Conjugated oligomeric electrolytes and methods of mitochondrial staining thereof

Abstract

The present disclosure relates to conjugated oligo electrolytes represented by Formula (I) and methods of using the same. The conjugated oligo electrolytes are suitable for staining mitochondria.

Description

Technical Field

[0001] This disclosure relates to conjugated oligoelectrolytes and methods of using them. Specifically, the conjugated oligoelectrolytes are suitable for staining mitochondria.

[0002] background Mitochondria play a crucial role in key biological processes. Therefore, mitochondrial dysregulation is associated with metabolic, neurological, and psychiatric diseases, including cancer, diabetes, and Alzheimer's disease. This correlation is reflected in the range of technologies developed to study different aspects of mitochondrial biology, which are essential for the diverse conditions required for the unique pathological states associated with mitochondrial damage. Long-term live-cell compatible probes and super-resolution dyes allow for the understanding of dynamic processes. Other types of functional biosensors are also used to monitor parameters such as voltage, cation concentration, temperature, polarity, and viscosity, and to image reactive small molecules such as reactive oxygen species and thiols. The negative mitochondrial membrane potential (MMP), essential for maintaining normal cellular homeostasis, forms the basis for the specific accumulation of cationic lipophilic fluorescent reporter molecules. For example, triphenylphosphonium conjugation is a common strategy for directing other nonspecific compounds to mitochondria. Small cationic dyes such as rhodamine 123 and TMRM are located within the mitochondrial matrix and are driven by MMP and intrinsic membrane permeability. Therefore, the correlation between dye accumulation and membrane potential can be used as an indicator of mitochondrial membrane health, with dye brightness reflecting the degree of depolarization. These examples demonstrate how crucial it is to develop chemical tools for probing mitochondrial function and dynamics for gaining a deeper understanding of intracellular biochemical processes.

[0003] Membrane-intercalated conjugated oligoelectrolytes (MICOEs) are a class of molecules characterized by an optically active conjugated core with ion-placing dangling groups. The lipid bilayer-like distribution of hydrophobic and hydrophilic domains facilitates spontaneous membrane intercalation and environment-specific fluorescence emission. The high biocompatibility and tunable physicochemical and photophysical properties of MICOEs have allowed for biological applications, including labeling or killing microorganisms, tracking extracellular vesicles, and cell imaging. Despite these applications, the molecular signatures influencing the interactions of COEs with mammalian cells, particularly subcellular organelles, are largely unknown. In the case of COE-S6 (a representative cationic oligo-phenylenevinylene (oligo-PV) COE), previous colocalization studies have demonstrated the absence of mitochondrial association, with its localization occurring only within lysosomes upon internalization. Figure 1 As shown, prolonged retention within the plasma membrane leads to endocytosis with other lipids, thereby locking the molecule into the endocytic pathway.

[0004] There is a lack of molecules that target mitochondria. There is an urgent need for molecules that are specific to mitochondria.

[0005] Therefore, it is hoped that at least one of the above problems can be overcome or improved.

[0006] Overview This disclosure relates to compounds of formula (I) or their salts or solvates: (I) in R1 is independently selected from H, optionally substituted alkyl groups, and optionally substituted alkoxy groups; R2 is independently selected from cyano, oxo, and optionally substituted alkyl groups; Ht is independently selected from optionally substituted heterocyclic groups and optionally substituted heteroaryl groups; and n is an integer independently selected from 2 to 5.

[0007] In some embodiments, the compound of formula (I) comprises a phenylene vinylene backbone, wherein the phenyl moiety is in a trans configuration around the alkenyl moiety.

[0008] In some embodiments, R1 is independently selected from optionally substituted C1-C5 alkyl or optionally substituted C1-C5 alkoxy.

[0009] In some implementations, R2 is a cyano group.

[0010] In some embodiments, Ht is independently selected from optionally substituted heterocyclic groups and optionally substituted heteroaryl groups, said heterocyclic and heteroaryl groups being selected from oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indazine, isoindole, indole, indazole, purine, quinazine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cyclophosphine, pteridine, carbazole, caroline, phenanthridine, acridine, phenanthridine, isoquinoline, isoquinoline, etc. Thiazoles, phenazines, isoxazoles, isothiazoles, phenazines, phenothiazines, imidazoline, imidazoline, piperidine, piperazine, dihydroindole, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazoles, thiadiazoles, oxadiazoles, oxtriazoles, tetrazolium, thiazoline, thiophene, benzo[b]thiophene, morpholino, piperidinyl, pyrrolidine, tetrahydrofuranyl, and triazoles.

[0011] In some embodiments, Ht is selected from optionally substituted N-heterocyclic groups and optionally substituted N-heteroaryl groups.

[0012] In some implementations, Ht is linked to the compound of formula (I) via its heteroatom.

[0013] In some embodiments, the optional substituents on Ht are selected from halogen, alkyl, alkenyl, alkoxy, alkenyloxy, alkynyloxy, acyl, oxyacyl, acyloxy, amino, aminoacyl, acylamino, aminoacyloxy, oxyacylamino, oxyacyloxy, thio, and phosphoryl.

[0014] In some embodiments, the compound of formula (I) or its salt or solvate is .

[0015] This disclosure also relates to compositions or kits comprising a compound of formula (I) configured for staining mitochondria in cells, or a salt or solvation thereof.

[0016] In some embodiments, the composition or kit further comprises a conjugated oligoelectrolyte configured for staining the lipid membrane of cells and / or a dye configured for staining another component of the cells.

[0017] This disclosure also relates to a method for staining mitochondria in cells, comprising contacting the cells with a compound of formula (I) or a salt or solvation thereof.

[0018] In some embodiments, the method further includes the step of incubating a compound of formula (I) or a salt or solvate thereof with cells.

[0019] In some embodiments, the method further includes the step of visualizing mitochondria by photoexciting the compound of formula (I) and detecting the emitted photons.

[0020] In some embodiments, the method further includes contacting the cells with a second dye configured to stain the lipid membrane of the cells.

[0021] In some embodiments, the second dye is a conjugated oligoelectrolyte configured to stain the lipid membrane.

[0022] This disclosure also relates to methods for distinguishing between damaged cells and healthy cells, including: a) Contacting the cells with the compound of formula (I) or a salt or solvate thereof, and a second dye; and b) Quantify the fluorescence emitted by the compound of formula (I) relative to the fluorescence emitted by the second dye to distinguish between damaged and healthy cells.

[0023] Brief description of the attached figures Embodiments of the invention will now be described by way of non-limiting examples with reference to the accompanying drawings, in which: Figure 1 A diagram illustrating the intracellular accumulation of COE-CN (mitochondria), COE-S6 (lysosomes), and COE-BO (lysosomes) and their chemical structures.

[0024] Figure 2 The synthetic route for COE-CN is shown.

[0025] Figure 3 shows the photophysical properties of COE-CN. (a) Normalized absorbance (solid line) and emission spectrum (dashed line) of 5 μM in PBS relative to the lipid bilayer. (b) Comparison of fluorescence intensity of COE-CN (5 μM) in PBS or SUV. Inset: Photograph of COE-CN solution illuminated with a UV lamp. (c) CLSM image of cells stained with COE-CN showing rapid internalization relative to COE-S6. (d) Cells co-stained with two different COEs, COE-CN and COE-BO, showing different spatial localization. Scale: 20 μm.

