Donor-acceptor conjugated oligoelectrolytes for cell labeling and their uses

Abstract

The present disclosure relates to compounds of formula (I) and methods of use thereof. [Formula 1] TIFF2024534774000114.tif13170 The compounds of formula (I) are conjugated oligoelectrolytes and are suitable for use as membrane probes for labeling and / or detecting cells and / or lipid vesicles, and are therefore suitable for flow cytometry applications. The present disclosure also relates to a flow system for detecting and / or quantifying cells and / or lipid vesicles.

Description

[Technical field]

[0001] Field FIELD OF THE DISCLOSURE The present disclosure relates to conjugated oligoelectrolytes and methods of use thereof. In particular, the conjugated oligoelectrolytes are suitable for use as membrane probes and therefore for flow cytometry applications. [Background technology]

[0002] background Flow cytometry is a high-throughput laboratory technique for the rapid counting, recognition, and sorting of individual cells, microorganisms, and particles. It is used in clinical and research characterization in many disciplines, including cancer biology, immunology, microbiology, and virology. Flow cytometry utilizes a microfluidic system in which individual cells or particles enter a stream, rapidly pass a laser light source, and are then analyzed by fluorescence or light scattering. Fluorescent proteins, fluorescently labeled antibodies, or structure-specific dyes, such as those specific for DNA or lipid membranes, can be used to measure the unique properties of individual cells or particles. Fluorescent labeling is important for a variety of purposes, including understanding cell viability, identifying different cell types in a heterogeneous mixture, measuring antigen or protein expression, cell cycle analysis, and understanding membrane integrity, to name a few.

[0003] Given that flow cytometry relies heavily on fluorescent tags, technicians may face many problems depending on the fluorescent labels used or when multiple fluorescent labels are required. For example, the fluorescence intensity may be weak or the fluorescent labels may be degraded upon exposure to light. Furthermore, the recognition probes may have few fluorophore binding sites or an effective number of dyes. The binding of the fluorescent labels to the desired cellular components may be weak. When the fluorescent labels are not properly internalized by the cells, the fluorescent signal may become overly saturated. Under improper incubation conditions, the fluorescent labels may aggregate and thus self-quench. Also, high background or nonspecific staining may interfere with the test method. When two or more fluorescent labels are used, the emitted signals may overlap, making the results confusing and even uninterpretable. Also, some fluorescent labels are toxic to cells, and therefore only a short working window is set up for performing flow cytometry.

[0004] Thus, there is a need for molecules that can act as fluorescent labels or dyes for use in flow cytometry. Further, there is a need for fluorescent molecules that can preferentially target microorganisms, particularly bacterial cells.

[0005] It would therefore be desirable to overcome or ameliorate at least one of the above problems. Summary of the Invention

[0006] overview The present invention is based on the discovery that certain conjugated oligoelectrolytes (COEs) exhibit distinct membrane binding and are therefore advantageous for use as fluorescent membrane probes. In particular, the inventors have discovered that when the conjugated moieties along the backbone of the COE are modified, the emitted fluorescent signal can be tuned to a specific wavelength. Furthermore, the Stokes shift (the difference between peak excitation and peak emission) can be tuned. The pendant chains at the ends of the conjugated backbone can be further modified to tune selectivity for specific cell membranes. The penetration of these COE compounds into cell membranes allows the analysis of cells, liposomes, vesicles, and other membrane-containing macrostructures using flow cytometry.

[0007] The present invention provides a compound of formula (I), or a salt or solvate thereof: [ka] During the ceremony, each R1 is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy, or optionally substituted thio, or optionally substituted phosphoryl; q is an integer selected from 1 to 5; q' is an integer selected from 1 to 5; Each L2 is independently selected from optionally substituted ethylene or optionally substituted phenylethylene; L1 is a π-conjugated core comprising monomeric unit A and monomeric unit D: [ka] During the ceremony, each A is independently selected from optionally substituted alkenylene, optionally substituted monocyclic heteroarylene, or optionally substituted fused heteroarylene; each D is independently selected from alkenylene, phenylene, optionally substituted fused arylene, optionally substituted monocyclic heteroarylene, or optionally substituted fused heteroarylene; n is an integer selected from 1 to 5; m is an integer selected from 1 to 5; * represents a bond to another monomeric unit or to L2; Monomer units A and D are alternately linked to each other; Compounds of formula (I) exhibit a substantially linear topology; L1 is not butadienylene, polyalkenylene, phenylalkenylene, or polyphenylalkenylene.

[0008] The conjugated compounds of formula (I) contain a composition of alternating donor (D) / acceptor (A) structural units, so that the emission, quantum yield, and Stokes shift can be tuned over a much wider range than those relying on π-conjugation of polyalkenylenes or polyphenylalkenylenes alone. Furthermore, the molecular topology of the molecules allows for rapid self-assembly in membranes of various cell and lipid types. A particular molecular fragment, e.g., phenylene, can be a monomeric unit D or a monomeric unit A, depending on the electron affinity or ionization potential of the adjacent groups.

[0009] In some embodiments, A is an electron accepting moiety.

[0010] In some embodiments, A is [ka] is selected from the group consisting of [ka] represents a bond to D or to L2; each X1 is independently selected from C, O, N, S, and Se; each X2, if present, is independently selected from C, O, N, S, and Se; When X2 is present, at least one of X1 and X2 is O, N, or S; Each R is independently selected from H, halo, cyano, and optionally substituted alkyl.

[0011] In some embodiments, A is a moiety of formula (II): [ka] During the ceremony, [ka] represents a bond to D or to L2; R2, R3, R4, and R5 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R2 and R3 are linked to form an optionally substituted heterocyclyl, an optionally substituted heteroaryl; or R4 and R5 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl.

[0012] In some embodiments, A is

[0013] [ka] where R4 and R5 are as disclosed herein.

[0014] In some embodiments, A is [ka] It is.

[0015] In some embodiments, D is an electron donating moiety.

[0016] In some embodiments, D is [ka] is a moiety selected from [ka] represents a bond to A or to L2; each X1 is independently selected from C, O, N, S, and Se; each X2, if present, is independently selected from C, O, N, S, and Se; When X2 is present, at least one of X1 and X2 is O, N, or S; R is independently selected from H, halo, cyano, and optionally substituted alkyl.

[0017] In some embodiments, D is an optionally substituted 5-membered heteroarylene.

[0018] In some embodiments, D is a moiety of formula (III): [ka] wherein Y is NR, O, or S; R6 and R7 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R6 and R7 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl; R is selected from H, halo, cyano, and optionally substituted alkyl.

[0019] In some embodiments, D is [ka] where R6 and R7 are as disclosed herein.

[0020] In some embodiments, D is [ka] It is.

[0021] In some embodiments, L1 is [ka] is selected from.

[0022] In some embodiments, each R1 is independently selected from optionally substituted alkyl, optionally substituted alkoxy.

[0023] In some embodiments, each R1 is independently selected from alkyl and alkoxy, each optionally substituted with amino or alkylamino.

[0024] In some embodiments, each R1 is independently C3-C8 alkoxy substituted with amino or alkylamino.

[0025] In some embodiments, the optional substituents of L are independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0026] In some embodiments, the compound of formula (I) is a compound of formula (Ia): [ka] wherein L1, A, D, R1, n, m, and q are as disclosed herein.

[0027] In some embodiments, the compound of formula (I) is a compound of formula (Ib): [ka] wherein L1, A, D, R1, n, m, and q are as disclosed herein.

[0028] In some embodiments, the compound of formula (I) is a compound of formula (Ib), wherein: A is a moiety of formula (II): [ka] During the ceremony, [ka] represents a bond to D or to L2; R2, R3, R4, and R5 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R2 and R3 are linked to form an optionally substituted heterocyclyl, an optionally substituted heteroaryl; or R4 and R5 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl; D is a moiety of formula (III): [ka] wherein Y is NR, O, or S; R6 and R7 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R6 and R7 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl.

[0029] In some embodiments, the compound of formula (I) is a compound of formula (Ib), wherein: A is, [ka] where R4 and R5 are as disclosed herein; D is [ka] where R6 and R7 are as disclosed herein.

[0030] In some embodiments, the compound of formula (I) is a compound of formula (Ic): [ka] each R1 is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy, or optionally substituted thio, or optionally substituted phosphoryl; R2, R3, R4, and R5 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R2 and R3 are linked to form an optionally substituted heterocyclyl, an optionally substituted heteroaryl; or R4 and R5 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl; R6 and R7 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R6 and R7 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl; each R8 is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; Y is NR, O, or S; each p is an integer independently selected from 0 to 4; q is an integer selected from 1 to 5; and q' is an integer selected from 1 to 5.

[0031] In some embodiments, the compound of formula (I) is a compound of formula (Id): [ka] wherein R1, R4, R5, R6, R7, R8, Y, p, q, and q' are as disclosed herein.

[0032] In some embodiments, the compound of formula (I) is a compound of formula (Ie): [ka] wherein R1, R4, R5, R6, R7, R8, p, q, and q' are as disclosed herein.

[0033] In some embodiments, the compound of formula (I) is a compound of formula (If): [ka] wherein R4, R5, R6, R7, R8, R9, p, q, and q' are as disclosed herein; Each R9 is independently H or optionally substituted alkyl; Each t is an integer independently selected from 1 to 8.

[0034] In some embodiments, the compound of formula (I) is [ka] is selected from.

[0035] The present invention also provides a compound of formula (Ig), or a salt or solvate thereof: [ka] During the ceremony, each R1 is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy, or optionally substituted thio, or optionally substituted phosphoryl; q is an integer selected from 1 to 5; q is an integer selected from 1 to 5; and r is an integer selected from 1 to 5.

[0036] The present invention also provides a method for labeling cells and / or lipid vesicles, comprising the steps of: a) incubating a compound of formula (I), sub-formulas (Ia-Ig), or a salt or solvate thereof with cells and / or lipid vesicles.

[0037] The present invention also provides a method for detecting cells and / or lipid vesicles using a fluorescence detector, comprising: a) incubating a compound of formula (I), sub-formulas (Ia-Ig), or a salt or solvate thereof with cells and / or lipid vesicles; and b) passing the cells through a fluorescence detector The present invention provides a method comprising:

[0038] The present invention also provides a method for detecting cells and / or lipid vesicles using a flow cytometer, comprising: a) Formula (IV); [ka] (In the formula, R1 is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy, or optionally substituted thio, or optionally substituted phosphoryl; R2 is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; u is an integer selected from 3 to 15; each s is an integer independently selected from 0 to 4; q is an integer selected from 2 to 5; q' is an integer selected from 2 to 5. or a salt or solvate thereof with cells and / or lipid vesicles; and b) running the cells and / or lipid vesicles through a flow cytometer The present invention provides a method comprising:

[0039] In some embodiments, the cells and / or lipid vesicles are in suspension.

[0040] In some embodiments, the cells are adherent cells.

[0041] In some embodiments, the incubation period is from about 1 minute to about 12 days.

[0042] In some embodiments, the cells and / or lipid vesicles are run through a flow cytometer without a purification step.

[0043] In some embodiments, compounds of formula (I), subformulas (Ia-Ig), and / or formula (IV) exhibit fluorescence excitation at wavelengths between about 300 nm and about 1000 nm.

[0044] In some embodiments, compounds of formula (I), subformulas (Ia-Ig), and / or formula (IV) exhibit fluorescent emission at wavelengths from about 300 nm to about 2000 nm.

[0045] In some embodiments, cells and / or lipid vesicles incubated with a compound of Formula (I), subformulae (Ia-Ig), and / or Formula (IV) exhibit luminescence intensity that is from about 2-fold to about 500-fold greater than a control sample of a compound of Formula (I), subformulae (Ia-Ig), and / or Formula (IV).

[0046] In some embodiments, the method further comprises contacting the cells and / or lipid vesicles with another dye.

[0047] The present invention also provides a flow system for detecting and / or quantifying cells and / or lipid vesicles, comprising: a) compounds of formula (I), sub-formulae (Ia-Ig) and / or formula (IV), or salts or solvates thereof, for labelling cells and / or lipid vesicles; b) an inlet for introducing labeled cells and / or lipid vesicles into the flow system; c) a detection means in fluid communication with the inlet for detecting fluorescent emissions from the labeled cells and / or lipid vesicles; and Optionally d) providing a flow system including counting means for quantifying the labeled cells and / or lipid vesicles.

[0048] Embodiments of the invention will now be described, by way of non-limiting example, with reference to the drawings, in which: [Brief description of the drawings]

