Polymer tandem dye having a spacing linker group

Abstract

Compounds useful as fluorescent dyes or chromogenic dyes are disclosed. Such compounds have the following structure (I): 【Chemical 1】 JPEG2025523426000093.jpg47150(I) or a stereoisomer, tautomer or salt thereof. (In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 1a , L 1b , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , M 1 , M 2 , m, n, q and w are as defined herein) Methods related to the preparation and use of such compounds are also provided.

Description

Technical Field

[0001] The present disclosure generally relates to dimers and polymeric fluorescent dyes or chromogenic tandem dyes having a spacing group for brightness enhancement, methods for their preparation, and their use in various analytical methods.

Background Art

[0002] Description of Related Art Fluorescent dyes and / or chromogenic dyes are known to be particularly suitable for applications where highly sensitive detection reagents are desired. By dyes that can preferentially label specific components or constituents in a sample, researchers can determine the presence, amount, and / or location of that specific component or constituent. Furthermore, in certain systems, monitoring of their spatial and temporal distribution in various environments can be performed. Fluorescence and colorimetric quantification methods are very widespread in chemistry and biology. These methods provide useful information regarding the presence, structure, distance, orientation, complex formation, and / or location of biomolecules. Furthermore, time-resolved methods are increasingly being used for the measurement of kinetics and rates. As a result, numerous strategies for the fluorescent or chromogenic labeling of biomolecules such as nucleic acids and proteins have been developed. Since the analysis of biomolecules is usually carried out in an aqueous environment, emphasis has been placed on the development and use of water-soluble dyes.

[0003] The use of highly fluorescent or chromogenic dyes is highly desirable because it increases the signal-to-noise ratio and realizes other related advantages. Therefore, attempts have been made to increase the signals derived from known fluorescent and / or chromogenic moieties. Specifically, Förster resonance energy transfer ("FRET" - sometimes used interchangeably with fluorescence resonance energy transfer) techniques generate information that reliably measures changes in the distance and interaction of biomolecules. Resonance energy transfer techniques are relatively inexpensive and can obtain measurement values quickly. However, FRET is subject to several constraints related to the masking of energy transfer due to the orientation and positioning of chromophores, as well as free fluorophores and undesirable pH sensitivity. Accordingly, there is a need in the art for water-soluble dyes, particularly resonance energy transfer dyes, having increased molar brightness and / or increased FRET emission signals. Ideally, such dyes and biomarkers should have strong chromogenic or fluorescent properties and be available in a variety of colors and fluorescence wavelengths. The present invention meets this need and realizes further related advantages. SUMMARY OF THE INVENTION

[0004] Briefly stated, embodiments of the present disclosure generally relate to water-soluble fluorescent dyes and / or chromogenic dyes and / or compounds useful as probes that enable visual detection of analyte molecules such as biomolecules, and reagents for preparing them. In particular, in some embodiments, the compounds of the present disclosure are useful because they enable FRET fluorescence emission when bound to this compound. Methods for visually detecting analyte molecules using dyes are also described. Embodiments of the disclosed dyes are two or more fluorescent and / or chromogenic moieties (i.e., FRET acceptor M 1 and the corresponding FRET donor M 2 ) covalently linked by a linker having the following structure.

Chemical formula

[0005] Specifically, the ratio of the FRET acceptor M 1 to the corresponding FRET donor M 2 is 1:1, 1:2, 1:3 or 2:3. In contrast to previous reports of dimeric dyes and / or polymeric dyes, this dye is considerably brighter than the corresponding monomeric dye compound, and as a result of intramolecular interactions, FRET absorption and emission are enabled. Without wishing to be bound by theory, the FRET acceptor M 1 separated by a linker from the corresponding FRET donor M 2 is at a specific ratio, bringing the fluorescent moiety and / or chromogenic moiety sufficiently close together, and thus intramolecular FRET is considered to be optimized. Embodiments of the disclosed dyes include a linker having one of the following structures that achieves sufficient proximity between two FRET donors.

Chemical formula

[0006] Furthermore, embodiments of the disclosed dyes have a fluorescent moiety and / or chromogenic moiety (i.e., the FRET acceptor M 1 and the corresponding FRET donor M 2 ) covalently linked to the '5 end' 3 end by a linker having the following structure.

Chemical formula

[0007] In some embodiments, there is provided a compound having the following structure (I) or a stereoisomer, tautomer or salt thereof. [Chemical formula] (wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1a , L 1b , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , M 1 , M 2 , m, n, q, and w are as defined herein) The compounds of structure (I) are found to have several uses in various analytical methods, including use as fluorescent dyes and / or chromogenic dyes.

[0008] In yet another embodiment, there is provided a method for staining a sample, the method comprising adding to the sample a compound of structure (I) in an amount sufficient to produce an optical response when the sample is irradiated at an appropriate wavelength. In yet another embodiment, the present disclosure provides a method for visually detecting an analyte molecule, (a) preparing a compound disclosed herein, and (b) detecting the compound by its visible characteristics comprising the method. Another disclosed method is a method for visually detecting a biomolecule, (a) mixing a compound disclosed herein with one or more biomolecules, and (b) detecting the compound by its visible characteristics comprising the method. Another embodiment is a method for visually detecting an analyte, (a) R 1 or R 2preparing a compound disclosed herein that comprises a linker that includes a covalent bond to a target-directed moiety that has specificity for an analyte; (b) mixing the compound and the analyte, thereby binding the target-directed moiety and the analyte; and (c) detecting the compound by its visible properties to provide a method.

[0009] In yet other embodiments, the disclosure is a method for increasing the luminance of a dye, comprising: (a) preparing a dye solution comprising a compound disclosed herein; and (b) aging the dye solution for a period of time to provide a method. Other embodiments are directed to a composition comprising a compound disclosed herein and one or more analyte molecules, such as one or more biomolecules. Use of such a composition in an analytical method for detecting one or more biomolecules is also provided. These and other aspects of the disclosure will become apparent with reference to the following detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0010] In the following description, certain specific details are set forth in order to provide a thorough understanding of the various embodiments of the disclosure. However, one skilled in the art will understand that the disclosure may be practiced without these details. Unless the context requires a different interpretation, throughout this specification and the claims, the word "comprise", and variations such as "comprises" and "comprising", are to be construed in an open, inclusive sense, that is, as "including, but not limited to". Throughout this specification, when we refer to "one embodiment" or "an embodiment", it means that the specific functions, structures, or features described in connection with this embodiment are included in at least one embodiment of the present disclosure. Therefore, when the phrases "in one embodiment" or "in an embodiment" appear at various positions throughout this specification, they do not necessarily all refer to the same embodiment. Furthermore, the specific functions, structures, or features can be combined in any suitable manner in one or more embodiments.

[0011] "Amino" refers to the -NH2 group. "Carboxy" refers to the -CO2H group. "Cyano" refers to the -CN group. "Formyl" refers to the -C(=O)H group. "Hydroxyl" or "hydroxyl" refers to the -OH group. "Imino" refers to the =NH group. "Nitro" refers to the -NO2 group. "Oxo" refers to the =O substituent. "Sulfhydryl" refers to the -SH group. "Thioxo" refers to the =S group. "Alkyl" refers to a straight-chain or branched hydrocarbon chain group consisting only of carbon atoms and hydrogen atoms, having no unsaturation and having 1 to 12 carbon atoms (C1-C 12 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl), and being a hydrocarbon chain group bonded to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless specifically specified otherwise in this specification, the alkyl group may be substituted.

[0012] "Alkylene" or "alkylene chain" refers to a divalent straight-chain or branched hydrocarbon chain that contains no unsaturation, consists only of carbon and hydrogen, has 1 to 12 carbon atoms, and connects the rest of the molecule to a radical group, such as methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is bonded to the rest of the molecule via a single bond and to the radical group via a single bond. The bonding points of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within this chain. Unless specifically specified otherwise in this specification, alkylene may be substituted. "Alkenylene" or "alkenylene chain" refers to a divalent straight-chain or branched hydrocarbon chain that contains at least one carbon-carbon double bond, consists only of carbon and hydrogen, has 2 to 12 carbon atoms, and connects the rest of the molecule to a radical group, such as ethenylene, propenylene, n-butenylene, etc. The alkenylene chain is bonded to the rest of the molecule via a single bond and to the radical group via a double bond or a single bond. The bonding points of the alkenylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within this chain. Unless specifically specified otherwise in this specification, alkenylene may be substituted.

[0013] "Alkynylene" or "alkynylene chain" refers to a divalent straight-chain or branched hydrocarbon chain that contains at least one carbon-carbon triple bond, consists only of carbon and hydrogen, has 2 to 12 carbon atoms, and connects the rest of the molecule to a radical group, such as ethenylene, propenylene, n-butenylene, etc. The alkynylene chain is bonded to the rest of the molecule via a single bond and to the radical group via a double bond or a single bond. The bonding points of the alkynylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within this chain. Unless specifically specified otherwise in this specification, alkynylene may be substituted. "Alkyl ether" refers to any alkyl group as defined above in which at least one carbon-carbon bond is replaced by a carbon-oxygen bond. The carbon-oxygen bond may be present at the terminus (as in an alkoxy group) or the carbon-oxygen bond may be internal (i.e., C-O-C). An alkyl ether contains at least one carbon-oxygen bond, but may contain more than one. For example, polyethylene glycol (PEG) is included within the meaning of alkyl ether. Unless specifically stated otherwise herein, an alkyl ether group may be substituted. For example, in some embodiments, the alkyl ether is substituted by alcohol or -OP(=R a )(R b )R c , where R a , R b and R c are as defined for the compound of structure (I), respectively.

[0014] "Alkoxy" refers to a group of the formula -OR a , where R a is an alkyl group as defined above containing 1 to 12 carbon atoms. Unless specifically stated otherwise herein, an alkoxy group may be substituted. "Alkoxyalkyl ether" refers to a group of the formula -OR a R b , where R a is an alkylene group as defined above containing 1 to 12 carbon atoms and Rb is an alkyl ether group as defined herein. Unless specifically stated otherwise herein, an alkoxyalkyl ether group may be substituted, for example, by alcohol or -OP(=R a )(R b )R c , where R a , R b and R c are as defined for the compound of structure (I), respectively.

[0015] "Heteroalkyl" refers to an alkyl group as defined above that contains at least one heteroatom (e.g., N, O, P, or S) within the alkyl group or at the terminus of the alkyl group. In some embodiments, the heteroatom is present within the alkyl group (i.e., the heteroalkyl contains at least one carbon-[heteroatom] x -carbon bond, and x is 1, 2, or 3). In other embodiments, the heteroatom is present at the terminus of the alkyl group and thus serves to attach the alkyl group to the remainder of the molecule (e.g., M1-H-A), where M1 is part of the molecule, H is the heteroatom, and A is the alkyl group. Unless specifically stated otherwise herein, heteroalkyl groups may be substituted. Exemplary heteroalkyl groups include ethylene oxide (e.g., polyethylene oxide), which may contain a phosphorus-oxygen bond such as a phosphodiester bond. "Heteroalkoxy" refers to a group of the formula -OR a wherein R a is a heteroalkyl group as defined above containing from 1 to 12 carbon atoms. Unless specifically stated otherwise herein, heteroalkoxy groups may be substituted.

[0016] "Heteroalkylene" refers to an alkylene group as defined above that contains at least one heteroatom (e.g., N, O, P, or S) within the alkylene chain or at the end of the alkylene chain. In some embodiments, the heteroatom is present within the alkylene chain (i.e., the heteroalkylene contains at least one carbon-[heteroatom]-carbon bond, and x is 1, 2, or 3). In other embodiments, the heteroatom is present at the end of the alkylene and thus serves to attach the alkylene to the rest of the molecule (e.g., M1-H-A-M2, where M1 and M2 are part of the molecule, H is the heteroatom, and A is the alkylene). Unless specifically stated otherwise herein, the heteroalkylene group may be substituted. Exemplary heteroalkylene groups include ethylene oxide (e.g., polyethylene oxide), as well as the linking groups "C", "HEG", "TEG", "PEG 1K", and variations thereof, as exemplified below. [Chemical formula] Multimers of the above-described C linker, HEG linker, and / or PEG1K linker are included in various embodiments of the heteroalkylene linker.

[0017] In some embodiments of the PEG 1K linker, n is 25. The multimer may, for example, include the following structures. [Chemical formula] (wherein x is 0 or an integer greater than 0, for example, x ranges from 0 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)) "Heteroalkenylene" is a heteroalkylene as defined above that contains at least one carbon-carbon double bond. Unless specifically stated otherwise herein, the heteroalkenylene group may be substituted. "Heteroalkynylene" is a heteroalkylene that contains at least one carbon-carbon triple bond. Unless specifically stated otherwise herein, the heteroalkynylene group may be substituted.

[0018] The "heteroatom" related to the "heteroatom linker" refers to a linker group consisting of one or more heteroatoms. Exemplary heteroatom linkers include a single atom selected from the group consisting of O, N, P, and S, as well as multiple heteroatoms, for example, a linker and multimer having the formula -P(O - )(=O)O- or -OP(O - )(=O)O-, and combinations thereof. "Phosphate" refers to the -OP(=O)(R a )R b group. (In the formula, R a is OH, O - or OR c , and R b is OH, O - , OR c , a thiophosphate group or a further phosphate group (R c is a counter ion (e.g., Na+, etc.))

[0019] "Phosphoalkyl" refers to the -OP(=O)(R a )R b group. (In the formula, R a is OH, O - or OR c , and R b is -Oalkyl, and R c is a counter ion (e.g., Na+, etc.)) Unless specifically stated otherwise herein, the phosphoalkyl group may be substituted. For example, in certain embodiments, the -Oalkyl portion of the phosphoalkyl group is hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether, or -OP(=R a )(R b )R c (R a , R b and R c are each as defined for the compound of structure (I)) and may be substituted by one or more thereof.

