Near-infrared response type asymmetric polymethine cyanine dye as well as preparation method and application thereof

Abstract

The invention provides a near-infrared response type asymmetric polymethine cyanine dye as well as a preparation method and application thereof. The dye has a structure as shown in a general formula I. The probe is combined with H < + > through an azaindole part in an asymmetric polymethine cyanine dye structure, so that rapid and reversible response to pH change is realized. The probe has high sensitivity and high specificity on pH, the pKa value is 6.46, the pH response linear range is 5.0-7.0, and the probe is suitable for monitoring the pH value in mitochondria. Due to inherent positive charges of the structure, the material can be gathered in mitochondria in a targeted manner and is kept stable in an autophagy process. The probe has the characteristics of high sensitivity, reliability and visualization, and is suitable for the fields of basic scientific research, drug action mechanism evaluation, cell physiological function and disease model analysis, environmental pollutant effect monitoring, regenerative medicine and the like. In addition, the probe is good in biocompatibility and high in light stability, is compatible with common fluorescence imaging equipment, and is convenient to popularize and apply.

Description

Technical Field

[0001] The invention relates to the technical field of pH fluorescent probes, and in particular to a cyanine pH fluorescent probe for monitoring mitochondrial autophagy and a preparation and application thereof. Background Art

[0002] Mitochondria are two-layer membrane-enclosed organelles present in most eukaryotic cells. They produce reactive oxygen and adenosine triphosphate through aerobic respiration and are the site of many key metabolic processes. Maintaining mitochondrial homeostasis is essential for cell growth. Mitochondria can depolarize and be severely damaged under the action of a large amount of reactive oxygen, nutrient deficiency, cell aging, etc. In order to maintain the number and quality of mitochondria, cells will initiate the process of mitochondrial autophagy, encapsulate the damaged mitochondria into autophagosome vesicles and fuse with lysosomes, complete mitochondrial degradation and recycling of nutrients, thereby maintaining the stability of the intracellular microenvironment. Abnormalities in this process can lead to certain diseases, including neurodegenerative diseases, cardiovascular diseases, and metabolic diseases. Therefore, real-time monitoring of mitochondrial autophagy is of great significance for studying diseases associated with it. The process of mitochondrial autophagy causes the pH of mitochondria to drop from pH ~8.0 in the normal physiological state to pH ~5.5-6.5, which makes pH an ideal target for monitoring the process of mitochondrial autophagy. Developing a method that can accurately detect the pH value of mitochondria in real time is of great significance for understanding the key functions of mitochondria under physiological and pathological conditions.

[0003] Small molecule fluorescent probes have the advantages of being non-invasive, highly sensitive, and highly specific, and have been widely used to image physiological processes in living cells. Probes designed to monitor the mitophagy process in living cells should meet three "basic requirements": excellent mitochondrial targeting, response in the pH range of mitophagy, and emission wavelength in the "near-infrared window" (650nm-900nm) of biological imaging. Currently reported mitophagy probes mainly use naphthylimide or rhodamine as the parent, but the emission wavelengths of these two structures are mostly below 650nm. In contrast, the inherent cationic structure of cyanine dyes is conducive to mitochondrial targeting, and the emission wavelength can be adjusted to the "near-infrared window", which is an ideal fluorescent probe parent structure for monitoring mitophagy processes in living cells. The most widely used polymethine cyanine dyes do not have the ability to bind to protons because the nitrogen atoms of the indole at both ends are replaced. The response of cyanine to pH can be achieved by exposing the indole nitrogen atom at one end of the structure through an asymmetric synthesis strategy, but this method destroys the inherent mitochondrial targeting ability of the dye.

[0004] Although some progress has been made in the development of fluorescent probes for mitophagy in recent years, especially the use of cyanine dyes to try to solve the needs of near-infrared imaging and mitochondrial targeting, most of these advances have focused on improving a single performance and have failed to fully integrate all the necessary features, often facing the following problems:

[0005] Dilemma of comprehensive performance integration: In the process of pursuing enhanced pH response characteristics, many probe designs have to sacrifice their inherent mitochondrial targeting properties or fluorescence signal intensity. This balance is particularly difficult to grasp when introducing new functional groups to achieve pH sensitivity. How to add new functions without compromising the original performance has become a technical bottleneck that needs to be broken through.

[0006] Balance between specificity and sensitivity: In practical applications, existing probes often have low signal resolution due to nonspecific binding or cross-response to other changes in the cell microenvironment. Especially in complex physiological or pathological conditions, how to ensure that the probe only responds to pH changes related to mitochondrial autophagy without interference from other factors is an urgent problem to be solved.

[0007] The contradiction between biocompatibility and long-term monitoring: Although some probes have shown good biocompatibility in the short term, their stability and safety are still challenged when conducting long-term or in vivo monitoring. How to ensure that the probes can continue to play a monitoring role while not causing cytotoxicity or biological function interference when they remain in cells or organisms for a long time is the key to promoting the clinical application of such technologies.

[0008] Difficulties in accurate quantification and real-time monitoring: Existing technologies often lack sufficient sensitivity and accuracy in accurately quantifying changes in mitochondrial pH, especially in dynamically tracking subtle changes during autophagy. This limits our ability to deeply understand the relationship between the mitochondrial autophagy mechanism and disease.

[0009] Therefore, it is necessary to develop new mitochondrial autophagy probes with polymethine cyanine dyes as the skeleton to address these problems, so as to overcome the current defects and enable them to have better imaging, labeling and detection capabilities. Summary of the invention

[0010] In order to overcome a series of defects of existing mitochondrial autophagy probes, a new type of asymmetric polymethine cyanine near-infrared fluorescent dye, a synthesis method and application thereof are provided.

