A large Stokes shift fluorescent dye targeting endoplasmic reticulum and its preparation method and application

Abstract

The present invention discloses a large Stokes shift fluorescent dye targeting the endoplasmic reticulum, and its preparation method and application. It is a new fluorescent dye based on the classic ESIPT dye and incorporating 4-phenylpyridinium salt, specially designed for endoplasmic reticulum targeting, and has both a large Stokes shift and a high viscosity response signal-to-noise ratio. Excellent endoplasmic reticulum targeting is ensured by adjusting the lipophilic-amphiphilic balance of the dye. The Stokes shift exceeds 200nm, eliminating self-absorption interference and enhancing imaging quality. In addition, the unique electron donor-acceptor structure makes the fluorescent signal sensitive to solvent viscosity, enabling accurate reporting of changes in the endoplasmic reticulum microenvironment. Therefore, the present invention not only improves the accuracy and efficiency of endoplasmic reticulum imaging, but also provides a powerful tool for understanding endoplasmic reticulum dynamics in cell and medical research, especially in monitoring changes in endoplasmic reticulum viscosity in damaged cells, showing significant advantages.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical functional dyes, and in particular relates to a large Stokes shift fluorescent dye targeting the endoplasmic reticulum, and a preparation method and application thereof. Background Art

[0002] The endoplasmic reticulum (ER) is a key subcellular organelle in eukaryotic cells and a crucial site for protein synthesis, post-transcriptional modification, and lipid metabolism. Many diseases manifest as dysfunctional ER function and altered microenvironmental processes (viscosity, polarity, pH, and protein metabolism). For example, the viscosity of damaged liver cells and tumor cells is typically significantly elevated. Fluorescence imaging offers high sensitivity, high spatiotemporal resolution, and excellent biocompatibility. Combined with increasingly advanced fluorescence imaging systems, it offers unique advantages for in situ investigation of cellular function in vivo. Fluorescent dyes are a core element of fluorescence imaging technology. Currently commercially available and reported ER-targeted dyes primarily target the well-developed ER membrane, the abundant sulfonylurea and sulfonamide receptors on the membrane, or the abundant metabolic enzymes within the ER. However, current ER-targeted fluorescent dyes, such as cyanine, fluorescein, rhodamine, and BODIPY, have small Stokes shifts and are susceptible to self-absorption and fluorescence inner filtering, which reduces fluorescence brightness. Modulating the dye's molecular structure to increase its Stokes shift is a key approach to improving fluorescence imaging performance.

[0003] Excited-state intramolecular proton-transfer (ESIPT) is a unique four-stage photochemical process. The ground state of ESIPT fluorescent dyes typically exists in the enol (E) form. Upon photoexcitation, electrons and charge redistribute within the dye molecule, increasing the acidity of the proton donor and the basicity of the proton acceptor in the E form. This leads to proton transfer, and the excited-state E-form undergoes photoisomerization to the excited-state keto form, which then returns to the ground-state E-form via radiative transition and reverse proton transfer. This unique photochemical process causes ESIPT fluorescent dyes to typically exhibit very large Stokes shifts (>200 nm). Currently, ESIPT-based fluorescent dyes that can target the endoplasmic reticulum (ER) are extremely scarce. One approach to creating novel ER-targeted fluorescent dyes with large Stokes shifts is to rationally optimize the molecular structure, manipulate lipophilicity, and efficiently target the ER membrane based on the classic ESIPT fluorescent dye. Summary of the Invention

[0004] In response to the above technical problems, the present invention introduces 4-phenylpyridinium salt (electron acceptor) through a condensation reaction on the basis of the classic ESIPT dye matrix (electron donor), regulates the dye's lipophilicity and amphiphilicity, and provides a large Stokes shift fluorescent dye targeting the endoplasmic reticulum and its preparation method and application, so as to provide a high-performance imaging tool for endoplasmic reticulum-related biological and medical research.

[0005] In view of this, the first aspect of the present invention provides a large Stokes shift fluorescent dye targeting the endoplasmic reticulum, which has the following structural formula 1:

[0006]

[0007] Furthermore, in the general formula 1:

[0008] R1 is selected from one of hydrogen, fluorine, chlorine, bromine and iodine atoms;

[0009] R2 is selected from one of an oxygen atom, a sulfur atom, a selenium atom, and a nitrogen atom;

[0010] R3 is selected from one of hydrogen atom, methyl group and methoxy group;

[0011] R4 is selected from one of fluorine, chlorine, bromine and iodine atoms;

[0012] Furthermore, the viscosity-responsive near-infrared fluorescent dye has an excitation wavelength of 350-430 nm, an emission wavelength of 650-750 nm, and a Stokes shift of 200-300 nm. More preferably, the viscosity-responsive near-infrared fluorescent dye has an excitation wavelength of 380-390 nm, and an emission wavelength of 680-720 nm.

