Polymer tandem dyes with spacer linker groups

Abstract

Compounds useful as fluorescent or colored dyes are disclosed. The compounds have the following structure (I): # imgabs0 # or a stereoisomer, tautomer or salt thereof, wherein R1, R2, R3, R4, R5, L1, L1a, L1b, L2, L3, L4, L5, L6, L7, M1, M2, m, n, q and w are as defined herein. Methods related to making and using such compounds are also provided.

Description

background Technical Field

[0002] The present disclosure generally relates to dimeric and polymeric fluorescent or colored tandem dyes with spacer groups for brightness enhancement, methods for their preparation, and their use in various analytical methods.

[0003] Related technical notes

[0004] Known fluorescence and / or colored dyes are particularly suitable for use in the application of highly sensitive detection reagents of expectation. The dye of a specific component or component in the preferably labeled sample enables the researcher to determine the existence, amount and / or position of the specific component or component. In addition, a specific system can be monitored in different environments in terms of its spatial and temporal distribution.

[0005] Fluorescence and colorimetry are extremely common in chemistry and biology. These methods provide available information about the existence, structure, distance, orientation, compound and / or position of biomolecules. In addition, time-resolved methods are increasingly used in the measurement of dynamics and kinetics. Therefore, many strategies for fluorescence or color labeling of biomolecules such as nucleic acids and proteins have been developed. Since the analysis of biomolecules typically occurs in aqueous environments, the development and use of water-soluble dyes are focused on.

[0006] Highly fluorescent or colored dyes are desirable because the use of such dyes increases the signal-to-noise ratio and provides other related benefits. Therefore, attempts have been made to increase the signal from known fluorescent and / or colored moieties. Specifically, fluorescence resonance energy transfer (FRET) Resonance energy transfer ("FRET" - sometimes also used interchangeably with fluorescence resonance energy transfer) technology produces information that reliably measures changes in biomolecular distances and interactions. Resonance energy transfer technology is relatively inexpensive and can obtain measurements rapidly; however, FRET is subject to several limitations related to the orientation and positioning of the chromophore and masking of the energy transfer due to free fluorophores and undesirable pH sensitivity.

[0007] Therefore, the art needs water-soluble dyes, especially resonance energy transfer dyes, with increased molar brightness and / or increased FRET emission signals. Ideally, such dyes and biomarkers should be intensely colored or fluorescent and should be available in a variety of colors and fluorescence wavelengths. The present invention meets this need and provides other related advantages.

[0008] Overview

[0009] In short, embodiments of the present disclosure generally relate to compounds that can be used as water-soluble, fluorescent and / or colored dyes and / or probes that can visually detect analyte molecules such as biomolecules and reagents for their preparation. In particular, in some embodiments, compounds of the present disclosure are useful because they can achieve FRET fluorescence emission associated therewith. Methods for visually detecting analyte molecules using dyes are also described.

[0010] Embodiments of the dyes disclosed herein include two or more fluorescent and / or colored moieties (ie, FRET acceptor M) covalently linked via a linker having the structure 1 and the corresponding FRET donor M 2 ):

[0011]

[0012] Specifically, the FRET acceptor M 1 With the corresponding FRET donor M 2 The ratio of is 1:1, 1:2, 1:3, or 2:3. In contrast to previous reports of dimeric and / or polymeric dyes, the dyes of the present invention are significantly brighter than the corresponding monomeric dye compounds and enable FRET absorption and emission due to intramolecular interactions. While not wishing to be bound by theory, it is believed that the FRET receptor M 1 The corresponding FRET donor M separated from the linker 2 The specific ratio of provides sufficient proximity between the fluorescent and / or colored moieties so that intramolecular FRET is optimized. Embodiments of the dyes disclosed herein include a linker having one of the following structures between the two FRET donors, which provides sufficient proximity:

[0013]

[0014] In addition, embodiments of the dyes disclosed herein include a fluorescent and / or coloring moiety (ie, a FRET acceptor M) covalently linked to the '5' end and the '3' end via a linker having the structure 1 and the corresponding FRET donor M 2 ):

[0015]

[0016] The water-soluble, fluorescent or colored dyes of the embodiments of the present disclosure are strongly colored and / or fluorescent, can realize FRET process (for example, light absorption, emission light, Stokes shift), and can be easily observed by visual inspection or other means. In some embodiments, compounds can be observed without prior illumination or chemical or enzyme activation. As described herein, by the appropriate selection of dye, visually detectable analyte molecules of various colors can be obtained.

[0017] In some embodiments, a compound having the following structure (I) is provided:

[0018]

[0019] or a stereoisomer, tautomer or salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1a , L 1b , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 、M 1 、M 2 , m, n, q and w are as defined herein.

[0020] The compounds of structure (I) find utility in a number of applications, including use as fluorescent and / or coloring dyes in various analytical methods.

[0021] In yet other embodiments, methods for staining a sample are provided, the method comprising adding to the sample a compound of structure (I) in an amount sufficient to produce an optical response when the sample is illuminated at an appropriate wavelength.

[0022] In yet other embodiments, the present disclosure provides a method for visually detecting an analyte molecule comprising:

[0023] (a) providing a compound as disclosed herein; and

[0024] (b) Detecting compounds by their visible properties.

[0025] Other disclosed methods include a method for visually detecting a biomolecule, the method comprising:

[0026] (a) admixing a compound as disclosed herein with one or more biomolecules; and

[0027] (b) Detecting compounds by their visible properties.

[0028] Other embodiments provide a method for visually detecting an analyte, the method comprising:

[0029] (a) providing a compound as disclosed herein, wherein R 1 or R 2 including a linker comprising a covalent bond to a targeting moiety specific for the analyte;

[0030] (b) admixing the compound with the analyte, thereby associating the targeting moiety with the analyte; and

[0031] (c) Detecting compounds by their visible properties

[0032] In yet other embodiments, the present disclosure provides a method for increasing the brightness of a dye, comprising:

[0033] (a) providing a dye solution comprising a compound as disclosed herein; and

[0034] (b) allowing the dye solution to age for a certain period of time.

