Conjugated oligoelectrolytes with diketopyrrolopyrrole cores and methods thereof

Abstract

The present disclosure relates to conjugated oligoelectrolytes with diketopyrrolopyrrole cores and their methods of use thereof.

Description

[0001] Conjugated Oligoelectrolytes with Diketopyrrolopyrrole Cores and Methods Thereof

[0002] Technical Field

[0003] The present disclosure relates to conjugated oligoelectrolytes with diketopyrrolopyrrole cores and their methods of use thereof.

[0004] Background

[0005] The cell membrane is a biological membrane that separates and protects the interior of all cells from the outside environment. The cell membrane consists of a lipid bilayer, made up of two layers of phospholipids with cholesterols (a lipid component) interspersed between them, maintaining appropriate membrane fluidity at various temperatures. The membrane also contains membrane proteins, including integral proteins that span the membrane and serve as membrane transporters, and peripheral proteins that loosely attach to the outer (peripheral) side of the cell membrane, acting as enzymes to facilitate interaction with the cell's environment. Glycolipids embedded in the outer lipid layer serve a similar purpose. The cell membrane controls the movement of substances in and out of cells and organelles, being selectively permeable to ions and organic molecules. In addition, cell membranes are involved in a variety of cellular processes such as cell adhesion, ion conductivity, and cell signalling and serve as the attachment surface for several extracellular structures, including the cell wall and the carbohydrate layer called the glycocalyx, as well as the intracellular network of protein fibers called the cytoskeleton. In this regard, the physiology of the cell membrane is highly varied and complicated. New drugs may be developed with better understanding of the cell membrane.

[0006] In particular, lipid bilayer membranes are essential structural elements in cellular systems. Beyond compartmentalizing cellular components from the surrounding environment, they play important roles in many subtle, yet vital cellular functions. Some major examples include molecular transport, signal transmission, intercell communication, and cell-cell and cell-extracellular matrix interactions. Membrane dyes have therefore been developed to visualize membranes and to gain insights into their properties and functions, such as inter-organelle communication and membrane trafficking. Fluorescent probes that "light up" membranes, however, typically provide information on location in space with little additional insights into relevant biophysical properties. For example, membrane tension is a dynamic physical property that plays an essential role in regulating cell division and remodeling. Physics-centric tools, such as micropipette aspiration, atomic force microscopy, and optical tweezers have been developed to examine mechanical properties and membrane tension. These approaches are invasive and difficult to configure for monitoring dynamic processes throughout a large population of cells. Chemical biology tools that interrogate tension in subcellular structures involve protein or DNA engineering, which are highly specific to the system under investigation. Only a few molecular fluorophores have been successfully reported to measure membrane tension of the plasma membrane or organelles within the cell. Of note is the Flipper series of reporters, which has opened opportunities to measure membrane tension of various organelles, intracellular vesicle formation, and osmotic shock response. Flipper probes comprise a hydrophobic chromophore that inserts into the bilayer and experiences different levels of planarization as a function of membrane tension. This intramolecular feature ultimately modulates polarization and fluorescence lifetimes.

[0007] Conjugated oligoelectrolytes (COEs) are a class of fluorescent molecules defined by a hydrophobic conjugated core bearing terminal polar ionic pendants. The hydrophobic backbone consisting of n-delocalized repeat units. The overall molecular structures are similar to the organization of hydrophilic and hydrophobic domains in lipid bilayers. As such, the similarities between the hydrophilic-hydrophobic distribution and molecular dimensions of these amphipathic molecules and the lipid bilayer allows COEs to spontaneously intercalate and partition into membranes, which is driven by electrostatic and hydrophobic interactions between the COEs and the lipids. The affinity of certain cationic COEs towards negatively charged phospholipid bilayers has enabled diverse applications including bioelectrochemical systems, biosensing, and antimicrobial candidates. It has also been determined that membrane-intercalated COEs reside in a perpendicular orientation relative to the lipid bilayer plane. Upon partition into lipid bilayers from aqueous solution, the COEs emission will enhance significantly. This "light up" mechanism will confer COE dyes to achieve a high signal-to-noise ratio upon localizing within the hydrophobic environment of lipid bilayers.

[0008] There is a lack of molecules that target the cell membrane or lipid bilayer. There is further a need for molecules with specificity for certain regions of the lipid bilayer.

[0009] Accordingly, it would be desirable to overcome or ameliorate at least one of the abovedescribed problems.

[0010] Summary The present disclosure relates to a compound of Formula (I) or a salt or solvate thereof: wherein

[0011] Ri and R2 are independently selected from H, optionally substituted alkyl, and optionally substituted alkenyl; each R3 and R4 are independently selected from 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; each Rs and Rs is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; n and m are independently an integer selected from 1 to 5; p and q are independently an integer selected from 1 to 3; and r and t are independently an integer selected from 0 to 4.

[0012] In some embodiments, Ri and R2 are independently selected from H and optionally substituted C1-C5 alkyl.

[0013] In some embodiments, each R3 and R4 are independently selected from alkyl and alkoxy, each optionally substituted with amino, or alkylamino.

[0014] In some embodiments, each Rs and Rs is independently selected from halogen, cyano, or optionally substituted C1-C5 alkyl.

[0015] In some embodiments, R3 and R4 are independently at a meta and / or para position relative to the ethylene moiety.

[0016] In some embodiments, the compound of Formula (I) is a compound of Formula (la) :

[0017] In some embodiments, the compound is selected from

[0018] In some embodiments, the compound is characterised by an absorbance of about 600 nm to about 650 nm. In some embodiments, the compound is characterised by an emission of about 600 nm to about 750 nm. In some embodiments, the compound is characterised by a Stokes' shift of about 600 cm to about 850 cm when a solvent orientation polarizability (Af) is 0 to about 0.35.

[0019] In some embodiments, the compound is characterised by an increase in fluorescence emission intensity of more than 800 times when integrated into a lipid bilayer.

[0020] In some embodiments, the compound is characterised by a shift in emission from about 800 nm to about 650 nm when integrated into a lipid bilayer.

[0021] In some embodiments, the compound is characterised by a partition coefficient between an aqueous medium and a lipid bilayer of about 2 x 106M’1to about 3 x 106M’1.

[0022] The present disclosure relates to a method of staining a lipid bilayer, comprising contacting the lipid bilayer with a compound of Formula (I) as disclosed herein.

[0023] The present disclosure relates to a method of imaging a lipid bilayer using stimulated emission depletion (STED) microscopy, comprising contacting the lipid bilayer with a compound of Formula (I) as disclosed herein or a compound of Formula (II): wherein each R? and Rs are 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; u is an integer selected from 1 to 5; v is an integer selected from 1 to 5; wherein each L2 is independently selected from optionally substituted ethylene, or optionally substituted phenylethylene;

[0024] Li is a n-conjugated core comprising monomeric unit A and monomeric unit D: (A) (D) wherein 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; w is an integer selected from 1 to 5; x is an integer selected from 1 to 5; wherein * represents a bond to another monomeric unit or to L2; and wherein monomeric units A and monomeric units D are alternatively bonded to each other.

[0025] In some embodiments, L2 is independently selected from: wherein * represents a bond to a monomeric unit and to a terminal phenyl moiety in compound of Formula (II).

[0026] In some embodiments, wherein ''' represents a bond to D or to L2;

[0027] Ra, Rb, Rc and Rd 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

[0028] Ra and Rb are linked to form optionally substituted heterocyclyl, optionally substituted heteroaryl; or

[0029] Rc and Rd are linked to form optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl; wherein ''' represents a bond to D or to L2;

[0030] Re is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl;

[0031] Rf, Ra, RK and Ri are independently selected from H and optionally substituted alkyl.

[0032] In some embodiments, D is independently selected from wherein Y is NR, 0, S, or Se;

[0033] Rj and Rk 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

[0034] Rj and Rk are linked to form optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl.

[0035] In some embodiments, each R? and Rs are independently selected from optionally substituted alkyl, optionally substituted alkoxy.

[0036] Brief description of the drawings

[0037] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which :

[0038] Figure 1 shows (A) chemical structures of COEs used for bioimaging (C0E-S5, COEBY) and an embodiment of the present disclosure, COE-KP. (B) Topology mimicking nature of COE-KP compared to traditional dyes used to probe the membrane.

[0039] Figure 2 shows a synthetic route towards COE-KP.

[0040] Figure 3 shows (a) absorbance and (b) emission spectra of COE-KP (or its unquaternized precursor) in small unilamellar vesicle (SUV) and various solvents.

[0041] Figure 4 shows Lippert-Mataga plot for COE-KP or KP-I in various organic solvents.

[0042] Figure 5 shows excitation spectra of COE-KP or KP-I in various organic solvents.

[0043] Figure 6 shows comparison of raw (left) and normalized (right) fluorescence emission spectra of COE-KP in PBS and when integrated into the lipid bilayer of SUVs.

[0044] Figure 7 shows (left) photographs of COE-KP in vials of various solutions (left to right: DMSO, PBS, MeOH, and SUVs). Right: representative image of a GUV stained with COE- KP imaged with a polarized filter.

[0045] Figure 8 shows lipid bilayer preference of COE-KP as demonstrated through changes in fluorescence intensity upon titration with SUVs with varying lipid concentrations.

[0046] Figure 9 shows changes in fluorescence intensity of SUVs stained with COE-KP over 2 hours of continuous illumination (Aex= 600 nm, Aex = 652 nm).

[0047] Figure 10 shows CLSM images of HeLa cells stained with COE-KP and co-labeled with either GFP-tagged LAMP-1 (Lyso-GFP, lysosome targeting), calreticulin-KDEL-GFP (ER- GFP, endoplasmic reticulum targeting) or commercial organelle dyes (MitoView Red, mitochondria; LipidTOX Green, lipid droplets; or DAPI, nucleus).

[0048] Figure 11 shows flow cytometry analysis of the fluorescence intensity of HeLa cells stained with COE-KP, detected in the NIR (710 nm) region.

[0049] Figure 12 shows HeLa cells stained with COE-KP images by either confocal microscopy or STED.

[0050] Figure 13 shows confocal (left) and STED (right) images of COE-KP labeled vesicles and their corresponding line plot profiles. The relative resolution of the images was done by fitting of the fluorescence intensity along the white line to a Lorentzian function.