[0020] "Phosphoalkyl ether" refers to an -OP(=O)(R a )R b group. (In the formula, R a is OH, O - or OR c and R b is -O alkyl ether, and R c is a counter ion (for example, Na + etc.)). Unless otherwise specifically specified in this specification, the phosphoalkyl ether group may be substituted. For example, in certain embodiments, the -O alkyl ether moiety in the phosphoalkyl ether group is hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether or -OP(=R a )(R b )R c (R a , R b and R c are each as defined for the compound of structure (I)) and may be substituted by one or more of them. "Thiophosphate" refers to an -OP(=R a )(R b )R c group. (In the formula, R a is O or S, and R b is OH, O - , S - , OR d or SR d and R c is OH, SH, O - , S - , OR d , SR d , a phosphate group or even a thiophosphate group, and R d is a counter ion (for example, Na+ etc.), provided that i) R a is S, ii) R b is S - or SR d and iii) R c is SH, S - or SRd or is a combination of i), ii) and / or iii))

[0021] "Thiophosphoalkyl" refers to an -OP(=R a )(R b )R c group. (In the formula, R a is O or S, R b is OH, O - , S - , OR d or SR d , R c is -Oalkyl, R d is a counter ion (e.g., Na+ etc.), provided that i) R a is S, ii) R b is S - or SR d , or iii) R a is S, R b is S - or SR d ). Unless specifically specified otherwise herein, the thiophosphoalkyl group may be substituted. For example, in certain embodiments, the -Oalkyl moiety in the thiophosphoalkyl group is hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether or -OP(=R a )(R b )R c (R a , R b and R c are each as defined for the compounds of structure (I)) and may be substituted by one or more of them. "Thiophosphoalkyl ether" refers to an -OP(=R a )(R b )R c group. (In the formula, R a is O or S, R b is OH, O - , S - , OR d or SRd and R c is an -O alkyl ether, and R d is a counter ion (e.g., Na+, etc.), provided that i) R a is S, ii) R b is S - or SR d or iii) R a is S, and R b is S - or SR d (provided that) Unless specifically specified otherwise herein, the thiophosphoalkyl ether group may be substituted. For example, in certain embodiments, the -O alkyl ether moiety in the thiophosphoalkyl group is hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether or -OP(=R a )(R b )R c (R a , R b and R c are each as defined for the compounds of structure (I)) and may be substituted by one or more of them.

[0022] "Carbocyclic" refers to a stable 3- to 18-membered aromatic or non-aromatic ring containing 3 to 18 carbon atoms. Unless specifically specified otherwise herein, the carbocyclic ring can be a monocyclic ring system, a bicyclic ring system, a tricyclic ring system or a tetracyclic ring system, which may include a fused ring system or a bridged ring system and may be partially saturated or fully saturated. Non-aromatic carbocyclic radicals include cycloalkyl, while aromatic carbocyclic radicals include aryl. Unless specifically specified otherwise herein, the carbocyclic group may be substituted. "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic carbocyclic ring, which is a saturated or unsaturated fused ring system or bridged ring system having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, and is bonded to the rest of the molecule by a single bond, and may include a fused ring system or a bridged ring system. Monocyclic cycloalkyl includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl (cycloheptly), and cyclooctyl. Polycyclic cycloalkyl includes, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo-[2.2.1]heptanyl, and the like. Unless specifically specified otherwise herein, the cycloalkyl group may be substituted.

[0023] "Aryl" refers to a ring system containing at least one carbocyclic aromatic ring. In some embodiments, aryl contains 6 to 18 carbon atoms. The aryl ring may be a monocyclic ring system, a bicyclic ring system, a tricyclic ring system, or a tetracyclic ring system, which may include a fused ring system or a bridged ring system. Aryl includes, but is not limited to, aryls derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, preiadene, pyrene, and triphenylene. Unless specifically specified otherwise herein, the aryl group may be substituted.

[0024] "Heterocyclic" refers to a stable 3- to 18-membered aromatic or non-aromatic ring containing 1 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless specifically stated otherwise herein, the heterocyclic ring may be a monocyclic ring system, bicyclic ring system, tricyclic ring system, or tetracyclic ring system, which may include a fused ring system or a bridged ring system. The nitrogen atom, carbon atom, or sulfur atom in the heterocyclic ring may be oxidized, the nitrogen atom may be quaternized, and the heterocyclic ring may be partially saturated or fully saturated. Examples of aromatic heterocyclic rings are listed below in the definition of heteroaryl (i.e., heteroaryl is a subset of heterocyclic). Examples of non-aromatic heterocyclic rings include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless specifically stated otherwise herein, the heterocyclic group may be substituted.

[0025] "Heteroaryl" refers to a 5- to 14-membered ring system containing 1 to 13 carbon atoms, 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. For the purposes of certain embodiments of the present disclosure, the heteroaryl radical may be a monocyclic ring system, a bicyclic ring system, a tricyclic ring system, or a tetracyclic ring system, which may include a fused ring system or a bridged ring system, and the nitrogen atoms, carbon atoms, or sulfur atoms in the heteroaryl radical may be oxidized, and the nitrogen atoms may be quaternized.Examples include azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4 - benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2 - a]pyridinyl, benzoxazolinonyl, benzimidazolothionyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2 - oxoazepinyl, oxazolyl, oxiranyl, 1 - oxidopyridinyl, 1 - oxidopyrimidinyl, 1 - oxidopyrazinyl, 1 - oxidopyridazinyl, 1 - phenyl - 1H - pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, pteridinononyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyridinononyl, pyrazinyl, pyrimidinyl, purimidinononyl, pyridazinyl, pyrrolyl, pyrido[2,3 - d]pyrimidinononyl, quinazolinyl, quinazolinononyl, quinoxalinyl, quinoxalinononyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, thieno[3,2 - d]pyrimidin - 4 - onyl, thieno[2,3 - d]pyrimidin - 4 - onyl, triazolyl, tetrazolyl, triazinyl and thiophenyl (i.e., thienyl). Unless specifically stated otherwise herein, heteroaryl groups may be substituted.

[0026] The suffix “-ene” refers to a specific structural feature (e.g., alkyl, aryl, heteroalkyl, heteroaryl) that is attached to the remainder of the molecule via a single bond and to a radical group via a single bond. In other words, the suffix “-ene” refers to a linker having the structural feature of the moiety to which it is attached. The points of attachment of the “-ene” chain to the remainder of the molecule and to the radical group can be through one atom or any two atoms within this chain. For example, heteroarylene refers to a linker containing a heteroaryl moiety as defined herein.

[0027] “Fused” refers to a ring system containing at least two rings, where the two rings share at least one common ring atom, e.g., two common ring atoms. When the fused ring is a heterocyclyl ring or a heteroaryl ring, the shared ring atom can be carbon or nitrogen. Fused rings include bicyclic, tricyclic, tetracyclic, etc.

[0028] As used herein, the term "substituted" means any of the above groups (e.g., alkyl, alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, alkoxy, alkyl ether, alkoxyalkyl ether, heteroalkyl, heteroalkoxy, phosphoalkyl, phosphoalkyl ether, thiophosphoalkyl, thiophosphoalkyl ether, carbocyclic, cycloalkyl, aryl, heterocyclic and / or heteroaryl), wherein at least one hydrogen atom (e.g., one, two, three or all hydrogen atoms) is replaced by a non-hydrogen atom such as, but not limited to, a halogen atom such as F, Cl, B and I; an oxygen atom in a group such as a hydroxyl group, an alkoxy group and an ester group; a sulfur atom in a group such as a thiol group, a thioalkyl group, a sulfone group, a sulfonyl group and a sulfoxide group; a nitrogen atom in a group such as an amine, an amide, an alkylamine, a dialkylamine, an arylamine, an alkylarylamine, a diarylamine, an N-oxide, an imide and an enamine; a silicon atom in a group such as a trialkylsilyl group, a dialkylarylsilyl group, an alkyldiarylsilyl group and a triarylsilyl group; and other heteroatoms in various other groups, by a bond to the non-hydrogen atom. "Substituted" also means any of the above groups wherein one or more hydrogen atoms are replaced by a higher order bond (e.g., a double bond or a triple bond) to an oxygen in an oxo group, a carbonyl group, a carboxyl group and an ester group, and a nitrogen in a group such as an imine, an oxime, a hydrazone and a nitrile. For example, "substituted" includes one or more hydrogen atoms being replaced by -NR g R h 、 -NR g C(=O)R h 、 -NR g C(=O)NR g R h 、 -NR g C(=O)OR h 、 -NR g SO2R h 、 -OC(=O)NR g R h 、 -OR g 、 -SR g 、 -SOR g, -SO2R g , -OSO2R g , -SO2OR g , =NSO2R g and -SO2NR g R h is replaced by, and any of the above groups contains. "Substituted" also means that one or more hydrogen atoms are -C(=O)R g , -C(=O)OR g , -C(=O)NR g R h , -CH2SO2R g , -CH2SO2NR g R h means any of the above groups replaced by. In the above, R g and R h are the same or different and independently are hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N - heterocyclyl, heterocyclylalkyl, heteroaryl, N - heteroaryl and / or heteroarylalkyl. "Substituted" further means that one or more hydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N - heterocyclyl, heterocyclylalkyl, heteroaryl, N - heteroaryl and / or heteroarylalkyl group, any of the above groups. In some embodiments, the optional substituent is -OP(=R a )(R b )R c , where R a , R b and R c are as defined for the compounds of structure (I). Further, any of the above substituents may also be substituted by one or more of the above substituents.

[0029] The term "electron-withdrawing group" refers to a functional group that, when occupied at the same position in a molecule, withdraws electrons from itself more than a hydrogen atom. These terms are well understood by those skilled in the art and are discussed in J. March, John Wiley & Sons, New York, N.Y., pp. 16-18 (1985), the discussion of which is incorporated herein by reference. Examples of electron-withdrawing groups include, but are not limited to, halo, halo (e.g., F, Cl, Br, I), -NO2, -CN, -SO3H, -SO2R a , -SO3R a , -COOH, -COR a , -COOR a , -CONHR a , -CON(R a )2, haloalkyl groups, and 5- to 14-membered electron-deficient heteroaryl groups, where R a is an alkyl group, an alkenyl group, or an alkynyl group.

[0030] "Conjugation" refers to the presence of overlap between one p-orbital and another p-orbital throughout the intervening sigma bonds. Conjugation can occur in cyclic or acyclic compounds. The "degree of conjugation" refers to the presence of overlap between at least one p-orbital and another p-orbital throughout the intervening sigma bonds. For example, 1,3-butadiene has one degree of conjugation, while benzene and other aromatic compounds typically have multiple degrees of conjugation. Fluorescent and chromogenic compounds typically contain at least one degree of conjugation. "Fluorescent" refers to a molecule that is capable of absorbing light at a specific frequency and emitting light at a different frequency. Fluorescence is well known to those skilled in the art. "Chromogenic" refers to a molecule that absorbs light within the chromogenic spectrum (i.e., red, yellow, blue, etc.).

[0031] "FRET" refers to Förster resonance energy transfer, which refers to the physical interaction by which energy resulting from the excitation of one moiety (e.g., a first chromophore or "donor") is transferred to an adjacent moiety (e.g., a second chromophore or "acceptor"). "FRET" is sometimes used interchangeably with fluorescence resonance energy transfer (i.e., when each chromophore is a fluorescent moiety). Generally, for FRET to occur, (1) the excitation or absorption spectrum of the acceptor chromophore must overlap the emission spectrum of the donor chromophore, (2) the transition dipole moments of the acceptor chromophore and the donor chromophore must be substantially parallel (i.e., approximately 0° or 180°), and (3) the acceptor chromophore and the donor chromophore must share a proximate space (i.e., be close to each other). Energy transfer from the donor to the acceptor occurs by non-radiative dipole-dipole coupling, and the distance between the donor chromophore and the acceptor chromophore is generally much shorter than the wavelength of light.

[0032] "Donor" or "donor chromophore" refers to a chromophore (e.g., a fluorophore) that can be induced or is induced into an excited electronic state and can non-radiatively transfer its excitation energy or absorption energy to a neighboring acceptor chromophore by long-range dipole-dipole interactions. Without wishing to be bound by theory, it is thought that energy transfer occurs because the vibrational dipoles of the individual chromophores have similar resonance frequencies. Donors and acceptors having these similar resonance frequencies are referred to as a "donor-acceptor pair", which is used interchangeably with "FRET moiety", "FRET pair", "FRET dye", or similar terms. "Acceptor" or "acceptor chromophore" refers to a chromophore (e.g., a fluorophore) to which excitation energy or absorption energy is transferred from a donor chromophore via non-radiative transfer by long-range dipole-dipole interactions.

[0033] "Stokes shift" refers to the difference between the position of the maximum band of excitation or absorption (e.g., wavelength) and the emission spectrum of an electronic transition (e.g., from an excited state to a non-excited state, or vice versa). In some embodiments, the compound has a Stokes shift of greater than 25 nm, greater than 30 nm, greater than 35 nm, greater than 40 nm, greater than 45 nm, greater than 50 nm, greater than 55 nm, greater than 60 nm, greater than 65 nm, greater than 70 nm, greater than 75 nm, greater than 80 nm, greater than 85 nm, greater than 90 nm, greater than 95 nm, greater than 100 nm, greater than 110 nm, greater than 120 nm, greater than 130 nm, greater than 140 nm, greater than 150 nm, greater than 160 nm, greater than 170 nm, greater than 180 nm, greater than 190 nm, or greater than 200 nm. "J value" is calculated as an integer value of spectral overlap between the emission spectrum of a donor chromophore and the excitation spectrum or absorption spectrum of an acceptor chromophore. The emission spectrum of the donor chromophore is generated when the donor chromophore is excited at a preferred excitation wavelength or absorption wavelength. The preferred excitation wavelength or absorption wavelength of the donor chromophore is at or near their respective excitation maxima or absorption maxima (e.g., Pacific Blue has an excitation maximum or absorption maximum at about 401 nm, and FITC has an excitation maximum or absorption maximum at about 495 nm), which are well known to those skilled in the art.

[0034] "Linker" refers to a continuous chain consisting of at least one atom such as carbon, oxygen, nitrogen, sulfur, phosphorus, and combinations thereof, which connects one part of a molecule to another part of the same molecule, or to a different molecule, moiety, or solid support (e.g., microparticle). The linker can be attached to the molecule via a covalent bond or other means, such as an ionic bond interaction or a hydrogen bond interaction. The term "biomolecule" refers to any of a variety of biological substances, including nucleic acids, carbohydrates, amino acids, polypeptides, glycoproteins, hormones, aptamers, and mixtures thereof. More specifically, this term is intended to include, without limitation, RNA, DNA, oligonucleotides, modified or derivatized nucleotides, enzymes, receptors, prions, receptor ligands (including hormones), antibodies, antigens, and toxins, as well as bacteria, viruses, blood cells, and tissue cells. The visually detectable biomolecules of the present disclosure (e.g., a compound of structure (I) having a biomolecule linked thereto) are prepared, as further described herein, by contacting the biomolecule with a compound having a reactive group that enables attachment of the biomolecule to the above-mentioned compound of the biomolecule via any available atom or functional group such as an amino, hydroxy, carboxyl, or sulfhydryl group on the biomolecule.