[0011] The first aspect of the present application is to protect a novel type of asymmetric polymethine cyanine near-infrared fluorescent dye, wherein the dye has a structure of general formula I:

[0012]

[0013] In the general formula I:

[0014] R1 is selected from one of hydrogen, halogen, methoxy, amino, nitro, hydroxyl, carboxyalkyl having 1-18 carbon atoms, alkylsulfonic acid having 1-18 carbon atoms, ester having 1-18 carbon atoms, and amide having 1-18 carbon atoms;

[0015] R2 or R3 are each independently selected from one of hydrogen, an alkyl group having 1 to 18 carbon atoms, a carboxyalkyl group having 1 to 18 carbon atoms, an aryl group, an arylcarboxylic acid group, an alkyl sulfonate group having 1 to 18 carbon atoms, an aryl sulfonate group, an alkyl sulfonate salt or an aryl sulfonate salt;

[0016] R4 is a non-positional substituent selected from one of hydrogen, halogen, carboxyalkyl having 1-18 carbon atoms, hydroxyalkyl having 1-18 carbon atoms, alkylsulfonic acid having 1-18 carbon atoms, ester having 1-18 carbon atoms, and amide having 1-18 carbon atoms;

[0017] n is selected from 2, 3 or 4;

[0018] X - Selected from halogen ions, ClO4 - 、BF4 - 、CH3COO - CF3COO - or OTs - One; more preferably selected from fluoride ion, chloride ion, bromide ion, iodide ion, ClO4 - 、CH3COO - One of a kind.

[0019] For the technical solution described above, R1 is more preferably selected from one of hydrogen, halogen, an ester group having 1 to 8 carbon atoms, and an amide group having 1 to 8 carbon atoms;

[0020] The ester group having 1 to 8 carbon atoms is -COOR; more preferably one selected from a C1-C8 alkyl ester group, a C1-C8 cycloalkyl ester group, and a C1-C8 aryl ester group:

[0021] C1-C8 alkyl ester group, for example: methyl ester (-COOCH3), ethyl ester (-COOCH2CH3), etc.

[0022] C1-C8 cycloalkyl ester group: such as cyclopropyl ester, cyclobutyl ester, etc.

[0023] C1-C8 aryl ester group: R is phenyl or aryl, etc.

[0024] The amide group having 1-8 carbon atoms refers to a -C(O)NR2 functional group, more preferably one selected from a C1-C8 alkylamide group, a C1-C8 cycloalkylamide group, and a C1-C8 arylamide group:

[0025] C1-C8 alkylamide: R is hydrogen or a hydrocarbon group, i.e., formamide (-CONH2), acetamide (-CONHCH3), etc.

[0026] C1-C8 cycloalkylamide: R is a cyclic structure, for example, cyclopropyl or cyclohexyl.

[0027] C1-C8 arylamide: R is phenyl or aryl, for example: methylphenyl, ethylphenyl, naphthalene, anthracene, etc.

[0028] For the technical solution described above, R2 or R3 is independently more preferably one selected from hydrogen, an alkyl group having 1 to 8 carbon atoms, an alkyl sulfonate group having 1 to 8 carbon atoms, and an aryl group;

[0029] The C1-C8 alkyl group includes, for example, methyl and ethyl;

[0030] C1-C8 alkylsulfonic acid group, for example: methylsulfonic acid group, ethylsulfonic acid group;

[0031] C1-C8 alkylaryl, for example: methylbenzene, ethylbenzene, methylnaphthalene, ethylnaphthalene, methylanthracene, ethylanthracene.

[0032] R2 or R3 are each independently more preferably selected from one of hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methyl formate, ethyl formate, ethyl acetate, propyl propionate, butyl butyrate, formamide, acetamide, propionamide, butyramide, methylsulfonyl, ethylsulfonyl, propylsulfonyl, butylsulfonyl, and benzoyl.

[0033] For the technical solution described above, R4 is preferably selected from one of hydrogen, a carboxyalkyl group having 1 to 8 carbon atoms, an ester group having 1 to 8 carbon atoms, and an amide group having 1 to 8 carbon atoms;

[0034] C1-C8 carboxyalkyl, for example: formate, acetate;

[0035] The ester group having 1 to 8 carbon atoms is -COOR; more preferably one selected from a C1-C8 alkyl ester group, a C1-C8 cycloalkyl ester group, and a C1-C8 aryl ester group:

[0036] C1-C8 alkyl ester group, for example: methyl ester (-COOCH3), ethyl ester (-COOCH2CH3), etc.

[0037] C1-C8 cycloalkyl ester group: such as cyclopropyl ester, cyclobutyl ester, etc.

[0038] C1-C8 aryl ester group: R is phenyl or aryl, etc.

[0039] The amide group having 1 to 8 carbon atoms refers to a -C(O)NR2 functional group, more preferably one of the following substituents:

[0040] C1-C8 alkylamide: R is hydrogen or a hydrocarbon group, i.e., formamide (-CONH2), acetamide (-CONHCH3), etc.

[0041] C1-C8 cycloalkylamide: R is a cyclic structure, for example, cyclopropyl or cyclohexyl.

[0042] C1-C8 arylamide: R is phenyl or aryl, for example: methylphenyl, ethylphenyl, naphthalene, anthracene, etc.

[0043] The second aspect of the present application is to protect a method for synthesizing a novel asymmetric polymethine cyanine near-infrared fluorescent dye, comprising the following steps:

[0044]

[0045] Step 1: at 60-120° C., Y-1 and 3-methyl-2-butanone are added to an organic solvent I, wherein the molar ratio of Y-1 to 3-methyl-2-butanone is 1:1-5; more preferably 1:1.5, after stirring for 3-12 hours, the solvent is evaporated, and the residue is added to an acid solution, stirred at 80-150° C. for 0.5-4 hours, the pH is adjusted to alkaline with a base, extracted, concentrated, and purified to obtain an intermediate product Y-2;

[0046] Step 2: In an organic solvent II, at 40-100° C., react Y-2 with an N-alkylating agent for 3-24 hours, and obtain an intermediate Y-3 after recrystallization, wherein the molar ratio of compound Y-2 to the N-alkylating agent is 1:1-10; more preferably 1:3-8;

[0047] Step 3: In an organic solvent III, at 60-120°C, stir and react Y-3 and S-1 for 0.5-5h, and obtain the intermediate S-2 by recrystallization; the molar ratio of Y-3 to S-1 is 1:1-5, more preferably 1:2; m is selected from 2, 3 or 4

[0048] Step 4: In an organic solvent IV, at 40-100° C., react Y-4 with an N-alkylating agent for 3-24 h, and obtain an intermediate Y-5 after recrystallization, wherein the molar ratio of compound Y-4 to the N-alkylating agent is 1:1-10; more preferably 1:3-8.

