Designing Spacing Linker Groups to Enhance Brightness in Dimeric or Polymeric Dyes

Abstract

Compounds useful as fluorescent or chromogenic dyes are disclosed. 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 , M 1 , M 2 wherein m, n, q, and w are as defined herein, or a stereoisomer, tautomer, or salt thereof. Related methods for the preparation and use of such compounds are also provided.

Description

[Technical Field]

[0001] This disclosure generally covers dimers and polymer fluorescent dyes or chromogenic dyes having spacing groups for brightness enhancement, methods for preparing them, and their use in various analytical methods. [Background technology]

[0002] Fluorescent and / or chromogenic dyes are known to be particularly suitable for applications where highly sensitive detection reagents are desired. Dyes capable of preferentially labeling specific components or constituents in a sample allow researchers to determine the presence, quantity, and / or location of those specific components or constituents. Furthermore, certain systems can be monitored for their spatial and temporal distribution in diverse environments. Fluorescence and colorimetric quantitative methods are widely used in chemistry and biology. These methods provide useful information about biomolecules regarding their presence, structure, distance, orientation, complex formation, and / or position. Furthermore, time-resolved methods are increasingly being used for measuring kinetics and velocity. As a result, numerous strategies have been developed for the fluorescence or color-labeling of biomolecules such as nucleic acids and proteins. Since the analysis of biomolecules is usually performed in an aqueous environment, emphasis has been placed on the development and use of water-soluble dyes. The use of highly fluorescent or chromogenic dyes is highly desirable because it increases the signal-to-noise ratio and realizes other related benefits. Therefore, attempts have been made to increase the signal from known fluorescent and / or chromogenic moieties. For example, dimeric and polymer compounds containing two or more fluorescent and / or chromogenic moieties have been prepared with the expectation that such compounds will be brighter dyes. However, as a result of intramolecular fluorescence quenching, known dimeric and polymer dyes do not achieve the desired improvement in brightness. [Overview of the Initiative]

[0003] Therefore, water-soluble dyes with increased molar brightness are needed in this field. Ideally, such dyes and biomarkers should be highly chromogenic or fluorescent and available in a variety of colors and fluorescence wavelengths. This disclosure satisfies this need and also realizes related advantages. In short, embodiments of this disclosure generally relate to compounds useful as water-soluble fluorescent dyes and / or chromogenic dyes and / or probes that enable the visual detection of analyte molecules such as biomolecules, as well as reagents for preparing them. Methods for visually detecting analyte molecules using dyes are also described. Embodiments of the dyes disclosed herein have the following structure: [ka] The compound contains two or more fluorescent and / or chromogenic moieties covalently linked by linkers having (wherein L4 and L5 are linker groups different from the L6 group). In contrast to previous reports of dimers and / or polymer dyes having only an L6 linker group, the introduction of additional linker groups L4 and L5 surrounding L6 helps to increase the rigidity of the linker and the spacing between adjacent fluorescent and / or chromogenic moieties. This dye compound is considerably brighter than the corresponding dye compound containing only an L6 linker group. The brightness of this dye compound also increases over time. Although we do not wish to be bound by theory, it is thought that the addition of additional linker groups around L6 provides greater spatial isolation and spatial isolation stability between the fluorescent and / or chromogenic moieties, thus reducing and / or eliminating intramolecular fluorescence quenching.

[0004] In some embodiments, the compounds of this disclosure are useful because they enable FRET fluorescence emission bound to the compound. A method for visually detecting analyte molecules using dyes is also described. Embodiments of the disclosed dyes include two or more fluorescent moieties and / or chromogenic moieties (i.e., chromophores or FRET donors / acceptors) covalently linked by linkers (e.g., "L1" or "L1c" and "L1d"). In contrast to previous reports of protein-based dimeric and / or polymeric dyes, significantly brighter FRET absorption and emission are enabled as a result of intramolecular interactions and are reliably reproducible using facile methods known in the art (i.e., automated DNA synthesis methods).

[0005] The water-soluble fluorescent or chromogenic dyes of embodiments of the present disclosure strongly emit color and / or fluorescence, enable FRET processes (e.g., absorbance, emission, Stokes shift), and can be readily observed by visual inspection or other means. In some embodiments, the compounds can be observed without prior irradiation or chemical or enzymatic activation. By appropriate selection of the dyes, various chromogenic visually detectable analyte molecules can be obtained as described herein. In some embodiments, a compound having the following structure (I): [Chemical formula] (I) or a stereoisomer, tautomer or salt thereof (wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , M 1 , M 2 , m and n are as defined herein) is provided. In some other embodiments, a compound having the following structure (A): [Chemical formula] (A) or its stereoisomers, tautomers, or salts (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 M 1 M 2 A (where m, n, and q are as defined herein) is provided. In other embodiments, compounds having the following structure (II): [ka] (II) or its stereoisomers, tautomers, or salts (wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1c , L 1d , L 2 , L 3 , L 4 , L 5 , L 6 M 1 M 2 A (where m, n, and q are as defined herein) is provided.

[0006] Compounds of structure (I), (A), or (II) have been found to have several applications, including use as fluorescent and / or chromogenic dyes in various analytical methods. In yet another embodiment, a method for staining a sample is provided, comprising the step of adding to the sample a sufficient amount of a compound of structure (I), (A), or (II) to produce an optical response when the sample is irradiated at an appropriate wavelength.

[0007] In yet another embodiment, the present disclosure is a method for visually detecting an analyte molecule, (a) the step of preparing the compound disclosed herein, and (b) A step of detecting the compound by its visible properties. This provides a method that includes [something]. Other disclosed methods are methods for visually detecting biomolecules, (a) the step of mixing one or more biomolecules with the compounds disclosed herein, and (b) A step of detecting the compound by its visible properties. This includes methods that include [specific methods]. Another embodiment is a method for visually detecting the object to be analyzed, (a)R 2 or R 3 However, the step of preparing a compound as disclosed herein, which includes a linker having a covalent bond to a target-directing moiety having specificity for the analyte, (b) The step of mixing the compound and the analyte to bind the target-directing portion and the analyte, (c) A step of detecting the compound by its visible properties. This provides a method that includes [something]. In yet another embodiment, the present disclosure is a method for increasing the brightness of a dye, (a) the step of preparing a dye solution containing the compound disclosed herein, and (b) A step of aging the dye solution for a certain period of time. This provides a method that includes [something].

[0008] Other embodiments relate to compositions comprising the compounds disclosed herein and one or more analyte molecules, such as one or more biomolecules. Use of such compositions in analytical methods for detecting one or more biomolecules is also provided. In some other different embodiments, the compound of structure (III): [ka] (III) or its stereoisomers, salts, or tautomers (wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1’ , L 2 , L 3 , L 4 , L 5 , L 6 , G 1 , G 2 A compound of structure (III) is provided, where m and n are as defined herein. The compound of structure (III) finds use in several applications, including its use as an intermediate for preparing the fluorescent and / or chromogenic dye of structure (I). In some other different embodiments, the compound of structure (IV): [ka] (IV) or its stereoisomers, salts, or tautomers (wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1c , L 1d’ , L 2 , L 3 , L 4 , L 5 , L 6 , G 1 , G 2 A compound of structure (III) is provided, where m and n are as defined herein. The compound of structure (III) finds use in several applications, including its use as an intermediate for preparing the fluorescent and / or chromogenic dyes of structure (II).

[0009] In yet another embodiment: (a)R 2 or R 3 However, the step of mixing a compound of structure (III) or (IV), which is a linker containing Q or a covalent bond to Q, with the molecule to be analyzed, (b) The step of forming a conjugate of the compound and the molecule to be analyzed, (c) The conjugate is given the formula ML 1b The step of reacting with the compound of -G', thereby forming at least one covalent bond through the reaction of G and G' (R 2 , R 3 , Q, G and ML 1b -G' is as defined herein. A method for labeling analyte molecules, including the above, is provided.

[0010] In some different embodiments, an alternative method for labeling the molecule to be analyzed is as follows: (a)R 2 or R 3 However, a compound of structure (III) or (IV) that is a linker containing Q, or a covalent bond to Q, is defined as formula ML 1b The steps include mixing with the compound of -G', thereby forming at least one covalent bond through the reaction of G and G', and (b) The product of step (A) is reacted with the analyte molecule to obtain a conjugate (R) of the product of step (A) and the analyte molecule. 2 , R 3 , Q, G and ML 1b -G' is as defined herein) step A method including this is provided.

[0011] In more different embodiments, a method for preparing a compound of structure (I), wherein the compound of structure (III) is of formula ML 1b - Mix with the compound G', thereby forming at least one covalent bond through the reaction of G and G' (G and ML 1b A method is provided which includes -G' as defined herein. In more different embodiments, a method for preparing a compound of structure (II), wherein the compound of structure (IV) is of formula ML 1b - Mix with the compound G', thereby forming at least one covalent bond through the reaction of G and G' (G and ML 1bA method is provided which includes -G' as defined herein.

[0012] These and other aspects of the Disclosure will become apparent from the following detailed description. In the drawings, the same reference number is used to identify similar elements. The size and relative position of elements in the drawings are not necessarily drawn to scale, and some of these elements are arbitrarily enlarged and positioned to improve the readability of the drawing. Furthermore, the specific shapes of the elements shown are not intended to convey any information about the actual shape of those specific elements, but are simply selected for ease of recognition in the drawing. [Brief explanation of the drawing]

[0013] [Figure 1] This figure shows the staining indices for representative compounds with various M moieties compared to a control compound. [Figure 2] This is a schematic diagram showing the increase in the time-dependent sequencing effectiveness of representative compounds in NaCl and KCl solutions. [Figure 3] This figure shows in vitro analyses of the target compound construct and a representative compound construct of structure (I). [Figure 4] This figure shows the effect of the buffer on the brightness of the control compound construct and the construct of a representative compound of structure (I). [Modes for carrying out the invention]

[0014] In the following description, certain specific details are provided to give a full understanding of the various embodiments of this disclosure. However, those skilled in the art will understand that this disclosure can be made without these details. Unless the context requires a different interpretation, throughout this specification and the claims, the word “comprise,” and its variations such as “comprises” and “comprising,” should be interpreted in an open, inclusive sense, that is, “including, but not limited to, the following.” Throughout this specification, when we refer to "one embodiment" or "a particular embodiment," we mean that any specific feature, structure, or characteristic described in relation to those embodiments is included in at least one embodiment of this disclosure. Therefore, where the phrase "in one embodiment" or "in a particular embodiment" appears in various places throughout this specification, it does not necessarily refer to the same embodiment. Furthermore, any specific feature, structure, or characteristic may be combined in any preferred manner in one or more embodiments.

[0015] "Amino" refers to the -NH2 group. "Carboxylate" refers to the -CO2H group. "Cyano" refers to the -CN group. "Formyl" refers to the -C(=O)H group. "Hydroxy" or "hydroxyl" refers to the -OH group. "Imino" refers to the NH group. "Nitro" refers to the -NO2 group. "Oxo" refers to an =O substituent. "Sulfhydryl" refers to the -SH group. "Thioxo" refers to the sulfur group. "Alkyl" refers to a group that does not contain unsaturated carbon atoms (C1-C1). 12This refers to a group of a linear or branched hydrocarbon chain consisting only of carbon and hydrogen atoms, having 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl), and being bonded to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise specifically stated herein, alkyl groups may be substituted. "Alkylene" or "alkylene chain" refers to a divalent linear or branched hydrocarbon chain consisting only of carbon and hydrogen, having 1 to 12 carbon atoms and not containing unsaturated atoms, with the rest of the molecule linked 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 single bonds and to the radical group via single bonds. The bonding points of the alkylene chain to the rest of the molecule and the radical group can be via one carbon or any two carbons within the chain. Unless otherwise specifically stated herein, alkylenes may be substituted. "Alkenylene" or "alkenylene chain" refers to a divalent linear or branched hydrocarbon chain consisting only of carbon and hydrogen, having 2 to 12 carbon atoms and containing at least one carbon-carbon double bond, with the remainder of the molecule linked to a radical group, such as etenylene, propenylene, n-butenylene, etc. The alkenylene chain is bonded to the remainder of the molecule via single bonds and to the radical group via double or single bonds. The bonding points of the alkenylene chain to the remainder of the molecule and the radical group can be via one carbon or any two carbons within the chain. Unless otherwise specifically stated herein, alkenylenes may be substituted. "Alkynylene" or "alkynylene chain" refers to a divalent straight-chain or branched hydrocarbon chain consisting only of carbon and hydrogen, having at least one carbon-carbon triple bond, having 2 to 12 carbon atoms, and connecting the rest of the molecule to a radical group, for example, ethenylene, propenylene, n-butenylene, etc. The alkynylene chain is bonded to the rest of the molecule through a single bond and to the radical group through a double bond or a single bond. The bonding points of the alkynylene chain to the rest of the molecule and 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 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 end (like an alkoxy group) or the carbon-oxygen bond may be present internally (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 specified otherwise in this specification, an alkyl ether group may be substituted. For example, in some embodiments, an 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 compounds of structure (I) respectively. "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 specified otherwise in this specification, 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 R bis an alkyl ether group as defined herein. Unless otherwise specifically stated herein, the alkoxyalkyl ether group may be substituted, for example, an alcohol or OP(=R a )(R b )R c It is replaced by R a , R b and R c These are defined as they are for the compound of structure (I). "Hyperalkyl" refers to the alkyl group as defined above, which contains at least one heteroatom (e.g., N, O, P, or S) within or at the terminus of the alkyl group. In some embodiments, the heteroatom is located within the alkyl group (i.e., the heteroalkyl group contains at least one carbon-[heteroatom] x -Includes a carbon bond, where x is 1, 2, or 3). In other embodiments, the heteroatom is located at the end of the alkyl group and therefore acts to bond the alkyl group to the rest of the molecule (e.g., M1-HA), where M1 is part of the molecule, H is the heteroatom, and A is the alkyl group. Unless otherwise specifically stated herein, the heteroalkyl groups may be substituted. Exemplary heteroalkyl groups include ethylene oxides (e.g., polyethylene oxide), which may include a phosphorus-oxygen bond such as a phosphodiester bond. "Heteroalkoxy" refers to a compound of the formula -OR a It refers to the base of R a This is a heteroalkyl group as defined above, containing 1 to 12 carbon atoms. Unless otherwise specifically stated herein, the heteroalkoxy group may be substituted. A "heteroalkylene" refers to an alkylene group as defined above, which contains at least one heteroatom (e.g., N, O, P, or S) within or at the end of an alkylene chain. In some embodiments, the heteroatom is located within the alkylene chain (i.e., the heteroalkylene contains at least one carbon-[heteroatom]-carbon bond, where x is 1, 2, or 3). In other embodiments, the heteroatom is located at the end of the alkylene and thus acts to bond this alkylene to the rest of the molecule (e.g., M1-HA-M2, where M1 and M2 are parts of the molecule, H is the heteroatom, and A is the alkylene). Unless otherwise specifically stated herein, heteroalkylene groups may be substituted. Exemplary heteroalkylene groups include ethylene oxide (e.g., polyethylene oxide), and the linking groups exemplified below, namely "C", "HEG", "TEG", "PEG 1K", and their variants.

[0016] [ka] The above-mentioned C-linker, HEG linker, and / or PEG 1K linker polymers are included in various embodiments of the heteroalkylene linker.

[0017] In some embodiments of the PEG 1K linker, n is 25. The polymer has, for example, the following structure: [ka] (wherein x is an integer greater than 0, for example, x may be in the range of 0 to 100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)). A "heteroalkenylene" is a heteroalkylene as defined above, containing at least one carbon-carbon double bond. Unless otherwise specifically stated herein, the heteroalkenylene group may be substituted. A "heteroalkylene" is a heteroalkylene containing at least one carbon-carbon triple bond. Unless otherwise specifically stated herein, the heteroalkylene group may be substituted. In relation to "heteroatom linkers," "heteroatom" refers to a linker group consisting of one or more heteroatoms. Examples of heteroatom linkers include single atoms selected from the group consisting of O, N, P, and S, as well as multiple heteroatoms, for example, formula -P(O - )(=O)O- or OP(O - This includes linkers and polymers having )(=O)O-, as well as combinations thereof. "phosphate" is -OP(=O)(R a )R b Group (R a OH, O - OR c And R b OH, O - , OR c (is), a thiophosphate group, or a further phosphate group (R c This refers to the counterion (for example, Na+). "Phosphoalkyl" refers to -OP(=O)(R a )R b It refers to the base, R a OH, O - OR c And R b It is an -O alkyl group, and R c is a counterion (for example, Na + (etc.). Unless otherwise specifically stated herein, phosphoalkyl groups may be substituted. For example, in certain embodiments, the -O alkyl portion of the phosphoalkyl group may be hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether or OP(=R a )(R b )R c It may be replaced by one or more of the following: R a , R b and R cThese are defined as they are for the compound of structure (I). "Phosphoalkyl ether" refers to -OP(=O)(R a )R b It refers to the base, R a OH, O - OR c And R b is an -O alkyl ether, and R c is a counterion (e.g., Na+). Unless otherwise specifically stated herein, 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 It may be replaced by one or more of the following: R a , R b and R c These are defined as they are for the compound of structure (I). What is "thiophosphate"? 、 -OP(=R a )(R b )R c It refers to the base, R a is O or S, R b OH, O - S - , OR d or SR d And R c OH, SH, O - S - , OR d , SR d , a phosphate group, or a further thiophosphate group, R d is a counterion (e.g., Na+), however, i)R a ii) R b is S - Or SR d iii)R c SH, S- Or SR d This is subject to the condition that it is either i) i), ii) and / or iii). "Thiophosphoalkyl" refers to -OP(=R a )(R b )R c It refers to the base, R a is O or S, R b OH, O - S - , OR d or SR d And R c It is an -O alkyl group, and R d is a counterion (e.g., Na+), however, i)R a ii) R b is, S - Or SR d is, or iii) R a S is R b is, S - Or SR d This is subject to the condition that the thiophosphoalkyl group is substituted unless otherwise specifically stated herein. For example, in certain embodiments, the -O alkyl portion of the thiophosphoalkyl group may be hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether, or OP(=R a )(R b )R c It may be replaced by one or more of the following: R a , R b and R c These are defined as they are for the compound of structure (I). "Thiophosphoalkyl ether" refers to -OP(=R a )(R b )R c It refers to the base, R a is O or S, R b OH, O - S - , OR d or SR dAnd R c is an -O alkyl ether, and R d is a counterion (e.g., Na+), however, i)R a ii) R b is, S - Or SR d is, or iii) R a S is R b is, S - Or SR d This is subject to the condition that the thiophosphoalkyl ether group is substituted unless otherwise specifically stated herein. For example, in certain embodiments, the -O alkyl ether portion of the thiophosphoalkyl group may be hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether, or OP(=R a )(R b )R c It may be replaced by one or more of the following: R a , R b and R c These are defined as they are for the compound of structure (I). A "carbocyclic" refers to a stable 3-18 member aromatic or non-aromatic ring containing 3-18 carbon atoms. Unless otherwise specifically stated herein, carbocyclic rings can be monocyclic, dicyclic, tricyclic, or tetracyclic ring systems, which may include condensed or bridging ring systems and may be partially or fully saturated. Non-aromatic carbocyclyl radicals include cycloalkyl groups, while aromatic carbocyclyl radicals include aryl groups. Unless otherwise specifically stated herein, carbocyclic groups may be substituted. "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic carbocyclic ring, which may include saturated or unsaturated condensed or crosslinked ring systems having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, and being bonded to the rest of the molecule by single bonds. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyls include adamantyl, norbornyl, dekalinyl, and 7,7-dimethyl-bicyclo-[2.2.1]heptanyl. Unless otherwise specifically stated herein, cycloalkyls may be substituted. "Aryl" refers to a ring system containing at least one carbocyclic aromatic ring. In some embodiments, the aryl contains 6 to 18 carbon atoms. The aryl ring may be monocyclic, bicyclic, tricyclic, or tetracyclic, and these may include condensed or bridging ring systems. Aryls include, but are not limited to, aryls derived from acetantrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluorantene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless otherwise specifically stated herein, the aryl group may be substituted.