[0035] A "reactive group" is a moiety that can react with a second reactive group (e.g., a "complementary reactive group") to form one or more covalent bonds, for example, by a substitution, oxidation, reduction, addition, or cycloaddition reaction. Exemplary reactive groups are presented in Table 1 and include, for example, nucleophiles, electrophiles, dienes, dienophiles, aldehydes, oximes, hydrazones, alkynes, amines, azides, acyl azides, acyl halides, nitriles, nitrones, sulfhydryls, disulfides, sulfonyl halides, isothiocyanates, imido esters, activated esters, ketones, α,β-unsaturated carbonyls, alkenes, maleimides, α-haloimides, epoxides, aziridines, tetrazines, tetrazoles, phosphines, biotin, thietanes, and the like. The terms "visible" and "visually detectable" are used herein to refer to substances that can be observed by visual inspection without prior irradiation, or chemical or enzymatic activation. Such visually detectable substances absorb and emit light in the spectral region of about 300 to about 900 nm. Preferably, such substances are strongly colored and preferably have a molar absorptivity of at least about 40,000 M -1 cm -1, more preferably, at least about 50,000 M -1 cm -1 , even more preferably, at least about 60,000 M -1 cm -1 , even more preferably, at least about 70,000 M -1 cm -1 and most preferably, at least about 80,000 M -1 cm -1 and having a molar extinction coefficient. The compounds of the present disclosure can be detected by naked-eye observation or, without limitation, using an optical-based detection device including an absorption spectrophotometer, a transmission optical microscope, a digital camera, and a scanner. Substances detectable visually are not limited to those that emit and / or absorb light in the visible spectrum. Substances that emit and / or absorb light in the ultraviolet (UV) region (from about 10 nm to about 400 nm), the infrared (IR) region (from about 700 nm to about 1 mm), and other regions of the electromagnetic spectrum are also included within the scope of "substances detectable visually".

[0036] For the purposes of the embodiments of the present disclosure, the term "photo-stable visible dye" refers to a chemical moiety that is visually detectable and that is not significantly modified or decomposed upon exposure to light, as defined above in this specification. Preferably, the photo-stable visible dye shows no significant decolorization or decomposition after at least 1 hour of exposure to light. More preferably, the visible dye is stable after at least 12 hours, even more preferably at least 24 hours, even more preferably at least 1 week, and most preferably at least 1 month of exposure to light. Non-limiting examples of photo-stable visible dyes suitable for use in the compounds and methods of the present disclosure include azo dyes, thioindigo dyes, quinacridone pigments, dioxazines, phthalocyanines, perinones, diketopyrrolopyrroles, quinophthalones, and triarylcarbonium. As used herein, the term "perylene derivative" is intended to include any substituted perylene that is visually detectable. However, this term is not intended to include perylene itself. The terms "anthracene derivative", "naphthalene derivative", and "pyrene derivative" are used similarly. In some preferred embodiments, the derivative (e.g., a perylene, pyrene, anthracene, or naphthalene derivative) is an imide, bisimide, or hydrazamimide derivative of perylene, anthracene, naphthalene, or pyrene.

[0037] The visually detectable molecules of various embodiments of the present disclosure are useful in a wide range of analytical applications such as biochemical and biomedical applications where it is necessary to determine the presence, location, or amount of a particular analyte (e.g., a biomolecule). Thus, in another aspect, the present disclosure provides a method for visually detecting a biomolecule, the method comprising: (a) providing to a biological system a visually detectable biomolecule comprising a compound of structure (I) linked to the biomolecule; and (b) detecting the biomolecule by its visible properties. For the purposes of the present disclosure, the phrase "detecting a biomolecule by its visible properties" means that the biomolecule is observed by the naked eye or, without limitation, using an optically based detection device including an absorption spectrophotometer, a transmission optical microscope, a digital camera, and a scanner, without irradiation, or chemical or enzymatic activation. A densitometer can be used to quantify the amount of visually detectable biomolecule present. For example, the relative amount of biomolecule in two samples can be determined by measuring the relative optical density. If the stoichiometry of the dye molecule per biomolecule is known and the extinction coefficient of the dye molecule is known, the absolute concentration of the biomolecule can also be determined from the measured optical density. As used herein, the term "biological system" is used to refer to any solution or mixture containing one or more biomolecules in addition to the visually detectable biomolecule. Non-limiting examples of such biological systems include cells, cell extracts, tissue samples, electrophoresis gels, assay mixtures, and hybridization reaction mixtures.

[0038] "Solid support" refers to any solid substrate known in the art as a solid-phase support for molecules. For example, "microparticles" refers to any of several small particles useful for binding to the compounds of the present disclosure, including but not limited to glass beads, magnetic beads, polymer beads, non-polymer beads, etc. In certain embodiments, the microparticles include polystyrene beads. "Solid support residue" refers to a functional group that remains attached to a molecule when the molecule is cleaved from the solid support. Solid support residues are known in the art and can be readily derivatized based on the structure of the solid support and the groups that link to its molecule.

[0039] "Target-directed moiety" refers to a moiety that selectively binds to or associates with a specific target, such as an analyte molecule. "Selectively" binding or associating means that the target-directed moiety preferentially associates with or binds to the desired target compared to other targets. In some embodiments, the compounds disclosed herein include a linking group to a target-directed moiety for the compound to selectively bind or associate with the intended analyte (i.e., the target of the target-directed moiety), thus enabling detection of the analyte. Exemplary target-directed moieties include, but are not limited to, antibodies, antigens, nucleic acid sequences, enzymes, proteins, cell surface receptor antagonists, etc. In some embodiments, the target-directed moiety is a moiety such as an antibody that selectively binds to or associates with a target feature on the cell surface or intracellularly, such as a target feature on the cell membrane or the surface of other cell structures, thus enabling detection of the target cell. Small molecules that selectively bind to or associate with the desired analyte are contemplated as target-directed moieties in certain embodiments. Those skilled in the art will understand other analytes and corresponding target-directed moieties useful in various embodiments. The "base pair moiety" refers to a heterocyclic moiety capable of hybridizing with a complementary heterocyclic moiety via hydrogen bonding (e.g., Watson-Crick base pairs). Base pair moieties include natural and unnatural bases. Non-limiting examples of base pair moieties are RNA and DNA bases such as adenosine, guanosine, thymidine, cytosine, and uridine, and their analogs.

[0040] Embodiments of the present disclosure as disclosed herein are also intended to encompass all compounds of structure (I) that are isotopically labeled by replacement of one or more atoms with atoms having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include, respectively, 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I and 125 I and other isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine. Isotopically labeled compounds of structure (I) can generally be prepared by conventional techniques known to those of ordinary skill in the art, or by methods similar to those described below, and in the following examples using appropriate isotopically labeled reagents in place of the unlabeled reagents previously used.

[0041] "Stable compounds" and "stable structures" are intended to refer to compounds that are sufficiently robust so as not to decompose to a useful degree of purity from the reaction mixture and upon formulation into an effective therapeutic agent. "Optional" or "may (optionally)" means that the event or situation described thereafter may or may not occur, and such description includes both the case where the event or situation occurs and the case where it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and such description includes both substituted alkyl groups and alkyl groups having no substitution. "Salt" includes both acid addition salts and base addition salts. "Acid addition salt" refers to salts formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, borneolic acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid.

[0042] "Basic addition salt" refers to a salt prepared from the addition of an inorganic base or an organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, salts of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc. Salts derived from organic bases include, but are not limited to, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins (ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resin, etc.). In particular, preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0043] Crystallization may result in solvates of the compounds described herein. Embodiments of the present disclosure include all solvates of the compounds described. As used herein, the term "solvate" refers to an aggregate that includes one or more molecules of a compound of the present disclosure together with one or more molecules and a solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present disclosure may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., as well as corresponding solvated forms. The compounds of the present disclosure may be true solvates, while in other cases, the compounds of the present disclosure may simply retain adventitious water or another solvent, or may be a mixture of water and an adventitious solvent.

[0044] Embodiments of the compounds of the present disclosure (e.g., compounds of Structure I or II), or salts, tautomers or solvates thereof, may contain one or more chiral centers and thus, with respect to absolute stereochemistry, may give rise to enantiomers, diastereomers and other stereoisomers which can be defined as (R)- or (S)-, or in the case of amino acids, (D)- or (L)-. Embodiments of the present disclosure are intended to include all such possible isomers, as well as their racemic and optically pure forms. The optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers can be prepared using a chiral synthon or chiral reagent or resolved using conventional techniques such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of a racemate (or a racemate of a salt or derivative) using, for example, chiral high performance liquid chromatography (HPLC). When the compounds described herein contain an olefinic double bond or other center of geometric asymmetry and unless otherwise specified, the compounds are intended to include both E and Z geometric isomers. Similarly, all tautomeric forms are also intended to be included.

[0045] "Stereoisomers" refer to compounds that are composed of the same atoms bonded by the same bonds but have different three-dimensional structures that cannot be interchanged with each other. The present disclosure contemplates various stereoisomers and mixtures thereof and includes "enantiomers", which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other. "Tautomers" refer to the transfer of a proton from one atom of a molecule to another atom of the same molecule. The present disclosure includes all tautomers of the said compounds. The various tautomeric forms of the compounds can be readily derived by those skilled in the art. The chemical nomenclature protocol and structural schematics used herein are a modified version of the I.U.P.A.C. nomenclature system using the ACD / Naming version 9.07 software program and / or the ChemDraw Ultra version 11.0 software nomenclature program (CambridgeSoft). Common names well known to those skilled in the art are also used.

[0046] As noted above, in one embodiment of the present disclosure, compounds useful as fluorescent dyes and / or chromogenic dyes in various analytical methods are provided. In other embodiments, compounds useful as synthetic intermediates for preparing compounds useful as fluorescent dyes and / or chromogenic dyes are provided. In general terms, embodiments of the present disclosure are directed to dimers or higher polymers of fluorescent moieties and / or chromogenic moieties. The fluorescent moiety and / or chromogenic moiety are linked by a linker. Without wishing to be bound by theory, the linker is thought to help maintain a sufficient spatial distance between the fluorescent moiety and / or chromogenic moiety, thus reducing or eliminating internal quenching and thus resulting in dye compounds having high molar "brightness" (e.g., high fluorescence emission). Accordingly, in some embodiments, the compounds of the present disclosure have the following structure (I) or a stereoisomer, salt or tautomer thereof.

Chemical formula

[0047]

Chemical formula

[0048] In some embodiments, at least one of the occurring L 1a is an optionally substituted 5- to 7-membered heteroarylene linker. In some more specific embodiments, L 1a is, independently for each occurrence, an optionally substituted 5- to 7-membered heteroarylene linker. In some embodiments, L 1a is a 6-membered heteroarylene. In some embodiments, L 1a contains 2 N atoms and 2 O atoms. In certain embodiments, L 1a is substituted for each occurrence. In some related embodiments, L 1a is substituted, for example, by oxo, alkyl (such as methyl, ethyl, etc.) or combinations thereof. In more specific embodiments, L 1ais replaced by at least one oxo at each occurrence. In some embodiments, L 1a has one of the following structures.

[0049]

Chemical formula

[0050] In some embodiments, the compounds of the present disclosure have one of the following structures (IA) or (IA’), or a stereoisomer, salt or tautomer thereof.

Chemical formula

[0051] In some embodiments, L 4 z is an integer from 1 to 30, such as 3 to 8, 15 to 30 or 22 to 26. In some embodiments, z is 22, 23, 24, 25 or 26. In some embodiments, z is 3, 4, 5, 6, 7 or 8. In some particular embodiments, z is 6. In some embodiments, L 4 when q is the integer 2, in the case of the first occurrence,

Chemical formula

Chemical formula

[0052]

Chemical formula

Chemical formula

Chemical formula

[0053] In some embodiments, the compounds of the present disclosure have one of the following structures (IB) or (IB’), or a stereoisomer, salt or tautomer thereof. [Chemical formula]

[0054] In some embodiments, at least one of the Ls that appear 5 or L 6 is alkylene. In some embodiments, L 5 and L 6 are, independently for each occurrence, C1-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene. For example, in some embodiments, L 5 and L 6 are, independently for each occurrence, C1-C6 alkylene. In some embodiments, at least one of the Ls that appear 3 is alkylene. In some embodiments, L 3 is, independently for each occurrence, C1-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene. For example, in some embodiments, L 3 is, independently for each occurrence, C1-C6 alkylene.

[0055] In some embodiments, the compounds of the present disclosure have one of the following structures (IC) or (IC’), or a stereoisomer, salt or tautomer thereof. [Chemical formula] (wherein y 1 , y 2 and y 3 are, independently for each occurrence, an integer from 1 to 6. In some embodiments, y 1 is the integer 1, 2, 3, 4, 5 or 6) In some embodiments, y 2is an integer 1, 2, 3, 4, 5 or 6. In some embodiments, y 3 is an integer 1, 2, 3, 4, 5 or 6. In some more specific embodiments, y 1 is the integer 1. In some other specific embodiments, y 2 is the integer 1. In some other specific embodiments, y 3 is the integer 1.

[0056] The various linkers and substituents (e.g., R 1 , R 2 , R 3 , R 4 , R 5 , L 1a , L 1b , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , M 1 , M 2 , R c and Q) in the compound of structure (I) may be substituted by another substituent. For example, in some embodiments, the optional substituent is selected to optimize the water solubility or other properties of the compound of structure (I). In certain embodiments, the alkyl, alkoxy, alkyl ether, alkoxyalkyl ether, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether and thiophosphoalkyl ether in the compound of structure (I) are each optionally substituted by another substituent selected from the group consisting of hydroxyl, alkoxy, alkyl ether, alkoxyalkyl ether, sulfhydryl, amino, alkylamino, carboxyl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether and thiophosphoalkyl ether. In certain embodiments, the optional substituent is -OP(=R a )(R b )R c , where R a , R b and R c are as defined for the compound of structure (I).

[0057] Optional linker L 1b can be used as the attachment point for the remainder of the compound. For example, in some embodiments, a synthetic precursor to the compound of structure (I) is prepared and the M 1 moiety is attached to the synthetic precursor using any number of facile methods known in the art, such as methods referred to as "click chemistry". For this purpose, any reaction that is rapid and substantially irreversible can be used to attach M 1 to the synthetic precursor to form the compound of structure (I). Exemplary reactions include copper-catalyzed reactions that form triazoles from azides and alkynes (Huisgen's 1,3-dipolar cycloaddition), reactions of dienes with dienophiles (Diels-Alder), strain-promoted alkyne-nitrone cycloadditions, reactions of strained alkenes with azides, tetrazines or tetrazoles, [3+2] cycloadditions of alkenes with azides, inverse-demand Diels-Alder of alkenes with tetrazines, photoreactions of alkenes with tetrazoles, and various substitution reactions such as substitution of a leaving group by nucleophilic attack on an electrophilic atom. Exemplary substitution reactions include reactions of amines with activated esters; N-hydroxysuccinimide esters; isocyanates; isothioscyanates, etc. In some embodiments, the reaction to form L 1 1b may be carried out in an aqueous environment.