[0049] Step 5: S-2 and Y-5 are dissolved in an organic solvent V at 60-120°C, and a condensation reaction occurs under the catalysis of a base. After recrystallization and silica gel column purification, a novel asymmetric polymethine cyanine near-infrared fluorescent dye I is obtained.

[0050] For the technical solution described above, it is further preferred that the organic solvent I is selected from one of toluene, ethanol, methanol, isopropanol, acetonitrile and tetrahydrofuran;

[0051] For the technical solution described above, it is further preferred that the acid in step 1 is selected from any one of acetic acid, hydrochloric acid, sulfuric acid, and polyphosphoric acid;

[0052] For the technical solution described above, it is further preferred that the organic solvent II is selected from one of acetone, acetonitrile, methanol, toluene, o-dichlorobenzene, and DMF;

[0053] For the technical solution described above, it is further preferred that the organic solvent III is selected from one or a mixed solvent of ethanol, acetic acid, and acetic anhydride;

[0054] For the technical solution described above, it is further preferred that the organic solvent IV is selected from one of acetone, acetonitrile, methanol, toluene, o-dichlorobenzene, and DMF;

[0055] For the technical solution described above, it is further preferred that the organic solvent V is selected from one of DMF, ethanol, isopropanol and acetic anhydride;

[0056] For the technical solution described above, it is further preferred that the N-alkylating agent is selected from the group consisting of methyl halogenated hydrocarbon, ethyl halogenated hydrocarbon, propyl halogenated hydrocarbon, methyl formate halogenated hydrocarbon, ethyl formate halogenated hydrocarbon, ethyl halogenated hydrocarbon, propyl propionate halogenated hydrocarbon, butyl butyrate halogenated hydrocarbon, formamide halogenated hydrocarbon, acetamide halogenated hydrocarbon, propionamide halogenated hydrocarbon, butyramide halogenated hydrocarbon, methylsulfonyl halogenated hydrocarbon, ethylsulfonyl halogenated hydrocarbon, propylsulfonyl halogenated hydrocarbon, butylsulfonyl halogenated hydrocarbon, and benzoyl halogenated hydrocarbon;

[0057] For the technical solution described above, it is further preferred that in the above step 1 or 5, the base is selected from one of sodium hydroxide, potassium hydroxide, ammonia water, and sodium bicarbonate;

[0058] For the technical solution described above, it is further preferred that in the above steps 2, 3, and 4, the recrystallization solvent is selected from any one of methanol, ethanol, acetonitrile, ethyl acetate, ether, acetone, and propanol, or a mixed solvent of several combinations thereof.

[0059] The applications of the probe of the present invention include applications in basic scientific research, evaluation of drug action mechanisms, analysis of cell physiological functions and disease models, monitoring of environmental pollutant effects, regulation of mitochondrial function in regenerative medicine, and discovery and verification of biomarkers.

[0060] Basic scientific research: In the field of cell biology, the probe described in the present invention can be used as a powerful tool to help scientists conduct in-depth research on the molecular mechanism of mitochondrial autophagy, including its triggering factors, regulatory pathways, and its role in cellular stress response, energy metabolism, and cell death.

[0061] Cell function assessment: Applied to evaluate the dynamic changes of mitochondrial autophagy in different cell types or under different physiological and pathological conditions. For example, in the study of cell aging, stress response, metabolic disorders, etc., the mitochondrial health status and autophagy activity can be evaluated by monitoring the changes in mitochondrial pH.

[0062] Drug screening and toxicity testing: In the process of new drug development, this probe can be used to evaluate the effects of compounds on the mitochondrial autophagy process. It can be used as a tool for early screening of drugs with the potential to regulate mitochondrial autophagy activity. It can also be used to detect the cytotoxicity of drug candidates in in vitro models, especially those that may affect mitochondrial function.

[0063] Biomarker discovery: Monitoring the dynamic changes of mitochondrial autophagy in cell models or tissue samples can help identify biomarkers associated with mitochondrial dysfunction, thereby promoting the understanding of the mechanisms of complex diseases. Although it does not directly diagnose the disease, it can provide important clues for the study of disease mechanisms.

[0064] Cell Engineering and Regenerative Medicine: In the research of stem cell differentiation, tissue engineering and regenerative medicine, this probe can assist in monitoring and regulating mitochondrial status to optimize cell culture conditions, improve cell viability and differentiation efficiency, and has potential value for the development of cell therapy and tissue repair strategies.

[0065] Environmental and toxicology research: used to evaluate the effects of environmental pollutants or chemicals on mitochondrial autophagy, providing a new analytical method for environmental pollution monitoring and chemical safety assessment.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] (1) The asymmetric polymethine cyanine pH fluorescent probe for monitoring mitochondrial autophagy described in the present invention is the first to directly use polymethine cyanine dyes with excellent photophysical properties to respond to pH. The synthesis steps are simple, the cost is low, and it is easy to mass produce, and it has potential commercial application value.

