Lysosome-targeted calcium ion fluorescent probe and application thereof

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a lysosome-targeted calcium ion fluorescent probe and application thereof. The calcium ion fluorescent probe provided by the present application has high sensitivity fluorescent response to calcium ions, and is not interfered by pH, and can be used for monitoring dynamic changes of lysosome calcium ions. Meanwhile, the lysosome calcium ion fluorescent probe in the present application has good biocompatibility, low background fluorescent interference and other advantages, makes up for the deficiency of traditional commercialized calcium ion fluorescent probes, and has wide application prospect in the field of acute liver injury and other liver diseases in vivo.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to lysosome-targeted calcium ion fluorescent probes and their applications. Background Technology

[0002] Lysosomes are organelles within cells that play a crucial role in the degradation and recycling of macromolecules, and are also central hubs for cellular metabolic regulation, signal transduction, and homeostasis. Previously, with the help of small-molecule fluorescent probes, nano / DNA probes, and gene-encoding probes, a relatively systematic understanding of calcium signaling in the cytoplasm, mitochondria, and endoplasmic reticulum has been established. However, the role of calcium in lysosomes... 2+ The dynamic regulation of concentration and its relationship with physiological and pathological processes remain unexplored areas.

[0003] lysosomal Ca 2+ The concentration is approximately 500 μM, which is higher than that of Ca in the cytoplasm. 2+ The concentration is more than 5000 times higher. Lysosomal Ca... 2+ The release process is mediated by multiple channels and transport proteins, including the mucin family transient receptor potential cation channel (TRPML), the two-pore channel (TPC), and the trimer Ca2+. 2+ Double transmembrane channel (P2X4). Traditional calcium ion probes can only monitor cytoplasmic calcium. 2+ Lysosomal calcium homeostasis is dynamic, but early fluctuations cannot be precisely captured, leaving the key mechanisms by which lysosomes regulate cellular calcium homeostasis unexplained. Furthermore, lysosomal calcium homeostasis imbalance is closely associated with several major diseases. In Alzheimer's disease and Parkinson's disease, calcium... 2+ Disorders mediate pathological protein aggregation and mitochondrial dysfunction. In the progression of malignant tumors, lysosomal calcium homeostasis imbalances drive malignant phenotypic transformation and remodel the tumor microenvironment by regulating chemotherapy resistance in tumor cells. The inability to visualize lysosomal calcium signaling in real time will hinder the elucidation of key molecular switches in the early stages of disease, thereby limiting the development of precision intervention strategies.

[0004] Most commercially available probes, such as Rhod-2 and Fluo-4, lack lysosomal localization ability and have low affinity. Kd Difficult to match lysosomal high Ca 2+Even though chemically modified nanoprobes incorporating lysosomal targeting sequences can solve the localization problem, issues such as transmembrane leakage, lysosomal swelling, or immune responses still exist due to the acidic microenvironment and high hydrolytic activity of lysosomes. Therefore, developing novel calcium ion probes with both excellent acid stability and high sensitivity to achieve precise monitoring of lysosomal calcium dynamics is of great significance for in-depth exploration of the regulatory mechanisms of lysosomal calcium homeostasis and the pathological processes of related diseases. Compared with other analytical methods such as high-performance liquid chromatography, mass spectrometry, capillary electrophoresis, or electrochemical analysis, fluorescence technology has the characteristics of high sensitivity, low detection limit, and rapid response, and has great potential in visualizing cellular biochemical processes and biological imaging. It is currently widely used in many fields such as genomics, proteomics, clinical diagnosis and treatment, drug screening, and microscopic imaging technology.

[0005] Disadvantages of existing technologies: (1) pH sensitivity: Most small molecule probes are protonated in the acidic environment of lysosomes, leading to fluorescence quenching or Ca2+. 2+ Decreased affinity makes it impossible to accurately reflect the true intracavitary Ca 2+ Level; (2) Biocompatibility and toxicity: Nanoprobes are prone to causing lysosomal swelling or immune reactions, and gene-encoded probes require transfection, which limits their application in vivo, especially in deep liver tissues; (3) Insufficient targeting specificity and dynamic range: Most probes lack strict lysosomal localization and have low affinity ( Kd Difficult to match lysosomal high Ca 2+ Concentration alone cannot distinguish between physiological homeostasis and pathological overload.

[0006] Therefore, a lysosomal localization, pH insensitive, high-Ca content is provided. 2+ Selectivity and good biocompatibility of small molecule fluorescent probes, their synthesis methods and applications, are of great research value and clinical application prospects for the precise monitoring of lysosomal calcium dynamics in acute liver injury. Summary of the Invention

[0007] In view of this, the present invention provides a lysosome-targeted calcium ion fluorescent probe and its application.

[0008] A lysosome-targeted calcium ion fluorescent probe includes a calcium ion chelating unit precursor as shown in Formula Ia; and a fluorescent unit as shown in Formula Ib connected to the calcium ion chelating unit precursor.