[0026] Figure 4 Images of HeLa cells stained with COE-CN (5 μM) and (a) Mito Red (mitochondria), (b) Syto Deep Red (nucleus), (c) CellMaskDeep Red (plasma membrane), or (d) LipidTOX Red (lipid droplets) are shown. Scale: 10 μm.

[0027] Figure 5 Photostableness is evaluated using repeated CLSM imaging of HeLa cells stained with COE-CN (5 μM), MitoTracker Green (MTG), MitoTracker Red (MTR), and MitoTracker Deep Red (MTDR) before and after 400 scans. Scale: 10 μm.

[0028] Figure 6 Cells stained with COE-CN are shown to exhibit distinct mitochondrial-lysosomal localization patterns imaged by CLSM in (a) untreated and (b) CCCP-treated cells (20 μM), allowing visualization of damaged mitochondria engulfed by lysosomes during mitophagy. Scale: 10 micrometers Figure 7 Flow cytometry analysis of depolarized mitochondria in cells based on COE-CN staining is shown. (a) Flow cytometry of HeLa cells stained with 1 μM COE-CN or (b) Mito-Tracker Red for 1 h, followed by mitochondrial membrane depolarization (30 min) using CCCP. Histograms of mean cellular fluorescence intensity after depolarization using (c) COE-CN or (d) Mito-Tracker Red at different CCCP treatment concentrations. (e) Changes in fluorescence intensity of cells stained with COE-BO and (f) COE-CN.

[0029] Figure 8The retention of COE-CN fluorescence intensity relative to other commercial mitochondrial dyes / cells is shown (mean ± sd, n = 10).

[0030] Figure 9 Cell viability (n = 3, mean ± sd) of mammalian cell lines HeLa (cervix), A549 (lung), and HepG2 (liver) stained with COE-CN is shown.

[0031] Detailed Explanation "Alkyl" refers to a monovalent alkyl group that can be straight-chain or branched and preferably has 1 to 10 carbon atoms, or more preferably 1 to 6 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-hexyl, etc.

[0032] "Alkenyl" refers to a monovalent alkenyl group that can be straight-chain or branched and preferably has 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and has at least one, preferably one to two, carbon-carbon double bonds. Examples include vinyl (-CH=CH2), n-propenyl (-CH2CH=CH2), isopropenyl (-C(CH3)=CH2), but-2-alkenyl (-CH2CH=CHCH3), etc.

[0033] "Alynyl" refers to an alkynyl group that preferably has 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and has at least one, preferably 1 to 2, carbon-to-carbon triple bonds. Examples of alkynyl groups include ethynyl (-C≡CH), propynyl (-CH2C≡CH), pentyl-2-alkynyl (-CH2C≡CCH2-CH3), etc.

[0034] "Alkoxy" refers to the alkyl-O- group, wherein the alkyl group is as described above. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, etc.

[0035] "Alkenyloxy group" refers to the alkenyl-O- group, as described above.

[0036] "Alkyne group" refers to the alkynyl group -O-, as described above.

[0037] "Halogen" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0038] “Acyl” refers to the groups HC(O)-, alkyl-C(O)-, cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)-, and heterocyclic-C(O)-, wherein alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as described herein.

[0039] “Oxyacyl” refers to the groups HOC(O)-, alkyl-OC(O)-, cycloalkyl-OC(O)-, aryl-OC(O)-, heteroaryl-OC(O)-, and heterocyclic-OC(O)-, wherein alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as described herein.

[0040] "Amino" refers to the group -NR"R", wherein each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic, and wherein each of the alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is as described herein.

[0041] "Aminoacyl" refers to the group -C(O)NR"R", wherein each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic, and each of the alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is as described herein.

[0042] "Acylamino" refers to the group -NR"C(O)R", wherein each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic, and wherein each of the alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic groups is as described herein.

[0043] “Acyloxy” refers to the groups -OC(O)-alkyl, -OC(O)-aryl, -C(O)O-heteroaryl, and -C(O)O-heterocyclic, wherein alkyl, aryl, heteroaryl, and heterocyclic are as described herein.

[0044] "Aminoacyloxy" refers to the groups -OC(O)NR''-alkyl, -OC(O)NR"-aryl, -OC(O)NR''-heteroaryl and -OC(O)NR"-heterocyclic, wherein R'' is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic, and wherein the alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic are each as described herein.

[0045] "Cyano" refers to the -CN group.

[0046] “Oxylammonium” refers to the groups -NR”C(O)O-alkyl, -NR”C(O)O-aryl, -NR''C(O)O-heteroaryl and NR''C(O)O-heterocyclic, wherein R'' is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic, and wherein the alkyl, cycloalkyl, aryl, heteroaryl and heterocyclic are each as described herein.

[0047] “Oxykoxy” refers to the groups -OC(O)O-alkyl, -OC(O)O-aryl, -OC(O)O-heteroaryl, and -OC(O)O-heterocyclic, wherein the alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic groups are as described herein.

[0048] "Thio" refers to the groups HS-, alkyl-S-, cycloalkyl-S-, aryl-S-, heteroaryl-S-, and heterocyclic-S-, where alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclic are as described herein.

[0049] "Phosphoyl" refers to the group -P(O)(R”')(OR”'), where R”' represents OR”' or is hydroxyl, alkyl or amino, and R”' is alkyl, cycloalkyl, aryl or aralkyl, wherein alkyl, amino, alkenyl, aryl, cycloalkyl and aralkyl are as described herein.

[0050] "Aryl" refers to an unsaturated aromatic carbocyclic group having a monocyclic (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracene), preferably having 6 to 14 carbon atoms. Examples of aryl groups include phenyl, naphthyl, etc.

[0051] "Heteroaryl" refers to a monovalent aromatic heterocyclic group that satisfies Hückel's aromaticity rule (i.e., contains 4n + 2 π electrons) and preferably has 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen, selenium, and sulfur (and includes oxides of sulfur, selenium, and nitrogen). Such heteroaryl groups can have a single ring (e.g., pyridyl, pyrroleyl, or their N-oxide or furanyl) or multiple fused rings (e.g., indazinyl, benzimidazolyl, coumarinyl, quinolinyl, isoquinolinyl, or benzothiopheneyl).

[0052] Examples of heteroaryl compounds include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indazine, isoindole, indole, indazole, purine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cyclophosphine, pteridine, carbazole, caroline, phenanthridine, acridine, phenanthroxazole, isoxazole, phenanthridine, phenothiazine, phenothiazine, thiazole, thiadiazole, oxadiazole, oxtriazole, tetrazolium, thiophene, benzo[b]thiophene, triazole, imidazopyridine, etc.

[0053] "Aneryl" refers to a divalent aryl group, where the aryl group is as described above.

[0054] "Heteroarylene" refers to a divalent heteroarylene, wherein the aryl group is as described above.

[0055] "Heterocyclic group" refers to a monovalent saturated or unsaturated group having a single ring or multiple fused rings, preferably having 1 to 8 carbon atoms and 1 to 4 heteroatoms selected from nitrogen, sulfur, oxygen, selenium, or phosphorus within the ring. The most preferred heteroatom is nitrogen. It should be understood that when, for example, R2 or R' is an optionally substituted heterocyclic group having one or more ring heteroatoms, the heterocyclic group can be linked to the core molecule of the compound of the present invention via C-CC or C-heteroatom bonds, particularly CN bonds.