[0068] (2) The recognition principle of the probe for pH change: The probe uses a symmetrical polymethine cyanine dye as the matrix, and synthesizes an asymmetrical polymethine cyanine dye structure by replacing one end of the symmetrical cyanine dye with an azaindole structure, releasing the indole nitrogen atom as H + The binding site of H +After binding to the indole nitrogen atom reaction site, the original intramolecular charge transfer effect of the asymmetric polymethine cyanine dye structure is greatly weakened, resulting in a decrease in the absorbance of the original structure and fluorescence quenching. + The dissociated structure will have an increase in absorbance and enhanced fluorescence. Through clever structural design, the probe achieves a rapid and reversible response to pH changes.

[0069] (3) The absorbance and fluorescence intensity of Cy5.5-H-CyN at different wavelengths changed significantly as the pH value decreased from 9.0 to 4.0. Specifically, the absorbance at 680nm gradually decreased, and the absorbance at 526nm gradually increased; at the same time, the fluorescence intensity at 710nm gradually decreased, while the fluorescence brightness at 662nm gradually increased. In the presence of various biological interferents (such as metal ions, reactive oxygen species, biological thiols and amino acids, etc.), Cy5.5-H-CyN showed good specificity, that is, its fluorescence intensity did not change significantly due to the presence of these interferents. The probe has high sensitivity and specificity to pH, can achieve rapid response to pH, and is not affected by environmental polarity, viscosity and other substances in the biological system.

[0070] (4) The probe has a pKa value of 6.46 and a linear range of pH response of 5.0-7.0, which matches the pH change during mitochondrial autophagy and is very suitable for monitoring the pH value in mitochondria during mitochondrial autophagy.

[0071] (5) The probe utilizes the inherent positive charge structure of the structure combined with the electrostatic effect of the negative membrane potential of the mitochondria to make the probe targeted and aggregated in the mitochondria. At the same time, the structural characteristics of the probe itself further fix the probe in the mitochondria. When used to monitor the changes in mitochondrial pH during mitochondrial autophagy, it can effectively resist the influence of microenvironment changes during the autophagy process (such as mitochondrial alkalinization and membrane depolarization causing a decrease in mitochondrial membrane potential, etc.). It has the characteristics of high sensitivity, reliability, visualization, rapidity and convenience, indicating that it has good application prospects in mitochondrial-related biological research and medical diagnosis.

[0072] (6) The detection method is simple and efficient, and is compatible with commonly used fluorescence imaging equipment. It only needs to include a fluorescence spectrophotometer and a laser confocal microscope, which is easy to popularize and apply.

[0073] (7) Biocompatibility and low toxicity: The probe of the present invention was designed with biosafety in mind. Cytotoxicity tests have shown that it has little effect on cell growth and metabolic activity, making it suitable as a long-term monitoring tool, which is particularly important for in vivo research and potential clinical transformation.

[0074] (8) Photostability: Compared with other fluorescent probes, the probe of the present invention has excellent photostability and can maintain stable fluorescence output under long-term illumination or repeated irradiation conditions, ensuring the reliability and repeatability of the data. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 This is the nuclear magnetic resonance characterization of the probe Cy5.5-H-CyN of the present invention. 1 H-NMR spectrum;

[0076] Figure 2 This is the nuclear magnetic resonance characterization of the probe Cy5.5-H-CyN of the present invention. 13 C-NMR spectrum;

[0077] Figure 3 High-resolution mass spectrometry characterization of the probe Cy5.5-H-CyN of the present invention, HR-MS spectrum;

[0078] Figure 4 The probe Cy5.5-H-CyN of the present invention was 0.01 mol·L at different pH values ​​(4.0-9.0). -1 Absorption spectrum in PBS buffer, the inset is the color change of the corresponding pH solution, from blue to pink;

[0079] Figure 5a and Figure 5b The probe Cy5.5-H-CyN of the present invention was 0.01 mol·L at different pH values ​​(4.0-9.0). -1 Fluorescence spectra at different excitation wavelengths in PBS buffer;

[0080] Figure 6 For comparison, the molecule Cy5.5-H-Cy5 was exposed to 0.01 mol·L -1 Absorption spectrum in PBS buffer;

[0081] Figure 7 For comparison, the molecule Cy5.5-H-Cy5 was exposed to 0.01 mol·L -1 Color change diagram of solution in PBS buffer;

[0082] Figure 8 For comparison, the molecule Cy5.5-H-Cy5 was exposed to 0.01 mol·L -1 Fluorescence spectra in PBS buffer;

[0083] Fig. 9 The probe Cy5.5-H-CyN of the present invention was 0.01 mol·L at different pH values ​​(4.0-9.0). -1In PBS buffer, the pH and the fluorescence intensity at 640 nm / fluorescence intensity at 488 nm were fitted to an “S”-shaped curve according to the Henderson-Hasselbalch equation;

[0084] Fig.10 The probe Cy5.5-H-CyN of the present invention was 0.01 mol·L at different pH values ​​(4.0-9.0). -1 In PBS buffer, a linear fitting graph was made between pH and 640nm excitation fluorescence intensity / 488nm excitation fluorescence intensity;

[0085] Fig.11 The fluorescence spectra of the probe Cy5.5-H-CyN of the present invention in the acid-base cycle test under the conditions of pH=8.0 and pH=4.0;

[0086] Fig.12 The fluorescence spectrum of the probe Cy5.5-H-CyN of the present invention is selective for common biological interferences (including metal cations, anions, active oxygen species, biological thiols and amino acids, etc.) under the condition of PBS buffer at pH = 7.4;

[0087] Fig.13 The fluorescence emission spectra of the probe Cy5.5-H-CyN of the present invention in ethanol-pentanediol (0%, 20%, 40%, 80% and 100%) mixed solvents with different 1,5-pentanediol volume contents;

[0088] Fig.14 This is a subcellular organelle co-localization fluorescence imaging image of the probe Cy5.5-H-CyN of the present invention and a commercially available green mitochondria-specific selective dye (MitoTrackerGreen FM) incubated with Hela cells for 30 minutes under the condition of pH 7.4;

[0089] Fig.15 The probe Cy5.5-H-CyN of the present invention and the commercially available green mitochondria-specific selective dye (MitoTrackerGreen FM) were co-stained in Hela cells at pH 7.4, and the subcellular organelle co-localization fluorescence imaging images of the two were obtained before and after CCCP treatment to reduce the cell membrane potential;

[0090] Fig.16 The commercially available mitochondria-specific selective dye Rhodamine 123 and the commercially available green mitochondria-specific selective dye (MitoTrackerGreenFM) were co-stained in Hela cells at pH 7.4, and the subcellular organelle co-localization fluorescence imaging of the two dyes was performed before and after CCCP treatment to reduce the cell membrane potential.