[0013] Furthermore, the dye structure prepared in the embodiment of the present invention is as follows:

[0014]

[0015] Fluorescence imaging offers unique advantages for in situ studies of cellular function at the in vivo level, with its high sensitivity, high spatiotemporal resolution, and high biocompatibility. Fluorescent dyes are a core element of fluorescence imaging technology. The fluorescent dyes described in this invention possess excellent endoplasmic reticulum targeting and large Stokes shifts, while also providing high signal-to-noise ratio feedback viscosity responses. Specific advantages are as follows:

[0016] First, excellent endoplasmic reticulum targeting performance: The present invention optimizes the endoplasmic reticulum targeting performance of the dye by regulating the lipophilicity and amphipathicity of the dye molecule. The oil-water partition coefficient (logP) and amphipathic index (AI) of the dye molecule were preliminarily predicted by the software ChemBioDraw Ultra 14.0. The dyes provided by the present invention all satisfy 6>logP>0 and 6>AI>0 (this is a regular method for predicting the endoplasmic reticulum targeting performance of the dye reported in the literature). In the embodiment, the Pearson colocalization coefficient of the colocalization imaging of dye 1 and the commercial endoplasmic reticulum targeting dye ER-Tracker Green is as high as 0.91, showing excellent endoplasmic reticulum targeting performance.

[0017] Second, large Stokes shift: The present invention designs the dye molecular structure based on the ESIPT effect. The Stokes shift of the disclosed dyes is >200nm, which can effectively avoid the influence of dye self-absorption and fluorescence inner filter effect, and has excellent fluorescence imaging performance.

[0018] Third, high signal-to-noise ratio fluorescence imaging of viscosity changes: The present invention uses the classic ESIPT dye matrix as the electron donor and introduces 4-phenylpyridinium salt as the electron acceptor through a condensation reaction. The electron donor and electron acceptor structures can be twisted around the connected vinyl groups, thereby achieving high signal-to-noise ratio feedback of the viscosity changes of the solvent environment.

[0019] In summary, the dye of the present invention has a large Stokes shift, can specifically target the endoplasmic reticulum, and can also perform high signal-to-noise ratio fluorescence imaging of viscosity changes in the endoplasmic reticulum.

[0020] A second aspect of the present invention provides a method for preparing the aforementioned endoplasmic reticulum-targeting large Stokes shift fluorescent dye. The method is used to prepare the aforementioned endoplasmic reticulum-targeting large Stokes shift fluorescent dye. The synthesis route of the endoplasmic reticulum-targeting large Stokes shift fluorescent dye is as follows:

[0021]

[0022] The synthesis process of the large Stokes shift fluorescent dye targeting the endoplasmic reticulum is as follows:

[0023] In an organic solvent, under alkaline conditions, an intermediate 3 having a structure of the general formula 3 undergoes a condensation reaction with an intermediate S1 at 50-85° C., and the dye represented by the general formula 1 is obtained after purification by column chromatography, wherein the molar ratio of the intermediate 3 to S1 is 1:(1-2), the reaction organic solvent is selected from one of ethanol, acetonitrile, toluene, N,N-dimethylformamide, and dimethyl sulfoxide, and the alkaline agent providing alkaline conditions is selected from one of piperidine, diethylamine, and piperazine.

[0024] More preferably, the reaction temperature in the above synthesis process is 75-80° C., and the molar ratio of intermediate 3 to S1 is 1:(1-1.2).

[0025] Further, the synthetic route of intermediate 3 is as follows:

[0026]

[0027] The synthesis process of intermediate 3 is:

[0028] In a DMF solvent, sodium metabisulfite is used as a catalyst to react an intermediate 4 having a structure of the general formula 4 with an intermediate 5 having a structure of the general formula 5 at 100-150°C. After 2-5 hours, deionized water is added to obtain a solid precipitate of an intermediate 2 having a structure of the general formula 2. The molar ratio of the intermediate 4 to the intermediate 5 is 1:1-2. Subsequently, in a trifluoroacetic acid solvent, an intermediate 2 and hexamethylenetetramine (HMTA) are formylated at 70-90°C. After 4-7 hours, the pH is adjusted to 7 by adding HCl, and a solid precipitates to obtain an intermediate 3 having a structure of the general formula 3. The molar ratio of the intermediate 2 to the HMTA is 1:1.5-4.0.