[0056] Examples of heterocyclic and heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indazine, isoindole, indole, indazole, purine, quinazine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cyclophosphine, pteridine, carbazole, caroline, phenanthridine, acridine, phenanthroxaline, isothiazine, phenazine, isoxazole, isothiazine, phenoxazine, phenthiazine, imidazoline, imidazoline, piperidine, piperazine, dihydroindole, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiadiazole, oxadiazole, oxtriazole, tetrazolium, thiazoline, thiophene, benzo[b]thiophene, morpholino, piperidinyl, pyrrolidine, tetrahydrofuranyl, triazole, etc.

[0057] "Optionally substituted" means that the group may or may not be further substituted or fused (thus forming a fused polycyclic group) by one or more groups selected from the following: hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenoxy, alkynyl, alkynoxy, amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, carboxyl, amide, cyano, halogen, nitro, phosphonyl, sulfonyl, phosphorylamino, oxophosphonyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclic, heterocyclic alkyl, heterocyclic, oxime, oxime ether, hydrazone, oxoamide Aminosulfonylamino, aminoacyloxy, trihalomethyl, trialkylsilyl, pentafluoroethyl, trifluoromethoxy, difluoromethoxy, trifluoromethylthio, trifluorovinyl, mono- and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclic amino, and asymmetric disubstituted amines with different substituents selected from alkyl, aryl, heteroaryl, and heterocyclic groups, and may also include bonds with a solid carrier material (e.g., substitution onto a polymer resin). For example, "optionally substituted amino" groups may include amino acid and peptide residues.

[0058] "Hydrophilic" refers to a molecule or part that has a greater affinity for water than an organic solvent and therefore a greater solubility in water. For example, the hydrophilicity of a compound can be quantified by measuring its partition coefficient between water (or a buffered aqueous solution) and a water-immiscible organic solvent such as octanol, ethyl acetate, dichloromethane, or methyl tert-butyl ether. A compound is considered hydrophilic if it is present in water at a higher concentration than in the organic solvent after equilibrium.

[0059] "Hydrophobic" refers to a molecule or part of an organic solvent that has a greater affinity for it than water and therefore a greater solubility in that solvent. For example, the hydrophobicity of a compound can be quantified by measuring its partition coefficient between water (or a buffered aqueous solution) and a water-immiscible organic solvent such as octanol, ethyl acetate, dichloromethane, or methyl tert-butyl ether. A compound is considered hydrophobic if, after equilibrium, it is present in a higher concentration in the organic solvent than in water.

[0060] Conjugated oligoelectrolytes (COEs) are a class of molecules defined by a hydrophobic conjugated core with terminal polar ionic side groups. The hydrophobic and hydrophilic moieties of COEs can be rationally designed to reflect the organization of hydrophilic and hydrophobic regions in the lipid bilayer. This structural design typically involves only unbranched internal structures with charged groups at both ends, facilitating the spontaneous embedding of COEs into the cell membrane driven by electrostatic and hydrophobic interactions between the COE and lipids.

[0061] Characteristically, the fluorescence emission of COEs is significantly enhanced when they are embedded in the lipid bilayer from aqueous solution. This "lighting-up" mechanism, where COEs are located in the more hydrophobic environment of the lipid bilayer, endows them with a high signal-to-noise ratio. Furthermore, COEs possess a chemical structure distinct from many commercially available lipophilic dyes, which typically contain surfactant-like structures—one side of the molecule is hydrophobic, while the other is hydrophilic. These surfactant-like structures induce micellar-like aggregation in aqueous solutions. For example, the commonly used membrane dye PKH-26 has been shown to form aggregates with similar size and fluorescence intensity to small particles such as exosomes, leading to false-positive signals. These phenomena can be avoided in the case of COEs, provided their emission is significantly enhanced upon embedding in the lipid bilayer; i.e., a high signal-to-noise ratio.

[0062] Unwilling to be bound by theory, the inventors hypothesize that modulating the hydrophobic-hydrophilic distribution within the molecular scaffold will allow for the design of relevant chemical structures with reduced affinity for cell membrane binding, while retaining the ability to integrate with the cell. For this purpose, and relatively speaking, shorter cationic COEs will bind less tightly to the plasma membrane due to less pronounced hydrophobic effects, thus allowing for easier dissociation. However, sufficient hydrophobicity is still required to drive membrane intercalation and cellular uptake. Accumulation within intracellular structures with higher transmembrane potentials will subsequently be expected to have a more significant effect through electrostatic interactions. To obtain satisfactory photophysical properties with a succinyl styrene framework, the inventors investigated the introduction of chromophores with minimal changes in molecular size. For example, oligophenylenevinylene can be substituted with cyano groups to impart enhanced charge separation relative to the polymer-PV, thereby causing the compound to luminesce.

[0063] Based on these design criteria, the inventors describe compounds herein (e.g., COE-CN as an embodiment) characterized by a methoxy-substituted cyanovinyl backbone having suitable physicochemical properties for intracellular labeling. Such compounds have been found to be located exclusively within mitochondria, even under different staining conditions, and have been successfully used to image subcellular changes caused by stress resulting from induced depolarization. Furthermore, flow cytometry analysis of stained cell populations demonstrated the ability to distinguish between cell populations with healthy and damaged mitochondria when used together with other COE compounds such as COE-BO as quantitative standards.

[0064] Therefore, this disclosure relates to compounds of formula (I) or their salts or solvates: (I) in R1 is independently selected from H, optionally substituted alkyl groups, and optionally substituted alkoxy groups; R2 is independently selected from cyano, oxo, and optionally substituted alkyl groups; Ht is independently selected from optionally substituted heterocyclic groups and optionally substituted heteroaryl groups; and n is an integer independently selected from 2 to 5.

[0065] The compound of formula (I) has a substantially linear topological structure. Topological structure refers to the molecular structure of a compound within the constraints of three-dimensional (3D) space. This linear topology has two nodes as ends and no connecting nodes. Linear topology facilitates mitochondrial embedding.

[0066] The compounds of formula (I) are linear to accommodate their position within the lipid bilayer. At this point, the phenyl moiety is in a trans (or E) configuration around the alkenyl moiety.

[0067] In some embodiments, the compound is not branched; that is, the monomeric unit extends only along a single chain. However, this does not preclude the optional substitution of the monomeric unit. In some embodiments, the compound of formula (I) is inherently symmetrical. The symmetry of the compound can be described by at least one of 32 point groups. Point groups describe all symmetry operations that can be performed on the molecule, resulting in conformations indistinguishable from the original conformation. In this regard, in some embodiments, the compound of formula (I) has C1... 2v Group chat.

[0068] In some embodiments, R1 is independently selected from optionally substituted alkyl or optionally substituted alkoxy. In some embodiments, R1 is independently selected from optionally substituted C1-C5 alkyl or optionally substituted C1-C5 alkoxy. In some embodiments, R1 is independently selected from optionally substituted C1-C3 alkyl or optionally substituted C1-C3 alkoxy. In some embodiments, R1 is optionally substituted C1-C3 alkoxy. In some embodiments, R1 is optionally substituted methoxy.

[0069] In some embodiments, the optional substituents on R1 are selected from halogen, alkyl, alkenyl, alkoxy, alkenyloxy, amino, cyano, or nitro.

[0070] Preferably, R1 is an electron donor.

