Near-infrared responsive asymmetric polymethine cyanine dyes, methods of making and use thereof

Abstract

The application provides a near-infrared responsive asymmetric polymethine cyanine dye, a preparation method and application thereof. + The probe realizes rapid and reversible response to pH change in combination with the nitrogen heteroindole part in the asymmetric polymethine cyanine dye structure. The probe has high sensitivity and high specificity to pH, the pKa value is 6.46, the linear pH response range is 5.0-7.0, and is suitable for monitoring the pH value in mitochondria. The inherent positive charge of the structure enables it to target and accumulate in mitochondria and remain stable during the autophagy process. The probe has high sensitivity, reliability and visualization characteristics, and is suitable for basic scientific research, drug mechanism evaluation, cell physiological function and disease model analysis, environmental pollutant effect monitoring, regenerative medicine and the like. In addition, the probe has good biocompatibility, strong light stability, is compatible with commonly used fluorescence imaging equipment, and is convenient for popularization and application.

Description

Technical Field

[0001] This invention relates to the field of pH fluorescent probe technology, specifically to cyanin-based pH fluorescent probes for monitoring mitophagy, their preparation, and applications. Background Technology

[0002] Mitochondria are organelles found in most eukaryotic cells, enclosed by two membranes. They produce reactive oxygen species (ROS) and adenosine triphosphate (ATP) through aerobic respiration and are the site of many key metabolic processes. Maintaining mitochondrial homeostasis is crucial for cell growth. Mitochondria can be severely damaged by depolarization under conditions such as high levels of ROS, nutrient deficiency, and cellular senescence. To maintain the quantity and quality of mitochondria, cells initiate mitophagy, encapsulating damaged mitochondria in autophagosomes and fusing them with lysosomes. This process completes mitochondrial degradation and nutrient recycling, 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 mitophagy is of great significance for studying related diseases. Mitophagy causes the mitochondrial pH to decrease from the normal physiological pH of ~8.0 to ~5.5-6.5, making pH an ideal target for monitoring this process. Developing a method for real-time and accurate detection of mitochondrial pH is crucial for understanding the key functions of mitochondria under physiological and pathological conditions.

[0003] Small molecule fluorescent probes, with their advantages of being non-invasive, highly sensitive, and specific, have been widely used for imaging physiological processes in living cells. Probes designed to monitor mitophagy in living cells should meet three basic requirements: excellent mitochondrial targeting, a response within the pH range of mitophagy, and an emission wavelength within the "near-infrared window" (650 nm-900 nm) for bioimaging. Currently reported mitophagy probes mainly use naphthalimide or rhodamine as parent compounds; however, the emission wavelengths of these two structures are mostly below 650 nm. In contrast, the inherent cationic structure of cyanine dyes facilitates mitochondrial targeting, and the emission wavelength can be tuned to the "near-infrared window," making them an ideal parent structure for monitoring mitophagy in living cells. The most widely used polymethyl cyanine dye, due to the substitution of indole nitrogen atoms at both ends, lacks the ability to bind to protons. While an asymmetric synthesis strategy can expose the indole nitrogen atoms at one end of the structure to achieve a pH response, this approach destroys the dye's inherent mitochondrial targeting ability.

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

[0005] The challenge of integrating comprehensive performance: In the pursuit of enhanced pH response characteristics, many probe designs have to sacrifice their inherent mitochondrial targeting properties or fluorescence signal intensity, especially when introducing new functional groups to achieve pH sensitivity, this balance is particularly difficult to maintain. How to add new functions without compromising the original performance has become a critical technical bottleneck that urgently needs to be overcome.

[0006] Balancing specificity and sensitivity: In practical applications, existing probes often suffer from low signal resolution due to non-specific binding or cross-response to other changes in the cellular microenvironment. Especially under complex physiological or pathological conditions, ensuring that probes respond only to pH changes related to mitophagy, without interference from other factors, is a pressing issue that needs to be addressed.

[0007] The contradiction between biocompatibility and long-term monitoring: While some probes demonstrate good biocompatibility in the short term, their stability and safety remain challenging when used for long-term or in vivo monitoring. Ensuring that probes can continuously perform their monitoring function while remaining in cells or organisms for extended periods without causing cytotoxicity or interference with biological functions is crucial for advancing the clinical application of this technology.