[0058] Thus, in some embodiments, L 1b is a linker that includes a functional group that can be formed by the reaction of two complementary reactive groups upon each occurrence, for example, a functional group that is a product of one of the above-described "click" reactions. In various embodiments, L 1b ​When at least one appears, the functional group can be formed by the reaction of an aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imido ester, activated ester (e.g., N-hydroxysuccinimide ester), ketone, α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin or thiirane functional group with a complementary reactive group. For example, the reaction of an amine with an N-hydroxysuccinimide ester or isothiocyanate.

[0059] In other embodiments, L 1b When at least one appears, the functional group can be formed by the reaction of an alkyne and an azide. In other embodiments, L 1b When at least one appears, the functional group can be formed by the reaction of an amine (e.g., a primary amine) and an N-hydroxysuccinimide ester or isothiocyanate. In more embodiments, L 1b When at least one appears, the functional group contains an alkene, ester, amide, thioester, disulfide, carbocyclic, heterocyclic or heteroaryl group. In more embodiments, L 1b When at least one appears, the functional group contains an alkene, ester, amide, thioester, thiourea, disulfide, carbocyclic, heterocyclic or heteroaryl group. In other embodiments, the functional group contains an amide or thiourea. In some more specific embodiments, L 1b When at least one appears, L 1b is a linker containing a triazolyl functional group. In other embodiments, L 1b When at least one appears, L 1b is a linker containing an amide functional group or a thiourea functional group.

[0060] In still other different embodiments of structure (I), L 1bis, independently for each occurrence, an alkylene linker or a heteroalkylene linker. In some embodiments, the occurring L 1b at least one of which is heteroalkylene. In other embodiments, the occurring L 1b at least one of which contains a functional group formed by reaction with a complementary reactive group of an aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imido ester, activated ester, ketone, α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin or thietane. In other embodiments, the occurring L 1b at least one of which contains a functional group formed by reaction of an alkyne and an azide. For example, the occurring L 1b at least one of which is a linker containing a triazolyl functional group.

[0061] In still other embodiments, when at least one occurrence of L 1b -M 1 occurs, L 7 -M 2 has one of the following structures.

Chemical formula

Chemical formula

[0062] In the various embodiments described above, L 1c or L 1d is absent, or both are absent. In other embodiments, L 1c or L 1d is present, or both are present. In some embodiments, L c and L d , when present, are each independently alkylene or heteroalkylene. For example, in some embodiments, L c and L d each independently have one of the following structures.

Chemical formula

[0063] In other embodiments, L 1b includes one of the following structures.

Chemical formula

[0064] In some embodiments, at least one of M 1 -L 1b in which structure (I) appears has one of the following structures among them.

Chemical formula

[0065] In some embodiments, M of structure (I) 1 -L 1b has, for each occurrence, one of the following structures.

Chemical formula

[0066] In some embodiments, at least one of the occurrences of L 7 is an optionally substituted heteroalkylene linker. In other embodiments, L 7 is, for each occurrence independently, an optionally substituted heteroalkylene. In some embodiments, L 7 contains an amide functional group. For example, in some embodiments, at least one of the occurrences of L 7 has one of the following structures.

[0067]

Chemical formula

[0068] In other embodiments, L 7 has, for each occurrence, one of the following structures.

Chemical formula

[0069] In some particular embodiments, at least one of the occurrences of L 7 has one of the following structures.

Chemical formula

[0070] In some other particular embodiments, L 7 has, for each occurrence, one of the following structures.

Chemical formula

[0071] In still other embodiments of the compounds of structure (I), (IA), (IA'), (IB), (IB'), (IC) or (IC'), R 5 is, independently for each occurrence, OH, O - or OR d is. "OR d " and "SR d " are intended to refer to O - and S - bonded to a cation. For example, the disodium salt of a phosphate group can be represented as follows.

Chemical formula

[0072] In still more different embodiments of any of the above-described compounds of structure (I), (IA), (IA'), (IB), (IB'), (IC) or (IC'), R1 and R 2 is, independently of each other, -OP(=R a )(R b )R c is. In some of these embodiments, R c is OL’. In other embodiments, R 1 and R 2 is, independently of each other, -OP(=R a )(R b )OL’, where L’ is Q, a target-directed moiety, an analyte (e.g., an analyte molecule), a solid support, a solid support residue, a nucleoside, or an alkylene linker or heteroalkylene linker to a further compound of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’). In some embodiments, the analyte molecule is a nucleic acid, an amino acid or a polymer thereof. In some other embodiments, the analyte molecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer or a prion. In some embodiments, the target-directed moiety is an antibody or a cell surface receptor antagonist. In some other embodiments, the solid support is a polymeric bead or a non-polymeric bead.

[0073] The linker L’ can be any linker suitable for attaching Q, a target-directed moiety, an analyte (e.g., an analyte molecule), a solid support, a solid support residue, a nucleoside, or a further compound of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’) to a compound of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’). Advantageously, certain embodiments include the use of an L’ moiety that is selected to increase or optimize the water solubility of the compound. In certain embodiments, L’ is a heteroalkylene moiety. In some other certain embodiments, L’ comprises an alkylene oxide or phosphodiester moiety, or a combination thereof. In certain embodiments, L’ has the following structure.

Chemical formula

[0074] In some embodiments, m” is an integer from 4 to 10, such as 4, 6 or 10. In other embodiments, n” is an integer from 3 to 6, such as 3, 4, 5 or 6. In some embodiments, n” is an integer from 18 to 28, such as 21 to 23. In some other embodiments, L” is an alkylene, an alkyleneheterocyclylene, an alkyleneheterocyclylenealkylene, an alkylenecyclylene, an alkylenecyclylenealkylene, a heteroalkylene, a heteroalkyleneheterocyclylene, a heteroalkyleneheterocyclyleneheteroalkylene, a heteroalkylenecyclylene or a heteroalkylenecycleneheteroalkylene moiety. In some other certain embodiments, L” comprises an alkylene oxide, a phosphodiester moiety, a sulfhydryl, a disulfide or a maleimide moiety, or a combination thereof. In certain of the above embodiments, the target-directed moiety is an antibody or a cell surface receptor antagonist.

[0075] In some embodiments, the antibodies include CD3, CD4, FoxP3, TNF-α, IFN-γ, clone 4S.B3, clone 206D, CD8α (D8A8Y) rabbit mAb, vimentin (D21H3) XP® rabbit mAb, phospho-RB-Ser608, phospho-RB-Ser612, phospho-RB-Ser780, phospho-RB-Ser795, phospho-RB-Ser807 or phospho-RB-Ser811, anti-human IL17A, integrin alpha E / CD103, CCR9 or MOPC-21. In yet more particular embodiments of any of the above-described compounds of Structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’), R 1 or R 2 has one of the following structures. [Chemical formula] [Chemical formula]

[0076] In yet more particular embodiments of any of the above-described compounds of Structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’), R 1 or R 2 has one of the following structures. [Chemical formula]

[0077] Certain specific embodiments of the compounds of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’) can be prepared according to solid-phase synthesis methods similar to those known in the art with respect to the preparation of oligonucleotides. Thus, in some embodiments, L’ is a solid support, a solid support residue or a linking group to a nucleoside. Solid supports containing an activated deoxythymidine (dT) group are readily available and, in some embodiments, can be used as starting materials for the preparation of the compounds of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’). Thus, in some embodiments, R 1 or R 2 has the following structure.

[0078] [Chemical formula] One of ordinary skill in the art will understand that the dT group illustrated above is included only to facilitate synthesis and for economic efficiency only and is not necessary. Other solid supports can be used and different nucleosides or solid support residues can be present on L’, or the nucleoside or solid support residue can be removed or modified after synthesis.

[0079] In still other embodiments, Q is, independently for each occurrence, a moiety comprising a reactive group capable of forming a covalent bond with an analyte molecule or a solid support. In other embodiments, Q is, independently for each occurrence, a moiety comprising a reactive group capable of forming a covalent bond with a complementary reactive group Q’. For example, in some embodiments, Q’ is present in a further compound of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’) (e.g., R 2 or R 3at the position of), Q and Q' contain complementary reactive groups, and thus, by reaction of the compound of structure (I) with a further compound of structure (I), (IA), (IA'), (IB), (IB'), (IC) or (IC'), a covalently bound dimer of the compound of structure (I), (IA), (IA'), (IB), (IB'), (IC) or (IC') is brought about. Multimeric compounds of structure (I), (IA), (IA'), (IB), (IB'), (IC) or (IC') can also be prepared in a similar manner and are included within the scope of embodiments of the present disclosure.

[0080] The type of Q group and the binding of the Q group to the remainder of the compound of structure (I), (IA), (IA'), (IB), (IB'), (IC) or (IC') are not limited, provided that Q contains a moiety having the appropriate reactivity to form the desired bond. In certain embodiments, Q is a moiety that is resistant to hydrolysis under aqueous conditions but is sufficiently reactive to form a bond with a corresponding group (e.g., amine, azide or alkyne) on the molecule to be analyzed or on the surface of the solid support. Certain embodiments of the compounds of structure (I), (IA), (IA'), (IB), (IB'), (IC) or (IC') include Q groups that are commonly used in the field of bioconjugates. For example, in some embodiments, Q includes a nucleophilic reactive group, an electrophilic reactive group or a cycloaddition reactive group. In some more specific embodiments, Q includes a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino or maleimide functional group. In some embodiments, the activated ester is an N-succinimide ester, an imido ester or a polyfluorophenyl ester. In other embodiments, the alkyne is an alkyl azide or an acyl azide.

[0081] The Q group can be conveniently provided in a protected form that increases storage stability or other desired properties, and then the protecting group is removed, for example, when appropriate for the target-directed moiety or conjugate with the analyte. Thus, the Q group includes a "protected form" of a reactive group, including any of the reactive groups in Table 1 above and below. The "protected form" of Q refers to a moiety that has lower reactivity under certain reaction conditions for Q, but preferably does not decompose or react with other moieties of the compounds of formula (I), (IA), (IA'), (IB), (IB'), (IC) or (IC'), and can be converted to Q. One of ordinary skill in the art can derive an appropriate protected form of Q based on the specific Q, as well as the desired end use and storage conditions. For example, when Q is SH, the protected form of Q includes disulfide, and the disulfide can exhibit the SH moiety when reduced using generally known techniques and reagents.

[0082] Exemplary Q moieties are presented in Table I below.

Table 1-1

Table 1-2

Table 1-3

[0083] It should be noted that in some embodiments, when Q is SH, the SH moiety tends to form a disulfide bond with another sulfhydryl group on another compound of formula (I), (IA), (IA'), (IB), (IB'), (IC) or (IC'). Thus, some embodiments include compounds of formula (I), (IA), (IA'), (IB), (IB'), (IC) or (IC') in the form of a disulfide dimer, and the disulfide bond is derived from the Q group that is SH. Similarly, the compounds of formula (I) are included within the scope of certain embodiments, where R 1 and R 2One or both of them contain a linking group to a further compound of structure (I). For example, R 1 and R 2 one or both of which are -OP(=R a )(R b )R c , where R c is OL’, and L’ is a linker containing a covalent bond to a further compound of structure (I). Such compounds can be prepared, for example, by preparing a compound of the first structure (I) having about 10 “M” moieties (i.e., n = 9) and having a “Q” suitable for reaction with a complementary Q’ group on a compound of the second structure (I). Thus, compounds of structure (I) having any number, for example 100 or more, of “M” moieties can be prepared without the need to sequentially couple each monomer.

[0084] The value for m is another variable that can be selected based on the desired fluorescence intensity and / or color intensity. In some embodiments, m is an integer of 1 or more for each occurrence. In some embodiments, m is independently an integer from 1 to 10 for each occurrence. In other embodiments, m is independently an integer from 1 to 6, for example, 1, 2, 3, 4, 5, or 6 for each occurrence. In some particular embodiments, m is an integer of 1. In some embodiments, m is an integer of 2. In some embodiments, m is an integer of 3. In some embodiments, m is an integer of 4. In some embodiments, m is an integer of 5. In some embodiments, m is an integer of 6. The fluorescence intensity can also be adjusted by selecting different values of n. In some embodiments, n is an integer of 1 or more for each occurrence. In certain embodiments, n is an integer from 1 to 100. In other embodiments, n is an integer from 1 to 10. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

[0085] The value for q is another variable that can be selected based on the desired fluorescence intensity and / or color intensity. In some embodiments, q is an integer of 1 or more for each occurrence. In some embodiments, q is independently an integer from 1 to 5 for each occurrence. For example, in some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, q is 4. In some embodiments, q is 5. The value for w is another variable that can be selected based on the desired fluorescence intensity and / or color intensity. In some embodiments, w is an integer of 1 or more for each occurrence, provided that q is an integer greater than w when n is 1. In some embodiments, w is an integer from 1 to 10. In some embodiments, w is an integer from 1 to 5. In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, w is 3. In some embodiments, w is 4. In some embodiments, w is 5. The values for q, w, n, and m are variables that can be selected based on the desired fluorescence intensity and / or color intensity. In some more specific embodiments, q is an integer of 2, w is an integer of 1, n is an integer of 1, and m is an integer of 1. In some other more specific embodiments, q is an integer of 3, w is an integer of 1, n is an integer of 1, and m is an integer of 1. In some other more specific embodiments, q is an integer of 1, w is an integer of 1, n is an integer of 2, and m is an integer of 1 for each occurrence. In some other more specific embodiments, q is an integer of 1 for the first occurrence and 2 for the second occurrence, w is an integer of 1 for each occurrence, n is an integer of 2, and m is an integer of 1 for each occurrence. In some other more specific embodiments, q is an integer of 2 for each occurrence, w is an integer of 1 for each occurrence, n is an integer of 2, and m is an integer of 1 for each occurrence. In some other more specific embodiments, q is an integer of 1 for the first occurrence and 2 for the second occurrence, w is an integer of 1 for each occurrence, n is an integer of 2, and m is an integer of 1 for each occurrence.