[0071] In some embodiments, R2 is independently selected from cyano, oxo, and optionally substituted C1-C5 alkyl groups. In some embodiments, R2 is independently selected from cyano and oxo. In some embodiments, R2 is cyano.

[0072] Preferably, R2 is an electron acceptor.

[0073] In some implementations, Ht is linked to the compound of formula (I) via its heteroatom.

[0074] In some embodiments, Ht is independently selected from optionally substituted heterocyclic groups and optionally substituted heteroaryl groups, said heterocyclic and heteroaryl groups being selected from oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indazine, isoindole, indole, indazole, purine, quinazine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cyclophosphine, pteridine, carbazole, caroline, phenanthridine, acridine, phenanthridine, isoquinoline, isoquinoline, etc. Thiazoles, phenazines, isoxazoles, isothiazoles, phenazines, phenothiazines, imidazolines, imidazolines, piperidines, piperazines, dihydroindole, phthalimides, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazoles, thiadiazoles, oxadiazoles, oxtriazoles, tetrazolium, thiazoline, thiophene, benzo[b]thiophene, morpholino, piperidinyl, pyrrolidine, tetrahydrofuranyl, and triazoles. In some embodiments, Ht is selected from optionally substituted N-heterocyclic groups and optionally substituted N-heteroaryl groups.

[0075] In some embodiments, Ht is independently an optionally substituted heterocyclic group. The heterocyclic group may be selected from indazoles, quinazines, phenazines, phenothiazines, imidazolines, imidazolines, piperidines, piperazines, dihydroindole, phthalimides, 1,2,3,4-tetrahydroisoquinoline, thiazoles, thiadiazoles, thiazoline, thiophene, morpholine, pyrrolidine, tetrahydrofuran, and triazoles. In some embodiments, Ht is an optionally substituted N-heterocyclic group.

[0076] Optional substituents may be substituted once, twice, or three times on the group. In some embodiments, the optional substituents on Ht are selected from halogens, alkyl, alkenyl, alkoxy, alkenyloxy, alkynyl, acyl, oxyacyl, acyloxy, amino, aminoacyl, amide, aminoacyloxy, oxyamide, oxyacyloxy, thio, and phosphoryl. In some embodiments, the optional substituents on Ht are selected from halogens and C1-C5 alkyl groups.

[0077] Ht can be a hydrophilic group. In other embodiments, Ht is a charged group, or can be configured to be charged. In this regard, optional substituents can be charged groups. Examples of hydrophilic and / or charged groups are trialkylammonium halides. For example, Ht can contain a charged group, which can be trimethylammonium iodide. This imparts a positive charge to the compound of formula (I). Other cationicly charged groups include, but are not limited to, pyridinium, pyrrolidineium, imidazolium, guanidineium, sulfonium, thioureaium, and phosphonium. Other anionicly charged groups include, but are not limited to, chlorates, sulfates, phosphates, acetates, carboxyl groups, and hydroxides. The hydrophilic and / or charged group can also be in zwitterionic form, containing both cationic and anionic charged groups via covalent bonds. Excess charge can be neutralized by an acceptable cation or anion.

[0078] Basic nitrogen-containing groups can be quaternized using reagents such as lower alkyl halides; dialkyl sulfates and other reagents, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates such as dimethyl sulfate and diethyl sulfate. Examples of optional substituents on Ht can be selected from: .

[0079] In some implementations, n is an integer independently selected from 1 to 5. In some implementations, n is an integer selected from 2 to 5. In some implementations, n is an integer selected from 3 to 5. In some implementations, n is 4.

[0080] In order for the compound of formula (I) to have a balance of hydrophilic and hydrophobic properties to facilitate its insertion into mitochondria, at least one side chain should be present at each end of the main chain.

[0081] In some embodiments, the compound of formula (I) is the compound of formula (Ia): (Ia).

[0082] In some embodiments, the compound of formula (I) or its salt or solvate is

[0083] In some embodiments, the compound of formula (I) is its salt. The salt form can be a protonated salt, or it can be produced by alkylating the compound of formula (I) with a haloalkanes. For example, alkyl halides (e.g., CH3Br or CH3I) can be used. In some embodiments, the compound of formula (I) or its salt or solvate is a quaternary ammonium salt. In this respect, Ht can be an N-heterocyclic group, wherein the N-heteroatom is configured to provide at least a positive charge. For example, Ht can comprise a quaternary ammonium salt.

[0084] In some embodiments, the salt of the compound of formula (I) is ; Where X- is a halide.

[0085] In some embodiments, the compound of formula (I) is characterized by an extinction coefficient of about 1 × 10⁻⁶. -4 M -1 cm -1 To approximately 2 × 10 -4 M -1 cm -1 .

[0086] In some embodiments, the compound of formula (I) is characterized by a quantum yield (ΦF) of about 30% to about 50% when it is embedded in a lipid membrane.

[0087] In some embodiments, the compound of formula (I) is characterized by a lipid bilayer partition coefficient (Ksuv) of approximately 2 × 10⁻⁶. -6 From approximately 4 × 10 -6 .

[0088] The compounds of the present invention may be in crystalline form as free compounds or as solvates (e.g., hydrates), and both forms are intended to be within the scope of the present invention. Methods of solvation are generally known in the art.

[0089] The compounds of the present invention can be provided in solid or solution form. For example, the compounds can be provided as lyophilized powders.

[0090] The compounds disclosed herein can be provided as compositions. The composition may also contain another conjugated oligoelectrolyte configured to stain a lipid membrane. Therefore, when used in combination with compounds of formula (I), a ratio standard is provided that can distinguish cell populations with healthy and damaged mitochondria. The conjugated oligoelectrolyte configured to stain a lipid membrane may be COE-S6, COE-BO, etc. Further conjugated oligoelectrolytes are disclosed in US2009230362 and WO2023022658, which are incorporated herein by reference.

[0091] The composition may also contain another dye for staining individual components of the cell. The dye can be used to stain the cell membrane, nucleus, DNA, RNA, or other organelles within the cell. The dye can be a fluorescent probe, such as FM4-64, FM2-10, FM1-43, propidium iodide, SYTO 82, SYTO 83, SYTO 84, SYTO 85, YOYO®-3 iodide, or YO-PRO. TM -3 iodide, BOBO TM -3 iodide, ethidium homodimer-1, ethidium homodimer-2, ethidium monoazide, acridine orange, CellMask TM Plasma membrane staining agent or 2-4-ANEPPS.

[0092] This composition can be contained as a single entity of a compound in a polar medium. As used herein, a “polar medium” includes polar protons and polar aprotic solvents. Polar solvents have large dipole moments or partial charges and contain bonds between atoms with very different electronegativity, such as oxygen and hydrogen. Proton solvents have OH or NH bonds. Such bonds allow participation in hydrogen bonding. Furthermore, these OH or NH bonds can act as protons (H+). + The source of the solvent is not specified. Aprotic solvents may have hydrogen atoms somewhere on them, but they do not have OH or NH bonds and therefore cannot form hydrogen bonds with themselves. Polar solvents include, but are not limited to, dichloromethane, tetrahydrofuran, ethyl acetate, acetone, N,N-dimethylformamide, acetonitrile, dimethyl sulfoxide, ammonia, butanol, propanol, ethanol, methanol, acetic acid, and water. This definition also includes solvent mixtures in which the main component is a polar solvent. For example, aqueous solvents or solvent systems can also include dissolved ions, salts, and molecules such as amino acids, proteins, sugars, and phospholipids. Such salts can be, but are not limited to, sodium chloride, potassium chloride, ammonium acetate, magnesium acetate, magnesium chloride, magnesium sulfate, potassium acetate, potassium chloride, sodium acetate, sodium citrate, zinc chloride, sodium HEPES, calcium chloride, ferric nitrate, sodium bicarbonate, potassium phosphate, and sodium phosphate. Therefore, biological fluids, physiological solutions, and culture media are also within the scope of this definition.