[0051] Figure 14 shows (A) STED (left) and confocal (right) images of cells grown under serum starvation and stained with COE-KP. (B) Zoomed in regions of interest (Image size: 2 mm) highlighting the multilamellar vesicles that are clearly visible through STED. (C) Line plot of the fluorescence intensity for COE-KP stained multilamellar vesicles.

[0052] Figure 15 shows relative size of vesicles that were measured using the membrane boundary from COE-KP derived signal. Size was measured from at least 50 vesicles, n = 5)

[0053] Detailed Description

[0054] "Alkyl" refers to monovalent alkyl groups which may be straight chained or branched and preferably have from 1 to 10 carbon atoms or more preferably 1 to 6 carbon atoms. Examples of such alkyl groups include methyl, ethyl, n-propyl, / so-propyl, n-butyl, isobutyl, n-hexyl, and the like.

[0055] "Alkenyl" refers to a monovalent alkenyl group which may be straight chained or branched and preferably have from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and have at least 1 and preferably from 1-2, carbon to carbon, double bonds. Examples include ethenyl (-CH=CH2), n-propenyl (-CH2CH=CH2), / so-propenyl (-C(CH3)=CH2), but-2-enyl (-CH2CH=CHCH3), and the like. "Alkynyl" refers to alkynyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1, and preferably from 1-2, carbon to carbon, triple bonds. Examples of alkynyl groups include ethynyl (-C= CH), propargyl (-CH2C= CH), pent-2-ynyl (-CH2CSCCH2-CH3), and the like.

[0056] "Alkoxy" refers to the group alkyl-O- where the alkyl group is as described above. Examples include, methoxy, ethoxy, n-propoxy, / so-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.

[0057] "Alkenyloxy" refers to the group alkenyl-O- wherein the alkenyl group is as described above.

[0058] "Alkynyloxy" refers to the group alkynyl-O- wherein the alkynyl groups is as described above.

[0059] "Halo" or "halogen" refers to fluoro, chloro, bromo and iodo.

[0060] "Acyl" refers to groups H-C(O)-, alkyl-C(O)-, cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl- C(O)- and heterocyclyl-C(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

[0061] "Oxyacyl" refers to groups HOC(O)-, alkyl-OC(O)-, cycloalkyl-OC(O)-, aryl-OC(O)-, heteroaryl-OC(O)-, and heterocyclyl-OC(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

[0062] "Amino" refers to the group -NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.

[0063] "Aminoacyl" refers to the group -C(O)NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.

[0064] "Acylamino" refers to the group -NR"C(O)R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein. "Acyloxy" refers to the groups -OC(O)-alkyl, -OC(O)-aryl, -C(O)O-heteroaryl, and -C(O)O-heterocyclyl where alkyl, aryl, heteroaryl and heterocyclyl are as described herein.

[0065] "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 and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.

[0066] 'Cyano' refers to the group -CN.

[0067] "Oxyacylamino" refers to 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 and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.

[0068] "Oxyacyloxy" refers to the groups -OC(O)O-alkyl, -O-C(O)O-aryl, -OC(O)O- heteroaryl, and -OC(O)O-heterocyclyl where alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are as described herein.

[0069] "Thio" refers to groups H-S-, alkyl-S-, cycloalkyl-S-, aryl-S-, heteroaryl-S-, and heterocyclyl-S-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

[0070] "Phosphoryl" refers to the groups -P(O)(R'")(OR"") 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.

[0071] "Aryl" refers to an unsaturated aromatic carbocyclic group having a single ring (eg. phenyl) or multiple condensed rings (eg. naphthyl or anthryl), preferably having from 6 to 14 carbon atoms. Examples of aryl groups include phenyl, naphthyl and the like.

[0072] "Heteroaryl" refers to a monovalent aromatic heterocyclic group which fulfils the Hiickel criteria for aromaticity (ie. contains 4n + 2 n electrons) and preferably has from 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen, selenium, and sulfur within the ring (and includes oxides of sulfur, selenium and nitrogen). Such heteroaryl groups can have a single ring (eg. pyridyl, pyrrolyl or N- oxides thereof or furyl) or multiple condensed rings (eg. indolizinyl, benzoimidazolyl, coumarinyl, quinolinyl, isoquinolinyl or benzothienyl).

[0073] 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, thiadiazoles, oxadiazole, oxatriazole, tetrazole, thiophene, benzo[b]thiophene, triazole, imidazopyridine and the like.

[0074] "Arylene" refers to a divalent aryl group wherein the aryl group is as described above.

[0075] "Heteroarylene" refers to a divalent heteroaryl group wherein the aryl group is as described above.

[0076] "Heterocyclyl" refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, preferably from 1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur, oxygen, selenium or phosphorous within the ring. The most preferred heteroatom is nitrogen. It will be understood that where, for instance, R2 or R' is an optionally substituted heterocyclyl which has one or more ring heteroatoms, the heterocyclyl group can be connected to the core molecule of the compounds of the present invention, through a C-C or C-heteroatom bond, in particular a C-N bond.

[0077] Examples of heterocyclyl and heteroaryl groups include, but are not limited to, 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, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiadiazoles, oxadiazole, oxatriazole, tetrazole, thiazolidine, thiophene, benzo[b]thiophene, morpholino, piperidinyl, pyrrolidine, tetra hydrofuranyl, triazole, and the like.

[0078] "Optionally substituted" is taken to mean that a group may or may not be further substituted or fused (so as to form a condensed polycyclic group) with one or more groups selected from 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, 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-heterocyclyl amino, and unsymmetric di-substituted amines having different substituents selected from alkyl, aryl, heteroaryl and heterocyclyl, and the like, and may also include a bond to a solid support material, (for example, substituted onto a polymer resin). For instance, an "optionally substituted amino" group may include amino acid and peptide residues.

[0079] "Hydrophilic" refers to molecules or moieties which have a greater affinity for, and thus solubility in, water as compared to organic solvents. For example, the hydrophilicity of a compound can be quantified by measuring its partition coefficient between water (or a buffered aqueous solution) and a water-immiscible organic solvent, such as octanol, ethyl acetate, methylene chloride, or methyl tert-butyl ether. If after equilibration a greater concentration of the compound is present in the water than in the organic solvent, then the compound may be considered to be hydrophilic.

[0080] "Hydrophobic" refers to molecules or moieties which have a greater affinity for, and thus solubility in, organic solvents as compared to water. For example, the hydrophobicity of a compound can be quantified by measuring its partition coefficient between water (or a buffered aqueous solution) and a water-immiscible organic solvent, such as octanol, ethyl acetate, methylene chloride, or methyl tert-butyi ether. If after equilibration a greater concentration of the compound is present in the organic solvent than in the water, then the compound may be considered to be hydrophobic.

[0081] Conjugated oligoelectrolytes (COEs) are a class of molecules defined by a hydrophobic conjugated core bearing terminal polar ionic pendants. The hydrophobic and hydrophilic moieties in COEs may be rationally designed such that they mirror the organization of hydrophilic and hydrophobic domains in lipid bilayers. This structural design may involve only unbranched internal structures with charged groups at the two termini so that it favours the spontaneous intercalation of COEs into cellular membranes, which is driven by electrostatic and hydrophobic interactions between the COEs and the lipids. Characteristically, the fluorescence emission of COEs enhance significantly upon their intercalation into lipid bilayers from the aqueous solution. This "light up" mechanism confers a high signal-to-noise ratio for COEs when they are localized within the more hydrophobic environment of lipid bilayers. Additionally, COEs have a distinct chemical structure from many commercially available lipophilic dyes, which usually contain a surfactant-like structure, i.e., one side of the molecule is hydrophobic, and the other side is hydrophilic. These surfactant-like structures will induce micelle-like aggregation in the aqueous solutions. For example, the commonly used membrane dye, PKH-26, has been shown to form aggregates, which have a similar size and fluorescence intensity compared to small particles such as the exosomes, thereby leading to false-positive signals. These phenomenona can be avoided in the case of COEs given that their emission has been shown to greatly intensify after intercalation into the lipid bilayer; i.e. high signal to noise ratio.

[0082] The present disclosure is predicated on the understanding that the intrinsic emissive nature and modular designability from simple subunits make COEs a flexible molecular platform for the design of bioimaging probes. In contrast to various commercially available dyes, the hydrophobic chromophore of these molecules span the whole bilayer, and allow for imaging and probing of membrane properties over extended period (Figure lb). By integrating and modifying the conjugated backbone, the photophysical properties may be tailored as required.

[0083] It was found that when the conjugated backbone comprises a diketopyrrolopyrrole core, the resultant COEs are highly fluorogenic, with significant emission enhancement only upon membrane labeling, and thus may be used as high resolution imaging modalities and as environmental-sensitive reporters, in applications as optical probes for visualizing and interrogating biological systems.

[0084] In particular, the presently disclosed COE compound having a diketopyrrolopyrrole core (COE-KP) is highly responsive to protic environments, and demonstrate the possibility of using these lipid bilayer probes for super resolution imaging using stimulated emission depletion (STED). By further introduction of a carbonyl-containing acceptor within the highly emissive conjugated core allowed for significant modulation of excited state properties upon exposure to a hydrogen bonding environment. As a result, COE- KP features an unprecedented fluorogenic turn-on ratio of an over 1000-fold increase in emission intensity upon integration into the lipid bilayer compared to when in aqueous buffer. In addition, analysis of the fluorescence lifetime of COE-KP in a variety of organic solvents including chloroform, dimethyl sulfoxide and ethanol, showed a notable response to protic environments, pointing towards efficient emission quenching of the chromophore by the local environment. Using STED microscopy, it is shown that COE- KP localizes within the vesicle membrane within cells, down to a resolution of 74 nm.

[0085] Accordingly, the present disclosure relates to a compound of Formula (I) or a salt or solvate thereof: wherein

[0086] Ri and Rz are independently selected from H, optionally substituted alkyl, and optionally substituted alkenyl; each Rs and R4 are independently selected from 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; each Rs and Re is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; n and m are independently an integer selected from 1 to 5; p and q are independently an integer selected from 1 to 3; and r and t are independently an integer selected from 0 to 4.