[0018] A "heterocyclic" refers to a stable 3-18 member aromatic or non-aromatic ring containing 1-12 carbon atoms and 1-6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless otherwise specifically stated herein, heterocyclic rings may be monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, which may include fused or bridging ring systems, and the nitrogen, carbon, or sulfur atoms in the heterocyclic ring may be oxidized. The nitrogen atom may be quaternized. Heterocyclic rings may be partially or completely saturated. Examples of aromatic heterocyclic rings are listed below in the definition of heteroaryls (i.e., heteroaryls are a subset of heterocyclic rings). Examples of non-aromatic heterocyclic rings include, but are not limited to, dioxolanil, thienyl[1,3]dithianil, decahydroisoquinolyl, imidazolinil, imidazolidinil, isothiazolidinil, isoxazolidinil, morpholinil, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinil, 2-oxopiperidinil, 2-oxopyrrolidinil, oxazolidinil, piperidinil, piperazinil, 4-piperidonyl, pyrrolidinil, pyrazolidinil, pyrazolopyrimidinil, quinuclidinil, thiazolidinil, tetrahydrofuryl, trioxanil, trithianil, triazinanil, tetrahydropyranil, thiomorpholinil, thiamorpholinil, 1-oxo-thiomorpholinil, and 1,1-dioxo-thiomorpholinil. Unless otherwise specifically stated herein, heterocyclic groups may be substituted. "Heteroaryl" refers to a 5-14 membered ring system comprising 1-6 heteroatoms selected from the group consisting of 1-13 carbon atoms, nitrogen, oxygen, and sulfur, and at least one aromatic ring. For the purposes of certain embodiments of this disclosure, the heteroaryl radical may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include condensed or bridging ring systems, and the nitrogen, carbon, or sulfur atoms in the heteroaryl radical may be oxidized, and the nitrogen atom may be quaternized.Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzoindolyl, benzodioxolyl, benzofuranil, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanil, benzonaphthofuranil, benzoxazolyl, benzodioxolyl, benzodioxynil, benzopyranil, benzopyranonil, benzofuranil, benzof Lanonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, benzoxazolinonyl, benzimidazolethionyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indazolyl, indazolyl, isoindolyl, indolinyl, isoindolyl, isoquinolyl, indolidinyl, isoox Sazolyl, naphthilidinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxyranil, 1-oxidepyridinyl, 1-oxidepyrimidinyl, 1-oxidepyradinyl, 1-oxidepyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxadinyl, phthalazinyl, pteridinyl, pteridinonyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyridinonyl, pyridinyl, pyrimidinyl, pyrimidinyl This includes (pryrimidinonyl), pyridadinyl, pyrrolyl, pyrido[2,3-d]pyrimidinonyl, quinazolinyl, quinazolinonyl, quinoxalinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, thieno[3,2-d]pyrimidine-4-onyl, thieno[2,3-d]pyrimidine-4-onyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless otherwise specifically stated herein, heteroaryl groups may be substituted.

[0019] The suffix "-ene" refers to a specific structural feature (e.g., alkyl, aryl, heteroalkyl, heteroaryl) that is bonded to the rest 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 that possesses the structural feature of the part to which it is bonded. The bond points of the "-ene" chain to the rest of the molecule and to the radical group can be via one atom of any two atoms in the chain, or any two atoms. For example, heteroarylene refers to a linker containing a heteroaryl moiety as defined herein. "Condensed" refers to a ring system containing at least two rings, where the two rings share at least one common ring atom, for example, two common ring atoms. If the fused rings are heterocyclyl or heteroaryl rings, the common ring atom can be carbon or nitrogen. Condensed rings include bicyclic, tricyclic, and tetracyclic rings.

[0020] As used herein, the term "substituted" means any of the above groups (e.g., alkyl, alkylene, alkenylene, alkylylene, heteroalkylene, heteroalkenylene, heteroalkylynylene, alkoxy, alkyl ether, alkoxyalkyl ether, heteroalkyl, heteroalkoxy, phosphoalkyl, phosphoalkyl ether, thiophosphoalkyl, thiophosphoalkyl ether, carbocyclic, cycloalkyl, aryl, heterocyclic and / or heteroaryl) in which at least one hydrogen atom (e.g., one, two, three or all hydrogen atoms) is replaced by F, Cl, etc. Halogen atoms such as B and I; oxygen atoms in groups such as hydroxyl, alkoxy, and ester groups; sulfur atoms in groups such as thiol, thioalkyl, sulfone, sulfonyl, and sulfoxide groups; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; silicon atoms in groups such as trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, and triarylsilyl groups; and non-hydrogen atoms such as other heteroatoms in various other groups are replaced by bonds to these groups. "Substituted" also means any of the above groups in which one or more hydrogen atoms are replaced by higher-order bonds (e.g., double or triple bonds) to heteroatoms such as oxygen in oxo, carbonyl, carboxyl, and ester groups, and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, "substituted" means that one or more hydrogen atoms are replaced by bonds to -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 -SORg , -SO2R g , -OSO2R g , -SO2OR g ,=NSO2R g and SO2NR g R h It includes any of the above groups which are replaced by -C(=O)R. "Replaced" also means that one or more hydrogen atoms are replaced by -C(=O)R g , -C(=O)OR g -C(=O)NR g R h -CH2SO2R g -CH2SO2NR g R h This means any of the above bases that are replaced by R. g and R h These are identical or different, and independently, hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl. "Substituted" further means any of the above groups in which one or more hydrogen atoms are replaced by bonding to 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. In some embodiments, an optional substituent may be -OP(=R a )(R b )R c And R a , R b and R c These are defined as they are for the compound of structure (I). Furthermore, any of the above substituents may also be substituted with one or more of the above substituents.

[0021] An "electron-withdrawing group" refers to a functional group that, when occupied in the same position in a molecule, attracts electrons to itself rather than to a hydrogen atom. These terms are well understood by those skilled in the art and are discussed in Advanced Organic Chemistry, by J. March, John Wiley & Sons, New York, NY, pp. 16-18 (1985), the discussion in that document is incorporated herein by reference. Examples of electron-withdrawing groups include, but are not limited to, halos, halos (e.g., F, Cl, Br, I), -NO2, -CN, -SO3H, -SO2R a , -SO3R a -COOH, -COR a ,-COOR a -CONHR a , -CON(R a )2, containing haloalkyl groups and 5-14 member electron-deficient heteroaryl groups, R a This is an alkyl group, an alkenyl group, or an alkynyl group.

[0022] "Conjugation" refers to the overlap between one p-orbital and another in the entire intervening sigma bond. Conjugation can occur in cyclic or acyclic compounds. "Degree of conjugation" refers to the overlap between at least one p-orbital and another in the entire intervening sigma bond. For example, 1,3-butadiene has a degree of conjugation of 1, while benzene and other aromatic compounds typically have multiple degrees of conjugation. Fluorescent and chromogenic compounds typically contain at least a degree of conjugation of 1. "Fluorescence" refers to molecules that can absorb light of a specific frequency and emit light of a different frequency. Fluorescence is well known to those skilled in the art. "Coloration" refers to molecules that absorb light within the colored spectrum (i.e., red, yellow, blue, etc.).

[0023] "FRET" refers to Forster resonance energy transfer, a physical interaction resulting from the transfer of energy from the excitation of one part (e.g., the first chromophore or "donor") to an adjacent part (e.g., the second chromophore or "acceptor"). "FRET" is sometimes used interchangeably with fluorescence resonance energy transfer (i.e., each chromophore is a fluorescent part). Generally, FRET requires that (1) the excitation or absorption spectrum of the acceptor chromophore overlaps with the emission spectrum of the donor chromophore, (2) the transition dipole moments of the acceptor and donor chromophore are substantially parallel (i.e., about 0° or 180°), and (3) the acceptor and donor chromophore share near-field (i.e., are close to each other). Energy transfer from the donor to the acceptor occurs via non-radiative dipole-dipole coupling, and the distance between the donor and acceptor chromophores is generally considerably shorter than the wavelength of light.

[0024] A "donor" or "donor chromophore" refers to a chromophore (e.g., a fluorophore) that is induced to, or can be induced to, an excited electronic state, and can non-radiatively transfer its excitation or absorption energy to a nearby acceptor chromophore via long-range dipole-dipole interactions. While we do not wish to be constrained by theory, it is thought that energy transfer occurs because the vibrational dipoles of individual chromophores have similar resonance frequencies. These donors and acceptors with 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. An "acceptor" or "acceptor chromophore" refers to a chromophore (e.g., a fluorophore), to which excitation or absorption energy from a donor chromophore is transferred to this chromophore via non-radiative transport through long-range dipole-dipole interactions.

[0025] "Stoke shift" refers to the difference between the position (e.g., wavelength) of the maximum excitation or absorbance band 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 stoke shift greater than 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm.

[0026] The "J value" is calculated as an integer value of the spectral overlap between the emission spectrum of the donor chromophore and the excitation or absorption spectrum of the acceptor chromophore. The emission spectrum of the donor chromophore is generated when the donor chromophore is excited at a preferred excitation or absorption wavelength. The preferred excitation or absorption wavelength of the donor chromophore is at or near its individual excitation or absorption maximum, which are well known to those skilled in the art (for example, Pacific blue has an excitation or absorption maximum at approximately 401 nm, and FITC has an excitation or absorption maximum at approximately 495 nm). A "linker" refers to a continuous chain of at least one atom, such as carbon, oxygen, nitrogen, sulfur, phosphorus, or combinations thereof, that links one part of a molecule to another part of the same molecule, or to a different molecule, part, or solid support (e.g., microparticles). Linkers can be linked to molecules via covalent bonds or other means, such as ionic or hydrogen bonding interactions.

[0027] The term “biomolecule” refers to any of the various biomolecules, including nucleic acids, carbohydrates, amino acids, polypeptides, glycoproteins, hormones, aptamers, and mixtures thereof. More specifically, the term is intended to include, non-limitingly, 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 this disclosure (e.g., compounds of structure (I) having biomolecules linked to the compound of structure (I)) are prepared, as further described herein, by contacting the biomolecule with a compound having a reactive group that allows the biomolecule to be linked to the compound via any available atom or functional group on the biomolecule, such as an amino, hydroxy, carboxyl, or sulfhydryl group. 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 substitution, oxidation, reduction, addition, or cycloaddition reactions. Exemplary reactive groups are shown in Table 1 and include, for example, nucleophiles, electrophiles, dienes, dienephiles, aldehydes, oximes, hydrazones, alkynes, amines, azides, acyl azides, acyl halides, nitriles, nitrones, sulfhydryls, disulfides, sulfonyl halides, isothiocyanates, imide esters, activated esters, ketones, α,β-unsaturated carbonyls, alkenes, maleimides, α-haloimides, epoxides, aziridines, tetrazine, tetrazole, phosphine, biotin, thiirane, and the like.

[0028] The terms “visible to the naked eye” and “detectable by visual inspection” 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 in the range of about 300 to about 900 nm. Preferably, such substances exhibit strong color, preferably at least about 40,000, more preferably at least about 50,000 M, even more preferably at least about 60,000, even more preferably at least about 70,000 and most preferably at least about 80,000 M. -1 cm -1 It has a molar extinction coefficient of . The compounds of this disclosure can be detected by naked-eye observation or, not limited to, by optical detection devices including absorption spectrophotometers, transmission optical microscopes, digital cameras and scanners. Substances detectable by sight 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 (about 10 nm to about 400 nm), the infrared (IR) region (about 700 nm to about 1 mm), and other regions of the electromagnetic spectrum are also included in the range of substances that are “detectable by sight.”

[0029] For the purposes of the embodiments of this disclosure, the term “photostable visible dye” means a chemical portion that is visually detectable and does not significantly alter or decompose upon exposure to light, as defined herein. Preferably, the photostable visible dye does not show significant decolorization or decomposition after at least one 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 one week, and most preferably at least one month of exposure to light. Non-limiting examples of photostable visible dyes suitable for use in the compounds and methods of this disclosure include azo dyes, thioindigo dyes, quinacridone pigments, dioxazines, phthalocyanines, perinones, diketopyrrolopyrroles, quinophthalones, and triarylcarbonium.

[0030] As used herein, the term “perylene derivative” is intended to include any substituted perylene that is visually detectable. However, the 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., perylene, pyrene, anthracene, or naphthalene derivative) is an imide, bisimide, or hydrazamimide derivative of perylene, anthracene, naphthalene, or pyrene.

[0031] The visually detectable molecules of the various embodiments of this disclosure are useful for a wide range of analytical applications, such as biochemical and biomedical applications, where it is necessary to determine the presence, location, or quantity of a specific analyte (e.g., a biomolecule). Accordingly, in another aspect, this disclosure provides a method for visually detecting a biomolecule, comprising the steps of (a) providing a visually detectable biomolecule to a biological system, comprising a compound of structure (I) linked to the biomolecule, and (b) detecting the biomolecule by its visible properties. For the purposes of this disclosure, the phrase "detecting a biomolecule by its visible properties" means that the biomolecule is observed, without irradiation or chemical or enzymatic activation, by the naked eye or not, using an optically-based detection device, including absorption spectrophotometers, transmission optical microscopes, digital cameras, and scanners. The amount of visually detectable biomolecule present can be quantified using a densimeter. For example, the relative amounts of biomolecules in two samples can be determined by measuring the relative optical density. If the stoichiometry of a pigment molecule per biomolecule is known and the extinction coefficient of the pigment molecule is known, the absolute concentration of the biomolecule can also be determined from the optical density measurement. As used herein, the term “biological system” is used to refer to any solution or mixture containing one or more biomolecules in addition to biomolecules detectable by visual inspection. Non-limiting examples of such biological systems include cells, cell extracts, tissue samples, electrophoretic gels, assay mixtures, and hybridization reaction mixtures.

[0032] "Solid support" means any solid substrate known in the art for supporting molecules in a solid phase, and "microparticles" means any of several small particles useful for binding to the compounds of this disclosure, including, but not limited to, glass beads, magnetic beads, polymer beads, and non-polymer beads. In certain embodiments, the microparticles include polystyrene beads. A "solid support residue" refers to a functional group that maintains its binding state to a molecule when the molecule is cleaved from a solid support. Solid support residues are well known in this field and can be easily derived based on the structure of the solid support and the groups linked to its molecule.

[0033] A “target-directed moiety” is a portion 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 linkage to a target-directed moiety to selectively bind or associate with the target analyte (i.e., the target of the target-directed moiety), thus enabling the 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, and the like. In some embodiments, the target-directed moiety is a portion, such as an antibody, that selectively binds to or associates with a target feature on the cell surface or within a cell, for example, a target feature on the cell membrane or the structural surface of another cell, thus enabling the detection of the target cell. Small molecules that selectively bind to or associate with a desired analyte are also intended to be target-directed moieties in certain embodiments. Those skilled in the art will understand other analytes and corresponding target-directed moieties that are useful in various embodiments. A "base pair moiety" refers to a heterocyclic moiety that can hybridize with a complementary heterocyclic moiety via hydrogen bonding (e.g., Watson-Crick base pairs). Base pair moieties include both native and non-native bases. Non-limited examples of base pair moieties include RNA and DNA bases such as adenosine, guanosine, thymidine, cytosine, and uridine, and their analogs. The embodiments of the disclosure disclosed herein are also intended to encompass all compounds of structure (I) or (II) that are isotope-labeled by replacing one or more atoms with atoms having different atomic masses or mass numbers. Examples of isotopes that may be incorporated into the disclosed compounds include, 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 It contains isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as I. Isotope-labeled compounds of structure (I) or (II) can generally be prepared by conventional techniques known to those skilled in the art, or by using appropriate isotope-labeling reagents instead of previously used unlabeled reagents, in a manner similar to that described below and in the following examples. "Stable compounds" and "stable structures" are intended to represent compounds that are robust enough to survive isolation from the reaction mixture to a useful degree of purity and formulation into effective therapeutic agents.

[0034] The phrases "optional" or "optionally" indicate that the event or situation described thereafter may or may not occur, and that this statement includes both the cases in which such event or situation occurs and the cases in which it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and that this statement includes both substituted alkyl groups and unsubstituted alkyl groups. The term "salt" includes both acid addition salts and base addition salts. "Acid addition salts" include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, and, but are not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, sodium acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfate, 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, and gluconic acid. This refers to salts formed with organic acids such as taric acid, 2-oxo-glutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucoic 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, and undecylenic acid.

[0035] A "base addition salt" refers to a salt prepared by adding an inorganic or 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, and aluminum. Salts derived from organic bases include, but are not limited to, salts of ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydravamin, choline, betaine, benetamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and other salts of primary, secondary, and tertiary amines and substituted amines, including naturally substituted amines, cyclic amines, and basic ion exchange resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0036] Crystallization may result in solvates of the compounds described herein. Embodiments of this disclosure include all solvates of the compounds described herein. As used herein, the term “solvate” means an aggregate containing one or more molecules of the compounds of this disclosure and one or more molecules of 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 this disclosure may exist as hydrates, including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, and the corresponding solvate forms. While the compounds of this disclosure may be true solvates, in other cases the compounds of this disclosure may simply hold incidental water or another solvent, or may be a mixture of water and some incidental solvent.

[0037] Embodiments of the compounds of this disclosure (e.g., compounds of structure I or II), or their salts, tautomers, or solvates, may contain one or more chiral centers, and thus enantiomers, diastereomers, and other stereoisomers may arise, which can be defined with respect to absolute stereochemistry as (R)- or (S)-, or in the case of amino acids, (D)- or (L)-. Embodiments of this disclosure are intended to include all such possible isomers, as well as their racemates and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or they can be resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). Where a compound described herein contains an olefinic double bond or another center of geometric chirality, and unless otherwise specified, the compound is intended to include both E and Z geometric isomers. Similarly, all tautomers are also intended to be included.

[0038] A "stereoisomer" refers to a compound that is composed of the same atoms bonded together by the same bonds, but has different three-dimensional structures that cannot be interconverted. This disclosure envisions various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers whose molecules are mirror images of each other and do not overlap. "Tautomerism" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. This disclosure includes tautomers of any of the aforementioned compounds. Various tautomers of the compounds can be readily derived by those skilled in the art. The chemical nomenclature protocols and structural diagrams used herein are modified versions of the IUPAC nomenclature system using the ACD / Nomenclature version 9.07 software program and / or the ChemDraw Ultra version 11.0 software nomenclature program (CambridgeSoft). Common names familiar to those skilled in the art are also used.

[0039] As described above, one embodiment of this disclosure provides a compound useful as a fluorescent and / or chromogenic dye in various analytical methods. Another embodiment provides a compound useful as a synthetic intermediate for preparing a compound useful as a fluorescent and / or chromogenic dye. In general terms, the embodiments of this disclosure concern polymers of dimers or more of fluorescent and / or chromogenic moieties. The fluorescent and / or chromogenic moieties are linked by linkers. While we do not wish to be bound by theory, it is thought that the linkers help maintain sufficient spatial distance between the fluorescent and / or chromogenic moieties, thus reducing or eliminating intramolecular quenching, and thus resulting in a dye compound having high molar "luminance" (e.g., high fluorescence emission).

[0040] Therefore, in some embodiments, the compounds of the present disclosure have the following structure (A): [ka] (A) or its stereoisomer, salt, or tautomer (in the formula, M 1 and M 2 Each instance is an independent chromophore, however M 1 and M 2 At least one of them is a FRET donor, M 1 and M 2 The other one is a corresponding FRET acceptor, L 1a Each instance is independently a heteroarylene linker. L 1 , L 1b , L 2 and L 3Each instance may be used independently and arbitrarily as an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, heteroarylene, or heteroatom linker. L 4 and L 5 Each instance is independently alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker. L 6 Each instance is independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker, provided that the L that appears 6 At least one of them is L 4 and L 5 Each of these is subject to the condition that they are different from each of the following: R 1 Each instance is independently H, alkyl, or alkoxy. R 2 and R 3 These are, independently, H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, and -OP(=R a )(R b )R c , Q or its protective form or L', R 4 Each instance of these is independently represented as OH, SH, O - S - , OR d or SR d And, R 5 Each instance is independently either oxo, thioxo, or absent. R a is either O or S, R b OH, SH, O - S - , OR d or SR d And, R c OH, SH, O - S - , OR d ,OL',SR dAlkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether, R d It is a counterion, Q is a moiety containing a reactive group or its protected form, which is capable of independently forming a covalent bond with the analyte molecule, target-directing moiety, solid support, or complementary reactive group Q' each time it appears. L' is, independently with each occurrence, a linker containing a covalent bond to Q, a linker containing a covalent bond to a target-directing moiety, a linker containing a covalent bond to the 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 (A). Each instance of m is an independent integer greater than or equal to zero, provided that at least one instance of m is an integer greater than or equal to 1. n is an integer greater than or equal to 1, q and w are independent integers between 0 and 3 in each occurrence, provided that at least one of either q or w is 1. It holds.

