A lysosomal targeted fluorescent dye based on the BODIPY structure and its synthesis method

Abstract

This invention relates to a lysosome-targeting fluorescent dye based on the BODIPY structure and its synthesis method, belonging to the field of organic fluorescent dyes. The invention designs and synthesizes the lysosome-targeting fluorescent dye BDP. The dye molecule uses BODIPY as the parent structure, and a novel lysosome-targeting fluorescent dye molecule is synthesized by introducing diphenylphosphine salt and tert-butyloxycarbonyl groups. The synthetic route of this dye is simple, it possesses excellent photostability, and exhibits good resistance to interference from ions, amino acids, and viscosity. Furthermore, this dye demonstrates superior fluorescence performance in a variety of common solvents, with fluorescence quantum yields exceeding 0.70. Experimental results show that this fluorescent dye can precisely target lysosomes in living cells, demonstrating promising applications in the field of bioimaging.

Description

Technical Field

[0001] This invention relates to the field of organic fluorescent dyes, and more particularly to a lysosomal targeted fluorescent dye based on the BODIPY structure and its synthesis method. Background Technology

[0002] Lysosomes are membrane-bound organelles with an acidic environment, containing approximately 60 hydrolytic enzymes. They degrade biomolecules such as proteins, nucleic acids, and polysaccharides, as well as aging organelles and endogenous and exogenous foreign substances, playing a central role in intracellular digestion, waste recycling, and catabolism. Therefore, they have long been considered the cell's "garbage disposal station." Furthermore, lysosomes are widely involved in various key life activities such as cell proliferation, apoptosis, signal transduction, plasma membrane repair, and cholesterol homeostasis, making them crucial organelles for maintaining cellular homeostasis. Lysosomes are highly dynamic within the cell, rapidly moving in response to changes in the cellular microenvironment, such as nutritional status. Their dysfunction is closely related to various diseases, including Alzheimer's disease and cancer. Therefore, developing tools for lysosome visualization is of great significance for exploring lysosomal physiological functions.

[0003] In recent years, fluorescent dyes have attracted much attention in lysosome research due to their advantages such as high sensitivity, convenient detection, and minimal damage, making them an effective means of organelle imaging. BODIPY (4,4-difluoro-4-boron-3a,4a-diaza-s-indane) derivatives have become ideal materials for constructing high-performance sub-organelle-targeting dyes because of their high fluorescence quantum yield, excellent photostability, and precisely tunable emission wavelengths. Furthermore, multiple sites in their core structure can be functionalized, facilitating the introduction of targeting groups or environmentally responsive units. However, traditional BODIPY dyes lack natural sub-organelle targeting capabilities; their intracellular distribution mainly depends on lipophilicity, easily leading to non-specific localization. In addition, BODIPY derivatives are prone to aggregation fluorescence quenching (ACQ) in high-water-content biological environments, affecting imaging quality and signal intensity, thus limiting their application in complex biological systems. Therefore, developing high-performance lysosome-targeting BODIPY fluorescent dyes remains challenging. Summary of the Invention

[0004] The purpose of this invention is to solve the aforementioned problems in the prior art and provide a lysosome-targeting fluorescent dye based on the BODIPY structure and its synthesis method. This fluorescent dye has advantages such as good photostability, strong anti-interference ability, and high fluorescence quantum yield in aqueous solution, and can target lysosomes in living cells.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A lysosomal targeting fluorescent dye based on the BODIPY structure, having the following compound BDP:

[0007] .

[0008] The fluorescent dye has aggregation-induced emission properties and an absolute fluorescence quantum yield of not less than 0.8 in water.

[0009] The method for synthesizing a lysosomal-targeting fluorescent dye based on the BODIPY structure is as follows:

[0010] .

[0011] The intermediate M1 was prepared as follows: n-Butyllithium was added dropwise to a tetrahydrofuran solution of diphenylphosphine at -78°C under a protective gas atmosphere, and the reaction was stirred; a tetrahydrofuran solution of N-Boc-3-aminopropylbromine was slowly added dropwise, and after the addition was complete, the temperature was raised to room temperature and the reaction was stirred; after post-treatment, tert-butyl(3-diphenylphosphinopropyl)carbamate (M1) was obtained.

[0012] The intermediate L1 was prepared as follows: a dichloromethane solution of 2-methylpyrrole was slowly added dropwise to a dichloromethane solution of bromoacetyl chloride under a protective gas at 0°C. After the addition was complete, the temperature was raised to room temperature and the reaction was stirred. The mixture was then cooled to 0°C, and triethylamine and boron trifluoride diethyl ether were added sequentially. The reaction was continued to be stirred at room temperature. After post-treatment, 8-bromomethyl-4,4-difluoro-1,7-dimethyl-4-boraza-3a,4a-diaza-s-indane (L1) was obtained.