[0049] [Figure 1] Examples of conjugated oligoelectrolytes of the present invention are given below. [Diagram 2] 1 shows flow cytometry measurements using compounds of formula (I) and / or formula (IV) for selective labeling of gram-positive from gram-negative bacteria. [Diagram 3] 1 shows flow cytometry measurements of compounds of formula (I) and / or formula (IV) (af) and unlabeled exosomes (h). [Figure 4] 1 shows flow cytometry measurements of exosomes labeled with compounds of formula (I) and / or formula (IV). [Diagram 5] TEM images of exosomes (a), exosomes labeled with compounds of Formula (I) and / or Formula (IV) (b), and exosomes labeled with compounds of Formula (I) and / or Formula (IV) and collected after FACS (c) are shown. [Figure 6] 1 shows flow cytometry measurements of RBCs labeled with compounds of formula (I) and / or formula (IV). [Figure 7] 1 shows the percentage of flow cytometry events occurring in gated regions of RBCs labeled with various ratios of compounds of Formula (I) and / or Formula (IV) versus unstained RBCs. [Figure 8] 1 shows flow cytometry measurements of Hep-G2 cells labeled with compounds of formula (I) and / or formula (IV). [Figure 9] 1 shows the percentage of flow cytometry events occurring in gated regions of Hep-G2 stained with various ratios of compounds of formula (I) and / or formula (IV) versus unstained Hep-G2. [Figure 10]1 shows flow cytometry measurements of two separate populations of A549 cells labeled with compounds of formula (I) and / or formula (IV), as well as unlabeled cells. [Figure 11] 1 shows flow cytometry measurements of passages of A549 cells labeled with compounds of formula (I) and / or formula (IV). [Figure 12] Confocal micrographs of HepG2 cells after staining with 4 μM of the compound of formula (I) and 4 μM of the commercial membrane dye FM 4-64 are shown: (a) compound of formula (I) channel, (b) FM 4-64 channel, (c) bright field channel, (d) merged channel. Scale bar is 20 μm. [Figure 13] Fluorescence microscopy images of A549 cells stained with compounds of formula (I) and / or formula (IV) are shown. [Figure 14] FIG. 1 shows (a-b) photoluminescence (PL) spectra of SUV, compounds of formula (I) and / or formula (IV), and the combination of SUV and the compounds in PBS buffer, (c) three compounds of formula (I) and / or formula (IV) showing various emission peaks after addition of SUV in PBS. [Figure 15] 1 shows various emission wavelengths of compounds of formula (I) and / or formula (IV). [Figure 16] Dynamic light scattering intensity size distribution curves are shown for liposomes not labeled with a compound of Formula (I) and / or Formula (IV) (solid grey line) and for liposomes labeled with the compound (dark dashed line). [Figure 17] Particle size distribution plots of dye-only controls and their corresponding gated dot plots are shown. [Figure 18] Particle size distribution plots of COE-labeled SW480 exosomes (10 μM) and their corresponding gated dot plots are shown. [Figure 19]FIG. 13 shows particle size distribution plots of dye-labeled SW480 exosomes (10 μM and 20 μM) after removal of excess dye using ultracentrifugation, and their corresponding gated dot plots. [Figure 20] 1 shows imaging flow cytometry images of A549 cells stained with COE-Ben stained exosomes at various treatments and time points, and their corresponding flow cytometry analyses. [Figure 21] Colocalization micrographs of A549 cells incubated with 2 μM COE-BT and then stained with early or late endosome-GFP reagents (BacMam 2.0) or 100 nM lysosome-specific dye LysoTracker® Green DND-26 are shown. [Figure 22] Transmission electron microscopy images of EVs secreted by COE-BT stained A549 cells and flow cytometry analysis of EVs secreted by COE-BT stained A549 cells after the first 24 h of incubation are shown. [Figure 23] Figures 23a-b show photographs and absorption spectra of 50 μM COE or DiR solutions in PBS before and after ultrafiltration. [Figure 24] Correlation coefficient curves of pure PBS, 1 μM COE-BT in PBS, 1 μM DiR or 1 mM SUV, or 1 μM of other COEs in PBS measured by dynamic light scattering (DLS) and average count rates derived by DLS measurements are shown. [Diagram 25] Photographs of the Tyndall effect of pure PBS or 10 μM COE in PBS after illumination using a red laser pointer are shown. [Figure 26] Photographs of 200 μL of COE solution and 200 μL of dye solution in PBS in a 96-well microplate before and after standing at room temperature for 16 hours are shown. [Figure 27]Flow cytometry measurements of mixtures of COE-BT and COE-Ben stained SUVs (130 nm) in PBS at various mixing ratios, incubated for 1 and 24 hours and analyzed on Cytoflex. [Figure 28] The percentage of the SUV population from FIG. 27 in the various gates after mixing for 1 hour or 24 hours is shown. [Figure 29] POPC liposomes labeled with 0.5 mol % COE-Ben and COE-BT and having particle sizes of 100 nm, 200 nm, 400 nm, and 800 nm were analyzed on a Cytoflex, showing dot plot profiles of dye-positive events. [Diagram 30] Confocal micrographs of 6.25 mg mL-1 LMVs (large multilamellar vesicles) after staining with 15 μM COE-Ben and 15 μM FM 4-64 for 30 min at room temperature in PBS are shown, and the grey-value curves represent the fluorescence intensity profile of the white lines in the fluorescence micrographs of both channels on the left. [Diagram 31] Photographs of COEs untreated (-) or treated (+) with 1 mM SUV in PBS under UV light (365 nm) irradiation using a portable UV lamp are shown. [Diagram 32] Zeta potential measurements of 1 mM POPC-only SUVs stained with 5 μM of various COEs in DI water, as well as DLS measured Z-average diameter and PDI of 1 mM POPC-only SUVs stained with 5 μM of various COEs in DI water are shown. [Diagram 33] Confocal micrographs of fresh red blood cells (RBCs) are shown: (a) unstained, (b) labeled with 2 μM COE-S6, and (c) labeled with 1 μg / mL Cell Mask Deep Red. [Diagram 34] Confocal micrographs of red blood cells (RBCs) stored for 22 days are shown, which were (a) unstained, (b) labeled with 2 μM COE-S6, and (c) labeled with 1 μg / mL Cell Mask Deep Red. [Diagram 35](a-c) FSC / SSC dot plots of unstained, 1 μM COE-S6 labeled, and 0.5 μg mL-1 Cell Mask Deep Red labeled red blood cells (RBCs) measured by flow cytometry, as well as (d) histograms showing the increase in fluorescence when RBCs were labeled with increasing concentrations of COE-S6, and (e) histograms showing the decrease in fluorescence due to the quenching effect when RBCs were labeled with increasing concentrations of Cell Mask Deep Red. The coefficient of variation (CV) of the histogram data is shown in (f). [Diagram 36] Flow cytometry measurements of COE-BBT stained A549 cells with excitation using an 808 nm laser and emission collected using a 950 nm long pass filter. Unstained A549 cells were used as a negative control. [Figure 37] Measured cytotoxicity of COEs against A549 cells is shown. [Figure 38] Measured hemolysis of COE against bovine red blood cells in PBS is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Detailed Description "Alkyl" refers to a monovalent alkyl group which may be linear or branched and preferably has 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. Examples of these alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-hexyl, and the like.

[0051] "Alkenyl" refers to a monovalent alkenyl group, which may be linear or branched, preferably having 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and having at least one, preferably 1 to 2 carbon-to-carbon double bonds. Examples include ethenyl (-CH=CH2), n-propenyl (-CH2CH=CH2), isopropenyl (-C(CH3)=CH2), but-2-enyl (-CH2CH=CHCH3), and the like.

[0052] "Alkynyl" refers to an alkynyl group having preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and at least one, preferably 1 to 2 carbon-to-carbon triple bonds. Examples of alkynyl groups include ethynyl (-C≡CH), propargyl (-CH2C≡CH), pent-2-ynyl (-CH2C≡CCH2-CH3), and the like.

[0053] "Alkoxy" means an alkyl-O- group in which the alkyl group is as previously described. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

[0054] "Alkenyloxy" means an alkenyl-O- group in which the alkenyl group is as previously described.

[0055] "Alkynyloxy" means an alkynyl-O- group in which the alkynyl group is as previously described.

[0056] "Halo" or "halogen" means fluoro, chloro, bromo, and iodo.

[0057] "Acyl" means an HC(O)-, alkyl-C(O)-, cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)- and heterocyclyl-C(O)- groups, in which alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.

[0058] "Oxyacyl" means an HOC(O)-, alkyl-OC(O)-, cycloalkyl-OC(O)-, aryl-OC(O)-, heteroaryl-OC(O)- and heterocyclyl-OC(O)- groups, in which alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.

[0059] "Amino" refers to the group --NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, each of which is as described herein.

[0060] "Aminoacyl" means the group -C(O)NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, each of which is as described herein.

[0061] "Acylamino" means the group --NR"C(O)R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, each of which is as described herein.

[0062] "Acyloxy" means an --OC(O)-alkyl group, an --OC(O)-aryl group, a --C(O)O-heteroaryl group, and a --C(O)O-heterocyclyl group, in which alkyl, aryl, heteroaryl, and heterocyclyl are as described herein.

[0063] "Aminoacyloxy" refers to the groups -OC(O)NR"-alkyl, -OC(O)NR"-aryl, -OC(O)NR"-heteroaryl, and -OC(O)NR"-heterocyclyl, where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, each of which is as defined herein.

[0064] "Cyano" refers to the radical -CN.

[0065] "Oxyacylamino" means the groups -NR"C(O)O-alkyl, -NR"C(O)O-aryl, -NR"C(O)O-heteroaryl, and NR"C(O)O-heterocyclyl, where R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, each of which is as defined herein.

[0066] "Oxyacyloxy" means an -OC(O)O-alkyl group, an -OC(O)O-aryl group, an -OC(O)O-heteroaryl group, and an -OC(O)O-heterocyclyl group, in which alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.

[0067] "Thio" means an HS-, alkyl-S-, cycloalkyl-S-, aryl-S-, heteroaryl-S- and heterocyclyl-S- group, in which alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

[0068] "Phosphoryl" means a -P(O)(R''')(OR'''') group, where R''' represents OR'''' or is hydroxyl, alkyl, or amino, and R'''' is alkyl, cycloalkyl, aryl, or arylalkyl, where alkyl, amino, alkenyl, aryl, cycloalkyl, and arylalkyl are as described herein.

[0069] "Aryl" refers to an unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), preferably having 6 to 14 carbon atoms. Examples of aryl groups include phenyl, naphthyl, and the like.

[0070] "Heteroaryl" refers to a monovalent aromatic heterocyclic group that satisfies Hückel's rule for aromaticity (i.e., contains 4n+2 pi-electrons) and preferably has 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen, selenium, and sulfur in the ring (including the oxides of sulfur, selenium, and nitrogen). These heteroaryl groups can have a single ring (e.g., pyridyl, pyrrolyl, or their N-oxides, or furyl) or multiple condensed rings (e.g., indolizinyl, benzimidazolyl, coumarinyl, quinolinyl, isoquinolinyl, or benzothienyl).

[0071] Examples of heteroaryl groups include, but are not limited to, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, phenoxazine, phenothiazine, thiazole, thiadiazole, oxadiazole, oxatriazole, tetrazole, thiophene, benzo[b]thiophene, triazole, imidazopyridine, and the like.

[0072] "Arylene" means a divalent aryl group, in which the aryl group is as previously described.

[0073] "Heteroarylene" means a divalent heteroaryl group in which the aryl groups are as previously described.

[0074] "Heterocyclyl" refers to a monovalent saturated or unsaturated group having a ring or multiple condensed rings, preferably having from 1 to 8 carbon atoms and from 1 to 4 heteroatoms selected from nitrogen, sulfur, oxygen, selenium, or phosphorus in the ring. The most preferred heteroatom is nitrogen. When, for example, R2 or R' is an optionally substituted heterocyclyl having one or more ring heteroatoms, it will be understood that the heterocyclyl group may be connected to the core molecule of the compounds of the invention through a CC or C-heteroatom bond, particularly a CN bond.

[0075] Examples of heterocyclyl and heteroaryl groups include oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, isothiazole, benzoylpyridine, benzoic acid, benzoylpyridine ... These include, but are not limited to, azole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiadiazole, oxadiazole, oxatriazole, tetrazole, thiazolidine, thiophene, benzo[b]thiophene, morpholino, piperidinyl, pyrrolidine, tetrahydrofuranyl, triazole, and the like.

[0076] "Optionally substituted" means that one group is hydroxyl, acyl, alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl, alkynyloxy, amino, aminoacyl, thio, arylalkyl, arylalkoxy, aryl, aryloxy, carboxyl, acylamino, cyano, halogen, nitro, phosphono, sulfo, phosphorylamino, phosphinyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, oxyacyl, oxime, oxime ether, hydrazone, oxyacylamino, oxysulfonylamino, aminoacyloxy, trihalomethyl, trialkylsilyl, pentafluoroethyl, or trifluoromethoxy. , difluoromethoxy, trifluoromethanethio, trifluoroethenyl, mono- and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclylamino, and asymmetric disubstituted amines having various substituents selected from alkyl, aryl, heteroaryl, and heterocyclyl, and the like, which may or may not be further substituted with, or fused thereto (to form a fused polycyclic group), and which may or may not include a bond to a solid support material (e.g., substituted to a polymer resin). For example, an "optionally substituted amino" group may include amino acid and peptide residues.

[0077] "Hydrophilic" refers to a molecule or moiety that has a greater affinity, and therefore solubility, for water than for organic solvents. For example, the hydrophilicity of a compound can be quantified by measuring its partition coefficient between water (or an aqueous buffer solution) and a water-immiscible organic solvent, such as octanol, ethyl acetate, methylene chloride, or methyl tert-butyl ether. A compound can be considered hydrophilic if, after equilibration, the concentration of the compound in water is greater than the concentration in the organic solvent.

[0078] "Hydrophobic" refers to a molecule or moiety that has a greater affinity, and therefore solubility, in organic solvents than in water. For example, the hydrophobicity of a compound can be quantified by measuring its partition coefficient between water (or an aqueous buffer solution) and a water-immiscible organic solvent such as octanol, ethyl acetate, methylene chloride, or methyl tert-butyl ether. A compound can be considered hydrophobic if, after equilibration, the compound's concentration in the organic solvent is greater than its concentration in water.

[0079] Conjugated oligoelectrolytes (COEs) are a class of molecules defined by a hydrophobic conjugated core with terminal polar ionic pendants. In certain embodiments, the hydrophobic and hydrophilic moieties in a COE can be rationally designed as molecules in which both moieties reflect the organization of hydrophilic and hydrophobic domains in a lipid bilayer. Typically, the structural design encompasses only unbranched internal structures with charged groups at the two termini to facilitate spontaneous insertion of the COE into cell membranes triggered by electrostatic and hydrophobic interactions between the COE and lipids. We hypothesize that the fluorescence resulting from π-π conjugation in the backbone can be tuned to occur at a variety of wavelengths across the electromagnetic spectrum, including the ultraviolet, visible, and infrared regions. This can be achieved by fine-tuning the optoelectronic properties of the conjugated core through structural guidance. For example, the backbone conjugated π system can be tuned to achieve an emission wavelength range of 300-2000 nm based on the donor and acceptor moieties. Fluorescence excitation, emission, and Stokes shift can be tuned toward this end. Furthermore, COEs can be modified to exhibit selectivity for specific membranes through functionalization with various chemical groups that control their membrane insertion ability, resulting in target-specific functionality.

[0080] Characteristically, the fluorescence emission of COEs is significantly enhanced when they are inserted into lipid bilayers from aqueous solutions. This "light-up" mechanism results in a high signal-to-noise ratio for COEs when they are localized within the more hydrophobic environment of the lipid bilayer. Furthermore, COEs have a different chemical structure than many commercially available lipophilic dyes (which usually contain detergent-like structures, i.e., one side of the molecule is hydrophobic and the other side is hydrophilic). These detergent-like structures induce micelle-like aggregation in aqueous solutions. For example, the commonly used membrane dye PKH-26 has been shown to form aggregates with similar particle size and fluorescence intensity as small particles such as exosomes, thereby giving rise to false-positive signals. These phenomena can be avoided in the case of COEs, since their emission is greatly enhanced after insertion into lipid bilayers, i.e., a high signal-to-noise ratio.

[0081] Thus, the present invention provides a compound of formula (I), or a salt or solvate thereof: [ka] During the ceremony, each R1 is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy, or optionally substituted thio or optionally substituted phosphoryl; q is an integer selected from 1 to 5; q' is an integer selected from 1 to 5; Each L2 is independently selected from optionally substituted ethylene or optionally substituted phenylethylene; L1 is a π-conjugated core comprising monomeric unit A and monomeric unit D: [ka] During the ceremony, each A is independently selected from optionally substituted alkenylene, optionally substituted arylene, or optionally substituted heteroarylene; each D is independently selected from optionally substituted alkenylene, optionally substituted arylene, or optionally substituted heteroarylene; n is an integer selected from 1 to 5; m is an integer selected from 1 to 5; * represents a bond to another monomeric unit or to L2; Monomer units A and D are alternately linked to each other; The compounds of formula (I) exhibit a substantially linear topology.

[0082] In some embodiments, A and D are not both alkenylene. In other embodiments, A and D are not both phenylene. In other embodiments, A and D are not both alkenylene and phenylene. In some embodiments, L1 is not butadienylene, polyalkenylene, phenylalkenylene, or polyphenylalkenylene.

[0083] In some embodiments, a compound of formula (I), or a salt or solvate thereof: [ka] During the ceremony, each R1 is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy, or optionally substituted thio or optionally substituted phosphoryl; q is an integer selected from 1 to 5; q' is an integer selected from 1 to 5; Each L2 is independently selected from optionally substituted ethylene or optionally substituted phenylethylene; L1 is a π-conjugated core comprising monomeric unit A and monomeric unit D: [ka] During the ceremony, each A is independently selected from alkenylene substituted with cyano, optionally substituted monocyclic heteroarylene, or optionally substituted fused heteroarylene; each D is independently selected from alkenylene, phenylene, optionally substituted fused arylene, optionally substituted monocyclic heteroarylene, or optionally substituted fused heteroarylene; n is an integer selected from 1 to 5; m is an integer selected from 1 to 5; * represents a bond to another monomeric unit or to L2; Monomer units A and D are alternately linked to each other; The compounds of formula (I) exhibit a substantially linear topology.

[0084] A further advantage is that these compounds do not require bioconjugation chemistry to be used as fluorescent labels, such as those required to label antibodies.

[0085] The compound of formula (I) exhibits a substantially linear topology. By topology is meant the molecular structure of the compound within the constraints of three-dimensional (3D) space. This linear topology has two nodes as termini and no junction nodes. The linear topology is advantageous for facilitating lipid membrane insertion.

[0086] In some embodiments, the bond connecting monomer unit A and monomer unit D in L1 is substantially aligned with the longitudinal axis of the compound. In other embodiments, monomer unit A and monomer unit D in L1 are substantially aligned with the longitudinal axis of the compound. In this regard, it is within the scope of the present invention for the bond connecting monomer unit A and monomer unit D to be off-center with respect to the longitudinal axis of the compound.