[0087] The compound of Formula (I) has a substantially linear topology. The topology refers to a molecular structure of a compound within the constraints of three-dimensional (3D) space. Such linear topology has two nodes as the termini without any junction nodes. The linear topology is advantageous for facilitating lipid membrane intercalation.

[0088] The compounds of Formula (I) are linear in order to accommodate its position within the lipid bilayer. In this regard, the phenyl moieties are in a trans (or E) configuration about the alkenyl moiety. In some embodiments, the compounds are not branched; i.e. the monomeric units only extend along a single chain. This however does not exclude the monomeric units from being optionally substituted. In some embodiments, the compounds of Formula (I) are symmetrical in nature. The symmetry of a compound can be described by at least one of the 32 point groups. A Point Group describes all the symmetry operations that can be performed on a molecule that result in a conformation indistinguishable from the original. In this regard, in some embodiments, the compounds of Formula (I) have a C2v point group.

[0089] It was found that the photophysical properties of the compound of Formula (I) is conferred by the encapsulation of the compound in the lipid bilayer, protecting it from the solvent. In an aqueous medium or a solvent comprising OH groups, fluorescence quenching of the compound is believed to be through a reduced non-radiative decay processes, such as solvent induced quenching and / or with chemical groups that interact more strongly with the solvent through effects such as hydrogen bonding. By a rationale design, the compounds of the present disclosure features a fluorogenic turn-on ratio of an over 1000-fold increase in emission intensity upon integration into the lipid bilayer compared to when in aqueous buffer.

[0090] In some embodiments, Ri and R2 are independently selected from H, optionally substituted C1-C5 alkyl, or optionally substituted C2-C5 alkenyl. In some embodiments, Ri and R2 are independently selected from H, optionally substituted C1-C5 alkyl. In some embodiments, Ri and R2 are independently selected from H, optionally substituted methyl, ethyl, propyl, t-butyl or n-butyl.

[0091] In some embodiments, the optional substituent on Ri and R2 is independently selected from halo, alkyl, alkenyl, alkoxy, alkenyloxy, amino, cyano, or nitro. In some embodiments, the optional substituent on Ri and 2 is independently selected from, alkyl, alkoxy, amino, cyano, or nitro.

[0092] In some embodiments, each 3 and 4 are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, or optionally substituted acylamino. In some embodiments, each R3 and 4 are independently selected from optionally substituted alkyl, optionally substituted alkoxy, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted aminoacyl, or optionally substituted acylamino. In some embodiments, each Rs and R4 are independently selected from optionally substituted alkyl, or optionally substituted alkoxy. In some embodiments, each R3 and R4 are independently selected from optionally substituted C2-C12 alkyl, or optionally substituted C2-C12 alkoxy. In some embodiments, each R3 and R4 are independently selected from alkyl and alkoxy, each optionally substituted with amino, or alkylamino. In some embodiments, R3 and R4 are independently Cs-Cs alkoxy substituted with amino, or alkylamino.

[0093] In some embodiments, the optional substituent on Rs and R4 is independently selected from halo, alkyl, alkenyl, alkoxy, alkenyloxy, and amino. In some embodiments, when R3 and R4 are independently selected from optionally substituted acyl, optionally substituted oxyacyl, optionally substituted acyloxy, optionally substituted amino, optionally substituted aminoacyl, or optionally substituted acylamino, the optional substituent on R3 and R4 is independently selected from alkyl, alkenyl, alkoxy, and alkenyloxy. In some embodiments, the optional substituent on R3 and R4 is independently selected from C2-C12 alkyl, C2-C12 alkenyl, C2-C12 alkoxy, and C2-C12 alkenyloxy.

[0094] In some embodiments, each Rs and Re is independently selected from halogen, cyano, optionally substituted alkyl, or optionally substituted alkenyl. In some embodiments, each Rs and Re is independently selected from halogen, cyano, or optionally substituted alkyl. In some embodiments, each Rs and Re is independently selected from halogen, cyano, or optionally substituted C1-C5 alkyl.

[0095] In some embodiments, n is an integer selected from 1 to 5. In some embodiments, n is an integer selected from 1 to 4. In some embodiments, n is an integer selected from 1 to 3. In some embodiments, n is 3.

[0096] In some embodiments, m is an integer selected from 1 to 5. In some embodiments, m is an integer selected from 1 to 4. In some embodiments, m is an integer selected from 1 to 3. In some embodiments, m is 3.

[0097] For compound of Formula (I) to have a balance of hydrophilic and hydrophobic properties to facilitate its intercalation within the lipid bilayer, at least one side chain should be present at each terminus of the backbone. In some embodiments, n is at least 1 and m is at least 1. In some embodiments, n is 3 and m is 3.

[0098] In some embodiments, R3 and R4 are independently at a meta and / or para position relative to the ethylene moiety. In some embodiments, when n and / or m is 1, R3 and R4 are independently at a meta position relative to the ethylene moiety. In some embodiments, when n and / or m is 1, Rs and R4 are independently at a para position relative to the ethylene moiety.

[0099] In some embodiments, p is an integer selected from 1 to 3. In some embodiments, p is an integer selected from 1 to 2. In some embodiments, p is 1.

[0100] In some embodiments, q is an integer selected from 1 to 3. In some embodiments, q is an integer selected from 1 to 2. In some embodiments, q is 1.

[0101] In some embodiments, p and q are the same integer. In some embodiments, p is 1 and q is 1.

[0102] In some embodiments, r is an integer selected from 0 to 4. In some embodiments, r is an integer selected from 0 to 3. In some embodiments, r is an integer selected from 0 to 2. In some embodiments, r is 0.

[0103] In some embodiments, t is an integer selected from 0 to 4. In some embodiments, t is an integer selected from 0 to 3. In some embodiments, t is an integer selected from 0 to 2. In some embodiments, t is 0.

[0104] In some embodiments, r and t are 0.

[0105] When p and q are independently more than 1, Rs and Rs are independently present and selected on the phenyl moiety. For example, when p is 2, Rs may be present on one phenyl moiety (r= 1) and absent on the other phenyl moiety (r=0). For example, when p is 2, Rs may be halo on one phenyl moiety (r= l) and may be alkyl on the other phenyl moiety (r= l).

[0106] In some embodiments, the compound of Formula (I) is a compound of Formula (la) : wherein

[0107] Ri and R2 are independently selected from H, optionally substituted alkyl, and optionally substituted alkenyl; each R3 and R4 are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkoxy, and optionally substituted alkenyloxy; n and m are independently an integer selected from 1 to 5.

[0108] In some embodiments, each R3 and R4 are independently optionally substituted C4-C8 alkyl, optionally substituted C4-C8 alkenyl, optionally substituted C4-C8 alkoxy, and optionally substituted C4-C8 alkenyloxy.

[0109] The optional substituent at R3 and R4 may be a hydrophilic moiety. In other embodiments, the optional substituent at Rs and R4 is a charged moiety. Examples of hydrophilic and / or charged moieties are trialkylammonium halide. For example, the charged moiety can be trimethylammonium iodide. In this embodiment, R3 and R4 terminates with trimethylammonium, and thereby imparts a positive charge to the terminal ends of the COE compound. Other cationic charged groups include but are not limited to pyridinium, pyrrolidinium, imidazolium, guanidinium, sulfonium, thiouronium, and phosphonium. Other anionic charged groups include but not limited to chlorate, sulphate, phosphate, acetate, carboxyl, hydroxide. The hydrophilic and / or charged moieties can also in zwitterionic form that contains both cationic and anionic charged groups through covalent bonds. The excess charges can be neutralized by acceptable cations or anions.

[0110] In some embodiments, each R3 and R4 are independently optionally substituted with amino.

[0111] Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others. Examples of the optional substituents on Rs or R4 can be selected from:

[0112]

[0113] In some embodiments, the compound of Formula (I) or salt or solvate thereof is selected from

[0114]

[0115] In some embodiments, the compound of Formula (I) is a salt thereof. The salt form can be a protonated salt, or can be generated by alkylating compound of Formula (I) with halocarbons. For example alkylhalide (such as CHsBr or CH3I) can 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 Ri is optionally substituted amino, each of Ri can be alkylated to provide at least a positive charge at their respective ends.

[0116] For example, quaternary ammonium salts of compound of Formula (I) can be:

[0117]

[0118] The compound of the invention may be in crystalline form either as the free compound or as a solvate (e.g. hydrate) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art.

[0119] The compounds of the present invention can be provided as a solid or as a solution. For example, the compound can be provided as a lyophilised powder.

[0120] In some embodiments, the compound of Formula (I) or (la) is characterised by an absorbance of about 600 nm to about 650 nm.

[0121] In some embodiments, the compound of Formula (I) or (la) is characterised by an emission of about 600 nm to about 750 nm. In some embodiments, the compound of Formula (I) or (la) is characterised by a Stokes' shift of about 600 cm to about 850 cm4when a solvent orientation polarizability (Af) is 0 to about 0.35. The Stokes' shift may be measured in water, methanol, ethanol, acetone, DMF, DMSO, THF, ethyl acetate, chloroform, toluene and dioxane.

[0122] In some embodiments, the compound of Formula (I) or (la) is characterised by a fluorescence emission lifetime of about 1.1 ns to about 1.8 ns at a viscosity of about 900 cP to about 1200 cP. In other embodiments, the fluorescence lifetime of about 1.1 ns to about 1.7 ns at a viscosity of about 1200 cP, or about 1.1 ns to about 1.6 ns, about 1.1 ns to about 1.5 ns, about 1.1 ns to about 1.4 ns, about 1.1 ns to about 1.3 ns, or about 1.2 ns to about 1.3 ns. In some embodiments, the compound of Formula (I) is characterised by a fluorescence emission lifetime of about 1.1 ns to about 3 ns at a viscosity of about 900 cP to about 1200 cP.

[0123] In some embodiments, the compound of Formula (I) or (la) is characterised by a bandgap of about 2.4 eV to about 2.7 eV.