[0029] Preferably, the condensation reaction temperature is 110-120°C.

[0030] Preferably, the molar ratio of the condensation reaction intermediate 4 to the intermediate 5 is 1:(1-1.2).

[0031] Preferably, the formylation reaction temperature is 75-85°C.

[0032] Preferably, the molar ratio of the formylation reaction intermediate 2 to HMTA is 1:(1.5-2.0).

[0033] Furthermore, the synthetic route of intermediate S1 is as follows:

[0034]

[0035] Among them, the synthesis process of S1 is:

[0036] 1-Chloro-2,4-dinitrobenzene reacts with 4-methylpyridine in ethanol at 50-80°C. After cooling, the reaction is concentrated, filtered, and washed with ethanol to prepare S1-1. S1-1 is then reacted with aniline in ethanol overnight at 50-80°C and purified by column chromatography to yield S1.

[0037] More preferably, the reaction temperature involved in the above-mentioned synthesis process of S1-1 is 70-80°C.

[0038] The third aspect of the present invention is to protect the use of the aforementioned large Stokes shift fluorescent dye in non-disease diagnosis and treatment directions, and in biological and medical diagnostic preparations related to the endoplasmic reticulum.

[0039] Furthermore, the above applications include but are not limited to: targeted fluorescence imaging of the endoplasmic reticulum, imaging of changes in endoplasmic reticulum viscosity, fluorescence imaging of damaged cells and tissues, and preparations related to light-driven tumor therapy.

[0040] The beneficial effects of the present invention compared with the prior art are as follows:

[0041] The fluorescent dye described in the present invention has excellent cellular endoplasmic reticulum targeting performance, and has an excitation wavelength of approximately 350-430nm, an emission wavelength of approximately 650-750nm, and a Stokes shift of approximately 200-300nm. It can effectively avoid dye self-absorption and fluorescence inner filtering effects, and at the same time can feedback viscosity changes with a high signal-to-noise ratio, thereby accurately feedback viscosity changes of damaged cell endoplasmic reticulum. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The nuclear magnetic resonance of the dye 1 of the present invention is 1 H-graph;

[0043] Figure 2 The nuclear magnetic resonance of the dye 1 of the present invention is 13 C spectrum;

[0044] Figure 3 is a high-resolution mass spectrum of the dye 1 of the present invention;

[0045] Figure 4 These are the absorption and emission spectra of the dye 1 of the present invention in PBS and glycerol solutions;

[0046] Figure 5 is the cytotoxicity evaluation result of the dye 1 of the present invention;

[0047] Figure 6 This is a subcellular organelle fluorescence imaging image of the dye 1 of the present invention;

[0048] Figure 7 This is an image of the dye 1 of the present invention used for fluorescent detection of damaged cells;

[0049] Figure 8 This is an image of the dye 1 of the present invention being used for fluorescent detection of liver fibrosis. DETAILED DESCRIPTION

[0050] The following will be combined with the accompanying drawings in the embodiments of the present application to clearly describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of this application.

[0051] In the description of this application, it should be noted that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments of this application. The viscosity-responsive near-infrared fluorescent dye, its preparation method, and its application according to the present invention are illustrated below by way of specific examples:

[0052] Example 1

[0053] The synthesis route of dye 1 is as follows:

[0054]

[0055] The synthetic process of dye 1 is as follows:

[0056] (1) Synthesis of intermediate 3-1

[0057] 2-Aminothiophenol (1.36 g, 10 mmol), 2-hydroxy-5-methylbenzaldehyde (1.25 g, 10 mmol), and sodium metabisulfite (2.88 g, 15 mmol) were added to a 20 ml DMF solution and reacted at 110°C for 5 hours. After the reaction was completed and cooled to room temperature, 50 ml of H2O was added to the solution to precipitate a solid powder, which was filtered to obtain a white intermediate 2-1 (1.87 g, 77.6%). Subsequently, intermediate 2-1 (482 mg, 2 mmol) and HMTA (1680 mg, 4 mmol) were added to 10 ml of trifluoroacetic acid and reacted at 80°C for 5 hours. After the reaction was completed, the pH was adjusted to 7 with sodium hydroxide to precipitate the precipitate, which was filtered to obtain the crude product. This was further purified by column chromatography to obtain intermediate 3-1 (273.5 mg, 50.8%).