[0087] The compound of formula (I) is linear in order to accommodate its location within the lipid bilayer. In some embodiments, the compound is unbranched, i.e., the monomeric units extend along only one chain. In some embodiments, the compound of formula (I) is symmetric. The symmetry of the compound can be described by at least one of 32 point groups. A point group describes all symmetry operations that can be performed on a molecule that result in a conformation that is indistinguishable from the original conformation. In this regard, in some embodiments, the compound of formula (I) is C 2v It has a point cloud.

[0088] In some embodiments, each L2 is independently selected from optionally substituted ethylene or optionally substituted phenylethylene. In other embodiments, L2 is independently [ka] where * represents the bond to the monomeric unit and to the terminal phenyl moiety in the compound of formula (I).

[0089] L1 is the pi-conjugated core. A conjugated system is a system of connected p-orbitals with delocalized electrons in a molecule, which generally lowers the overall energy of the molecule and increases its stability. Lone pairs, radicals, or carbenium ions can be part of this system. The system can be cyclic, acyclic, linear, or mixed.

[0090] In some embodiments, when L1 comprises a 6-membered aryl or heteroaryl, or when L1 comprises a fused aryl or heteroaryl having a 6-membered ring, the monomer units are 1,4-conjugated on the 6-membered ring. In other embodiments, when L1 comprises a 5-membered aryl or heteroaryl, or when L1 comprises a fused aryl or heteroaryl having a 5-membered ring, the monomer units are 1,4-conjugated or 2,5-conjugated on the 5-membered ring.

[0091] Alternatively, L1 may be represented by at least one monomer unit A and at least one monomer unit D. In some embodiments, the combination of n and m is an integer selected from 2-10, 3-10, 3-9, 3-8, 3-10, or 3-7.

[0092] In some embodiments, L1 is [ka] is selected from.

[0093] As stated above, A and D may each be the same moiety such that L1 is an alternating pi-conjugated core. Alternatively, A and D may each be different. Because L1 includes a composition of alternating donor / acceptor structural units, structures not attached with alternating donor / acceptor compositions are excluded from the scope of the present invention. For example, butadienylene, polyalkenylene, phenylalkenylene, and polyphenylalkenylene are excluded.

[0094] In some embodiments, the optional substituents of L1 are selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, and optionally substituted alkynyloxy. In other embodiments, the optional substituents of D are selected from halogen, cyano, alkyl, alkenyl, alkoxy, and alkenyloxy. In some embodiments, the optional substituents of L1 are independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0095] As used herein, monomer unit D is an electron donating (electron rich) moiety relative to monomer unit A. In this regard, monomer unit A is an electron withdrawing / accepting (electron poor) moiety. When ordered, a DA combination is a permutation of similar moieties that are in conjugation (-D n -or-A n -) gives rise to an intramolecular charge-transfer excited state whose absorption and emission are further towards the red.

[0096] In some embodiments, A is an electron accepting moiety. An electron acceptor is a chemical entity that accepts electrons transferred from another moiety or compound. In some embodiments, A has an electron accepting substituent. In other embodiments, A has an electron withdrawing substituent.

[0097] In some embodiments, A is independently selected from an optionally substituted alkenylene or an optionally substituted heteroarylene. In some embodiments, A is independently selected from a cyano-substituted alkenylene or an optionally substituted heteroarylene. In some embodiments, the cyano-substituted alkenylene is a mono-substituted alkenylene or a di-substituted alkenylene. In other embodiments, the optionally substituted heteroarylene is an optionally substituted monocyclic heteroarylene or an optionally substituted fused heteroarylene. The heteroarylene can be a 5-membered or 6-membered heteroarylene. The heteroarylene can be a fused heteroarylene. In some embodiments, the heteroarylene is a fused 5,5-membered heteroarylene, a fused 5,6-membered heteroarylene, a fused 6,6-membered heteroarylene, a fused 5,5,6-membered heteroarylene, a fused 5,6,6-membered heteroarylene, a fused 6,6,6-membered heteroarylene, a fused 5,5,6,6-membered heteroarylene, a fused 5,6,6,6-membered heteroarylene, or a fused 6,6,6,6-membered heteroarylene.

[0098] In some embodiments, the optional substituents on A are selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, and optionally substituted alkynyloxy. In other embodiments, the optional substituents on A are selected from halogen, cyano, alkyl, alkenyl, alkoxy, and alkenyloxy.

[0099] In some embodiments, A is [ka] is selected from the group consisting of [ka] represents a bond to D or to L2; each X1 is independently selected from C, O, N, S, and Se; each X2, if present, is independently selected from C, O, N, S, and Se; When X2 is present, at least one of X1 and X2 is O, N, or S; R is independently selected from H, halo, cyano, and optionally substituted alkyl.

[0100] In some embodiments, A is a moiety of formula (II): [ka] During the ceremony, [ka] represents a bond to D or to L2; R2, R3, R4, and R5 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R2 and R3 are linked to form an optionally substituted heterocyclyl, an optionally substituted heteroaryl; or R4 and R5 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl.

[0101] In some embodiments, R2 and R3 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl. In other embodiments, R2 and R3 are independently selected from H, halogen, optionally substituted alkyl, and optionally substituted alkenyl. In other embodiments, R2 and R3 are independently selected from H, halogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C1-C6 alkoxy, and optionally substituted C2-C6 alkenyloxy. In other embodiments, R2 and R3 are independently selected from H, halogen, and C1-C6 alkyl.

[0102] In some embodiments, A is a moiety of formula (II): [ka] During the ceremony, [ka] represents a bond to D or to L2; R2 and R3 are linked to form an optionally substituted heterocyclyl, an optionally substituted heteroaryl; R4 and R5 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R4 and R5 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl.

[0103] In some embodiments, R2 and R3 are linked to form an optionally substituted heteroaryl that forms a conjugated π system with the phenyl moiety.

[0104] In some embodiments, R4 and R5 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl. In other embodiments, R4 and R5 are independently selected from H, halogen, optionally substituted alkyl, and optionally substituted alkenyl. In other embodiments, R4 and R5 are independently selected from H, halogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C1-C6 alkoxy, and optionally substituted C2-C6 alkenyloxy. In other embodiments, R4 and R5 are independently selected from H, halogen, and C1-C6 alkyl.

[0105] In some embodiments, R4 and R5 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl. In other embodiments, R4 and R5 are linked to form an optionally substituted cycloalkyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl. In some embodiments, R4 and R5 are linked to form an optionally substituted aryl or an optionally substituted heteroaryl. In some embodiments, R4 and R5 are linked to form an optionally substituted heteroaryl that forms a conjugated π system with the phenyl moiety.

[0106] In some embodiments, A is [ka] where R4 and R5 are as disclosed herein.

[0107] In some embodiments, A is [ka] where R4 and R5 are as disclosed herein.

[0108] In some embodiments, A is [ka] It is.

[0109] In some embodiments, n is an integer selected from 1-4, 1-3, 1-2, 2-4, 3-4, or 3-5.

[0110] In some embodiments, D is an electron donating moiety. An electron donor is a chemical entity that donates electrons that are transferred to another moiety or compound. In some embodiments, D has an electron donating substituent.

[0111] In some embodiments, D is independently selected from an optionally substituted alkenylene, an optionally substituted arylene, or an optionally substituted heteroarylene. In some embodiments, D is independently selected from an alkenylene, an arylene, or an optionally substituted heteroarylene. In other embodiments, the arylene is phenylene. In other embodiments, the optionally substituted heteroarylene is an optionally substituted monocyclic heteroarylene or an optionally substituted fused heteroarylene. The heteroarylene can be a 5-membered or 6-membered heteroarylene. The heteroarylene can be a fused heteroarylene. In some embodiments, the heteroarylene is a fused 5,5-membered heteroarylene, a fused 5,6-membered heteroarylene, a fused 6,6-membered heteroarylene, a fused 5,5,6-membered heteroarylene, a fused 5,6,6-membered heteroarylene, a fused 6,6,6-membered heteroarylene, a fused 5,5,6,6-membered heteroarylene, a fused 5,6,6,6-membered heteroarylene, or a fused 6,6,6,6-membered heteroarylene.

[0112] In some embodiments, the optional substituents on D are selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, and optionally substituted alkynyloxy. In other embodiments, the optional substituents on D are selected from halogen, cyano, alkyl, alkenyl, alkoxy, and alkenyloxy.

[0113] In some embodiments, D is [ka] is a moiety selected from [ka] represents a bond to A or to L2; each X1 is independently selected from C, O, N, S, and Se; each X2, if present, is independently selected from C, O, N, S, and Se; When X2 is present, at least one of X1 and X2 is O, N, or S; R is independently selected from H, halo, cyano, and optionally substituted alkyl.

[0114] In some embodiments, D is an optionally substituted 5-membered heteroarylene.

[0115] In some embodiments, D is a moiety of formula (III): [ka] wherein Y is NR, O, S, or Se; R6 and R7 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R6 and R7 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl; R is selected from H, halo, cyano, and optionally substituted alkyl.

[0116] In some embodiments, R6 and R7 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkoxy, or optionally substituted alkenyloxy. In other embodiments, R6 and R7 are independently selected from H, halogen, optionally substituted alkyl, or optionally substituted alkoxy. In other embodiments, R6 and R7 are independently selected from H, halogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C1-C6 alkoxy, or optionally substituted C2-C6 alkenyloxy. In other embodiments, R6 and R7 are independently selected from H, halogen, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 alkoxy. In other embodiments, R6 and R7 are independently selected from H, halogen, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 alkoxy. In other embodiments, R6 and R7 are independently selected from H, halogen, or optionally substituted C1-C6 alkyl.

[0117] In some embodiments, R6 and R7 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl. In other embodiments, R6 and R7 are linked to form an optionally substituted cycloalkyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl.

[0118] In some embodiments, D is [ka] where R6 and R7 are as disclosed herein.

[0119] In some embodiments, D is [ka] It is.

[0120] In some embodiments, m is an integer selected from 1-4, 1-3, 1-2, 2-4, 3-4, or 3-5.

[0121] In some embodiments, n and m together add up to at least 3. In other embodiments, n and m together add up to at least 4 or 5.

[0122] In some embodiments, each R1 is independently selected from optionally substituted alkyl, optionally substituted alkoxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted oxyacyloxy, or optionally substituted thio, or optionally substituted phosphoryl. In other embodiments, R1 is optionally substituted alkoxy, optionally substituted oxyacyl, or optionally substituted amino. In another embodiment, R1 is an optionally substituted polyethoxy having 3 to 10 monomer units. In another embodiment, the chain length of R1 is about 3 to about 10. In another embodiment, R1 is an optionally substituted C3 to C6 10 Alkoxy, optionally substituted C3-C 10 Alkylamino, optionally substituted C3-C 10 Dialkylamino, optionally substituted C3-C 10 In some embodiments, R1 is independently selected from optionally substituted alkyl, optionally substituted alkoxy. In some embodiments, R1 is independently selected from optionally substituted alkyl and optionally substituted alkoxy, each of which may be substituted with amino or alkylamino.

[0123] In some embodiments, the optional substituents of R1 are independently selected from oxy, oxyacyl, acyl, amino, phosphoryl, thiol, alkyl, alkenyl, alkynyl, oxyalkyl, alkylacyloxy, sulfonyl, chlorate, or a charged species thereof. In some embodiments, the optional substituents of R1 are independently selected from hydroxyl, carboxyl, phosphate, amino, alkylamino, dialkylamino, chlorate, sulfate, acetate, or a charged species thereof. In some embodiments, the optional substituents of R1 are tertiary amino. The tertiary amino can be neutralized with a counterion, which can be a halide.

[0124] In other embodiments, the optional substituents of R1 are hydrophilic moieties. In other embodiments, the optional substituents of R1 are charged moieties. Examples of hydrophilic moieties and / or charged moieties include trialkylammonium halides. For example, the charged moiety can be trimethylammonium iodide. In this embodiment, R1 is terminated with trimethylammonium, which imparts a positive charge to R1 (e.g., alkyl) when substituted. Other cationic charged groups include, but are not limited to, pyridinium, pyrrolidinium, imidazolium, guanidinium, sulfonium, thiouronium, and phosphonium. Other anionic charged groups include, but are not limited to, chlorate, sulfate, phosphate, acetate, carboxyl, and hydroxide. The hydrophilic moiety and / or charged moiety may be in a zwitterionic form, containing both cationic and anionic charged groups through covalent bonds. Excess charge can be neutralized by acceptable cations or anions.

[0125] Basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides, e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, e.g., dimethyl and diethyl sulfate. Examples of optional substituents for R1 are: [ka] can be selected from:

[0126] For example, to achieve the functionality of "Gram" selectivity, the compounds of the invention may contain a charged ammonium group. To achieve a transmembrane configuration, one side is required to cross the hydrophobic bilayer core. Once this is achieved, the charged moiety "holds" the compound throughout the bilayer and does not easily detach. This allows visualization of Gram-positive bacterial cells for extended periods of time.

[0127] To balance hydrophilicity and hydrophobicity to facilitate insertion of the compound of formula (I) into the lipid bilayer, there should be at least one side chain at each end of the backbone. In this regard, in some embodiments, q is an integer from 1 to 4. In other embodiments, q is an integer from 1 to 3. In some embodiments, q' is an integer from 1 to 4. In other embodiments, q' is an integer from 1 to 3. In this regard, there are a total of at least 2 R1 groups, at least 3 R1 groups, at least 4 R1 groups, at least 5 R1 groups, or at least 6 R1 groups in a compound of formula (I) or subformulas (Ia-Ig).

[0128] In order for the compound of formula (I) to maintain its linear configuration, R1 is preferentially located at the meta and / or para positions of the terminal phenyl group. In some embodiments, R1 is located at the meta and para positions of the terminal phenyl group. In other embodiments, R1 is located at the meta or para positions of the terminal phenyl group.

[0129] In some embodiments, the compound of formula (I) is a compound of formula (Ia): [ka] wherein L1, A, D, R1, n, m, q, and q' are as disclosed herein.

[0130] In some embodiments, the compound of formula (I) is a compound of formula (Ib): [ka] wherein L1, A, D, R1, n, m, q, and q' are as disclosed herein.

[0131] In some embodiments, the compound of formula (I) is a compound of formula (Ib), wherein: A is a moiety of formula (II): [ka] During the ceremony, [ka] represents a bond to D or to L2; R2 and R3 are linked to form an optionally substituted heterocyclyl, an optionally substituted heteroaryl; R4 and R5 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R4 and R5 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl; D is a moiety of formula (III): [ka] wherein Y is NR, O, or S; R6 and R7 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R6 and R7 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl; R is selected from H, halo, cyano, and optionally substituted alkyl.

[0132] In some embodiments, the compound of formula (I) is a compound of formula (Ib), wherein: A is, [ka] where R4 and R5 are as disclosed herein; D is [ka] where R6 and R7 are as disclosed herein.

[0133] In some embodiments, the compound of formula (I) is a compound of formula (Ic): [ka] each R1 is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy, or optionally substituted thio, or optionally substituted phosphoryl; R2 and R3 are linked to form an optionally substituted heterocyclyl, an optionally substituted heteroaryl; R4 and R5 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R4 and R5 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl; R6 and R7 are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R6 and R7 are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, an optionally substituted heteroaryl; each R8 is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; Y is NR, O, or S; each p is an integer independently selected from 0 to 4; q is an integer selected from 1 to 5; q' is an integer selected from 1 to 5.

[0134] In some embodiments, the compound of formula (I) is a compound of formula (Id): [ka] wherein R1, R4, R5, R6, R7, R8, Y, p, q, and q' are as disclosed herein.

[0135] In some embodiments, the compound of formula (I) is a compound of formula (Ie): [ka] wherein R1, R4, R5, R6, R7, R8, p, q, and q' are as disclosed herein.

[0136] In some embodiments, the compound of formula (I) is a compound of formula (If): [ka] wherein R1, R4, R5, R6, R7, R8, p, q, and q' are as disclosed herein; Each R9 is independently H or optionally substituted alkyl; Each t is an integer independently selected from 1 to 8.