[0041] In some embodiments, w is 0. In some relevant embodiments, the compounds of this disclosure have the following structure (I): [ka] (I) or its stereoisomer, salt, or tautomer (in the formula, M 1 and M 2 Each instance independently comprises a portion containing two or more carbon-carbon double bonds and at least one degree of conjugation. L 1 , L 2 and L 3Each instance may be used independently and arbitrarily as an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, heteroarylene, or heteroatom linker. L 4 and L 5 Each instance is independently alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene. L 6 Each instance is independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker, provided that the L that appears 6 At least one of them is L 4 and L 5 Each of these is subject to the condition that they are different from each of the following: R 1 Each instance is independently H, alkyl, or alkoxy. R 2 and R 3 These are, independently, H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, and -OP(=R a )(R b )R c , Q or its protective form or L', R 4 Each instance of these is independently represented as OH, SH, O - S - , OR d or SR d And, R 5 Each instance is independently either oxo, thioxo, or absent. R a is either O or S, R b OH, SH, O - S - , OR d or SR d And, R c OH, SH, O - S - , OR d ,OL',SR dAlkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether, R d It is a counterion, Q is a moiety containing a reactive group or its protected form, which is capable of independently forming a covalent bond with the analyte molecule, target-directing moiety, solid support, or complementary reactive group Q' each time it appears. L' is, independently of its appearance, a linker containing a covalent bond to Q, a linker containing a covalent bond to a target-directing moiety, a linker containing a covalent bond to the 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). Each instance of m is an independent integer greater than or equal to zero, provided that at least one instance of m is an integer greater than or equal to 1. n is an integer greater than or equal to 1. It holds.

[0042] In some relevant embodiments, the compounds of this disclosure have the following structure (I): [ka] (I) or its stereoisomer, salt, or tautomer (in the formula, M 1 and M 2 Each instance is an independent chromophore, however M 1 and M 2 At least one of them is a FRET donor, M 1 and M 2 The other one is a corresponding FRET acceptor, L 1 , L 2 and L 3Each instance may be used independently and arbitrarily as an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, heteroarylene, or heteroatom linker. L 4 and L 5 Each instance is independently alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene. L 6 Each instance is independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker, provided that the L that appears 6 At least one of them is L 4 and L 5 Each of these is subject to the condition that they are different from each of the following: R 1 Each instance is independently H, alkyl, or alkoxy. R 2 and R 3 These are, independently, H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, and -OP(=R a )(R b )R c , Q or its protective form or L', R 4 Each instance of these is independently represented as OH, SH, O - S - , OR d or SR d And, R 5 Each instance is independently either oxo, thioxo, or absent. R a is either O or S, R b OH, SH, O - S - , OR d or SR d And, R c OH, SH, O - S - , OR d ,OL',SR dAlkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether, R d It is a counterion, Q is a moiety containing a reactive group or its protected form, which is capable of independently forming a covalent bond with the analyte molecule, target-directing moiety, solid support, or complementary reactive group Q' each time it appears. L' is, independently of its appearance, a linker containing a covalent bond to Q, a linker containing a covalent bond to a target-directing moiety, a linker containing a covalent bond to the 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). Each instance of m is an independent integer greater than or equal to zero, provided that at least one instance of m is an integer greater than or equal to 1. n is an integer greater than or equal to 1. It holds.

[0043] Various linkers and substituents (e.g., M, Q, R) in the compound of structure (I) 1 , R 2 , R 3 , R 4 , R 5 , R c , L 1 , L 2 , L 3 , L 4 , L 5 and L 6) may be substituted with another substituent. For example, in some embodiments, the optionally used substituent is selected to optimize the water solubility or other properties of the compound of structure (I). In certain embodiments, each alkyl, alkoxy, alkyl ether, alkoxyalkyl ether, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether and thiophosphoalkyl ether in the compound of structure (I) may be substituted with one more 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 optionally used substituent is -OP(=Ra)(Rb)Rc, where Ra, Rb and Rc are as defined with respect to the compound of structure (I).

[0044] Linker L may be used at will 1The M moiety can be used as a bonding site for the M moiety to the rest of the compound. For example, in some embodiments, a synthetic precursor to the compound of structure (I) is prepared, and the M moiety is bonded to the synthetic precursor using any number of readily available methods known in the art, e.g., methods referred to as "click chemistry". For this purpose, M can be bonded to the synthetic precursor using any rapid and substantially irreversible reaction to form the compound of structure (I). Exemplary reactions include copper-catalyzed reactions forming triazoles of azides and alkynes (Huisgen's 1,3-dipolar cycloaddition), reactions of dienes with dienephiles (Diels-Alder), strain-promoted alkyne-nitrone cycloaddition, reactions of strained alkenes with azides, tetrazines or tetrazoles, [3+2] cycloaddition of alkenes and azides, reverse-demand Diels-Alder reactions of alkenes and tetrazines, photoreactions of alkenes and tetrazoles, and various substitution reactions such as substitution of leaving groups by nucleophilic attack on electrophilic atoms. Exemplary substitution reactions include reactions of amines with activated esters; N-hydroxysuccinimide esters; isocyanates; isothiocyanates, etc. In some embodiments, L 1 The reaction that forms the compound may be carried out in an aqueous environment.

[0045] Therefore, in some embodiments, L 1 Each occurrence is a linker containing a functional group that can be formed by the reaction of two complementary reactive groups, for example, a functional group that is one product of the "click" reaction described above. In various embodiments, the L that appears 1For at least one of these, the functional group may be formed by the reaction of an aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halogen, isothiocyanate, imide ester, activated ester (e.g., N-hydroxysuccinimide), 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 or isothiocyanate. In other embodiments, L appears 1 For at least one of them, the functional group can be formed by the reaction of an alkyne and an azide. In other embodiments, the L that appears 1 For at least one of these, the functional group may be formed by the reaction of an amine (e.g., a primary amine) with an N-hydroxysuccinimide ester or isothiocyanate.

[0046] In more embodiments, L appears 1 For at least one of the functional groups, the functional group includes an alkene, ester, amide, thioester, disulfide, carbocyclic group, heterocyclic group, or heteroaryl group. In more embodiments, the L that appears 1 For at least one of the functional groups, the functional group includes an alkene, ester, amide, thioester, thiourea, disulfide, carbocyclic group, heterocyclic group, or heteroaryl group. In other embodiments, the functional group includes an amide or thiourea. In some more specific embodiments, L appears. 1 For at least one of the following, L 1 This is a linker containing a triazolyl functional group. In other embodiments, the L that appears 1 For at least one of the following, L 1 It is a linker containing an amide or thiourea functional group. In yet another different embodiment of structure (I), L 1Each instance is independently an alkylene or heteroalkylene linker. In some embodiments, the L that appears 1 At least one of them is a heteroalkylene.

[0047] In some embodiments, the L that appears 1 At least one of them has the following structure: [ka] (In the formula, x 0 and y 0 Each of these elements is an independent integer greater than or equal to 1. In some embodiments, x 0 is 1, 2, 3, or 4. In certain embodiments, y 0 is 2, 3, 4, or 5. In some specific embodiments, x 0 is 1 or 2, and y 0 It is 2, 3, or 4.

[0048] In some other embodiments, L 1 It is one of the following structures: [ka] It holds.

[0049] In yet another embodiment, the L that appears 1 For at least one of the following, L 1 -M 1 or L 1 -M 2 This is one of the following structures: [ka] (In the formula, L 1a and L 1b Each of these is a linker that may be used independently and at will.

[0050] In various embodiments, L appears 1 For at least one of the following, L 1 -M1 or L 1 -M 2 This is one of the following structures: [ka] (In the formula, L 1a and L 1b Each of these is a linker that may be used independently and at will. In the various embodiments described above, L 1a Or L 1b , or neither of them exists. In other embodiments, L 1a Or L 1b , or both exist.

[0051] In some embodiments, L 1a and L 1b If present, each is independently an alkylene or a heteroalkylene. For example, in some embodiments, L 1a and L 1b If present, it is independently one of the following structures: [ka] It holds.

[0052] In some embodiments, structure (A) M 2 -L 1b The structure is as follows: [ka] It holds. In some embodiments, the L that appears 4 and L 5 At least one of them is the same. In some embodiments, L 4 and L 5 This is the same each time it appears.

[0053] In some embodiments, L 6 Each instance is independently a heteroalkylene linker. In other more specific embodiments, L 6Each instance is an alkylene oxide linker. In some embodiments, L 6 It is polyethylene oxide. In some related embodiments, the compound has the following structure (Ia): [ka] (Ia) (In the formula, L 4 and L 5 Each instance is independently alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker, provided that L 4 and L 5 This is provided that it is not polyethylene oxide. z is an integer between 1 and 100. It holds.

[0054] In some embodiments, z is an integer from 1 to 30, for example, from 15 to 30, or from 22 to 25. In some embodiments, z is 23. In some embodiments, z is 21, 22, 23, 24, or 25. In some embodiments, z is an integer from 1 to 10, for example, from 3 to 6. In some embodiments, z is 3, 4, 5, or 6. In some embodiments, L 4 and L 5 Each instance is independently an alkylene linker. In other more specific embodiments, L 4 and L 5 Each instance is independently a C3-C6 alkylene. In some embodiments, at least one instance is L 4 and L 5 In other embodiments, L 4 and L 5 This is the same each time it appears.

[0055] In some more specific embodiments, L 4 and L 5 Each instance is independently a C3-C6 alkylene. In some embodiments, L 4 and L5 Each instance is independently a C3 alkylene, a C4 alkylene, or a C6 alkylene. In some related embodiments, the compound has the following structure (Ib): [ka] (Ib) (In the formula, R 7 , R 8 , R 9 and R 10 Each instance is independently either H or alkyl. y 1 and y 2 (Each instance is an integer between 1 and 6, independently of the others.) It holds.

[0056] In some embodiments, L 2 and L 3 Each instance is independently a C1-C6 alkylene, a C2-C6 alkenylene, or a C2-C6 alkynylene. For example, in some embodiments, L 2 and L 3 Each instance is independently a C1-C6 alkylene, and R 7 , R 8 , R 9 and R 10 Each instance is independently H. In some related embodiments, the compound has the following structure (Ic):

[0057] [ka] (I C) (In the formula, x 1 , x 2 , x 3 and x 4 Each occurrence is an independent integer between 0 and 6. y 1 and y 2 Each occurrence is an independent integer between 1 and 6. z is an integer between 1 and 100. It holds.

[0058] In some embodiments, x 1 and x 3 Each occurrence is 0, and x 2 and x 4 Each occurrence is 1. In other embodiments, x 1 , x 2 , x 3 and x 4 Each instance of this value is 1. In some embodiments, y 1 and y 2 Each occurrence is 3. In other embodiments, y 1 and y 2 Each occurrence is 4. In yet another embodiment, y 1 and y 2 Each instance of this is 6.

[0059] In some embodiments, z is an integer between 1 and 6. In other embodiments, z is an integer between 15 and 30. In yet another embodiment, z is an integer between 22 and 25. In some embodiments of the above-described compounds of structure (I), the R that appears 1 At least one of them is H. In a more specific embodiment, R 1 Each instance of this is H. In some embodiments, q is 0. In some related embodiments, the compound of the present disclosure has the following structure (II): [ka] (II) or its stereoisomer, salt, or tautomer (in the formula, M 1 and M 2 Each instance independently comprises a portion containing two or more carbon-carbon double bonds and at least one degree of conjugation. L 1c Each instance is independently a heteroarylene linker. L 1d , L 2 and L 3Each instance may be used independently and arbitrarily as an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkylynylene linker. L 4 and L 5 Each instance is independently alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkylynylene linker. L 6 Each instance is independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker, provided that the L that appears 6 At least one of them is L 4 and L 5 Each of these is subject to the condition that they are different from each of the following: R 2 and R 3 These are, independently, H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, and -OP(=R a )(R b )R c , Q or its protective form or L', R 4 Each instance of these is independently represented as OH, SH, O - S - , OR d or SR d And, R 5 Each instance is independently either oxo, thioxo, or absent. R a is either O or S, R b OH, SH, O - S - , OR d or SR d And, R c OH, SH, O - S - , OR d ,OL',SR dAlkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether, R d It is a counterion, Q is a moiety containing a reactive group or its protected form, which is capable of independently forming a covalent bond with the analyte molecule, target-directing moiety, solid support, or complementary reactive group Q' each time it appears. L' is, independently of its appearance, a linker containing a covalent bond to Q, a linker containing a covalent bond to a target-directing moiety, a linker containing a covalent bond to the 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). Each instance of m is an independent integer greater than or equal to zero, provided that at least one instance of m is an integer greater than or equal to 1. n is an integer greater than or equal to 1. It holds.

[0060] In some embodiments, the compounds of this disclosure have the following structure (II): [ka] (II) or its stereoisomer, salt, or tautomer (in the formula, M 1 and M 2 Each instance is an independent chromophore, however M 1 and M 2 At least one of them is a FRET donor, M 1 and M 2 The other one is a corresponding FRET acceptor, L 1c Each instance is independently a heteroarylene linker. L 1d , L 2 and L3 Each instance may be used independently and arbitrarily as an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkylynylene linker. L 4 and L 5 Each instance is independently alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkylynylene linker. L 6 Each instance is independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker, provided that the L that appears 6 At least one of them is L 4 and L 5 Each of these is subject to the condition that they are different from each of the following: R 2 and R 3 These are, independently, H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, and -OP(=R a )(R b )R c , Q or its protective form or L', R 4 Each instance of these is independently represented as OH, SH, O - S - , OR d or SR d And, R 5 Each instance is independently either oxo, thioxo, or absent. R a is either O or S, R b OH, SH, O - S - , OR d or SR d And, R c OH, SH, O - S - , OR d ,OL',SR dAlkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether, R d It is a counterion, Q is a moiety containing a reactive group or its protected form, which is capable of independently forming a covalent bond with the analyte molecule, target-directing moiety, solid support, or complementary reactive group Q' each time it appears. L' is, independently of its appearance, a linker containing a covalent bond to Q, a linker containing a covalent bond to a target-directing moiety, a linker containing a covalent bond to the 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 (II). Each instance of m is an independent integer greater than or equal to zero, provided that at least one instance of m is an integer greater than or equal to 1. n is an integer greater than or equal to 1. It holds.

[0061] Various linkers and substituents (e.g., M, Q, R) in the compound of structure (II) 2 , R 3 , R c , L 1c , L 1d , L 2 , L 3 , L 4 , L 5 and L 6) may be substituted with another substituent. For example, in some embodiments, the optionally used substituent is selected to optimize the water solubility or other properties of the compound of structure (II). In certain embodiments, the chromophore, alkyl, alkoxy, alkyl ether, heteroarylene, heteroalkyl, alkylene, alkenylene, alkylylene, heteroalkylene, heteroalkenylene, heteroalkylynylene, alkoxyalkyl ether, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether and thiophosphoalkyl ether in the compound of structure (II) may each be substituted with one more 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 optionally used substituent is -OP(=Ra)(Rb)Rc, where Ra, Rb and Rc are as defined with respect to the compound of structure (II).

[0062] In some embodiments, the L that appears 1c At least one of them is a 5- to 7-member heteroarylene linker, which may be substituted. In some more specific embodiments, L 1c Each instance is independently substituted, or may be substituted, of a 5-7 member heteroarylene linker. In some embodiments, L 1c is a 6-membered heteroarylene. In some embodiments, L 1c It contains two N atoms and two O atoms. In certain embodiments, L 1c It is replaced each time it appears. In some related embodiments, L 1c L is substituted, for example, by oxo, alkyl (e.g., methyl, ethyl, etc.), or a combination thereof. In a more specific embodiment, L 1c Each instance is replaced by at least one oxo. In some embodiments, L 1c The structure is as follows: [ka] Take one of them.

[0063] In some embodiments, L 1d Alkylen, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkylynylene, alkylene heteroarylenealkylene, alkylene heterocyclylenealkylene, alkylene carbocyclylenealkylene, heteroalkylene heteroarylenealkylene, heteroalkylene heterocyclylenealkylene, heteroalkylene carbocyclylenealkylene, heteroalkylene heteroarylene heteroalkylene, heteroalkylene heterocyclylene heteroalkylene, heteroalkylene heterocyclylene heteroalkylene, heteroalkylene carbocyclylene heteroalkyl The linker is either a heteroatom linker, an alkylene heteroarylene heteroalkylene, an alkylene heterocyclylene heteroalkylene, an alkylene carbocyclylene heteroalkylene, a heteroarylene, a heterocyclylene, a carbocyclylene, an alkylene heteroarylene, an alkylene heterocyclylene, a heteroarylene alkylene, an alkylene carbocyclylene, a carbocyclylene alkylene, a heteroarylene heteroarylene, a heteroarylene heterocyclylene, a heteroarylene heteroalkylene, a heteroarylene carbocyclylene, a carbocyclylene heteroalkylene, or a heteroatom linker. In some embodiments, L 1d This is a heteroalkenylene linker that may be substituted. In some embodiments, the L that appears 1d At least one of them is replaced. In certain embodiments, L 1d L is replaced each time it appears. In some more specific embodiments, L 1d It is substituted with oxo. In other embodiments, L 1d Each instance independently contains a linker that can be formed by the reaction of two complementary reactive groups (e.g., triazolyl, amide, etc.), such as the Q group.

[0064] Linker L may be used at will 1d The M moiety can be used as a binding site for the M moiety to the rest of the compound. For example, in some embodiments, a synthetic precursor to the compound of structure (II) is prepared, and the M moiety is attached to the synthetic precursor using any number of easy methods known in the art, for example, methods referred to as "click chemistry." For this purpose, the M moiety can be attached to the synthetic precursor to form the compound of structure (II) using any rapid and substantially irreversible reaction. Exemplary reactions include copper-catalyzed reactions forming triazoles with azides and alkynes (Huisgen's 1,3-dipolar cycloaddition), reactions of dienes with dienephiles (Diels-Alder), strain-promoted alkyne-nitrone cycloaddition, strain-promoted cycloalkyne-azide cycloaddition (Cu-free click), reactions of strained alkenes with azides, tetrazines, or tetrazoles, [3+2] cycloaddition of alkenes and azides, reverse-promoted Diels-Alder reactions of alkenes and tetrazines, photoreactions of alkenes and tetrazoles, and various substitution reactions such as substitution of leaving groups by nucleophilic attack on electrophilic atoms. Exemplary substitution reactions include reactions of amines with activated esters; N-hydroxysuccinimide esters; isocyanates; isothiocyanates, etc. In some embodiments, L 1d The reaction that forms the compound may be carried out in an aqueous environment.

[0065] Therefore, in some embodiments, L 1d L is a linker containing a functional group that can be formed by the reaction of two complementary reactive groups independently with each occurrence, for example, a functional group that is one product of the "click" reaction described above. In various embodiments, L 1dIf at least one of these appears, the functional group may be formed by the reaction of an aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, sulfhydryl, disulfide, halogenated sulfonyl, isothiocyanate, imide ester, activated ester (e.g., N-hydroxysuccinimide), 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 or isothiocyanate.