[0124] In some embodiments, when the compound of Formula (I), (la) or a salt or solvate thereof is inserted into the cellular and / or lipid membrane, the compound of Formula (I) or a salt or solvate thereof has an emission intensity of more than about 500 times to about 1000 times relative to a control sample of the compound of Formula (I) or a salt or solvate thereof. In other embodiments, the emission intensity is more than about 600 times to about 1000 times, about 700 times to about 1000 times, about 800 times to about 1000 times, or about 900 times to about 1000 times. In some embodiments, the compound is characterised by an increase in fluorescence emission intensity of more than 500 times, 600 times, 700 times, 800 times, 900 times or 1000 times when integrated into a lipid bilayer.

[0125] A control in an experiment is a group separated from the rest of the experiment, where the independent variable being tested cannot influence the results. When testing a sample of cells and / or lipid vesicles, the at least one control sample may comprise a compound of Formula (I) 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.

[0126] Advantageously, compounds of Formula (I) or a salt or solvate thereof have a low (or negligible) photoluminescence when dissolved in an aqueous medium. However, when partitioned into the lipid bilayer, the photoluminescence of compound of Formula (I) or a salt or solvate thereof is enhanced. It is believed that the change in local environment to a hydrophobic one (alkyl chains of the lipid bilayer) allows for the enhancement of fluorescence. Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. In general, the emitted light is of a longer wavelength than the absorbed light.

[0127] In some embodiments, the compound is characterised by a shift in emission from about 800 nm to about 650 nm when integrated into a lipid bilayer.

[0128] Because compound of Formula (I) or a salt or solvate thereof are advantageously nontoxic (or have a low toxicity) and / or have stable to excitation, when under constant excitation, the photoluminescence intensity may be maintained for a period of time. In this regard, the photoluminescence intensity does not decrease for some time when the compounds are excited under the appropriate wavelength for imaging. This is believed to be due to the conjugation system, which allows for the dissipation and transfer of energy, thus preventing localised heating and degradation of the compound. In some embodiments, the photoluminescence intensity may be maintained for at least 20 min, at least 30 min, at least 40 min, at least 50 min, at least 60 min, at least 1.5 h, at least 2 h, at least 3 h, at least 4 h, at least 6 h, at least 10 h or at least 24 h.

[0129] In some embodiments, the compound of Formula (I) is characterised by a partition coefficient (Kp) between an aqueous medium and a lipid bilayer of about 4 x 106to about 7 x 10sat 25 °C. In some embodiments, the partition coefficient is about 2 x 106M-1to about 3 x 106M’1.

[0130] In some embodiments, the compound of Formula (I) is characterised by a two photon absorption cross section area of about 150 GM to about 500 GM.

[0131] In some embodiments, the compound of Formula (I) is characterised by a IC50 of more than 50 pM.

[0132] The compounds of the present invention can be provided as a composition. The composition can comprise the compound in a polar medium as a single entity. As used herein, 'polar medium' includes polar protic and polar aprotic solvents. Polar solvents have large dipole moments or partial charges and contain bonds between atoms with very different electronegativities such as oxygen and hydrogen. Protic solvents have O- H or N-H bonds (a hydrogen atom bound to an oxygen, nitrogen or fluoride). Such bonds allow for participation in hydrogen bonding. Additionally, these O-H or N-H bonds can serve as a source of protons (H+). In general, any solvent that contains a labile H+is a protic solvent. Aprotic solvents may have hydrogens on them somewhere, but they lack O-H or N-H bonds, and therefore cannot hydrogen bond with themselves. Polar solvents include, but is not limited to, dichloromethane, tetrahydrofuran, ethyl acetate, acetone, N,N-dimethylformamide, acetonitrile, dimethylsulfoxide, ammonia, butanol, propanol, ethanol, methanol, acetic acid and water. Included within this definition are also solvent mixtures, wherein the major component of the solvent mixture is a polar solvent. For example, water based solvent or solvent systems can also include dissolved ions, salts and molecules such as amino acids, proteins, sugars and phospholipids. Such salts may be, but 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, HEPES sodium, calcium chloride, ferric nitrate, sodium bicarbonate, potassium phosphate and sodium phosphate. As such, biological fluids, physiological solutions and culture medium also falls within this definition.

[0133] In some embodiments, the composition comprises a compound of Formula (I) or subFormula (la) and a polar medium. For example, the final concentration of the compound of Formula (I) or sub-Formula (la) may be about 100 pM to about 300 pM. In other embodiments, the concentration is about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 100 pM, about 150 pM, about 200 pM, about 250 pM, about 300 pM, about 350 pM, or about 400 pM. In other embodiments, the concentration is not more than 10 pM, not more than 20 pM, not more than 30 pM, not more than 40 pM, not more than 50 pM, not more than 100 pM, not more than 150 pM, not more than 200 pM, not more than 250 pM, not more than 300 pM, not more than 350 pM, or not more than 400 pM.

[0134] Alternatively, the compounds can be provided as a kit. The kit can comprise the compound and the polar medium. The compound and the polar medium can be in separate vessels or as separately packaged components, to be mixed before use. Alternatively, the kit can comprise a composition of the compound in a first polar medium and separately a second medium, both components contained in separate vessels. The kit can additionally comprise another dye for staining a separate component of the bacterial cell. For example, the kit can additionally comprise FM 4-64. The kit can additionally comprise an excipient. The excipient can act to further stabilise the compound, and / or reduce the background noise by further quenching the fluorescence of the compound before its penetration into the bacterial cell membrane.

[0135] Compounds of the present disclosure are suitable for use as a dye or a fluorescence probe. The compounds may be used in membrane labelling, for viscosity sensing in membranes, for bioimaging and / or flow cytometry.

[0136] In particular, the compounds (and other COE compounds) may be preferentially used in stimulated emission depletion (STED) microscopy. STED microscopy is one type of super resolution microscopy techniques that have recently been developed to bypass the diffraction limit of light microscopy to increase resolution. STED is a deterministic functional technique that exploits the non-linear response of fluorophores commonly used to label biological samples in order to achieve an improvement in resolution, that is to say STED allows for images to be taken at resolutions below the diffraction limit. It creates super-resolution images by the selective deactivation of fluorophores, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system.

[0137] It was found that COE compounds are photostability and highly fluorogenic, thus advantageous for a sensitive technique such as STED. Further, as the emission of COE compounds after lipid intercalation may be maintained within a tight range, the sensitivity of STED is not lost.

[0138] The present disclosure relates to a method of imaging a lipid bilayer using stimulated emission depletion (STED) microscopy, comprising contacting the lipid bilayer with a compound of Formula (I) as disclosed herein or a compound of Formula (II) : wherein each R? and Rs are 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; u is an integer selected from 1 to 5; v is an integer selected from 1 to 5;wherein each Lz is independently selected from optionally substituted ethylene, or optionally substituted phenylethylene;

[0139] Li is a n-conjugated core comprising monomeric unit A and monomeric unit D: *“(© * x (D) wherein 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; w is an integer selected from 1 to 5; x is an integer selected from 1 to 5; wherein * represents a bond to another monomeric unit or to L2; wherein monomeric units A and monomeric units D are alternatively bonded to each other.

[0140] In some embodiments, the compound of Formula (II) is: wherein each R? and Rs are 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; u is an integer selected from 1 to 5; v is an integer selected from 1 to 5; wherein each L2 is independently selected from optionally substituted ethylene, or optionally substituted phenylethylene;

[0141] Li is a n-conjugated core comprising monomeric unit A and monomeric unit D: wherein 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; w is an integer selected from 1 to 5; x is an integer selected from 1 to 5; wherein * represents a bond to another monomeric unit or to L2; wherein monomeric units A and monomeric units D are alternatively bonded to each other.

[0142] In some embodiments, the compound of Formula (II) has a substantially linear topology. 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, Li is not butadienylene, polyalkenylene, phenylalkenylene, polyphenylalkenylene.

[0143] In some embodiments, the bonds connecting monomeric units A and monomeric units D in Li are substantially aligned along a longitudinal axis of the compound. In other embodiments, the monomeric units A and monomeric units D in Li are substantially aligned along a longitudinal axis of the compound. In this regard, bonds connecting monomeric units A and monomeric units D when offset from the longitudinal axis of the compound are within the scope of the invention.

[0144] The compounds of Formula (II) are linear in order to accommodate its position within the lipid bilayer. In some embodiments, the compounds are not branched; i.e. the monomeric units only extend along a single chain. In some embodiments, the compounds of Formula (II) are symmetrical in nature. The symmetry of a compound can be described by at least one of the 32 point groups. A Point Group describes all the symmetry operations that can be performed on a molecule that result in a conformation indistinguishable from the original. In this regard, in some embodiments, the compounds of Formula (II) have a C2v point group.

[0145] In some embodiments, L2 is independently selected from optionally substituted ethylene, or optionally substituted phenylethylene. In other embodiments, L2 is independently selected from: wherein * represents a bond to a monomeric unit and to a terminal phenyl moiety in compound of Formula (II).

[0146] Li is a n-conjugated core. A conjugated system is a system of connected p orbitals with delocalized electrons in a molecule, which in general lowers the overall energy of the molecule and increases stability. Lone pairs, radicals or carbenium ions may be part of the system, which may be cyclic, acyclic, linear or mixed.

[0147] In some embodiments, when Li comprises 6 membered aryl or heteroaryl, or when Li comprises fused aryl or heteroaryl having a 6 membered ring, the monomeric units are 1,4 conjugated on the 6 membered ring. In other embodiments, when Li comprises 5 membered aryl or heteroaryl, or when Li comprises fused aryl or heteroaryl having a 5 membered ring, the monomeric units are 1,4 conjugated or 2,5 conjugated on the 5 membered ring.

[0148] Alternatively, Li can be represented by at least one monomeric unit A and at least one monomeric unit D. In some embodiments, w and x in combination is an integer selected from 2 to 10, 3 to 10, 3 to 9, 3 to 8, 3 to 10, or 3 to 7.

[0149] In some embodiments, Li is selected from:

[0150] As mentioned, each A and D can be the same moiety such that Li is an alternating n- conjugated core. Alternatively, each A and D can be different. As Li comprises an alternating donor / acceptor composition of structural units (relative to each other), structures in which the alternating donor / acceptor composition is not adhered to are excluded from the scope of this invention. For example, butadienylene, polyalkenylene, phenylalkenylene and polyphenylalkenylene are excluded.