[0137] In some embodiments, each R8 is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl. In other embodiments, each R8 is independently selected from halogen, cyano, or optionally substituted alkyl. In other embodiments, each R8 is independently selected from halogen, cyano, methyl, ethyl, or propyl.

[0138] In some embodiments, each R8 is independently selected from H or optionally substituted C1-C5 alkyl. In other embodiments, each R8 is independently selected from H or C1-C5 alkyl. In other embodiments, each R8 is independently selected from H, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, sec-butyl, isobutyl, or tert-butyl.

[0139] In some embodiments, each R9 is optionally substituted alkyl. In other embodiments, each R9 is C1-C5 alkyl. In other embodiments, each R9 is independently selected from methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, sec-butyl, isobutyl, or tert-butyl.

[0140] In some embodiments, the compound of formula (I), or a salt or solvate thereof, [ka] is selected from.

[0141] In some embodiments, the compound of formula (I) is a salt thereof. The salt form may be a protonated salt or may be produced by alkylating the compound of formula (I) with a halocarbon. For example, an alkyl halide (e.g., CH3Br or CH3I) may be used. In some embodiments, the compound of formula (I), or a salt or solvate thereof, is a quaternary ammonium salt. In this regard, when R1 is an optionally substituted amino, each R1 may be alkylated to exhibit at least a positive charge at each end.

[0142] For example, the quaternary ammonium salt of the compound of formula (I) is [ka] It is possible.

[0143] The compounds of the present invention may be in crystalline form, either 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.

[0144] The compounds of the invention can be provided as a solid or a solution, for example, the compounds can be provided as a lyophilized powder.

[0145] The compound of the present invention can be provided as a composition. The composition can include the compound as one entity in a polar medium. As used herein, "polar medium" includes polar protic solvents and polar aprotic solvents. Polar solvents have a large dipole moment or partial charge and contain bonds between atoms with a wide range of electronegativity, such as oxygen and hydrogen. Protic solvents have OH or NH bonds. These bonds allow for participation in hydrogen bonds. Furthermore, these OH or NH bonds can carry protons (H +) can act as a source of hydrogen. An aprotic solvent may have hydrogen somewhere in it, but lacks an OH or NH bond and therefore cannot form a hydrogen bond with itself. Polar solvents include, but are not limited to, dichloromethane, tetrahydrofuran, ethyl acetate, acetone, N,N-dimethylformamide, acetonitrile, dimethylsulfoxide, ammonia, butanol, propanol, ethanol, methanol, acetic acid, and water. Solvent mixtures in which the majority of the solvent mixture is a polar solvent are also included within this definition. For example, a water-based solvent or solvent system may contain dissolved ions, salts, and molecules such as amino acids, proteins, sugars, and phospholipids. These 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. Thus, biological fluids, physiological solutions, and media are also within this definition.

[0146] In some embodiments, the composition comprises a compound of formula (I) or sub-formulas (Ia-Ig) and a polar medium. For example, in other embodiments, when the MIC value is 256 μM, the composition comprises a compound of formula (I) or sub-formulas (Ia-Ig) and a polar medium, and the final concentration of the compound of formula (I) or sub-formulas (Ia-Ig) is about 130 μM. In other embodiments, the concentration is about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 100 μM, about 150 μM, about 200 μM, about 250 μM, about 300 μM, about 350 μM, or about 400 μM. In other embodiments, the concentration is 10 μM or less, 20 μM or less, 30 μM or less, 40 μM or less, 50 μM or less, 100 μM or less, 150 μM or less, 200 μM or less, 250 μM or less, 300 μM or less, 350 μM or less, or 400 μM or less.

[0147] Alternatively, the compound can be provided as a kit. The kit can include the compound and a polar medium. The compound and the polar medium can be in separate containers or as separate packaged components to be mixed before use. Alternatively, the kit can include a composition of the compound in a first polar medium and, separately, a second medium as separate containerized components. The kit can further include another dye for staining another component of the bacterial cell. For example, the kit can further include FM 4-64. The kit can further include an excipient. The excipient can act to further stabilize the compound and / or to further quench the fluorescence of the compound before it enters the bacterial cell membrane, thereby reducing background noise.

[0148] Flow cytometry is a technique for investigating biological cells, bacterial cells, and extracellular vesicles (e.g., lipid vesicles, exosomes) and is used in laboratories in both academic and industrial facilities. These biological samples are characterized by the essential presence of lipid bilayers (membranes), and the COEs are designed to maintain high affinity with lipid bilayers (membranes). Thus, the physicochemical and optoelectronic properties of the compounds of the present invention when bound within lipid bilayers allow them to be used as dyes for flow cytometry applications.

[0149] The compounds of the present invention are suitable for use as fluorescent probes. For example, the backbone of the compounds can be tailored to have a certain length (about 3.4 nm) and also to have a topology suitable for a particular membrane insertion. This extended backbone length can accommodate the thickness of the lipid bilayer (about 4 nm) and therefore can be negligibly toxic to cells and bacteria such as E. coli and S. aureus (MIC values ​​above 256 μM). In this regard, the compounds can be used as dyes in biological systems without reducing cell viability. Secondly, to achieve sufficient solubility in aqueous media, six positively charged side chains can be included as terminal groups in the compounds. It has been found that as the length of the hydrophobic conjugated core increases, the number of charged side chains must be balanced to give the COE compounds good water solubility. An unfavorable hydrophilic / hydrophobic profile and strong aggregation tendency will affect effective membrane insertion and affect fluorescence stability (prone to fluorescence quenching). For example, a five-benzene ring oligophenylenevinylene backbone with four positively charged side chains at both ends shows some tendency to aggregate. -1 In this case, the aqueous solution would be visible to the naked eye as being turbid, suggesting the presence of many aggregates in the incompletely dissolved suspension. When modified with six charged side chains, the COE compounds have excellent solubility in aqueous solution (>50 mg mL -1 ) are shown. The feature that the aqueous solutions of COE compounds are transparent and homogeneous enhances their practical value, especially when they are required to be stored at high concentrations before dilution and use. Thirdly, the extended backbone of COE compounds is expected to promote improved membrane stability, while the increased hydrophilic groups are expected to increase water solubility. In addition, it was assumed that the combination of these physical features would increase both hydrophobic and electrostatic interactions, thereby facilitating insertion into the bilayer. Therefore, when used as membrane labeling dyes, COE compounds can be stably incorporated into lipid bilayers by strong binding forces for long-term staining.

[0150] Figure 16 shows the change in fluorescence emission when the A and D units of this compound are varied. With reference to COE-Quin, where alternating phenyl moieties exhibit weak electron-accepting and electron-donating properties, the fluorescence excitation, and therefore emission, can be calibrated to a specific wavelength by tuning the electron-accepting and electron-donating properties of the A and D units. The choice of these units also narrows the FWHM and therefore improves specificity.

[0151] Thus, the present invention also provides a compound of formula (Ig), or a salt or solvate thereof: [ka] During the ceremony, each R1 is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy, or optionally substituted thio, or optionally substituted phosphoryl; q is an integer selected from 1 to 5; q' is an integer selected from 1 to 5; and r is an integer selected from 1 to 5.

[0152] These compounds can be applied to stain bacterial cells, mammalian cells (including but not limited to A549 cancer cells and red blood cells), and exosomes (especially unbound exosomes).

[0153] Thus, the present invention provides a method for labeling cells and / or lipid vesicles, comprising the steps of: a) incubating a compound of formula (I), sub-formulas (Ia-Ig), or a salt or solvate thereof with cells and / or lipid vesicles.

[0154] The cells can be mammalian cells or bacterial cells. 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 the group consisting of Escherichia coli, P. aeruginosa, Staphylococcus aureus, E. faecalis, Shewanella oneidensis, B. megaterium, or combinations thereof. The mammalian cells can be from a cell line or from a sample obtained from a subject.

[0155] In some embodiments, the sample of bacterial cells is a sample of planktonic bacterial cells.

[0156] As used herein, "plankton" refers to free-flowing bacterial cells that are in suspension. This is in contrast to sessile (or biofilm) cells, which are structured aggregates of bacterial cells encapsulated in a self-generated polymeric matrix and attached to an inert or biological surface. In this regard, plankton are freely growing bacteria that constitute the populations that grow in test tube and flask cultures in the laboratory.

[0157] A lipid vesicle is a structure inside or outside a cell that consists of liquid or cytoplasm enclosed by a lipid bilayer. Vesicles form naturally during the processes of secretion (exocytosis), uptake (endocytosis), and transport of substances within the plasma membrane. Alternatively, they may be prepared artificially, in which case they are called liposomes. Unilamellar lipid vesicles have one phospholipid bilayer, while multilamellar lipid vesicles have two or more bilayers. Vesicles may also fuse with other organelles within the cell. Vesicles released from cells are extracellular vesicles. For example, lipid vesicles can be exosomes. Exosomes are membrane-bound extracellular vesicles produced in the endosomal compartment of most eukaryotic cells. These lipid vesicles are included within the scope.

[0158] In some embodiments, the lipid vesicle is an extracellular vesicle, hi other embodiments, the lipid vesicle is an exosome.

[0159] In some embodiments, the method comprises: contacting a sample of cells and / or lipid vesicles under defined conditions with a compound of formula (I), sub-formulae (Ia-Ig), or a salt or solvate thereof; Includes.

[0160] In some embodiments, cells and / or lipid vesicles are incubated with a compound of formula (I), sub-formulas (Ia-Ig), or a salt or solvate thereof at a compound concentration that is less than its MIC value. In other embodiments, cells and / or lipid vesicles are incubated with a compound of formula (I), sub-formulas (Ia-Ig), or a salt or solvate thereof at a compound concentration that is less than half its MIC value. In some embodiments, cells and / or lipid vesicles are incubated with a compound of formula (I), sub-formulas (Ia-Ig), or a salt or solvate thereof at a compound concentration that is less than one-third its MIC value. The minimum inhibitory concentration (MIC) is the lowest concentration of a chemical that prevents visible growth of one or more bacteria. The MIC is microorganism and chemical dependent. The MIC of a compound can be determined by incubating cells with various concentrations of the compound in liquid medium or on solid growth medium (e.g., agar) plates and identifying the compound concentration at which bacteria did not grow and the next lowest dose that allowed bacterial growth. This information can also be derived from a plot of turbidity versus compound concentration.Other methods of determining MIC values ​​are available, for example the Etest test or Kirby-Bauer test may be used.

[0161] In some embodiments, the MIC value is greater than about 1 μM. In other embodiments, the MIC value is greater than about 5 μM, greater than about 10 μM, greater than about 20 μM, greater than about 30 μM, greater than about 40 μM, greater than about 50 μM, greater than about 60 μM, greater than about 70 μM, greater than about 80 μM, greater than about 90 μM, greater than about 100 μM, greater than about 150 μM, greater than about 200 μM, greater than about 250 μM, greater than about 300 μM, greater than about 350 μM, greater than about 400 μM, or greater than about 500 μM.

[0162] In some embodiments, the compound of formula (I), subformulae (Ia-Ig), or a salt or solvate thereof 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 ... nM ~ about 900nM, about 1nM - about 800nM, about 1nM - about 700nM, about 1nM - about 600nM, about 1nM - about 500nM, about 1nM - 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.

[0163] In some embodiments, the compound of formula (I), subformulae (Ia-Ig), or a salt or solvate thereof is incubated with cells and / or lipid vesicles 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 or ambient temperature.

[0164] In some embodiments, the compound of formula (I), subformulae (Ia-Ig), or a salt or solvate thereof is incubated with cells and / or lipid vesicles for about 5 minutes to about 120 minutes. In other embodiments, the period is about 5 minutes to about 110 minutes, about 5 minutes to about 100 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 80 minutes, about 5 minutes to about 70 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 50 minutes, about 5 minutes to about 40 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, or about 5 minutes to about 10 minutes.

[0165] The compound of formula (I), sub-formulas (Ia-Ig), or a salt or solvate thereof, and the cells and / or lipid vesicles may be incubated in an aqueous medium.

[0166] The term "aqueous medium" as used herein refers to water-based solvents or solvent systems, and solvents or solvent systems that are primarily composed of water. These solvents can be polar or non-polar, and / or protic or aprotic. A solvent system refers to a combination of solvents that results in one final phase. Both "solvent" and "solvent system" can include, but are not limited to, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, dioxane, chloroform, diethyl ether, dichloromethane, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethylsulfoxide, nitromethane, propylene carbonate, formic acid, butanol, isopropanol, propanol, ethanol, methanol, acetic acid, ethylene glycol, diethylene glycol, or water. A water-based solvent or solvent system may contain dissolved ions, salts, and molecules such as amino acids, proteins, sugars, and phospholipids. These 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. Thus, biological fluids, physiological solutions, and media are also within this definition.

[0167] The present invention also provides a method for detecting cells and / or lipid vesicles using a fluorescence detector, comprising: a) incubating a compound of formula (I), sub-formulas (Ia-Ig), or a salt or solvate thereof with cells and / or lipid vesicles; and b) passing the cells and / or lipid vesicles through a fluorescence detector The present invention provides a method comprising:

[0168] Because the compounds of formula (I), sub-formulas (Ia-Ig), or salts or solvates thereof fluoresce when inserted into cell membranes or lipid bilayers, fluorescence-based techniques can be used to detect cells and / or lipid vesicles that have incorporated the compounds of formula (I), sub-formulas (Ia-Ig), or salts or solvates thereof. 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).

[0169] In some embodiments, the method of detecting cells and / or lipid vesicles using a flow cytometer comprises: a) incubating a compound of formula (I), sub-formulas (Ia-Ig), or a salt or solvate thereof with cells and / or lipid vesicles; and b) running the cells and / or lipid vesicles through a flow cytometer Includes.

[0170] Cells and / or lipid vesicles into which a compound of formula (I), sub-formulae (Ia-Ig), or a salt or solvate thereof has been incorporated, can be excited by electromagnetic radiation (source) in the wavelength range of about 300 nm to about 1000 nm.

[0171] The present invention also provides a method for detecting cells and / or lipid vesicles using a flow cytometer, comprising: a) Formula (IV); [ka] (In the formula, R1 is independently selected from halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, optionally substituted acylamino, optionally substituted aminoacyloxy, optionally substituted oxyacylamino, optionally substituted oxyacyloxy, or optionally substituted thio or optionally substituted phosphoryl; R2 is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; u is an integer selected from 3 to 15; each s is an integer independently selected from 0 to 4; q is an integer selected from 2 to 5; q' is an integer selected from 2 to 5. or a salt or solvate thereof with cells and / or lipid vesicles; and b) running the cells and / or lipid vesicles through a flow cytometer The present invention provides a method comprising:

[0172] In some embodiments, R2 is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl. In other embodiments, R2 is independently selected from halogen, cyano, or optionally substituted alkyl. In other embodiments, R2 is independently selected from halogen, cyano, methyl, ethyl, or propyl.

[0173] In some embodiments, u is an integer selected from 3 to 14. In other embodiments, u is an integer selected from 3 to 12, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 4 to 7, or 4 to 6. In other embodiments, u is 3, 4, 5, 6, or 7. In some embodiments, u is 4, 5, or 6. In other embodiments, u is 4.

[0174] Examples of salt or solvate forms of compounds of formula (IV) are shown below: These examples of compounds of formula (IV) are also within the scope of the present disclosure. [ka] TIFF2024534774000067.tif229170TIFF2024534774000068.tif230170TIFF2024534774000069.tif228170 TIFF2024534774000070.tif229170TIFF2024534774000071.tif229170TIFF2024534774000072.tif198170

[0175] The cells may be attached cells or may be in suspension. The lipid vesicles may be exosomes (membrane-bound extracellular vesicles produced in the endosomal compartment of eukaryotic cells), synthetic and non-synthetic liposomes, or lipid nanoparticles (spherical vesicles made of ionizable lipids that are positively charged at low pH and neutral at physiological pH). Advantageously, it has been found that after cell membrane insertion by the fluorescent probe, the fluorescent probe can enter the subsequent cell population.