[0066] In other embodiments, L 1d If at least one of these appears, the functional group may be formed by the reaction of an alkyne and an azide. In other embodiments, L 1d If at least one of these appears, the functional group can be formed by the reaction of an amine (e.g., a primary amine) with an N-hydroxysuccinimide ester or isothiocyanate. In more embodiments, L appears 1d For at least one of the functional groups, the functional group includes an alkene, ester, amide, thioester, disulfide, carbocyclic, heterocyclic, or heteroaryl group. In more embodiments, L 1d If at least one of the functional groups appears, the functional group includes an alkene, ester, amide, thioester, thiourea, disulfide, carbocyclic group, heterocyclic group, or heteroaryl group. In other embodiments, the functional group includes an amide or thiourea. In some more specific embodiments, L 1d , L b If at least one of them appears, L 1d , L b L is a linker containing a triazolyl functional group. In some related embodiments, L 1d Each instance independently contains a triazolyl functional group. In other embodiments, L 1d At least one occurrence of is a linker containing an amide or thiourea functional group.

[0067] In yet another embodiment, the L that appears 1d For at least one of the following, L 1d -M 1 or L 1d -M 2 This is one of the following structures: [ka] (In the formula, L 1a and L 1b Each of these is a linker that may be used independently and at will.

[0068] In different embodiments, L appears 1d For at least one of the following, L 1d -M 1 or L 1d -M 2 This is one of the following structures: [ka] (In the formula, L 1a and L 1b Each of these is a linker that may be used independently and at will. In the various embodiments described above, L 1a Or L 1b , or neither of them exists. In other embodiments, L 1a Or L 1b , or both exist.

[0069] In some embodiments, L 1a and L 1b If present, each is independently an alkylene or a heteroalkylene. For example, in some embodiments, L 1a and L 1b If present, it is independently one of the following structures: [ka] It holds.

[0070] In yet another different embodiment of structure (I), L1d L is an alkylene or heteroalkylene linker that may be used independently and arbitrarily each time it appears. In certain embodiments, L 1d This is one of the following structures: [ka] (In the formula, a, b, c, and d are each independent integers in the range of 1 to 6. It holds. In some embodiments, the L that appears 2 At least one of them is an alkylene linker. In a more specific embodiment, L 2 Each instance is an alkylene linker. In certain embodiments, the alkylene linker is a methylene linker. In some embodiments, the L that appears 3 At least one of them does not exist. In a more specific embodiment, L 3 It does not exist each time it appears. In some embodiments, L occurs at least once per occurrence. 4 and L 5 They are the same. In some embodiments, L 4 and L 5 It is the same each time it appears. In some embodiments, the L that appears 6 At least one of these comprises an alkylene oxide. In some of the embodiments described above, the alkylene oxide is an ethylene oxide, for example, polyethylene oxide. In some relevant embodiments, the compound has the following structure (IIa):

[0071] [ka] (IIa) (In the formula, L 4 and L 5 Each instance is independently alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker, provided that L 4 and L5 This is provided that it is not polyethylene oxide. z is an integer between 1 and 100. It holds.

[0072] In some embodiments, z is an integer from 1 to 30, for example, from 15 to 30, or from 22 to 25. In some embodiments, z is 23. In some embodiments, z is 21, 22, 23, 24, or 25. In some embodiments, z is an integer from 1 to 10, for example, from 3 to 6. In some embodiments, z is 3, 4, 5, or 6.

[0073] In some embodiments, L 4 and L 5 Each instance is an alkylene linker independently. In some more specific embodiments, L 4 and L 5 Each instance is independently a C3-C6 alkylene. In some embodiments, L 4 and L 5 Each instance is independently a C3 alkylene, a C4 alkylene, or a C6 alkylene. In some related embodiments, the compound has the following structure (IIb):

[0074] [ka] (IIb) (In the formula, R 7 , R 8 , R 9 and R 10 Each instance is independently either H or alkyl. y 1 and y 2 (Each instance is an integer between 1 and 6, independently of the others.) It holds.

[0075] In some embodiments, R 7 , R 8 , R 9 and R 10 Each instance is independently H and L 1c This is one of the following structures: [ka] It holds. In some related embodiments, the compound has the following structures: (IIc), (IId), (IIe), or (IIf): [ka] (IIc); JPEG2023552448000033.jpg47160(IId); JPEG2023552448000034.jpg48159(IIe); or JPEG2023552448000035.jpg47158(IIf) (In the formula, L 1d Each instance is independently a substituted alkylene or a substituted heteroalkylene linker. It holds.

[0076] In some embodiments, L 1d Each instance independently contains either an amide functional group or a triazolyl functional group. In yet another embodiment of any compound of structure (A), (I), or (II), R 4 Each instance of occurrence is independent of OH, O - OR d That is. "OR d " and "SR d " is O accompanied by a positive ion - and S - It is understood that this is intended to refer to the following. For example, the disodium salt of a phosphate group can be expressed as follows: [ka] , (In the formula, R d Sodium (Na + ) is ).

[0077] In any other embodiment of the compound of structure (A), (I), or (II), the R that appears 5 At least one of them is an oxo. In any other embodiment of the compound of structure (A), (I), or (II), R 5 Each time it appears, it is oxo. In various other embodiments, R 2 and R 3 These are, independently, OH or OP (=R a )(R b )R c In some different embodiments, R 2 or R 3 OH or OP (=R a )(R b )R c And R 2 or R 3 The other is a linker that includes Q, or a covalent bond to Q. In any further different embodiment of the compound of the above-described structure (A), (I), or (II), R 2 and R 3 These are, independently, -OP(=R a )(R b )R c In some of these embodiments, R c She is an office lady (OL). In other embodiments, R 2 and R 3 These are, independently, -OP(=R a )(R b )OL', where L' is an alkylene or heteroalkylene linker to Q, a target-directing moiety, an analyte (e.g., an analyte molecule), a solid support, solid support residues, a nucleoside, or a further compound of structure (A), (I), or (II).

[0078] Linker L' can be any linker suitable for linking 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 (A), (I), or (II) to the compound of structure (A), (I), or (II). Advantageously, certain embodiments involve the use of an L' moiety 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' includes an alkylene oxide or phosphodiester moiety, or a combination thereof.

[0079] In certain embodiments, L' has the following structure: [ka] , (In the formula, m'' and n'' are independent integers between 1 and 10. R e H is an electron pair or counterion, L is R e (or Q, a target-directed moiety, an analyte (e.g., an analyte), a solid support, a solid support residue, a nucleoside, or a direct bond or linking group to a further compound of structure (A), (I), or (II)) It holds.

[0080] In some embodiments, m'' is an integer between 4 and 10, for example, 4, 6, or 10. In other embodiments, n'' is an integer between 3 and 6, for example, 3, 4, 5, or 6. In some embodiments, n'' is an integer between 18 and 28, for example, 21 and 23. In some other embodiments, L'' is an alkylene, alkylene heterocyclylene, alkylene heterocyclylene alkylene, alkylene cyclylene, alkylene cyclylene alkylene, heteroalkylene, heteroalkylene heterocyclylene, heteroalkylene heterocyclylene heteroalkylene, heteroalkylene cyclylene, or heteroalkylene cyclene heteroalkylene moiety. In some other embodiments, L'' includes an alkylene oxide, a phosphodiester moiety, a sulfhydryl, a disulfide or maleimide moiety, or a combination thereof. In certain embodiments described above, the target-directing portion is an antibody or a cell surface receptor antagonist.

[0081] 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(registered trademark) 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.

[0082] In any other more specific embodiment of any of the above compounds of structure (A), (I), or (II), R 2 or R 3 This is one of the following structures: [ka] It holds.

[0083] In any further specific embodiment of the compound of the above-described structure (A), (I), or (II), R 1 or R 2 The structure is as follows: [ka] Take one of them.

[0084] Certain embodiments of the compounds of structure (A), (I), or (II) can be prepared by solid-phase synthesis methods similar to those known in the art for the preparation of oligonucleotides. Therefore, in some embodiments, L' is a solid support, a solid support residue, or a linker 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 (A), (I), or (II). Therefore, in some embodiments, R 2 or R 3 The structure is as follows: [ka] It holds.

[0085] Those skilled in the art will understand that the dT group shown above is included solely for the purpose of facilitating synthesis and economic efficiency, and is not necessary. Other solid supports can be used, resulting in different nucleosides or solid support residues present on L', or the nucleosides or solid support residues can be removed or modified after synthesis.

[0086] In further embodiments, Q is a moiety comprising a reactive group capable of independently forming a covalent bond with the analyte molecule or solid support each time it appears. In other embodiments, Q is a moiety comprising a reactive group capable of independently forming a covalent bond with a complementary reactive group Q' each time it appears. For example, in some embodiments, Q' is present in a further compound of structure (A), (I), or (II) (e.g., R 2 or R 3 (at position), Q and Q' contain complementary reactive groups, and thus the reaction of the compound of structure (I) with further compounds of structure (A), (I), or (II) yields a covalent dimer of the compound of structure (A), (I), or (II). The polymer compounds of structure (A), (I), or (II) can also be prepared by similar methods and are included within the scope of embodiments of this disclosure.

[0087] The type of Q group and its ability to bind to the rest of the compound of structure (A), (I), or (II) are not limited, provided that Q contains a moiety that is sufficiently reactive to form the desired bond. In certain embodiments, Q is a portion that is not easily hydrolyzed under aqueous conditions but is sufficiently reactive to form bonds with the analyte molecule or the corresponding group on the surface of the solid support (e.g., an amine, azide, or alkyne). Certain embodiments of the compounds of structure (A), (I), or (II) include a Q group 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, dienephile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, or maleimide functional group. In some embodiments, the activated ester is an N-succinimide ester, imide ester, or polyfluorophenyl ester. In other embodiments, the alkyne is an alkyl azide or acyl azide.

[0088] The Q group can be conveniently provided in a protected form that enhances storage stability or other desired properties, and this protecting group can then be removed, for example, when appropriate for a target-directed moiety or conjugate with an analyte. Thus, the Q group includes a “protected form” of a reactive group, which includes any of the reactive groups in Table 1 above and below. A “protected form” of Q refers to a moiety that, under given reaction conditions, is less reactive than Q and can be converted to Q under conditions that preferably do not decompose other parts of the compound of structure (A), (I), or (II) or react with the compound of structure (A), (I), or (II). Those skilled in the art can derive appropriate protected forms of Q based on a specific Q and desired end-use and storage conditions. For example, if Q is SH, the protected form of Q includes a disulfide, which can be reduced using common known techniques and reagents to reveal the SH moiety. An exemplary Q portion is shown in Table I below.

[0089] [Table 1] TIFF2023552448000042.tif254160 TIFF2023552448000043.tif96160 It should be noted that in some embodiments, when Q is SH, the SH portion tends to form a disulfide bond with another sulfhydryl group of another compound of structure (A), (I), or (II), for example. Thus, some embodiments include compounds of structure (A), (I), or (II) in the form of disulfide dimers, where the disulfide bond is derived from the Q group which is SH.

[0090] Similarly, the compound of structure (I) is included within the scope of certain embodiments, in which case R 2 and R 3 One or both of these involve linking structure (I) to a further compound. For example, R 2 and R 3 One or both of -OP(=R a )(Rb )R c And R c OL' 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 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 structure (I) second to structure (I). In this method, compounds of structure (I) having any number, e.g., 100 or more "M" moieties, can be prepared without the need to sequentially couple each monomer. An exemplary embodiment of such a compound of structure (I) is shown below: structure (I') [ka] (I') (In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 , L 4 , L 5 , L 6 M 1 M 2 m and n, as defined independently with respect to the compound of structure (I), L'' is a linker containing a functional group resulting from the reaction between the Q portion and the corresponding Q' portion. α is an integer greater than or equal to 1, for example, 1 to 100 or 1 to 10. It holds. Compounds of structure (I') can be derived by those skilled in the art, for example, by dimerizing or polymerizing a compound of structure (I) provided herein.

[0091] Similarly, compounds of structure (II) are included within the scope of certain embodiments, in which case R 2 and R 3 One or both of these involve linking structure (II) to a further compound. For example, R2 and R 3 One or both of -OP(=R a )(R b )R c Here, Rc is OL', and L' is a linker containing a covalent bond to a further compound of structure (II). Such compounds can be prepared, for example, by preparing a compound of structure (II) 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 structure (I). In this method, compounds of structure (II) having any number, e.g., 100 or more "M" moieties, can be prepared without the need to sequentially couple each monomer. An exemplary embodiment of such a compound of structure (II) is shown below as structure (II'):

[0092] [ka] (II') (In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , L 1c , L 1d , L 2 , L 3 , L 4 , L 5 , L 6 M 1 M 2 m and n, as defined independently with respect to the compound of structure (II) in each instance, L'' is a linker containing a functional group resulting from the reaction between the Q portion and the corresponding Q' portion. α is an integer greater than 1, for example, between 1 and 100, or between 1 and 10. It holds. Compounds of structure (II') can be derived by those skilled in the art, for example, by dimerizing or polymerizing compounds of structure (II) provided herein.

[0093] In other embodiments, the Q moiety can be conveniently masked (e.g., protected) as a disulfide moiety, which can then be reduced to yield an activated Q moiety for binding to a desired analyte molecule or target-directed moiety. For example, the Q moiety may be a disulfide having the following structure: [ka] (wherein R is an optionally substituted alkyl group) can be masked as follows: For example, in some embodiments, Q is a disulfide moiety having the following structure: [ka] (In the formula, n is an integer between 1 and 10, for example, 6.) It will be offered as such.

[0094] In some other embodiments, R 2 or R 3 One of them is OH or OP (=R a )(R b )R c And R 2 or R 3 The other linker is a linker that includes a covalent bond to the analyte molecule, or a linker that includes a covalent bond to a solid support. For example, in some embodiments, the analyte molecule is a nucleic acid, an amino acid, or a polymer thereof. In other embodiments, the analyte molecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion. In yet another embodiment, the solid support is a polymer bead or a non-polymer bead. The value for m is another variable factor that can be selected based on the desired fluorescence intensity and / or color intensity. In some embodiments, m is an integer from 1 to 10, independently for each occurrence. In other embodiments, m is an integer from 1 to 5, for example, 1, 2, 3, 4, or 5, independently for each occurrence.

[0095] In other embodiments, m is an integer greater than 2, independently of each occurrence, and z is an integer between 15 and 30. For example, in one embodiment, m is an integer greater than 2, independently of each occurrence, such as 3, 4, 5, or 6, and z is an integer between 22 and 25. The fluorescence intensity can also be adjusted by selecting different values ​​for n. In some 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.

[0096] The value of q is a variable factor (ban) that can be selected based on the desired fluorescence intensity and / or color intensity. In some embodiments, q is an integer between 0 and 3, independently for each occurrence. For example, in some embodiments, q is 0. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. The value of w is a variable factor (ban) that can be selected based on the desired fluorescence intensity and / or color intensity. In some embodiments, w is an integer between 0 and 3, independently for each occurrence. For example, in some embodiments, w is 0. In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, w is 3.

[0097] M 1 and M 2 The element is selected based on desired optical properties, for example, based on desired color and / or fluorescence emission wavelength. In some embodiments, M 1 and M 2 This is the same each time it appears. However, M 1 Or M 2 Each occurrence is the same M 1 Or M 2It does not need to be M 1 and M 2 It is important to note that each occurrence may contain different compounds. For example, in some embodiments, M 1 and M 2 These are not the same, and each has different M values ​​to have absorbance and / or emission for use in fluorescence resonance energy transfer (FRET) spectroscopy. 1 Part and M 2 A portion is selected. For example, in such an embodiment, the FRET mechanism selects different M portions to form FRET donor-acceptor pairs such that the absorption of irradiation at one wavelength causes the emission of irradiation at a different wavelength. Exemplary M 1 Part and M 2 The parts can be appropriately selected by those skilled in the art based on the desired end use. An example M for the FRET method. 1 Part and M 2 The portion contains fluorescein and 5-TAMRA (5-carboxytetramethylrhodamine, succinimidyl ester) dye. M 1 and M 2 M 1 and M 2 It can bond to the rest of the molecule from any position on top (i.e., an atom). Those skilled in the art will understand M 1 and M 2 It will recognize the means for binding to the rest of the molecule. Exemplary methods include the “click” reaction described herein.

[0098] In some embodiments, M 1 and M 2M is a fluorescent or chromogenic moiety. Any fluorescent and / or chromogenic moiety may be used, for example, those known in the art and those commonly used in colorimetric assays, UV assays and / or fluorescence assays. Examples of M moieties useful in various embodiments of this 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); squaline derivatives and ring-substituted squalines (including ceta, cetau and square dyes); naphthalene derivatives (e.g., dansyl and porodane derivatives); coumarin derivatives; oxadiazole derivatives (e.g., pyridyloxazole, These include 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., Proflavin, Acridine Orange, Acridine Yellow); arylmethine derivatives: Auramine, Crystal Violet, Malachite Green; and tetrapyrrole derivatives (e.g., Porfin, Phthalocyanine, or Bilirubin). Other exemplary M portions include cyanine dyes, xanthate dyes (e.g., Hex, Vic, Nedd, Joe, or Tet); Yakima Yellow; Redmond Red; Tamura; 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® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, or Alexa Fluor® 680.

[0099] The compounds of this disclosure have been found to be useful as fluorescent dyes and / or chromogenic dyes with high quantum efficiency. This is partly due to the donor portion (e.g., M 1 ) emission spectrum and acceptor part (for example, M 2This is due to overlap with the absorbance or excitation spectrum of ). Therefore, some embodiments provide FRET donors having excitation maxima between 300 and 900 nm and emission maxima between 350 and 900 nm. For example, in some embodiments (embodiemtn), the FRET donor includes 2,5-diphenyloxazole having excitation maxima at 311 nm and emission maxima at 375 nm. In another example, in some embodiments, the FRET donor includes dansyl fluorophore having excitation maxima at 333 nm and emission maxima at 518 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 350 having excitation maxima at 346 nm and emission maxima at 442 nm. In yet another example, in some embodiments, the FRET donor includes pyrene having excitation maxima at 340 nm and emission maxima at 376 nm. In further examples, in some embodiments, the FRET donor includes coumarin 343 having an excitation maximum at 437 nm and an emission maximum at 477 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 430 having an excitation maximum at 430 nm and an emission maximum at 539 nm. In yet another example, in some embodiments, the FRET donor includes 5-carboxyfluorescein (FAM) having an excitation maximum at 495 nm and an emission maximum at 519 nm. In yet another example, in some embodiments, the FRET donor includes a cyanide dye (CY3) having an excitation maximum at 550 nm and an emission maximum at 615 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 555 having an excitation maximum at 555 nm and an emission maximum at 572 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 568 having an excitation maximum at 578 nm and an emission maximum at 603 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 633 having an excitation maximum at 630 nm and an emission maximum at 650 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor® 647 having an excitation maximum at 650 nm and an emission maximum at 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.

[0100] Some embodiments provide a FRET acceptor having an excitation maximum between 400 and 800 nm and an emission maximum between 500 and 500 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 at 555 nm and an emission maximum at 572 nm. In yet another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 568 having an excitation maximum at 578 nm and an emission maximum at 603 nm. In yet another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 594 having an excitation maximum at 590 nm and an emission maximum at 617 nm. In yet another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 633 having an excitation maximum at 630 nm and an emission maximum at 650 nm. In yet another example, in some embodiments, the FRET acceptor includes Alexa Fluor® 660 having an excitation maximum at 663 nm and an emission maximum at 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 includes Alexa Fluor® 750 having an excitation maximum at 756 nm and an emission maximum at 776 nm.

[0101] Embodiments of this disclosure enable various combinations of FRET donor / acceptor pairs for enhancing brightness as a sensor. 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, 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, 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, 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 or Alexa Fluor® 633 as the FRET acceptors. 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 acceptors. 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 acceptors. 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.

[0102] In any further embodiment described above, M 1 and M 2 However, this also includes three or more aryl or heteroaryl rings, or combinations thereof, for example, 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 However, it includes six aryl or heteroaryl rings, or combinations thereof. In further embodiments, the rings are fused. For example, in some embodiments, M 1 and M 2However, this includes three or more fused rings, four or more fused rings, five or more fused rings, or even six or more fused rings.