[0093] This disclosure also relates to a kit comprising a compound and a medium of formula (I). The medium may be a polar medium. The compound and the polar medium can be mixed before use in separate containers or as separately packaged components. Alternatively, the kit can comprise a component of the compound in a first polar medium and a separate second medium, both components being contained in separate containers. The kit can additionally comprise another dye as a separate component for staining cells. For example, the kit can additionally include a conjugated oligoelectrolyte, nuclear dye, or vacuolar membrane dye, such as FM 4-64, configured to stain lipid membranes. The kit can additionally comprise an excipient. The excipient can be used to further stabilize the compound and / or reduce background noise by further quenching the fluorescence of the compound before it penetrates the bacterial cell membrane.

[0094] The kit may also include instructions for applying the compound of formula (I) to a sample.

[0095] This disclosure also relates to a method for staining mitochondria in cells, including contacting the cells with a compound of formula (I) or a salt or solvate thereof.

[0096] In some embodiments, the method further includes the step of incubating the cell with a compound of formula (I) or a salt or solvation thereof. In other embodiments, the incubation step is performed for at least about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes.

[0097] In some embodiments, the method further includes the step of visualizing mitochondria by photoexciting a compound of formula (I) and detecting the emitted photons. Since a compound of formula (I) or its salts or solvates emit fluorescence when intercalated with mitochondria, fluorescence-based techniques can be used. Examples of fluorescence-based techniques include, but are not limited to, fluorescence microscopy, confocal microscopy, plate readers, fluorometers, fluorescence spectroscopy, and flow cytometry (e.g., fluorescence-activated cell sorting).

[0098] When combined with a compound of formula (I) or its salt or solvate, cells can be excited by electromagnetic radiation (source) in the wavelength range of about 300 nm to about 1000 nm.

[0099] In some embodiments, the compound of formula (I) can be in contact with cells for an extended period of time without substantially reducing cell viability. This period can be from about 1 day to about 30 days, from about 5 days to about 30 days, from about 10 days to about 30 days, from about 15 days to about 30 days, or from about 20 days to about 30 days.

[0100] The compound of formula (I), or its salts or solvates, can be combined with other dyes such as membrane dyes. For example, the commercially available dye FM4-64 can be added to identify bacterial envelope types in situ in a bacterial mixture. In this regard, dual-dye systems capable of identifying multi-bacterial samples are also disclosed. These methods are easy to use, requiring only the simple application of a dye mixture that does not require fixation or other pretreatment, and the compound of formula (I), or its salts or solvates, is stable in aqueous solutions and can be used to monitor cells in a living system.

[0101] In some embodiments, the method further includes contacting the cells with a dye configured to stain the lipid membrane of the cells. The dye may be a conjugated oligoelectrolyte configured to stain the lipid membrane. The lipid membrane may be a lipid bilayer. For example, COE-S6 or COE-BO may be used. Other dyes may also be used to stain cell membranes, cell nuclei, DNA, RNA, or other organelles within the cell. The dye may be a fluorescent probe, such as FM4-64, FM2-10, FM1-43, propidium iodide, SYTO 82, SYTO 83, SYTO 84, SYTO 85, YOYO®-3 iodide, or YO-PRO. TM -3 iodide, BOBO TM -3 iodide, ethidium homodimer-1, ethidium homodimer-2, ethidium monoazide, acridine orange, CellMask TM Plasma membrane staining agent or 2-4-ANEPPS.

[0102] These compounds can be used for staining bacterial and mammalian cells (including, but not limited to, A549 cancer cells and erythrocytes). In some embodiments, the bacterial cells are Gram-positive or Gram-negative bacterial cells. In other embodiments, the Gram-negative or Gram-positive bacterial cells are selected from *Escherichia coli* (E. coli). E. coli ), Pseudomonas aeruginosa ( P. aeruginosa Staphylococcus aureus S. aureus ), Enterococcus faecalis ( E. faecalis ), Shewanella olivaceus ( S. oneidensis ), Bacillus megaterium ( B. megaterium (or a combination thereof.) Mammalian cells can be derived from cell lines or from samples taken from individuals.

[0103] In some embodiments, the compound of formula (I) or its salt or solvate is provided at a concentration of about 1 nM to about 100 μM. In other embodiments, the concentration is about 1 nM to about 90 μM, about 1 nM to about 80 μM, about 1 nM to about 70 μM, about 1 nM to about 60 μM, about 1 nM to about 50 μM, about 1 nM to about 40 μM, about 1 nM to about 30 μM, about 1 nM to about 20 μM, about 1 nM to about 10 μM, about 1 nM to about 5 μM, about 1 nM to about 1 μM, about 1 nM to about 900 nM, about 1 nM to about 800 nM, about 1 nM to about 700 nM, about 1 nM to about 600 nM, about 1 nM to about 500 nM, about 1 nM to about 400 nM, about 1 nM to about 300 nM, about 1 nM to about 200 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, or about 1 nM to about 20 nM.

[0104] In some embodiments, the compound of formula (I) or its salt or solvate is incubated with cells at about 5 °C to about 50 °C. In other embodiments, the temperature is about 5 °C to about 45 °C, about 5 °C to about 40 °C, about 5 °C to about 35 °C, about 10 °C to about 35 °C, about 15 °C to about 35 °C, or about 15 °C to about 30 °C. In other embodiments, the temperature is room temperature or ambient temperature.

[0105] In some embodiments, when a compound of formula (I) or a salt or solvate thereof is inserted into mitochondria, the compound of formula (I) or a salt or solvate thereof has an emission intensity greater than about 2 to about 500 times that of a control sample of the compound of formula (I) or a salt or solvate thereof. In other embodiments, the emission intensity is greater than about 10 to about 500 times, about 20 to about 500 times, about 30 to about 500 times, about 40 to about 500 times, about 50 to about 500 times, about 60 to about 500 times, about 70 to about 500 times, about 80 to about 500 times, about 90 to about 500 times, about 100 to about 500 times, about 100 to about 450 times, about 150 to about 450 times, about 200 to about 450 times, about 250 to about 450 times, about 300 to about 450 times, or about 350 to about 450 times.

[0106] Advantageously, compounds of formula (I), or their salts or solvates, exhibit low (or negligible) photoluminescence when dissolved in an aqueous medium. However, when partitioned into a lipid bilayer, the photoluminescence of compounds of formula (I), or their salts or solvates, is enhanced. It is believed that the change in the local environment to a hydrophobic environment (the alkyl chains of the lipid bilayer) allows for this enhanced fluorescence. Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Typically, the emitted light has a longer wavelength than the absorbed light.

[0107] Because the compounds of formula (I) or their salts or solvates are advantageously non-toxic (or have low toxicity) and / or stable to excitation, the photoluminescence intensity can be maintained for a period of time under constant excitation. At this point, when the compound is excited at an appropriate wavelength for imaging, the photoluminescence intensity does not decrease over a period of time. This is thought to be due to the conjugated system, which allows for energy dissipation and transfer, thereby preventing localized heating and degradation of the compound. In some embodiments, the photoluminescence intensity can be maintained for at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 1.5 hours, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 10 hours, or at least 24 hours.