[0009] Formula Ia;

[0010] Formula Ib; Wherein, R1 is selected from C1~C4 hydrocarbon groups; R2, R3, R4, and R5 are independently selected from C1 to C4 hydrocarbon groups; X is selected from -O-, -C(Me)2-, or -Si(Me)2-.

[0011] CMe2, or -C(Me)2-, refers to two methyl groups (Me) attached to the same carbon atom; SiMe2, or -Si(Me)2-, refers to two methyl groups attached to the same silicon atom.

[0012] Furthermore, in the calcium ion fluorescent probe, the benzene ring in the compound of formula Ia is connected to the benzene ring containing X in the compound of formula Ib via a carbon-carbon triple bond; even further, the carbon at position 4 of the benzene ring in the compound of formula Ia is connected to the carbon at position 9 of the benzene ring containing X in the compound of formula Ib via a carbon-carbon triple bond, and its structure is as shown in Formula I:

[0013] Formula I; Wherein, X, R1, R2, R3, R4, and R5 are selected from the groups mentioned above, and will not be described in detail here.

[0014] More preferably, R1, R2, R3, R4, and R5 are independently selected from C1 to C4 alkyl groups, such as any one of methyl, ethyl, propyl, isopropyl, n-butyl, and isobutyl.

[0015] Furthermore, the specific structure of the calcium ion fluorescent probe of the present invention is as follows: .

[0016] The calcium ion fluorescent probe (denoted as BPY) provided by this invention exhibits a highly sensitive fluorescence response to calcium ions and is unaffected by pH interference, making it suitable for monitoring dynamic changes in lysosomal calcium ions. Furthermore, the BPY probe possesses advantages such as good biocompatibility and low background fluorescence interference, overcoming the shortcomings of traditional calcium ion fluorescent probes and showing broad application prospects in the field of liver diseases such as acute liver injury in vivo.

[0017] This invention also provides a method for preparing the lysosome-targeting calcium ion fluorescent probe, comprising the following steps: The calcium ion chelating unit precursor shown in Formula Ia is linked to the fluorescent unit shown in Formula Ib via a carbon-carbon triple bond through the Sonogashira coupling reaction to obtain the calcium ion fluorescent probe shown in Formula I.

[0018] In some implementations, a triple bond is introduced into the calcium ion chelating unit precursor of Formula Ia via a Sonogashira coupling reaction and a deprotection step to obtain an alkynyl-modified compound of Formula Ia, i.e., the calcium ion chelating unit:

[0019] Calcium ion chelating unit.

[0020] In some embodiments, the calcium ion chelating unit shown is prepared from 5-bromo-2-nitrophenol via a nucleophilic substitution reaction, a Sonogashira coupling reaction, a deprotection reaction, a nitro reduction reaction, and an N-alkylation reaction.

[0021] In some embodiments, the fluorescent unit represented by Formula Ib is introduced with a Cl atom before the Sonogashira coupling reaction to obtain a chlorinated fluorescent unit. Specifically, in the chlorinated fluorescent unit, the hydrogen on the 9-position carbon of the X-containing benzene ring is replaced by chlorine.

[0022] This invention involves performing a Sonogashira coupling reaction and deprotection on the calcium ion chelating unit precursor shown in Formula Ia, introducing a carbon-carbon triple bond at the 4-position of the benzene ring in Formula Ia to obtain a calcium ion chelating unit (a compound of Formula Ia containing a terminal alkyne group), and obtaining a chlorinated fluorescent unit by chlorination modification of the fluorescent unit. Then, the calcium ion chelating unit and the chlorinated fluorescent unit are coupled via a Sonogashira reaction to obtain the calcium ion fluorescent probe described in this invention.

[0023] In some embodiments, the catalyst for the Sonogashira coupling reaction includes bis(triphenylphosphine)palladium dichloride or cuprous iodide.

[0024] In some embodiments, the solvent for the Sonogashira coupling reaction includes tetrahydrofuran.

[0025] The present invention also provides the application of the calcium ion fluorescent probe in any of the following: (1) Preparation of products for detecting lysosomal calcium ions; (2) Prepare products for monitoring dynamic changes in lysosomal calcium ions and / or imaging of calcium ion levels; (3) Prepare products for screening drugs.

[0026] The drugs include those that improve calcium ion imbalance and / or those that treat diseases related to lysosomal calcium imbalance.

[0027] In some embodiments, the lysosomal calcium imbalance includes lysosomal calcium imbalance caused by liver disease and lysosomal calcium imbalance caused by other diseases. The liver disease includes acute liver injury; acute liver injury includes drug-induced liver injury (such as APAP) and / or liver injury caused by viral infection; the other diseases include at least one of myocardial injury, neurodegenerative disease, and tumor.

[0028] The present invention also provides a method for detecting lysosomal calcium ion concentration or observing lysosomal calcium disorders in liver diseases for non-diagnostic purposes, comprising: incubating the calcium ion fluorescent probe of the present invention with the test cells and then performing fluorescence imaging.