[0086] M 1 and M 2 are selected based on the desired optical properties, for example, based on the desired color development and / or fluorescence emission wavelength. In some embodiments, M 1 and M 2 are different for each occurrence. For example, in some embodiments, M 1 and M 2 are each different, and different M 1 parts and M 2 parts are selected to have absorption and / or emission for use in the fluorescence resonance energy transfer (FRET) method. For example, in such embodiments, different M parts are selected to form a FRET donor-acceptor such that absorption of irradiation at one wavelength causes emission of irradiation at a different wavelength by the FRET mechanism. In this regard, M 1 and M 2 parts form a FRET pair. Exemplary M 1 parts and M2 The moieties can be appropriately selected by those skilled in the art based on the desired end use. Exemplary M moieties for the FRET method 1 moieties and M 2 moieties include fluorescein and Alexa Fluor® 594 dye. In some other embodiments, M moieties for the FRET method 1 and M 2 moieties include fluorescein and Alexa Fluor® 555 dye. In some other embodiments, M moieties for the FRET method 1 and M 2 moieties include fluorescein and Alexa Fluor® 568 dye. In some other embodiments, M moieties for the FRET method 1 and M 2 moieties include fluorescein and Alexa Fluor® 532 dye. In some other embodiments, M moieties for the FRET method 1 and M 2 moieties include fluorescein and Alexa Fluor® 546 dye. In some other embodiments, M moieties for the FRET method 1 and M 2 moieties include Cy3 and Alexa Fluor® 680 dye. M 1 and M 2 can be attached to the rest of the molecule from any position (i.e., atom) on M 1 and M 2 . Those skilled in the art recognize the means by which M and M are attached to the rest of the molecule. 1 and M 2

[0087] In some embodiments, M 1 and M 2is a fluorescent or chromogenic moiety. Any fluorescent and / or chromogenic moiety can be used, such as those known in the art and commonly used in colorimetric assays, UV assays, and / or fluorescence assays. Examples of M moieties useful in various embodiments of the present disclosure include, but are not limited to, xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, or Texas Red); cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarboL1cyanine, thiacarbocyanine, or merocyanine); squaraine derivatives and ring-substituted squaraines (including Seta dyes, SeTau dyes, and Square dyes); naphthalene derivatives (e.g., dansyl and prodan derivatives); coumarin derivatives; oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, or benzoxadiazole); anthracene derivatives (e.g., anthraquinones including DRAQ5, DRAQ7, and CyTRAK Orange); pyrene derivatives such as cascade blue; oxazine derivatives (e.g., Nile Red, Nile Blue, cresyl violet, oxazine 170); acridine derivatives (e.g., proflavine, acridine orange, acridine yellow); arylmethine derivatives: auramine, crystal violet, malachite green; and tetrapyrrole derivatives (e.g., porphyrin, phthalocyanine, or bilirubin).Other exemplary M moieties include cyanine dyes, xanthene dyes (e.g., Hex, Vic, Nedd, Joe, or Tet); Yakima yellow; Redmond red; tamra; texas red, and Alexa Fluor® dyes such as Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, or Alexa Fluor® 750.

[0088] The compounds of the present disclosure are found to be useful as fluorescent dyes and / or chromogenic dyes having high quantum efficiency. This is in part due to the emission spectrum of the donor moiety (e.g., M 1 ) and the acceptor moiety (e.g., M 2) due to overlap with the absorption or excitation spectrum. Thus, some embodiments provide a FRET donor having an excitation maximum between 300 and 900 nm and an emission maximum between 350 and 900 nm. For example, in some embodiments, the FRET donor includes 2,5-diphenyloxazole having an excitation maximum of 311 nm and an emission maximum of 375 nm. In another example, in some embodiments, the FRET donor includes a dansyl fluorophore having an excitation maximum of 333 nm and an emission maximum of 518 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 350 having an excitation maximum of 346 nm and an emission maximum of 442 nm. In yet further examples, in some embodiments, the FRET donor includes pyrene having an excitation maximum of 340 nm and an emission maximum of 376 nm. In yet further examples, in some embodiments, the FRET donor includes coumarin 343 having an excitation maximum of 437 nm and an emission maximum of 477 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 430 having an excitation maximum of 430 nm and an emission maximum of 539 nm. In yet another example, in some embodiments, the FRET donor includes 5-carboxyfluorescein (FAM) having an excitation maximum of 495 nm and an emission maximum of 519 nm. In yet another example, in some embodiments, the FRET donor includes cyanine dye (CY3) having an excitation maximum of 550 nm and an emission maximum of 615 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 555 having an excitation maximum of 555 nm and an emission maximum of 572 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 568 having an excitation maximum of 578 nm and an emission maximum of 603 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 633 having an excitation maximum of 630 nm and an emission maximum of 650 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 647 having an excitation maximum of 650 nm and an emission maximum of 668 nm.In yet another example, in some embodiments, the FRET donor includes MB800 having an excitation maximum at 774 nm and an emission maximum at 798 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 800 having an excitation maximum at 801 nm and an emission maximum at 814 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 810 having an excitation maximum at 812 nm and an emission maximum at 826 nm. In yet another example, in some embodiments, the FRET donor includes CF820 having an excitation maximum at 820 nm and an emission maximum at 830 nm. In yet another example, in some embodiments, the FRET donor includes iFluor® 820 having an excitation maximum at 820 nm and an emission maximum at 849 nm. In yet another example, in some embodiments, the FRET donor includes PromoFluor840 / iFluor® 840 having an excitation maximum at 838 nm and an emission maximum at 880 nm. In yet another example, in some embodiments, the FRET donor includes iFluor® 860 having an excitation maximum at 852 nm and an emission maximum at 877 nm.

[0089] Some embodiments provide a FRET acceptor having an excitation maximum between 400 and 800 nm and an emission maximum between 500 and 550 nm. For example, in some embodiments, the FRET acceptor includes 5-carboxyfluorescein (FAM) having an excitation maximum of 495 nm and an emission maximum of 519 nm. In another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 543 having an excitation maximum of 548 nm and an emission maximum of 566 nm. In yet another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 532 having an excitation maximum of 532 nm and an emission maximum of 554 nm. In yet another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 546 having an excitation maximum of 554 nm and an emission maximum of 570 nm. In yet another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 555 having an excitation maximum of 555 nm and an emission maximum of 572 nm. In yet another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 568 having an excitation maximum of 578 nm and an emission maximum of 603 nm. In yet another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 594 having an excitation maximum of 590 nm and an emission maximum of 617 nm. In yet another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 633 having an excitation maximum of 630 nm and an emission maximum of 650 nm. In yet another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 660 having an excitation maximum of 663 nm and an emission maximum of 690 nm. In yet another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 647 having an excitation maximum of 650 nm and an emission maximum of 668 nm. In yet another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 680 having an excitation maximum of 679 nm and an emission maximum of 702 nm.In yet another example, in some embodiments, the FRET acceptor comprises Alexa Fluor® 750 having an excitation maximum of 756 nm and an emission maximum of 776 nm.

[0090] Embodiments of the present disclosure enable various combinations of FRET donor / acceptor pairs that enhance luminance as sensors. For example, in some embodiments, the FRET donor / acceptor pair is 2,5-diphenyloxazole as the FRET donor and Alexa Fluor® 430 as the FRET acceptor. In another example, in some embodiments, the FRET donor / acceptor pair is dansyl fluorophore as the FRET donor and Alexa Fluor® 543 or Alexa Fluor® 532 as the FRET acceptor. In yet another example, in some embodiments, the FRET donor / acceptor pair is Alexa Fluor® 350 as the FRET donor and Alexa Fluor® 430 as the FRET acceptor. In yet another example, in some embodiments, the FRET donor / acceptor pair is pyrene as the FRET donor and Alexa Fluor® 430 as the FRET acceptor. In yet another example, in some embodiments, the FRET donor / acceptor pair is coumarin 343 as the FRET donor and FAM as the FRET acceptor. In yet another example, in some embodiments, the FRET donor / acceptor pair is Alexa Fluor® 430 as the FRET donor and Alexa Fluor® 543, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568 or Alexa Fluor® 594 as the FRET acceptor. In yet another example, in some embodiments, the FRET donor / acceptor pair is FAM as the FRET donor and Alexa Fluor® 532, Alexa Fluor® 555, Alexa Fluor® 546, Alexa Fluor® 568 or Alexa Fluor® 594 as the FRET acceptor.In yet another example, in some embodiments, the FRET donor / acceptor pair is CY3 as the FRET donor and Alexa Fluor® 532 and Alexa Fluor® 633 as the FRET acceptor. In yet another example, in some embodiments, the FRET donor / acceptor pair is Alexa Fluor® 555 as the FRET donor and Alexa Fluor® 633 or Alexa Fluor® 660 as the FRET acceptor. In yet another example, in some embodiments, the FRET donor / acceptor pair is Alexa Fluor® 568 as the FRET donor and Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660 or Alexa Fluor® 680 as the FRET acceptor. In yet another example, in some embodiments, the FRET donor / acceptor pair is Alexa Fluor® 633 as the FRET donor and Alexa Fluor® 680 as the FRET acceptor. In yet another example, in some embodiments, the FRET donor / acceptor pair is Alexa Fluor® 647 as the FRET donor and Alexa Fluor® 680 or Alexa Fluor® 750 as the FRET acceptor.

[0091] In any of the further other embodiments described above, M 1 and M 2 comprise three or more aryl or heteroaryl rings or combinations thereof, such as four or more aryl or heteroaryl rings or combinations thereof, or even five or more aryl or heteroaryl rings, or combinations thereof. In some embodiments, M 1 and M 2 comprise six aryl or heteroaryl rings, or combinations thereof. In further embodiments, the rings are fused. For example, in some embodiments, M 1 and M 2contains three or more fused rings, four or more fused rings, five or more fused rings, or even six or more fused rings.

[0092] In some embodiments, M 1 or M 2 is cyclic. For example, in some embodiments, M 1 or M 2 is carbocyclic. In other embodiments, M 1 or M 2 is heterocyclic. In still other embodiments described above, M 1 or M 2 independently for each occurrence, contains an aryl moiety. In some of these embodiments, the aryl moiety is polycyclic. In other further specific examples, the aryl moiety is a fused polycyclic aryl moiety, for example, this fused polycyclic aryl moiety may contain at least three, at least four, or even more than four aryl rings. In any other embodiment of the compounds of structures (I), (IA), (IA'), (IB), (IB'), (IC) or (IC') described above, M 1 or M 2 independently for each occurrence, contains at least one heteroatom. For example, in some embodiments, the heteroatom is nitrogen, oxygen or sulfur.

[0093] In any further embodiments of any of the above, M 1 or M 2 independently for each occurrence, contains at least one substituent. For example, in some embodiments, the substituent is fluoro, chloro, bromo, iodo, amino, alkylamino, arylamino, hydroxy, sulfhydryl, alkoxy, aryloxy, phenyl, aryl, methyl, ethyl, propyl, butyl, isopropyl, t-butyl, carboxy, sulfonate, amide or formyl group. In some even more specific embodiments of the above, M 1 or M 2is, independently for each occurrence, dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, bis-fluorophenyl-BODIPY, acridine, terrylene, sexiphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl, squaraine, squarylium, 9,10-ethynylanthracene or ter-naphthyl moiety. In other embodiments, M 1 or M 2 is, independently for each occurrence, p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthene, chrysene, rubrene, coronene, cyanine, perylene imide or perylene amide, or derivatives thereof. In still more embodiments, M 1 or M 2 is, independently for each occurrence, a coumarin dye, a resorufin dye, a dipyrrometheneboron difluoride dye, a ruthenium bipyridyl dye, an energy transfer dye, a thiazole orange dye, a polymethine or an N-aryl-1,8-naphthalimide dye.

[0094] In still more embodiments of any of the above, M 1 or M 2 are each different. In still more embodiments, one or more M 1 or M 2 are the same and one or more M 1 or M 2 are different. In some embodiments, M 1 or M 2 is pyrene, perylene, perylene monoimide, 5-carboxyfluorescein (FAM), 6-FAM, 6-FITC, 5-FITC or derivatives thereof.

[0095] In some embodiments, M 1 and M 2In one or more occurrences, independently includes a fused polycyclic aryl moiety or heteroaryl moiety containing at least 4 fused rings. In some embodiments, M 1 or M 1 -L 1b independently has, for each occurrence, one of the following structures.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0096] In some embodiments, M 2 independently has, for each occurrence, one of the following structures.

Chemical formula

[0097] The M 1 moiety or the M 2 moiety containing a carboxylic acid group is illustrated in the above anionic form (CO2 - ), but those skilled in the art understand that this can vary depending on the pH and that the protonated form (CO2H) is included in various embodiments. In some specific embodiments, the compound of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’) is a compound selected from Table 2. The compounds in Table 2 were prepared according to the procedures described in the examples and their identity was confirmed by mass spectrometry.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

[0098] As used in Table 2 above and throughout the present disclosure, M 1 and M 2 are, each appearance independently, a fluorescent moiety or a chromogenic moiety as described above. One of M 1 and M 2 is a FRET donor and the other of M 1 and M 2 is a FRET acceptor. In some embodiments, M 1 is Alexa Fluor® 594 (AF594) and M 2 is FAM. In some embodiments, M 1 is Alexa Fluor® 555 (AF555) and M 2 is FAM. In some embodiments, M 1 is Alexa Fluor® 568 (AF568) and M 2 is FAM. In some embodiments, M 1 is Alexa Fluor® 680 (AF680) and M 2 is Cy3.

[0099] FAM refers to a moiety having one of the following structures.

Chemical formula

[0100] AF594 refers to a moiety having the following structure.

Chemical formula

[0101] AF555 refers to a moiety having the following structure.

Chemical formula

[0102] AF568 refers to a moiety having the following structure.

Chemical formula

[0103] AF680 refers to a moiety having one of the following structures.

Chemical formula

[0104] Cy3 refers to a moiety having the following structure.

Chemical formula

[0105] As used throughout Table 2 above and in the present disclosure, dT refers to the following structure.

Chemical formula

[0106] The FRET donor-acceptor ratio is another variable that can be selected based on the desired fluorescence intensity and / or color intensity. In some embodiments, the FRET acceptor M 1The ratio to the corresponding FRET donor M 2 is 1:1. In other words, the polymeric dye contains one FRET acceptor M 2 for each one of the FRET donors M 1 . In some embodiments, the ratio of the FRET acceptor M 1 to the corresponding FRET donor M 2 is 1:2. In other words, the polymeric dye contains one FRET acceptor M 2 for every two of the FRET donors M 1 . In some embodiments, the ratio of the FRET acceptor M 1 to the corresponding FRET donor M 2 is 1:3. In other words, the polymeric dye contains one FRET acceptor M 2 for every three of the FRET donors M 1 . In some embodiments, the ratio of the FRET acceptor M 1 to the corresponding FRET donor M 2 is 2:3. In other words, the polymeric dye contains two FRET acceptor M 2 for every three of the FRET donors M 1 .