[0008] The challenge of precise quantification and real-time monitoring: Current technologies often lack sufficient sensitivity and accuracy in accurately quantifying mitochondrial pH changes, especially when dynamically tracking subtle changes during autophagy. This limits our ability to gain a deeper understanding of the link between mitophagy mechanisms and disease.

[0009] Therefore, it is necessary to develop novel mitochondrial autophagy probes with polymethyl cyanine dye as the backbone to address these issues, thereby overcoming the current shortcomings and enabling them to have better imaging, labeling, and detection capabilities. Summary of the Invention

[0010] To overcome a series of shortcomings of existing mitochondrial autophagy probes, a novel class of asymmetric polymethyl cyanine near-infrared fluorescent dyes, their synthesis methods, and applications are presented.

[0011] The first aspect of this application is to protect a novel class of asymmetric polymethyl cyanine near-infrared fluorescent dyes, said dyes having the structure of general formula I:

[0012]

[0013] In general formula I:

[0014] R1 is selected from one of hydrogen, halogen, methoxy, amino, nitro, hydroxyl, carboxylalkyl having 1-18 carbons, alkylsulfonic acid having 1-18 carbons, ester having 1-18 carbons, and amide having 1-18 carbons.

[0015] R2 or R3 is independently selected from one of hydrogen, alkyl with 1-18 carbons, carboxylalkyl with 1-18 carbons, aryl, arylcarboxylic acid, alkylsulfonate with 1-18 carbons, arylsulfonate, alkyl sulfonate or aryl sulfonate.

[0016] R4 is a substituent at an indeterminate position, selected from hydrogen, halogen, carboxyl group having 1-18 carbons, hydroxyalkyl group having 1-18 carbons, alkylsulfonic acid group having 1-18 carbons, ester group having 1-18 carbons, and amide group having 1-18 carbons.

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

[0018] X - Selected from halide ions, ClO4 - BF4 - CH3COO - CF3COO - or OTs - One; more preferably, from fluoride ions, chloride ions, bromide ions, iodide ions, ClO4 - CH3COO - One of them.

[0019] For the technical solution described above, R1 is more preferably one of hydrogen, halogen, ester group having 1-8 carbons, or amide group having 1-8 carbons;

[0020] The ester group having 1-8 carbons is -COOR; more preferably, it is one of C1-C8 alkyl ester group, C1-C8 cycloalkyl ester group, and C1-C8 aryl ester group:

[0021] C1-C8 alkyl ester groups, such as methyl esters (-COOCH3), ethyl esters (-COOCH2CH3), etc.

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

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

[0024] Amide groups having 1-8 carbons refer to the -C(O)NR2 functional group, more preferably one of C1-C8 alkylamide groups, C1-C8 cycloalkylamide groups, and C1-C8 arylamide groups:

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

[0026] C1-C8 cycloalkylamide groups: R is a cyclic structure, such as cyclopropyl or cyclohexyl.

[0027] C1-C8 aryl amide groups: R is phenyl or aryl, such as methylphenyl, ethylphenyl, naphthalene, anthracene, etc.

[0028] For the technical solutions described above, R2 or R3 is more preferably one of hydrogen, an alkyl group having 1-8 carbons, an alkyl sulfonate group having 1-8 carbons, or an aryl group.

[0029] The alkyl group having C1-C8, such as methyl or ethyl;

[0030] C1-C8 alkyl sulfonic acid groups, such as: methanesulfonic acid group, ethyl sulfonic acid group;

[0031] C1-C8 alkyl aryl groups, such as methylbenzene, ethylbenzene, methylnaphthalene, ethylnaphthalene, methylanthracene, and ethylanthracene.

[0032] R2 or R3 is more preferably derived from one of the following groups: hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, methyl formate, ethyl formate, ethyl acetate, propyl propionate, butyl butyrate, formamide, acetamide, propionamide, butyramide, methanesulfonic acid group, ethylsulfonic acid group, propylsulfonic acid group, butylsulfonic acid group, and benzoic acid group.

[0033] For the technical solution described above, R4 is preferably one of hydrogen, a carboxyl group having 1-8 carbons, an ester group having 1-8 carbons, or an amide group having 1-8 carbons;

[0034] C1-C8 carboxyl groups, such as formic acid group and acetate group;

[0035] The ester group having 1-8 carbons is -COOR; more preferably, it is one of C1-C8 alkyl ester group, C1-C8 cycloalkyl ester group, and C1-C8 aryl ester group:

[0036] C1-C8 alkyl ester groups, such as methyl esters (-COOCH3), ethyl esters (-COOCH2CH3), etc.