[0151] In some embodiments, the optional substituent on Li is 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 substituent on D is selected from halogen, cyano, alkyl, alkenyl, alkoxy, and alkenyloxy. In some embodiments, the optional substituent on Li is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl.

[0152] As used herein, monomeric unit D is an electron donating (rich) moiety relative to monomeric unit A. In this regard, monomeric unit A is an electron withdrawing / accepting (poor) moiety. When in sequence the D-A combination gives rise to intramolecular charge transfer excited states with optical absorption and emission further into the red, as compared to a sequence of similar moieties (-Dn- or -An-) in conjugation.

[0153] In some embodiments, A is an electron accepting moiety. An electron acceptor is a chemical entity that accepts electrons transferred to it from another moiety or compound. In some embodiments, A has electron accepting substituents. In other embodiments, A has electron withdrawing substituents.

[0154] In some embodiments, A is independently selected from optionally substituted alkenylene or optionally substituted heteroarylene. In some embodiments, A is independently selected from cyano substituted alkenylene or optionally substituted heteroarylene. In some embodiments, the cyano substituted alkenylene is monosubstituted alkenylene or di-substituted alkenylene. In other embodiments, the optionally substituted heteroarylene is optionally substituted monocyclic heteroarylene or optionally substituted fused heteroarylene. The heteroarylene can be 5 membered heteroarylene or a 6 membered heteroarylene. The heteroarylene can be a fused heteroarylene. In some embodiments, the heteroarylene is a fused 5,5 membered heteroarylene, fused 5,6 membered heteroarylene, fused 6,6 membered heteroarylene, fused 5,5,6 membered heteroarylene, fused 5,6,6 membered heteroarylene, fused 6,6,6 membered heteroarylene, fused 5, 5, 6, 6 membered heteroarylene, fused 5, 6, 6, 6 membered heteroarylene, or fused 6, 6, 6, 6 membered heteroarylene.

[0155] In some embodiments, the optional substituent on A is 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 substituent on A is selected from halogen, cyano, alkyl, alkenyl, alkoxy, and alkenyloxy. In some embodiments, A is selected from wherein ''' represents a bond to D or to L2; each Xi 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 Xi and X2 is 0, N or S;

[0156] R is independenly selected from H, halo, cyano, optionally substituted alkyl, optionally substituted aryl and optionally substituted heteroaryl. wherein ''' represents a bond to D or to L2;

[0157] Ra, Rb, Rc and Rd 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

[0158] Ra and Rb are linked to form optionally substituted heterocyclyl, optionally substituted heteroaryl; or

[0159] Rc and Rd are linked to form optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl. In some embodiments, Raand Rb are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl. In other embodiments, Ra and Rb are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl. In other embodiments, Raand Rb are independently selected from H, halogen, optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted Ci-Cs alkoxy, and optionally substituted C2-C6 alkenyloxy. In other embodiments, Ra and Rb are independently selected from H, halogen, and Ci-Cs alkyl.

[0160] In some embodiments, wherein ''' represents a bond to D or to L2;

[0161] Ra and Rb are linked to form optionally substituted heterocyclyl, optionally substituted heteroaryl;

[0162] Rc and Rd 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

[0163] Rc and Rd are linked to form optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl.

[0164] In some embodiments, Raand Rb are linked to form optionally substituted heteroaryl such that it forms a conjugated n system with the phenyl moiety.

[0165] In some embodiments, Rcand Rd are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl. In other embodiments, R4 and Rs are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl. In other embodiments, Rc and Rd are independently selected from H, halogen, optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted Ci-Cs alkoxy, and optionally substituted C2-C6 alkenyloxy. In other embodiments, Rc and Rd are independently selected from H, halogen, and Ci-Ce alkyl.

[0166] In some embodiments, Rcand Rd are linked to form optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl. In other embodiments, Rcand Rd are linked to form optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl. In some embodiments, Rc and Rd are linked to form optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, Rc and Rd are linked to form optionally substituted heteroaryl such that it forms a conjugated n system with the phenyl moiety. w herein '' represents a bond to D or to L2;

[0167] Re is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl;

[0168] Rf, Rfl, Rh and Ri are independently selected from H and optionally substituted alkyl.

[0169] In some embodiments, Reis selected from C1-C5 alkyl and optionally substituted phenyl. The phenyl may be optionally substituted with halo, alkyl, alkenyl, alkoxy, alkenyloxy, amino, cyano, or nitro. In some embodiments, the optional substituent is selected from, alkyl, alkoxy, amino, cyano, or nitro.

[0170] In some embodiments, Rr, Rg, Rh and Ri are each independently H. In some embodiments, Rf, Rg, Rh and Ri are each optionally substituted alkyl. In some embodiments, Rf, Rg, Rh and Ri are each independently selected from methyl, ethyl, propyl, t-butyl or n-butyl.

[0171] In some embodiments, w is an integer selected from 1 to 4, 1 to 3, 1 to 2, 2 to 4, 3 to

[0172] 4 or 3 to 5.

[0173] In some embodiments, D is an electron donating moiety. An electron donor is a chemical entity that donates electrons transferred from it to another moiety or compound. In some embodiments, D has electron donating substituents.

[0174] In some embodiments, D is independently selected from optionally substituted alkenylene, optionally substituted arylene or optionally substituted heteroarylene. In some embodiments, D is independently selected from alkenylene, arylene or optionally substituted heteroarylene. In other embodiments, the arylene is phenylene. In other embodiments, the optionally substituted heteroarylene is optionally substituted monocyclic heteroarylene or optionally substituted fused heteroarylene. The heteroarylene can be 5 membered heteroarylene or a 6 membered heteroarylene. The heteroarylene can be a fused heteroarylene. In some embodiments, the heteroarylene is a fused 5,5 membered heteroarylene, fused 5,6 membered heteroarylene, fused 6,6 membered heteroarylene, fused 5,5,6 membered heteroarylene, fused 5,6,6 membered heteroarylene, fused 6,6,6 membered heteroarylene, fused 5, 5, 6, 6 membered heteroarylene, fused 5, 6, 6, 6 membered heteroarylene, or fused 6, 6, 6, 6 membered heteroarylene.

[0175] In some embodiments, the optional substituent on D is 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 substituent on D is selected from halogen, cyano, alkyl, alkenyl, alkoxy, and alkenyloxy.

[0176] In some embodiments, D is a moiety selected from w herein '' represents a bond to A or to La; each Xi 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 Xi and X2 is 0, N or S;

[0177] R is independently selected from H, halo, cyano, and optionally substituted alkyl.

[0178] In some embodiments, D is an optionally substituted 5 membered heteroarylene.

[0179] In some embodiments, wherein Y is NR, 0, S, o

[0180] Rj and Rk 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

[0181] Rj and Rk are linked to form optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl.

[0182] In some embodiments, Rj and kare independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkoxy, or optionally substituted alkenyloxy. In other embodiments, j and Rk are independently selected from H, halogen, optionally substituted alkyl, or optionally substituted alkoxy. In other embodiments, Rj and Rkare independently selected from H, halogen, optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, optionally substituted Ci- Ce alkoxy, or optionally substituted C2-C6 alkenyloxy. In other embodiments, Rj and Rk are independently selected from H, halogen, optionally substituted Ci-Cs alkyl, or optionally substituted Ci-Ce alkoxy. In other embodiments, Rj and Rkare independently selected from H, halogen, or optionally substituted Ci-Ce alkyl.

[0183] In some embodiments, Rj and Rk are linked to form optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl. In other embodiments, Rj and Rk are linked to form optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0184] In some embodiments, D is independently selected from

[0185] In some embodiments, x is an integer selected from 1 to 4, 1 to 3, 1 to 2, 2 to 4, 3 to 4, or 3 to 5.

[0186] In some embodiments, w and x together is at least 3. In other embodiments, w and x together is at least 4 or 5.

[0187] In some embodiments, each R? and Rs are 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, each R? and Rs are independently optionally substituted alkoxy, optionally substituted oxyacyl or optionally substituted amino. In another embodiment, each R? and Rs are independently optionally substituted polyethoxy, wherein the monomeric unit is from 3 to 10. In other embodiments, the chain length of each R? and Rs are independently from about 3 to about 10. In other embodiments, each R? and Rs are independently optionally substituted C3-C10 alkoxy, optionally substituted C3-C10 alkylamino, optionally substituted C3-C10 dialkylamino, optionally substituted C3-C10 alkyloxyacyl or optionally substituted polyethoxy. In some embodiments, each R7 and Rs are independently selected from optionally substituted alkyl, optionally substituted alkoxy. In some embodiments, each R7 and Rs are independently selected from alkyl and alkoxy, each optionally substituted with amino, or alkylamino.

[0188] In some embodiments, the optional substituent at each R? and Rs are independently selected from oxy, oxyacyl, acyl, amino, phosphoryl, thiol, alkyl, alkenyl, alkynyl, oxyalkyl, alkylacyloxy, sulfonyl, chlorate or its charged species thereof. In some embodiments, the optional substituent at each R7 and Rs are independently selected from hydroxyl, carboxyl, phosphate, amino, alkylamino, dialkylamino, chlorate, sulphate, acetate or its charged species thereof. In some embodiments, the optional substituent at each R7 and Rs are independently tertiary amino. The tertiary amino may be neutralised by a counterion, which can be a halide.

[0189] In other embodiments, the optional substituent at each R? and Rs are independently a hydrophilic moiety. In other embodiments, the optional substituent at each R7 and Rs are independently a charged moiety. Examples of hydrophilic and / or charged moieties are trialkylammonium halide. For example, the charged moiety can be trimethylammonium iodide. In this embodiment, each R7 and Rs independently terminates with trimethylammonium, and thereby imparts a positive charge when substituted to Ri (for example, alkyl). Other cationic charged groups include but are not limited to pyridinium, pyrrolidinium, imidazolium, guanidinium, sulfonium, thiouronium, and phosphonium. Other anionic charged groups include but not limited to chlorate, sulphate, phosphate, acetate, carboxyl, hydroxide. The hydrophilic and / or charged moieties can also in zwitterionic form that contains both cationic and anionic charged groups through covalent bonds. The excess charges can be neutralized by acceptable cations or anions. Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others. Examples of the optional substituents on each R? and Rs can independently be selected from:

[0190] For compound of Formula (II) to have a balance of hydrophilic and hydrophobic properties to facilitate its intercalation within the lipid bilayer, at least one side chain should be present at each terminus of the backbone. In this regard, in some embodiments, u is an integer from 1 to 4. In other embodiments, u is an integer from 1 to 3. In some embodiments, v is an integer from 1 to 4. In other embodiments, v is an integer from 1 to 3. In this regard, there are on compound of Formula (II) a total of at least 2 R? groups, at least 3 R? groups, at least 4 R? groups, at least 5 R? groups or at least 6 R? groups. There are on compound of Formula (II) a total of at least 2 Rs groups, at least 3 Rs groups, at least 4 Rs groups, at least 5 Rs groups or at least 6 Rs groups.