[0107] Some embodiments are presented in Table 2 and include any of the above-described compounds, including specific compounds conjugated to a target-directed moiety such as an antibody. In some embodiments, the antibody includes CD3, CD4, FoxP3, TNF-α, IFN-γ, clone 4S.B3, clone 206D, CD8α (D8A8Y) rabbit mAb, vimentin (D21H3) XP® rabbit mAb, phospho-RB-Ser608, phospho-RB-Ser612, phospho-RB-Ser780, phospho-RB-Ser795, phospho-RB-Ser807 or phospho-RB-Ser811, anti-human IL17A, integrin alpha E / CD103, CCR9 or MOPC-21. The present disclosure generally provides compounds with increased fluorescence emission compared to previously known compounds. Thus, in certain embodiments, there is provided a fluorescent compound comprising Y fluorescent moieties M, which, when excited by ultraviolet light of a predetermined wavelength, has a peak fluorescence emission amount that is at least 85% of Y (where Y is an integer of 2 or more) times less than the peak fluorescence emission amount of a single M moiety when excited by ultraviolet light of the same wavelength. Fluorescent compounds include compounds that emit a fluorescent signal upon excitation by light such as ultraviolet light.

[0108] Also provided are compositions comprising a fluorescent compound of any one of structures (I), (IA), (IA'), (IB), (IB'), (IC) or (IC') and an analyte. The presently disclosed compounds are "tunable", which means that one of ordinary skill in the art can reach a compound having a desired and / or predetermined molar fluorescence (molar luminance) by appropriately selecting the variables in any of the above-described compounds. Due to the "tunability" of the present compounds, the user can easily reach a compound having a desired fluorescence and / or coloration for use in a specific assay or for identifying a specific analyte of interest. All variables can affect the molar fluorescence of the present compounds, but M 1 , M 2 , L 4 , L 5 , L 6 , m, n, q, w and z are thought to play an important role in the molar fluorescence of the present compounds. Thus, in one embodiment, there is provided a method for obtaining a compound having a desired molar fluorescence, the method comprising the steps of selecting an M moiety having a known fluorescence, preparing a compound of structure (I), (IA), (IA'), (IB), (IB'), (IC) or (IC') comprising the M moiety, and selecting appropriate variables for L 4 , L 5 , L 6 , m, n, q, w and z to reach the desired molar fluorescence.

[0109] Molar fluorescence can, in certain embodiments, be expressed in terms of the doubling or reduction factor relative to the fluorescence emission of the parent fluorophore (e.g., monomer). In some embodiments, the molar fluorescence of the present compound is 1.1x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9×10x or even higher compared to the parent fluorophore. Various embodiments 4 , L 5 , L 6 , m, n, q, w and z by appropriate selection, to prepare a compound having a desired doubling factor of fluorescence relative to the parent fluorophore. For ease of illustration, various compounds containing a phosphorus moiety (e.g., phosphate, etc.) are depicted in the anionic state (e.g., -OPO(OH)O - , -OPO3 2- ). Those skilled in the art will readily understand that the charge is pH-dependent and that the uncharged (e.g., protonated or salt such as sodium or other cation) forms are also within the scope of the embodiments of the present disclosure.

[0110] Compositions comprising any of the above-described compounds and one or more analyte molecules (e.g., biomolecules) are provided in various other embodiments. In some embodiments, it is also provided to use such a composition in an analytical method for detecting one or more analyte molecules. In yet other embodiments, the present compound is useful in various analytical methods. For example, in certain embodiments, the present disclosure provides a method of staining a sample, wherein the sample is irradiated at an appropriate wavelength with an amount sufficient to produce an optical response, and the sample is, for example, R 1 or R 2 wherein one of them is a linker containing a covalent bond to an analyte molecule (e.g., biomolecule) or microparticle, and the other of R 1 or R 2 is H, OH, alkyl, alkoxy, alkyl ether or -OP(=R a )(R b )R cProvided is a method comprising the step of adding a compound of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’). In some embodiments of the above method, R 1 or R 2 is a linker containing a covalent linking group to an analyte molecule, such as a biomolecule, e.g., a nucleic acid, an amino acid or a polymer thereof (e.g., a polynucleotide or a polypeptide). In more embodiments, the biomolecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer or a prion.

[0111] In still other embodiments of the above method, R 1 or R 2 is a linker containing a covalent linking group to a solid support, such as a microparticle. For example, in some embodiments, the microparticle is a polymeric bead or a non-polymeric bead. In even more embodiments, the optical response is a fluorescence response. In other embodiments, the sample contains cells, and some embodiments further include the step of observing the cells by flow cytometry. In more embodiments, the method further includes the step of distinguishing the fluorescence response from the fluorescence response of a second fluorophore capable of detecting different optical properties. In other embodiments, the present disclosure is a method for visually detecting an analyte molecule, such as a biomolecule, comprising (a) preparing a compound of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’) wherein, for example, one of R 1 or R 2 is a linker containing a covalent bond to the analyte molecule and the other of R 1 or R 2 is H, OH, alkyl, alkoxy, alkyl ether or -OP(=R a )(R b )R c , and (b) detecting the compound by its visible properties A method including is provided.

[0112] In some embodiments, the molecule to be analyzed is a nucleic acid, an amino acid, or a polymer thereof (e.g., a polynucleotide or a polypeptide). In still more embodiments, the molecule to be analyzed is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion. In other embodiments, a method for visually detecting a molecule to be analyzed, such as a biomolecule, (a) mixing any of the above-described compounds with one or more molecules to be analyzed, and (b) detecting the compound by its visible characteristics A method including is provided. In other embodiments, a method for visually detecting a molecule to be analyzed, (a) R 1 or R 2 is a linker containing a covalent bond to Q or Q, and mixing a compound of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’) with a molecule to be analyzed, (b) forming a conjugate of the compound and the molecule to be analyzed, and (c) detecting the conjugate by its visible characteristics A method including is provided.

[0113] Another exemplary method is a method for detecting an object to be analyzed: (a) preparing a compound of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’) in which R 1 or R 2 is a linker containing a covalent bond to a target-directed moiety having specificity for the object to be analyzed, (b) mixing the compound and the object to be analyzed, thereby binding the target-directed moiety and the object to be analyzed, and (c) detecting the compound, for example, by its visible characteristics or fluorescence characteristics A method including is included. In certain embodiments of the above method, the analyte is a particle such as a cell, and the method includes the use of flow cytometry. For example, the compound is provided with a targeting moiety such as an antibody to selectively bind to a desired cell, and thus the cell can be made detectable by any number of techniques such as visual detection or fluorescence detection. In some embodiments, the antibody is a polyclonal antibody. In other embodiments, the antibody is a monoclonal antibody. Suitable antibodies can be selected by those skilled in the art depending on the desired end use. In certain embodiments, exemplary antibodies for use include CD3 (clone UCHT1), CD4 (clone OKT4), FoxP3, TNF-α, IFN-γ, clone 4S.B3, clone 206D, CD8α (D8A8Y) rabbit mAb, vimentin (D21H3) XP® rabbit mAb, phospho-RB antibody (such as phospho-RB-Ser608, phospho-RB-Ser612, phospho-RB-Ser780, phospho-RB-Ser795, phospho-RB-Ser807 or phospho-RB-Ser811), anti-human IL17A, integrin alpha E / CD103, CCR9 and MOPC-21.

[0114] In certain embodiments, the conjugation efficiency of forming a conjugate comprising a compound of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’) and an analyte is higher than about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 98.5% or 99%. In yet other embodiments, the present disclosure is a method for increasing the luminance of a dye, comprising: (a) preparing a dye solution comprising a compound of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’), and (b) aging the dye solution for a certain period of time is provided. In some embodiments, the dye solution is aged for at least one week. For example, in some embodiments, the dye solution is aged for about three weeks before use.

[0115] The pigment solution may contain various buffers. In some embodiments, the pigment contains ETOH. In some embodiments, the pigment solution contains BD brilliant. In some embodiments, the pigment solution contains sodium chloride or potassium chloride. Thus, embodiments of the present compounds are found to be useful in any number of methods including, but not limited to, cell counting, cell sorting, biomarker detection, quantification of apoptosis, determination of cell viability, identification of cell surface antigens, determination of total DNA and / or RNA content, identification of specific nucleic acid sequences (e.g., as nucleic acid probes), and diagnosis of diseases such as blood cancers. In addition to the above methods, embodiments of the compounds of formula (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’) are found to be useful in various areas and methods including, but not limited to, imaging in endoscopic procedures for identifying cancerous and other tissues, single cell and / or single molecule analysis methods, e.g., detection of polynucleotides with little or no proliferation, e.g., cancer imaging by including a target-directed moiety such as an antibody or sugar, or other moiety that preferentially binds to cancer cells, in a compound of formula (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’), imaging in surgery, histone binding for identifying various diseases, e.g., drug delivery by replacing the M moiety in a compound of formula (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’) with an active pharmaceutical moiety, and / or contrast agents in dental procedures, and other procedures including, e.g., preferential binding of compounds of formula (I) to various microbiota and / or organisms.

[0116] Any embodiment of the compound of formula (I) described above, and the variables R in the compound of formula (I) described above 1 , R 2 , R 3 , R 4 , R 5 , L’, L 1a , L 1b , L 2 , L 3 , L4 , L 5 , L 6 , L 7 , M 1 , M 2 , m, n, q, and w, any specific selection described herein may be combined independently of the variables of other embodiments and / or of the compounds of structure (I) to form embodiments of the disclosure not specifically described above. Further, a list of selections, for any specific variable R in a particular embodiment and / or claim 1 , R 2 , R 3 , R 4 , R 5 , L’, L 1a , L 1b , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , M 1 , M 2 , m, n, q, and w, if listed, each individual selection may be deleted from a particular embodiment and / or claim, and the remaining list of selections is considered to be within the scope of the disclosure.

[0117] It is understood in this description that combinations of substituents and / or variables of the formulas shown are only permissible if such contributions result in stable compounds. It will also be appreciated by those skilled in the art that in the methods described herein, the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl and the like. Suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl and the like. Suitable protecting groups for mercapto include -C(O)-R” (wherein R” is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups are known to those skilled in the art and can be added or removed according to standard techniques described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. Protecting groups can also be polymeric resins such as Wang resin, Rink resin or 2-chlorotrityl chloride resin as understood by those skilled in the art.

[0118] Furthermore, all of the compounds of the present disclosure that exist in the free base form or free acid form can be converted to their salts by treatment with a suitable inorganic base or organic base or inorganic acid or organic acid by methods known to those skilled in the art. The salts of the compounds of the present disclosure can be converted to their free base form or free acid form by standard techniques. The following reaction scheme illustrates an exemplary method for preparing compounds of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’) of the present disclosure. It is understood that one of ordinary skill in the art may be able to prepare these compounds by similar methods or by combining other methods known to one of ordinary skill in the art. One of ordinary skill in the art can use appropriate starting components and modify the synthetic parameters as needed to prepare other compounds of structure (I), (IA), (IA’), (IB), (IB’), (IC) or (IC’) not specifically exemplified below by similar methods described herein. Generally, the starting components can be obtained from suppliers such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI and Fluorochem USA, or can be synthesized according to information sources known to one of ordinary skill in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)), or can be prepared as described in the present disclosure.

[0119] Reaction Scheme I

Chemical formula

[0120] Reaction Scheme II

Chemical formula

[0121] The compound of structure (I) can be prepared from one of structures b or d by reaction with a phosphoramidite compound having the following structure (e) under well-known automated DNA synthesis conditions.

Chemical formula

[0122] DNA synthesis methods are well known in the art. Briefly, two alcohol groups, for example R in the above intermediates b or d 2 and R 3 are each functionalized with a dimethoxytrityl (DMT) group and a 2-cyanoethyl-N,N-diisopropylaminophosphoramidite group, respectively. The phosphoramidite group is usually coupled with the alcohol group in the presence of an activator such as tetrazole, and then the phosphorus atom is oxidized with iodine. The dimethoxytrityl group is removed with an acid (e.g., chloroacetic acid) to expose the free alcohol, which can then be reacted with the phosphoramidite group. The 2-cyanoethyl group can be removed after oligomerization by treatment with aqueous ammonia. The preparation of phosphoramidites used in the oligomerization method is also well known in the art. For example, a primary alcohol (e.g., R 3 ) can be protected as a DMT group by reaction with DMT-Cl. Next, a secondary alcohol (e.g., R 2 ) is functionalized as a phosphoramidite by reaction with a suitable reagent such as 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. Methods for the preparation of phosphoramidites and their oligomerization are well known in the art and are described in more detail in the examples. The compounds of structure (I) are prepared by oligomerization of intermediates b or d and e according to the well-known phosphoramidite chemistry described above. By repeating the phosphoramidite coupling the desired number of times, the desired number of m and n repeating units are incorporated into the molecule.

[0123] Furthermore, the compounds of the present disclosure can be prepared according to the methods described in PCT Publication Numbers WO2016 / 183185; WO2017 / 173355; and WO2017 / 177065, each of which is incorporated herein by reference. The efficiency of the FRET process depends in part on the characteristics of the chromophores. Specifically, high-efficiency FRET requires a large overlap between the absorption spectrum of the donor chromophore and the emission spectrum of the acceptor chromophore. Furthermore, the distance and orientation of the chromophores play important roles. The FRET efficiency is inversely proportional to the sixth power of the distance between the two chromophores, and the angle of the transition dipole moments should be substantially parallel (i.e., close to 0° or 180°). Thus, in certain embodiments, the covalent attachment of the first and second chromophores to the polymer backbone is selected such that the distance between the first chromophore and the second chromophore is minimized and the transition dipole moments are substantially aligned. The efficiency of FRET can be represented by the following equation. [Equation] (where E FRET is the FRET efficiency, R is the distance between the two chromophores, and Ro is represented by the following equation. [Equation] (where J is the overlap spectrum of the absorption spectrum of the acceptor and the emission spectrum of the donor, Q o is the quantum efficiency of the donor, n -4 is the refractive index (constant) of the medium between the donor and the acceptor, and K 2 is the dipole orientation alignment))

[0124] Accordingly, one embodiment provides a polymeric compound having an acceptor transition dipole moment and an acceptor chromophore covalently linked to the polymer backbone, and a donor transition dipole moment and a donor chromophore covalently linked to the polymer backbone, wherein in a solution under physiological conditions, the effective distance between the acceptor chromophore and the donor chromophore is less than about 50.0 nm, and the acceptor transition dipole and the donor transition dipole assume a configuration that is substantially parallel. In some embodiments, the effective distance between the acceptor chromophore and the donor chromophore is less than about 25.0 nm. In some embodiments, the effective distance between the acceptor chromophore and the donor chromophore is less than about 10.0 nm. In some embodiments, the effective distance between the acceptor chromophore and the donor chromophore is less than about 30.0 nm, less than about 27.0 nm, less than about 22.0 nm, less than about 20.0 nm, less than about 17.0 nm, less than about 15.0 nm, less than about 12.0 nm, less than about 11.0 nm, less than about 9.0 nm, less than about 8.0 nm, less than about 7.0 nm, less than about 6.0 nm, less than about 5.0 nm, less than about 4.0 nm, less than about 3.0 nm, less than about 2.0 nm or less than about 1.0 nm.