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

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

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

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

[0041] C1-C8 cycloalkylamide groups: R is a cyclic structure, such as cyclopropyl or cyclohexyl.

[0042] C1-C8 aryl amide groups: R is phenyl or aryl, such as methylphenyl, ethylphenyl, naphthalene, anthracene, etc.

[0043] The second aspect of this application relates to a method for synthesizing a novel class of asymmetric polymethyl cyanine near-infrared fluorescent dyes, comprising the following steps:

[0044]

[0045] Step 1: At 60-120℃, Y-1 and 3-methyl-2-butanone are added to 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 h, the solvent is evaporated, and the residue is added to an acid solution. The mixture is stirred at 80-150℃ for 0.5-4 h, and the pH is adjusted to alkaline with alkali. The mixture is then extracted, concentrated, and purified to obtain intermediate product Y-2.

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

[0047] Step 3: In organic solvent III, Y-3 and S-1 are stirred and reacted at 60-120°C for 0.5-5 hours, and then recrystallized to obtain intermediate S-2; 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 organic solvent IV, Y-4 is reacted with N-alkylating agent at 40-100°C for 3-24 h, and the intermediate Y-5 is obtained by recrystallization, wherein the molar ratio of compound Y-4 to N-alkylating agent is 1:1-10; more preferably 1:3-8.

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

[0050] For the technical solution described above, more preferably, the organic solvent I is selected from toluene, ethanol, methanol, isopropanol, acetonitrile, and tetrahydrofuran;

[0051] For the technical solution described above, more preferably, 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, more preferably, the organic solvent II is selected from one of acetone, acetonitrile, methanol, toluene, o-dichlorobenzene, and DMF;

[0053] For the technical solution described above, more preferably, the organic solvent III is selected from one or a mixture of several of ethanol, acetic acid, and acetic anhydride;

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

[0055] For the technical solution described above, more preferably, the organic solvent V is selected from DMF, ethanol, isopropanol, and acetic anhydride;

[0056] For the technical solution described above, more preferably, the N-alkylating agent is selected from one of methyl halogenated hydrocarbons, ethyl halogenated hydrocarbons, propyl halogenated hydrocarbons, methyl formate halogenated hydrocarbons, ethyl formate halogenated hydrocarbons, ethyl acetate halogenated hydrocarbons, propyl propionate halogenated hydrocarbons, butyl butyrate halogenated hydrocarbons, formamide halogenated hydrocarbons, acetamide halogenated hydrocarbons, propionamide halogenated hydrocarbons, butyramide halogenated hydrocarbons, methanesulfonic acid halogenated hydrocarbons, ethylsulfonic acid halogenated hydrocarbons, propylsulfonic acid halogenated hydrocarbons, butylsulfonic acid halogenated hydrocarbons, and benzoic acid halogenated hydrocarbons.

[0057] For the technical solution described above, in a further preferred embodiment, in step 1 or 5 above, the alkali is selected from one of sodium hydroxide, potassium hydroxide, ammonia, and sodium bicarbonate.

[0058] In a further preferred embodiment of the above-described technical solution, in steps 2, 3, and 4, the recrystallization solvent is selected from any one or a mixture of several of methanol, ethanol, acetonitrile, ethyl acetate, diethyl ether, acetone, and propanol.

[0059] The probes described in this invention have applications in basic scientific research, drug mechanism assessment, cell physiological function and disease model analysis, environmental pollutant effect monitoring, mitochondrial function regulation in regenerative medicine, and biomarker discovery and validation.

[0060] Basic scientific research: In the field of cell biology, the probe described in this invention can serve as a powerful tool to help scientists delve into the molecular mechanisms of mitophagy, including its triggering factors, regulatory pathways, and its role in cellular stress response, energy metabolism, and cell death.

[0061] Cellular function assessment: It is used to evaluate the dynamic changes of mitochondrial autophagy in different cell types or under different physiological and pathological conditions. For example, in studies of cell aging, stress response, and metabolic disorders, the health status and autophagy activity of mitochondria are assessed by monitoring changes in mitochondrial pH.

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

[0063] Biomarker discovery: By monitoring the dynamic changes in mitophagy in cell models or tissue samples, it is helpful to identify biomarkers associated with mitochondrial dysfunction, thereby promoting the understanding of complex disease mechanisms. Although it does not directly diagnose diseases, it can provide important clues for disease mechanism research.