[0029] This invention has at least the following advantages: (1) The lysosome-targeting calcium ion fluorescent probe provided by the present invention modifies the BAPTA calcium ion chelating agent with an alkyne group and then combines it with the pyronin fluorescent group. This method can increase the variety of fluorophores to be selected. By screening the fluorescent probes, probe molecules that meet the requirements can be obtained, thus expanding the types of probes. At the same time, the introduction of the calcium ion chelating agent increases the conjugated structure of the probe and achieves lysosome-specific localization through the charge delocalization effect.

[0030] (2) Compared with existing calcium ion probes, the lysosome-targeted calcium ion fluorescent probe prepared in this invention has Ca 2+ It exhibits high selectivity, is unaffected by pH, and has good biocompatibility. It maintains stability and sensitivity in acidic lysosomes, enabling accurate and reliable fluorescence detection during autophagy, apoptosis, and ferroptosis. Furthermore, it achieves high penetration depth, resolution, and fidelity.

[0031] (3) The lysosome-targeting calcium ion fluorescent probe prepared in this invention can specifically bind to calcium ions and has high stability and sensitivity. At the same time, it has low background interference and weak scattering, avoiding the influence of the complex biological environment in vivo, and has broad application prospects in fields such as in vivo acute liver injury monitoring. Attached Figure Description

[0032] Figure 1 For the target molecule BPY 1 H NMR spectrum; Figure 2 For the target molecule BPY 13 C NMR spectrum; Figure 3 For probe BPY to Ca 2+ Fluorescence response diagram; Figure 4 This is a selectivity test plot for the BPY probe; Figure 5 This is a diagram showing the pH operating range of the BPY probe. Figure 6 The graph shows the cytotoxicity assay of the probe BPY, where A represents the effect on HeLa cell survival and B represents the effect on HUH7 cell survival. Figure 7 Organelle colocalization map of probe BPY; Figure 8 For endogenous calcium in lysosomes2+ The fluorescence imaging results are shown, where A is the fluorescence imaging image and B is the fluorescence intensity statistical result. Figure 9 For lysosomal calcium during liver injury 2+ The changing fluorescence imaging images, where A is the fluorescence imaging image and B is the fluorescence intensity statistical result; Figure 10 This is a diagram showing the biocompatibility test of the probe BPY; Figure 11 The probe BPY was used to detect Ca in liver tissue sections from mice with acute liver injury. 2+ Imaging images, where A is a fluorescence imaging image and B is the statistical result of fluorescence intensity; Figure 12 BPY probe for detecting intralysosomal Ca² + The fluorescence response mechanism diagram. Detailed Implementation

[0033] This invention provides lysosome-targeted fluorescent probes for calcium ions and their applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0034] In this document, the terms "including", "comprising", and "having" describe both closed-loop technical solutions consisting of the listed features and open-loop technical solutions that include the listed features.

[0035] In this document, the term “and / or” as used includes any and all combinations of one or more of the related listed items.

[0036] In this document, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when a range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0037] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0038] This invention provides a lysosome-targeting calcium ion fluorescent probe, the structural formula of which is shown in Formula I:

[0039] Wherein, X, R1, R2, R3, R4, and R5 are selected from the groups mentioned above, and will not be repeated here.

[0040] In some preferred embodiments, the fluorescent probe has the following structure: ; where X is selected from the groups described above.

[0041] In some specific embodiments, the fluorescent probe is specifically BPY, and its structure is as follows:

[0042] This invention also provides a method for synthesizing the above-mentioned lysosomal-targeting calcium ion fluorescent probe, which includes the following steps:

[0043] The specific conditions for each step are as follows: (a) 1,2-dibromoethane, potassium carbonate, acetonitrile, reflux; (b) 2-nitrophenol, potassium carbonate, acetonitrile, reflux; (c) 2-methyl-3-butyn-2-ol, tetra(triphenylphosphine)palladium, potassium carbonate, cuprous iodide, acetonitrile, 70°C; (d) reduced iron powder, ammonium chloride, ethanol / water, reflux; (e) potassium hydroxide, toluene, reflux; (f) ethyl bromoacetate, sodium carbonate, anhydrous sodium iodide, acetone, reflux; (g) copper chloride, potassium chloride, acetonitrile, stirring at room temperature; (h) bis(triphenylphosphine)dipalladium chloride, cuprous iodide, triethylamine, tetrahydrofuran, 60°C.

[0044] The present invention will be further illustrated below with reference to the embodiments: Example 1: Synthesis of the fluorescent probe BPY of the present invention

[0045] 1. Synthesis of compound (2)

[0046] 5-Bromo-2-nitrophenol (1.0 g), potassium carbonate (1.9 g), and 1,2-dibromoethane (5.0 mL) were dissolved in 25.0 mL of acetonitrile. The reaction mixture was heated under reflux for 4 hours, with the reaction progress monitored by thin-layer chromatography (TLC) during heating. After the reaction was complete, the mixture was filtered under reduced pressure, and the filter cake was washed three times with ethyl acetate (10 mL each time). The combined filtrates were concentrated under reduced pressure to remove the solvent, and the crude product was purified by rapid column chromatography using a mixture of n-hexane and ethyl acetate (10:1, v / v) as the eluent to give 1.3 g of a pale yellow solid, in 87.3% yield.