[0125] In some embodiments, the acceptor chromophore is a fluorescent dye moiety. In certain embodiments, the donor chromophore is a fluorescent dye moiety. In certain related embodiments, both the acceptor chromophore and the donor chromophore are fluorescent dye moieties. In some embodiments, the angle between the acceptor transition dipole moment and the donor transition dipole moment ranges from 120° to 180°. For example, in some embodiments, the angle between the acceptor transition dipole moment and the donor transition dipole moment ranges from 125° to 180°, from 130° to 180°, from 140° to 180°, from 150° to 180°, from 160° to 180°, from 170° to 180°, from 172° to 180°, from 175° to 180° or from 177° to 180°. In certain embodiments, the angle between the acceptor transition dipole moment and the donor transition dipole moment is in the range of 0° to 60°. For example, in some embodiments, the angle between the acceptor transition dipole moment and the donor transition dipole moment is in the range of 0° to 50°, 0° to 40°, 0° to 30°, 0° to 20°, 0° to 10°, 0° to 8°, 0° to 5°, 0° to 3°, or 0° to 2°.

[0126] In some more specific embodiments, the polymer compound further comprises a first acceptor chromophore covalently linked at the proximal end of the polymer backbone, a second acceptor chromophore covalently linked at the distal end of the polymer backbone, and a donor chromophore covalently linked between the proximal and distal ends of the polymer backbone. In certain embodiments, the polymer backbone comprises a phosphate linker. In some embodiments, the polymer backbone comprises a plurality of phosphate linkers. In some related embodiments, the polymer backbone comprises an alkylene oxide linker. In some more specific embodiments, the alkylene oxide is ethylene oxide. In some specific embodiments, the polymer backbone comprises an HEG linker, a C linker, or a combination thereof.

[0127] In some embodiments, the polymer compound has a molecular weight of less than 20,000 g / mol. In some embodiments, the polymer compound has a molecular weight of less than 19,000 g / mol, 18,500 g / mol, 18,000 g / mol, 17,500 g / mol, 17,000 g / mol, 16,500 g / mol, 16,000 g / mol, 15,500 g / mol, 15,000 g / mol, 14,500 g / mol, 14,000 g / mol, 13,500 g / mol, 13,000 g / mol, 12,500 g / mol, 11,500 g / mol, 11,000 g / mol, 10,500 g / mol, 10,000 g / mol, 9,500 g / mol, 9,000 g / mol, 8,500 g / mol, 8,000 g / mol, 7,500 g / mol, 7,000 g / mol, 6,500 g / mol, 6,000 g / mol, 5,500 g / mol, 5,000 g / mol, 4,500 g / mol, 4,000 g / mol, 3,500 g / mol, 3,000 g / mol, 2,500 g / mol, 2,000 g / mol, 1,500 g / mol or 1,000 g / mol. In some embodiments, the polymer compound is neither a peptide nor a protein. In some other embodiments, the polymer backbone does not have an amide bond. The following examples are presented for illustrative purposes only and not for limitation.

Examples

[0128] General Method Mass spectrometry analysis was performed on a Waters / Micromass Quattro micro MS / MS system (MS mode only) using MassLynx 4.1 acquisition software. The mobile phase used for LC / MS with respect to the dyes was 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6 mM triethylamine (TEA), pH 8. Phosphoramidites and precursor molecules were also analyzed using a Waters Acquity UHPLC system equipped with a 2.1 mm x 50 mm Acquity BEH-C18 column maintained at 45 °C using a gradient of an acetonitrile / water mobile phase. The molecular weights of the monomer intermediates were obtained using a Waters / Micromass Quattro micro MS / MS system (MS mode only) with tropylium cation injection enhanced ionization. Excitation and emission profile experiments were recorded on a Cary Eclipse spectrophotometer. All reactions were carried out in oven-dried glassware under a nitrogen atmosphere, unless otherwise specified. Commercially available DNA synthesis reagents were purchased from Glen Research (Sterling, VA). Anhydrous pyridine, toluene, dichloromethane, diisopropylethylamine, triethylamine, acetic acid, pyridine, and THF were purchased from Aldrich. All other chemicals were purchased from Aldrich or TCI and used without further purification.

[0129] (Example 1) Synthesis of Dyes with Alkylene-Polyethylene Glycol-Alkylene Spacers Compounds having an alkylene-polyethylene oxide-alkylene linker were prepared as follows: The oligofluoroside constructs (i.e., the compounds of structure (I)) were synthesized on a 1 μmol scale using an Applied Biosystems 394 DNA / RNA synthesizer and had a 3'-phosphate group, or a 3'-S2-(CH2)6-OH group, or any other group described herein. The synthesis was carried out directly on CPG beads or a polystyrene solid support using standard phosphoramidite chemistry. The oligofluorides were synthesized in the 3' to 5' direction using standard solid-phase DNA methods, and the coupling was carried out using standard β-cyanoethyl phosphoramidite chemistry. The fluoroside phosphoramidites and spacers (e.g., polyethylene glycol phosphoramidite, propane-diol phosphoramidite, butane-diol phosphoramidite, and hexane-diol phosphoramidite) and linkers (e.g., 5'-amino-modified phosphoramidite and thiol-modified S2 phosphoramidite) were dissolved in acetonitrile to make a 0.1 M solution and added in a sequential order using the following synthesis cycle: 1) removal of the 5'-dimethoxytrityl protecting group with dichloroacetic acid in dichloromethane, 2) coupling of the next phosphoramidite with an activator reagent in acetonitrile, 3) oxidation of P(III) to form stable P(V) with iodine / pyridine / water, and 4) capping of any unreacted 5'-hydroxyl groups with acetic anhydride / 1-methylimidazole / acetonitrile. The synthesis cycle was repeated until the full-length oligofluoroside construct was assembled. At the end of the chain assembly, the monomethoxytrityl (MMT) group or the dimethoxytrityl (DMT) group was removed with dichloroacetic acid in dichloromethane.

[0130] The compound was provided on a pore-controlled glass (CPG) support at a scale of 0.2 μmol in labeled Eppendorf tubes. 400 μL of 20 - 30% NH₄OH was added and gently mixed. The open tubes were placed at 55 °C for about 5 minutes or until excess gas was released, then tightly sealed and incubated for 2 hours (+ / - 15 minutes). The tubes were removed from the heating block, allowed to reach room temperature, then centrifuged at 13,400 RPM for 30 seconds, and the supernatant and solid were combined. The supernatant was carefully removed and placed in a labeled tube, then 150 μL of acetonitrile was added to wash the support. After adding the wash solution to the tube, it was placed in a CentriVap device at 40 °C until dry. The product was characterized by ESI-MS, UV-absorbance, and fluorescence spectroscopy.

[0131] (Example 2) General flow cytometry method Unless otherwise specified, the following general procedure was used throughout the following examples: Lysis of whole blood: Buffered ammonium chloride method. For staining of live cells, normal human blood anticoagulated with ethylenediaminetetraacetate (EDTA) was bulk lysed in ammonium chloride solution (ACK) at room temperature (RT) for 15 minutes with 15 mL of blood per 35 mL of lysis solution. The cells were washed twice with 50% Hank's balanced salt solution (HBSS) containing 0.02% sodium azide and 50% 1% fetal bovine serum (FBS) in 1x Dulbecco's phosphate buffered saline (PBS). The cells were then resuspended in donor plasma at 100 μL / test article / 0.1 - 1x10e6. In a 96-well HTS plate made of polypropylene, 100 μL of 1% bovine serum albumin (BSA) containing 0.02% sodium azide and V of 1×DPBS f of pre-diluted antibody were added to the cells in plasma. After incubation at room temperature for 45 minutes, the cells were washed twice with 50% HBSS containing 0.02% sodium azide and 50% - 1% FBS 1x DPBS.

[0132] Lysis / Fixation method. Blood was lysed with 100 - 15 mL of blood per 35 mL of lysis solution with 1.0 mL of RBC lysis solution (ammonium chloride) for 15 minutes at room temperature. Next, it was washed twice with 50% HBSS containing 0.02% sodium azide and 50% - 1% FBS 1xDPBS. Next, the cells were resuspended in donor plasma at 100 μL / sample / 1x10e6. Pre-diluted antibody was added into 100 μL of 1% BSA and 1xDPBS containing 0.02% sodium azide. 100 μL of cells were added to a 96-well HTS plate made of polypropylene (test size with a total volume of 200 μL). After incubation at room temperature for 45 minutes, it was washed twice with 50% HBSS containing 0.02% sodium azide and 50% of 1% FBS 1xDPBS.

[0133] Preparation of Antibody Conjugate The antibody conjugate was prepared by reacting a compound of Structure (I) containing a Q moiety having the following structure with the desired antibody.

Chemical formula

Chemical formula

[0134] The antibody conjugate is designated by the name of the antibody according to the compound number. For example, UCHT1-I-1 indicates the conjugate formed between the UCHT1 antibody and the compound of Structure (I) I-1. When the number of the referenced compound does not contain the Q moiety in Table 1 above, it is understood that this Q moiety was introduced and the conjugate was prepared from the compound resulting from having the Q moiety. Dilution of Conjugate The antibody was brought to room temperature. The antibody conjugate was diluted in cell staining buffer (1X DPBS, 1% BSA, 0.02% sodium azide) to concentrations in the range of 0.1 - 540 nM (≤ 8.0 micrograms per test). In some examples, serial dilutions of each sample were initiated with a 269 nM antibody in cell staining buffer and the antibody dilutions were kept protected from light until use. In other experiments, dilutions were initiated with 4.0 μg of antibody per test size, where the test size ranged from 100 - 200 μL. Titrations were performed in 2-fold or 4-fold dilutions to generate binding curves. In some cases, 8.0 or 2.0 μg of antibody per test size was used in the first well of the dilution series.

[0135] Flow cytometry using the conjugate: After physical characterization, the activity and function of the conjugate (antibody binding affinity and dye brightness) were tested and compared to reference antibody staining. Next, the quality of resolution was determined by examining the brightness compared to autofluorescent negative controls and other non-specific binding using a flow cytometer. Whole blood screening to test the conjugate was made most routine. Bridging studies were performed since new constructs were formed. Performance of free dye flow cytometry: After molecular and physical characterization, the potential affinity of the dye for cells was also tested compared to staining with a reference dye. Since the dye may also function as a cell probe and has the potential to bind to cellular material, the dye was generally screened against blood at high concentrations (> 100 nM - 10,000 nM) to confirm specific characteristics. Next, any unexpected or expected off-target binding was quantified by evaluating the brightness and linearity upon dilution compared to autofluorescent negative controls and other dye controls using a flow cytometer.

[0136] Flow cytometry workflow: Cells were cultured and observed for visual signs of metabolic burden related to pigment screening or off-target binding (data not shown), or new healthy cells were used for conjugate screening. Cells were counted regularly to confirm cell density (1×10e5 and 1×10e6 viable cells / mL). After diluting the antibody conjugate (preferably in plates or tubes), cells in staining buffer (DPBS, 0.1% BSA, 0.02% sodium azide) were harvested. Cells with a viability range of 80 - 85% were used. The cells were washed twice by centrifugation and washing the cells with buffer to remove pH indicator and block cells containing Ig and other proteins present in FBS. The cell density was adjusted and sized in staining buffer. Cells were plate-cultured for one test per well, or pigment (pre-diluted) was applied to the cells in the plate. These cells were then incubated at 23°C for 45 minutes. The cells were centrifuged and washed twice by washing the cells with wash buffer, and then the plate was aspirated. The cells were resuspended in acquisition buffer. 5000 intact cells were obtained by flow cytometry. The fluorescence of the pigment was detected by flow cytometry with a peak emission (521 nM) using a 525 / 50 bandpass filter and detected by a 488 nM blue laser line. Aiming for the acquisition of 3000 - 5000 intact cells, at least 1500 intact cells were obtained by flow cytometry and analyzed to identify viable cells present in the cell preparation.

[0137] Data analysis method: Descriptive statistics. With the EC-800 software, the user can collect numerous statistical data regarding the acquisition of each sample. The brightness of the antibody-pigment reagent was measured by flow cytometry using the mean or median fluorescence intensity (MFI) in the FL1-A channel and when examining the noise. Other statistics were evaluated to determine the characteristics of the pigment and the overall quality of the reagent, including the median signal-to-noise and absolute fluorescence (median or geometric mean). Histogram. Flow cytometry events were gated by size with respect to forward scatter versus side scatter (cell volume versus cell granularity). Next, they were gated by fluorescence emission at 515 nm with respect to the mean fluorescence intensity (MFI) of those cells. The collected data were represented as a dual-parameter histogram plotted as the number of events versus fluorescence intensity on the y-axis and represented on a logarithmic scale with respect to the x-axis. The data can be summarized by an affinity curve or a histogram of relative fluorescence intensity.

[0138] Binding curve. When determined by FCM, MFI is the parameter that best measures the brightness of the antibody-dye reagent, so MFI is selected, which can be expressed as the geometric mean, median, or mean value and represents the absolute fluorescence measurement. For comparison, when noise can be very characteristic, the signal-to-noise ratio S / N is reported as MFI. Bivariate dual-parameter histogram. In some cases, to examine the quantitative output, FCM events were not gated and the data were represented by cell granularity (SSC) versus dye fluorescence. This method allows for a comprehensive evaluation of all populations recovered in whole blood. (Example 3) Flow cytometry analysis of Compounds I-1 and I-2 was conjugated to a CD4 (clone OKT4) antibody and eluted with 1x d-PBS (phosphate-buffered saline). The dyes used included FAM and Alexa Fluor® 594 (AF594). Whole blood was stained with 0.5 μg of the antibody conjugate and screened on a spectral instrument.

[0139] (Example 4) Preparation of Phosphoramidites and Compounds Exemplary compounds were prepared using a protocol for standard solid-phase oligonucleotide synthesis and a fluorescein-containing phosphoramidite having the following structure purchased from ChemGenes (Catalog #CLP-9780). [Chemical formula]

[0140] Exemplary linker (L 6 ) was also incorporated into the compound by coupling with a commercially available phosphoramidite having the following structure. [Chemical formula] Exemplary linker (L 7 / L 1b ) was also incorporated into the compound by coupling with a phosphoramidite having one of the following commercially available structures. [Chemical formula]

[0141] Other exemplary compounds were prepared using phosphoramidites prepared according to the following scheme. [Chemical formula]

[0142] Final deprotection generates the desired Fx moiety. Various moieties of this compound were introduced using other commercially available phosphoramidite reagents as appropriate. The Q moiety having the following structure: [Chemical formula] was

[0143] [Chemical formula] introduced by reaction with free sulfhydryl. Other Q moieties are introduced by similar methods according to the knowledge of those skilled in the art.