[0064] Cell Engineering and Regenerative Medicine: In stem cell differentiation, tissue engineering, and regenerative medicine research, this probe can help monitor and regulate 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 toxicological studies: used to assess the effects of environmental pollutants or chemicals on cellular mitochondrial autophagy, providing a new analytical approach 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 polymethyl cyanine pH fluorescent probe for monitoring mitochondrial autophagy described in this invention is the first to realize the direct application of polymethyl cyanine dye with excellent photophysical properties to respond to pH. The synthesis steps are simple, the cost is low, and it is easy to produce on a large scale, which has potential commercial application value.

[0068] (2) The principle of pH change recognition by the probe: The probe uses a symmetrical polymethyl cyanine dye as the parent material. By replacing one end of the symmetrical cyanine dye with an aza-indole structure, an asymmetrical polymethyl cyanine dye structure is synthesized, releasing indole nitrogen atoms as H atoms. + The binding site. When H +Upon binding to the indole nitrogen atom reaction site, the original intramolecular charge transfer effect of the asymmetric polymethyl cyanine dye structure is significantly weakened, leading to a decrease in the absorbance of the original structure and fluorescence quenching. + The dissociated structure exhibits increased absorbance and enhanced fluorescence. Through ingenious structural design, the probe achieves a rapid and reversible response to pH changes.

[0069] (3) As the pH value decreased from 9.0 to 4.0, the absorbance and fluorescence intensity of Cy5.5-H-CyN changed significantly at different wavelengths. Specifically, the absorbance at 680 nm gradually decreased, while the absorbance at 526 nm gradually increased; simultaneously, the fluorescence intensity at 710 nm gradually decreased, while the fluorescence intensity at 662 nm gradually increased. In the presence of various biological interfering substances (such as metal ions, reactive oxygen species, biothiols, and amino acids), Cy5.5-H-CyN exhibited good specificity, meaning its fluorescence intensity did not change significantly due to the presence of these interfering substances. The probe has high sensitivity and specificity to pH, enabling rapid pH response and is unaffected by environmental polarity, viscosity, or interference from other substances in the biological system.

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

[0071] (5) The probe utilizes the inherent positive charge structure of the structure to combine with the electrostatic effect of the negative membrane potential of the mitochondria, so that the probe is 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 applied to monitor the changes in mitochondrial pH during autophagy, it can effectively resist the influence of changes in the microenvironment during autophagy (such as the decrease in mitochondrial membrane potential caused by mitochondrial alkalization and membrane depolarization). It has the characteristics of high sensitivity, reliability, visualization, speed and convenience, indicating that it has good application prospects in mitochondrial-related biological research and medical diagnosis.

[0072] (6) The detection method described is simple and efficient, compatible with commonly used fluorescence imaging equipment, and only requires a fluorescence spectrophotometer and a laser confocal microscope, which is convenient for widespread application.

[0073] (7) Biocompatibility and low toxicity: The probe of this invention is designed with biosafety in mind. After cytotoxicity testing, it has been shown to have little impact on cell growth and metabolic activities, making it suitable as a long-term monitoring tool. This is especially important for conducting in vivo studies and potential clinical translation.

[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 light exposure or repeated irradiation, ensuring the reliability and repeatability of data. Attached Figure Description

[0075] Figure 1 For the NMR characterization of the probe Cy5.5-H-CyN of this invention, 1 H-NMR spectrum;

[0076] Figure 2 For the NMR characterization of the probe Cy5.5-H-CyN of this invention, 13 C-NMR spectrum;

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

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

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

[0080] Figure 6 To compare the 0.01 mol·L⁻¹ molecule Cy5.5-H-Cy5 at different pH values ​​(4.0-9.0), -1 Absorption spectrum of PBS buffer;

[0081] Figure 7 To compare the 0.01 mol·L⁻¹ molecule Cy5.5-H-Cy5 at different pH values ​​(4.0-9.0), -1 Graph showing the color change of the solution in PBS buffer;

[0082] Figure 8 To compare the 0.01 mol·L⁻¹ molecule Cy5.5-H-Cy5 at different pH values ​​(4.0-9.0), -1 Fluorescence spectrum in PBS buffer;

[0083] Figure 9 The probe Cy5.5-H-CyN of this invention was tested at 0.01 mol·L⁻¹ at different pH values ​​(4.0-9.0). -1In PBS buffer, pH was used to fit an "S"-shaped curve with the excitation fluorescence intensity at 640 nm / excitation fluorescence intensity at 488 nm according to the Henderson-Hasselbalch equation;

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

[0085] Figure 11 The fluorescence spectra of the probe Cy5.5-H-CyN of this invention under acid-base cycling tests at pH=8.0 and pH=4.0 are shown.