[0047] 1 H NMR (300 MHz, CDCl3, ppm), δ7.76 (d, J = 9.1 Hz, 1H), 7.25 (dd, J = 9.1, 1.8 Hz, 2H), 7.24 (d, J = 1.8 Hz, 1H), 4.42 (t, J = 6.5 Hz, 2H), 3.69(t, J = 6.5 Hz, 2H).

[0048] 2. Synthesis of compound (3)

[0049] 2-Nitrophenol (0.5 g), potassium carbonate (1.3 g), and compound 2 (1.0 g) were dissolved in 15.0 mL of acetonitrile. The reaction mixture was heated under reflux for 10 hours, with the reaction progress monitored by TLC during heating. After the reaction was complete, the mixture was filtered under reduced pressure, and the filter cake was washed three times with ethyl acetate (10 mL each time). The combined filtrates were concentrated under reduced pressure, and the crude product was dissolved in 30 mL of ethyl acetate. The organic phase was washed three times with saturated sodium carbonate solution (10 mL each time), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by rapid column chromatography using a mixture of dichloromethane and n-hexane (3:1, v / v) as the eluent to give 0.9 g of a brownish-yellow solid, with a yield of 78.6%.

[0050] Mp: 165.0-168.0 ℃. IR (film, cm -1 ): 3399, 2919, 2341, 1606, 1521,1364, 1047, 1132. 1 H NMR (300 MHz, CDCl3, ppm), δ 7.86 (dd, J = 8.6, 1.7 Hz, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.64–7.56(m, 1H), 7.39 (d, J = 1.7 Hz, 1H), 7.25 (dd, J = 8.6, 1.7 Hz, 2H), 7.16–7.09 (m, 1H), 4.53 (s, 4H). 13 C NMR (75MHz, CDCl3, ppm), δ: 152.4, 151.8, 134.2, 128.5, 126.8, 125.6, 124.5, 121.5,119.0, 116.0, 68.9, 68.6, 68.4. LC-MS(ESI + ): m / z [M] + Calculated value: 383.1; Found value: [M] + : 383.1.

[0051] 3. Synthesis of compound (4)

[0052] Under argon protection, compound (3) (1.0 g), tetrakis(triphenylphosphine)palladium (253 mg), potassium carbonate (1.1 g), and cuprous iodide (42 mg) were dissolved in 10 mL of dehydrated and deoxygenated acetonitrile, followed by the slow dropwise addition of 2-methyl-3-butyn-2-ol (1.3 mL). The reaction mixture was heated under reflux for 10 hours, with the reaction progress monitored by TLC during heating. After the reaction was complete, the mixture was filtered under reduced pressure, and the filter cake was washed with ethyl acetate (5 mL × 3). The combined filtrates were concentrated under reduced pressure, and the crude product was dissolved in 15 mL of ethyl acetate. The organic phase was washed three times with saturated sodium chloride solution (10 mL / time), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by rapid column chromatography using a mixture of dichloromethane and ethyl acetate (10:1, v / v) as the eluent to give 707 mg of a pale yellow solid, with a yield of 70.7%.

[0053] Mp: 165.0-168.0 ℃. IR (film, cm -1 ): 2356, 2331, 1600, 1525, 1346,1288, 1220. 1 H NMR (500 MHz, CDCl3, ppm), δ 7.78 (dd, J = 8.4, 1.7 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.53–7.48 (m, 1H), 7.24 (d, J = 1.7 Hz, 1H), 7.15(dd, J = 8.4, 1.7 Hz, 1H), 7.04–7.00 (m, 2H), 4.47 (d, J = 1.7 Hz, 4H), 1.58 (s, 6H). 13 C NMR (75 MHz, CDCl3, ppm), δ : 151.8, 151.8, 140.2, 139.4, 134.4,129.4, 125.7, 125.7, 124.3, 121.4, 118.9, 115.6, 98.3, 80.3, 68.8, 68.7,65.5, 31.1. LC-MS(ESI + ): m / z [M] + Calcd: 386.3; Found: [M+Na] + : 409.2.

[0054] 4. Synthesis of compound (5)

[0055] Under argon protection, iron powder (1.0 g), ammonium chloride (1.9 g), and compound (4) (0.7 g) were dissolved in 15 mL of ethanol / water (3:1, v / v). The reaction mixture was heated under reflux for half an hour, and the reaction progress was monitored by TLC during heating. After the reaction was complete, the mixture was filtered under reduced pressure, and the filter cake was washed with 15 mL of hot ethanol. The combined filtrates were concentrated under reduced pressure, and the crude product was dissolved in 30 mL of dichloromethane. The organic phase was washed three times with deionized water (10 mL each time), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by rapid column chromatography using a mixture of dichloromethane and ethyl acetate (10:1, v / v) as eluent to give 408 mg of yellow solid, with a yield of 69.9%.