[0103] In some embodiments, M 1 Or M 2 It is a ring structure. For example, in some embodiments, M 1 Or M 2 It is a carbocyclic compound. In other embodiments, M 1 Or M 2 This is a complex algebra. In yet another embodiment described above, M 1 Or M 2 Each instance independently includes an aryl moiety. In some of these embodiments, the aryl moiety is polycyclic. In other further specific examples, the aryl moiety is a condensed polycyclic aryl moiety, which may include, for example, at least three, at least four, or even more than four aryl rings. In other embodiments of any of the compounds of the above structures (A), (I), (II), (I'), or (I''), M 1 Or M 2 Each instance independently contains at least one heteroatom. For example, in some embodiments, the heteroatom is nitrogen, oxygen, or sulfur. In any of the further embodiments described above, M 1 Or M 2 Each instance independently contains at least one substituent. For example, in some embodiments, the substituents are 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 groups.

[0104] In some of the more specific embodiments described above, M 1 Or M 2Each instance independently corresponds to dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine, terylene, sexiphenyl, porphyrin, benzopyrene (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthrasyl, squaline, squallium, 9,10-ethynylanthracene, or ter-naphthyl moiety. In other embodiments, M 1 Or M 2 Each instance is independently p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, peryleneimide or peryleneamide, or derivatives thereof. In more embodiments, M 1 Or M 2 Each of these is independently a coumarin dye, resorphine dye, dipyrometheneborone difluoride dye, ruthenium bipyridyl dye, energy transfer dye, thiazole orange dye, polymethine, or N-aryl-1,8-naphthalimide dye. In any of the further embodiments described above, M 1 Or M 2 It is the same each time it appears. In other embodiments, M 1 Or M 2 Each of these is different. In more embodiments, one or more M 1 Or M 2 These are the same, one or more M 1 Or M 2 They are different.

[0105] In some embodiments, M 1 Or M 2 These are pyrene, perylene, perylene monoimide, 5-carboxyfluorescein (FAM), 6-FAM, 6-FITC, 5-FITC, or derivatives thereof. In some embodiments, M 1 Or M2 The structure is as follows: [ka] Take one of them.

[0106] M containing a carboxylic acid group 1 Part or M 2 The part is the anionic form (CO2) - As illustrated in the diagram, those skilled in the art will understand that this varies depending on the pH, and that the protonated form (-CO2H) is included in various embodiments.

[0107] In some embodiments, M 1 Or M 2 This is one of the following structures: [ka] TIFF2023552448000050.tif226169 TIFF2023552448000051.tif230170 It has TIFF2023552448000052.tif106102. In some specific embodiments, the compound of structure (A), (I), or (II) is a compound selected from Table 2. The compounds in Table 2 were prepared according to the procedures described in the examples, and their structures (identities) were confirmed by mass spectrometry.

[0108] [Table 2] TIFF2023552448000054.tif207170 TIFF2023552448000055.tif181170 TIFF2023552448000056.tif196170 TIFF2023552448000057.tif169170 TIFF2023552448000058.tif229170 TIFF2023552448000059.tif210170 TIFF2023552448000060.tif224170 TIFF2023552448000061.tif225170

[0109] As used in Table 2 and throughout this disclosure, R 2 , R 3 Unless otherwise specified, z and n have the definitions presented for compounds of structure (A), (I), or (II).

[0110] As used in Table 2 and throughout this disclosure, Fx, in each instance, independently represents one of the following structures: [ka] This refers to a fluorescent (fluorescent) or color-emitting portion that has [the characteristic of fluorescent properties].

[0111] In some embodiments, Fx is fluorescein. It is well known in the art that the fluorescein moiety tautomerizes between quinoid, zwitterionic, and lactoid forms. Those skilled in the art will readily understand that this form is pH-dependent, and that each form (e.g., quinoid, zwitterionic, and lactoid) is also within the scope of embodiments of this disclosure.

[0112] As used in Table 2 above and throughout this disclosure, M 1 and M 2 Each instance is independently of the fluorescent or colored portion of M as described above. 1 and M 2 One of them is a FRET donor, M 1 and M 2 The other is a FRET acceptor. In some embodiments, M 1 FAM and M 2This is AF594. In some embodiments, M 1 It is Cy3, and M 2 It is the AF680.

[0113] FAM is one of the following structures: [ka] This refers to the part that has [something]. The AF594 has the following structure: [ka] AF594 This refers to the part that has [something]. Cy3 is one of the following structures: [ka] Cy3 This refers to the part that has [something]. The AF680 has the following structure: [ka] AF680 This refers to the part that has [something]. As used in Table 2 above and throughout this disclosure, dT has the following structure: [ka] dT (In the formula, R refers to H (or a direct bond). Some embodiments are shown in Table 2 and include any of the above, including specific compounds conjugated to a target-directing portion 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.

[0114] This disclosure generally provides compounds that exhibit increased fluorescence emission compared to previously known compounds. Accordingly, certain embodiments relate to fluorescent compounds comprising Y fluorescent moieties M, wherein when excited by ultraviolet light of a predetermined wavelength, the peak fluorescence emission is at least 85% of the emission amount that is Y times higher than the peak fluorescence emission of a single M moiety when excited by ultraviolet light of the same wavelength, and Y is an integer of 2 or more. Fluorescent compounds include compounds that emit a fluorescence signal when excited by light such as ultraviolet light. In some embodiments, the fluorescent compound has a peak fluorescence emission amount that is at least 90% of, 95% of, 97% of, or 99% of the peak fluorescence emission amount that is Y times higher than that of a single M portion. In some embodiments, Y is an integer between 2 and 100, for example, between 2 and 10.

[0115] In some embodiments, the Y M portions independently have the following structure: [ka] (In the formula, [ka] (This indicates the binding site to the fluorescent compound.) Take one of them.

[0116] In other embodiments, a single M portion independently has the following structure: [ka] Take one of them.

[0117] In a more specific embodiment, the fluorescent compound has the following structure: [ka] (In the formula, [ka] This indicates the binding site to the fluorescent compound, and the single M portion has the following structure: [ka] (has) It contains Y M parts, each independently having one of the following.

[0118] In some embodiments, the Y M portions are independently one of the following structures: [ka] TIFF2023552448000075.tif227170 TIFF2023552448000076.tif232170 It has TIFF2023552448000077.tif10699. In other embodiments, peak fluorescence emission occurs at wavelengths in the range of approximately 500 to 550 nm. In many more embodiments, the fluorescent compound comprises at least one ethylene oxide moiety. Compositions and analytes containing a fluorescent compound of any one of the structures (A), (I), or (II) are also provided.

[0119] The compounds currently disclosed are "adjustable," meaning that those skilled in the art can obtain compounds with a desired and / or predetermined molar fluorescence (molar brightness) by appropriately selecting the variable factors in any of the compounds described above. The "adjustability" of these compounds allows users to easily obtain compounds with the desired fluorescence and / or color for use in specific assays or for identifying specific target analytes. All variable factors may affect the molar fluorescence of these compounds, but M, L 4 , L 5 , L 6 The appropriate selection of m, n, and z is considered to play an important role in the molar fluorescence of the compound. Therefore, in one embodiment, a method for obtaining a compound having a desired molar fluorescence is provided, comprising the steps of: selecting an M moiety having a known fluorescence; preparing a compound of structure (A), (I), or (II) containing the M moiety; and L to arrive at the desired molar fluorescence. 4 , L 5 , L 6 A method is provided which includes the step of selecting appropriate variable factors for m, n, and z.

[0120] In certain embodiments, molar fluorescence intensity can be expressed in terms of an increase or decrease ratio compared to the fluorescence emission of a parent fluorophore (e.g., monomer). In some embodiments, the molar fluorescence intensity of the compound is 1.1×, 1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10× or even greater than that of the parent fluorophore. Various embodiments are described below. 4 , L 5 , L 6 The method involves preparing a compound having a desired increase in fluorescence intensity compared to a fluorophore by appropriately selecting m, n, and z. To facilitate the illustration, various compounds containing a phosphorus portion (e.g., phosphate) are shown in an 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 uncharged forms (e.g., protonated or salts such as sodium or other cations) are also included within the scope of embodiments of this disclosure. Compositions comprising any of the above-mentioned compounds and one or more analyte molecules (e.g., biomolecules) are provided in various other embodiments. In some embodiments, the use of such compositions in analytical methods for detecting one or more analyte molecules is also provided.

[0121] In other embodiments, the compound is useful in various analytical methods. For example, in certain embodiments, the disclosure is a method for staining a sample, wherein the sample is stained with, for example, R 2 or R 3 One of the linkers is a linker containing a covalent bond to the analyte molecule (e.g., biomolecule) or microparticle, R 2 or R 3 The other is H, OH, alkyl, alkoxy, alkyl ether or OP(=R a )(R b )R c The present invention provides a method comprising the step of adding a compound having structure (A), (I), or (II) in an amount sufficient to produce an optical response when the sample is irradiated at an appropriate wavelength. In some embodiments of the above-described method, R 2 The linker is a linker that includes a covalent linkage to the analyte molecule, such as a biomolecule. For example, it may be a nucleic acid, an amino acid, or a polymer thereof (e.g., a polynucleotide or polypeptide). In more embodiments, the biomolecule may be an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion. In yet another embodiment of the above method, R 2 This linker includes covalent bonding to a solid support such as microparticles. For example, in some embodiments, the microparticles are polymer beads or non-polymer beads.

[0122] In even more embodiments, the optical response is a fluorescence response. In other embodiments, the sample includes 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 having different optical properties in a detectable manner. In other embodiments, the present disclosure relates to a method for visually detecting analyte molecules such as biomolecules, (a) For example, R 2 or R 3 One of them is a linker containing a covalent bond to the molecule to be analyzed, R 2 or R 3 The other side is H, OH, alkyl, alkoxy, alkyl ether or OP(=R a )(R b )R c The steps include: preparing a compound having structure (A), (I), or (II), and (b) A step of detecting the compound by its visible properties. This provides a method that includes [something]. In some embodiments, the analyte molecule is a nucleic acid, an amino acid, or a polymer thereof (e.g., a polynucleotide or polypeptide). In even more embodiments, the analyte molecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion.

[0123] In other embodiments, the following: (a) The step of mixing any of the above-mentioned compounds with one or more molecules to be analyzed, and (b) A step of detecting the compound by its visible properties. A method is provided for visually detecting target molecules, such as biomolecules, including those mentioned above. In another embodiment, the method involves visually detecting the molecule to be analyzed, (a)R 2 or R 3 A step of mixing a compound of structure (A), (I), or (II), in which is a linker containing Q or a covalent bond to Q, with the molecule to be analyzed. (b) The step of forming a conjugate of the compound and the molecule to be analyzed, (c) Step of detecting the conjugate by its visibility characteristics. A method including this is provided.

[0124] Other exemplary methods include methods for detecting the object of analysis, as follows: (a)R 2 or R 3 The step of preparing a compound of structure (A), (I), or (II) that includes a linker containing a covalent bond to a target-directing moiety having specificity for the analyte, (b) a step of mixing the compound and the analyte to associate the target-directing portion with the analyte, and (c) A step of detecting the compound, for example, by its visible properties or fluorescence properties. This includes methods that include [specific methods]. In certain embodiments of the method described above, the analyte is a particle such as a cell, and the method includes the use of flow cytometry. For example, the compound may be provided with a target-directing moiety, such as an antibody, to selectively associate with desired cells, thereby making the cells 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. A suitable antibody 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 antibodies (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.

[0125] In certain embodiments, the conjugate rate forming a conjugate containing the compound of structure (A), (I), or (II) and the analyte is higher than approximately 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 98.5%, or 99%. In yet another embodiment, the present disclosure is a method for increasing the brightness of a dye, (a) the step of preparing a dye solution containing a compound of structure (A), (I), or (II), and (b) A step of aging the dye solution for a certain period of time. This provides a method that includes [something]. 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. The dye solution may contain various buffers. In some embodiments, the dye contains ETOH. In some embodiments, the dye solution contains BD brilliant. In some embodiments, the dye solution contains sodium chloride or potassium chloride. Therefore, the embodiments of this compound are not limited to: applications in any number of methods, including but are limited to: cell counting, cell sorting, biomarker detection, apoptosis quantification, cell viability determination, cell surface antigen identification, determination of total DNA and / or RNA contents, identification of specific nucleic acid sequences (e.g., as nucleic acid probes), and diagnosis of diseases such as hematological cancers.

[0126] In addition to the above methods, embodiments of the compounds of structure (A), (I) or (II) include, but are 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 amplification; imaging of cancer, e.g., by including a targeting moiety such as an antibody or sugar or other moiety that preferentially binds to cancer cells in the compounds of structure (A), (I) or (II); imaging in surgery; histone binding for identifying various diseases; drug delivery, e.g., by replacing the M moiety in the compounds of structure (A), (I) or (II) with an active drug moiety; and / or use in various fields and methods including contrast agents in dental work and other procedures such as preferential binding of the compounds of structure (I) to various microbiota and / or organisms.

[0127] Any embodiment of the compounds of structure (I) described above, and the variable factor R in the compounds of structure (I) described above 1 , R 2 , R 3 , R 4 , R 5 , L’, L 1 , L 2 , L 3 , L 4 , L 5 , L 6 It is understood that any particular option described herein with respect to M, m, n and / or z may be combined independently of other embodiments and / or variable factors of the compounds of structure (I) to form embodiments of the present disclosure not specifically described above. Further, the list of options for any particular variable factor R in a particular embodiment and / or claim 1 , R 2 , R 3 , R 4 , R 5 , L’, L 1 , L 2 , L 3 , L 4 , L 5 , L 6Where listed with respect to M, m, n and / or z, it is understood that each individual option may be removed from the scope of a particular embodiment and / or claim, and the remaining list of options is considered to be within the scope of this disclosure.

[0128] Any embodiment of the compound of structure (II) described above, and the variable factor R in the compound of structure (II) described above 1 , R 2 , R 3 , R 4 , R 5 , L', L 1c , L 1d , L 2 , L 3 , L 4 , L 5 , L 6 It is understood that any particular option described herein with respect to M, m, n and / or z may be combined independently with other embodiments and / or variable factors of the compound of structure (II) to form embodiments of the present disclosure not specifically described above. Furthermore, the list of options may include any particular variable factor R in any particular embodiment and / or claim. 1 , R 2 , R 3 , R 4 , R 5 , L', L 1c , L 1d , L 2 , L 3 , L 4 , L 5 , L 6 Where listed with respect to M, m, n and / or z, it is understood that each individual option may be removed from the scope of a particular embodiment and / or claim, and the remaining list of options is considered to be within the scope of this disclosure. In this description, it is understood that combinations of substituents and / or variable factors in the illustrated formulas are acceptable only if such contributions result in a stable compound.

[0129] Those skilled in the art will also understand 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 acids. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, and benzyl. Suitable protecting groups for amino, amidino, and guanidino include t-butoxycarbonyl and benzyloxycarbonyl. Suitable protecting groups for mercapto include -C(O)-R'' (wherein R'' is alkyl, aryl, or arylalkyl), p-methoxybenzyl, and trityl. Suitable protecting groups for carboxylic acids include alkyl, aryl, or arylalkyl esters. Protecting groups are known to those skilled in the art and can be added or removed in accordance with standard techniques described herein. The use of protecting groups is detailed in Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As those skilled in the art will understand, the protecting group may also be a polymer resin such as Wang resin, Rink resin, or 2-chlorotrityl chloride resin. Furthermore, all compounds of the Disclosure that exist in free base or acid form can be converted to salts thereof by treatment with appropriate inorganic or organic bases or acids, by methods known to those skilled in the art. Salts of the compounds of the Disclosure can be converted to their free base or acid form by standard techniques.

[0130] The following reaction schemes illustrate exemplary methods for preparing compounds of structures (A), (I), or (II) of the present disclosure. Those skilled in the art will understand that these compounds may be prepared by similar methods or by combining them with other methods known to those skilled in the art. Those skilled in the art will also understand that other compounds of structures (A), (I), or (II) not specifically illustrated below may be prepared by similar methods described below, using appropriate starting components and, as necessary, modifying the parameters of the synthesis. Generally, starting components can be obtained from suppliers such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)), or prepared as described in the present disclosure.

[0131] Reaction Scheme I [ka]

[0132] Reaction scheme I is R 1 , L 2 , L 3 And M is as defined above, R 2 and R 3Exemplary methods for preparing intermediates useful for preparing the compound of structure (I), where is as defined above or a protected variant thereof, and L is an optional linker, are illustrated. Referring to reaction scheme 1, the compound of structure can be purchased or prepared by methods well known to those skilled in the art. The compound of structure b is obtained by reaction with MX, where x is a halogen such as bromo, under the Suzuki coupling conditions known in the art. The compound of structure b can be used in the preparation of the compound of structure (I) as described below.

[0133] Reaction Scheme II [ka] Reaction scheme II illustrates an alternative method for preparing intermediates useful for preparing the compound of structure (I). 1 , L 1 , L 2 , L 3 , G and M are as defined above, R 2 and R 3 As defined above, or their protected variants, a compound of structure c, which can be purchased or prepared by known techniques, reacts with M-G' to produce a compound of structure d, with reference to reaction scheme II. Here, G and G' represent complementary reactive functional groups (i.e., functional groups that react to form a covalent bond). G' may be a pendant group to M or part of the structural main chain of M. G and G' may be any number of functional groups as described herein, such as alkynes and azides, amines and activated esters, or amines and isothiocyanates, respectively.

[0134] The compound of structure (I) is obtained from either structure b or d under well-known automated DNA synthesis conditions, resulting in the following structure (e): [ka] , (e) It can be prepared by reaction with a phosphoramidite compound having (wherein L is an optional linker).

[0135] DNA synthesis methods are well known in this field. Briefly speaking, two alcohol groups, for example, R in intermediate b or d above, 2 and R 3 These groups are functionalized with a dimethoxytrityl (DMT) group and a 2-cyanoethyl-N,N-diisopropylaminophosphoramidite group, respectively. The phosphoramidite group is usually coupled with an alcohol group in the presence of an activator such as tetrazole, and then the phosphorus atom is oxidized with iodine. The dimethoxytrityl group can be removed with an acid (e.g., chloroacetic acid), exposing 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.

[0136] The preparation of phosphoramidites used in oligomerization is also well known in this field. For example, primary alcohols (e.g., R 3 ) can be protected as a DMT group by reaction with DMT-Cl. Next, a secondary alcohol (e.g., R) 2 The ) is functionalized as a phosphoramidite by reaction with a suitable reagent such as 2-cyanoethyl N,N-diisopropyl chlorophosphoamidite. The preparation of phosphoramidites and the methods for oligomerizing them are well known in the art and will be described in more detail in the examples.

[0137] The compound of structure (I) is prepared by oligomerization of intermediates b or d and e according to the well-known phosphoramidite chemical reaction described above. By repeating the phosphoramidite coupling a desired number of times, the desired number of m and n repeating units are incorporated into the molecule. It will be understood that the compound of structure (III) can be prepared by a similar method, as described below.

[0138] Furthermore, each of the compounds of this disclosure may be prepared according to the methods described in PCT publication numbers WO2016 / 183185, WO2017 / 173355, and WO2017 / 177065, which are incorporated herein by reference. In various other embodiments, compounds useful for preparing compounds of structure (I) are provided. These compounds can be prepared in monomer, dimer, and / or oligomer forms as described above, and the M portion is then covalently bonded to the compound by any number of synthetic methods (e.g., the "click" reaction described above) to form the compound of structure (I). Thus, in various embodiments, compounds having the following structure (III): [ka] (III) or its stereoisomer, salt, or tautomer (in the formula, G 1 and G 2 This is a portion containing a reactive group or a protecting analog thereof that is capable of independently forming a covalent bond with a complementary reactive group each time it appears. L 1 , L 2 and L 3 Each instance may be used independently and arbitrarily as an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, heteroarylene, or heteroatom linker. L 4 and L 5 Each instance is independently alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkylynylene linker. L 6 Each instance is independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker, provided that the L that appears 6 At least one of them is L 4 and L 5 Each of these is subject to the condition that they are different from each of the following: R 1 Each instance is independently H, alkyl, or alkoxy. R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a )(R b )R c , Q or its protected form or L’, R 4 is independently, each time it appears, OH, SH, O - , S - , OR d or SR d . R 5 is independently, each time it appears, oxo, thioxo, or absent, R a is O or S, R b is OH, SH, O - , S - , OR d or SR d . 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 counterion, Q is, each time it appears, independently, a moiety comprising a reactive group or its protected form that can form a covalent bond with an analyte molecule, a target-directed moiety, a solid support or a complementary reactive group Q’, L’ is, each time it appears, independently, a linker comprising a covalent bond to Q, a linker comprising a covalent bond to a target-directed moiety, a linker comprising a covalent bond to an analyte molecule, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, a linker comprising a covalent bond to a nucleoside, or a linker comprising a covalent bond to a further compound of structure (III), Each instance of m is an independent integer greater than or equal to zero, provided that at least one instance of m is an integer greater than or equal to 1. n is an integer greater than or equal to 1. It will be provided.