[0035] Other embodiments relate to compositions comprising a compound as disclosed herein and one or more analyte molecules (such as one or more biomolecules). Also provided are uses of such compositions in analytical methods for detecting the one or more biomolecules.

[0036] These and other aspects of the disclosure will be apparent upon reference to the following detailed description.

[0037] Details

[0038] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the present disclosure. However, it will be understood by those skilled in the art that the present disclosure can be practiced without these details.

[0039] Unless the context requires otherwise, throughout this specification and claims, the word "comprise" and variations such as "comprises" and "comprising" are to be interpreted in an open-ended, inclusive sense, that is, as meaning "including, but not limited to."

[0040] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in conjunction with that embodiment is included in at least one embodiment of the present disclosure. Thus, the appearance of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, in one or more embodiments, the particular features, structures, or characteristics may be combined in any suitable manner.

[0041] "Amino" refers to -NH 2 Group.

[0042] "Carboxyl" refers to -CO 2 H group.

[0043] "Cyano" refers to a -CN group.

[0044] "Formyl" refers to a -C(=O)H group.

[0045] "Hydroxy" or "hydroxyl" refers to an -OH group.

[0046] "Imino" refers to a =NH group.

[0047] "Nitro" refers to -NO 2 Group.

[0048] "Oxo" refers to the =0 substituent group.

[0049] "Sulhydryl" refers to a -SH group.

[0050] "Thioxo" refers to a =S group.

[0051] "Alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to twelve carbon atoms (C 1 -C 12 Alkyl), one to eight carbon atoms (C 1 -C 8 Alkyl) or one to six carbon atoms (C 1 -C 6 Alkyl) and it is attached to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted.

[0052] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a radical, consisting solely of carbon and hydrogen, containing no unsaturation, and having from one to twelve carbon atoms, for example, methylene, ethylene, propylene, n-butylene, vinylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is attached to the rest of the molecule and to the radical by a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical can be through one carbon or any two carbons within the chain. Unless otherwise specifically stated in the specification, alkylene is optionally substituted.

[0053] "Alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a radical, consisting only of carbon and hydrogen, containing at least one carbon-carbon double bond and having two to twelve carbon atoms, for example, vinylene, propenylene, n-butenylene, etc. The alkenylene chain is attached to the rest of the molecule by a single bond and to the radical by a double bond or a single bond. The points of attachment of the alkenylene chain to the rest of the molecule and to the radical can be through one carbon or any two carbons within the chain. Unless otherwise specifically stated in the specification, alkenylene is optionally substituted.

[0054] "Alkynylidene" or "alkynylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a radical, consisting only of carbon and hydrogen, containing at least one carbon-carbon triple bond and having two to twelve carbon atoms, for example, vinylidene, propenylene, n-butenylene, etc. The alkynylene chain is attached to the rest of the molecule by a single bond and to the radical by a double bond or a single bond. The points of attachment of the alkynylene chain to the rest of the molecule and to the radical can be through one carbon or any two carbons within the chain. Unless otherwise specifically stated in the specification, alkynylene is optionally substituted.

[0055] "Alkyl ether" refers to any alkyl group as defined above, in which at least one carbon-carbon bond is replaced by a carbon-oxygen bond. The carbon-oxygen bond can be at the terminal end (as in an alkoxy group) or the carbon-oxygen bond can be internal (i.e., COC). Alkyl ethers include at least one carbon-oxygen bond, but may include more than one. For example, polyethylene glycol (PEG) is included within the meaning of alkyl ethers. Unless otherwise specifically stated in the specification, alkyl ether groups are optionally substituted. For example, in some embodiments, alkyl ethers are replaced by alcohols or -OP (=R a )(R b )R c Substitution, where R a , R b and R c Each of which is as defined for the compound of structure (I).

[0056] "Alkoxy" refers to a group of the formula -OR a A group in which Ra is an alkyl group as defined above containing from one to twelve carbon atoms. Unless stated otherwise specifically in the specification, an alkoxy group is optionally substituted.

[0057] "Alkoxyalkyl ether" refers to a alkyl ether of the formula -OR a R b A group in which R a R is an alkylene group as defined above containing from one to twelve carbon atoms, and R is an alkyl ether group as defined herein. Unless otherwise specifically stated in the specification, an alkoxyalkyl ether group is optionally substituted, for example by an alcohol or -OP(=R a )(R b )R c Substitution, where R a , R b and R c Each of which is as defined for the compound of structure (I).

[0058] "Heteroalkyl" refers to an alkyl group as defined above that contains at least one heteroatom (e.g., N, O, P, or S) within the alkyl group or at the terminus of the alkyl group. In some embodiments, the heteroatom is within the alkyl group (i.e., the heteroalkyl group contains at least one carbon-[heteroatom] x -carbon bond, wherein x is 1, 2 or 3). In other embodiments, the heteroatom is at the end of the alkyl group and is therefore used to connect the alkyl group to the rest of the molecule (e.g., M1-HA), wherein M1 is part of the molecule, H is a heteroatom and A is an alkyl group). Unless otherwise specifically stated in the specification, heteroalkyl groups are optionally substituted. Exemplary heteroalkyl groups include ethyleneoxy (e.g., polyethyleneoxy), optionally including a phosphorus-oxygen bond, such as a phosphodiester bond.

[0059] "Heteroalkoxy" refers to a group of the formula -OR a A group in which R a is a heteroalkyl group as defined above containing from one to twelve carbon atoms. Unless stated otherwise specifically in the specification, a heteroalkoxy group is optionally substituted.