[0013] The target product BDP was prepared as follows: Under a protective gas atmosphere, a toluene solution of intermediate L1 was mixed with a toluene solution of intermediate M1; the temperature was raised to 100-120°C and the mixture was stirred for 10-14 hours; the mixture was cooled to room temperature and post-processed to obtain the target product BDP.

[0014] The application of the fluorescent dye in the preparation of reagents for lysosomal targeted imaging.

[0015] The fluorescent dye is used for specific labeling and imaging of lysosomes in living cells.

[0016] The fluorescent dye exhibits excellent photostability and anti-interference ability, and demonstrates high fluorescence quantum yield in an aqueous environment, making it suitable for targeting lysosomes in living cells.

[0017] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0018] 1. The fluorescent dye synthesized in this invention uses inexpensive and readily available raw materials, involves fewer reaction steps, and is simple to purify.

[0019] 2. The fluorescent dye synthesized in this invention has excellent optical properties, and its fluorescence quantum yield in common solvents exceeds 0.70.

[0020] 3. This invention transforms BODIPY, which has aggregation fluorescence quenching properties, into a dye with aggregation-induced emission properties through simple structural modification, greatly assisting BODIPY derivatives in biological research related to living cells.

[0021] 4. The fluorescent dye synthesized in this invention has good photostability, strong resistance to interference from ions, amino acids and viscosity, and can specifically target lysosomes in cells, laying the foundation for long-term observation of lysosomal life activities. Attached Figure Description

[0022] Figure 1 The above is the 1H NMR spectrum of the fluorescent dye BDP described in this invention.

[0023] Figure 2 This is a high-resolution mass spectrum of the fluorescent dye BDP described in this invention;

[0024] Figure 3 The aggregate fluorescence behavior of the fluorescent dye BDP described in this invention;

[0025] Figure 4 Photostability test of the fluorescent dye BDP described in this invention;

[0026] Figure 5 This is a test of the anti-interference ability of the fluorescent dye BDP described in this invention;

[0027] Figure 6 Viscosity response test of the fluorescent dye BDP described in this invention;

[0028] Figure 7 This is a cellular colocalization assay for the fluorescent dye BDP described in this invention. Detailed Implementation

[0029] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0030] Example 1: Chemical Synthesis Method of BDP

[0031] The synthesis route is shown below:

[0032]

[0033] Step 1, tert-butyl (3-diphenylphosphinopropyl) carbamate (M1)

[0034] Under nitrogen protection at -78°C, 0.96 mL (2.4 mmol) of n-butyllithium was slowly added dropwise to a tetrahydrofuran solution of 0.35 mL (2.0 mmol) of diphenylphosphine (15 mL), and stirred for 30 min. Then, a tetrahydrofuran solution of 474 mg (2.0 mmol) of N-Boc-3-aminopropyl bromide (5 mL) was slowly added dropwise. After the addition was complete, the temperature was raised to room temperature, and stirring was continued for 10 h. Excess solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography. After recrystallization from petroleum ether, 186 mg of white crystals were obtained, with a yield of 27.2%. 1 H NMR (500 MHz, CDCl3): δ = 7.35~7.46 (m, 10 H), 4.60 (s, 1H), 3.22~3.23 (m, 2H), 2.08 (t, J = 8.1 Hz, 3 H), 1.59~1.67 (m, 2H), 1.45(m, 9H).

[0035] Step 2, 8-bromomethyl-4,4-difluoro-1,7-dimethyl-4-boraza-3a,4a-diaza-s-indane (L1)

[0036] Under nitrogen protection, a 20 mL solution of 2-methylpyrrole (810 mg, 10.0 mmol) in dichloromethane was slowly added dropwise to a solution of bromoacetyl chloride (410 μL, 5.0 mmol). After the addition was complete, the mixture was heated to room temperature and stirred for 2.5 h. Then, it was cooled to 0 °C, and triethylamine (2.8 mL, 20.0 mmol) was added and stirred for 5 min. Then, boron trifluoride diethyl ether (2.5 mL, 10.0 mmol) was added, and the mixture was stirred at room temperature for 1 h. Excess solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography to give 34 mg of a reddish-brown solid, with a yield of 21.7%. 1 H NMR (500 MHz, CDCl3): δ = 7.15 (d, J = 3.5Hz, 2H), 6.32 (d, J = 3.9 Hz, 2H), 4.61 (s, 2H), 2.62 (s, 2H).