[0139] In some embodiments, L 6 Each instance is an independent alkylene oxide linker. In some embodiments, L 4 and L 5 Each instance is an alkylene linker independently. In some embodiments, L 4 and L 5 Each instance is independently a C3-C6 alkylene. In some embodiments, at least one instance is L 4 and L 5 They are the same.

[0140] In some embodiments, L 6 Each instance is independently a heteroalkylene linker. In other more specific embodiments, L 6 Each instance is an alkylene oxide linker. In some embodiments, L 6 It is polyethylene oxide. In some related embodiments, the compound has the following structure (IIIa): [ka] (IIIa) (In the formula, L 4 and L 5 Each instance is independently alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker, provided that L 4 and L 5 This is provided that it is not polyethylene oxide. z is an integer between 1 and 100. It holds.

[0141] In some embodiments, L 4 and L5 Each instance is independently alkylene. In some related embodiments, the compound has the following structure (IIIb): [ka] (IIIb) (In the formula, R 7 , R 8 , R 9 and R 10 Each instance is independently either H or alkyl. y 1 and y 2 Each occurrence is an independent integer between 1 and 6. z is an integer between 1 and 100. It holds. In some embodiments, R 7 , R 8 , R 9 and R 10 Each instance is independently H.

[0142] In some embodiments, L 2 and L 3 Each instance is independently a C1-C6 alkylene, a C2-C6 alkenylene, or a C2-C6 alkynylene. For example, in some embodiments, L 2 and L 3 Each instance is independently a C1-C6 alkylene, and R 7 , R 8 , R 9 and R 10 Each instance is independently H. In some related embodiments, the compound has the following structure (IIIc):

[0143] [ka] (IIIc) (In the formula, x 1 , x 2 , x 3 and x 4 Each occurrence is an independent integer between 0 and 6. y 1and y 2 Each occurrence is an independent integer between 1 and 6. z is an integer between 1 and 100. It holds.

[0144] In some embodiments, x 1 and x 3 Each occurrence is 0, and x 2 and x 4 Each instance of this value is 1. In some embodiments, x 1 , x 2 , x 3 and x 4 Each instance of this value is 1. In some embodiments, y 1 and y 2 Each occurrence is 3. In some embodiments, y 1 and y 2 Each occurrence is 4. In some embodiments, y 1 and y 2 Each instance of this is 6. In some embodiments, z is an integer between 15 and 30. In other embodiments, z is an integer between 22 and 25.

[0145] In some embodiments, L 1 ' has the following structure: One of them: [ka] It holds. In some embodiments, R 1 Each instance of this is H. In some embodiments, L 2 and L 3 Each instance is independently a C1-C6 alkylene, a C2-C6 alkenylene, or a C2-C6 alkynylene.

[0146] Reaction Scheme III [ka] Reaction scheme III illustrates a method for preparing an intermediate useful for preparing the compound of structure (II). 1c , L 1d’ , L 2 , L 3 , G and M are as defined above, R 2 and R 3 However, a compound of structure f, which can be purchased or prepared by known techniques, as defined above or a protected variant thereof, as shown in Reaction Scheme III, reacts with M-G' to produce a compound of structure g, where G and G' represent complementary functional groups (i.e., functional groups that react to form a covalent bond). G' may be a pendant group to M or part of the structural main chain of M. G and G' can be any number of functional groups described herein, such as alkynes and azides, amines and activated esters, or amines and isothiocyanates, respectively.

[0147] Compounds of structure f can be prepared by oligomerization using a well-known phosphoramidite chemical reaction. The applicants have discovered an intermediate compound useful for the synthesis of compounds of structure f. Therefore, one embodiment provides a compound having the following structure h: [ka] (h) (In the formula, n1 is an integer between 1 and 6. n2 is an integer between 1 and 3. X is O or a direct bond, R 1 "and R 2 Each instance of " is independently H, a protecting group, or an activated phosphorus moiety. R 3 " is H or alkyl, R 4 (where " is an alkoxy, haloalkyl, alkyl, optionally substituted aryl, or optionally substituted aralkyl) To provide.

[0148] In some embodiments of compound (h), n1 is 2. In some embodiments, n is 4. In some related embodiments, n2 is 1. In certain embodiments, n1 is 2 and n2 is 1. In other embodiments, n1 is 4 and n2 is 1. In some of the embodiments described above, X is a direct bond. In some embodiments of compound (h), n1 is 2. In certain related embodiments, n2 is 2. In some of the embodiments described above, X is O. In some embodiments of compound (h), X is a direct bond. In some embodiments, X is O. In some embodiments of compound (h), R 1 " is H. In certain embodiments, the protecting group is, for example, a trityl protecting group. In some embodiments, R 1 '' is trityl. In some embodiments, R 1 '' is 4-methoxytrityl. In a more specific embodiment, R 1 '' is 4,4'-dimethoxytrityl.

[0149] In some embodiments, R 2 '' is H. In some embodiments, R 2 '' is the activated phosphorus moiety. For example, in some embodiments, R 2 '' has the following structure: [ka] (In the formula, R 5 '' is H or cyanoalkyl, R 6 (Each instance is independently a C1-C6 alkyl group.) Includes. In some embodiments of compound (h), R 5 '' is H. In other embodiments, R 5 '' is 2-cyanoethyl. In some embodiments, the appearing R 6At least one of the '' is isopropyl. In some embodiments, R 6 Each instance of '' is isopropyl.

[0150] In certain specific embodiments, R 2 '' has the following structure: [ka] It holds. In some embodiments of compound (h), R 5 '' is H. In some embodiments of compound (h), R 4 '' is an aryl compound containing one, two, or three aromatic rings, for example, R 4 '' contains one or two aromatic rings. In some embodiments, R 4 '' does not contain silicon. In some embodiments, R 4 '' is a C1-C4 haloalkyl. In a more specific embodiment, R 4 '' is -CF3. In some embodiments, R 4 '' is a C1-C4 alkoxy. In a more specific embodiment, R 4 '' is tert-butoxy.

[0151] In various other embodiments, compounds useful for preparing compounds of structure (II) are provided. These compounds can be prepared in monomer, dimer, and / or oligomer forms as described above, and the M portion is then covalently bonded to the compound by any number of synthetic methods (e.g., the "click" reaction described above) to form compounds of structure (II). Thus, in various embodiments, compounds having the following structure (IV):

[0152] [ka] (IV) (In the formula, G 1 and G 2This is a portion containing a reactive group or a protecting analog thereof that is capable of independently forming a covalent bond with a complementary reactive group each time it appears. L 1c Each instance is independently a heteroarylene linker. L 1d’ , L 2 and L 3 Each instance may be independently and arbitrarily used alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker. L 4 and L 5 Each instance is independently alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkylynylene linker. L 6 Each instance is independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker, provided that the L that appears 6 At least one of them is L 4 and L 5 Each of these is subject to the condition that they are different from each of the following: R 2 and R 3 These are, independently, H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, and -OP(=R a )(R b )R c , Q or its protective form or L', R 4 Each instance of these is independently represented as OH, SH, O - S - , OR d or SR d And, R 5 Each instance is independently either oxo, thioxo, or absent. R a is either O or S, R b OH, SH, O - S - , OR d or SR d And, Rc 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 It is a counterion, Q is a moiety containing a reactive group or its protected form, which is capable of independently forming a covalent bond with the analyte molecule, target-directing moiety, solid support, or complementary reactive group Q' each time it appears. L' is, independently of its appearance, a linker containing a covalent bond to Q, a linker containing a covalent bond to a target-directing moiety, a linker containing a covalent bond to the 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 (IV). Each instance of m is an independent integer greater than or equal to zero, provided that at least one instance of m is an integer greater than or equal to 1. n is an integer greater than or equal to 1. It will be provided.

[0153] In some embodiments, the L that appears 1c At least one of them is a 5- to 7-member heteroarylene linker, which may be substituted. In some embodiments, L 1c Each instance is independently substituted, or may be substituted, of a 5-7 member heteroarylene linker. In some embodiments, L 1c It is replaced each time it appears. For example, in some embodiments, L 1c Each instance is replaced by at least one oxo. In some embodiments, L 1c This is one of the following structures:

[0154] [ka] It holds. In some embodiments, the L that appears 1d’ At least one of them is replaced. For example, in some embodiments, L 1d’ It is substituted with oxo. In some embodiments, the L that appears 2 At least one of them is an alkylene linker. In some embodiments, L 2 Each time it appears, it is an alkylene linker. In some embodiments, the L that appears 3 At least one of them does not exist. In some embodiments, L 3 It does not exist each time it appears.

[0155] In some embodiments, L 4 and L 5 Each instance is an alkylene linker independently. In some embodiments, L 4 and L 5 Each instance is independently a C3-C6 alkylene. In some embodiments, at least one instance is L 4 and L 5 They are the same.

[0156] In some embodiments, L 6 Each instance is independently a heteroalkylene linker. In some embodiments, L 6 Each instance is an alkylene oxide linker. In some embodiments, L 6 Each instance is independently a polyethylene oxide. In some related embodiments, the compound has the following structure (IVa): [ka] (IVa) (In the formula, L 4 and L 5 Each instance is independently alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker, provided that L4 and L 5 This is provided that it is not polyethylene oxide. z is an integer between 1 and 100. It holds.

[0157] In some embodiments, L 4 and L 5 Each instance is an alkylene linker independently. In some more specific embodiments, L 4 and L 5 Each instance is independently a C3-C6 alkylene. In some embodiments, L 4 and L 5 Each instance is independently a C3 alkylene, a C4 alkylene, or a C6 alkylene. In some related embodiments, the compound has the following structure (IVb): [ka] (IVb) (In the formula, R 7 , R 8 , R 9 and R 10 Each instance is independently either H or alkyl. y 1 and y 2 Each occurrence is an independent integer between 1 and 6. z is an integer between 1 and 100. It holds.

[0158] In some embodiments, R 7 , R 8 , R 9 and R 10 Each instance is independently H. In some related embodiments, the compound is one of the following structures (IVc), (IVd), (IVe), or (IVf): [ka] (IVc); JPEG2023552448000095.jpg48161(IVd); JPEG2023552448000096.jpg49160(IVe) or JPEG2023552448000097.jpg48161(IVf) (In the formula, L 1d ' independently contains an amide functional group or a triazolyl functional group each time it appears, y 1 and y 2 Each occurrence is an independent integer between 1 and 6. z is an integer between 1 and 100. It holds. In some embodiments, z is an integer between 15 and 30. In other embodiments, z is an integer between 22 and 25.

[0159] In some embodiments, L 1d’ The structure is as follows: One of them: [ka] (In the formula, a, b, c, and d are each independent integers in the range of 1 to 6. It holds.

[0160] For example, in certain embodiments, L 1d’ This is one of the following structures: [ka] It holds. In some specific embodiments, L 1d’ These are alkylenes, such as ethylene, propylene, butylene, or pentylene. In some embodiments, L 2 Each instance is an alkylene linker (e.g., methylene). In some specific embodiments of compound (II), L 3 It does not exist each time it appears.

[0161] In other embodiments of structure (III) or (IV), G is a moiety containing a reactive group that, each instance, is independently capable of forming a covalent bond with a complementary reactive group. G in the compound of structure (III) or (IV) 1 or G 2 The portion can be selected from any portion that includes a group having a suitable reactive group for forming a covalent bond with a complementary group on the M portion. In an exemplary embodiment, G 1 or G 2 The portion can be selected from any of the Q portions described herein, including the specific examples shown in Table 1. In some embodiments, G 1 or G 2 Each instance independently includes a portion suitable for various substitution reactions, such as copper-catalyzed reactions forming triazoles with azides and alkynes (Huisgen's 1,3-dipolar cycloaddition), reactions of dienes with dienephiles (Diels-Alder), strain-promoted alkyne-nitrone cycloaddition, reactions of strained alkenes with azides, tetrazines, or tetrazoles, [3+2] cycloaddition of alkenes and azides, reverse demand Diels-Alder reactions of alkenes and tetrazines, photoreactions of alkenes and tetrazoles, and substitution of leaving groups by nucleophilic attack on electrophilic atoms. In some embodiments, G 1 or G 2 However, each instance independently contains an aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halogen, isothiocyanate, imide ester, activated ester, ketone, α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin, or thiirane functional group.

[0162] In other embodiments, G 1 or G 2 Each instance independently contains an alkyne group or an azide group. In other embodiments, G 1 or G 2Each instance independently contains an amino group, an isothiocyanate group, or an activated ester group. In different embodiments, G 1 or G 2 Each instance independently contains a reactive group capable of reacting with a complementary reactive group to form a functional group including an alkene, ester, amide, thioester, disulfide, carbocyclic, heterocyclic, or heteroaryl group. For example, in some embodiments, the heteroaryl is a triazolyl.

[0163] In either of the above embodiments of structure (III) or (IV), G 1 or G 2 Each instance has the following independent structure: [ka] Take one of them. In some embodiments of compound (III) or (IV), G 1 or G 2 Each instance is independent, [ka] That is the case.

[0164] In some embodiments of compound (III) or (IV), the G that appears 1 or G 2 At least one of them is a protected form of the amine. In some embodiments, G 1 or G 2 In multiple appearances, it is independently a protective form of the amine. In certain embodiments, G 1 or G 2 Each instance is an independent protective form of the amine.

[0165] In some embodiments, the G that appears 1 At least one of them [ka] In some embodiments, G 1 Each instance is independent, [ka] In some embodiments, the G that appears 2 At least one of them [ka] In some embodiments, G 2 Each instance is independent, [ka] In some embodiments, G 1 Each time it appears [ka] G 2 Each time it appears, [ka] That is the case.

[0166] In some of the embodiments described above, the amine protection form is a trifluoroacetate-protected amine. In some embodiments, the amine protection form is a BOC-protected amine. In some embodiments, the amine protection form is an Fmoc-protected amine. For example, in certain embodiments, the G that appears 1 or G 2 At least one of them has the following structure: [ka] Take one of them. In a more specific embodiment, G independently has the following structure for each occurrence: [ka] Take one of them. In some embodiments, R 2 , R 3 , R 4 , R 5 , L 2 , L3 , L 4 , L 5 or L 6 This is as defined in any one of the embodiments described above. For example, in some embodiments of compound (III) or (IV), R 4 Each instance is independently represented as OH, O-, or OR. d In some embodiments of compound (II) or (IV), R 5 Each time it appears, it is oxo.

[0167] In various other embodiments of the compound of structure (III) or (IV), R 2 and R 3 These are, independently, OH or -OP (=R a )(R b )R c In some different embodiments, R 2 or R 3 OH or -OP (=R a )(R b )R c And R 2 or R 3 The other is a linker that includes Q, or a covalent bond to Q. In any of the above-mentioned compounds of structure (III) or (IV), R 2 and R 3 These are, independently, -OP(=R a )(R b )R c In some of these embodiments, R c She is an office lady (OL). In other embodiments of structure (III) or (IV), R 2 and R 3 These are, independently, -OP(=R a )(R b )OL', where L' is a heteroalkylene linker to Q, a target-directing moiety, an analyte (e.g., an analyte molecule), a solid support, a solid support residue, a nucleoside, or a further compound of structure (III) or (IV). Linker L' can be any linker suitable for linking 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 (III) or (IV) to a compound of structure (III) or (IV). Advantageously, certain embodiments involve the use of an L' moiety selected to increase or optimize the water solubility of the compound. In some certain embodiments, L' includes an alkylene oxide or phosphodiester moiety, or a combination thereof.

[0168] In certain embodiments, L' has the following structure: [ka] (In the formula, m'' and n'' are independent integers between 1 and 10. R e H is an electron pair or counterion, L is R e (or Q, a target-directed moiety, an analyte (e.g., an analyte molecule), a solid support, a solid support residue, a nucleoside, or direct bonding or linkage to a further compound of structure (III) or (IV)) It holds.

[0169] In certain embodiments described above, the target-directing moiety is an antibody or a cell surface receptor antagonist. 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.

[0170] In any other more specific embodiment of the above-mentioned compounds of structure (III) or (IV), R 2or R 3 This is one of the following structures: [ka] It holds.

[0171] Certain embodiments of the compounds of structure (III) or (IV) can be prepared by solid-phase synthesis methods similar to those known in the art for the preparation of oligonucleotides. Therefore, in some embodiments, L' is a solid support, a solid support residue, or a linkage to a nucleoside. Solid supports containing activated deoxythymidine (dT) groups are readily available and, in some embodiments, can be used as starting materials for the preparation of the compounds of structure (III) or (IV). Therefore, in some embodiments, R 2 or R 3 The structure is as follows:

[0172] [ka] It holds.

[0173] In yet another embodiment of the compound of structure (III) or (IV), Q is a moiety comprising a reactive group that can independently form a covalent bond with the analyte molecule or solid support, each instance of which is independent. In another embodiment, Q is a moiety comprising a reactive group that can independently form a covalent bond with a complementary reactive group Q', each instance of which is independent. For example, in some embodiments, Q' is present in further compounds of structure (III) or (IV) (e.g., R 2 or R 3 At position (1), Q and Q' contain complementary reactive groups, and thus the reaction of a compound of structure (III) or (IV) with a further compound of structure (III) or (IV) yields a covalent dimer of the compound of structure (III) or (IV). Multimeric compounds of structure (III) or (IV) can also be prepared by similar methods and are included within the scope of embodiments of this disclosure.

[0174] The type of Q group and its ability to bind to the rest of the compound of structure (III) or (IV) are not limited, provided that Q contains a moiety that is sufficiently reactive to form the desired bond. In certain embodiments of the compound of structure (III) or (IV), Q is a portion that is not easily hydrolyzed under aqueous conditions but is sufficiently reactive to form a bond with the analyte molecule or the corresponding group on the surface of the solid support (e.g., an amine, azide, or alkyne).

[0175] Certain embodiments of the compounds of structure (III) or (IV) include a Q group 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, dienephile, acid halide, sulfonyl halogen, phosphine, α-haloamide, biotin, amino, or maleimide functional group. In some embodiments, the activated ester is an N-succinimide ester, imide ester, or polyfluorophenyl ester. In other embodiments, the alkyne is an alkyl azide or acyl azide. Exemplary Q portions relating to compounds of structure (III) or (IV) are shown in Table I above.

[0176] Similar to compounds of structures (A), (I), or (II), in some embodiments of compounds of structure (III) or (IV) where Q is SH, the SH moiety tends to form a disulfide bond with another sulfhydryl group of another compound of structure (III) or (IV). Thus, some embodiments include compounds of structure (III) or (IV) in the form of disulfide dimers, where the disulfide bond is derived from the SH Q group. In other embodiments of some compounds of structure (III) or (IV), R 2 or R 3 One of them is OH or -OP (=R a)(R b )R c And R 2 or R 3 The other linker is a linker containing a covalent bond to the analyte molecule, a linker containing a covalent bond to a target-directing moiety, or a linker containing a covalent bond to a solid support. For example, in some embodiments, the analyte molecule is a nucleic acid, an amino acid, or a polymer thereof. In 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-directing moiety is an antibody or a cell surface receptor antagonist. In yet another embodiment, the solid support is a polymer bead or a non-polymer bead.