[0060] "Heteroalkylene" refers to an alkylene as defined above that includes at least one heteroatom (e.g., N, O, P, or S) within an alkylene chain or at the end of an alkylene chain. In some embodiments, the heteroatom is within the alkylene chain (i.e., the heteroalkylene includes at least one carbon-[heteroatom]-carbon bond, wherein x is 1, 2, or 3). In other embodiments, the heteroatom is at the end of the alkylene and is therefore used to connect the alkylene to the rest of the molecule (e.g., M1-HA-M2, wherein M1 and M2 are part of the molecule, H is a heteroatom and A is an alkylene). Unless otherwise specifically stated in the specification, heteroalkylene is optionally substituted. Exemplary heteroalkylenes include ethyleneoxy (e.g., polyethyleneoxy) and "C", "HEG", "TEG", "PEG 1K" and variants thereof, and the linking group is shown below:

[0061]

[0062] "PEG 1K Linker"

[0063] Multimers of the above C-linkers, HEG linkers, and / or PEG 1K linkers are included in the various embodiments of the heteroalkylene linker.

[0064] In some embodiments of the PEG 1K linker, n is 25. The polymer may comprise, for example, the following structure:

[0065]

[0066] wherein x is an integer of 0 or greater, for example, x ranges from 0-100 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0067] "Heteroalkenylene" is a heteroalkylene group as defined above containing at least one carbon-carbon double bond. Unless stated otherwise specifically in the specification, a heteroalkenylene group is optionally substituted.

[0068] "Heteroalkynylene" is a heteroalkylene group containing at least one carbon-carbon triple bond. Unless stated otherwise specifically in the specification, a heteroalkynylene group is optionally substituted.

[0069] The term "heteroatom" in reference to a "heteroatom linker" refers to a linker group consisting of one or more heteroatoms. Exemplary heteroatom linkers include single atoms selected from the group consisting of O, N, P, and S, as well as multiple heteroatoms, such as a group having the formula -P(O - )(=O)O-or-OP(O - )(=O)O- linking groups and multimers and combinations thereof.

[0070] "Phosphate" refers to -OP(=O)(R a)R b Group, where R a It is OH, O - OR c ; and R b It is OH, O - , OR c , a phosphorothioate group or another phosphate group, wherein R c It is a counter ion (e.g., Na+, etc.).

[0071] "Phosphate alkyl" refers to -OP(=O)(R a )R b Group, where R a It is OH, O - OR c ; and R b is -O alkyl, where R c is a counterion (e.g., Na+, etc.). Unless otherwise specifically stated in the specification, the phosphoalkyl group is optionally substituted. For example, in certain embodiments, the -Oalkyl portion of the phosphoalkyl group is optionally substituted with a hydroxyl, an amino, a sulfhydryl, a phosphate, a thiophosphate, a phosphate alkyl, a thiophosphate alkyl, a phosphate alkyl ether, a thiophosphate alkyl ether, or -OP (=R a )(R b )R c One or more substitutions in which R a , R b and R c Each of which is as defined for the compound of structure (I).

[0072] "Alkyl phosphate ether" refers to -OP(=O)(R a )R b Group, where R a It is OH, O - OR c ; and R b is -O alkyl ether, where R c is a counterion (e.g., Na+, etc.). Unless otherwise specifically stated in the specification, the phosphoalkyl ether group is optionally substituted. For example, in certain embodiments, the -O alkyl ether portion of the phosphoalkyl ether group is optionally substituted with a hydroxyl, an amino, a sulfhydryl, a phosphate, a thiophosphate, a phosphate alkyl, a thiophosphate alkyl, a phosphoalkyl ether, a thiophosphate alkyl ether, or -OP (=R a )(R b )R c One or more substitutions in which R a , R b and R c Each of which is as defined for the compound of structure (I).

[0073] "Phosphorothioate" refers to -OP(=R a )(R b )R c Group, where R a O or S, R b It is OH, O - , S - , OR d or SR d ; and R c OH, SH, O-, S-, OR d , SR d , a phosphate group or another phosphorothioate group, wherein R d is a counterion (e.g., Na+, etc.), and provided that: i) R a is S; ii) R b Is S- or SR d iii) R c SH, S- or SR d ; or iv) a combination of i), ii) and / or iii).

[0074] "Phosphorothioate alkyl" refers to -OP(=R a )(R b )R c Group, where R a O or S, R b It is OH, O-, S-, OR d or SR d ; and R c is -O alkyl, where R d is a counterion (e.g., Na+, etc.), and provided that: i) R a is S; ii) R b Is S- or SR d or iii) R a is S and R b Is S- or SR d Unless otherwise specifically stated in the specification, the phosphorothioate alkyl group is optionally substituted. For example, in certain embodiments, the -O alkyl portion of the phosphorothioate alkyl group is optionally substituted with a hydroxyl, an amino, a sulfhydryl, a phosphate, a thiophosphate, a phosphate alkyl, a thiophosphate alkyl, a phosphate alkyl ether, a phosphorothioate alkyl ether, or an -OP(=R a )(R b )R c One or more substitutions in which R a , R b and R c Each of which is as defined for the compound of structure (I).

[0075] "Thiophosphate alkyl ether" refers to -OP(=R a )(R b )R c Group, where R a O or S, R b It is OH, O - , S - , OR d or SR d ; and R c is -O alkyl ether, where R d is a counterion (e.g., Na+, etc.), and provided that: i) R a is S; ii) R b YesS - or SR d or iii) R a is S and R b Is S- or SR d Unless otherwise specifically stated in the specification, the phosphorothioate alkyl ether group is optionally substituted. For example, in certain embodiments, the -O alkyl ether portion of the phosphorothioate alkyl group is optionally substituted with a hydroxyl, an amino, a sulfhydryl, a phosphate, a thiophosphate, an alkyl phosphate, an alkyl thiophosphate, an alkyl phosphate ether, an alkyl thiophosphate ether, or an -OP(=R a )(R b )R c One or more substitutions in which R a , R b and R c Each of which is as defined for the compound of structure (I).