[0176] The cells and / or lipid vesicles are stained with a compound of formula (I), subformulae (Ia-Ig), and / or formula (IV), or a salt or solvate thereof, after the incubation step. In some embodiments, the incubation period is from about 1 minute to about 12 days, from about 1 minute to about 10 days, from about 1 minute to about 8 days, from about 1 minute to about 6 days, from about 1 minute to about 5 days, from about 1 minute to about 4 days, from about 1 minute to about 3 days, from about 1 minute to about 2 days, from about 1 minute to about 24 hours, from about 1 minute to about 20 hours, from about 1 minute to about 16 hours, from about 1 minute to about 12 hours, from about 1 minute to about 10 hours, from about 1 minute to about 9 hours, from about 1 minute to about 8 hours, from about 1 minute to about 7 hours, In another embodiment, the incubation period is about 1 minute to about 6 hours, about 5 minutes to about 6 hours, about 5 minutes to about 5.5 hours, about 5 minutes to about 5 hours, about 5 minutes to about 4.5 hours, about 5 minutes to about 4 hours, about 5 minutes to about 3.5 hours, about 5 minutes to about 3 hours, about 5 minutes to about 2.5 hours, about 5 minutes to about 2 hours, about 5 minutes to about 1.5 hours, about 5 minutes to about 1 hour, about 5 minutes to about 50 minutes, about 5 minutes to about 40 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, or about 5 minutes to about 10 minutes. In another embodiment, the incubation period is about 10 minutes.

[0177] The concentration of the compound of formula (I), sub-formulae (Ia-Ig), and / or formula (IV), or salts or solvates thereof, can be controlled to allow a sufficient amount of time for analysis of the sample. In some embodiments, the cells and / or lipid vesicles are passed through the flow cytometer within 30 minutes of contacting the compound of formula (I) and / or formula (IV) with the sample. In other embodiments, the time is within 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 8 days, 10 days, or 12 days. In other embodiments, the time period is greater than about 30 minutes, greater than about 1 hour, greater than about 2 hours, greater than about 3 hours, greater than about 4 hours, greater than about 5 hours, greater than about 6 hours, greater than about 8 hours, greater than about 10 hours, greater than about 12 hours, greater than about 16 hours, greater than about 20 hours, greater than about 24 hours, greater than about 2 days, greater than about 3 days, greater than about 4 days, greater than about 5 days, greater than about 6 days, greater than about 8 days, greater than about 10 days, or greater than about 12 days.

[0178] In some embodiments, cells and / or lipid vesicles can be run through a flow cytometer without a purification step. This is possible because the free compounds of formula (I), subformulae (Ia-Ig), and / or formula (IV), or salts or solvates thereof, are weakly luminescent and produce relatively low background.

[0179] The cells and / or lipid vesicles can be flowed through a flow cytometer, whereby the cells and / or lipid vesicles can be excited by electromagnetic radiation and subsequently detected by a detector.Thus, the method can further include exposing the cells and / or lipid vesicles to electromagnetic radiation having a wavelength of less than about 2500 nm or less than about 1000 nm.

[0180] In some embodiments, the compounds of formula (I), subformulae (Ia-Ig), and / or formula (IV), or salts or solvates thereof, exhibit fluorescence excitation at wavelengths from about 300 nm to about 1000 nm, or from about 400 nm to about 700 nm. Depending on the combination of D and A, the excitation may be tunable within this wavelength range.

[0181] In some embodiments, the compounds of Formula (I), subformulae (Ia-Ig), and / or Formula (IV), or salts or solvates thereof, exhibit a fluorescence excitation peak with a full width at half maximum (FWHM) of about 10 nm to about 200 nm. In other embodiments, the FWHM is about 10 nm to about 190 nm, about 10 nm to about 180 nm, about 10 nm to about 170 nm, about 10 nm to about 160 nm, about 10 nm to about 150 nm, about 10 nm to about 140 nm, about 10 nm to about 130 nm, about 10 nm to about 120 nm, about 10 nm to about 110 nm, or about 10 nm to about 100 nm. In other embodiments, the FWHM is about 20 nm to about 200 nm, about 30 nm to about 200 nm, about 40 nm to about 200 nm, about 50 nm to about 200 nm, about 60 nm to about 200 nm, about 70 nm to about 200 nm, about 80 nm to about 200 nm, about 90 nm to about 200 nm, or about 100 nm to about 200 nm.

[0182] In some embodiments, the compounds of formula (I), subformulae (Ia-Ig), and / or formula (IV), or salts or solvates thereof, exhibit fluorescence emission at wavelengths from about 300 nm to about 2000 nm. In other embodiments, the range is from about 300 nm to about 1900 nm, from about 300 nm to about 1800 nm, from about 300 nm to about 1700 nm, from about 300 nm to about 1600 nm, or from about 300 nm to about 1500 nm. Depending on the combination of D and A, excitation may be tunable within this wavelength range.

[0183] In some embodiments, the compounds of formula (I), subformulae (Ia-Ig), and / or formula (IV), or salts or solvates thereof, exhibit a fluorescence emission peak with a full width at half maximum (FWHM) of about 10 nm to about 200 nm. In other embodiments, the FWHM is about 10 nm to about 190 nm, about 10 nm to about 180 nm, about 10 nm to about 170 nm, about 10 nm to about 160 nm, about 10 nm to about 150 nm, about 10 nm to about 140 nm, about 10 nm to about 130 nm, about 10 nm to about 120 nm, about 10 nm to about 110 nm, or about 10 nm to about 100 nm. In other embodiments, the FWHM is about 20 nm to about 200 nm, about 30 nm to about 200 nm, about 40 nm to about 200 nm, about 50 nm to about 200 nm, about 60 nm to about 200 nm, about 70 nm to about 200 nm, about 80 nm to about 200 nm, about 90 nm to about 200 nm, or about 100 nm to about 200 nm.

[0184] In some embodiments, when a compound of Formula (I), sub-formulae (Ia-Ig), and / or Formula (IV), or a salt or solvate thereof is inserted into a cell membrane and / or a lipid membrane, the compound of Formula (I), sub-formulae (Ia-Ig), and / or Formula (IV), or a salt or solvate thereof exhibits a luminescence intensity that is from about 2-fold to about 500-fold greater than a control sample of the compound of Formula (I), sub-formulae (Ia-Ig), and / or Formula (IV), or a salt or solvate thereof. In other embodiments, the luminescence intensity is about 10 times to about 500 times, about 20 times to about 500 times, about 30 times to about 500 times, about 40 times to about 500 times, about 50 times to about 500 times, about 60 times to about 500 times, about 70 times to about 500 times, about 80 times to about 500 times, about 90 times to about 500 times, about 100 times to about 500 times, about 100 times to about 450 times, about 150 times to about 450 times, about 200 times to about 450 times, about 250 times to about 450 times, about 300 times to about 450 times, or about 350 times to about 450 times.

[0185] A control in an experiment is a group separated from the rest of the experiment in which the independent variable being tested cannot affect the results. When testing samples of cells and / or lipid vesicles, at least one control sample may contain a compound of formula (I), subformulas (Ia-Ig), and / or formula (IV), or a salt or solvate thereof, in an aqueous / water medium. In this regard, the control sample does not contain cells and / or lipid vesicles.

[0186] Advantageously, the compounds of formula (I), sub-formulae (Ia-Ig) and / or formula (IV), or their salts or solvates, when dissolved in aqueous media, show low (or negligible) photoluminescence. However, when partitioned into lipid bilayers, the photoluminescence of the compounds of formula (I), sub-formulae (Ia-Ig) and / or formula (IV), or their salts or solvates, is enhanced. It is believed that the fluorescence is enhanced by a change in the local environment to a hydrophobic environment (the alkyl chains of the lipid bilayer). Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. In general, the emitted light has a longer wavelength than the absorbed light.

[0187] Advantageously, the compounds of formula (I), sub-formulae (Ia-Ig), and / or formula (IV), or salts or solvates thereof, are non-toxic (or exhibit low toxicity) and / or stable to excitation, so that the photoluminescence intensity can be maintained for a certain period of time under constant excitation. In this regard, when the compounds are excited under a suitable wavelength for imaging, the photoluminescence intensity does not decrease for a certain period of time. This is believed to be due to the conjugated system, which dissipates and transfers energy, thereby preventing localized heating and decomposition of the compounds. 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.

[0188] The compounds of formula (I), sub-formulae (Ia-Ig), and / or formula (IV), or salts or solvates thereof, may work in combination with other dyes, such as membrane dyes. For example, the commercially available dye FM4-64 can be added to recognize the type of bacterial envelope in situ in a bacterial mixture. In this regard, dual dye systems capable of recognizing polymicrobial samples are also disclosed. These methods are easy to use, requiring only simple application of the dye mixture, no fixation or other pretreatment, and the compounds of formula (I), sub-formulae (Ia-Ig), and / or formula (IV), or salts or solvates thereof, are stable in aqueous solution and can be used to monitor cells in biological systems.

[0189] Thus, in one embodiment, the method further comprises contacting the cells and / or lipid vesicles with another dye. The dye can be used to stain cell membranes, nuclei, DNA, RNA, or other organelles in the cells. The dye can be a fluorescent probe, such as FM4-64, FM 2-10, FM 1-43, propidium iodide, SYTO 82, SYTO 83, SYTO 84, SYTO 85, YOYO®-3 iodide, YO-PRO™-3 iodide, BOBO™-3 iodide, ethidium homodimer 1, ethidium homodimer 2, ethidium monoazide, acridine orange, CellMask™ plasma membrane stain, or Di-4-ANEPPS.

[0190] The present invention also provides a flow system for detecting and / or quantifying cells and / or lipid vesicles, comprising: a) compounds of formula (I), sub-formulae (Ia-Ig) and / or formula (IV), or salts or solvates thereof, for labelling cells and / or lipid vesicles; b) an inlet for introducing labeled cells and / or lipid vesicles into the flow system; c) a detection means in fluid communication with the inlet for detecting fluorescent emissions from the labeled cells and / or lipid vesicles; and Optionally d) providing a flow system including counting means for quantifying the labeled cells and / or lipid vesicles.

[0191] The flow system can be, for example, a microfluidic chip.

[0192] In some embodiments, the flow system further comprises an incubation means by which the compound of formula (I), subformulas (Ia-Ig), and / or formula (IV), or a salt or solvate thereof, is inserted into a cell membrane or into a lipid bilayer.

[0193] In some embodiments, the detection means is a fluorescence detector.

[0194] It will be recognized that many further modifications and permutations of various aspects of the described embodiments are possible, and the described aspects are therefore intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

[0195] Throughout the following specification and claims, unless the context requires otherwise, the term "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps.

[0196] Reference in this specification to any prior publication (or information derived therefrom) or to any matter which is publicly known is not, and should not be construed as, an acknowledgment or admission, or any form of suggestion, that that prior publication (or information derived therefrom) or public knowledge forms part of the common general knowledge in the business field to which this specification pertains. EXAMPLES

[0197] General Protocol for the Synthesis of Compounds of Formula (I)

[0198] [ka]

[0199] [ka]

[0200] [ka]

[0201] (E)-1,2,3-Tris((6-bromohexyl)oxy)-5-(4-bromostyryl)benzene (Compound 3) Compound 1 (5.69 g, 8.46 mmol), compound 2 (4.07 g, 13.27 mmol), potassium tert-butoxide (0.99 g, 8.46 mmol), and 100 mL of dry THF were added to a round-bottom flask under the protection of nitrogen atmosphere. After 16 h of reaction under stirring at room temperature, the reaction mixture was poured into water and extracted with chloroform. The clear organic phase was dried over Na2SO4, and then the organic solvent was removed by rotary evaporator. After the crude product was purified by column chromatography using hexane and dichloromethane as eluents, the product was obtained as a white solid (6.63 g, 94% yield). 1 H NMR (400 MHz, chloroform-d) δ 7.49 - 7.44 (m, 2H), 7.37 - 7.33 (m, 2H), 6.99 (d, J = 16.2 Hz, 1H), 6.90 (d, J = 16.2 Hz, 1H), 6.70 (s, 2H), 4.06 - 4.00 (m, 4H), 3.99 - 3.95 (m, 2H), 3.49 - 3.39 (m, 6H), 1.96 - 1.71 (m, 12H), 1.60 - 1.47 (m, 12H). 13 C NMR (101 MHz, CDCl3) δ 153.56, 138.68, 136.62, 132.69, 132.12, 129.82, 128.18, 126.95, 121.51, 105.65, 73.58, 69.27, 34.25, 34.12, 33.20, 33.07, 30.48, 29.61, 28.44, 28.27, 25.70, 25.68.

[0202] (E)-2-(4-(3,4,5-tris((6-bromohexyl)oxy)styryl)phenyl)thiophene (Compound 5) Compound 3 (4.44 g, 5.58 mmol), compound 4 (4.16 g, 11.15 mmol), and Pd(PPh3)2Cl2 (157 mg, 0.223 mmol) were added to a round-bottom flask under the protection of nitrogen atmosphere. After purging using nitrogen, 50 mL of dry toluene was added to the reaction mixture, which was then heated at 120 °C under stirring for 16 h. After cooling to room temperature, the reaction mixture was poured into water and extracted with chloroform. The organic phase was dried over Na2SO4, and then the organic solvent was removed by rotary evaporator. The crude product was purified by silica gel column chromatography using hexane and dichloromethane as eluents to give the product as a light yellow solid (4.02 g, 90% yield). 1 H NMR (400 MHz, chloroform-d) δ 7.63 - 7.58 (m, 2H), 7.52 - 7.48 (m, 2H), 7.33 (dd, J = 3.6, 1.2 Hz, 1H), 7.28 (dd, J = 5.1, 1.1 Hz, 1H), 7.09 (dd, J = 5.1, 3.6 Hz, 1H), 7.03 (d, J = 16.2 Hz, 1H), 6.97 (d, J = 16.3 Hz, 1H), 6.72 (s, 2H), 4.09 - 3.95 (m, 6H), 3.49 - 3.39 (m, 6H), 1.97 - 1.73 (m, 12H), 1.62 - 1.48 (m, 12H). 13 C NMR (101 MHz, CDCl3) δ 153.55, 144.48, 138.57, 136.85, 133.85, 133.03, 129.11, 128.44, 127.64, 127.20, 126.45, 125.15, 123.34, 105.65, 73.59, 69.29, 34.24, 34.11, 33.21, 33.09, 30.50, 29.64, 28.46, 28.28, 25.72, 25.70.

[0203] (E)-Trimethyl(5-(4-(3,4,5-tris((6-bromohexyl)oxy)styryl)phenyl)thiophen-2-yl)stannane (Compound 6) Compound 5 (2.88 g, 3.60 mmol) and 40 mL of dry THF were added to the flask under nitrogen protection. The mixture was cooled to -80°C using a cryogenic reaction bath. Then, 3.6 mL of n-butyllithium in cyclohexane (2 M, 7.2 mmol) was added dropwise to the reaction mixture. After stirring at -80°C for 2 h, 14.4 mL of trimethyltin chloride in hexane (1 M, 14.4 mmol) was added, and then the reaction mixture was allowed to warm to room temperature. After stirring at room temperature overnight (about 16 h), the reaction mixture was poured into water and extracted with hexane. The organic phase was washed four times using water and then dried over Na2SO4. After the solvent was removed using a rotary evaporator and a vacuum pump, the crude product was obtained as a colorless oil. This was used in the next reaction without further purification (3.34, 96% yield). 1 H NMR (400 MHz, chloroform-d) δ 7.63 - 7.58 (m, 2H), 7.52 - 7.47 (m, 2H), 7.44 (d, J = 3.3 Hz, 1H), 7.17 (d, J = 3.3 Hz, 1H), 7.05 - 6.93 (m, 2H), 6.72 (s, 2H), 4.09 - 3.92 (m, 6H), 3.51 - 3.36 (m, 6H), 1.97 - 1.71 (m, 12H), 1.62 - 1.47 (m, 12H), 0.40 (s, 9H). 13 C NMR (101 MHz, CDCl3) δ 153.54, 150.19, 138.51, 138.18, 136.61, 136.59, 133.97, 133.09, 128.92, 127.74, 127.17, 126.47, 124.59, 105.60, 73.59, 69.27, 34.25, 34.12, 33.22, 33.09, 30.50, 29.64, 28.46, 28.29, 25.72, 25.70, -7.87.