[0177] In other embodiments of the compound of structure (III) or (IV), m is an integer from 0 to 10, independently of each occurrence. For example, in some embodiments, m is an integer from 1 to 5, such as 1, 2, 3, 4, or 5, independently of each occurrence. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In further different embodiments of the compound of structure (III) or (IV), n is an integer from 1 to 100. For example, in some 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. In some specific embodiments of the compounds of structure (III) or (IV), n is 3, and for each n value, the m values ​​are 1, 1, and 0 (e.g., compound IV-7). In other embodiments of the compounds of structure (III) or (IV), n is 2, and for each n value, the m values ​​are 1 and 0 (e.g., compound IV-8).

[0178] In further different embodiments of the compound of structure (III) or (IV), z is an integer from 1 to 30, e.g., 15 to 30, or 22 to 25. In some embodiments, z is 23. In some embodiments, z is 21, 22, 23, 24, or 25. In some embodiments, z is an integer from 1 to 10, e.g., 3 to 6. In some embodiments, z is 3, 4, 5, or 6.

[0179] In other different embodiments, the compound of structure (III) or (IV) is selected from Table 3. [Table 3] TIFF2023552448000114.tif197170 TIFF2023552448000115.tif250170 TIFF2023552448000116.tif228170 TIFF2023552448000117.tif212170 TIFF2023552448000118.tif245170

[0180] In various embodiments, G in the compounds in Table 3 1 or G 2 This is an alkynyl such as ethinyl. In other embodiments, G in the compounds in Table 3 1 or G 2 This is an azide. In other embodiments, G in the compounds in Table 3 1 or G 2 In other embodiments, G is present in the compounds in Table 3. 1 or G 2 is an isothiocyanate. In other embodiments, G in the compounds in Table 3 1 or G 2 These are activated esters such as esters of N-hydroxysuccinimide.

[0181] Compounds of structure (III) or (IV) can be used in a variety of ways, for example, in the embodiment, (a)R 2 or R 3 A step of mixing one of the compounds described in structure (III) or (IV), in which Q is a linker containing a covalent bond to Q, with the molecule to be analyzed or a target-directing moiety. (b) The step of forming a conjugate of the compound and the molecule to be analyzed or a target-directed moiety, and (c) The conjugate is given the formula ML 1b -G 1’ or G 2 By reacting with the compound of ', thereby at least one G 1 or G 2 and at least one G 1’ or G 2 The step of forming at least one covalent bond through the reaction of ' A method for labeling an analyte, such as an analyte molecule or a target-directing moiety, including, During the ceremony, M 1 and M 2 This is a moiety containing two or more carbon-carbon double bonds and at least one degree of conjugation. L 1b This is an alkylene, heteroalkylene, or heteroatom linker that may be used as desired. G 1’ or G 2 ' are, G 1 or G 2 A reactive group that is complementary to it, A method is provided.

[0182] Different embodiments include: (a) A compound of structure (III) or (IV) disclosed herein (wherein R 2 or R 3 (This is a linker that includes Q, or a covalent bond to Q) 1b The steps include mixing with the compound of -G', thereby forming at least one covalent bond through the reaction of G and G', and (b) A step in which the product of step (A) is reacted with the analyte molecule or target-directed moiety to form a conjugate between the product of step (A) and the analyte molecule or target-directed moiety. A method for labeling an analyte, such as an analyte molecule or a target-directing moiety, including, During the ceremony, M 1 and M 2 This is a moiety containing two or more carbon-carbon double bonds and at least one degree of conjugation. L 1b This is an alkylene, heteroalkylene, or heteroatom linker that may be used as desired. G 1’ or G 2 ' are, G 1 or G 2 A reactive group that is complementary to it, It is a method.

[0183] Furthermore, as stated above, the compound of structure (III) is useful for preparing the compound of structure (I), and the compound of structure (IV) is useful for preparing the compound of structure (II). Therefore, in one embodiment, a method for preparing the compound of structure (I) is provided, wherein the compound of structure (III) is prepared using the formula ML 1b A method is provided comprising the step of mixing a compound of -G' with a compound of formula ML, thereby forming at least one covalent bond through a reaction between G and G'. In another embodiment, a compound of structure (IV) is used with formula ML 1b A method for preparing a compound of structure (II), comprising the step of mixing it with a compound of -G', thereby forming at least one covalent bond through the reaction of G and G', During the ceremony, M 1 and M 2 This is a moiety containing two or more carbon-carbon double bonds and at least one degree of conjugation, or M 1 and M 2 Each instance is an independent chromophore, however M 1 and M 2 At least one of them is a FRET donor, M 1 and M2 The other one is a corresponding FRET acceptor, L 1b This is an alkylene, heteroalkylene, or heteroatom linker that may be used as desired. G' is a reactive group complementary to G. A method is provided. The following examples are provided for illustrative purposes only and are not limiting. [Examples]

[0184] General method Mass spectroscopy was performed using a Waters / Micromass Quattro micro-MS / MS system (MS mode only) with MassLynx 4.1 acquisition software. For LC / MS analysis of the dyes, the mobile phase used was 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6 mM triethylamine (TEA), pH 8. Phosphoramidite and precursor molecules were also analyzed using a Waters Acquity UHPLC system with a 2.1 mm x 50 mm Acquity BEH-C18 column maintained at 45°C, using an acetonitrile / water mobile phase gradient. Molecular weights of monomer intermediates were obtained using tropylium cation implantation-enhanced ionization on a Waters / Micromass Quattro micro-MS / MS system (MS mode only). Excitation and emission profile experiments were recorded using a Cary Eclipse spectrophotometer. All reactions were carried out under a nitrogen atmosphere in oven-dried glassware unless otherwise specified. Commercial 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.

[0185] (Example 1) Synthesis of dyes containing alkylene-polyethylene glycol-alkylene spacers Compounds containing alkylene-polyethylene oxide-alkylene linkers were prepared as follows: Oligofluoroside constructs (i.e., compounds of structure (I) or (II)) were synthesized on a 1 μmol scale using an Applied Biosystems 394 DNA / RNA synthesizer and possessed either a 3'-phosphate group, a 3'-S2-(CH2)6-OH group, or any other group described herein. Synthesis was carried out directly on CPG beads or polystyrene solid supports using standard phosphoramidite chemistry. Oligofluorosides were synthesized in the 3'-to-5' direction using the standard solid-phase DNA method, and coupling was performed using the standard β-cyanoethyl phosphoramidite reaction. 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 prepare 0.1 M solutions, which were then added in a sequential order using the following synthesis cycle: 1) Remove the 5'-dimethoxytrityl protecting group with dichloroacetic acid in dichloromethane; 2) Couple the next phosphoramidite with an activating reagent in acetonitrile; 3) Oxidize P(III) with iodine / pyridine / water to form stable P(v); and 4) Cap any unreacted 5'-hydroxyl groups with acetic anhydride / 1-methylimidazole / acetonitrile. The synthesis cycle was repeated until a full-length oligofluoroside construct was assembled. At the end of chain assembly, the monomethoxytrityl (MMT) group or dimethoxytrityl (DMT) group was removed with dichloroacetic acid in dichloromethane.

[0186] These compounds were supplied to a controlled-pore glass (CPG) support in a labeled Eppendorf tube at a scale of 0.2 μL. 400 μL of 20-30% NH4OH was added and gently mixed. The open tube was left at 55°C for approximately 5 minutes, or until excess gas was released, then tightly sealed and incubated for 2 hours (+ / - 15 minutes). The tube was removed from the heating block, allowed to reach room temperature, and then centrifuged at 13,400 RPM for 30 seconds to obtain the supernatant and solid. The supernatant was carefully removed and placed in the labeled tube, and then the support was washed with 150 μL of acetonitrile. After adding the washing solution to the tube, it was placed in a CentriVap apparatus at 40°C until dry. The products were characterized by ESI-MS, UV absorbance, and fluorescence spectroscopy.

[0187] (Example 2) Cumulative staining index from flow cytometry measurements Representative compounds of structure (I), which include propylene / phosphate / polyethylene glycol / phosphate / propylene spacer (spacder) (C3-PEG-C3) and 3, 5, 7, or 10 fluorophore moieties (referred to as C3-3xFAM, C3-5xFAM, C3-7xFAM, and C3-10xFAM, respectively), were prepared in accordance with Example 1. Flow cytometry measurements were performed on representative compounds of structure (I), as well as control compounds (PEG-5xFAM), fluorescein (FITC), and Alexa Fluor® 488 dye (AF488). The results are shown in Figure 1. Structure (I) (C3-3xFAM, C3-5xFAM, C3-7xFAM, and C3-10xFAM), control compound (PEG-5xFAM), and representative compounds of FITC and AF488 are presented in Table 4.

[0188] [Table 4] TIFF2023552448000120.tif202170

[0189] As used in Table 4, R 2 and R 3 Each of them (respectfully) has the following structure: [ka] This refers to the part that has [something].

[0190] Data on staining index indicate that, by introducing a linker containing a propylene (C3) linker group around the PEG linker group, the compounds according to the embodiments of this disclosure have a brightness approximately 4 to 7 times higher than that of FITC and AF488. The compounds according to the embodiments of this disclosure also exhibit higher brightness than the corresponding control compound (PEG-5xFAM) containing the same amount of fluorophore (i.e., dye). The increase in brightness may be due to greater spatial isolation between fluorophore portions by introducing a propylene linker group around the PEG.

[0191] (Example 3) Comparative fluorescence emission response over time The fluorescence emission stability of compounds of structure (I), including a C3-PEG-C3 spacer and 3, 5, 7, or 10 fluorophore moieties (i.e., compounds C3-3xFAM, C3-5xFAM, C3-7xFAM, and C3-10xFAM, as presented in Table 4), was compared with the corresponding control compounds FITC and AF488. The results are summarized in Table 5. The data show that the compounds according to the embodiments of this disclosure increase their fluorescence emission over time. After 3 weeks, the brightness of all compounds according to the embodiments of this disclosure increased, while the brightness of the control compounds, FITC, and AF488 remained unchanged. The increase in brightness over time may be due to increased sequestration efficiency as the compounds of structure (I) "age" over time (Figure 2).

[0192] [Table 5]

[0193] (Example 4) General flow cytometry methods Unless otherwise specified, the following general procedures were used throughout the following examples. Dissolution of total 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) using 15 mL of blood in 35 mL of solvent at room temperature (RT) for 15 minutes. These cells were washed twice with 50% Hanks buffer solution (HBSS) containing 0.02% sodium azide, 50% 1% fetal bovine serum (FBS), and 1x Dulbecco's phosphate-buffered saline (PBS). Next, these cells were resuspended in donor plasma at a rate of 100 μL / test sample / 0.1~1x10 e6. In a 96-well polypropylene HTS plate, 100 μL of 1% bovine serum albumin (BSA) containing 0.02% sodium azide and 1x DPBS were used for fluoride staining. f Cells in plasma were added to the pre-diluted antibody. 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.

[0194] Dissolution / Fixation Method. Blood was dissolved in 1.0 mL of RBC dissolution solution (ammonium chloride) in 35 mL of solubil, in 100-15 mL of blood, at room temperature for 15 minutes. Next, the cells were washed twice with 50% HBSS containing 0.02% sodium azide and 50% 1% FBS 1x DPBS. Then, the cells were resuspended in donor plasma at a rate of 100 μL / test sample / 1x10 e6. The pre-diluted antibody was added to 100 μL of 1% BSA and 1x DPBS containing 0.02% sodium azide. 100 μL of cells were added to a 96-well polypropylene HTS plate (total volume 200 μL test size). 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.

[0195] Preparation of antibody conjugates The antibody conjugate has a Q portion with the following structure: [ka] Compounds of structure (I) or (II) containing the above were prepared by reacting them with a desired antibody. The compound and the antibody were then conjugated by the reaction of S on the antibody having the Q moiety, forming the following linked structure:

[0196] [ka] Antibody conjugates are indicated by the antibody name, which follows the compound number. For example, UCHT1-C3-3xFx indicates a conjugate formed between the UCHT1 antibody and a compound of structure (I) containing a C3 linker and three fluorophos (Fx). If the referenced compound number does not contain the upper Q portion in Table 1, it is understood that this Q portion was introduced, and the conjugate was prepared from the resulting compound having the Q portion.

[0197] 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 ranging from 0.1 to 540 nM (less than 8.0 micrograms per test). In some cases, serial dilutions of each sample were started with 269 nM antibody in cell staining buffer, and the antibody dilutions were kept protected from light until use. In other experiments, dilutions were started with 4.0 μg antibody / test size, and the test size ranged from 100 to 200 μL. Titration was performed with 2-fold or 4-fold dilutions to generate binding curves. In some cases, 8.0 or 2.0 μg / test size was used in the first well of the dilution series.

[0198] Flow cytometry using conjugates: Following physical characterization, the activity and functionality (antibody binding affinity and dye brightness) of the conjugates were tested and compared with reference antibody staining. Next, the quality of resolution was determined by brightness compared to an automated fluorescence-negative control and by examining other nonspecific bindings using a flow cytometer. A standard whole blood screening was performed to test the conjugates. Once the novel constructs were formed, bridging studies were conducted.

[0199] Perform free dye flow cytometry: Following molecular and physical characterization, the potential affinity of the above dyes to cells was also tested compared to staining with reference dyes. Since dyes also function as cell probes and have the potential to bind to cellular material, dyes can generally be screened against blood at high concentrations (>100 nM to 10,000 nM) to confirm specific characteristics. Next, using a flow cytometer, expected or unexpected off-target binding was quantified by evaluating brightness and linearity at dilution compared to autofluorescence-negative controls and other dye controls.

[0200] Flow cytometry workflow: Cells were cultured and observed for visual signs of metabolic load related to dye screening or off-target binding (data not shown), or fresh, healthy cells were used for conjugate screening. Cells were periodically counted to confirm cell density (1 × 10⁵ and 1 × 10⁶ viable cells / mL). After diluting the antibody conjugate (preferably in a plate or tube), cells were collected in staining buffer (DPBS, 0.1% BSA, 0.02% sodium azide). Cells with viability in the range of 80–85% were used. Cells were washed twice by centrifugation and washing with buffer to remove pH indicators and block cells containing Ig and other proteins present in FBS. Cell density was adjusted and size was tested in staining buffer. Cells were plate-cultured and tested once per well or per dye (pre-diluted) on the cells in the plate. These cells were then incubated at 23°C for 45 minutes. These cells were centrifuged and washed twice with washing 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 these dyes was detected by flow cytometry using a 488 nM blue laser beam, with peak emission (521 nM) detected using a 525 / 50 bandpass filter. With a goal of obtaining 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.

[0201] Data analysis methods: Descriptive statistics. The EC-800 software allows users to correct numerous statistical data related to the acquisition of each sample. The brightness of the antibody-dye reagent was measured by flow cytometry, using the mean or median fluorescence intensity (MFI) in the FL1-A channel, after scrutinizing for noise. Other statistics were evaluated to determine the characteristics of the dye and the overall quality of the reagent, including median signal vs. noise and absolute fluorescence (median or geometric mean). Histogram. Flow cytometry events were gated by size relative to forward scatter versus side scatter (cell volume versus cell granularity). Next, the mean fluorescence intensity (MFI) of those cells was gated by fluorescence emission at 515 nm. The collected data were represented as a dual-parameter histogram plotted as the number of events against fluorescence intensity on the y-axis, and this was plotted on a logarithmic scale with respect to x-axis. The data can be summarized by affinity curves or histograms of relative fluorescence intensity.

[0202] The binding curve. The Multi-Fibrosis Index (MFI) is selected because, when examined by FCM, it best measures the brightness of the antibody-dye reagent. This MFI can be expressed as a geometric mean, median, or average value, and represents an absolute fluorescence measurement. For comparison, if noise can be highly characteristic, the signal-to-noise ratio (S / N) is reported as the MFI. Bivariate dual-parameter histogram. In some cases, to examine qualitative output values, FCM events were not gated, and data were represented by cell size (SSC) versus dye fluorescence intensity. This method allows for a comprehensive assessment of the entire population collected in whole blood.

[0203] (Example 5) Flow cytometry analysis of control compounds (spacer = PEG) and structure (I) sequence constructs (spacer = propylene / phosphate / PEG / phosphate / propylene (C3-PEG-C3)) against various dyes was performed by conjugating CD4 (clone OKT4) antibody and eluting in 1xd-PBS (phosphate-buffered saline). Dyes used included FAM, Pacific Blue (PB), Alexa Fluor® 532 (AF532), and Alexa Fluor® 660 (AF660). Whole blood was stained using 0.5 ug of antibody conjugate and screened using a spectral instrument. Table 6 shows the difference in staining index (SI) between a control compound with a PEG spacer and a compound with structure (I) having a C3-PEG-C3 spacer. Compounds with structure (I) having a C3-PEG-C3 spacer have higher SI values ​​than the corresponding control compound with a PEG spacer and existing commercially available compounds. Compounds with structure (I) having a C3-PEG-C3 spacer also show an improvement in SI value as the number of fluorophores on the main chain increases.

[0204] [Table 6] TIFF2023552448000126.tif241170 TIFF2023552448000127.tif251167 TIFF2023552448000128.tif149170

[0205] As used in Table 6, R 2 and R 3 Each has the following structure: [ka] This refers to the part that has [something]. FAM is one of the following structures: [ka] This refers to the part that has [something]. PB has the following structure: [ka] This refers to the part that has [something]. The AF532 has the following structure: [ka] This refers to the part that has [something]. The AF660 has the following structure: [ka] This refers to the part that has [something].

[0206] (Example 6) A CD3 (clone UCHT1) antibody was conjugated to either the control compound (PEG-5x-FAM) or a construct of the compound with structure (I) (C3-3x-FAM, C3-5x-FAM, C3-7x-FAM, or C3-10x-FAM). 0.5 ug of antibody conjugate was diluted in Dulbecco 1xPBS, mixed using reverse pipetting, and analyzed with a Synergy plate reader at 488 nm emission. The degree of labeling (DOL) was calculated by measuring the signal intensity and dividing by the DOL, and then graphed. The results of the in vitro analysis of the control compound (PEG-5xFAM) and the compounds with structure (I) (C3-3xFAM, C3-5xFAM, C3-7xFAM, or C3-10xFAM) are summarized in Figure 3. As shown in Figure 3, the C3-10xFAM construct is as bright as the PEG-5xFAM construct. As the number of fluorophores on the back chain of the compound in structure (I) increases, the signal intensity of the corresponding compound improves. For the C3-7xFAM and C3-10xFAM constructs, the signal is so bright that it is off the scale at 0.5 ug.

[0207] (Example 7) The dye constructs were conjugated with CD3 (clone UCHT1) antibody and eluted in 1xd-PBS. Whole blood was stained using 0.5 ug of antibody conjugate and screened using a spectral instrument. Previous studies have shown that cyclodextrin (CD) increases the brightness of FAM when analyzed in aqueous solution. The constructs of the compound of structure (I) (C3-5xFAM and C3-7xFAM) are brighter than the control compound construct (PEG-5x-FAM), even when cyclodextrin (CD) is added to the maleimide of the control compound construct (PEG-5x-FAM). The effect of buffers on the staining index of the control compound and the construct of the compound of structure (I) is summarized in Table 7. The structures of the compounds PEG-5xFAM, C3-5xFAM, and C3-7xFAM are shown in Table 6.

[0208] [Table 7] Next, 2 mM NaPO4 buffer or 2 mM NaPO4 buffer + 5 mM cyclodextrin (CD) was lyophilized. Then, the maleimide sample was resuspended in water and conjugated with CD3 antibody, and eluted in 1xd-PBS. 0.5 ug of antibody conjugate was diluted in Dulbecco 1xPBS, mixed using reverse pipetting, and analyzed with a Synergy plate reader at 488 nm emission. Degree of Labelling (DOL) was calculated by measuring the signal intensity and dividing by DOL, and then graphed. The effect of buffer on the control compound construct PEG-5xFAM and the constructs C3-5xFAM and C3-7xFAM of the compound of structure (I) is illustrated in Figure 4. As shown in Figure 4, the constructs C3-5xFAM and C3-7xFAM of the compound of structure (I) are brighter than the control compound construct PEG-5xFAM, even with the addition of CD.