[0076] "Carbocycle" refers to a stable 3- to 18-membered aromatic or non-aromatic ring containing 3 to 18 carbon atoms. Unless otherwise specified in the specification, the carbocycle can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include a fused ring or a bridged ring system, and can be partially or completely saturated. Non-aromatic carbocyclic groups include cycloalkyl, while aromatic carbocyclic groups include aryl. Unless otherwise specified in the specification, the carbocyclic group is optionally substituted.

[0077] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic carbocyclic ring which may include a fused or bridged ring system having three to fifteen carbon atoms, preferably three to ten carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond. Monocyclic cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyls include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo-[2.2.1]heptanyl, and the like. Unless otherwise specifically stated in the specification, cycloalkyls are optionally substituted.

[0078] "Aryl" refers to a ring system containing at least one carbocyclic aromatic ring. In some embodiments, the aryl group contains 6 to 18 carbon atoms. The aryl ring can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems. Aryl groups include, but are not limited to, aryl groups derived from: aceanthrene, acenaphthene, acephenanthrene, anthracene, azulene, benzene, Fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene and triphenylene. Unless stated otherwise specifically in the specification, aryl groups are optionally substituted.

[0079] "Heterocycle" refers to a stable 3- to 18-membered aromatic or non-aromatic ring containing one to twelve carbon atoms and one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless otherwise specifically stated in the specification, the heterocycle can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include a fused ring or a bridged ring system; and the nitrogen, carbon or sulfur atoms in the heterocycle can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocycle can be partially or completely saturated. Examples of aromatic heterocycles are listed below in the definition of heteroaryl (i.e., heteroaryl is a subset of heterocycle). Examples of non-aromatic heterocycles include, but are not limited to, dioxolanyl, thienyl [1,3] dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trioxanyl, trithianyl, triazinyl, tetrahydropyranyl, thiomorpholinyl, thiomorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless otherwise specifically stated in the specification, the heterocyclic group is optionally substituted.

[0080] "Heteroaryl" refers to a 5- to 14-membered ring system containing one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring. For the purposes of certain embodiments of the present disclosure, heteroaryl can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl can be optionally oxidized; the nitrogen atom can be optionally quaternized. Examples include, but are not limited to, aza benzo[b][1,4]dioxolyl, benzo[b][1,4]benzo[ ... 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, benzoxazolinone, benzimidazolylthione, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanone, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolyl, isoindolyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepine 1-O-pyridyl, 1-O-pyrimidyl, 1-O-pyrazinyl, 1-O-pyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, pteridinone, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyridone, pyrazinyl, pyrimidinyl, pyrimidone, pyridazinyl, pyrrolyl, pyrido[2,3

[0063] The heteroaryl groups are optionally substituted, unless otherwise specifically stated in the specification, thioaryl groups are optionally substituted, and thioaryl groups are optionally substituted.

[0081] The suffix "-ene" refers to a specific structural feature (e.g., alkyl, aryl, heteroalkyl, heteroaryl) that is attached to the rest of the molecule and to the group by a single bond. In other words, the suffix "-ene" refers to a linker having the structural features of the moiety to which it is attached. The points of attachment of the "-ene" chain to the rest of the molecule and to the group can be through one atom or any two atoms within the chain. For example, heteroarylene refers to a linker that contains a heteroaryl moiety as defined herein.

[0082] "Fused" refers to a ring system comprising at least two rings, wherein the two rings share at least one common ring atom, e.g., two common ring atoms. When the fused ring is a heterocyclyl ring or a heteroaryl ring, one or more of the common ring atoms may be carbon or nitrogen. Fused rings include bicyclic, tricyclic, tetracyclic, and the like.

[0083] As used herein, the term "substituted" means any of the above groups (e.g., alkyl, alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, alkoxy, alkyl ether, alkoxyalkyl ether, heteroalkyl, heteroalkoxy, phosphate alkyl, phosphate alkyl ether, thiophosphate alkyl, thiophosphate alkyl ether, carbocycle, cycloalkyl, aryl, heterocycle, and / or heteroaryl) in which at least one hydrogen atom (e.g., 1, 2, 3, or all of the hydrogen atoms) is replaced with a bond to a non-hydrogen atom, such as, but not limited to, a halogen atom. , such as F, Cl, Br and I; oxygen atoms in groups such as hydroxyl, alkoxy and ester groups; sulfur atoms in groups such as thiol groups, thioalkyl, sulfone groups, 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; and other heteroatoms in various other groups. "Substituted" also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., double or triple bond) with a heteroatom, such as oxygen in oxo, carbonyl, carboxyl and ester groups; and nitrogen in groups such as imines, oximes, hydrazones and nitriles. For example, "substituted" includes groups in which one or more hydrogen atoms are replaced by -NR g R h 、-NR g C(=O)R h 、-NR g C(=O)NR g R h 、-NR g C(=O)OR h 、-NR g SO 2 R h 、-OC(=O)NRg R h 、-OR g 、-SR g 、-SOR g 、-SO 2 R g 、-OSO 2 R g 、-SO 2 OR g , =NSO 2 R g and-SO 2 NR g R h "Substituted" also means any of the above groups replaced by -C(=O)R g 、-C(=O)OR g 、-C(=O)NR g R h 、-CH 2 SO 2 R g 、-CH 2 SO 2 NR g R h Any of the above groups substituted. In the foregoing, R g and R h are the same or different and are 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 a bond to amino, cyano, hydroxy, imino, nitro, oxo, thio, halo, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl. In some embodiments, the optional substituent is -OP(=R a )(R b )R c , where R a , R b and R c Each of the above substituents is as defined for the compound of structure (I). In addition, each of the aforementioned substituents may also be optionally substituted with one or more of the above substituents.