[0091] Fig.17The fluorescent imaging diagrams of the fluorescent probe Cy5.5-H-CyN of the present invention when incubated with HeLa cells for 60 minutes at pH=8.0 to pH=4.0 respectively;

[0092] Fig.18 The linear fitting diagram of the ratio channel fluorescence intensity of the fluorescent probe Cy5.5-H-CyN in HeLa cells and pH (5.0-7.0)

[0093] Fig.19 The fluorescence imaging diagram of HeLa cells at different time periods (0-180 min) under the action of FCCP (a mitochondrial uncoupler) of the probe Cy5.5-H-CyN of the present invention;

[0094] Fig. 20 The fluorescence imaging diagram of HeLa cells at different times (0-12h) under different mitophagy induction conditions (rapamycin, starvation and hypoxia) of the probe Cy5.5-H-CyN of the present invention; DETAILED DESCRIPTION

[0095] Hereinafter, the present invention will be described in further detail.

[0096] Unless otherwise specified, the terms used herein have the following meanings.

[0097] The solvent in the preparation method of the present invention is preferably a dehydrating solvent.

[0098] The purification method in the above preparation method of the present invention adopts conventional methods without special restrictions, preferably dichloromethane-methanol as eluent chromatographic column separation, recrystallization or a combination of the two. And the intermediates and final products obtained can be recovered by separation and purification techniques known in the art to achieve the required purity.

[0099] The raw materials used in the above-mentioned preparation method of the present invention can be commercially available or prepared by methods known in the art.

[0100] The structures of the compounds synthesized in the above-mentioned preparation method of the present invention are confirmed by high-resolution mass spectrometry, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum.

[0101] The instruments and equipment used in the examples are:

[0102] The dye absorption and emission spectra were measured using Agilent's Cary 60 UV-visible spectrophotometer and CaryEclipse fluorescence spectrophotometer. Cell fluorescence imaging was obtained using an Olympus FV3000 single-photon confocal microscope.

[0103] MitoTracker Green FM, also known as MitoTracker Green, is a mitochondrial green fluorescent probe that can be used for mitochondrial-specific fluorescent staining of living cells. Mito-Tracker Green is a Mito-Tracker fluorescently labeled with carbocyanine from Molecular Probes. Its molecular formula is C 34 H 28 Cl5N3O, molecular weight is 671.88, CAS number is 201860-17-5. Compared with Rhodamine 123 or JC-1, Mito-Tracker Green staining of mitochondria is not dependent on mitochondrial membrane potential.

[0104] Example 1

[0105] 1. Preparation and characterization of cyanine pH fluorescent probe Cy5.5-H-CyN for detecting mitophagy

[0106]

[0107] (1) 1 (692 mg, 2.19 mmol), 2 (600 mg, 2.33 mmol) and NaOAc (200 mg, 2.44 mmol) were added to a 50 ml round-bottom flask, and 10 ml Ac2O was added. The reactants were heated at 50 °C for 5 h. After the reaction, the mixture was cooled to room temperature and added dropwise to a 500 ml beaker containing 150 ml ether. The precipitated solid was collected and redissolved in DCM. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (DCM: MeOH = 50: 1, v / v). The brown-yellow band was collected to obtain a brown solid 3 with a yield of 42% (448 mg). HRMS (ESI) was calculated for C 23 H 26 N3O + [MI] + 360.2070, found 360.2079.

[0108] (2) 3 (244 mg, 0.5 mmol), 4 (219 mg, 0.6 mmol) and NaOAc (82 mg, 1 mmol) were added to a 50 ml round-bottom flask, and 10 ml of EtOH was added. The reactants were heated at 80°C for 1 h. After the reaction, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography (DCM: MeOH = 75: 1, v / v) to obtain Cy5.5-H-CyN as a green solid with a yield of 20% (116 mg). 1H NMR (400MHz, DMSO-d6) δ8.34(d,J=7.0Hz,1H),8.28(t,J=13.2Hz,1H),8.16(d,J=8.6Hz,1H),8.12(d,J =7.2Hz,1H),8.08(d,J=13.1Hz,1H),7.99(d,J=8.7Hz,2H),7.59(t,J=8.6Hz,2H),7.46–7.38(m,1H),7 .31(t,J=6.8Hz,1H),6.48(t,J=12.6Hz,1H),6.33(d,J=14.0Hz,1H),6.08(d,J=13.2Hz,1H),4.60(q,J =7.2Hz,2H),4.14–4.08(m,2H),1.90(s,6H),1.53(t,J=7.2Hz,3H),1.45(s,6H),1.28(t,J=7.1Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ192.62,169.15,162.67,152.40,149.27,144.51,140.44,138.42,133.65,131.89,131.03,130.50,130.33,128.4 2,127.87,124.40,124.24,122.27,118.11,111.24,100.16,51.54,49.98,49.08,38.24,27.36,24.08,15.56,12.58.HRMS(ESI)calcedfor C 32 H 36 N3 + [MI] + 462.2904, found 462.2910.