[0108] This disclosure also relates to methods for distinguishing between damaged cells and healthy cells, including: a) Contacting the cell with the compound of formula (I) or its salt or solvate, along with the second dye; and b) Quantify the fluorescence emitted by the compound of formula (I) relative to the fluorescence emitted by the second dye to distinguish between damaged and healthy cells.

[0109] Example Synthesis and Characterization .exist Figure 2 The synthetic route for COE-CN is provided below. A Knoevenagel condensation between alkylbenzyl cyanide 2 and dimethoxytetraphenyl phthalaldehyde provides the conjugated backbone. Subsequent quaternization with N,N-dimethylimidazolium yields COE-CN in 76% yield. The aqueous solubility of COE-CN was determined to be >2 mM.

[0110] 2-(4-(4-bromobutoxy)phenyl)acetonitrile (2) In a 250 mL round-bottom flask equipped with a magnetic stir bar, 2-(4-hydroxyphenyl)acetonitrile (1) (3.08 g, 23 mmol), K₂CO₃ (16 g, 116 mmol), and 1,4-dibromobutane (15 g, 69 mmol) were dissolved in acetone. The reaction mixture was then heated to reflux and stirred for 48 hours. After cooling, excess solvent was removed under vacuum, and the reaction mixture was extracted with DCM (2 × 50 mL). The organic phase was washed with water (2 × 100 mL) and brine (100 mL), dried over sodium sulfate, and the solvent was removed by rotary evaporation under vacuum. The crude product was purified by silica gel column chromatography (EA / hexane 1:5) and dried under high vacuum overnight to give a product as a colorless solid (4.08 g, 67% yield). ¹H NMR (400 MHz, chloroform) d ) δ 7.22 (d,2H), 6.88 (d, J = 8.7Hz, 2H), 3.99 (s, 2H), 3.68 (d, J = 0.8Hz, 2H), 3.49 (s,2H), 2.11-2.02 (m, 2H), 1.95 (m, J = 9.0, 5.5Hz, 2H).

[0111] ( 2Z,2'Z )-3,3'-(2,5-dimethoxy-1,4-phenylene)bis(2-(4-(4-bromobutoxy)phenyl)acrylonitrile)(4) Compound 2 (1 g, 3.7 mmol) and compound 3 (0.33 g, 1.7 mmol) were weighed into a round-bottom flask under argon atmosphere and dissolved in anhydrous MeOH (5 mL). The mixture was then cooled to 0 °C in an ice bath. Potassium hydroxide (0.21 g, 3.7 mmol) was dissolved in anhydrous MeOH (1 mL) and added dropwise to the reaction mixture, which rapidly turned bright yellow-orange. The reaction mixture was stirred at room temperature for 16 hours, followed by quenching with 1 M HCl. The reaction mixture was then extracted with DCM (50 mL), and the organic phase was dried over sodium sulfate to give a bright orange solution. The solvent was removed by rotary evaporation under vacuum, and the crude product was purified by silica gel column chromatography (DCM / hexane 1:1) and recrystallization (DCM / MeOH) to give compound 4 (0.55 g, 50% yield) as a bright orange solid. ¹H NMR (400 MHz, chloroform) d ) δ 7.88 (d, J = 4.1Hz, 4H), 7.64 (d, J =8.9Hz, 4H), 6.96 (d, J= 8.9Hz, 4H), 4.06 (t, J = 6.0Hz, 4H), 3.95 (s, 6H), 3.51 (t, J = 6.5Hz, 4H), 2.12-2.06 (m, 4H), 1.99 (d, J = 8.6Hz, 4H). 13C NMR (126MHz, chloroform-d) δ 159.80, 133.85, 130.43, 127.52, 127.20, 118.54, 114.91, 111.49, 110.18, 67.06, 56.35, 33.33, 29.39, 27.81.

[0112] In a round-bottom flask equipped with a magnetic stir bar, compound 4 (50 mg) and 1,2-dimethyl-1H-imidazole (500 mg) were added under an argon atmosphere and dissolved in CHCl3 (5 mL). The reaction was stirred for 24 hours, and an oily orange precipitate was observed. The solvent was then removed under vacuum, and the residue was redissolved in a minimal amount of CHCl3 / MeOH. The reaction was continued for 24 hours. The solvent was removed under vacuum, and the residue was thoroughly washed with CHCl3 and diethyl ether. After drying under high vacuum, CoE-CN (76% yield) was obtained as a bright orange solid. HRMS C44H50BrN6O4 (M-Br)+: Calculated value 805.3071, measured value 805.3069. ¹H NMR (400MHz, DMSO-d6) δ 7.92 (s, ¹H), 7.73 (s, ¹H), 7.71–7.66 (m, ³H), 7.64 (d, J = 2.1Hz, 1H), 7.08 (d, J = 9.0Hz, 2H), 4.21 (s, 2H), 4.08 (s, 2H), 3.89(s, 3H), 3.75 (s, 3H), 2.61 (s, 3H), 1.89 (d, J = 7.4Hz, 2H), 1.77 (d, J=7.3Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.03, 151.65, 144.83, 134.98,127.83, 126.52, 125.64, 122.84, 121.36, 118.42, 115.71, 112.44, 111.41,67.66, 56.71, 47.69, 35.20, 26.49, 25.78, 9.77.

[0113] Density functional theory (DFT) calculations revealed a total molecular length of 3.6 nm for COE-CN, which also indicates a relatively flat core. We infer that the reduced length is related to the 55 Å core of COE-S6. 2 In comparison, the increased COE-CN has a 10 Å 2 The total polar surface area (TPSA) will weaken the hydrophobic interactions that retain COE-CN within the membrane. These properties are expected to reduce the tendency of the plasma membrane to stain.

[0114] Photophysical properties and lipid bilayer selectivity Figure 3a shows the relevant optical properties of COE-CN in phosphate-buffered saline (PBS), when embedded in a lipid bilayer of monolayer vesicles (SUVs) composed of 1-palmitoyl-2-oleoyl-glycerol-3-phosphocholine (POPC). In PBS, COE-CN exhibits maximum absorption (λ) at 392 nm. abs When inside an SUV, it shifts to 435 nm. The emission spectrum of COE-CN in PBS is characterized by emission centered at 525 nm, which undergoes a blue shift due to interaction within the lipid bilayer. The resulting emission spectrum is similar to that observed in methanol or DMSO. These observations are consistent with the expected change in molecular environment, reflecting the transition from a more polar aqueous medium to a less polar lipid bilayer. Membrane intercalation also leads to an increase in fluorescence intensity (Fig. 3b), which can be discerned by the naked eye, ultimately reaching a quantum yield (ΦF) of 35%. An overview of the light absorption and emission parameters is provided in Table 1 below. The increase in emission intensity is used to estimate the lipid bilayer partition coefficient (K). suv This reflects the SUV-related preference for COE-CN. CoE-CN has a high preference for lipid bilayers (K). suv = 2.4±0.3 × 10 6 K suv This order of magnitude is close to that observed using established membrane-accumulating dyes.