[0084] "Electron withdrawing group" refers to a functional group that is more capable of attracting electrons to itself than a hydrogen atom if it occupies the same position in a molecule. These terms are well understood by those skilled in the art and are discussed in J. March's Advanced Organic Chemistry, John Wiley & Sons, New York, NY, pp. 16-18 (1985), and the discussion therein is incorporated herein by reference. Examples of electron withdrawing groups include, but are not limited to, halo, halogenated (e.g., F, Cl, Br, I), -NO 2 、-CN、-SO 3 H, -SO 2 R a 、-SO 3 R a , -COOH, -CO R a 、-COOR a ,-CONHR a 、-CON(R a ) 2 , halogenated alkyl and 5-14 membered electron-deficient heteroaryl, wherein R a is an alkyl, alkenyl, or alkynyl group.

[0085] "Conjugation" means that one p-orbital is in the middle of another p-orbital. σ bond on overlapping Conjugation can occur in cyclic or acyclic compounds. "Degree of conjugation" refers to the overlap of at least one p-orbital with another p-orbital across an intervening sigma bond. For example, 1,3-butadine has one degree of conjugation, while benzene and other aromatic compounds typically have multiple degrees of conjugation. Fluorescent and coloring compounds typically contain at least one degree of conjugation.

[0086] "Fluorescence" refers to molecules that absorb light of a particular frequency and emit light of a different frequency. Fluorescence is well known to those of ordinary skill in the art.

[0087] "Colored" refers to molecules that absorb light within the colored spectrum (ie, red, yellow, blue, etc.).

[0088] "FRET" refers to fluorescence resonance energy transfer, which refers to a physical interaction whereby energy from the excitation of one moiety (e.g., a first chromophore or "donor") is transferred to an adjacent moiety (e.g., a second chromophore or "acceptor"). "FRET" is also sometimes used interchangeably with fluorescence resonance energy transfer (i.e., when each chromophore is a fluorescent moiety). In general, 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 chromophores are substantially parallel (i.e., at about 0° or 180°); and (3) the acceptor and donor chromophores share spatial proximity (i.e., are close to each other). Energy transfer from the donor to the acceptor occurs via nonradiative dipole-dipole coupling, and the distance between the donor and acceptor chromophores is typically much smaller than one or more wavelengths of light.

[0089] "Donor" or "donor chromophore" refers to a chromophore (e.g., a fluorophore) that is or can be induced into an excited electronic state and can transfer its excitation or absorption energy to a nearby acceptor chromophore in a non-radiative manner via long-range dipole-dipole interactions. Without wishing to be bound by theory, it is believed that energy transfer occurs because the oscillating dipoles of the corresponding chromophores have similar resonant frequencies. Donors and acceptors with these similar resonant frequencies are referred to as "one or more donor-acceptor pairs," which are used interchangeably with "FRET moiety," "FRET pair," "FRET dye," or the like.

[0090] "Acceptor" or "acceptor chromophore" refers to a chromophore (eg, fluorophore) to which excitation or absorption energy from a donor chromophore is transferred nonradiatively via long-range dipole-dipole interactions.

[0091] "Stokes shift" refers to the difference between the position of the excitation or absorption band maximum and the emission spectrum for an electronic transition (e.g., from an excited state to a non-excited state, or vice versa). In some embodiments, the compound has a Stokes shift of greater than 25 nm, greater than 30 nm, greater than 35 nm, greater than 40 nm, greater than 45 nm, greater than 50 nm, greater than 55 nm, greater than 60 nm, greater than 65 nm, greater than 70 nm, greater than 75 nm, greater than 80 nm, greater than 85 nm, greater than 90 nm, greater than 95 nm, greater than 100 nm, greater than 110 nm, greater than 120 nm, greater than 130 nm, greater than 140 nm, greater than 150 nm, greater than 160 nm, greater than 170 nm, greater than 180 nm, greater than 190 nm, or greater than 200 nm.

[0092] The "J-value" is calculated as the integral 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 with a preferred excitation or absorption wavelength. The preferred excitation or absorption wavelength for the donor chromophore is equal to or close to the corresponding excitation or absorption maximum well known to those of ordinary skill in the art (e.g., Pacific Blue has an excitation or absorption maximum at about 401 nm, FITC has an excitation or absorption maximum at about 495 nm).

[0093] "Linker" refers to a continuous chain of at least one atom (such as carbon, oxygen, nitrogen, sulfur, phosphorus, and combinations thereof) that connects one part of a molecule to another part of the same molecule or to a different molecule, part, or solid support (e.g., microparticle). Linkers can connect molecules via covalent bonds or other means (such as ionic or hydrogen bonding interactions).

[0094] The term "biomolecule" refers to any of a variety of biological materials, including nucleic acids, carbohydrates, amino acids, polypeptides, glycoproteins, hormones, aptamers, and mixtures thereof. More specifically, the term is intended to include, but is not limited to, RNA, DNA, oligonucleotides, modified or derived nucleotides, enzymes, receptors, prions, receptor ligands (including hormones), antibodies, antigens, and toxins, as well as bacteria, viruses, blood cells, and tissue cells. As further described herein, visually detectable biomolecules of the present disclosure (e.g., compounds having structure (I) of a biomolecule attached thereto) are prepared by contacting the biomolecule with a compound having a reactive group capable of attaching the biomolecule to the compound via any available atom or functional group (such as an amino, hydroxyl, carboxyl, or sulfhydryl group on the biomolecule).

[0095] 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, such as by displacement, oxidation, reduction, addition, or cycloaddition reactions. Exemplary reactive groups are provided in Table 1 and include, for example, nucleophiles, electrophiles, dienes, dienophiles, aldehydes, oximes, hydrazones, alkynes, amines, azides, acyl azides, acyl halides, nitriles, nitrones, sulfhydryls, disulfides, sulfonyl halides, isothiocyanates, imidoesters, activated esters, ketones, α,β-unsaturated carbonyls, olefins, maleimides, α-haloimides, epoxides, aziridines, tetrazines, tetrazoles, phosphines, biotin, thiirane, and the like.