[0056] Mp: 115.5-116.5 ℃. IR (film, cm -1 ): 2969, 2961, 2341, 1612, 1504,1454, 1211. 1 H NMR (500 MHz, CDCl3, ppm), δ 6.94 (d, J = 6.6 Hz, 2H), 6.88(d, J = 8.3 Hz, 1H), 6.84 (d, J = 7.7 Hz, 1H), 6.76 (d, J = 7.7 Hz, 2H), 6.63(d, J = 8.3 Hz, 1H), 4.38 (s, 4H), 3.20 (br, 4H), 1.63 (s, 6H). 13 C NMR (75MHz, CDCl3, ppm), δ: 146.2, 145.5, 137.5, 136.8, 126.0, 122.0, 118.4, 115.5,115.4, 114.6, 112.5, 91.4, 82.7, 67.5, 67.4, 65.7, 31.7. LC-MS(ESI + ): m / z [M] + Calcd: 326.1; Found: [M+Na] + : 349.3.

[0057] 5. Synthesis of compound (6)

[0058] Under argon protection, compound (5) (400 mg) and potassium hydroxide (206 mg) were dissolved in 6.0 mL of toluene. The reaction mixture was heated under reflux for 2 hours, with the reaction progress monitored by TLC during heating. After the reaction was complete, the mixture was filtered under reduced pressure, and the filter cake was washed with 15 mL of dichloromethane. The combined filtrates were concentrated under reduced pressure, and the crude product was dissolved in 15 mL of dichloromethane. The organic phase was washed three times (10 mL each) with saturated sodium carbonate solution and deionized water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by rapid column chromatography using a mixture of dichloromethane and ethyl acetate (10:1, v / v) as eluent to give 280 mg of a yellow solid, with a yield of 85.3%.

[0059] Mp: 123.2-124.3 ℃. IR (film, cm -1 ): 3261, 2921, 2082, 1612, 1506,1220, 1143. 1 H NMR (500 MHz, CDCl3, ppm), δ 7.02 (d, J = 8.7, 1H), 7.00 (s,1H), 6.88 (d, J = 8.7 Hz, 1H), 6.85 (d, J = 7.7 Hz, 1H), 6.76 (d, J = 7.7 Hz, 2H), 6.64 (d, J = 8.7 Hz, 1H), 4.39 (s, 4H), 3.95 (s, 4H), 2.98 (s, 1H). 13 CNMR (126 MHz, CDCl3, ppm), δ:146.2, 145.4, 138.0, 136.8, 126.6, 122.0, 118.4,115.8, 115.4, 114.5, 112.5, 110.9, 84.5, 74.7, 67.6, 67.3. LC-MS(ESI + ): m / z[M] + Calculated value: 268.1; Found value: [M+H] + : 269.3.

[0060] 6. Synthesis of compound BAPTA-A

[0061] Under argon protection, compound (6) (100 mg), sodium carbonate (392 mg), sodium iodide (249 mg), and ethyl bromoacetate (800 μL) were dissolved in 5.0 mL of acetone. The reaction mixture was heated under reflux for 72 hours, and the reaction progress was monitored by TLC during heating. After the reaction was complete, the mixture was filtered under reduced pressure, and the filter cake was washed with 10 mL of dichloromethane. The combined filtrates were concentrated under reduced pressure, and the crude product was dissolved in 30 mL of deionized water. The aqueous phase was extracted three times with ethyl acetate (10 mL each time), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by rapid column chromatography using a mixture of n-hexane and ethyl acetate (3:1, v / v) as eluent to give 126 mg of white solid, in a yield of 55.3%.

[0062] Mp: 166.2-167.1 ℃. IR (film, cm -1 ): 2977, 2360, 2319, 1739, 1513,1247, 1169, 1025. 1 H NMR (500 MHz, CDCl3, ppm), δ 6.96 (d, J = 8.2 Hz, 1H), 6.89 (s, 1H), 6.83–6.73 (m, 4H), 6.61 (d, J = 8.2 Hz, 1H), 4.62 (s, 4H), 4.08(s, 8H), 4.00-3.94 (m, 8H), 2.93 (s, 1H), 1.09 (d, J = 7.6 Hz, 6H), 1.06 (d, J = 7.5 Hz, 6H). 13 C NMR (75 MHz, CDCl3, ppm), δ: 171.6, 171.2, 150.2, 149.4,140.3, 125.8, 122.1, 121.6, 118.9, 118.0, 116.3, 114.7, 113.2, 77.2, 75.9,67.2, 66.9, 60.9, 60.8, 53.5, 14.0, 13.9. LC-MS(ESI + ): m / z [M] + Calcd: 612.3; Found: [M+Na] + : 635.3.