[0209] (Example 8) Preparation of phosphoramidites and compounds The exemplary compound is based on a standard solid-phase oligonucleotide synthesis protocol and was purchased from ChemGenes, and has the following structure: [ka] It was prepared using a fluorescein-containing phosphoramidite (catalog number CLP-9780) containing [specific compound / component]. An example linker (L 6 ) is actually the commercially available structure as follows: [ka] It was incorporated into the compound by coupling with a phosphoramidite having the specified property.

[0210] An example linker (L 4 / L 5 ) is actually the commercially available structure as follows: [ka] It was incorporated into the compound by coupling with a phosphoramidite containing one of the following:

[0211] Other exemplary compounds are shown in the following scheme: [ka] It was prepared using phosphoramidite prepared according to the following method.

[0212] The final deprotection generates the desired Fx moiety. Various parts of this compound were introduced using other commercially available phosphoramidite reagents as appropriate. The Q moiety having the following structure: [ka] teeth,

[0213] [ka] It was introduced by reaction with free sulfidyls. The other Q portion is introduced by similar methods according to the knowledge of those skilled in the art.

[0214] Further embodiments are derived by combining the various embodiments described above. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein, including U.S. Provisional Patent Application No. 63 / 122,285 filed December 7, 2020, and U.S. Provisional Patent Application No. 63 / 219,706 filed July 8, 2021, are incorporated herein by reference. The aspects of the embodiments may be modified, where necessary, to produce further embodiments using various patent, application, and publication concepts. Such and other modifications may be made to the embodiments in light of the detailed description above. In general, the terms used in the following claims should not be construed as limiting the claims to specific embodiments disclosed herein and herein, but rather as including all possible embodiments encompassing the entire scope of equivalents granted to such claims. Therefore, the claims are not limited by this disclosure.

Claims

1. A compound having the following structure (A) or a stereoisomer, salt, or tautomer thereof: 【Chemistry 1】 (A) (In the formula, M 1 and M. 2 is, independently at each occurrence, a chromophore and contains three or more aryl or heteroaryl rings, or combinations thereof, with the proviso that M 1 and M. 2 At least one of is a FRET donor, M 1 and M. 2 the other being the corresponding FRET acceptor; L 1a is, independently at each occurrence, a heteroarylene linker; L 1 , L 1b , L 2 and L 3 is independently at each occurrence an optionally used alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, heteroarylene, or heteroatom linker; L 4 and L 5 is, independently at each occurrence, an alkylene linker; L 6 is, independently at each occurrence, an alkylene oxide or polyethylene oxide linker, provided that each occurrence of L 6 At least one of the following is O-L 4 and O-L 5 provided that each of the R 1 is independently at each occurrence H, alkyl or alkoxy; R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a ) (R b ) R c , Q or L′; R 4 are independently selected for each occurrence: OH, SH, O - , S - , OR d or S.R. d and R 5 is independently at each occurrence oxo, thioxo, or absent; R a is O or S, R b OH, SH, O - , S - , OR d or S.R. d and R c OH, SH, O - , S - , OR d , O.L.', S.R. d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counter ion, Q is, independently at each occurrence, a moiety that contains a sulfhydryl, disulfide, N-succinimide ester, imidoester, polyfluorophenyl ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, or maleimide functional group; L', at each occurrence, is a heteroalkylene linker to Q, a targeting moiety selected from an antibody and a cell surface receptor antagonist, an analyte molecule, a solid support, or a nucleoside; m is, independently at each occurrence, an integer greater than or equal to zero, provided that at least one occurrence of m is an integer greater than or equal to 1; n is an integer of 1 or more, q and w are, independently at each occurrence, an integer from 0 to 3, provided that at least one occurrence of either q or w is 1.

2. A compound having the following structure (I): or a stereoisomer, salt, or tautomer thereof: 【Chemistry 2】 (I) (In the formula, M 1 and M. 2 is, independently at each occurrence, a fluorescent moiety that comprises three or more aryl or heteroaryl rings, or a combination thereof; L 1 is, independently at each occurrence, an alkylene, heteroalkylene, heteroarylene, or a heteroatom linker; L 2 and L 3 is, independently at each occurrence, an optional alkylene linker; L 4 and L 5 is, independently at each occurrence, an alkylene linker; L 6 is, independently at each occurrence, an alkylene oxide or polyethylene oxide linker, provided that each occurrence of L 6 At least one of the following is O-L 4 and O-L 5 provided that each of the R 1 is independently at each occurrence H, alkyl or alkoxy; R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a ) (R b ) R c , Q or L′; R 4 are independently selected for each occurrence: OH, SH, O - , S - , OR d or S.R. d and R 5 is independently at each occurrence oxo, thioxo, or absent; R a is O or S, R b OH, SH, O - , S - , OR d or S.R. d and R c OH, SH, O - , S - , OR d , O.L.', S.R. d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counter ion, Q is, independently at each occurrence, a moiety that contains a sulfhydryl, disulfide, N-succinimide ester, imidoester, polyfluorophenyl ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, or maleimide functional group; L', at each occurrence, is a heteroalkylene linker to Q, a targeting moiety selected from an antibody and a cell surface receptor antagonist, an analyte molecule, a solid support, or a nucleoside; m is, independently at each occurrence, an integer greater than or equal to zero, provided that at least one occurrence of m is an integer greater than or equal to 1; n is an integer of 1 or more.

3. A) A compound having the following structure (Ia): 【Chemistry 3】 (Ia) (In the formula, L 4 and L 5 is independently at each occurrence a C 3 -C 6 alkylene and z is an integer from 1 to 100; B) a compound having the following structure (Ib): 【Chemistry 4】 (Ib) (In the formula, R 7 , R 8 , R 9 and R 10 is, independently at each occurrence, H or alkyl; y 1 and y 2 is, independently at each occurrence, an integer from 1 to 6, preferably R 7 , R 8 , R 9 and R 10 is, independently for each occurrence, H; or C) A compound having the following structure (Ic): 【Chemistry 5】 (I C) (In the formula, x 1 , x 2 , x 3 and x 4 is, independently at each occurrence, an integer from 0 to 6; y 1 and y 2 is, independently at each occurrence, an integer from 1 to 6, preferably i) x 1 and x 3 are 0 for each occurrence, and x 2 and x 4 are each 1 for each occurrence; or ii) x 1 , x 2 , x 3 and x 4 are 1 for each occurrence; and iii) y 1 and y 2 are 3 for each occurrence; iv) y 1 and y 2 is 4 for each occurrence; or v) y 1 and y 2 is 6 for each occurrence; and vi) z is an integer from 22 to 25; The compound according to claim 2.

4. A) Appearing L 1 for at least one of the functional groups comprises an alkene, ester, amide, thioester, thiourea, disulfide, carbocyclic, heterocyclic, or heteroaryl group; B) Appearing L 1 For at least one of 1 is a linker comprising a triazolyl functional group; C) L 1 but one of the following structures: 【Chemistry 6】 having D) Appearing L 1 For at least one of 1 -M 1 Or L 1 -M 2 but one of the following structures: 【Chemistry 7】 (In the formula: i) L 1a and L 1b are each independently an optional linker; ii) L 1a Or L 1b , or neither is present; iii) L 1a Or L 1b , or both are present; iv) L 1a and L 1b each, if present, is independently alkylene or heteroalkylene; or v) L 1a and L 1b is, when present, independently one of the following structures: 【Chemistry 8】 having or E) M 2 -L 1b has the following structure: 【Chemistry 9】 having and F) L 2 and L 3 But for each occurrence, C 1 -C 6 is alkylene; and G) R 1 A compound according to any one of claims 1 to 3, wherein each occurrence of is H.

5. A compound having the following structure (II): or a stereoisomer, salt, or tautomer thereof: 【Chemistry 10】 (II) (In the formula, M 1 and M. 2 is, independently at each occurrence, A) a fluorescent moiety comprising three or more aryl or heteroaryl rings, or a combination thereof, or B) a chromophore comprising three or more aryl or heteroaryl rings, or a combination thereof, provided that at least one of M 1 and M 2 is a FRET donor and the other of M 1 and M 2 is a corresponding FRET acceptor; L 1c is, independently at each occurrence, a heteroarylene linker; L 1d , L 2 and L 3 is independently at each occurrence an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; L 4 and L 5 is, independently at each occurrence, an alkylene linker; L 6 is, independently at each occurrence, an alkylene oxide or polyethylene oxide linker, provided that each occurrence of L 6 At least one of the following is O-L 4 and O-L 5 provided that each of the R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a ) (R b ) R c , Q or L′, R 4 are independently selected for each occurrence: OH, SH, O - , S - , OR d or S.R. d and R 5 is independently at each occurrence oxo, thioxo, or absent; R a is O or S, R b OH, SH, O - , S - , OR d or S.R. d and R c OH, SH, O - , S - , OR d , O.L.', S.R. d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counter ion, Q is, independently at each occurrence, a moiety that contains a sulfhydryl, disulfide, N-succinimide ester, imidoester, polyfluorophenyl ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, or maleimide functional group; L', at each occurrence, is a heteroalkylene linker to Q, a targeting moiety selected from an antibody and a cell surface receptor antagonist, an analyte molecule, a solid support, or a nucleoside; m is, independently at each occurrence, an integer greater than or equal to zero, provided that at least one occurrence of m is an integer greater than or equal to 1; n is an integer of 1 or more.

6. A) Appearing L 1d at least one of comprises an alkene, ester, amide, thioester, disulfide, carbocyclic, heterocyclic, or heteroaryl group; B) Appearing L 1d At least one of contains a triazolyl functional group; C) Appearing L 1d -M 1 Or L 1d -M 2 At least one of the following structures: 【Chemistry 11】 (In the formula, L a and L b are each independently an optional linker: i) L a Or L b , or neither is present; ii) L a Or L b , or both are present; iii) L a and L b each, if present, is independently alkylene or heteroalkylene; iv) L a and L b has the following structure: 【Chemistry 12】 (independently having one of the following): Contains one of the following: D) L 1d independently at each occurrence, comprises an alkylene or heteroalkylene linker; or E) L 1d has the following structure: 【Chemistry 13】 (In the formula, a, b, c and d are each independently an integer ranging from 1 to 6. and F) Appearing L 1c at least one of is an optionally substituted 5- to 7-membered heteroarylene linker; G) L 1c is independently at each occurrence an optionally substituted 5- to 7-membered heteroarylene linker; or H) L 1c has the following structure: 【Chemistry 14】 and I) Appearing L 2 at least one of is an alkylene linker; or J)L 2 is an alkylene linker; and K) Appearing L 3 is absent; or L) L 3 is not present for each occurrence; and M) L 4 and L 5 But for each occurrence, C 3 -C 6 is alkylene; or N) For at least one occurrence, L 4 and L 5 are the same; and O) L 6 is, independently at each occurrence, an alkylene oxide linker; or P)L 6 is, independently at each occurrence, polyethylene oxide; The compound according to claim 5.

7. A) A compound having the following structure (IIa): 【Chemistry 15】 (IIa) (In the formula, L 4 and L 5 is, independently at each occurrence, C 3 -C 6 alkylene and z is an integer from 1 to 100; B) A compound having the following structure (IIb): 【Chemistry 16】 (IIb) (In the formula, R 7 , R 8 , R 9 and R 10 is, independently at each occurrence, H or alkyl; y 1 and y 2 is, independently at each occurrence, an integer from 1 to 6; z is an integer from 1 to 100, preferably R 7 , R 8 , R 9 and R 10 is, independently at each occurrence, H; or C) A compound having one of the following structures (IIc), (IId), (IIe) or (IIf): 【Chemistry 17】 (IIc); 【change】 (IId); 【change】 (IIe); or 【change】 (IIf) (In the formula, L 1d contains, independently at each occurrence, an amide functionality or a triazolyl functionality; y 1 and y 2 is, independently at each occurrence, an integer from 1 to 6; z is an integer from 1 to 100, preferably z is an integer from 1 to 6; The compound according to any one of claims 5 to 6.

8. A) R 4 However, each occurrence is independent, OH, O - OR d is; B) R 5 is, at each occurrence, oxo; and C) R 2 and R 3 are each independently OH or OP(=R a ) (R b ) R c is; D) R 2 Or R 3 One of the groups is OH or OP (=R a ) (R b ) R c and R 2 Or R 3 the other is Q or a linker comprising a covalent bond to Q; E) R 2 and R 3 Each independently represents -OP(=R a ) (R b ) R c and preferably, R c is OL', i) L' comprises an alkylene oxide moiety or a phosphodiester moiety, or a combination thereof; or ii) L' has the structure: 【Chemistry 18】 (In the formula, m″ and n″ are independently integers from 1 to 10; R e is H, an electron pair or a counter ion, L” is R e or a direct bond or linkage to Q, a targeting moiety, an analyte molecule, a solid support, or a nucleoside, wherein the targeting moiety is an antibody, the antibody having a specific binding domain comprising 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; F) R 2 Or R 3 has the following structure: 【Chemistry 19】 【change】 ） and F) Q is the following: 【Chemistry 20】 wherein X is halo and EWG is an electron withdrawing group; The compound according to any one of claims 1 to 7.

9. A) M 1 and M. 2 independently at each occurrence, comprises a fused polycyclic aryl moiety comprising at least four fused rings; B) M 1 and M. 2 is independently at each occurrence a dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine, terylene, 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; C) M 1 and M. 2 is independently at each occurrence p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, or perylene amide, or a derivative thereof; D) M 1 and M. 2 is independently at 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; E) M 1 and M. 2 is independently at each occurrence pyrene, perylene, perylene monoimide, or 6-FAM, or a derivative thereof; F) M 1 and M. 2 may, independently at each occurrence, have the structure: 【Chemistry 21】 or G)M 1 and M. 2 may, independently for each occurrence, be one of the following structures: 【Chemical 22】 【change】 【change】 【change】 having one of: The compound according to any one of claims 1 to 8.

10. The compound of claim 1, wherein the compound has the following structure: 【Chemistry 23】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 and Fx independently for each occurrence has the structure: 【change】 having one of: M 1 is FAM, and M 2 Is AF594 or M 1 is Cy3, M 2 is AF680; FAM has the following structure: 【change】 AF594 has the following structure: 【change】 AF594 having Cy3 has the following structure: 【change】 Cy3 having AF680 has the following structure: 【change】 AF680 having R 2 has the following structure: 【change】 having R 3 has the following structure: 【change】 having dT has the following structure: 【change】 where R is H or a direct bond. having z is an integer from 22 to 25; The compound according to any one of claims 1 to 9, wherein n is an integer from 1 to 10.

11. 1. A method for visually detecting an analyte molecule, comprising: (a) A) R 2 Or R 3 is a linker to the analyte molecule; B) R 2 Or R 3 is a linker to Q or Q; or C) R 2 Or R 3 providing a compound according to any one of claims 1 to 10, wherein said compound comprises a linker to a targeting moiety having specificity for an analyte; (b) mixing the compound and the analyte molecule, thereby allowing the targeting moiety to associate with the analyte; (c) detecting the compound by its visible properties; The method of claim 1, wherein the targeting 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.

12. A compound having the following structure (III): or a stereoisomer, salt, or tautomer thereof: 【Chemistry 24】 (III) (In the formula, G 1 and G 2 comprises, independently at each occurrence, an aldehyde, oxime, hydrazone, alkyne, amino, 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 functional group; L1', L 2 and L 3 is independently at each occurrence an optionally used alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, heteroarylene, or heteroatom linker; L 4 and L 5 is, independently at each occurrence, an alkylene linker; L 6 is, independently at each occurrence, an alkylene oxide or polyethylene oxide linker, provided that each occurrence of L 6 At least one of the following is O-L 4 and O-L 5 provided that each of the R 1 is independently at each occurrence H, alkyl or alkoxy; R 2 and R 3 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 4 are independently selected for each occurrence: OH, SH, O - , S - , OR d or S.R. d and R 5 is independently at each occurrence oxo, thioxo, or absent; R a is O or S, R b OH, SH, O - , S - , OR d or S.R. d and R c OH, SH, O - , S - , OR d , O.L.', S.R. d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counter ion, Q is, independently at each occurrence, a sulfhydryl, disulfide, N-succinimide ester, imidoester, polyfluorophenyl ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, or maleimide functionality; L' is, independently at each occurrence, a heteroalkylene linker to Q, the targeting moiety, the analyte molecule, the solid support, the nucleoside; m is, independently at each occurrence, an integer greater than or equal to zero, provided that at least one occurrence of m is an integer greater than or equal to 1; n is an integer of 1 or more.

13. A) The following structure (IIIa): 【Chemistry 25】 (IIIa) (In the formula, L 4 and L 5 is expressed independently for each occurrence as C 3 -C 6 is alkylene, z is an integer from 1 to 100. A compound having the formula: B) The following structure (IIIb): 【Chemistry 26】 (IIIb) (In the formula, R 7 , R 8 , R 9 and R 10 is, independently at each occurrence, H or alkyl; y 1 and y 2 is, independently at each occurrence, an integer from 1 to 6; z is an integer from 1 to 100, preferably R 7 , R 8 , R 9 and R 10 is, independently for each occurrence, H) or a compound having C) The following structure (IIIc): 【Chemistry 27】 (IIIc) (In the formula, x 1 , x 2 , x 3 and x 4 is, independently at each occurrence, an integer from 0 to 6; y 1 and y 2 is, independently at each occurrence, an integer from 1 to 6; z is an integer from 1 to 100, preferably i) x 1 and x 3 are 0 for each occurrence, and x 2 and x 4 are each 1 for each occurrence; or ii) x 1 , x 2 , x 3 and x 4 are 1 for each occurrence; and iii) y 1 and y 2 are 3 for each occurrence; iv) y 1 and y 2 is 4 for each occurrence; or v) y 1 and y 2 are each 6 for each occurrence; and vi) z is an integer from 22 to 25. is a compound having the formula 13. The compound of claim 12.

14. A compound having the following structure (IV): 【Chemistry 28】 (IV) (In the formula, G 1 and G 2 comprises, independently at each occurrence, an aldehyde, oxime, hydrazone, alkyne, amino, 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 functional group; L 1c is, independently at each occurrence, a heteroarylene linker; L 1d’ , L 2 and L 3 is independently at each occurrence an optionally used alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker; L 4 and L 5 is, independently at each occurrence, an alkylene linker; L 6 is, independently at each occurrence, an alkylene oxide or polyethylene oxide linker, provided that each occurrence of L 6 At least one of the following is O-L 4 and O-L 5 provided that each of the R 2 and R 3 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 4 are independently selected for each occurrence: OH, SH, O - , S - , OR d or S.R. d and R 5 is independently at each occurrence oxo, thioxo, or absent; R a is O or S, R b OH, SH, O - , S - , OR d or S.R. d and R c OH, SH, O - , S - , OR d , O.L.', S.R. d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counter ion, Q is, independently at each occurrence, a moiety that contains a reactive group or a protected form thereof that is capable of forming a covalent bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q'; L', at each occurrence, is a heteroalkylene linker to Q, a targeting moiety selected from an antibody and a cell surface receptor antagonist, an analyte molecule, a solid support, or a nucleoside; m is, independently at each occurrence, an integer greater than or equal to zero, provided that at least one occurrence of m is an integer greater than or equal to 1; n is an integer of 1 or more.

15. A) The following structure (IVa): 【Chemical 29】 (IVa) (In the formula, L 4 and L 5 is expressed independently for each occurrence as C 3 -C 6 is alkylene, z is an integer from 1 to 100. A compound having the formula: B) The following structure (IVb): 【Chemistry 30】 (IVb) (In the formula, R 7 , R 8 , R 9 and R 10 is, independently at each occurrence, H or alkyl; y 1 and y 2 is, independently at each occurrence, an integer from 1 to 6; z is an integer from 1 to 100, preferably R 7 , R 8 , R 9 and R 10 is, independently for each occurrence, H) or a compound having C) The following structures (IVc), (IVd), (IVe) or (IVf): 【Chemistry 31】 (IVc) 【change】 (IVd) 【change】 (IVe) or 【change】 (IVf) (In the formula, L 1d ', independently at each occurrence, contains an amide functionality or a triazolyl functionality; y 1 and y 2 is, independently at each occurrence, an integer from 1 to 6; z is an integer from 1 to 100, preferably z is an integer from 22 to 25. is a compound having one of the following:

15. The compound of claim 14.

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