[0096] The terms "visible" and "visually detectable" are used herein to refer to substances that are observable by visual inspection without prior illumination or chemical or enzymatic activation. Such visually detectable substances absorb and emit light in the spectral region ranging from about 300 to about 900 nm. Preferably, such substances are intensely colored, preferably having a chromatin of at least about 40,000, more preferably at least about 50,000, still more preferably at least about 60,000, yet still more preferably at least about 70,000, and most preferably at least about 80,000 nm. -1 cm -1 The molar extinction coefficient of the present invention can be detected by naked eye observation or by means of optical detection devices (including but not limited to absorption spectrophotometers, transmission optical microscopes, digital cameras and scanners). Visually detectable substances 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 10nm to about 400nm), infrared (IR) region (about 700nm to about 1mm) and substances that emit and / or absorb in other regions of the electromagnetic spectrum are also included in the scope of "visually detectable" substances.

[0097] For the purpose of the embodiments of the present disclosure, the term "light-stable visible dye" refers to a chemical part that is visually detectable as defined above and does not significantly change or decompose when exposed to light. Preferably, the light-stable visible dye does not show significant bleaching or decomposition after being exposed to light for at least one hour. More preferably, the visible dye is stable after being exposed to light for at least 12 hours, more preferably at least 24 hours, more preferably at least one week and most preferably at least one month. Non-limiting examples of light-stable visible dyes suitable for use in the compounds and methods of the present disclosure include azo dyes, thioindigo dyes, quinacridone pigments, dioxazines, phthalocyanines, perinones, dipyrrolopyrroles, quinophthalones and trueycarbonium.

[0098] 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 similarly used. 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.

[0099] The visually detectable molecules of various embodiments of the present disclosure can be used for a variety of analytical applications, such as biochemical and biomedical applications, where it is necessary to determine the presence, location, or amount of a specific analyte (e.g., a biomolecule). Therefore, on the other hand, the present disclosure provides a method for visually detecting a biomolecule, comprising: (a) providing a biological system having a visually detectable biomolecule, the biomolecule comprising a compound of structure (I) connected to the biomolecule; and (b) detecting the biomolecule by its visible properties. For the purposes of the present disclosure, the phrase "detecting a biomolecule by its visible properties" means observing the biomolecule with the naked eye or with the aid of an optically based detection device (including but not limited to an absorption spectrophotometer, a transmission optical microscope, a digital camera, and a scanner) without illumination or chemical or enzyme activation. A densitometer can be used to quantify the amount of visually detectable biomolecules present. For example, the relative amount of biomolecules in two samples can be determined by measuring relative optical density. If the stoichiometry of the dye molecule of each biomolecule is known, and the extinction coefficient of the dye molecule is known, the absolute concentration of the biomolecule can also be determined from the measurement of optical density. As used herein, the term "biological system" is used to refer to any solution or mixture comprising one or more biomolecules in addition to visually detectable biomolecules. Non-limiting examples of such biological systems include cells, cell extracts, tissue samples, electrophoresis gels, assay mixtures, and hybridization reaction mixtures.

[0100] "Solid support" refers to any solid matrix known in the art for solid support of molecules, for example, "microparticles" refers to any of a number of small particles that can be used to attach to the compounds of the present disclosure, including but not limited to glass beads, magnetic beads, polymer beads, non-polymer beads, etc. In certain embodiments, the microparticles comprise polystyrene beads.

[0101] "Solid support residue" refers to a functional group that remains attached to a molecule when the molecule is cleaved from the solid support. Solid support residues are known in the art and can be readily derivatized based on the structure of the solid support and the group that attaches the molecule thereto.

[0102] "Targeting moiety" is a part that selectively binds or associates with a specific target (such as an analyte molecule). "Selectively" binding or association means a targeting moiety that preferentially associates or associates with a desired target relative to other targets. In some embodiments, for the purpose of selectively binding or associating a compound with an analyte of interest (i.e., the target of a targeting moiety), the compound disclosed herein includes a key to a targeting moiety, thereby allowing detection of the analyte. Exemplary targeting moieties include, but are not limited to, antibodies, antigens, nucleic acid sequences, enzymes, proteins, cell surface receptor antagonists, etc. In some embodiments, a targeting moiety is a part that selectively binds or associates with a target feature on or in a cell (e.g., a target feature on a cell membrane or other cell structure), such as an antibody, thereby allowing detection of a target cell. In certain embodiments, a small molecule that selectively binds or associates with a desired analyte is also envisioned as a targeting moiety. Those skilled in the art will appreciate other analytes and corresponding targeting moieties available in various embodiments.

[0103] "Base pairing moiety" refers to a heterocyclic moiety that is capable of hybridizing to a complementary heterocyclic moiety via hydrogen bonding (e.g., Watson-Crick base pairing). Base pairing moieties include natural and unnatural bases. Non-limiting examples of base pairing moieties are RNA and DNA bases such as adenosine, guanosine, thymidine, cytosine, and uridine and their analogs.

[0104] The embodiments of the present disclosure disclosed herein are also intended to encompass all compounds of structure (I) that are isotopically labeled by replacing one or more atoms with atoms having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 36 Cl, 123 I and 125 I.

[0105] Isotopically labeled compounds of structure (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described below and in the examples below using an appropriate isotopically labeled reagent in place of the non-labeled reagent previously employed.

[0106] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0107] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where said event or circumstance does not occur. For example, "optionally substituted alkyl" means that the alkyl may or may not be substituted, and that the description includes both substituted alkyl and unsubstituted alkyl.

[0108] "Salt" includes both acid addition salts and base addition salts.

[0109] "Acid addition salts" refers to those salts formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, Gluceptic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

[0110] "Base addition salts" refer to those salts prepared by adding an inorganic base or an organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0111] Crystallization can produce solvates of the compounds described herein. Embodiments of the present disclosure include all solvates of the compounds described. As used herein, the term "solvate" refers to an aggregate of one or more molecules of a compound of the present disclosure and one or more molecules of a solvent. The solvent can be water, in which case the solvate can be a hydrate. Alternatively, the solvent can be an organic solvent. Therefore, the compounds of the present disclosure can exist as hydrates (including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc.) and corresponding solvated forms. The compounds of the present disclosure can be true solvates, and in other cases, the compounds of the present disclosure can only retain exogenous water or other solvents or a mixture of water plus some exogenous solvents.