[0204] 4,7-Bis(5-(4-((E)-3,4,5-tris((6-bromohexyl)oxy)styryl)phenyl)thiophen-2-yl)benzo[c][1,2,5]thiadiazole (compound 8) Compound 6 (2.76 g, 2.87 mmol), compound 7 (281 mg, 0.88 mmol), and Pd(PPh3)2Cl2 (27 mg, 0.038 mmol) were added to a round-bottom flask under the protection of nitrogen atmosphere. After purging with nitrogen, 10 mL of dry toluene was added to the reaction mixture, which was then heated at 120 °C under stirring for 16 h. After cooling to room temperature, the reaction mixture was poured into water and extracted with chloroform. The organic phase was dried over Na2SO4, and then the organic solvent was removed by rotary evaporator. The crude product was purified by silica gel column chromatography using hexane and dichloromethane as eluents. The product was dissolved in chloroform and precipitated using methanol, and the precipitate was collected by filtration and washed with methanol. After drying under reduced pressure, the product was obtained as a deep red solid (1.29 g, 78% yield). 1 H NMR (500 MHz, chloroform-d) δ 8.12 (d, J = 3.9 Hz, 2H), 7.89 (s, 2H), 7.69 (d, J = 8.4 Hz, 4H), 7.53 (d, J = 8.5 Hz, 4H), 7.43 (d, J = 3.8 Hz, 2H), 7.05 (d, J = 16.2 Hz, 2H), 6.99 (d, J = 16.2 Hz, 2H), 6.73 (s, 4H), 4.08 - 4.02 (m, 8H), 4.01 - 3.96 (m, 4H), 3.47 - 3.40 (m, 12H), 1.96 - 1.74 (m, 24H), 1.60 - 1.47 (m, 24H). 13 C NMR (126 MHz, CDCl3) δ 153.55, 152.90, 145.65, 138.96, 138.57, 137.22, 133.48, 132.96, 129.31, 129.03, 127.54, 127.26, 126.30, 126.06, 125.64, 124.36, 105.60, 73.60, 69.26, 34.28, 34.15, 33.21, 33.09, 30.50, 29.64, 28.46, 28.29, 25.72, 25.71.

[0205] Compound COE-BT Compound 8 (557 mg, 0.322 mmol) and chloroform (40 mL) were added to a single-necked round-bottom flask. After compound 8 was dissolved, 5 mL of a 2 M solution of trimethylamine in THF was added to the reaction mixture and stirred at 55° C. for 16 h. After the reaction, the crude product was precipitated and attached to the bottom of the flask. The relatively light-colored solution was poured off and the solid precipitate was gently rinsed five times with chloroform. The solid precipitate was then dissolved using 40 mL of methanol. 5 mL of a 3.2 M solution of trimethylamine in methanol was added to the reaction mixture and stirred at 55° C. for another 16 h. The solvent was removed by rotary evaporation and the solid was dried under reduced pressure. The final product was obtained as a deep red solid (605 mg, 90% yield). 1 H NMR (500 MHz, DMSO-d6) δ 8.29 - 8.18 (m, 4H), 7.81 (d, J = 8.0 Hz, 4H), 7.75 (d, J = 3.9 Hz, 2H), 7.68 (d, J = 8.2 Hz, 4H), 7.27 (s, 4H), 6.96 (s, 4H), 4.12 - 4.00 (m, 8H), 3.95 - 3.87 (m, 4H), 3.37 - 3.27 (m, 12H), 3.09 (s, 54H), 1.84 - 1.62 (m, 24H), 1.58 - 1.47 (m, 12H), 1.42 - 1.28 (m, 12H). 13 C NMR (126 MHz, DMSO) δ 153.57, 152.56, 145.89, 138.53, 138.02, 137.88, 133.42, 133.28, 129.93, 129.76, 128.01, 127.88, 126.56, 126.49, 125.68, 125.60, 106.00, 73.27, 69.11, 66.13, 60.82, 53.09, 30.40, 29.56, 26.54, 26.42, 26.02, 25.97, 23.01.

[0206] compound 10 Compound 6 (2.54 g, 2.64 mmol), compound 9 (0.31 g, 0.88 mmol), and Pd(PPh3)2Cl2 (25 mg, 0.035 mmol) were added to a round-bottom flask under the protection of nitrogen atmosphere. After purging with nitrogen, 10 mL of dry toluene was added to the reaction mixture, which was then heated at 120 °C under stirring for 16 h. After cooling to room temperature, the reaction mixture was poured into water and extracted with chloroform. The organic phase was dried over Na2SO4, and then the organic solvent was removed by rotary evaporator. The crude product was purified by silica gel column chromatography using hexane and dichloromethane as eluents. The product was dissolved in chloroform and precipitated using methanol, and the precipitate was collected by filtration and washed with methanol. After drying under reduced pressure, the product was obtained as a brown solid (0.69 g, 44% yield). 1 H NMR (500 MHz, chloroform-d) δ 8.91 (d, J = 4.1 Hz, 2H), 7.71 (d, J = 8.0 Hz, 4H), 7.49 (d, J = 8.5 Hz, 4H), 7.46 (d, J = 4.2 Hz, 2H), 7.03 (d, J = 16.2 Hz, 2H), 6.96 (d, J = 16.1 Hz, 2H), 6.72 (s, 4H), 4.11 - 3.94 (m, 12H), 3.51 - 3.39 (m, 12H), 1.97 - 1.73 (m, 24H), 1.63 - 1.45 (m, 24H). 13 C NMR (126 MHz, CDCl3) δ 153.56, 151.49, 149.12, 138.58, 137.90, 137.43, 134.40, 133.48, 132.94, 129.36, 127.50, 127.23, 126.32, 124.54, 113.50, 105.58, 73.61, 69.26, 34.29, 34.17, 33.22, 33.11, 30.53, 29.66, 28.48, 28.32, 25.74, 25.72.

[0207] Compound COE-BBT Compound 10 (196 mg, 0.110 mmol) and chloroform (20 mL) were added to a single-necked round-bottom flask. After compound 10 was dissolved, 2 mL of a 2M solution of trimethylamine in THF was added to the reaction mixture and stirred at 55° C. for 16 h. After the reaction, the crude product precipitated and adhered to the bottom of the flask. The relatively light-colored solution was poured off and the solid precipitate was gently rinsed five times with chloroform. The solid precipitate was then dissolved using 20 mL of methanol. 2 mL of a 3.2M solution of trimethylamine in methanol was added to the reaction mixture and stirred at 55° C. for another 16 h. The solvent was removed by rotary evaporation and the solid was dried under reduced pressure. The final product was obtained as a brown solid (219 mg, 93% yield). 1 H NMR (400 MHz, DMSO-d6) δ 9.13 - 9.04 (m, 2H), 7.97 - 7.83 (m, 6H), 7.77 - 7.66 (m, 4H), 7.29 (br, 4H), 6.97 (br, 4H), 4.12 - 3.99 (m, 8H), 3.95 - 3.85 (m, 4H), 3.39 - 3.23 (m, 12H), 3.09 (s, 54H), 1.86 - 1.63 (m, 24H), 1.59 - 1.47 (m, 12H), 1.43 - 1.28 (m, 12H). 13 C NMR (126 MHz, DMSO) δ 153.57, 151.50, 148.78, 138.24, 138.06, 137.70, 134.93, 133.40, 133.29, 130.12, 128.08, 127.89, 126.72, 125.98, 113.24, 106.01, 73.27, 69.10, 66.11, 58.48, 55.32, 55.29, 55.26, 53.07, 30.41, 29.56, 26.55, 26.43, 26.02, 25.98, 23.00.

[0208] compound 12 The synthesis and purification procedures were the same as for compound 5. The reactant tributyl(thiophen-2-yl)stannane (compound 4) was changed to (3,4-ethylenedioxythien-2-yl)trimethylstannane (compound 11), but the feed ratio was kept the same. The product was obtained as a colorless oil (5.49 g, 86% yield). 1 H NMR (500 MHz, chloroform-d) δ 7.70 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 16.2 Hz, 1H), 6.96 (d, J = 16.2 Hz, 1H), 6.72 (s, 2H), 6.31 (s, 1H), 4.36 - 4.31 (m, 2H), 4.29 - 4.23 (m, 2H), 4.08 - 4.01 (m, 4H), 3.99 - 3.94 (m, 2H), 3.48 - 3.39 (m, 6H), 1.96 - 1.72 (m, 12H), 1.62 - 1.46 (m, 12H). 13 C NMR (126 MHz, CDCl3) δ 153.50, 142.61, 138.62, 138.36, 135.80, 133.13, 132.72, 128.67, 127.87, 126.93, 126.42, 117.68, 105.48, 98.04, 73.57, 69.22, 65.14, 64.79, 34.28, 34.16, 33.20, 33.07, 30.48, 29.61, 28.44, 28.27, 25.70, 25.69.

[0209] compound 13 The synthesis and purification procedures are the same as for compound 6. The crude product was obtained as a colorless oil, which was used in the next reaction without further purification (2.55 g, 94% yield). 1H NMR (500 MHz, chloroform-d) δ 7.72 - 7.68 (m, 2H), 7.49 - 7.45 (m, 2H), 6.99 - 6.95 (m, 2H), 6.71 (s, 2H), 4.34 - 4.20 (m, 4H), 4.07 - 3.94 (m, 6H), 3.47 - 3.39 (m, 6H), 1.96 - 1.79 (m, 12H), 1.60 - 1.45 (m, 12H), 0.38 (s, 9H).

[0210] compound 14 The synthesis and purification procedures were the same as for compound 8. The product was obtained as a dark green solid (431 mg, 40% yield). 1 H NMR (500 MHz, chloroform-d) δ 7.85 (d, J = 8.5 Hz, 4H), 7.53 (d, J = 8.6 Hz, 4H), 7.04 (d, J = 16.2 Hz, 2H), 6.98 (d, J = 16.1 Hz, 2H), 6.73 (s, 4H), 4.53 - 4.46 (m, 4H), 4.39 - 4.32 (m, 4H), 4.09 - 3.95 (m, 12H), 3.48 - 3.40 (m, 12H), 1.96 - 1.72 (m, 24H), 1.60 - 1.48 (m, 24H). 13 C NMR (126 MHz, CDCl3) δ 153.51, 152.84, 142.17, 138.93, 138.45, 136.43, 133.07, 132.35, 129.01, 127.80, 126.97, 126.90, 122.06, 113.41, 109.52, 105.55, 73.57, 69.23, 65.03, 64.87, 34.26, 34.14, 33.19, 33.07, 30.48, 29.61, 28.44, 28.27, 25.70, 25.68.

[0211] Compound COE-EDOT: The synthesis and purification procedures were the same as for compound COE-BBT. The product was obtained as a dark green solid (181 mg, 93% yield). 1 H NMR (500 MHz, DMSO-d6, 353K) δ 7.85 - 7.80 (m, 4H), 7.69 - 7.65 (m, 4H), 7.21 (br, 4H), 6.94 (br, 4H), 4.50 (br, 4H), 4.37 (br, 4H), 4.13 - 4.04 (m, 8H), 3.99 - 3.91 (m, 4H), 3.45 - 3.35 (m, 12H), 3.13 (s, 54H), 1.86 - 1.66 (m, 24H), 1.60 - 1.49 (m, 12H), 1.46 - 1.34 (m, 12H). 13 C NMR (126 MHz, DMSO-d6, 353K) δ 153.30, 152.36, 142.64, 139.29, 138.53, 136.80, 133.09, 132.01, 129.49, 127.77, 127.38, 126.53, 120.37, 112.86, 109.21, 106.52, 73.05, 69.29, 66.19, 65.34, 64.90, 53.04, 53.01, 30.05, 29.26, 26.23, 26.10, 25.58, 25.53, 22.70.

[0212] The above protocol can be used to similarly synthesize other COE compounds (e.g., COE-BO and COE-QX), the structures of which are shown in Figure 1.

[0213] Labeling and detection of bacterial cells in flow cytometry The application of COE dyes can be extended to labeling bacteria in flow cytometry measurements. As shown in Figure 2, Gram-positive bacteria Methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative bacteria Escherichia coli (E. coli) were premixed in PBS at various volume ratios of 5:0, 4:1, 3:2, 2:3, 1:4, and 0:5. These bacterial mixtures were stained using COE dyes, which have Gram selectivity and can specifically label Gram-positive bacteria. Two isolated populations were observed during flow cytometry measurements. As the proportion of Gram-positive bacteria decreased, the population showing positive signals gradually decreased and disappeared when the bacterial mixture contained only Gram-negative bacteria. These results not only prove that the compound has the ability to distinguish the bacterial ratio of each Gram type, but also reveal that the compound can be used to label bacterial samples in flow cytometry.

[0214] Labeling and detection of exosomes using flow cytometry Exosomes were prepared by the following protocol: Exosomes were purchased from ATCC, aliquoted and stored at -80°C. One aliquot was removed from -80°C and placed on ice. Exosomes were diluted 10x in cell culture grade PBS. 8μL of exosome suspension was mixed with 25-1μM COE compound. This was kept on ice for 10 minutes and then diluted 100x in PBS to yield 6x10 particles. 6 Approximate final concentrations of exosomes were 1000 / mL and COE 25–1 nM. Samples were vortexed briefly and used for flow cytometry. Side scatter of the violet laser (x-axis) was measured, and fluorescence intensity (y-axis: λ 励起 = 405 nm, 488 nm, λ 発光Flow cytometry measurements were obtained by measuring the wavelengths of the exosomes (at 525 nm, 690 nm). The experiments in Figures 3 and 4 show that exosomes can be successfully labeled and detected using COE compounds. Using COE compounds, exosomes can be stained with low concentrations of dye and can be used directly without further purification. Figure 1 shows the structure of the compound.

[0215] [Table 1]

[0216] [Table 2]

[0217] In another example, NanoFCM (UK) kindly provided us with exosomes isolated from SW480 colon cancer cell line and purified using size exclusion chromatography. Labeling and flow cytometry analysis were also performed by trained technicians at NanoFCM (UK). Briefly, exosomes were purified at approximately 10e particles per mL. 10 The exosomes were labeled at a particle concentration of 1 μM, where 1 μL of 10 μM COE dye solution or 1 μL of PKH-26 (in diluent C) was added to 9 μL of sample to achieve a final staining concentration of 1 μM. The mixtures were incubated at room temperature for 30 min, then diluted 100-fold using PBS and then analyzed with a NanoAnalyzer (NanoFCM). Detection of exosomes was performed at small threshold settings (68–155 S16M-Exo) and analysis of the labeled population was performed in the PC5 channel (Ex488 / Em670). A dye control sample was prepared using PBS instead of 9 μL of exosome solution, and then the same steps as for the exosome samples were performed for flow analysis. Results were confirmed by performing at least two replicate measurements. Also, in separate experiments, excess dye was removed from the labeled exosomes using standard ultracentrifugation methods before analysis as described above.

[0218] Figure 17 shows (a-e) particle size distribution plots of COE-BO, COE-BT, COE-QX, COE-BSe, and PKH26 dye-only controls (10 μM) and (f-j) their corresponding gated dot plots. Figure 18 shows (a-d) particle size distribution plots of COE-labeled SW480 exosomes (10 μM) and (e-h) their corresponding gated dot plots. Tables 3 and 4 summarize the results of the COE-only buffer control and COE-labeled exosomes when analyzed using the NanoAnalyzer.