[0086] Figure 12 The fluorescence spectrum of the probe Cy5.5-H-CyN of this invention selectively targets common biological interfering substances (including metal cations, anions, reactive oxygen species, biothiols, and amino acids) in PBS buffer at pH 7.4.

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

[0088] Figure 14 This is a subcellular organelle co-localization fluorescence imaging image of the probe Cy5.5-H-CyN of this invention co-incubated with HeLa cells for 30 min at pH 7.4 using a commercially available green mitochondrial-specific selective dye (MitoTrackerGreen FM).

[0089] Figure 15 The image shows the colocalization fluorescence imaging of subcellular organelles of the probe Cy5.5-H-CyN of this invention and commercially available green mitochondrial-specific selective dye (MitoTrackerGreen FM) co-stained HeLa cells at pH 7.4, before and after CCCP treatment to reduce the cell membrane potential.

[0090] Figure 16 HeLa cells were co-stained with the commercially available mitochondrial-specific selective dye rhodamine 123 and the commercially available green mitochondrial-specific selective dye (MitoTrackerGreenFM) at pH 7.4. The subcellular organelle colocalization fluorescence imaging images were obtained before and after CCCP treatment to reduce the cell membrane potential.

[0091] Figure 17The images show fluorescence images of the fluorescent probe Cy5.5-H-CyN of this invention, after being co-incubated with HeLa cells for 60 min at pH 8.0 to pH 4.0.

[0092] Figure 18 This is a linear fit plot of the ratio channel fluorescence intensity of the fluorescent probe Cy5.5-H-CyN of this invention in HeLa cells against pH (5.0-7.0).

[0093] Figure 19 The image shows fluorescence imaging of HeLa cells at different time points (0-180 min) under the action of FCCP (a mitochondrial uncoupling agent) using the probe Cy5.5-H-CyN of this invention.

[0094] Figure 20 The images show fluorescence images of HeLa cells at different times (0-12h) under different mitophagy induction conditions (rapamycin, starvation and hypoxia) for the probe Cy5.5-H-CyN of this invention. Detailed Implementation

[0095] The present invention will now be described in further detail.

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

[0097] In the preparation method described in this invention, the solvents are preferably dehydrated solvents.

[0098] The purification method described above in this invention employs conventional methods without particular limitations. Preferably, dichloromethane-methanol is used as the eluent for column chromatography, recrystallization, or a combination of both. Furthermore, the obtained intermediates and final products can be recovered using separation and purification techniques known in the art to achieve the required purity.

[0099] The raw materials used in the preparation methods described above in this invention can all be commercially available or prepared by methods known in the art.

[0100] The structures of the compounds synthesized in the above preparation methods of the present invention were confirmed by high-resolution mass spectrometry, proton nuclear magnetic resonance (NMR) spectroscopy, and carbon nuclear magnetic resonance (NMR) spectroscopy.

[0101] The instruments and equipment used in the embodiments are as follows:

[0102] Dye absorption and emission spectra were measured using an Agilent Cary 60 UV-Vis spectrophotometer and a CaryEclipse fluorescence spectrophotometer. Cell fluorescence images were obtained using an Olympus FV3000 single-photon confocal microscope.

[0103] Mito-Tracker Green FM, also known as MitoTracker Green, is a mitochondrial-targeting dye that is a green fluorescent probe for mitochondria and can be used for specific fluorescent staining of mitochondria in live cells. Mito-Tracker Green is a mitochondrial-tracker fluorescently labeled with carbocyanine from Molecular Probes, and its molecular formula is C0.05. 34 H 28 Cl5N3O, with a molecular weight of 671.88 and CAS number 201860-17-5, exhibits mitochondrial staining that is independent of mitochondrial membrane potential, unlike Rhodamine 123 or JC-1.

