furthermore, absorption occurs only at the focal plane, leading to high spatial resolution. Two-photon fluorescence imaging has already found widespread applications in the biological field. In particular, these superior characteristics of 2PM have prompted chemists to search for two-photon absorption (TPA) chromophores that are easy to synthesize and have a large absorption cross-section for the detection and imaging of biological processes. However, current two-photon fluorescence imaging dyes still face challenges such as limited variety and complex synthesis.

[0003] This invention discovers a 4-methylenemalonium-benzopyran derivative fluorescent dye with strong two-photon absorption. It has good biocompatibility, cell membrane permeability and targeting properties, and can be applied to confocal imaging of HeLa cells and two-photon excitation microscopy of HEK cells. It has potential application value in many fields and is therefore of great research significance. Summary of the Invention

[0004] Purpose of the invention: To address the shortcomings of existing technologies, the purpose of this invention is to provide an application of a 4-methylenemalonium-benzopyran derivative fluorescent dye in biofluorescence imaging.

[0005] Technical solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0006] The structure of a 4-methylenemalononitrile-benzopyran derivative fluorescent dye involved in this invention is as follows:

[0007]

[0008] The synthetic route of the 4-methylenemalonium-benzopyran derivative fluorescent dye (I) involved in this invention is as follows:

[0009]

[0010] The preparation process of a 4-methylenemalononitrile-benzopyran derivative fluorescent dye according to the present invention includes the following steps:

[0011] 9-Aldehyde julonidine and 2-(2-methyl-4H-benzopyran-4-yl)malonitrile were dissolved in acetonitrile, and piperidine was added dropwise to the solution. The mixture was heated to reflux at 82°C for 3 h under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, and the organic solvent was removed by rotary evaporation. The 4-ylmalonitrile-benzopyran derivative fluorescent dye (I) was obtained by silica gel column chromatography using dichloromethane-petroleum ether as the eluent.

[0012] In the above reaction steps, the molar ratio of 9-aldehyde julonidine to 2-(2-methyl-4H-benzopyran-4-yl)malononitrile is 1:1.2;

[0013] In the above reaction steps, the reaction temperature is 82℃ and the reaction time is 3 hours;

[0014] In the above separation and purification steps, the volume ratio of the eluent dichloromethane to petroleum ether is 4:1.

[0015] The application of the 4-methylenemalonium-benzopyran derivative fluorescent dye of the present invention includes the following steps:

[0016] Single-photon confocal fluorescence imaging:

[0017] HeLa cells were transferred to 35 mm culture dishes and incubated for two days in a DMEM incubator at 37°C and 5% CO2. Cells were then incubated for 60 minutes at 37°C with 1 μM 4-methylenemalononitrile-benzopyran derivative fluorescent dye (I). The culture medium was aspirated, and excess mixture was removed three times with washing buffer to prevent interference with subsequent detection. Single-photon fluorescence imaging was then performed using a Leica TCS SP8 laser confocal microscope (excitation wavelength: 488 nm) to obtain single-photon fluorescence images.

[0018] Two-photon biofluorescence imaging:

[0019] Two cell lines were cultured using the same method as for confocal microscopy. After culturing HEK cells for 24 hours, 1 μM of 4-methylenemalononitrile-benzopyran derivative fluorescent dye (I) was added to the cell culture dish. The cells were incubated at 37°C for 30 minutes, the culture medium was aspirated, and excess mixture was removed with washing buffer three times to prevent interference with subsequent detection. Confocal fluorescence imaging was then performed using a self-made two-photon microscope to obtain two-photon fluorescence images. Fluorescence intensity images were acquired (using a 63x water immersion objective and FLIM images using a single-photon counting unit).