[0058] 1 H NMR (400MHz, CDCl3) δ10.47 (s, 1H), 8.03 (d, J = 8.1Hz, 1H), 7.96–7.89 (m, 2H), 7.71 (s, 1H) ,7.54(t,J=7.7Hz,1H),7.45(t,J=7.6Hz,1H),2.41(s,3H).HRMS(ESI):m / z269.0510[M+H] + (calcd for C 15 H 12 NO2S +,270.0585)

[0059] (2) Synthesis of compound S1

[0060] 2,4-Dinitrochlorobenzene (10.13 g, 20 mmol) and 4-methylpyridine (2.79 g, 30 mmol) were added to 20 ml of ethanol and reacted at 85°C for 3 hours. After the reaction, 100 ml of ether was added to the solution to precipitate a solid, which was filtered to obtain crude intermediate S1-1 (9.6 g, 68.2%). Intermediate S1-1 (3 g, 10.15 mmol) and aniline (1.89 g, 20.29 mmol) were then added to 15 ml of ethanol and reacted at 85°C for 6 hours. After the reaction, compound S1 (430 mg, 20.7%) was purified by column chromatography.

[0061] 1 H NMR(400MHz, DMSO-d6)δ9.24(d,J=6.4Hz,2H),8.17(d,J=6.2Hz,2H),7.91–7.85(m,2H),7.74(m,3H),2.73(s,3H).HRMS(ESI):m / z 170.0965[M] + (calcdfor C 12 H 12 N + ,170.0972)

[0062] (3) Synthesis of Dye 1

[0063] To 15 ml of ethanol solution, intermediate 3-1 (269 mg, 1 mmol), compound S1 (205 mg, 1 mmol), and potassium carbonate (83 mg, 0.6 mmol) were added sequentially and refluxed at 80°C for 3 hours. After the reaction, the viscosity-responsive near-infrared fluorescent dye 1 was purified by column chromatography.

[0064] 1 H NMR(400MHz, DMSO-d6)δ9.20(d,J=6.5Hz,2H),8.43–8.34(m,3H),8.22(d,J=8.0Hz,1H),8.10(d,J=8 .1Hz,1H),7.94–7.79(m,5H),7.75(m,3H),7.60(t,J=7.6Hz,1H),7.52(t,J=7.5Hz,1H),2.41(s,3H). 13C NMR(101MHz,DMSO-d6)δ167.82,154.71,151.41,144.34,142.78,137.59,133.64,132.96,131.64,131.31 ,130.67,127.37,126.12,124.81,124.23,124.10,124.05,122.76,122.36,118.72,20.50.HRMS(ESI):m / z 421.1371[M+H](calcd for C27H21N2OS+,421.1369).

[0065] The obtained nuclear magnetic resonance of the endoplasmic reticulum-targeted large Stokes shift fluorescent dye 1 1 H Figure Figure 1 As shown, NMR 13 C spectrum Figure 2 As shown, the high-resolution mass spectrum is as follows Figure 3 shown.

[0066] (4) The present invention introduces 4-phenylpyridinium salt as a core structural unit through careful design. This unique design not only breaks through the design framework of traditional endoplasmic reticulum targeting dyes, but also achieves efficient endoplasmic reticulum targeting by finely regulating the balance between the lipophilicity and amphiphilicity of the molecule. Although 4-methylpyridinium salt and 4-ethylpyridinium salt have been previously reported for mitochondrial targeting, the present invention uses 4-phenylpyridinium salt to not only significantly improve the lipophilicity of the dye (ClogP value of 2.38, much higher than the 0.267 and 0.796 reported in similar studies), but more importantly, this change is combined with the optimization of the amphiphilicity of the molecule to form an unprecedented molecular configuration that can specifically interact with the endoplasmic reticulum membrane and exhibit efficient endoplasmic reticulum targeting performance.

[0067]

[0068] Example 2

[0069] Absorption fluorescence spectra of dye 1 in PBS buffer (low viscosity medium) and glycerol (high viscosity medium).