[0112] Embodiments of the compounds of the present disclosure (e.g., compounds of Structure I or II) or salts, tautomers or solvates thereof may contain one or more asymmetric centers and may therefore produce enantiomers, diastereomers and other stereoisomeric forms, which may be defined as (R)- or (S)- in terms of absolute stereochemistry, or as (D)- or (L)- for amino acids. Embodiments of the present disclosure are intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, e.g., chromatography and fractional crystallization. Conventional techniques for preparing / isolating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0113] "Stereoisomers" refers to compounds made up of the same atoms bonded by the same bonds but having different three-dimensional structures that are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0114] "Tautomer" refers to a proton transfer from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any of the described compounds. Various tautomeric forms of compounds are readily accessible to those of ordinary skill in the art.

[0115] The chemical naming conventions and structure diagrams used herein are a modification of the IUPAC naming system, using the ACD / Name Version 9.07 software program and / or the ChemDraw Ultra Version 11.0 software naming program (CambridgeSoft). Common names familiar to those of ordinary skill in the art are also used.

[0116] As noted above, in one embodiment of the present disclosure, compounds that can be used as fluorescence and / or colored dyes in various analytical methods are provided. In other embodiments, compounds that can be used as synthetic intermediates for the preparation of fluorescence and / or colored dyes are provided. Generally speaking, embodiments of the present disclosure relate to dimers and higher polymers of fluorescence and / or colored moieties. Fluorescence and or colored moieties are connected by linking groups. Do not wish to be bound by theory, think that linking groups help to maintain enough spatial distances between fluorescence and / or colored moieties, so as to reduce or eliminate intramolecular quenching, thereby produce dye compounds with high molar "brightness" (e.g., high fluorescence emission).

[0117] Thus, in some embodiments, the compounds of the present disclosure have the following structure (I):

[0118]

[0119] or a stereoisomer, salt or tautomer thereof, wherein:

[0120] M 1 and M 2 is independently a chromophore at each occurrence, provided that M 1 is a FRET acceptor and M 2 is the corresponding FRET donor, and M 1 and M 2 Forming a FRET pair;

[0121] L 1a is independently at each occurrence a heteroalkylene or heteroarylene linker;

[0122] L 1b , L 2 , L 3 , L 5 , L 6 and L 7 is independently at each occurrence an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene or heteroalkynylene linker;

[0123] L 4 Has one of the following structures at each occurrence:

[0124]

[0125] in:

[0126] z is an integer from 1 to 100; and

[0127] * indicates bonds to adjacent phosphorus atoms;

[0128] R 1 and R 2are 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';

[0129] R 3 is independently at each occurrence H, alkyl or alkoxy;

[0130] R 4 is, independently at each occurrence, OH, SH, O - , S-, OR d or SR d ;

[0131] R 5 is independently at each occurrence oxo, thio or absent;

[0132] R a is O or S;

[0133] R b OH, SH, O - , S - , OR d or SR d ;

[0134] R c OH, SH, O - , S - , OR d , OL', SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, alkyl phosphate, thiophosphate alkyl, phosphate alkyl ether, or thiophosphate alkyl ether;

[0135] R d is the counter ion;

[0136] Q is independently at each occurrence a moiety comprising a reactive group, or a protected form thereof, capable of forming a covalent bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q′;

[0137] L' is independently at each occurrence a linker comprising a covalent bond to Q, a linker comprising a covalent bond to a targeting 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 another compound of structure (I);

[0138] m, at each occurrence, is an integer of one or greater;

[0139] q is an integer of one or greater at each occurrence;

[0140] w is, in at least one occurrence, an integer of one or greater, provided that when n is an integer of 1, q is an integer greater than w; and

[0141] n is an integer of one or more.

[0142] In some embodiments, L 1a At least one occurrence of is an optionally substituted 5-7 membered heteroarylene linker. In some more specific embodiments, L 1a is independently at each occurrence an optionally substituted 5-7 membered heteroarylene linker. 1a is a 6-membered heteroarylene. 1a comprises two N atoms and two O atoms. In certain embodiments, L 1a is substituted at each occurrence. In some related embodiments, L 1a For example, it is substituted with oxo, alkyl (e.g., methyl, ethyl, etc.) or a combination thereof. In a more specific embodiment, L 1a is substituted with at least one oxo at each occurrence. In some embodiments, L 1a Has one of the following structures:

[0143]

[0144] In some embodiments, the compounds of the present disclosure have one of the following structures (IA) or (IA'):

[0145]

[0146] or a stereoisomer, a salt or a tautomer thereof.

[0147] In some embodiments, L 4 In some embodiments, z is an integer from 1 to 30, such as from 3 to 8, from 15 to 30, or from 22 to 26. In some embodiments, z is 22, 23, 24, 25, or 26. In some embodiments, z is 3, 4, 5, 6, 7, or 8. In some specific embodiments, z is 6.

[0148] In some embodiments, when q is an integer of 2, L 4 For the first occurrence and for the second occurrence In some embodiments, when q is an integer of 3, L 4 For the first occurrence For the second occurrence and for the third occurrence

[0149] In some embodiments, the compounds of the present disclosure have one of the following structures (IB) or (IB'):

[0150]

[0151] or a stereoisomer, a salt or a tautomer thereof.

[0152] In some embodiments, L 5 or L 6 At least one occurrence of is alkylene. In some embodiments, L 5 and L 6 C independently at each occurrence 1 -C 6 Alkylene, C 2 -C 6 Alkenylene, or C 2 -C 6 For example, in some embodiments, L 5 and L 6 C independently at each occurrence 1 -C 6 Alkylene.