[0020] The beneficial effects of this invention are:

[0021] Compared with the prior art, the advantages of the 4-methylenemalonium-benzopyran derivative fluorescent dye in the present invention for the application in biological fluorescence imaging are as follows: (1) The preparation conditions of the 4-methylenemalonium-benzopyran derivative fluorescent dye are simple, the reaction conditions are mild, and the yield is high; (2) The maximum fluorescence emission wavelength of the 4-methylenemalonium-benzopyran derivative in tetrahydrofuran solvent is about 680 nm, and it has a large Stokes shift (>125 nm) and a large two-photon absorption cross section; (3) The 4-methylenemalonium-benzopyran derivative has good cell permeability and biocompatibility, and can be applied to single-photon confocal microscopy imaging of HeLa cells and two-photon fluorescence imaging of HEK cells, and can be located to mitochondria, overcoming the problems of severe background interference and poor penetration in traditional biological imaging. Attached Figure Description

[0022] Figure 1 A fluorescent dye (I) of 4-methylenemalonium-benzopyran derivative. 1 H NMR spectrum.

[0023] Figure 2 The image shows the UV absorption spectrum of 4-methylenemalonium-benzopyran derivative fluorescent dye (I) in dichloromethane solution.

[0024] Figure 3 The fluorescence emission spectrum of 4-methylenemalonium-benzopyran derivative fluorescent dye (I) in dichloromethane solution is shown.

[0025] Figure 4 The image shows the two-photon excitation spectrum of 4-methylenemalonium-benzopyran derivative fluorescent dye (I) in THF solution.

[0026] Figure 5 This is a single-photon excitation confocal fluorescence imaging image of HeLa cells after co-incubation with 4-methylenemalononitrile-benzopyran derivative fluorescent dye (I).

[0027] Figure 6Two-photon excitation confocal fluorescence imaging of HEK cells after co-incubation with 4-methylenemalononitrile-benzopyran derivative fluorescent dye (I). Detailed Implementation

[0028] The present invention will be further illustrated below with specific examples, but these specific examples do not limit the present invention in any way.

[0029] The spectral properties of the compounds were determined using ultraviolet-visible spectroscopy and fluorescence spectroscopy. The instruments used for detection were a BioTech UVIkon XL ultraviolet-visible spectrophotometer and a Horiba Fluromax+ fluorescence spectrometer. For cell fluorescence imaging experiments, a Leica SP8 confocal microscope was used, and a self-made two-photon confocal microscope detection platform was employed.

[0030] Example 1

[0031] 9-Aldehyde julonidine (0.5 mmol) and 2-(2-methyl-4H-benzopyran-4-yl)malononitrile (0.6 mmol) were dissolved in acetonitrile. Piperidine was added dropwise to the solution, and the mixture was heated to reflux at 82 °C for 3 h under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, and the organic solvent was removed by rotary evaporation. The 4-ylmalononitrile-benzopyran derivative fluorescent dye (I) (173 mg, yield: 88%) was purified by silica gel column chromatography using dichloromethane-petroleum ether (v / v = 4:1) as the eluent. 1 H NMR (CDCl3, 600MHz): δ8.89 (d, J=8.4Hz, 1H), 7.69-7.66 (m, 1H), 7.49-7.46 (m, 1H), 7.46 (s, 1H), 7.41-7.39 (m, 1H), 7.05 (s, 2H), 6.73 (s, 1H), 6.49 (d, J=15.6Hz, 1H), 3.28-3.27 (m, 4H), 2.78-2.76 (m, 4H), 2.0-1.96 (m, 4H). 13 C NMR (150MHz, CDCl3): δ159.37, 152.72, 152.46, 145.47, 140.36, 133.99, 127.80, 125.76, 125.47, 121.4 5, 121.24, 118.34, 118.15, 116.69, 111.60, 105.50, 104.96, 50.02, 27.71, 21.47.ESI-HRMS: calculated forC 26 H 21 N3O: 391.1679; found: 391.1675.

[0032] Example 2: UV-Vis absorption spectrum of 4-methylenedionitrile-benzopyran derivative fluorescent dye (I) in dichloromethane solution.