[0109] 2. Synthesis of comparative molecule Cy5.5-H-Cy5

[0110] The comparative molecule Cy5.5-H-Cy5 was synthesized according to the aforementioned synthesis steps, and its structural formula is:

[0111]

[0112] Compared with Cy5.5-H-CyN, Cy5.5-H-Cy5 replaces the azaindole part with ordinary indole. Since the indole nitrogen atom is occupied, Cy5.5-H-Cy5 does not have pH response ability.

[0113] Example 2

[0114] Photophysical properties of the probe molecule Cy5.5-H-CyN

[0115] The probe molecule Cy5.5-H-CyN prepared in Example 1 after vacuum drying was accurately weighed with a 1 / 10,000 balance, and a 2 mmol / L mother solution was prepared with HPLC grade DMSO, placed in a brown sample bottle, and stored in a 4°C refrigerator for later use.

[0116] When testing the UV-visible absorption spectrum and fluorescence spectrum, use a micropipette to draw 4.5 μL of the mother solution and dissolve it in a quartz cuvette containing 3 mL of PBS buffer of different pH values. Mix well to obtain a molecular concentration of 3 μmol / L for testing the absorption spectrum and fluorescence emission spectrum. All tests were completed at 25°C. The instruments used for the tests were the AgIIlent 8453 UV spectrophotometer and the AgIIlent Cary EclIIpse fluorescence spectrophotometer. As the pH value decreased from 9.0 to 4.0, the absorbance of the probe at 680 nm gradually decreased, and the absorbance at 526 nm gradually increased, and the corresponding solution color gradually changed from blue to pink ( Figure 4 ). The excitation wavelength was set to 640 nm, and the fluorescence intensity at 710 nm (Fl.λ ex640nm ) gradually decreases( Figure 5a ). Set the excitation wavelength to 488nm and the fluorescence brightness at 662nm (Fl.λ ex488nm ) gradually rises( Figure 5b ). The comparative molecule Cy5.5-H-Cy5 has no pH response ability, so in PBS buffers of different pH values, the absorption wavelength ( Figure 6 ), fluorescence intensity ( Figure 7 ) and solution color ( Figure 8 ) had no significant changes. ex 640nm / Fl.λ ex 488nm The numerical value can be fitted according to the Henderson-Hasselbalch equation, and the pKa value of Cy5.5-H-CyN is calculated to be about 6.46 ( Fig. 9 ), the pH response linear range is 5.0~7.0( Fig.10 ). This property perfectly matches the pH change during mitochondrial autophagy, demonstrating the targetedness and effectiveness of the design. Both fluorescence peaks of the probe molecule are located in the near-infrared region, with good photophysical properties, making it suitable for biological imaging applications.

[0117] It is particularly noteworthy that the fluorescence peak of Cy5.5-H-CyN is located in the near-infrared region (662nm and 710nm), which not only reduces the autofluorescence interference of biological tissues, but also improves the imaging depth and signal-to-noise ratio, which is a major advantage in biological imaging applications. This feature is extremely critical in practical applications because it allows deeper tissue penetration and clearer signal identification, especially in in vivo imaging studies, which is far beyond the reach of traditional probes.

[0118] also, Figures 4 to 10 The data not only demonstrate the performance of the probe, but also indirectly prove the non-obviousness of the design of the present invention. Through the detailed spectral analysis of the pH response mechanism of Cy5.5-H-CyN, it can be seen that the response characteristics are based on the sophisticated molecular structure design and deep understanding of the biological environment. For example, Figure 4 The color change from blue to pink in the sample directly reflects the regulation of the charge transfer effect inside the molecule with pH changes. This is the result of careful design through the introduction of the azaindole structure and the asymmetry of the molecule, rather than the effect that can be expected from simple structural adjustments. These data and image materials together constitute strong evidence of the non-obviousness of the technology of the present invention, further establishing its potential value and application prospects in biomedical research.

[0119] Example 3

[0120] Specificity of the probe molecule Cy5.5-H-CyN in response to pH

[0121] The concentration of the probe molecule Cy5.5-H-CyN in Example 1 was kept at 3 μM, and the probe was investigated for its effect on H in the presence of common biological interferences (including metal cations, anions, reactive oxygen species, biothiols and amino acids, etc.) or mixed solvents of different viscosities. + After four cycles of testing, the probe molecule Cy5.5-H-CyN can achieve rapid response between pH = 4 and pH = 8, and the fluorescence intensity does not change significantly ( Fig.11 The probe molecule Cy5.5-H-CyN has almost no response to common biological interferences ( Fig.12, the order of substances in the figure is: NaCl, NH4Cl, K2SO4, MgCl2, CuCl2, CaCl2, FeCl3, KI, NaF, NaNO2, ZnNO3, NaClO4, Na2S2O3, H2O2, GSH, Cys, HCy, pH = 4PBS buffer. The concentrations are 0.1 mM except for PBS). In the ethanol-pentanediol mixed solvents with different 1,5-pentanediol volume contents (0%, 20%, 40%, 80% and 100%), the probe molecule Cy5.5-H-CyN did not show a specific response to viscosity ( Fig.13 ), which further emphasizes the stability and specificity of Cy5.5-H-CyN in complex biological media and provides a strong guarantee for its accurate pH monitoring.