[0063] 7. Synthesis of compound PY-Cl

[0064] Thione (PS, 200 mg), copper chloride (108 mg), and potassium chloride (500 mg) were dissolved in 10.0 mL of acetonitrile and reacted with stirring at room temperature for 1 hour, during which the reaction progress was monitored by TLC. After the reaction was complete, the mixture was filtered under reduced pressure, and the filter cake was washed with 15 mL of dichloromethane. The combined filtrates were concentrated under reduced pressure and recrystallized to give 170 mg of a purple-black solid, with a yield of 48.8%.

[0065] 1 H NMR (300 MHz, CDCl3, ppm), δ 8.15 (d, 2H, J = 8.4 Hz), 7.14 (d,2H, J = 8.4 Hz), 6.73 (s, 2H), 3.06 (s, 12H).

[0066] 8. Synthesis of the target molecule BPY

[0067] PY-Cl (100 mg) and Pd(PPh3)2Cl2 (7 mg) were dissolved in a mixture of dehydrated and deoxygenated tetrahydrofuran and triethylamine (4:1, v / v). The calcium ion chelating unit BAPTA-A (141 mg) prepared in step 6 and cuprous iodide (4 mg) were added sequentially. The reaction was heated under reflux for 9 hours, with the reaction progress monitored by TLC during heating. After the reaction was complete, the crude product was dissolved in 10 mL of dichloromethane. The organic phase was washed three times with deionized water (5 mL each time), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by rapid column chromatography using a mixture of dichloromethane and methanol (30:1, v / v) as the eluent. 112 mg of an orange-yellow solid was finally obtained, with a yield of 64.7%. Figures 1-2 These are the proton and carbon spectra of BPY, respectively.

[0068] Mp: 111.5-112.3 ℃. IR (film, cm -1 ): 2354.6, 1733.6, 1698.9, 1558.2,1454.0, 1384.6, 1259.2, 1120.4, 1024.0, 970.0, 865.8. 1 ¹H NMR (300 MHz, CDCl₃, ppm), δ 7.42 (d, J = 8.2 Hz, 2H), 7.00 (d, J = 8.5 Hz, 1H), 6.95 (s, 1H), 6.80 (m, 6H), 6.65 (d, J = 8.2 Hz, 1H), 6.37 (d, J = 2.4 Hz, 2H), 4.22 (s,4H), 4.10 (d, J = 3.5 Hz, 8H), 3.99 (m, 8H), 2.96 (s, 12H), 1.08 (m, 12H). 13 CNMR (75 MHz, CDCl3, ppm), δ : 170.61, 170.39, 151.85, 149.24, 148.59, 138.47,131.87, 124.07, 121.09, 120.52, 117.92, 117.18, 114.60, 112.18, 66.17, 65.97,59.82, 39.06, 13.02, 12.98. HRMS for C 49 H 57 N4O 11 , Calcd.: 877.4018, Found:[MH] - : 876.3935.

[0069] Example 2: Application of the fluorescent probe BPY of the present invention in an acute liver injury model

[0070] The specific detection method is as follows: The lysosome-targeting calcium ion fluorescent probe BPY prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution for later use. All tests were performed in an organic sulfonate buffer solution containing 300 mM MOPS, 1000 mM KCl, 100 mM EGTA, and 1 U / ml esterase. The final concentration of esterase was 1 U / ml; Ca 2+ The final concentration was 3.0 mM; Mg 2+ The final concentration of β-BY was 1.0 mM. The final concentration of BPY was 10.0 μM. In fluorescence measurements, the slit widths for both excitation and emission wavelengths were set to 5 nm.

[0071] 1. Probe BPY for Ca 2+ fluorescence response

[0072] In the presence of Ca 2+ (3.0 mM) or Mg 2+ In a mixed solution of (3.0 mM) esterase (1 U), the UV absorption and fluorescence spectra of probe BPY (10 μM) were measured under both the presence and absence of esterase (1 U). This confirmed that the fluorescence recovery of probe BPY depends on the esterase hydrolysis process and exhibits activity against Ca²⁺. + Specific response ( Figure 3 (See fluorescence response mechanism) Figure 12 All test systems consisted of 300 mM MOPS, 1000 mM KCl, and 100 mM EGTA (pH = 7.2), containing 10% DMSO (v / v). ex = 488 nm.

[0073] BPY for Ca 2+ The detection is performed by using different concentrations of Ca 2+ Solutions (0, 0.5, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0 mM) were added to systems containing 10.0 µM BPY probe, and the effect of BPY on Ca was measured. 2+ Response fluorescence spectrum ( Figure 3 All test systems consisted of 300 mM MOPS, 1000 mM KCl, 100 mM EGTA, and 1 U / ml esterase (pH = 7.2), containing 10% DMSO (v / v). ex = 488 nm. The results show that the probe fluorescence intensity increases with Ca. 2+ The fluorescence intensity increases with increasing Ca concentration, and its maximum emission intensity increases with increasing Ca concentration. 2+ The effect increases with increasing concentration.