[0104] Example 1

[0105] I. Preparation and characterization of Cy5.5-H-CyN, a cyanin-based pH fluorescent probe 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 together to a 50 mL round-bottom flask, and 10 mL of Ac₂O was added. The reaction mixture was heated at 50 °C for 5 h. After the reaction was completed, the mixture was cooled to room temperature and added dropwise to a 500 mL beaker containing 150 mL of diethyl ether. The precipitated solid was collected and redissolved with DCM. The solvent was removed under reduced pressure, and the crude product was purified by column chromatography (DCM:MeOH = 50:1, v / v). A brownish-yellow band was collected to give brown solid 3, with a yield of 42% (448 mg). HRMS (ESI) 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 together to a 50 mL round-bottom flask, and 10 mL of EtOH was added. The reaction mixture was heated at 80 °C for 1 h. After the reaction was completed, 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] II. Synthesis of the comparative molecule Cy5.5-H-Cy5

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

[0111]

[0112] Compared to Cy5.5-H-CyN, Cy5.5-H-Cy5 replaces the azaindole portion with common indole. Because the indole nitrogen atom is occupied, Cy5.5-H-Cy5 does not have pH-responsiveness.

[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 using a 0.01 g / L balance. A 2 mmol / L stock solution was prepared using HPLC-grade DMSO, placed in a brown sample vial, and stored in a refrigerator at 4°C for later use.

[0116] For testing UV-Vis absorption and fluorescence spectra, 4.5 μL of the stock solution was pipetted and dissolved in quartz cuvettes containing 3 mL of PBS buffer at different pH values. The mixture was thoroughly mixed to obtain a molecular concentration of 3 μmol / L, which was used for absorption and fluorescence emission spectra testing. All tests were performed at 25°C. The instruments used were an AgIIlent 8453 UV spectrophotometer and an 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, while the absorbance at 526 nm gradually increased, corresponding to a change in solution color from blue to pink. Figure 4 The excitation wavelength was set to 640 nm, and the fluorescence intensity (Fl.λ) at 710 nm was set accordingly. ex640nm ) gradually decrease ( Figure 5a The excitation wavelength was set to 488 nm, and the fluorescence intensity (Fl.λ) at 662 nm was set accordingly. ex488nm ) gradually rise ( Figure 5b The contrast molecule Cy5.5-H-Cy5, lacking pH-responsiveness, exhibits varying absorption wavelengths in PBS buffers at different pH values. Figure 6 ), fluorescence intensity ( Figure 7 ) and solution color ( Figure 8 There were no significant changes in either pH or the corresponding Fl.λ. ex 640nm / Fl.λ ex 488nm The curve can be fitted using the Henderson-Hasselbalch equation, and the calculated pKa value of Cy5.5-H-CyN is approximately 6.46. Figure 9 The linear pH response range is 5.0–7.0. Figure 10 This characteristic perfectly matches the pH changes during mitochondrial autophagy, demonstrating the targeted and effective design. Both fluorescence peaks of the probe molecule are located in the near-infrared region, exhibiting good photophysical properties suitable for bioimaging applications.

[0117] Of particular note is the fluorescence peak of Cy5.5-H-CyN located in the near-infrared region (662 nm and 710 nm), which not only reduces autofluorescence interference from biological tissues but also improves imaging depth and signal-to-noise ratio, representing a significant advantage in bioimaging applications. This characteristic is crucial in practical applications because it allows for deeper tissue penetration and clearer signal identification, especially in in vivo imaging studies, where it far surpasses the capabilities of traditional probes.

[0118] also, Figures 4 to 10 The data not only demonstrates the probe's performance but also indirectly proves the non-obviousness of the invention's design. Detailed spectroscopic analysis of the pH response mechanism of Cy5.5-H-CyN reveals that its response characteristics are underpinned by sophisticated molecular structure design and a profound understanding of the biological environment. For example, Figure 4 The color change from blue to pink visually reflects the regulation of charge transfer effects within the molecule with pH changes. This is the result of careful design through the introduction of the azidoindole structure and molecular asymmetry, rather than an effect that could be predicted by simple structural adjustments. These data and images together provide strong evidence for the non-obviousness of this invention, further establishing its potential value and application prospects in biomedical research.

[0119] Example 3

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

[0121] The concentration of the probe molecule Cy5.5-H-CyN from Example 1 was maintained at 3 μM. The effects of this probe on H+ were investigated in the presence of common biological interfering agents (including metal cations, anions, reactive oxygen species, biothiols, and amino acids) or in mixed solvents of different viscosities. + The selectivity was observed. After four cycles of testing, the probe molecule Cy5.5-H-CyN exhibited rapid response between pH 4 and pH 8, with no significant change in fluorescence intensity. Figure 11 The probe molecule Cy5.5-H-CyN showed almost no response to common biological disruptors. Figure 12The 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=4 PBS buffer (all concentrations except PBS are 0.1 mM). In 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. Figure 13 This further emphasizes the stability and specificity of Cy5.5-H-CyN in complex biological media, providing a strong guarantee for its accurate pH monitoring.