[0122] Example 4

[0123] In order to study the mitochondrial targeting ability of the probe molecule Cy5.5-H-CyN, it was co-stained with HeLa cells and MitoTracker Green FM (a commercial mitochondrial targeting dye). The adherent HeLa cells were incubated with Cy5.5-H-CyN (concentration 1 μmol / L) at pH 7.4 in an incubator at 37°C and 5% CO2 for 60 min, and then gently washed three times with PBS buffer (pH = 7.4) to remove excess dye. Fig.14 As shown in the figure, the fluorescence distribution of Cy5.5-H-CyN overlaps well with that of MitoTracker Green FM (Pearson coefficient: 0.946), and obvious mitochondrial filamentous structures can be observed in both, indicating that the probe molecule Cy5.5-H-CyN has excellent mitochondrial targeting ability. In order to further confirm that the probe can remain in the mitochondria for a long time without being affected by the mitochondrial membrane potential, the mitochondrial uncoupler carbonyl cyanidem-chlorophenylhydrazone (CCCP) was added to the cells to quickly reduce the mitochondrial membrane potential. It was found that the fluorescence overlap between the probe and MitoTracker Green FM did not decrease significantly ( Fig.15 ), while the fluorescence intensity of the commercially available mitochondrial-specific selective dye rhodamine 123 in cells almost completely disappeared after CCCP ( Fig.16 ), indicating that even with the decrease of membrane potential, the probe Cy5.5-H-CyN still firmly aggregates in the mitochondria. This result highlights the stability and membrane potential independence of Cy5.5-H-CyN. Even in the extreme microenvironment changes caused by cellular autophagy, it can be stably retained in the mitochondria, providing a reliable tool for the dynamic monitoring of the mitochondrial autophagy process.

[0124] Example 5

[0125] HeLa cells were pretreated with DMEM containing Cy5.5-H-CyN (1 μM) for 1 h at 37 °C. Then, the culture medium was replaced with 0.01 mol / L PBS buffer of various pH values ​​(pH 4.0–8.0) containing 10 μM nigericin. After incubation with the cells for 30 min, the cells were washed three times with 0.01 mol / L PBS buffer of the corresponding pH, and images of the red (700-730 nm) and green (620-670 nm) channels were recorded under 640 nm excitation through a 60x oil-immersion lens under 488 nm excitation. When the pH was reduced from 8.0 to 4.0, the fluorescence signal of the red channel gradually weakened, and the fluorescence signal of the green channel gradually increased. The merged channel gradually changed from red at pH = 8.0 to orange, and finally to green at pH = 4.0. The ratio fluorescence signal of the red channel / green channel also gradually changed from red-green at pH = 8.0 to blue-green, until the ratio fluorescence signal at pH = 4.0 basically disappeared ( Fig.17 The ratio of red to green fluorescence signals has a linear relationship with pH (5.0-7.0). Fig.18 ), indicating that Cy5.5-H-CyN can be used to quantitatively track and visualize changes in mitochondrial pH in cells within this range, providing intuitive and precise visual feedback for dynamic monitoring of mitophagy.

[0126] Example 6

[0127] Mitophagy was induced using various external stimulation conditions and monitored by Cy5.5-H-CyN. Before applying stimulation, HeLa cells were first pretreated with DMEM containing Cy5.5-H-CyN (1 μM) at 37°C, and then different external stimulations were applied: mitochondrial uncoupler FCCP (50 μM, incubated for 180 min, images collected every 30 min); rapamycin (5 μM, incubated for 12 h, images collected every 6 h); DMEM medium without FBS (incubated for 12 h, images collected every 6 h); 1% O2 culture conditions (incubated for 12 h, images collected every 6 h). Images of the red (700-730 nm) and green (620-670 nm) channels were recorded under 640 nm excitation and 488 nm excitation, respectively, through a 60x oil immersion lens.

[0128] After only 30 minutes of incubation with FCCP, the fluorescence intensity of the red channel decreased significantly. After 180 minutes of incubation, the fluorescence intensity of the red channel was already very low, and the green channel showed obvious fluorescence signals ( Fig.19). Based on the fluorescence intensity ratio of the two channels and the pH calibration curve, it can be calculated that the mitochondrial pH drops from about 6.10 at 30 min to about 5.21 at 180 min.

[0129] 5 μM rapamycin was added to induce mitochondrial autophagy. Compared with the control group, the fluorescence intensity of the red channel decreased significantly after 6 hours, and the green channel showed obvious fluorescence signals after 12 hours. According to the pH calibration curve, it can be calculated that the use of rapamycin to induce mitophagy in Hela cells, the mitochondrial pH value is about 6.02 after 6 hours, and the mitochondrial pH is about 5.59 after 12 hours. After 6 hours of application of both culture conditions, the fluorescence intensity of the red channel decreased significantly, and the green channel showed obvious fluorescence signals after 12 hours. According to the pH calibration curve, it can be calculated that under the culture conditions of nutrient deficiency or hypoxia, the mitochondrial pH is 6.42 and 6.17 after 6 hours, and 5.99 and 5.73 after 12 hours. All the above experimental results show that the application of Cy5.5-H-CyN can realize the quantitative display of mitochondrial pH, thereby realizing the monitoring of the mitophagy process of Hela cells ( Fig. 20 These data not only confirm the high accuracy and reliability of Cy5.5-H-CyN in tracking mitochondrial pH dynamics under various autophagy induction modes, but also provide a powerful tool for in-depth understanding of the autophagy mechanism and its role in disease states.

[0130] In summary, Cy5.5-H-CyN not only exhibits excellent pH responsiveness and specificity, but also exhibits stable performance in complex biological environments, especially the ability to accurately monitor pH changes during mitophagy. These characteristics make it an ideal tool for studying mitochondrial function and autophagy.

[0131] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A new type of asymmetric polymethine cyanine near-infrared fluorescent dye, characterized in that: The dye has a structure of general formula I: In the general formula I: R1 is selected from one of hydrogen, halogen, methoxy, amino, nitro, hydroxyl, carboxyalkyl having 1-18 carbon atoms, alkylsulfonic acid having 1-18 carbon atoms, ester having 1-18 carbon atoms, and amide having 1-18 carbon atoms; R2 or R3 are each independently selected from one of hydrogen, an alkyl group having 1 to 18 carbon atoms, a carboxyalkyl group having 1 to 18 carbon atoms, an aryl group, an arylcarboxylic acid group, an alkyl sulfonate group having 1 to 18 carbon atoms, an aryl sulfonate group, an alkyl sulfonate salt or an aryl sulfonate salt; R4 is a non-positional substituent selected from one of hydrogen, halogen, carboxyalkyl having 1-18 carbon atoms, hydroxyalkyl having 1-18 carbon atoms, alkylsulfonic acid having 1-18 carbon atoms, ester having 1-18 carbon atoms, and amide having 1-18 carbon atoms; n is selected from 2, 3 or 4; X - Selected from halogen ions, ClO4 - 、BF4 - 、CH3COO - CF3COO - or OTs - One of a kind.