[0115] Table 1: Photophysical properties and solubility of COE-CN in PBS (150 mM). Quantum yield was determined as the mean of three independent replicates (mean ± sem). Φ Solubility was estimated by turbidity screening at 25 °C. No visible precipitation was observed at the highest test concentration of 2 mM.

[0116]

[0117] Effect of structure on subcellular localization of CoE We first examined the cell staining patterns of COE-CN to further confirm our strategy of modulating organelle selectivity through molecular design. HeLa cells were stained with 5 μM COE-CN and imaged using confocal laser scanning microscopy (CLSM). Intracellular localization of COE-CN was evident, even with a relatively short staining duration of 15 minutes, under which COE-S6 staining was observed and thus resided within the cell boundary (Fig. 3c). Prolonged incubation of COE-S6 led to endocytosis and lysosomal membrane localization. Due to spectral overlap with COE-S6, we chose to co-stain cells with COE-CN and COE-BO (also red-glossing COEs that label lysosomes). Fig. 3d confirms that signals from the two COEs are located at mutually exclusive subcellular locations (R = 0.08). The difference in cell entry rate and organelle selectivity of COE-CN compared to the two elongated MICOEs examined in this paper thus points to a shorter residence within the plasma membrane, likely due to differences in structural and physicochemical properties.

[0118] We explored three parameters commonly used to evaluate membrane permeability: Log P, TPSA, and molecular length. Figure 1 The values ​​of these parameters for the three COEs were estimated using Molinjection. Interestingly, the calculated miLogP values ​​for COE-CN (-0.7), COE-S6 (-0.38), and COE-BO (-1.2) are all negative, providing an indication of hydrophilic behavior. Consistent with this observation is the water solubility of all molecules, which is conferred by charged ionic groups.

[0119] Although the high lipid bilayer preference and hydrophilicity of COE-CN initially appear anomalous, this can be reconciled by defining the lipid bilayer-mimicking topology of these membrane-enhancing MICOEs. Therefore, other structural features play an equal or greater role in controlling COE-lipid association. Compared to elongated MICOEs, the reduced hydrophobicity and higher TPSA derived from shorter conjugated cores (COE-CN - 3.6 nm, COE-BO - 5.2 nm, COE-S6 - 5.4 nm) may favor more reversible lipid association. Furthermore, the greater number of cationic charges in COE-S6 and COE-BO relative to COE-CN may act as an electrostatic "lock" for membrane components, leading to a less pronounced dynamic equilibrium in membrane partitioning.

[0120] Mitochondrial staining using CoE-CN To examine subcellular localization, cells stained with COE-CN were then studied along with dyes that showed specific organelle localization. Within 30 minutes, Mito-Tracker staining was observed. TM Bright intracellular staining of cells co-localized with Red (established mitochondrial staining) ( Figure 4 a) Wide-field images show uniform staining of all cells, with no evidence of aggregated COE-CN. This feature is advantageous for applications in fluorescence microscopy and flow cytometry analysis. Co-localization analysis using a set of organelle-targeting dyes (CellMask Deep Red - membrane, SYTO Deep Red - nucleus, LipidTOX Red - lipid droplets) is also shown. Figure 4 As shown in b, 4c, and 4d, COE-CN is internalized only in mitochondria. Most notably, unlike previous plasma membrane markers such as COE, ... Figure 4 As shown in c, COE-CN is not present within the plasma membrane (R = 0.03). Furthermore, efficient mitochondrial imaging was achieved at concentrations as low as 250 nM, further emphasizing molecular selectivity.

[0121] Repeated CLSM scans and irradiation of the dye demonstrated good photostability; mitochondria remained clearly visible after 400 images. (See [link to CLSM scans]). Figure 5 Furthermore, the resulting dye strength is comparable to commercially available dyes, despite a higher energy excitation wavelength ( Figure 8 This stability, along with the observation that there was no observable effect on cell viability at the imaging concentration, ( Figure 9 This highlights the potential of COE-CN for long-term live-cell imaging. Furthermore, repeated image acquisition and prolonged dye incubation for 24 hours did not appear to have a visible effect on cell morphology.

[0122] Effects of cells on CoE labelingWe sought to determine the extent to which COE-CN could be used to examine mitochondrial dynamics under pharmacological treatment conditions, which could affect staining efficiency and facilitate dye translocation to other organelles. As a typical depolarizer, carbonyl cyanochlorophenylhydrazone (CCCP) is a proton ionoporter that dissipates MMPs within minutes. Therefore, we investigated the MMP dependence of COE-CN staining by pretreating live cells with 20 μM CCCP followed by dye addition. Compared to untreated cells imaged under the same conditions, the fluorescence intensity of COE-CN in CCCP-pretreated cells decreased by approximately 25% relative to untreated cells, but still allowed for visualization of mitochondria. Similar results were observed using other mitochondrial-specific dyes. We also examined dye retention upon cell fixation, which also attenuates MMPs, but is a necessary step in microscopy techniques, including snapshot-specific dynamic processes. We imaged fixed HeLa cells co-stained with COE-CN and Mito Deep Red, both of which are known to remain in the mitochondria after fixation. Using structured illumination microscopy, COE-CN was identified as retaining, after chemical fixation, to reveal circular vesicle structures co-localized with Mito Deep Red, corresponding to mitochondrial membrane localization. Therefore, sufficient COE-CN remains associated with mitochondria, enabling imaging of structural changes under varying treatment conditions, where the dye might otherwise redistribute throughout the cell. Overall, these results indicate that COE-CN is retained within mitochondria under a variety of conditions (live, fixed, healthy, and depolarized cells), making it a universal dye for cell imaging.

[0123] Mitochondrial autophagy imaging Our next objective was to demonstrate the applicability of COE-CN in visualizing subcellular processes by inducing mitophagy in HeLa cells through 2-hour CCCP treatment. This treatment triggers mitophagic clearance of damaged mitochondria in an attempt to mitigate cellular damage. Cells were initially transfected with Lamp1-RFP labeled with lysosomal membranes, followed by COE-CN co-staining to observe organelle interactions. These cells were then retreated for 2 hours with medium containing CCCP (20 μM) or with fresh growth medium. Figure 6The observed healthy mitochondria with a regular rod-shaped morphology were found in untreated cells irradiated with COE-CN. Lysosomes were also visible as bright red vesicles, and the spatial distribution of the two organelles differed, further confirming the specific mitochondrial localization in healthy cells. In contrast, CCCP treatment induced mitochondrial swelling to form a ring-like structure. This structural change was combined with a visible increase in the number of staining spots caused by mitochondrial division. The green spots corresponding to COE-labeled mitochondria were clearly located within the lysosomal vesicles, reflecting the lysosomal engulfment of damaged mitochondria. Therefore, CoE-CN can serve as an effective fluorescent probe for monitoring changes in mitochondrial morphology and interactions within living cells.

[0124] COE is used for flow cytometry-based assays. This COE-CN accumulation is MMP-dependent, leading us to investigate its use in a flow cytometry-based assay designed to address mitochondrial health. To separate the effects of membrane depolarization from subsequent downstream degradation processes, cells were stained with a dye and then treated with CCCP. Analysis was performed by flow cytometry 30 minutes post-treatment. This time point appears to precede mitophagy, where previously observed depolarization had begun, albeit not at its peak. Figure 7 As shown in a and 7c, treatment with 20 μM CCCP resulted in cell population migration, characterized by a significantly lower COE-CN intensity compared to the untreated group. This cell population was observed to decrease proportionally to the CCCP concentration at 5 and 1 μM, respectively. In contrast, depolarization of cells stained with Mito-Tracker Red (a dye that remains within mitochondria even during depolarization) showed minimal change in intensity. Figure 7 (b, 7d). Detailed analysis of the mean fluorescence intensity at different treatment concentrations showed a dose-dependent decrease starting from 5 μM. Therefore, CoE-CN can be used to distinguish between damaged and healthy cell populations, which allows for its use in the screening of mitochondrial depolarizers.