[0191] For compound of Formula (II) to maintain its linear configuration, R? and Rs are preferentially positioned at the meta and / or para positions of the terminus phenyl groups. In some embodiments, R? and Rs are present at the meta and para positions of the terminus phenyl groups. In other embodiments, R? and Rs are present at the meta or para positions of the terminus phenyl groups.

[0192] In some embodiments, the compound of formula (II) is a compound of Formula (Ila) : (Ha).

[0193] In some embodiments, the compound of formula (II) is a compound of Formula (Ila), wherein

[0194] In some embodiments, the compound of formula (II) is a compound of Formula (lib) : (lib).

[0195] In some embodiments, the compound of formula (II) is a compound of Formula (lib), wherein

[0196] In some embodiments, the compound of formula (II) is a compound of Formula (lib), wherein

[0197] In some embodiments, the compound of formula (II) is a compound of Formula (Hb), wherein

[0198] In some embodiments, the compound of Formula (II) or a salt or solvate thereof is selected from

[0199]

[0200]

[0201] In some embodiments, the compound of Formula (II) is a salt thereof. The salt form can be a protonated salt, or can be generated by alkylating compound of Formula (II) with halocarbons. For example alkylhalide (such as CHaBr or CH3I) can be used. In some embodiments, the compound of Formula (II) or a salt or solvate thereof is a quaternary ammonium salt.

[0202] It was found that the elongated backbone of COE compounds favor greater membrane stability, while the enriched hydrophilic groups are expected to increase aqueous solubility. Besides, these combined physical features increase both hydrophobic and electrostatic interactions to promote intercalation within the bilayer. When the compounds are used as membrane labelling dye, they may stably be incorporated within the lipid bilayers via the strong binding forces for a long-time staining.

[0203] The present disclosure also relates to a method of staining a cell membrane and / or lipid vesicle, comprising contacting the compound of Formula (I) or (II) or a salt or solvate thereof with the cell membrane and / or lipid vesicle.

[0204] In some embodiments, the cell membrane is from an adherent cell. Adherent cells are cells which must be attached to a surface to grow. They are commonly used in laboratory environments.

[0205] In some embodiments, the contact period is about 1 min to about 12 days. In some embodiments, the compound of Formula (I) or (II) or a salt or solvate thereof with the cell and / or lipid vesicle are incubated for about 5 min to about 120 min. In other embodiments, the duration is about 5 min to about 110 min, about 5 min to about 100 min, about 5 min to about 90 min, about 5 min to about 80 min, about 5 min to about 70 min, about 5 min to about 60 min, about 5 min to about 50 min, about 5 min to about 40 min, about 5 min to about 30 min, about 5 min to about 20 min, or about 5 min to about 10 min.

[0206] The compounds may be used in flow cytometry to study biological cells, bacterial cells and extracellular vesicles (e.g., lipid vesicles, exosomes). These biological samples are defined by the essentiality of a lipid bilayer (membrane), of which COEs are designed to maintain a high affinity with. Hence, the physicochemical and optoelectronic properties of the compounds of the present disclosure when associated within a lipid bilayer allows them to be used a dye for flow cytometry applications or in flow systems.

[0207] The present disclosure also relates to a method of detecting a cell membrane and / or a lipid vesicle using a fluorescence detector, comprising : a) contacting a compound of Formula (I) or (II) or a salt or solvate thereof with the cell membrane and / or lipid vesicle; and b) passing the cell membrane and / or lipid vesicle through the fluorescence detector.

[0208] In some embodiments, the compound of Formula (I) or (II) is configured to exhibit an increasing fluorescence emission lifetime as it is internalised into the cell. In some embodiments, the compound of Formula (I) or (II) is characterised by a fluorescence emission lifetime of about 1 ns to about 1.5 ns about 0.5 h after uptake by the cell. In some embodiments, the compound of Formula (I) or (II) is characterised by a fluorescence emission lifetime of about 1 ns to about 1.5 ns when bound to a cell membrane of the cell.

[0209] In some embodiments, the compound of Formula (I) or (II) is characterised by a fluorescence emission lifetime of about 2 ns to about 2.5 ns about 1 h to about 2 h after uptake by the cell. In some embodiments, the compound of Formula (I) or (II) is characterised by a fluorescence emission lifetime of about 2 ns to about 2.5 ns when bound to an early endosome of the cell.

[0210] The application of these compounds can be used in the staining of bacterial cells, mammalian (including but not limited to A549 cancer cells and red blood cells), and exosomes (especially unbound exosomes).

[0211] The cell can be a mammalian cell or a bacterial cell. In some embodiments, the bacterial cell is a Gram-positive or Gram-negative bacterial cell. In other embodiments, the Gram-negative or Gram-positive bacterial cells is selected from the group consisting of E. coli, P. aeruginosa, S. aureus, E. faecalis, S. oneidensis, B. megaterium or a combination thereof. The mammalian cell can be from a cell line, or from a sample derived from a subject.

[0212] In some embodiments, the sample of bacterial cells is a sample of planktonic bacterial cells.

[0213] 'Planktonic bacterial cells' as used herein refers to free flowing bacterial cells in suspension. This is as opposed to the sessile state (or biofilm), in which a structured community of bacterial cells is enclosed in a self-produced polymeric matrix and adherent to an inert or living surface. In this regard, planktonic bacteria are free-living bacteria and makes up the populations that grow in test tubes and flask cultures in the laboratory.

[0214] The lipid vesicle is a structure within or outside a cell, consisting of a liquid or cytoplasm enclosed by a lipid bilayer. Vesicles form naturally during the processes of secretion (exocytosis), uptake (endocytosis) and transport of materials within the plasma membrane. Alternatively, they may be prepared artificially, in which case they are called liposomes. Unilamellar lipid vesicles has one phospholipid bilayer, while multilamellar lipid vesicles has more than one bilayer. Vesicles can also fuse with other organelles within the cell. A vesicle released from the cell is an extracellular vesicle. For example, the lipid vesicle can be an exosome. Exosomes are membrane-bound extracellular vesicles that are produced in the endosomal compartment of most eukaryotic cells. These lipid vesicles are included within the scope.

[0215] In some embodiments, the lipid vesicle is an extracellular vesicle. In other embodiments, the lipid vesicle is an exosome.

[0216] In some embodiments, the compound of Formula (I) or (II) or a salt or solvate thereof is provided at a concentration of about 1 nM to about 100 pM. In other embodiments, the concentration is about 1 nM to about 90 pM, about 1 nM to about 80 pM, about 1 nM to about 70 pM, about 1 nM to about 60 pM, about 1 nM to about 50 pM, about 1 nM to about 40 pM, about 1 nM to about 30 pM, about 1 nM to about 20 pM, about 1 nM to about 10 pM, about 1 nM to about 5 pM, about 1 nM to about 1 pM, about 1 nM to about 900 nM, about 1 nM to about 800 nM, about 1 nM to about 700 nM, about 1 nM to about 600 nM, about 1 nM to about 500 nM, about 1 nM to about 400 nM, about 1 nM to about 300 nM, about 1 nM to about 200 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, or about 1 nM to about 20 nM.

[0217] In some embodiments, the compound of Formula (I) or (II) or a salt or solvate thereof with the cell membrane and / or lipid vesicle are incubated at about 5 °C to about 50 °C. In other embodiments, the temperature is about 5 °C to about 45 °C, about 5 °C to about 40 °C, about 5 °C to about 35 °C, about 10 °C to about 35 °C, about 15 °C to about 35 °C, or about 15 °C to about 30 °C. In other embodiments, the temperature is room temperature or ambient temperature.

[0218] The compound of Formula (I) or (II) or a salt or solvate thereof and the cell and / or lipid vesicle can be incubated in an aqueous medium.

[0219] The term 'aqueous medium' used herein refers to a water based solvent or solvent system, and which comprises of mainly water. Such solvents can be either polar or nonpolar, and / or either protic or aprotic. Solvent systems refer to combinations of solvents which resulting in a final single phase. Both 'solvents' and 'solvent systems' can include, and is not limited to, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, dioxane, chloroform, diethylether, dichloromethane, tetra hydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, nitromethane, propylene carbonate, formic acid, butanol, isopropanol, propanol, ethanol, methanol, acetic acid, ethylene glycol, diethylene glycol or water. Water based solvent or solvent systems can also include dissolved ions, salts and molecules such as amino acids, proteins, sugars and phospholipids. Such salts may be, but 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, HEPES sodium, calcium chloride, ferric nitrate, sodium bicarbonate, potassium phosphate and sodium phosphate. As such, biological fluids, physiological solutions and culture medium also falls within this definition.

[0220] As the compound of Formula (I) or (II) or a salt or solvate thereof emits fluorescence when intercalated with the cell membrane or lipid bilayer, fluorescence based techniques can be used to detect cells and / or lipid vesicles incorporated with compound of Formula (I) or (II) or a salt or solvate thereof. Examples of fluorescence based techniques include, but is not limited to, fluorescence microscopy, confocal microscopy, plate readers, fluorometer, fluorescence spectroscopy, and flow cytometry (such as fluorescence activated cell sorting).

[0221] The cell and / or lipid vesicle when incorporated with compound of Formula (I) or (II) or a salt or solvate thereof can be excited by an electromagnetic radiation (source) in a wavelength range of about 300 nm to about 1000 nm.