[0074] 2. Selectivity and stability of probe BPY

[0075] In the presence of Mg 2+ (1 mM), Zn 2+ (10 μM), K + (130 mM), Na + (10 mM), Ni 2+ (1 μM), Mn 2+ (1 μM), Cu 2+ (1 μM), Co 2+ (1 μM) and Fe 2+ In a mixed solution of (1 μM) Ca, with and without 3.0 mM Ca 2+ Under two conditions, the fluorescence emission intensity of BPY (10 μM) at 544 nm was measured. Figure 4 All test systems consisted of 300 mM MPOPS, 1000 mM KCl, 100 mM EGTA, and 1 U Esterase (pH = 7.2), containing 10% DMSO (v / v). ex = 488 nm. The results show that BPY only exists in Ca 2+ The fluorescence intensity is significantly enhanced in its presence and is not affected by interference from other metal ions; when the concentration of these ions is within the physiological range, the fluorescence intensity of BPY is also unaffected by them.

[0076] With or without 3.0 mM Ca 2+ Under the condition of pH = 3.0–9.0, the relative fluorescence emission intensity of the esterase hydrolysate of BPY (10.0 μM) at 544 nm was measured (normalized to the intensity at pH = 7.0). Figure 5 All test systems consisted of 300 mM MOPS, 1000 mM KCl, 100 mM EGTA, and 1 U / ml esterase, containing 10% DMSO (v / v). ex = 488 nm. The results showed that within the normal physiological range (pH = 5.0-9.0), regardless of the presence of Ca... 2 + The fluorescence intensity of BPY remained almost constant. Furthermore, the probe maintained excellent fluorescence stability even under acidic conditions, laying the foundation for its application in lysosomes.

[0077] 3. Cytotoxicity and organelle localization of probe BPY

[0078] The effect of probe BPY on cell survival in human cervical cancer cell line (HeLa) and human hepatocellular carcinoma cell line (HUH7) was determined using the CCK-8 assay. Figure 6 (A and B). The results showed that the probe BPY had almost no toxicity to HeLa and HUH7 cells in the range of 0-20 μM, and it had good biocompatibility.

[0079] HeLa cells were co-incubated at 37 ℃ with BPY (10.0 μM) and lysosomal red dye (0.5 μM) or mitochondrial red dye (0.2 μM) for 30 min. Figure 7 The probe incubation system, Hanks' Balanced Salt Solution (HBSS), contained 1 mM probenecid and 0.05% poloxamer 407. Fluorescence imaging was performed using a Nikon C2 confocal laser scanning microscope with a 40x objective lens. BPY was excited at 488 nm and collected in the 500–550 nm range (green channel). A commercial dye was excited at 640 nm and collected in the 650–750 nm range (red channel). The results indicate that the probe BPY possesses specific lysosomal targeting capabilities.

[0080] 4. Endogenous calcium in cells 2+ Fluorescence imaging in lysosomes

[0081] HeLa cells were stained with 10.0 μM BPY for 30 minutes as a control group; cells in the ionomycin experimental group were first treated with 10.0 μM BPY for 30 minutes, then stained with 10.0 μM Ionomycin for 10 seconds; cells in the adenosine triphosphate (ATP) experimental group were first treated with 10.0 μM BPY for 30 minutes, then stained with 200.0 μM ATP for 10 seconds. Scale bar: 20 μm. Green channel: 500-550 nm, excitation wavelength: 488 nm (… Figure 8 (A and B in the text). Experimental results show that the probe BPY can track lysosomal Ca in real time. 2+ Transient changes provide a reliable means for studying calcium-dependent cellular processes.

[0082] 5. Lysosomal Ca during in vitro liver injury 2+ Changes

[0083] Control group: HUH7 cells were stained with 10.0 μM BPY for 30 minutes. APAP group: Cells were treated with 20.0 mM APAP for 12 hours, then stained with 10.0 μM BPY for 30 minutes. APAP + NAC group: Cells were co-incubated with 20.0 mM APAP and 2.0 mM NAC for 12 hours, then stained with 10.0 μM BPY for 30 minutes. Figure 9 (A and B). Green channel: 500-550 nm, excitation wavelength 488 nm. The results indicate that APAP-induced liver injury mainly involves ferroptosis, accompanied by lysosomal calcium deficiency. 2+ Abnormal accumulation occurs, and NAC can effectively reverse liver damage, inhibit ferroptosis, and restore lysosomal calcium homeostasis. The probe BPY can provide real-time visualization and monitoring of the entire process, offering a key monitoring tool for drug development.

[0084] 6. Lysosomal Ca during liver injury in mice 2+ Changes

[0085] BPY (200 μM, 100 μL) was injected into the tail vein of mice. Twenty-four hours later, tissue sections from the heart, liver, spleen, lung, and kidney were stained with hematoxylin and eosin (HE). Figure 10 This indicates that BPY exhibits low toxicity and good biocompatibility in the heart, liver, spleen, lungs, and kidneys of mice. Therefore, BPY can be used for further applications in the diagnosis and treatment of related diseases.