[0125] Evaluation of the ratio of COEs with different membrane localizations to mitochondrial health To more accurately assess mitochondrial damage, we compared the effect of depolarization on the fluorescence intensity of COE-CN and COE-BO in different cellular compartments. We first labeled HeLa cells with both COEs, then treated them with CCCP to depolarize the mitochondria. Figure 7 As observed in e, no significant changes were observed in the intensity distribution of COE-BO upon treatment. Conversely, CCCP treatment led to a decrease in the intensity of COE-CN in a CCCP-concentration-dependent manner. Figure 7f), this reflects the translocation of dye from mitochondria to the cytoplasm, where its brightness decreases as it transitions from the lipid environment to free dye. Conversely, the relatively constant intensity of COE-BO implies that the elongated MICOE remains stably embedded throughout the lipid bilayer. Compared to single-channel assessments, ratio analysis is a more objective strategy for assessing changes in biological processes and is less dependent on experimental parameters. Therefore, we plotted the emission ratio between red and green COE channels to evaluate mitochondrial health, whereby the response to depolarization occurs only in one channel. This ratio was observed to increase by 1.4-fold and 2-fold, respectively, upon treatment with 10 and 20 μM CCCP, consistent with the degree of depolarization across the entire cell population. Overall, these findings provide supporting evidence for designing assays reflecting mitochondrial damage using COEs with different chemical compositions and therefore different patterns of internal cellular accumulation.

[0126] in conclusion In summary, we designed and synthesized a novel COE derivative, COE-CN, which rapidly localizes within mitochondria without remaining in the plasma membrane. This specificity contrasts with previous generations of COEs that primarily stained the plasma membrane or intracellular vesicles via endocytosis. CoE-CN allows for selective visualization of mitochondria and features characteristic brightness and photostability suitable for practical applications. Furthermore, COE-CN maintains sufficient brightness under various cell staining experimental conditions, such as depolarization or fixation, to allow imaging of treated cells. Thus, COE-CN was successfully used to observe changes in mitochondrial morphology and cell localization during mitophagy. Moreover, its membrane potential-dependent accumulation allows for the identification of cells with mitochondrial damage using COE-CN, providing a novel and simple method for using optical reporters associated with rapid screening for mitochondrial dysfunction. Combined with the elongated MICOEs found to be non-transferable upon treatment, COE-CN is capable of assessing mitochondrial health. More broadly, based on these findings, we anticipate the development of COE molecular designs that serve as target-specific intracellular optical probes capable of reporting cellular parameters relevant to disease identification.

[0127] It should be understood that numerous further modifications and substitutions of the various aspects of the described embodiments are possible. Therefore, the described aspects are intended to cover all such changes, modifications, and variations that fall within the spirit and scope of the appended claims.

[0128] In this specification and the following claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” shall be understood to imply inclusion of the said whole or steps, but not to exclude any other whole or steps.

[0129] References to any prior publications (or information derived therefrom) or any known things in this specification are not and should not be construed as an admission or endorsement or any form of advice that such prior publications (or information derived therefrom) or known things form part of the common general knowledge in the field to which this specification is addressed.

Claims

1. A compound of formula (I) or a salt or solvate thereof: (I) in R1 is independently selected from H, optionally substituted alkyl groups, and optionally substituted alkoxy groups; R2 is independently selected from cyano, oxo, and optionally substituted alkyl groups; Ht is independently selected from optionally substituted heterocyclic groups and optionally substituted heteroaryl groups; and n is an integer independently selected from 2 to 5.

2. The compound of claim 1, wherein the compound of formula (I) comprises a phenylene vinylene backbone, wherein the phenyl moiety is in a trans configuration around the alkenyl moiety.

3. The compound of claim 1 or 2, wherein R1 is independently selected from optionally substituted C1-C5 alkyl or optionally substituted C1-C5 alkoxy.

4. The compound according to any one of claims 1 to 3, wherein R1 is independently methoxy.

5. The compound according to any one of claims 1 to 4, wherein R2 is a cyano group.

6. The compound according to any one of claims 1 to 5, wherein Ht is independently selected from optionally substituted heterocyclic groups and optionally substituted heteroaryl groups, said heterocyclic and heteroaryl groups being selected from oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indazine, isoindole, indole, indazole, purine, quinazine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cyclophosphine, pteridine, carbazole, carboline, phenanthridine, etc. Acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenthiazine, imidazoline, imidazoline, piperidine, piperazine, dihydroindole, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiadiazole, oxadiazole, oxtriazole, tetrazolium, thiazoline, thiophene, benzo[b]thiophene, morpholino, piperidinyl, pyrrolidine, tetrahydrofuranyl, and triazole.

7. The compound according to any one of claims 1 to 6, wherein Ht is selected from optionally substituted N-heterocyclic groups and optionally substituted N-heteroaryl groups.

8. The compound according to any one of claims 1 to 7, wherein Ht is connected to the compound of formula (I) via its heteroatom.

9. The compound according to any one of claims 1 to 8, wherein the optional substituents on Ht are selected from halogen, alkyl, alkenyl, alkoxy, alkenyloxy, alkynyloxy, acyl, oxyacyl, acyloxy, amino, aminoacyl, acylamino, aminoacyloxy, oxyacylamino, oxyacyloxy, thio, and phosphoryl.

10. The compound according to any one of claims 1 to 8, wherein Ht is independently an optionally substituted imidazole group.

11. The compound according to any one of claims 1 to 10, wherein the compound of formula (I) or its salt or solvation is 。 12. A composition or kit comprising a compound of formula (I) according to any one of claims 1 to 11, or a salt or solvation thereof, said compound or salt or solvation thereof configured for staining mitochondria in cells.

13. The composition or kit of claim 12, further comprising a conjugated oligoelectrolyte configured for staining the lipid membrane of the cells and / or a dye configured for staining another component of the cells.

14. A method for staining mitochondria in cells, comprising contacting the cells with a compound of formula (I) as claimed in any one of claims 1 to 11, or a salt or solvation thereof.

15. The method of claim 14, wherein the method further comprises the step of incubating the cell with a compound of formula (I) or a salt or solvate thereof.

16. The method of claim 14 or 15, wherein the method further comprises the step of visualizing the mitochondria by photoexciting the compound of formula (I) and detecting the emitted photons.

17. The method of any one of claims 14 to 16, wherein the method further comprises contacting the cell with a second dye configured to stain the lipid membrane of the cell.

18. The method of claim 17, wherein the second dye is a conjugated oligoelectrolyte configured to stain a lipid membrane.

19. Methods for distinguishing between damaged and healthy cells include: a) Contacting the cells with the compound of formula (I) as described in any one of claims 1 to 11, or a salt or solvate thereof, and the second dye; as well as b) Quantify the fluorescence emitted by the compound of formula (I) relative to the fluorescence emitted by the second dye to distinguish between the damaged cells and the healthy cells.

Images