[0222] The cells can be adherent cells, or can be in suspension. The lipid vesicles can be exosomes (membrane-bound extracellular vesicles that are produced in the endosomal compartment of eukaryotic cells), synthetic and non-synthetic liposomes, or lipid nanoparticles (spherical vesicles made of ionizable lipids, which are positively charged at low pH and neutral at physiological pH). Advantageously, it was found that after intercalation of cell membranes by the fluorescence probe, the fluorescence probe can perpetuate to later cell populations.

[0223] In some embodiments, the cell and / or lipid vesicle can be flowed through a flow system without a purification step. This is possible as the free compound of Formula (I) or (II) or a salt or solvate thereof are weakly emissive and will yield less background.

[0224] By flowing the cell and / or lipid vesicle through the flow system, the cell and / or lipid vesicle can be excited by an electromagnetic radiation and subsequently detected by a detector. Accordingly, the method may further include a step of exposing the cell and / or lipid vesicle to electromagnetic radiation having a wavelength of less than about 2500 nm, or less than about 1000 nm.

[0225] Compounds of Formula (I) or (II) or a salt or solvate thereof can work in combination with other dyes, for example membrane dye. For example, a commercial available dye, FM4-64, can be added to recognize bacterial envelope type in-situ in the bacteria mixture. In this regard, a dual-dye system that can recognise polymicrobial samples is also disclosed. These methods are easy-to-use requiring only a simple application of a dye mixture with no fixation or other pre-treatment requirement, and compound of Formula (I) or (II) or a salt or solvate thereof is stable in aqueous solution and can be used to monitor the cells in a living system.

[0226] Accordingly, in an embodiment, the method further comprises a step of contacting the cell and / or lipid vesicle with another dye. The dye can be used to stain cell membranes, nucleus, DNA, RNA, or other organelles in the cell. The dye can be a fluorescence probe, such as FM4-64, FM 2-10, FM 1-43, Propidium Iodide, SYTO 82, SYTO 83, SYTO 84, SYTO 85, YOYOS-3 iodide, YO-PRO™-3 Iodide, BOBO™-3 Iodide, Ethidium Homodimer- 1, Ethidium Homodimer-2, Ethidium monoazide, Acridine Orange, CellMask™ Plasma Membrane Stains or Di-4-ANEPPS.

[0227] The present disclosure also relates to a flow system for detecting and / or quantifying cells membrane and / or lipid vesicle, comprising: a) a compound of Formula (I) or (II) or a salt or solvate thereof for labelling the cell membrane and / or lipid vesicle; b) an inlet for introducing the labelled cell membrane and / or lipid vesicle into the flow system; c) a detection means in fluid communication with the inlet for detecting a fluorescence emission from the labelled cell membrane and / or lipid vesicle; and d) optionally a counter means for quantifying the labelled cell membrane and / or lipid vesicle.

[0228] The flow system can for example be a microfluidic chip.

[0229] In some embodiments, the flow system further comprises an incubation means. The incubation means allows the compound of Formula (I) or (II) or a salt or solvate thereof to intercalate with the cell membrane or within the lipid bilayer.

[0230] In some embodiments, the detection means is a fluorescence detector.

[0231] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

[0232] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "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.

[0233] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0234] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0235] Examples

[0236] General protocol for the synthesis of Compound of Formula (I)

[0237] Synthesis route of fa) COE-KP

[0238] The synthetic route towards COE-KP is described in Figure 2. In brief, assembly of the neutral chromophore leverages on a key Suzuki-Miyaura coupling between the stilbene "wing" 1 and the diketopyrrolopyrrole derivative 2. Subsequently, a Finkelstein reaction and quaternization with trimethylamine furnished COE-KP in 80% yield, which features high aqueous solubility (>20 mM).

[0239] Results

[0240] The absorption and emission spectra of COE-KP in different organic solvents are shown in Figure 3, with emission spectra taken from the neutral precursor KP-I (compound 4) as necessary. Small unilamellar vesicles (SUVs) composed of l-palmitoyl-2-oleoyl- glycero-3-phosphocholine (POPC) and l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(l'- rac-glycerol) sodium (POPG) (85: 15, mol / mol) were used as a model system to investigate the photophysical properties when embedded within a lipid bilayer, mimicking the probe environment when used for staining cellular membranes. SUV (Small Unilamellar Vesicle) is a type of artificially prepared lipid vesicle (liposome) that consists of a single lipid bilayer membrane (unilamellar) enclosing an aqueous core. COE-KP features a low-energy absorption band centered around 600 nm, see Figure 3a. When dissolved in water, this absorption maxima is red-shifted by approximately 50 nm to 650 nm. In organic solvents, both COE-KP and KP-I feature an emission envelope spanning 600 to 750 nm, with a maxima at 655 nm (Figure 3b). Furthermore, the Lippert-Mataga plot for KP-I and COE-KP, with the Stokes' shift expressed as a function of solvent orientation polarizability (Af), is shown in Figure 4. No notable change in Stokes' shift was observed in solvents of varying Af, reflecting a minimal solvatochromic effect. To further highlight the absence of any notable solvent effects, we recording the excitation spectra of KP-I and COE-KP in various solvents, see Figure 5. Similar to the Lippert-Mataga plot, no notable changes in spectral shape or excitation maxima (Aex) were observed.

[0241] As a prelude to applications in staining intracellular membranes, we first compared the fluorescence spectra of COE-KP in phosphate buffered saline (PBS), a commonly employed buffer used in biological applications, to that of within SUVs. Upon integrated into SUV bilayers, COE-KP features over a 1000-fold enhancement in fluorescence emission intensity, as shown in Figure 6, when detected at the emission maxima. This is attributable to the large blue-shift in emission maxima (Xem) from ~800 nm in PBS to 652 nm in SUVs, resulting in no detectable emission at 650 - 680 nm in aqueous solutions. This shift, coupled with efficient quenching of fluorescence signal in water, allows for an unprecedented turn-on effect compared to previous COEs. Most notably, this fluorogenic nature can easily directly observed in photographic images of COE-KP when within SUVs, see Figure 7. Bright red fluorescence only observed in organic solvents or when the chromophore is embedded within the lipid bilayer of SUVs, but not when in PBS. Furthermore, staining of giant unilamellar vesicles (GUVs) with COE-KP showed a 'halo-like' structure with a notable equatorial extinction line when imaged with a polarized light source (Figure 7, right), reflecting the membrane-intercalated orientation of the DPP chromophore.

[0242] To demonstrate the high affinity of COE-KP for lipid bilayers, we titrated a 1 pM COE solution against POPC SUVs used as a model membrane (Figure 8). Notably, we observed a sharp increase in fluorescence emission, even at low lipid concentrations of 10 pM. Fitting of this data to a non-linear regression model gave an estimated partition coefficient, Kp (SUV) of 2.4 ± 0.4 x 106M-1, indicating a high preference for the lipid phase. To evaluate the probe photostability, SUVs stained with COE-KP were continually excited in a fluorimeter and the resultant emission signal was recorded at regular intervals. As shown in Figure 9, minimal decrease was also observed in emission intensity even after 2 hours of measurements.The unprecedented fluorogenic character of COE-KP conferred by the efficient quenching of its emission within aqueous environments, spontaneous self-assembly within lipid bilayers, and large emission blue-shift within the hydrophobic lipid environment relative to water thus underscores its potential for use in bioimaging of cellular membranes.

[0243] The ability of COE-KP to label subcellular organelles was first studied by confocal laser scanning microscopy (CLSM). Colocalization with a panel of organelle specific fluorescent tags showed that COE-KP localizes primarily within LAMP-1 positive lysosomes, see Figure 10, as reflected by the high Pearson's correlation of 0.64 and the similar line profiles (Figure 10b). No significant correlation was observed within other subcellular organelles, as shown in Figure 10c, particularly those featuring similar vesicular structures, such as the mitochondria (R = 0.05), endoplasmic reticulum (R = 0.29), or lipid droplets (R = 0.01). That staining persists within the lysosomes over extended periods of labeling without notable redistribution to either cytosolic or mitochondrial compartments enables long-term imaging of the vesicles of interest. Flow cytometric analysis of cells stained with COE-KP and detected in the NIR region (710 nm) showed homogenous labelling (Figure 11).

[0244] The favorable photophysical properties of COE-KP, including photostability and its highly fluorogenic nature led us to investigate its use for super-resolution imaging using stimulated emission depletion (STED) microscopy. COE-KP was first used to stain the lysosomes of HeLa cells, prior to fixation and imaging. Deconvoluted STED images acquired with excitation at 640 nm and a depletion laser of 775 nm, revealed that the staining pattern of COE-KP within the lysosomes formed a halo-like structure, as shown in Figure 12. In contrast to the single puncta observed in the confocal images, the images obtained with STED microscopy visually confirms that the COE is indeed localized within the lipid bilayer membrane of the lysosome, as opposed to being within the lumen. Figure 13 shows two ROIs in greater detail, with line plots of the corresponding regions across specific vesicles. Fitting of the fluorescence intensity of the line plots to a Lorentzian function gave a full width at half maximum value (FWHM) of 61 ± 11 nm, narrower than the 526 ± 31 afforded by analysis of the CLSM image.

[0245] The increased definition of the vesicles stained by COE-KP provides a tool to analyze changes in vesicle morphology. Cells were first starved in serum-free media, which induces autophagy in cells due to limited nutrient availability. Figure 14a showcases the STED images for COE-KP stained cells in comparison to the image capture by confocal. A detailed examination of a subpopulation of the vesicles, as seen in Figure 14b, reveals a clear multilamellar structure. This fine structure is enabled by the increase in resolution and emission from the COE localized within the vesicle membrane. Furthermore, the fluorescence intensity line profile in Figure 14c shows clear peak to peak separation that corresponds to the individual vesicle membranes, with a resolution usually obtainable only through other imaging modalities such as electron microscopy. To further confirm this, we additionally treated the cells with chloroquine, which results in enlargement of vesicles. Measurement of the relative vesicle size for serum-starved and chloroquine treatment revealed a significant enlargement of the vesicles compared to the untreated control, as shown in Figure 15, from 216 ± 44 nm (control) to 378 ± 43 nm (starved) and 362 ± 25 nm (chloroquine) respectively. Taken together, these observations show that COE-KP is capable of visualizing and distinguishing subtle morphological changes within the lysosomes upon treatment at a resolution below the diffraction limit. These results open up additional possibilities for utilizing COEs as a platform for designing dyes localized within the lipid bilayer for super resolution imaging of subcellular membranes.