[0070] Dissolve dye 1 in organic solvent DMSO to prepare a stock solution with a concentration of 10mM and store it at 4°C until use. Take 1μL of dye 1 stock solution with a pipette, dilute it in 2mL PBS or glycerol solution, mix it thoroughly, and use Duetta TM Fluorescence and absorption spectrometers were used to measure the absorption and fluorescence spectra respectively. Figure 4As shown, the absorption peaks of Dye 1 at 385 nm and 550 nm in high-viscosity glycerol are significantly enhanced compared to those in PBS. Furthermore, when excited at 385 nm, the fluorescence intensity of Dye 1 at 705 nm in glycerol is 62 times that in PBS. These spectral data preliminarily demonstrate that Dye 1 can be used to detect changes in liquid viscosity.

[0071] Example 2 discloses the spectral behavior of dye 1 in different media, particularly showing significantly enhanced absorption and fluorescence characteristics in the high viscosity medium glycerol. Although dye 1-1 (4-methylpyridinium salt) and dye 1-2 (4-ethylpyridinium salt) reported in the literature have similar structures, they are mainly targeted at mitochondria. Dye 1 introduces 4-phenylpyridinium salt through structural innovation, regulates the lipophilicity of the dye molecule, and thus has good endoplasmic reticulum targeting performance. At the same time, dye 1 can highly sensitively feedback the viscosity change of the endoplasmic reticulum of the cell. These characteristics give dye 1 unique advantages in detecting the viscosity change of the endoplasmic reticulum of the cell, assessing the health status of the cell and studying the pathogenesis, which are not available with traditional dyes.

[0072] Example 3

[0073] Cytotoxicity evaluation of dye 1.

[0074] After MCF-7 cells and LO2 cells were incubated with 0-2.5 μM dye 1 for 24 hours, the cytotoxicity of fluorescent dye 1 was determined using the thiazole blue colorimetric method (MTTassay). Figure 5 As shown, after incubating MCF-7 cells and LO2 cells with 0-2.5 μM dye 1 for 24 hours, the survival rates were both above 90%, proving that dye 1 had no obvious cytotoxicity even at higher concentrations, had high biocompatibility, and could be safely used in biomedical and other fields.

[0075] Example 4

[0076] Evaluation of the endoplasmic reticulum targeting performance of dye 1

[0077] Dye 1 (1 μM) was co-stained with commercial subcellular organelle localization dyes ER-Tracker Green (1 μM), Lyso-Tracker Green (1 μM), and Mito-Tracker Green (1 μM) for 30 minutes in MCF-7 cells. Cell fluorescence signals were collected using a Leica STELLARIS 5 laser scanning confocal fluorescence microscope. The excitation wavelength of dye 1 was 405 nm, and the emission wavelength range was 680-750 nm. The excitation wavelength of the commercial subcellular organelle localization dye was 488 nm, and the emission wavelength range was 500-540 nm. Results Figure 6As shown in the figure, the Pearson colocalization coefficients of dye 1 and the three commercial subcellular organelle localization dyes were 0.91, 0.48 and 0.61, respectively, demonstrating that dye 1 can specifically and efficiently target the endoplasmic reticulum.

[0078] Example 5

[0079] Dye 1 for imaging viscosity changes in damaged cells

[0080] Prepare the culture medium of human breast cancer cells MCF-7, culture the MCF-7 cells in a laser confocal culture dish, and when the cell abundance reaches 60%, add Monensin (10μM, 1μL DMSO) and Nystatin (10μM, 1μL DMSO) to the MCF-7 cells in the two experimental groups, respectively. At the same time, add 1μL DMSO to the MCF-7 cells in the blank group and incubate for 3 hours. After washing the three groups of cells three times with pre-cooled PBS, add fresh culture medium containing dye 1 (1μM) to the three groups of cells. After incubation for 20 minutes, use Leica STELLARIS 5 laser scanning confocal fluorescence microscope to collect cell fluorescence signals (excitation wavelength 405nm, emission wavelength receiving range is 680-750nm). Monensin and Nystatin treatment can damage cells and lead to increased endoplasmic reticulum viscosity. The results are as follows Figure 7 As shown, the fluorescence signals of cells treated with Monensin and Nystatin were significantly stronger than those of cells in the blank group, demonstrating that fluorescent dye 1 can be used to image changes in endoplasmic reticulum viscosity in damaged cells.