[0122] Example 4

[0123] To investigate the mitochondrial targeting ability of the probe molecule Cy5.5-H-CyN, it was co-stained with HeLa cells and MitoTracker Green FM (a commercially available mitochondrial targeting dye). Adherent HeLa cells were incubated with Cy5.5-H-CyN (1 μmol / L) at pH 7.4 in a 37°C, 5% CO2 incubator for 60 min. Afterward, the cells were gently washed three times with PBS buffer (pH 7.4) to remove excess dye. Figure 14 As shown, the fluorescence distribution of Cy5.5-H-CyN overlapped well with that of MitoTracker Green FM (Pearson coefficient: 0.946), and both exhibited obvious mitochondrial filamentous structures, indicating that the probe molecule Cy5.5-H-CyN has excellent mitochondrial targeting ability. To further confirm that the probe can remain in mitochondria for a long time unaffected by mitochondrial membrane potential, the mitochondrial uncoupling agent carbonyl cyanidem-chlorophenylhydrazone (CCCP) was added to the cells to rapidly reduce the mitochondrial membrane potential. It was found that the fluorescence overlap between the probe and MitoTracker Green FM did not decrease significantly. Figure 15 The commercially available mitochondrial-specific selective dye rhodamine 123 showed almost complete disappearance of intracellular fluorescence intensity after CCCP. Figure 16 This indicates that even with a decrease in membrane potential, the probe Cy5.5-H-CyN remains firmly aggregated in the mitochondria. This result highlights the stability and membrane potential independence of Cy5.5-H-CyN, demonstrating that it can remain stably retained in the mitochondria even under extreme microenvironmental changes induced by autophagy, providing a reliable tool for the dynamic monitoring of the mitophagy process.

[0124] Example 5

[0125] HeLa cells were pretreated with DMEM containing Cy5.5-H-CyN (1 μM) at 37°C for 1 hour. Then, the culture medium was replaced with 0.01 mol / L PBS buffer at various pH values ​​(pH 4.0–8.0) containing 10 μM nigrain. After incubation for 30 min, the cells were washed three times with 0.01 mol / L PBS buffer at the corresponding pH. Images of the red (700-730 nm) and green (620-670 nm) channels were recorded using a 60x oil immersion lens under 640 nm excitation and 488 nm excitation, respectively. As the pH decreased from 8.0 to 4.0, the fluorescence signal of the red channel gradually decreased, while the fluorescence signal of the green channel gradually increased. The merged channel changed from red at pH 8.0 to orange, and finally to green at pH 4.0. The ratio fluorescence signal of the red / green channel also changed from red-green at pH 8.0 to blue-green, until the ratio fluorescence signal at pH 4.0 essentially disappeared. Figure 17 The ratio of red to green fluorescence signals showed a linear relationship with pH (5.0-7.0). Figure 18 This indicates 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 accurate visual feedback for dynamic monitoring of mitophagy.

[0126] Example 6

[0127] Mitophagy was induced in HeLa cells using various external stimuli, and monitored using Cy5.5-H-CyN. Before stimulation, HeLa cells were pretreated at 37°C with DMEM containing 1 μM Cy5.5-H-CyN. Different external stimuli were then applied: 50 μM FCCP (for 180 min co-incubation, images collected every 30 min); rapamycin (5 μM, for 12 h incubation, images collected every 6 h); FBS-free DMEM medium (for 12 h incubation, images collected every 6 h); and 1% O2 culture conditions (for 12 h incubation, images collected every 6 h). Images of the red (700-730 nm) channel were recorded using a 60x oil immersion lens under 640 nm excitation and the green (620-670 nm) channel under 488 nm excitation.

[0128] After incubation with FCCP for only 30 minutes, the fluorescence intensity of the red channel showed a significant decrease, and after 180 minutes of incubation, the fluorescence intensity of the red channel was already very low, while the green channel showed a clear fluorescence signal. Figure 19Based on the fluorescence intensity ratio of the two channels and the pH calibration curve, the pH of the mitochondria can be calculated to have decreased from approximately 6.10 at 30 min to approximately 5.21 at 180 min.