[0037] Step 3, (3-((tert-butoxycarbonyl)amino)propyl)((5,5-difluoro-3,7-dimethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborhexacyclopenta

[10] en-10-yl)methyl)diphenylphosphonium (BDP)

[0038] Under nitrogen protection, a toluene solution (5 mL) of M1 (68 mg, 0.2 mmol) was added dropwise to a toluene solution (5 mL) of L1 (62 mg, 0.2 mmol). After the addition was complete, the mixture was heated to 110 °C and stirred for 12 h. Then, it was cooled to room temperature, and the mixture was added to n-hexane (30 mL). The mixture was filtered and dried to give 47.5 mg of a red solid, with a yield of 41.2%. 1 H NMR (500 MHz, CDCl3), δ = 7.80~7.84 (m, 4H), 7.62~7.65 (m, 2H), 7.51~7.52 (m, 4H), 6.99 (d,J = 2.6 Hz, 2H), 5.90 (s, 1H), 5.73 (s, 2H) 5.25~5.28 (d, J = 15.9 Hz, 2H), 3.57 (s, 2H), 3.23~3.24 (m, 2H), 2.38 (s, 6H), 1.66~1.67 (m, 2H), 1.44 (s, 9H).

[0039] The 1H NMR spectrum and high-resolution mass spectra of the lysosome-targeting fluorescent dye BDP prepared in this embodiment are as follows: Figure 1 and Figure 2 As shown.

[0040] Example 2: Aggregate fluorescence behavior of BDP

[0041] like Figure 3 As shown, BDP exhibits strong fluorescence emission in pure DMSO solution, which increases with water content (f). w With increasing water content, the emission intensity decreases sharply, while the change in fluorescence emission peak position is relatively small. The emission intensity reaches its lowest point when the water content is 10%. When the water content exceeds 40%, the emission intensity of the compound increases, reaching a maximum at 99% water content, where the emission intensity is 1.16 times that in pure DMSO solvent. These results indicate that by rationally adjusting the structure of BODIPY, the ACQ effect is suppressed, enabling the dye to exhibit strong fluorescence emission in high-water-content systems.

[0042] Example 3: Absolute fluorescence quantum yield test of BDP

[0043] The DMSO stock solution of BDP was diluted to a test solution with an absorbance less than 0.1 using toluene (Tol), ethyl acetate (EA), dichloromethane (DCM), ethanol (EtOH), dimethyl sulfoxide (DMSO), and water (H2O) as solvents. The absolute fluorescence quantum yield (Φ) of BDP in different solvents was determined using a Quantaurus-QYPLUS C13534 fluorescence spectrometer. As shown in Table 1, the quantum yield of BDP in different solvents all exceeded 0.70. Among them, the absolute quantum yield of BDP in aqueous solution reached 0.81, which is suitable for fluorescence imaging in vivo.

[0044] Table 1. Absolute fluorescence quantum yield of BDP in different solvents

[0045]

[0046] Example 4: Photostability Test of BDP

[0047] BDP was dissolved in DMSO solution to prepare a 10 µM test solution. After continuous laser irradiation for 15 min, the relative fluorescence intensity changes were recorded every 1 min. Figure 4 As shown, the emission intensity of compound BDP remained relatively stable after 15 minutes of illumination, at 95% of its initial value, demonstrating that the dye exhibits good photostability.

[0048] Example 5: Anti-interference capability test of BDP

[0049] Select metal cations (Ni) 2+ Co 2+ Ca 2+ Fe 3+ Fe 2+ Al 3+ Cu 2+ Na + Cr 3+ Mg 2+ Zn 2+ Ba 2 + Mn 2+ ), anion (SH) - HPO4 2- I - S 2- SO4 2- F - PO4 3- NO2 - ,Br - HCO3 - CNS - SO3 2-HSO4 - Representative substances such as reactive oxygen species (·OH, H2O2, ROO·), amino acids (Cys, Lys, Ser, Glu, Thr, Gly, Phe, Pro, Tyr, Trp, His, Ala, Arg, Asn, Met, IIe, Val, Asp, Leu), DNA, and RNA were used to simulate bioactive molecules in organisms, and the fluorescence properties of BDP were tested in the presence of different interfering substances. Figure 5 As shown, the fluorescence emission intensity of BDP remains relatively stable in the presence of different interfering substances, indicating that the probe has good anti-interference performance and is suitable for bioimaging in complex biological samples.