2. The dye according to claim 1, characterized in that: The R1 is selected from one of hydrogen, halogen, an ester group having 1 to 8 carbon atoms, and an amide group having 1 to 8 carbon atoms.

3. The dye according to claim 2, characterized in that: The ester group having 1-8 carbon atoms is selected from one of a C1-C8 alkyl ester group, a C1-C8 cycloalkyl ester group, and a C1-C8 aryl ester group; the amide group having 1-8 carbon atoms is selected from one of a C1-C8 alkyl amide group, a C1-C8 cycloalkyl amide group, and a C1-C8 aryl amide group.

4. The dye according to claim 1, characterized in that: The R1 is selected from one of hydrogen, halogen, methyl ester, ethyl ester, cyclopropyl ester, cyclobutyl ester, phenyl, aryl, formamide, acetamide, cyclopropyl, cyclohexyl, methylphenyl, ethylphenyl, naphthalene and anthracene.

5. The dye according to claim 1, characterized in that: The R2 or R3 is independently selected from one of hydrogen, an alkyl group having 1 to 8 carbon atoms, an alkyl sulfonate group having 1 to 8 carbon atoms, and an aryl group.

6. The dye according to claim 1, characterized in that: The R2 or R3 is independently selected from one of hydrogen, methyl, ethyl, methylsulfonate, ethylsulfonate, methylbenzene, ethylbenzene, methylnaphthalene, ethylnaphthalene, methylanthracene and ethylanthracene.

7. The dye according to claim 1, characterized in that: The R2 or R3 is independently selected from one of hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methyl formate, ethyl formate, ethyl acetate, propyl propionate, butyl butyrate, formamide, acetamide, propionamide, butyramide, methylsulfonyl, ethylsulfonyl, propylsulfonyl, butylsulfonyl, and benzoyl.

8. The method for preparing a dye as claimed in claim 1, wherein: The following steps are involved: Step 1: at 60-120° C., Y-1 and 3-methyl-2-butanone are added to an organic solvent I, wherein the molar ratio of Y-1 to 3-methyl-2-butanone is 1:1-5; after stirring for 3-12 hours, the solvent is evaporated, and the residue is added to an acid solution, stirred at 80-150° C. for 0.5-4 hours, the pH is adjusted to alkaline with a base, extracted, concentrated, and purified to obtain an intermediate product Y-2; Step 2: In an organic solvent II, at 40-100° C., react Y-2 with an N-alkylating agent for 3-24 h, and obtain an intermediate Y-3 through recrystallization, wherein the molar ratio of compound Y-2 to the N-alkylating agent is 1:1-10; Step 3: In an organic solvent III, at 60-120° C., stir and react Y-3 and S-1 for 0.5-5 h, and obtain the intermediate S-2 through recrystallization; the molar ratio of Y-3 to S-1 is 1:1-5; m is selected from 2, 3 or 4; Step 4: In an organic solvent IV, at 40-100° C., react Y-4 with an N-alkylating agent for 3-24 hours, and obtain an intermediate Y-5 after recrystallization, wherein the molar ratio of compound Y-4 to the N-alkylating agent is 1:1-10; Step 5: S-2 and Y-5 are dissolved in an organic solvent V at 60-120° C., and a condensation reaction is carried out under the catalysis of a base. The dye having the general formula I is obtained through recrystallization and silica gel column purification.

9. The method for preparing a dye according to claim 1, wherein: The organic solvent I is selected from one of toluene, ethanol, methanol, isopropanol, acetonitrile and tetrahydrofuran; The organic solvent II is selected from one of acetone, acetonitrile, methanol, toluene, o-dichlorobenzene, and DMF; The organic solvent III is selected from one or more mixed solvents of ethanol, acetic acid, and acetic anhydride; The organic solvent IV is selected from one of acetone, acetonitrile, methanol, toluene, o-dichlorobenzene, and DMF; The organic solvent V is selected from one of DMF, ethanol, isopropanol and acetic anhydride; The N-alkylating agent is selected from the group consisting of methyl halogenated hydrocarbon, ethyl halogenated hydrocarbon, propyl halogenated hydrocarbon, methyl formate halogenated hydrocarbon, ethyl formate halogenated hydrocarbon, ethyl halogenated hydrocarbon, propyl propionate halogenated hydrocarbon, butyl butyrate halogenated hydrocarbon, formamide halogenated hydrocarbon, acetamide halogenated hydrocarbon, propionamide halogenated hydrocarbon, butyramide halogenated hydrocarbon, methylsulfonyl halogenated hydrocarbon, ethylsulfonyl halogenated hydrocarbon, propylsulfonyl halogenated hydrocarbon, butylsulfonyl halogenated hydrocarbon, and benzoyl halogenated hydrocarbon; In step 1 or 5, the base is selected from one of sodium hydroxide, potassium hydroxide, ammonia water, and sodium bicarbonate; In step 1, the acid is selected from any one of acetic acid, hydrochloric acid, sulfuric acid, and polyphosphoric acid; In the steps 2, 3 and 4, the recrystallization solvent is selected from any one of methanol, ethanol, acetonitrile, ethyl acetate, ether, acetone and propanol or a mixed solvent of several combinations thereof.

10. The use of the dye according to claim 1, characterized in that: This includes the application of the dye in basic scientific research, evaluation of drug action mechanisms, analysis of cell physiological functions and disease models, monitoring of the effects of environmental pollutants, regulation of mitochondrial function in regenerative medicine, and discovery and verification of biomarkers.

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