[0081] Example 6

[0082] Dye 1 for liver fibrosis imaging

[0083] First, a liver fibrosis mouse model was prepared. 6-week-old Kunming mice were intraperitoneally injected with 50 μL of olive oil containing 40% CCl4 twice a week for 4 and 8 weeks to establish a liver fibrosis mouse model. The livers of normal mice and mice with liver fibrosis stimulated by drugs for 4 and 8 weeks were incubated with 100 μM dye 1 for 10 minutes. After washing three times with PBS, the ex vivo liver fluorescence signal was collected using the NightOWL II LB 983 small animal in vivo imaging system (excitation wavelength 410 nm, emission wavelength receiving range 680-720 nm). The results are shown in Figure 2. Figure 8 The results showed that the fluorescence signal of the liver tissue of mice with liver fibrosis after 4 and 8 weeks of drug stimulation was significantly stronger than that of the liver tissue of normal mice, and the fluorescence signal also increased with the increase of drug stimulation time. This preliminarily proved that fluorescent dye 1 can be used for fluorescence imaging of liver fibrosis and early diagnosis of liver fibrosis.

[0084] Examples 5 and 6 not only demonstrate the high sensitivity of the dye to changes in the intracellular environment, but also foreshadow its broad application prospects in pathological research and clinical diagnosis, which is an innovative application that goes beyond the expected effects of general endoplasmic reticulum-targeted dyes.

[0085] The embodiments of the present application are described above in conjunction with the accompanying drawings. Unless there is a conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of this application, ordinary technicians in this field can also make many forms without departing from the purpose of this application and the scope of protection of the claims, all of which are within the protection of this application.

Claims

1. A fluorescent dye with a large Stokes shift targeting the endoplasmic reticulum, characterized in that: It has the following structural formula 1: In the general formula 1: R1 is selected from one of hydrogen, fluorine, chlorine, bromine and iodine atoms; R2 is selected from one of an oxygen atom, a sulfur atom, a selenium atom, and a nitrogen atom; R3 is selected from one of hydrogen atom, methyl group and methoxy group; R4 is selected from one of fluorine, chlorine, bromine and iodine atoms.

2. The dye according to claim 1, wherein: The excitation wavelength of the dye is 350-430 nm, and the emission wavelength is 650-750 nm.

3. The method for preparing a dye according to claim 1, wherein: The synthetic route is as follows: In an organic solvent, under alkaline conditions, an intermediate 3 having a structure of the general formula 3 undergoes a condensation reaction with an intermediate S1 at 50-85° C., and the dye represented by the general formula 1 is obtained by purification by column chromatography, wherein the molar ratio of the intermediate 3 to S1 is 1:(1-2).

4. The method for preparing a dye according to claim 3, wherein: The reaction organic solvent is selected from one of ethanol, acetonitrile, toluene, N,N-dimethylformamide and dimethyl sulfoxide.

5. The method for preparing a dye according to claim 3, wherein: The reagent providing alkaline conditions is selected from one of piperidine, diethylamine and piperazine.

6. The method for preparing a dye according to claim 3, wherein: The molar ratio of intermediate 3 to S1 is 1:(1-1.2).

7. The method for preparing a dye according to claim 3, wherein: The synthetic route of the intermediate 3 is as follows: In a solvent of DMF, using sodium metabisulfite as a catalyst, an intermediate 4 having a structure of the general formula 4 and an intermediate 5 having a structure of the general formula 5 undergo a condensation reaction at 100-150° C., and after 2-5 hours, deionized water is added to obtain a solid precipitate of an intermediate 2 having a structure of the general formula 2; the molar ratio of the intermediate 4 to the intermediate 5 is 1:(1-2); then, in a solvent of trifluoroacetic acid, the intermediate 2 and hexamethylenetetramine undergo a formylation reaction at 70-90° C., and after 4-7 hours, the pH is adjusted to 7, and a solid precipitates to obtain an intermediate 3 having a structure of the general formula 3.

8. The method for preparing a dye according to claim 3, wherein: The synthetic route of the intermediate S1 is as follows: In an organic solvent, ethanol, 1-chloro-2,4-dinitrobenzene reacts with 4-methylpyridine at 50-80°C, followed by concentration, filtration, and washing to prepare S1-1. Subsequently, S1-1 reacts with aniline in an organic solvent at 50-80°C overnight, and S1 is purified by column chromatography.

9. Use of the dye according to claim 1 in preparing a preparation for fluorescence imaging of endoplasmic reticulum targeting, imaging of endoplasmic reticulum viscosity changes, and fluorescence imaging of damaged cells and tissues.

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