[0050] Example 6: Viscosity Response Test of BDP

[0051] Methanol and glycerol solvent were mixed at different volume ratios, resulting in glycerol solvent comprising 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90% of the total solvent system. The mixtures were then ultrasonically vibrated for 45 min to remove air bubbles and ensure thorough mixing, yielding mixed solvent viscosities of 0.53 cp, 1.79 cp, 4.83 cp, 11.8 cp, 26.4 cp, 55.0 cp, 107 cp, 198 cp, 348 cp, and 584 cp. The BDP DMSO mother liquor was diluted to 10 µM test solutions using solvents of different viscosities, and the fluorescence spectra of BDP in these different viscosity systems were measured using a fluorescence spectroscopy instrument. Figure 6 As shown, the emission intensity and peak position of BDP do not change much with the increase of solution viscosity, proving that the dye has low sensitivity to viscosity.

[0052] Example 7 Cell Imaging Test of BDP

[0053] PC12 cells in logarithmic growth phase were seeded into 35 mm confocal culture dishes and cultured overnight. After the cells were fully adherent, 500 nM of the lysosome-targeting commercial dye Lyso Red Tracker was added and incubated for 20 min, followed by 500 nM of BDP and culturing for another 1 h. After incubation, the cells were washed three times with PBS, fresh culture medium was added, and the dye in the cells was imaged under a laser confocal microscope. Figure 7As shown, the excitation wavelength of the green channel was 488 nm, and fluorescence signals were collected from 500 to 540 nm. The excitation wavelength of the red channel was 594 nm, and fluorescence signals were collected from 600 to 640 nm. Comparing the fluorescence images of the two channels, it was found that the red fluorescence signal of the commercial dye had a high degree of overlap with the fluorescence signal of the dye molecule BDP, with a Pearson correlation coefficient (Rr) as high as 0.84, thus confirming that the dye can target lysosomes of living cells.

Claims

1. A lysosomal-targeting fluorescent dye based on the BODIPY structure, characterized in that, Compound BDP has the following structure: 。 2. The lysosomal targeting fluorescent dye based on the BODIPY structure as described in claim 1, characterized in that: The fluorescent dye has aggregation-induced emission properties and an absolute fluorescence quantum yield of not less than 0.8 in water.

3. A method for synthesizing a lysosomal-targeted fluorescent dye based on a BODIPY structure as described in any one of claims 1 to 2, characterized in that, The synthesis route is as follows: 。 4. The method for synthesizing a lysosomal-targeted fluorescent dye based on a BODIPY structure as described in claim 3, characterized in that, The intermediate M1 was prepared as follows: n-Butyllithium was added dropwise to a tetrahydrofuran solution of diphenylphosphine at -78°C under a protective gas atmosphere, and the reaction was stirred; a tetrahydrofuran solution of N-Boc-3-aminopropylbromine was slowly added dropwise, and after the addition was complete, the temperature was raised to room temperature and the reaction was stirred; after post-treatment, tert-butyl(3-diphenylphosphinopropyl)carbamate (M1) was obtained.

5. The method for synthesizing a lysosomal-targeted fluorescent dye based on a BODIPY structure as described in claim 3, characterized in that, The intermediate L1 was prepared as follows: a dichloromethane solution of 2-methylpyrrole was slowly added dropwise to a dichloromethane solution of bromoacetyl chloride under a protective gas at 0°C. After the addition was complete, the temperature was raised to room temperature and the reaction was stirred. The mixture was then cooled to 0°C, and triethylamine and boron trifluoride diethyl ether were added sequentially. The reaction was continued to be stirred at room temperature. After post-treatment, 8-bromomethyl-4,4-difluoro-1,7-dimethyl-4-boraza-3a,4a-diaza-s-indane (L1) was obtained.

6. The method for synthesizing a lysosomal-targeted fluorescent dye based on a BODIPY structure as described in claim 3, characterized in that, The target product BDP was prepared as follows: Under a protective gas atmosphere, a toluene solution of intermediate L1 was mixed with a toluene solution of intermediate M1; the temperature was raised to 100-120°C and the mixture was stirred for 10-14 hours; the mixture was cooled to room temperature and post-processed to obtain the target product BDP.

7. The use of the fluorescent dye according to any one of claims 1 to 2 in the preparation of reagents for lysosomal targeted imaging.

8. The application as described in claim 7, characterized in that: The fluorescent dye is used for specific labeling and imaging of lysosomes in living cells.

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