[0129] Mitochondrial autophagy was induced by the addition of 5 μM rapamycin. Compared with the control group, the fluorescence intensity of the red channel was significantly reduced after 6 h, and the green channel showed a clear fluorescence signal after 12 h. According to the pH calibration curve, the mitochondrial pH value of HeLa cells induced by rapamycin was approximately 6.02 after 6 h and approximately 5.59 after 12 h. Under both culture conditions, the fluorescence intensity of the red channel was significantly reduced after 6 h, and the green channel showed a clear fluorescence signal after 12 h. According to the pH calibration curve, the mitochondrial pH value of nutrient-deficient or hypoxic culture conditions was calculated to be 6.42 and 6.17 after 6 h, and 5.99 and 5.73 after 12 h, respectively. All the above experimental results indicate that Cy5.5-H-CyN can be used to quantitatively display mitochondrial pH, thereby enabling the monitoring of the mitochondrial autophagy process in HeLa cells. Figure 20 These data not only confirm the high accuracy and reliability of Cy5.5-H-CyN in tracking mitochondrial pH dynamics under multiple autophagy-induced modes, but also provide a powerful tool for a deeper understanding of autophagy mechanisms and their role in disease states.

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

[0131] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A novel class of asymmetric polymethine cyanine near-infrared fluorescent dyes, characterized in that: The dye has the structure of general formula I: In general formula I: R1 is selected from hydrogen; R2 or R3 is independently selected from hydrogen, alkyl of 1-8 carbons; R4 is a substituent at an indefinite position, selected from hydrogen; n is 2, 3 or 4; X - one selected from halide ions.

2. The dye of claim 1, wherein: The R2 or R3 is independently selected from hydrogen, methyl, ethyl.

3. The method for preparing the dye according to claim 1, characterized in that: The method comprises the following steps: Step 1: Y-1 and 3-methyl-2-butanone are added to an organic solvent I at 60-120℃, wherein the molar ratio of Y-1 and 3-methyl-2-butanone is 1:1-5; after stirring for 3-12h, the solvent is evaporated, and the residue is added to an acid solution, stirred at 80-150℃ for 0.5-4h, adjusted to alkaline with a base, extracted, concentrated, purified, and an intermediate Y-2 is obtained; Step 2: Y-2 is reacted with an N-alkylating agent in an organic solvent II at 40-100℃ for 3-24h, and an intermediate Y-3 is obtained by recrystallization, wherein the molar ratio of compound Y-2 and the N-alkylating agent is 1:1-10; Step 3: Y-3 is stirred with S-1 in an organic solvent III at 60-120℃ for 0.5-5h, and an intermediate S-2 is obtained by recrystallization; the molar ratio of Y-3 and S-1 is 1:1-5; Step 4: Y-4 is reacted with an N-alkylating agent in an organic solvent IV at 40-100℃ for 3-24h, and an intermediate Y-5 is obtained by recrystallization, wherein the molar ratio of compound Y-4 and 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℃, and a condensation reaction occurs under the catalysis of a base, and the dye having the structure of general formula I is obtained by recrystallization and silica gel column purification.

4. The method of claim 3, wherein: The organic solvent I is selected from one of toluene, ethanol, methanol, isopropanol, acetonitrile, tetrahydrofuran; The organic solvent II is selected from one of acetone, acetonitrile, methanol, toluene, o-dichlorobenzene, DMF; The organic solvent III is selected from one or a mixture of several of ethanol, acetic acid, acetic anhydride; The organic solvent IV is selected from one of acetone, acetonitrile, methanol, toluene, o-dichlorobenzene, DMF; The organic solvent V is selected from one of DMF, ethanol, isopropanol, acetic anhydride; The N-alkylating agent is selected from one of methyl halide, ethyl halide, propyl halide; In steps 1 or 5, the base is selected from one of sodium hydroxide, potassium hydroxide, ammonia, sodium bicarbonate; In step 1, the acid is selected from any one of acetic acid, hydrochloric acid, sulfuric acid, polyphosphoric acid; In steps 2, 3, 4, the recrystallization solvent is selected from any one or a combination of several of methanol, ethanol, acetonitrile, ethyl acetate, diethyl ether, acetone, propanol.

5. Use of the dye according to claim 1 for non-diagnostic and non-therapeutic purposes, characterized in that: The dye is used in the preparation of a pH fluorescent probe for monitoring mitochondrial autophagy.

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