[0219] [Table 3] [Table 4]

[0220] FIG. 19 and Table 5 show the results after removing excess COE compound from COE-labeled SW480 exosomes.

[0221] [Table 5]

[0222] Transmission electron microscopy (TEM) of exosomes Samples prepared for flow cytometry or collected by fluorescence-activated cell sorting (FACS) were immersed in liquid nitrogen and lyophilized. The powder was resuspended in 50 μL of cold MiliQ water. Glutaraldehyde was added to a final concentration of 2.5%. 10 μL of sample was added to a freshly glow-discharged 200 mesh Cu carbon / Formvar grid and allowed to adhere for 10 min. Excess liquid was blotted with filter paper and the grid was washed twice with MiliQ water. The grid was stained with 2.5% gadolinium triacetate stain for 1 min, after which excess liquid was blotted with filter paper. Samples were imaged on a Tecnai G2 at 100 kV and 30,000x magnification. Figure 5 shows TEM micrographs of (a) exosomes, exosomes labeled with COE-BT, and c) exosomes labeled with COE-S6 and collected after FACS.

[0223] Labeling and Detection of Red Blood Cells (RBCs) Using Flow Cytometry As shown in Figure 6, COE compounds can be used to label RBCs for flow cell cytometry. RBCs (isolated from bovine whole blood, Innovative Research, Inc.) were diluted 10-fold from stock in PBS and centrifuged at 3,000 RPM for 5 minutes. The supernatant was removed and the pellet was resuspended in PBS. This was repeated a total of three times. The final pellet was resuspended in PBS to 1% by volume. 10 μM COE compounds were added to the RBC suspension and incubated at room temperature for 20 minutes. The RBC COE compound mixture was then centrifuged again at 3,000 RPM for 5 minutes. The supernatant was removed and the pellet was resuspended in fresh PBS. COE compound stained RBCs and unstained RBCs were mixed at various volume ratios: 0:5, 1:4, 2:3, 3:2, 4:1, and 5:0, respectively. The resulting RBC mixture was diluted 100-fold in PBS for flow cytometry experiments. Forward scatter (x-axis) was measured, and fluorescence intensity ((y-axis: λ 励起 = 405 nm, λ 発光Flow cytometry measurements were obtained by measuring the fluorescence intensity (F = 525 nm). Figure 6 shows that two separate populations can be seen for COE compound labeled and unlabeled RBCs. As the percentage of RBCs stained with the COE compound increases, the percentage of the more fluorescent population also increases. This indicates successful labeling of the RBCs with the COE compound and successful detection by flow cytometry (Figure 6).

[0224] [Table 6]

[0225] Labeling and Detection of Mammalian Cells Using Flow Cytometry As shown in Figures 8-11, COEs can be used to label mammalian cells for cell flow cytometry. Hep-G2 and A549 (ATCC HB-8065, CCL-185) were purchased from ATCC. Upon receiving frozen cells, the cells were thawed by gentle agitation in a 37°C water bath and then transferred to a centrifuge tube containing 9 mL of warmed medium of DMEM + 10% FBS. The tube was then centrifuged at 200xg for 5 minutes. The supernatant was discarded and the cell pellet was resuspended in medium in a culture flask. The cells were incubated at 37°C and 5% CO2. When cell confluency reached approximately 90%, the cells were suspended with 1x trypsin and neutralized with an equal volume of medium. After centrifugation at 200xg for 5 minutes, the supernatant was discarded and the cell pellet was resuspended in warmed PBS containing 5-10 μM COE. The suspension was incubated at room temperature for 20 min and then centrifuged at 1000 rpm for 5 min to remove excess COE compounds. The supernatant was discarded, and the pellet was resuspended in warmed PBS. COE compound stained cells were mixed with unstained cells at various volume ratios: 0:5, 1:4, 2:3, 3:2, 4:1, and 5:0, respectively. The resulting suspension was used directly for flow cytometry experiments. Forward scatter (x-axis) was measured, and fluorescence intensity (y-axis: λ 励起 = 561 nm, λ 発光Flow cytometry measurements were obtained by measuring the fluorescence intensity (λ = 610 nm) of COE-labeled and unlabeled cells. Figure 8 shows that two separate populations can be seen for COE-labeled and unlabeled cells. As the percentage of cells stained with each COE compound increases, the percentage of the population with higher fluorescence intensity also increases. This indicates successful labeling of cells with COE and successful detection by flow cytometry (Figures 8 and 10). Figure 9 shows the percentage of flow cytometry events that occurred in the gated region of Hep-G2 stained with various ratios of compounds of formula (I) versus unstained Hep-G2. Figure 11 shows flow cytometry measurements of A549 cells labeled with 5 μM COE-BSe. Forward scatter (x-axis) was measured and fluorescence intensity (y-axis: λ = 610 nm) was measured. 励起 = 638 nm, λ 発光 Flow cytometry measurements were obtained by measuring the fluorescence intensity (F = 712 nm). Confluent cells were stained with 5 μM COE in PBS at room temperature for 20 min. The COE solution was aspirated and confluent cells were washed with fresh PBS. Cells were suspended with 1× trypsin and neutralized with an equal volume of medium. After centrifugation at 200×g for 5 min, the supernatant was discarded and the cell pellet was resuspended in warmed PBS and used for flow cytometry. 20% COE-labeled cell suspension was seeded and allowed to reach 90% confluency. Cells were harvested and used for flow cytometry without additional COE for 4 passages after seeding. (Figure 11)

[0226] [Table 7]

[0227] Fluorescence microscopy of mammalian cells The above experiments confirmed that COE dyes can be used to label extracellular vesicles (including exosomes), bacteria, RBCs, and mammalian cells in flow cytometry. All of these samples contain lipid bilayer structures in the membrane. To further confirm that the binding target of COE dyes is lipid membranes, confocal microscopy was used to colocalize COE compounds with the commercially available membrane dye FM 4-64. As shown in Figure 12, mammalian Hep-G2 cells were stained with both 4 μM COE compounds (green channel) and 4 μM FM 4-64 (red channel), and a clear staining pattern was observed delineating the cell membrane by collecting the emission from the COE compounds in the range of 450-490 nm after excitation at 405 nm. The well-matched colocalization of COE compounds and FM 4-64 proves that the binding target of COE dyes is lipid membranes. A549 cells were stained with 5 μM COE compounds in PBS and visualized by fluorescence microscopy after incubation at 37 °C and 5% CO2 for 1 h. (Fig. 13)

[0228] Assessment of COE insertion into lipid bilayers using a fluorimeter Liposomes were prepared using the following protocol. Chloroform solutions of phospholipids (e.g., POPC, POPE, and POPG) were mixed in appropriate molar ratios and dried under a gentle stream of nitrogen. The dried lipids were further dried overnight under reduced pressure to obtain a thin lipid film. To prepare small unilamellar vesicles (SUVs), phosphate-buffered saline (PBS) was added to a concentration of 5 mg mL -1 The dried films were rehydrated by adding 1000 mM NaCl to 1000 mM NaCl, followed by incubation at 35° C. for 2 h with constant agitation at approximately 300 rpm. The vesicles were then extruded 21 times at 45° C. using a 100 nm membrane to obtain the SUV samples. The vesicles were kept at 4° C. until further use.

[0229] After the COE is inserted into the lipid bilayer, the emission of the COE is significantly enhanced. Thus, the free dye in the aqueous phase has weak emission and low background, which is ideal for obtaining a high signal-to-noise ratio. As shown in Figure 14, both SUV (1 mg / mL) and COE-BT (10 μM) in PBS have weak emission (λ 励起 = 520 nm). When SUV and COE-BT are mixed, COE is inserted into the lipid bilayer. This is caused spontaneously by electrostatic and hydrophobic interactions between COE and the lipid bilayer. As a result, a significant emission enhancement is observed. For example, the emission intensity of the SUV-COE-BT complex increases by more than 400 times at 600-630 nm compared to the COE-BT solution. The same phenomenon is observed with COE-BBT as well (Figures 14 and 15). By adjusting various chemical structures, COE can achieve various emission wavelengths ranging from "green" to "red" and even "infrared" (Figures 14 and 15). Such a variety of color choices makes COE dyes highly compatible for the simultaneous use of multiple dyes.

[0230] Evaluation of COE addition on liposome particle size and uniformity To confirm whether COE treatment alters liposome morphology (e.g., induces liposome fusion or fission), dynamic light scattering (DLS) measurements with lipid SUV models were used (Figure 16). SUVs were obtained after extrusion through a 100 nm membrane. A Z-average diameter of 104.5 ± 29.45 nm and a PDI of 0.048 were observed for the 1 mg / mL SUV sample during DLS measurements. After adding 10 μM COE-BBT to the 1 mg / mL SUV sample, the Z-average diameter becomes 104.8 ± 32.65 nm and the PDI becomes 0.054. The DLS results indicate that COE addition does not change the liposome morphology.

[0231] The results show that the compounds of the present invention can be used across a wide range of samples, including exosomes, red blood cells, mammalian cells, and bacterial cells. The compounds show improved labeling efficiency and sensitivity through their increased fluorescence intensity. These results demonstrate that COEs can be used as fluorescent labels in flow cytometry, which relies primarily on interactions with lipid bilayers. The tunable molecular structure and ease of synthesis of COEs allows for extensive possibilities and flexibility in tailoring to luminescence detection needs.

[0232] COE-labeled exosomes and uptake of COE-labeled exosomes into mammalian cells using imaging flow cytometry A549 cells were cultured in DMEM medium containing 10% FBS in a 6-well plate at 1 × 10 5The cells were seeded at a concentration of 100 μg mL-1 and allowed to attach overnight before the experiment. The next day, 4.5 mL of PC-3 exosomes or control (PBS only) at 10 μg mL-1 were mixed with COE-Ben to a final dye concentration of 1 μM or with DiD to a final dye concentration of 2.5 μM. Samples were incubated for 1 hour at room temperature in the dark. Samples were washed by filtration using a 100 KDa cutoff centrifugal filter unit (Amicon® Ultra) and centrifuged at 2000×g for 15 minutes to remove free dye. Concentrated samples were collected, resuspended in DMEM medium without FBS, and divided into three aliquots. After rinsing the cells in the microplate using DMEM medium without FBS, stained exosomes or control (residual dye solution) were added at various time points, e.g., 2 hours, 4 hours, and 8 hours before harvesting the cells. After exosome or dye uptake, cells were washed with warmed PBS (37°C) and 1x trypsin-EDTA was added. Cells were incubated for up to 5 min at 37°C in a 5% CO2 chamber. DMEM containing 10% FBS was added into the microplate and cells were centrifuged at 200×g for 4 min. The supernatant was removed and cells were resuspended in DMEM medium (containing 10% FBS) and imaged flow cytometry was performed using an Amnis ImageStreamX Mk II imaging flow cytometer. Collection gates were set using the area-to-aspect ratio of the brightfield channel to select only intact cell events. In the COE-Ben channel (i.e. Ch02), cells were excited using a 405 nm laser and emission was collected in the range of 505–560 nm. In the DiD channel (i.e. Ch05), cells were excited using a 638 nm laser and emission was collected in the range of 642–745 nm. The bright field area of ​​the whole cell was used as an indicator of particle size, i.e., M04 or M01 channel. Data were processed using IDEAS v6.3. Gradient RMS (root mean square) was applied to the bright field channel to exclude out-of-focus cells. Flow cytometry results were presented using the M04 channel (particle size) versus the Ch02 channel (COE-Ben) or Ch05 channel (DiD). Gates were set according to unstained control samples.The "spot counting algorithm" function built into the IDEAS 6.3 software was used to count the number of bright spots in the cells, which was used as a metric for exosome uptake. At least 1000 cells were statistically counted in each group.

[0233] FIG. 20 shows (a) representative imaging flow cytometry images of A549 cells stained with COE-Ben stained exosomes at various treatments and time points, as well as (b-d) their corresponding flow cytometry analysis. (e) Average spot number of COE-Ben or DiD probe (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine 4-chlorobenzenesulfonate) in each cell. (f) Representative imaging flow cytometry images of A549 cells after staining with residues of COE-Ben or DiD dye after washing using ultrafiltration, as well as (g-h) their corresponding flow cytometry analysis. In imaging flow cytometry, the COE-Ben channel was excited using 405 nm and the emission was collected in the range of 505-560 nm, the DiD channel was excited using 638 nm and the emission was collected in the range of 642-745 nm, and the bright field area of ​​the whole cell was used as an indicator of particle size.

[0234] Labeling prestained exosomes using COE To distinguish exosomes produced by A549 from those derived from serum, a new cell medium for exosome production (CCM-Exo-Dep) was prepared by supplementing DMEM with exosome-depleted fetal bovine serum (Systems Biosciences, Cat: EXO-FBS-250A-1). A549 cells were cultured at 5 × 10 cells in 10 mL of CCM-Exo-Dep in a T75 flask. 5 pieces mL -1After seeding with 1 μM COE-BT and allowing it to attach for 1 h, 2 μM COE-BT was introduced into the cell culture. After incubating the cells for at least 24 h, the cell culture supernatant was collected for the first round of exosome production and stored at 4 °C, followed by purification. Without passaging the labeled cells, a new 10 mL of CCM-Exo-Dep was added to the cells and incubated for at least another 24 h. Then, after 24 h, the cell culture supernatant was collected for the second round of exosome production. The collected cell culture was first spun at 10,000 × g for 30 min at room temperature to remove all necrotic cell debris. Exosome isolation was then performed by ultracentrifugation (Optima™ XPN-100, Beckman Coulter) at 100,000 × g for 1 h at 4 °C. The supernatant was carefully removed, and the bottom of the centrifuge tube, where the pellet was expected to be located, was rinsed three times with PBS. After washing, the exosomes were pelleted again under the same conditions and then resuspended in 100 μL of PBS. Aliquots of purified exosomes were stored at -80°C prior to downstream analysis (flow cytometry or TEM).

[0235] Figure 21 shows colocalization micrographs of A549 cells stained with early or late endosome-GFP reagent (BacMam 2.0) or 100 nM lysosome-specific dye LysoTracker® Green DND-26 after incubation with 2 μM COE-BT. The COE-BT channel (represented in green) was observed by excitation at 561 nm and collection of emission in the range of 570-620 nm. The GFP (green fluorescent protein) and LysoTracker Green channels (represented in red) were observed by excitation at 488 nm and collection of emission in the range of 500-540 nm. Pearson's correlation coefficient (R) was analyzed using ImageJ as a metric of colocalization.

[0236] Exosome samples were analyzed using transmission electron microscopy (TEM). Briefly, exosomes obtained from pre-stained A549 cells were fixed with 2.5% glutaraldehyde in PBS for 30 min at room temperature. 10 μL of exosome solution was applied onto freshly glow-discharged Formvar carbon-coated 200 mesh copper EM grids for 15 min. Samples were blotted with filter paper and washed three times with MiliQ water. Samples were negatively stained with 2% gadolinium acetate for 30 s. Samples were blotted with filter paper and stored overnight in a grid box in the vacuum chamber of a desiccator before being imaged using 80 kV on a FEI TENCAI G2 instrument.

[0237] Figure 22 shows (a) transmission electron microscopy images of EVs secreted by COE-BT stained A549 cells. (b) Flow cytometry analysis of EVs secreted by COE-BT stained A549 cells after the first 24 hours of incubation. Excitation and emission wavelengths are 488 / 690 nm for the B690 channel.

[0238] Lack of nanoparticle formation by COEs Figure 23 shows (a) photographs and (b) absorption spectra of 50 μM COE or DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) solutions in PBS before and after ultrafiltration using 100K MWCO protein concentration tubes (Pierce™, Thermo Scientific™) at a relative centrifugal force of 4000. The slight decrease in the absorption spectrum of the COE sample is likely due to non-specific binding of the positively charged COE to the polyethersulfone-based ultrafiltration membrane.