[0033] 4-Methylenemalonium-benzopyran derivative fluorescent dye (I) was dissolved in dichloromethane to prepare a solution with a concentration of 1×10⁻⁶. -5 The UV-Vis absorption spectrum of compound (I) in a dichloromethane solution was measured; the results are as follows: Figure 2 As shown.

[0034] Example 3: Fluorescence emission spectrum of 4-methylenedionitrile-benzopyran derivative fluorescent dye (I) in dichloromethane solution.

[0035] Fluorescent dye (I) was dissolved in dichloromethane to prepare a solution with a concentration of 1×10⁻⁶. -6 The fluorescence emission spectrum of a mol / L dichloromethane solution was measured, and the results are as follows: Figure 3 As shown.

[0036] Example 4: The two-photon excitation spectrum of 4-methylenedionitrile-benzopyran derivative fluorescent dye (I) in THF solution was measured using a built-in SpectraPhysic femtosecond pulsed laser as the excitation source. The results are as follows: Figure 5 As shown, the two-photon absorption cross section at a wavelength of 1020 nm is 300 GM.

[0037] Example 5: Single-photon excitation confocal fluorescence imaging of HeLa cells after co-incubation with the 4-methylenedionitrile-benzopyran derivative fluorescent dye (I) described in this invention.

[0038] HeLa cells were placed in 35 mm culture dishes and incubated for two days in DMEM medium containing 0.1% DMSO at 37°C and 5% CO2. Cells were then incubated for 1 hour at 37°C with 1 μM 4-methylenedionitrile-benzopyran derivative fluorescent dye (I). The medium was aspirated, and excess mixture was removed with washing buffer three times to prevent interference with subsequent detection. Single-photon fluorescence imaging was then obtained using a Leica TCS SP8 laser confocal microscope (excitation wavelength: 488 nm). The results are shown below. Figure 6 As shown, the dye rapidly penetrates the cell and accumulates in the mitochondria.

[0039] Example 6: Two-photon excitation confocal fluorescence imaging of HEK cells after co-incubation with the 4-methylenedionitrile-benzopyran derivative fluorescent dye (I) described in this invention.

[0040] HEK cells were placed in 35 mm culture dishes and incubated for 24 hours in a 37°C, 5% CO2 incubator with DMEM medium containing 0.1% DMSO. Then, 1 μM of 4-methylenedionitrile-benzopyran derivative fluorescent dye (I) was added to the cell culture dishes. The cells were incubated at 37°C for 30 minutes, the medium was aspirated, and excess mixture was removed with washing buffer three times to prevent interference with subsequent detection. Confocal fluorescence imaging was then performed using a self-made two-photon microscope to acquire two-photon fluorescence images. Fluorescence images were acquired using fluorescence intensity and fluorescence lifetime. The results are as follows: Figure 6 As shown, the top row is fluorescence intensity imaging, and the second row is fluorescence lifetime imaging.

Claims

1. Use of a 4-alkylidenemalononitrile-benzopyran derivative fluorescent dye (I) in biofluorescence imaging, characterized in that, The application is not for disease diagnosis or treatment, but for single-photon excitation confocal imaging of HeLa cells. The fluorescent dye can accumulate in the mitochondrial organelle. The structure of the 4-methylenedionitrile-benzopyran derivative fluorescent dye (I) is as follows: 。 2. Use of a 4-alkylidenemalononitrile-benzopyran derivative fluorescent dye (I) in biofluorescence imaging, characterized in that, The application is not for disease diagnosis or treatment, but for two-photon excitation microscopy imaging of HEK cells. The structure of the 4-methylenediamine-benzopyran derivative fluorescent dye (I) is as follows: 。 3. Use according to claim 2, characterized in that: The 4-methylenedionitrile-benzopyran derivative fluorescent dye (I) exhibits good two-photon absorption performance, with a two-photon absorption cross-section of 200-250 GM in the 800-850 nm region and 270-300 GM in the 970-1020 nm region.

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