[0086] This study established three experimental groups: a normal control group of mice was intraperitoneally injected with phosphate-buffered saline (PBS); an acute liver injury model group was intraperitoneally injected with APAP (300 mg / kg); and the treatment group was simultaneously injected with APAP (300 mg / kg) and NAC (300 mg / kg). Eight hours after injection, all groups received a tail vein injection of BPY (200 μM, 100 μL), and liver tissue was harvested 30 minutes later for fluorescence imaging and pathological analysis. Twelve hours after model establishment, normal mice, model mice, and treatment mice were intravenously injected with BPY (200 μmol / L, 100 μL). 0.5 hours later, livers were harvested from normal mice, model mice, and treatment mice and cut into 10 μm thick sections. These sections were washed three times with PBS (pH 7.4) before fluorescence imaging. Figure 11 (A and B). The above results indicate that in the APAP-induced acute liver injury model, lysosomal Ca2+... 2+Closely correlated with the degree of liver injury, it suggests that NAC could serve as a potential biomarker for assessing the severity of liver damage. Meanwhile, NAC alleviates liver injury by regulating lysosomal calcium homeostasis, providing a novel target for clinical intervention. The probe BPY successfully achieved real-time visualization of lysosomal calcium dynamics during liver injury progression in in vivo experiments, providing a key monitoring tool for related drug development.

[0087] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A lysosome-targeted fluorescent probe for calcium ions, characterized in that, include: The calcium ion chelating unit precursor shown in Formula Ia; and, the fluorescent unit of formula Ib connected to the calcium ion chelating unit precursor; Formula Ia; Formula Ib; Wherein, R1 is selected from C1~C4 hydrocarbon groups; R2, R3, R4, and R5 are independently selected from C1 to C4 hydrocarbon groups; X is selected from -O-, -C(Me)2-, or -Si(Me)2-.

2. The calcium ion fluorescent probe according to claim 1, characterized in that, The benzene ring in the compound shown in Formula Ia is connected to the benzene ring containing X in the compound of Formula Ib by a carbon-carbon triple bond. And / or, R1, R2, R3, R4, and R5 are independently selected from C1 to C4 alkyl groups.

3. The calcium ion fluorescent probe according to claim 1, characterized in that, It has the structure shown in Equation I: Formula I; R1, R2, R3, R4, and R5 are selected from the groups described in any one of claims 1 to 2.

4. The calcium ion fluorescent probe according to any one of claims 1 to 3, characterized in that, It is a BPY, and the structure of the BPY is as follows: 。 5. The method for preparing the lysosomal-targeting calcium ion fluorescent probe according to any one of claims 1 to 4, characterized in that, Includes the following steps: Using the Sonogashira coupling reaction, the calcium ion chelating unit precursor shown in Formula Ia is connected to the fluorescent group shown in Formula Ib through a carbon-carbon triple bond to obtain the calcium ion fluorescent probe shown in Formula I.

6. The preparation method according to claim 5, characterized in that, The catalysts for the Sonogashira coupling reaction include bis(triphenylphosphine)palladium dichloride or cuprous iodide; And / or, the solvent for the Sonogashira coupling reaction includes tetrahydrofuran.

7. The preparation method according to claim 5, characterized in that, A calcium ion chelating unit is obtained by introducing a carbon-carbon triple bond into the calcium ion chelating unit precursor shown in Formula Ia via a Sonogashira coupling reaction and a deprotection step. The calcium ion chelating unit was prepared from 5-bromo-2-nitrophenol via a series of nucleophilic substitution, Sonogashira coupling, deprotection, nitro reduction, and N-alkylation reactions.

8. The application of the calcium ion fluorescent probe according to any one of claims 1 to 4 in the preparation of products for detecting calcium ions, characterized in that, The product includes at least one of (1) to (3): (1) Products for detecting lysosomal calcium ions; (2) Products that monitor the dynamic changes of lysosomal calcium ions and / or image calcium ion levels; (3) Screening products for drugs; The drugs include those that improve calcium ion imbalance, and / or those that treat diseases related to lysosomal calcium imbalance.

9. The application according to claim 8, characterized in that, The lysosomal calcium imbalance includes lysosomal calcium imbalance caused by liver disease and lysosomal calcium imbalance caused by other diseases. The liver disease includes at least one of acute liver injury; the acute liver injury includes drug-induced liver injury and / or liver injury caused by viral infection; The other diseases include at least one of myocardial injury, neurodegenerative diseases, and tumors.

10. A method for detecting lysosomal calcium ion concentration or observing lysosomal calcium disorders in liver diseases for non-diagnostic purposes, characterized in that, Fluorescence imaging was performed after incubating the calcium ion fluorescent probes described in claims 1 to 4 with the cells to be tested.

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