[0246] In summary, we disclose a COE compound, COE-KP, that is sensitive to the protic environment surrounding the diketopyrrolopyrrole fluorophore. COE-KP exhibits good brightness, two-photon cross section area, and emission into the NIR region, which are all highly favorable photophysical properties for bioimaging. STED nanoscopy with COE- KP enabled direct observation for the first time of membrane-bound COEs located within subcellular vesicles, representing a significant advance in the optical resolution that can be studied with COE-based lipid bilayer probes. More broadly, we envision that further applications and rational structural design will enable COEs to form a central class of optical probes both in nanoscopy and functional bioimaging of subcellular organelles and lipid bilayer-based nanoparticles, or vesicles such as exosomes.

[0247] Synthesis of Compound 3:

[0248] To a two-necked round-bottom flask, compound 5 (250 mg, 0.350 mmol) and 8 (100 mg, 0. 175 mmol) were weighed out and the flask was charged with argon. The reactants were then dissolved in toluene (10 mL), followed by the addition of Pd(PPha)4 under argon (115 mg, 0.1 mmol). A degassed aqueous solution of potassium carbonate (2M, 2.5 mL) was then added to the mixture and the reaction was heated to 100 °C and stirred for 16 hours. After cooling to room temperature, the reaction mixture was poured into water and extracted with dichloromethane (2 x 20 mL). The organic phases were combined and sequentially washed with water and brine. The solvents were removed under reduced pressure with a rotary evaporator and the crude product was purified by silica gel column chromatography (DCM / hexanes, 4: 1 to 9: 1). This was subsequently recrystallized in chloroform / methanol to give compound 9 as a red-black solid (220 mg, 40% yield).

[0249] NMR (400 MHz, Chloroform-d) 3 8.89 (s, 2H), 7.58 (d, J = 7.3 Hz, 4H), 7.49 - 7.37 (m, 6H), 6.94 (q, J = 16.2 Hz, 4H), 6.66 (s, 4H), 4.07 (t, J = 7.8 Hz, 4H), 3.94 (dt, J = 23.8, 6.4 Hz, 12H), 3.59 - 3.34 (m, 12H), 1.81 - 1.69 (m, 25H), 1.47 (s, 25H), 0.94 (t, J = 7.3 Hz, 6H).

[0250] Synthesis of Compound 4:

[0251] In a pressure vessel, compound 9 (50 mg, 0.032 mmol) and sodium iodide (713 mg, 4.8 mmol, 150 eqv.) were dissolved in acetone (10 mL) and heated to reflux at 80 °C. The reaction mixture was then stirred for 48 hours. The solvent was removed under vacuum with a rotary evaporator and the crude product was extracted with chloroform (3 x 10 mL). The organic phases were combined and washed with sodium thiosulfate (2 x 20 mL), water and brine. The solvents were then concentrated under vacuum and the crude product was purified with silica gel column chromatography (DCM / hexanes, 2: 1 to 9: 1), washed with methanol, and recrystallized in chloroform / methanol to give compound 10 as a red-black solid (60 mg, 90% yield).

[0252] JH NMR (400 MHz, Chloroform-d) 6 8.94 (d, J = 4.2 Hz, 1H), 7.66 - 7.58 (m, 2H), 7.53 - 7.41 (m, 3H), 7.07 - 6.90 (m, 2H), 6.71 (s, 2H), 4.11 (t, J = 7.8 Hz, 2H), 4.00 (dt, J = 20.0, 6.4 Hz, 6H), 3.21 (td, J = 7.0, 1.8 Hz, 6H), 1.87 (d, J = 7.0 Hz, 15H), 1.52 (s, 15H), 1.02 (t, J = 7.4 Hz, 3H).

[0253] 13C NMR (101 MHz, CDCI3) 6 161.27, 153.24, 149.42, 139.18, 138.38, 137.91, 136.79, 132.42, 132.00, 129.59, 128.71, 127.00, 126.86, 126.27, 124.44, 108.04, 105.33, 73.28, 68.94, 42.08, 33.62, 33.47, 32.13, 30.47, 30.30, 30.18, 29.30, 25.19, 25.15, 20.31, 13.86, 7.20, 7.03.

[0254] Synthesis of COE-KP:

[0255] A 20 mL scintillation vial was charged with compound 10 (25 mg) under argon, to which a minimal amount of CHCI3 was added to fully dissolve the solids. Trimethylamine (1 mL, 2M in THE) was added to the solution and the reaction was stirred at room temperature for 24 hours. The solution was then concentrated under vacuum and the solids were washed with CHCls. The solids were then redissolved in methanol (5 mL), and an additional portion of trimethylamine was added (2 mL, ca. 3.2M). The reaction was then continued for an additional 24 hours at 35°C. The solvents were then removed under vacuum with a rotary evaporator to dryness and the product was dissolved in a minimal amount of methanol and triturated in diethyl ether. This was further washed with CHCh, acetone, and ether followed by drying under high vacuum to afford COE-KP as a shiny black-red solid (23 mg, 80%).XH NMR (400 MHz, DMSO-d6) 6 8.92 (d, J = 4.1 Hz, 2H), 7.98 - 7.79 (m, 6H), 7.71 (d, J = 8.2 Hz, 4H), 7.29 (s, 4H), 6.97 (s, 4H), 4.06 (q, J = 11.4, 6.5 Hz, 12H), 3.90 (t, J = 6.2 Hz, 4H), 3.33 - 3.27 (m, 12H), 3.07 (s, 54H), 1.83 - 1.63 (m, 28H), 1.52 (s, 12H), 1.37 (s, 16H), 0.95 (t, J = 7.4 Hz, 6H).13C NMR (101 MHz, DMSO) 5 160.86, 153.13, 149.69, 149.68, 139.08, 139.00, 137.71, 133.75, 132.85, 131.69, 130.20, 128.45, 127.68, 126.74, 125.76, 107.65, 105.68, 79.41, 72.82, 68.69, 65.75, 52.70,

[0256] 32.03, 29.94, 29.10, 26.06, 25.94, 25.55, 25.50, 22.56, 20.09, 14.12. HRMS [M-3I]3+ : Calc'd 699.3077, Found 699.308.

Claims

Claims1. A compound of Formula (I) or a salt or solvate thereof:Ri and R2 are independently selected from H, optionally substituted alkyl, and optionally substituted alkenyl; each R3 and R4 are independently selected from 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; each Rs and Rs is independently selected from halogen, cyano, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; n and m are independently an integer selected from 1 to 5; p and q are independently an integer selected from 1 to 3; and r and t are independently an integer selected from 0 to 4.

2. The compound according to claim 1, wherein Ri and R2 are independently selected from H and optionally substituted C1-C5 alkyl.

3. The compound according to claim 1 or 2, wherein each R3 and 4 are independently selected from alkyl and alkoxy, each optionally substituted with amino, or alkylamino.

4. The compound according to any one of claims 1 to 3, wherein each Rs and Rs is independently selected from halogen, cyano, or optionally substituted C1-C5 alkyl.

5. The compound according to any one of claims 1 to 4, wherein R3 and R4 are independently at a meta and / or para position relative to the ethylene moiety.

6. The compound according to any one of claims 1 to 5, wherein the compound ofFormula (I) is a compound of Formula (la) :

7. The compound according to any one of claims 1 to 6, wherein the compound is selected from8. The compound according to any one of claims 1 to 7, wherein the compound is characterised by an absorbance of about 600 nm to about 650 nm.

9. The compound according to any one of claims 1 to 8, wherein the compound is characterised by an emission of about 600 nm to about 750 nm.

10. The compound according to any one of claims 1 to 9, wherein the compound is characterised by a Stokes' shift of about 600 cm to about 850 cm when a solvent orientation polarizability (Af) is 0 to about 0.35.

11. The compound according to any one of claims 1 to 10, wherein the compound is characterised by an increase in fluorescence emission intensity of more than 800 times when integrated into a lipid bilayer.

12. The compound according to any one of claims 1 to 11, wherein the compound is characterised by a shift in emission from about 800 nm to about 650 nm when integrated into a lipid bilayer.

13. The compound according to any one of claims 1 to 12, wherein the compound is characterised by a partition coefficient between an aqueous medium and a lipid bilayer of about 2 x 106M-1to about 3 x 106M’1.

14. A method of staining a lipid bilayer, comprising contacting the lipid bilayer with a compound of Formula (I) according to any one of claims 1 to 13.

15. A method of imaging a lipid bilayer using stimulated emission depletion (STED) microscopy, comprising contacting the lipid bilayer with a compound of Formula (I) according to any one of claims 1 to 13 or a compound of Formula (II):wherein each R? and Rs are 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; u is an integer selected from 1 to 5; v is an integer selected from 1 to 5;wherein each L2 is independently selected from optionally substituted ethylene, or optionally substituted phenylethylene;Li is a n-conjugated core comprising monomeric unit A and monomeric unit D:wherein 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; w is an integer selected from 1 to 5; x is an integer selected from 1 to 5; wherein * represents a bond to another monomeric unit or to L2; and wherein monomeric units A and monomeric units D are alternatively bonded to each other.

16. The method according to claim 15, wherein L2 is independently selected from:wherein * represents a bond to a monomeric unit and to a terminal phenyl moiety in compound of Formula (II).

17. The method according to claim 15 or 16, whereinwherein ''' represents a bond to D or to L2;Ra, Rb, Rc and Rd are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; orRa and Rb are linked to form optionally substituted heterocyclyl, optionally substituted heteroaryl; orRc and Rd are linked to form optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl;nd to D or to L2;Re is selected from optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl;Rf, Rg, Rh and Ri are independently selected from H and optionally substituted alkyl.

18. The method according to any one of claims 15 to 17, wherein D is independently selected fromwherein Y is NR, 0, S, or Se;Rj and Rk are independently selected from H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy; orRj and Rk are linked to form optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl.

19. The method according to any one of claims 15 to 18, wherein each R7 and Rs are independently selected from optionally substituted alkyl, optionally substituted alkoxy.

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