[0144] The various embodiments described above can be combined to obtain further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the application data sheet, including U.S. Provisional Patent Application No. 63 / 352,563 filed on June 15, 2022, are hereby incorporated by reference in their entirety. Aspects of the embodiments can, if necessary, be modified to present still further embodiments using the concepts of the various patents, applications, and publications. These and other changes can be made to the embodiments in light of the above detailed description. Generally, in the following claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed in this specification and the claims, but should be construed to include all possible embodiments with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.

Claims

1. A compound having the following structure (I), or a stereoisomer, salt or tautomer thereof. 【Chemical 1】 (I) (In the formula, M 1 and M 2 are, independently for each occurrence, chromophores, provided that M 1 is a FRET acceptor and M 2 is the corresponding FRET donor, and M 1 and M 2 form a FRET pair, provided that L 1a is, independently for each occurrence, a heteroalkylene linker or a heteroarylene linker, L 1b 、 L 2 、 L 3 、 L 5 、 L 6 and L 7 are, independently for each occurrence, optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene or heteroalkynylene linkers, L 4 has, for each occurrence, one of the following structures, 【Chemical 2】 (In the formula, z is an integer from 1 to 100, * indicates the bond to the adjacent phosphorus atom), R 1 and R 2 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a )(R b )R c , Q or a protected form thereof, or L'. R 3 is, independently for each occurrence, H, alkyl or alkoxy, R 4 is, independently for each occurrence, OH, SH, O - , S - , OR d or SR d and R 5 is, independently at each occurrence, oxo, thioxo or absent, R a is O or S, R b is OH, SH, O - , S - , OR d or SR d and R c is OH, SH, O - , S - , OR d , OL’, SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether, R d is a counter ion, Q is, independently for each occurrence, a reactive group or a moiety containing a protected form thereof, which can form a covalent bond with an analyte molecule, a target-directed moiety, a solid support or a complementary reactive group Q', L' is, independently for each occurrence, a linker containing a covalent bond to Q, a linker containing a covalent bond to a target-directed moiety, a linker containing a covalent bond to an analyte molecule, a linker containing a covalent bond to a solid support, a linker containing a covalent bond to a solid support residue, a linker containing a covalent bond to a nucleoside, or a linker containing a covalent bond to a further compound of structure (I), m is, for each occurrence, an integer of 1 or more, q is, for each occurrence, an integer of 1 or more, w is, in at least one occurrence, an integer of 1 or more, provided that q is an integer greater than w when n is an integer of 1, n is an integer of 1 or more)

2. L 1a The compound according to claim 1, wherein L is a 5- to 7-membered heteroarylene linker which may be independently substituted at each occurrence.

3. L 1a The compound according to claim 1 or 2, wherein L has one of the following structures. [Chemical Formula 3]

4. A compound according to any one of claims 1 to 3, having one of the following structures (IA) or (IA'), or a stereoisomer, salt or tautomer thereof. 【Chemical Formula 4】

5. The compound according to claim 1, wherein z is an integer from 3 to 8, an integer from 15 to 30 or an integer from 22 to 26.

6. A compound according to any one of claims 1 to 5, having one of the following structures (IB) or (IB'), or a stereoisomer, salt or tautomer thereof. 【Chemical Formula 5】

7. The L that appears 5 or L 6 The compound according to any one of claims 1 to 6, wherein at least one of them is alkylene.

8. L 5 or L 6 The compound according to any one of claims 1 to 7, wherein each occurrence of is an alkylene.

9. The L that appears 3 The compound according to any one of claims 1 to 8, wherein at least one of them is alkylene.

10. L 3 The compound according to any one of claims 1 to 9, wherein alkylene is present each time it appears.

11. A compound according to any one of claims 7 to 10, having one of the following structures (IC) or (IC'), or a stereoisomer, salt or tautomer thereof. [Chemical Formula 6] (wherein, y 1 , y 2 and y 3 are, independently for each occurrence, integers from 1 to 6)

12. The L that appears 1b The compound according to any one of claims 1 to 11, wherein at least one of which contains a functional group formed by reaction with a complementary reactive group of aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imido ester, activated ester, ketone, α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin or thiirane.

13. The L that appears 1b The compound according to claim 12, wherein at least one of them contains a functional group formed by the reaction of an alkyne and an azide.

14. The L that appears 1b The compound according to claim 13, wherein at least one of them is a linker containing a triazolyl functional group.

15. The L that appears 1b -M 1 The compound according to any one of claims 1 to 14, wherein at least one of them has the following structure. 【Chemical Formula 7】 (wherein L c and L d are each independently an optional linker)

16. L c or L d or neither is present, a compound according to claim 15

17. L c or L d or both are present, the compound according to claim 15.

18. L c and L d When present, each independently is alkylene or heteroalkylene, the compound according to claim 17.

19. L c and L d which independently has one of the following structures, the compound according to claim 17 [Chemical 8]

20. L 1b The compound according to any one of claims 1 to 19, wherein L contains one of the following structures. 【Chemical Formula 9】 (In the formula, a, b, c, d and e are each independently an integer in the range of 1 to 6)

21. The M that appears 1 -L 1b The compound according to any one of claims 1 to 20, wherein at least one of them has one of the following structures. 【Chemical 10】

22. M 1 -L 1b For each occurrence of, a compound according to any one of claims 1 to 21, having one of the following structures. 【Chemical 11】

23. The L that appears 7 The compound according to any one of claims 1 to 22, wherein at least one of them is a heteroalkylene linker which may be substituted.

24. L 7 The compound according to any one of claims 1 to 23, wherein L is a heteroalkylene which may be independently substituted each time it appears.

25. L 7 The compound according to any one of claims 1 to 24, wherein L contains an amide functional group.

26. The L that appears 7 A compound according to any one of claims 1 to 25, wherein at least one of them has one of the following structures. 【Chemical 12】

27. L 7 The compound according to any one of claims 1 to 26, having one of the following structures each time L appears. 【Chemical 13】

28. The L that appears 7 The compound according to any one of claims 1 to 27, wherein at least one of them has one of the following structures. 【Chemical Formula 14】

29. L 7 The compound according to any one of claims 1 to 28, having one of the following structures each time L appears. 【Chemical Formula 15】

30. The R that appears 3 The compound according to any one of claims 1 to 29, wherein at least one of them is H.

31. R 5 is, independently for each occurrence, OH, O - or OR d and is a compound according to any one of claims 1 to 30.

32. R 4 The compound according to any one of claims 1 to 31, wherein R is oxo each time it appears.

33. R 1 and R 2 are each independently OH or -OP(=R a )(R b )R c The compound according to any one of claims 1 to 32, wherein

34. R 1 or R 2 wherein one of them is OH or -OP(=R a )(R b )R c and the other of R 1 or R 2 is Q or a linker containing a covalent bond to Q, the compound according to any one of claims 1 to 32.

35. R 1 and R 2 are each independently -OP(=R a )(R b )R c The compound according to any one of claims 1 to 32, wherein

36. R c The compound according to any one of claims 33 to 35, wherein R is OL'.

37. The compound according to claim 36, wherein L’ is a heteroalkylene linker to Q, a target-directed moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside or a further compound of structure (I).

38. The compound according to claim 37, wherein the analyte molecule is a nucleic acid, an amino acid or a polymer thereof.

39. The compound according to claim 37, wherein the analyte molecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer or a prion.

40. The compound according to claim 37, wherein the target-directed moiety is an antibody or a cell surface receptor antagonist.

41. The compound according to claim 37, wherein the solid support is a polymeric bead or a non-polymeric bead.

42. The compound according to claim 37, wherein L’ comprises an alkylene oxide or a phosphodiester moiety or a combination thereof.

43. The compound according to claim 42, wherein L’ has the following structure. 【Chemical 16】 (wherein, m” and n” are each independently an integer from 1 to 10, R e is H, an electron pair or a counter ion, "L" is R e , or a direct bond, or a linking group to Q, a target-directed moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside or a further compound of structure (I))

44. R 1 or R 2 The compound according to any one of claims 1 to 43, wherein R has one of the following structures. 【Chemical 17-1】 【Chemical 17-2】

45. R 1 or R 2 is a compound according to any one of claims 1 to 44, having the following structure. 【Chemical Formula 18】

46. The compound according to any one of claims 1 to 45, wherein Q comprises a nucleophilic reactive group, an electrophilic reactive group or a cycloaddition reactive group.

47. The compound according to claim 46, wherein Q comprises a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino or maleimide functional group.

48. The compound according to claim 47, wherein the activated ester is an N-succinimide ester, an imido ester or a polyfluorophenyl ester.

49. The compound according to claim 47, wherein the azide is an alkyl azide or an acyl azide.

50. The compound according to any one of claims 1 to 49, wherein Q is a moiety selected from Table 1.

51. M 1 and M 2 wherein, in one or more occurrences, M is independently a moiety comprising four or more aryl or heteroaryl rings, or combinations thereof, according to any one of claims 1 to 50.

52. M 1 and M 2 The compound according to any one of claims 1 to 51, wherein M and M are, independently in one or more occurrences, fluorescent or chromogenic.

53. M 1 and M 2 The compound according to claim 52, wherein M and M are fluorescent.

54. M 1 and M 2 wherein, in one or more occurrences, M independently comprises a fused polycyclic aryl or heteroaryl moiety comprising at least four fused rings, a compound according to any one of claims 1 to 53.

55. M 1 or M 1 -L 1b wherein, each occurrence independently has one of the following structures, the compound according to any one of claims 1 to 54. 【Chemical Formula 19-1】 【Chemical Formula 19-2】 【Chemical Formula 19-3】 【Chemical Formula 19-4】

56. M 2 The compound according to any one of claims 1 to 54, wherein M independently has one of the following structures each time it appears. 【Chemical 20】

57. The polymeric dye is the FRET donor M 2 One FRET a per one Compound M 1 The compound according to any one of claims 1 to 56, comprising 1 .

58. The polymeric dye is the FRET donor M 2 and contains one FRET acceptor M for every two 1 The compound according to any one of claims 1 to 56

59. The polymeric dye is FRET donor M 2 with one FRET acceptor M per three 1 The compound according to any one of claims 1 to 56, comprising

60. The polymeric dye is the FRET donor M 2 For every three, two FRET acceptors M 1 The compound according to any one of claims 1 to 56, comprising

61. The compound according to any one of claims 1 to 60, wherein n is an integer from 1 to 100.

62. The compound according to any one of claims 1 to 61, wherein n is an integer from 1 to 10.

63. The compound according to any one of claims 1 to 62, wherein q is an integer from 1 to 10.

64. The compound according to any one of claims 1 to 63, wherein q is an integer from 1 to 5.

65. The compound according to any one of claims 1 to 64, wherein q is an integer of 1, w is an integer of 1, and n is an integer of 2.

66. The compound according to any one of claims 1 to 64, wherein q is an integer of 2, w is an integer of 1, and n is an integer of 1.

67. The compound according to any one of claims 1 to 64, wherein q is an integer of 3, w is an integer of 1, and n is an integer of 1.

68. The compound according to any one of claims 1 to 64, wherein q is an integer of 2, w is an integer of 1, and n is an integer of 2.

69. The compound according to any one of claims 1 to 68, wherein m is an integer from 1 to 6.

70. The compound according to any one of claims 1 to 68, wherein m is an integer of 1.

71. y 1 , y 2 and y 3 is 1 each time of appearance, the compound according to claim 11.

72. A compound selected from Table 2.

73. A method for staining a sample, comprising adding to the sample a compound according to any one of claims 1 to 72 in an amount sufficient to produce an optical response when the sample is irradiated at an appropriate wavelength.

74. The method according to claim 73, wherein the optical response is a fluorescence response.

75. The method according to claim 73 or 74, wherein the sample contains cells.

76. The method according to claim 75, further comprising observing the cells by flow cytometry.

77. The method according to claim 76, further comprising distinguishing the fluorescence response from the fluorescence response of a second fluorophore having detectably different optical properties.

78. A method for visually detecting an analyte molecule, comprising: (a) R 1 or R 2 is a linker containing a covalent bond to the molecule to be analyzed, the step of preparing the compound according to any one of claims 1 to 72, and (b) detecting the compound by its visible properties The method comprising.

79. A method for visually detecting an analyte molecule, comprising: (a) R 1 or R 2 Mixing the compound according to any one of claims 1 to 72, wherein R is a linker containing a covalent bond to Q or Q, with an analyte molecule (b) forming a conjugate of the compound and the analyte molecule, and (c) detecting the conjugate by its visible properties The method comprising.

80. A method for visually detecting an analyte, comprising: (a) R 1 or R 2 preparing a compound according to any one of claims 1 to 72, wherein R or R comprises a linker comprising a covalent bond to a target-directed moiety having specificity for the analyte (b) mixing the compound and the analyte, thereby binding the target-directed moiety and the analyte, and (c) detecting the compound by its visible properties The method comprising.

81. The method according to claim 80, wherein the target-directed moiety is an antibody selected from the group consisting of CD3, CD4, FoxP3, TNF-α, IFN-γ, clone 4S.B3, clone 206D, CD8α (D8A8Y) rabbit mAb, vimentin (D21H3) XP® rabbit mAb, phospho-RB-Ser608, phospho-RB-Ser612, phospho-RB-Ser780, phospho-RB-Ser795, phospho-RB-Ser807 or phospho-RB-Ser811, anti-human IL17A, integrin alpha E / CD103, CCR9 and MOPC-21.

82. A method for increasing the brightness of a dye, comprising: (a) preparing a dye solution containing the compound according to any one of claims 1 to 72; and (b) aging the dye solution for a certain period of time. A method comprising the steps of:

83. The method according to claim 82, wherein the step of aging the dye solution comprises storing the dye solution for at least one week.

84. The method according to claim 83, wherein the step of aging the solution comprises storing the dye solution for three weeks.

85. The method according to any one of claims 82 to 84, wherein the dye solution contains ETOH.

86. The method according to any one of claims 82 to 85, wherein the dye solution contains BD brilliant.

87. The method according to any one of claims 82 to 86, wherein the dye solution contains sodium chloride or potassium chloride.

88. A composition comprising the compound according to any one of claims 1 to 72 and one or more analyte molecules.

89. Use of the composition according to claim 88 in an analytical method for detecting one or more analyte molecules.

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