[0239] FIG. 24 shows (a) correlation coefficient curves of pure PBS, or 1 μM COE-BT or 1 μM DiR or 1 mM SUV in PBS, measured by dynamic light scattering (DLS). (b) Average count rates derived by DLS measurements of pure PBS, or 1 μM COE-BT or 1 μM DiR or 1 mM SUV in PBS; experiments were repeated five times. (c) Correlation coefficient curves of other COEs at 1 μM in PBS, measured by DLS. (d) Average count rates derived by DLS measurements of other COEs at 1 μM in PBS; experiments were repeated five times.

[0240] FIG. 25 shows pictures of the Tyndall effect of pure PBS or 10 μM COE in PBS after illumination using a red laser pointer.

[0241] Figure 26 shows (a) a photograph of 200 μL of COE solution in PBS in a 96-well microplate (Costar®, polystyrene-based, Ref: 3599) before and after standing for 16 hours at room temperature. (b) a photograph of 200 μL of dye solution in PBS in a 96-well microplate (Costar®, polystyrene-based, Ref: 3599) before and after standing for 16 hours at room temperature. The COE solution was obtained by dilution from a 1 mM stock solution in PBS. The DiI solution was obtained by dispersing a DiI DMSO solution (1 mM) in PBS under vigorous stirring. The same volume of PBS was used as a negative control.

[0242] Stability of COE in labeled vesicles using flow cytometry Liposomes containing 15% POPG and 85% POPC were prepared as 5 mg / mL (or 6.564 mM) stock in PBS as described above. Liposomes were extruded through a 200 nm membrane filter and further confirmed by DLS to have a particle size of approximately 140 nm. Separate aliquots of the stock liposomes were then diluted to 1 mM and treated with 20 μM COE-Ben (or DiO) and 40 μM COE-BT (or DiD) in PBS, respectively, with gentle heating to 60°C for 30 min. The labeled liposomes were then stored at 4°C before being used for flow cytometry experiments. To demonstrate the stability of the dye in the liposomes after insertion, two separate working solutions of COE-Ben labeled liposomes and COE-BT liposomes were first prepared at 10 particles per mL. 7 The results were normalized to the same particle concentration of 10000 / ml. These two standard solutions were then mixed in various ratios of 0:5, 1:4, 2:3, 3:2, 4:1, and 5:0, and the mixtures were incubated at room temperature for 1 h. The undiluted mixtures were then analyzed on a CytoFLEX LX (Beckman Coulter) and detection was initiated on violet side scatter (VSSC>4000). For unstained liposomes, gating was performed on both the V525 channel (Ex405 / Em525) and the B690 channel (Ex488 / Em690) for COE-Ben and COE-BT labeled liposomes, respectively. The percentage of events within the double positive region of the dot plot was used to analyze liposomes that may contain both COE-Ben and COE-BT dyes, suggesting transfer of dye from one population to another. Additionally, samples were incubated overnight (24 hours) at room temperature before the same flow cytometry analysis was performed to check the dye stability and the extent of dye cross-bleed. Experiments were repeated twice.

[0243] FIG. 27 shows flow cytometry measurements of mixtures of COE-BT and COE-Ben stained SUVs (130 nm) in PBS mixed at various mixing ratios, incubated for (a-f) 1 h and (g-l) 24 h, and analyzed by Cytoflex.

[0244] FIG. 28 shows the percentage of the SUV population from FIG. 27 in the various gates after mixing for (a) 1 hour or (b) 24 hours.

[0245] Labeling of liposomes of various sizes using COE The POPC solution in chloroform was mixed with a solution of 0.5 mol% COE-Ben or COE-BT in chloroform and then dried under a gentle argon stream in a glass vial. The dried lipid was further dried overnight under reduced pressure to obtain a thin lipid film. To prepare small unilamellar vesicles (SUVs), PBS buffer (phosphate buffered saline) was added to a concentration of 5 mg mL -1 The dried film was rehydrated by adding 100 to 1000 μl of ... 7 After dilution into individual cells, the cells were analyzed by Cytoflex.

[0246] FIG. 29 shows dot plot profiles of dye-positive events in POPC liposomes of 100 nm, 200 nm, 400 nm, and 800 nm diameters labeled with 0.5 mol % COE-Ben (a-d) and COE-BT (e-f) analyzed by Cytoflex.

[0247] Figure 30 shows (a) 6.25 mg mL after staining with 15 μM COE-Ben and 15 μM FM 4-64 in PBS at room temperature for 30 min. -1(b) Confocal micrographs of LMVs (large multilamellar vesicles) of 100 μM IgG1. The mixture was diluted 5-fold with PBS before confocal imaging. The COE-Ben fluorescence channel was observed by excitation at 405 nm and collection of emission in the range of 450-490 nm (represented in green), and the FM 4-64 fluorescence channel was observed by excitation at 561 nm and collection of emission in the range of 640-700 nm (represented in red). The scale bar is 20 μm. (b) The gray-value curves represent the fluorescence intensity profiles of the white lines in the fluorescence micrographs of both channels on the left.

[0248] Figure 31 shows the photographs of COEs without (-) or with (+) treatment with 1 mM SUV in PBS under UV (365 nm) irradiation using a portable UV lamp (UVP® UVLS-24 EL, 4 watts). The concentration of COE-Quin is 0.25 μM; the concentration of COE-S6 and COE-Ben is 0.5 μM; the concentration of COE-QX, COE-BO, and COE-BT is 1 μM; and the concentration of COE-BSe is 5 μM. The camera (SONY, α7R) parameters are set as follows: ISO value 8000, aperture F2.8, exposure time 1 / 20.

[0249] Figure 32 shows (a) Zeta potential measurements of 1 mM POPC-only SUVs stained with 5 μM of various COEs in DI water, and (b) DLS measured Z-average diameter and PDI of 1 mM POPC-only SUVs stained with 5 μM of various COEs in DI water.

[0250] Labeling red blood cells using COE for microscopy and flow cytometry analysis Red blood cells were obtained from donor blood after removal of plasma by centrifugation. RBCs were used fresh or stored for 22 days. RBC concentrates were diluted 500-fold in PBS and then labeled with 2 μM COE-S6 or 1 μg / mL Cell Mask Deep Red. Cells were incubated with the dye for 10 min and then centrifuged at 700g for 3 min to remove free dye. The labeled cell pellet was then resuspended in 1 mL of PBS. Cells were then further diluted 10-fold in PBS before being imaged on 8-well ibidi chamber slides and analyzed by flow cytometry (Cytoflex) for particle size and fluorescence.

[0251] 33 shows confocal micrographs of fresh red blood cells (RBCs) that were (a) unstained, (b) labeled with 2 μM COE-S6, and (c) labeled with 1 μg / mL Cell Mask Deep Red. Images were acquired in the Ex405 / Em525, Ex638 / Em660, and brightfield channels.

[0252] 34 shows confocal micrographs of red blood cells (RBCs) stored for 22 days, (a) unstained, (b) labeled with 2 μM COE-S6, and (c) labeled with 1 μg / mL Cell Mask Deep Red. Images were acquired in the Ex405 / Em525, Ex638 / Em660, and brightfield channels.

[0253] FIG. 35 shows FSC / SSC dot plots of (a) unstained, (b) labeled with 1 μM COE-S6, and (c) labeled with 0.5 μg mL-1 Cell Mask Deep Red red red blood cells (RBCs) measured by flow cytometry. COE-S6 labeling, unlike Cell Mask Deep Red, does not introduce artifacts into the RBC morphology. (d) Histogram showing the increase in fluorescence when RBCs are labeled with increasing concentrations of COE-S6, and (e) histogram showing the decrease in fluorescence due to the quenching effect when RBCs are labeled with increasing concentrations of Cell Mask Deep Red. The homogeneity and uniformity of labeling is also superior in favor of COE-S6, as can be seen from the coefficient of variation (Cv), or spread (f), of the histogram data.

[0254] NIR-II COE as a fluorescent probe for mammalian cells For cell detection using near-infrared fluorescence, cells were directly stained with 20 μM COE-BBT in DMEM medium and incubated overnight. After rinsing the stained cells using warmed PBS (37°C), 1× Trypsin-EDTA was added. The cells were incubated for up to 5 min at 37°C in a 5% CO2 chamber. DMEM medium containing 10% FBS was added, and the cells were centrifuged at 200×g for 4 min. After removing the supernatant, the cells were resuspended in medium and subjected to flow cytometry. COE-BBT stained cells were excited using an 808 nm laser. To collect the emission signal from COE-BBT, a 900 nm long-pass filter based on ultraviolet fused silica glass was customized according to the specifications of the CytoFLEX platform optical filter (length 14.5 ± 0.1 mm, width 6.1 ± 0.1 mm, thickness 2.0 ± 0.1 mm) (Chroma, INC., China). This filter glass was attached to a holder and used instead of the IR885 channel (Ex808 / Em885). This new channel for detecting COE-BBT fluorescence was named "IR1000."

[0255] Figure 36 shows flow cytometry measurements of COE-BBT stained A549 cells with excitation using an 808 nm laser and collection of emission using a 950 nm long pass filter designated as the IR1000 channel. Unstained A549 cells were used as a negative control.

[0256] Cytotoxicity and hemolytic activity of COEs A549 cells were routinely maintained in DMEM supplemented with 10% FBS, i.e., cell culture medium (CCM). Stock solutions of COE were prepared by dissolving solid COE in PBS to a stock concentration of 2 mM. The stock solution was further diluted in CCM to prepare a 256 μM working solution of COE. Stock solutions of DiR were prepared in ethanol due to the low water solubility of DiR. A 256 μM working solution was prepared by dilution in CCM, resulting in a final ethanol content of 12.8% (v / v). To test cytotoxicity, 1000 cells were seeded in a 96-well black plate with a clear optical bottom and left to attach overnight at 37 °C in a 5% CO2 chamber. The next day, COE / DiR solutions of various concentrations were obtained by two-fold serial dilution in CCM (concentrations ranged from 0.5 to 256 μM) and then used to replace the consumed CCM in the wells. To clarify the toxic effect from ethanol, identical serial dilutions were performed using pure ethanol instead of the DiR dye solution in ethanol to ensure similar ethanol content in each well. Cells were then incubated with the additives for 24 hours, after which cell viability was measured using CellTiter-Glo® (COE-Quin, COE-S6, COE-Ben, COE-BBT, DiR) or CCK-8 (COE-BT, COE-BO, COE-QX, COE-BSe) according to the manufacturer's protocol. Fluorescence (CellTiter-Glo® assay) and absorbance (CCK-8 assay) measurements were recorded using a TECAN plate reader (Spark®). Cytotoxicity assays were repeated three times.

[0257] Figure 37 shows the measured cytotoxicity of the COEs against A549 cells. The IC50 values ​​of the six COEs are all higher than 100 μM.

[0258] Bovine whole blood was purchased from Innovative Research, USA. 2 mL of blood was mixed with 10 mL of PBS and centrifuged at 1,000 rpm for 5 min. The red blood cell pellet was collected and subsequently washed with PBS three times, then diluted to a concentration of 2% (v / v) using PBS. Due to the slight absorption interference of COE-Quin, COE-S6, and COE-Ben at 540 nm, only COE-Quin, COE-S6, and COE-Ben were selected for this study. Each COE was dissolved in PBS and serially diluted two-fold in a 96-well microplate. 100 μL of red blood cell suspension was mixed with 100 μL of COE solution in each well and incubated at 37 °C for 1 h under shaking (200 rpm). The microplate was centrifuged at 1,000 rpm for 10 min. A 150 μL aliquot of the supernatant was transferred to a new 96-well microplate. The calculation of hemolytic activity was performed by measuring the absorbance at 540 nm using a multimode microplate reader (Spark®, Tecan). Triton X-100 (0.1% in PBS), which can completely lyse red blood cells, was used as a positive control, while bovine red blood cells in PBS were used as a negative control. The percentage of hemolysis was calculated using the following formula:

number

[0259] FIG. 38 shows the measurement of hemolysis of COE against bovine red blood cells in PBS.

Claims

1. A compound of formula (I), or a salt or solvate thereof: 【Chemical 1】 Wherein, Each R １ is independently selected from optionally substituted alkoxy, optionally substituted alkenyloxy, and optionally substituted alkynyloxy; q is an integer selected from 1 to 5; q' is an integer selected from 1 to 5; Each L ２ is independently selected from ethylene which may be substituted or phenylethylene which may be substituted; L １ is a π-conjugated core containing monomer unit A and monomer unit D: [Chemical Formula 2] Wherein, A is, [Chemical Formula 3] selected from, wherein, [Chemical Formula 4] represents a bond to D or to L2; D is, 【Chemical Formula 5】 selected from, wherein, 【Chemical Formula 6】 represents a bond to A or to L2; each X1 is independently selected from C, O, N, S, and Se; each X2, when present, is independently selected from C, O, N, S, and Se; when X2 is present, at least one of X1 and X2 is O, N, or S; each R is independently selected from H, halo, cyano, and optionally substituted alkyl; n is an integer selected from 1 to 5; m is an integer selected from 1 to 5; * represents a bond to another monomer unit or to L ２ ; the monomer unit A and the monomer unit D are bonded to each other in an alternating manner; L １ is not butadienylene, polyalkenylene, phenylalkenylene, or polyphenylalkenylene.

2. The compound according to claim 1, wherein A is an electron-withdrawing moiety.

3. A is a moiety of formula (II): 【Chemical Formula 7】 Wherein, 【Chemical 8】 represents the binding to D or L ２ ; R ２ 、 R ３ 、 R ４ 、 and R ５ are each independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R ２ and R ３ are linked to form an optionally substituted heterocyclyl or an optionally substituted heteroaryl; or R ４ and R ５ are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl, the compound according to claim 1.

4. A is, 【Chemical Formula 9】 or 【Chemical 10】 wherein R ４ and R ５ are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R ４ and R ５ are linked to form an optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, the compound according to claim 1.

5. A is, 【Chemical 11】 【Chemical Formula 12】 【Chemical 13】 or 【Chemical 14】 The compound according to claim 1.

6. The compound according to claim 1, wherein D is an electron-donating moiety.

7. The compound according to claim 1, wherein D is an optionally substituted 5-membered heteroarylene or phenylene.

8. D is a moiety of formula (III): 【Chemical Formula 15】 Wherein Y is NR, O, or S; R ６ and R ７ are each independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R ６ and R ７ are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; R is selected from H, halo, cyano, and optionally substituted alkyl, the compound according to claim 1.

9. D is, 【Chemical 16】 and in the formula, R ６ and R ７ are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; or R ６ and R ７ are linked to form an optionally substituted cycloalkyl, an optionally substituted cycloalkenyl, an optionally substituted cycloalkynyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl, the compound according to claim 1.

10. D is, 【Chemical 17】 【Chemical Formula 18】 or 【Chemical Formula 19】 The compound according to claim 1.

11. L １ is 【Chemical 20】 Selected from, the compound according to claim 1.

12. R １ The compound according to claim 1, wherein R is independently an optionally substituted alkoxy.

13. R １ is independently C substituted with amino or alkylamino ３ ~C ８ is alkoxy, the compound according to claim 1.

14. L １ The compound according to claim 1, wherein the optional substituents of L are independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

15. The compound of formula (I) is a compound of formula (Ia): 【Chemical 21】 The compound according to claim 1.

16. The compound of formula (I) is a compound of formula (Ib): 【Chemical 22】 The compound according to claim 1.

17. The compound of formula (I) is, 【Chemical 23】 【Chemical 24】 Selected from, the compound according to claim 1.

18. The compound of formula (I) is a compound of formula (Ig), or a salt or solvate thereof: 【Chemical 25】 Wherein r is an integer selected from 1 to 5, the compound according to claim 1.

19. A method for labeling cells and / or lipid vesicles, comprising a) incubating a compound of formula (I) according to claim 1, or a salt or solvate thereof, with cells and / or lipid vesicles A method comprising the same. **Claim 20**: A lipid vesicle and / or liposome comprising a compound of formula (I) according to claim 1, or a salt or solvate thereof.

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