[0153] In some embodiments, L 3 At least one occurrence of is alkylene. In some embodiments, L 3 C independently at each occurrence 1 -C 6 Alkylene, C 2 -C 6 Alkenylene, or C 2 -C 6 For example, in some embodiments, L 3 C independently at each occurrence 1 -C 6 Alkylene.

[0154] In some embodiments, the compounds of the present disclosure have one of the following structures (IC) or (IC'):

[0155]

[0156]

[0157] or a stereoisomer, salt or tautomer thereof, wherein y 1 ,y 2 and 3 is independently an integer from 1 to 6 at each occurrence. In some embodiments, y 1is an integer of 1, 2, 3, 4, 5, or 6. In some embodiments, y 2 is an integer of 1, 2, 3, 4, 5, or 6. In some embodiments, y 3 is an integer of 1, 2, 3, 4, 5, or 6. In some more specific embodiments, y 1 is an integer of 1. In some other specific embodiments, y 2 is an integer of 1. In some other specific embodiments, y 3 is the integer 1.

[0158] Various linkers and substituents (e.g., R 1 , R 2 , R 3 , R 4 , R 5 , L 1a , L 1b , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 、M 1 、M 2 , R c and Q) are optionally substituted with another substituent. For example, in some embodiments, the optional substituent is selected to optimize the water solubility or other characteristics of the compound of structure (I). In certain embodiments, each alkyl, alkoxy, alkyl ether, alkoxyalkyl ether, phosphate alkyl, thiophosphate alkyl, phosphate alkyl ether and thiophosphate alkyl ether in the compound of structure (I) is optionally substituted with one or more substituents selected from the group consisting of hydroxyl, alkoxy, alkyl ether, alkoxyalkyl ether, sulfhydryl, amino, alkylamino, carboxyl, phosphate, thiophosphate, phosphate alkyl, thiophosphate alkyl, phosphate alkyl ether and thiophosphate alkyl ether. In certain embodiments, the optional substituent is -OP(=R a )(R b )R c , where R a , R b and R c As defined for the compounds of structure (I).

[0159] Optional linker L 1b Can be used as M 1 For example, in some embodiments, a synthetic precursor of a compound of structure (I) is prepared and the M is attached to the M using any number of simple methods known in the art, such as a method known as "click chemistry". 1For this purpose, any reaction that is rapid and essentially irreversible can be used to attach M 1 Attached to a synthetic precursor to form a compound of structure (I). Exemplary reactions include the copper-catalyzed reaction of azides and alkynes to form triazoles (Huisgen 1,3-dipolar cycloaddition), the reaction of dienes and dienophiles (Diels-Alder), strain-promoted alkyne-nitrone cycloaddition, the reaction of strained olefins with azides, tetrazines or tetrazoles, olefins and azides [3+2] cycloaddition, olefins and tetrazines reverse demand Diels-Alder reaction (Diels-Alder), olefins and tetrazoles photoreactions and various displacement reactions, such as displacement of leaving groups by nucleophilic attack on electrophilic atoms. Exemplary displacement reactions include the reaction of amines with: activated esters; N-hydroxysuccinimide esters; isocyanates; isothiocyanates, etc. In some embodiments, the formation of L 1b The reaction can be carried out in an aqueous environment.

[0160] Therefore, in some embodiments, L 1b At each occurrence is a linker comprising a functional group that can be formed by reaction of two complementary reactive groups (e.g., a functional group that is the product of one of the aforementioned "click" reactions). 1b The functional group can be formed by the reaction of an aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imidate, activated ester (e.g., N-hydroxysuccinimide ester), ketone, α,β-unsaturated carbonyl, olefin, maleimide, α-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin or thiirane functional group with a complementary reactive group. For example, the reaction of an amine with an N-hydroxysuccinimide ester or an isothiocyanate.

[0161] In other embodiments, for L 1b In other embodiments, for L 1b The functional group can be formed by the reaction of an amine (eg, a primary amine) and an N-hydroxysuccinimide ester or an isothiocyanate.

[0162] In more embodiments, for L 1b In at least one occurrence of L, the functional group comprises an alkene, an ester, an amide, a thioester, a disulfide, a carbocycle, a heterocycle, or a heteroaryl. In further embodiments, for L 1bIn at least one occurrence of L, the functional group comprises an olefin, an ester, an amide, a thioester, a thiourea, a disulfide, a carbocycle, a heterocycle, or a heteroaryl. In other embodiments, the functional group comprises an amide or a thiourea. In some more specific embodiments, for L 1b At least one occurrence of L 1b is a linker comprising a triazole functional group. In other embodiments, for L 1b At least one occurrence of L 1b is a linker containing an amide or thiourea functional group.

[0163] In yet other various embodiments of Structure (I), L 1b is independently an alkylene or heteroalkylene linker at each occurrence. 1b is heteroalkylene.

[0164] In other embodiments, L 1b At least one occurrence of comprises a functional group formed by reaction of an aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imidate, activated ester, ketone, α,β-unsaturated carbonyl, olefin, maleimide, α-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin, or thiirane with a complementary reactive group.

[0165] In other embodiments, L 1b At least one occurrence of comprises a functional group formed by the reaction of an alkyne and an azide. For example, L 1b At least one occurrence of is a linker comprising a triazole functional group.

[0166] In yet other embodiments, for L 1b -M 1 , or L 7 -M 2 At least one occurrence of has one of the following structures:

[0167]

[0168] Where L 1c and L 1d Each is independently an optional linker.

[0169] In various embodiments, for L 1b -M 1 , or L 7 -M 2 At least one occurrence of has one of the following structures:

[0170]

[0171] Where L 1c and L 1d Each is independently an optional linker.

[0172] In the various embodiments described above, L 1c or L 1d , or both are absent. In other embodiments, L 1c or L 1d , or both. In some embodiments, when present, L c and L d Each is independently alkylene or heteroalkylene. For example, in some embodiments, L c and L d Independently has one of the following structures:

[0173]

[0174] In other embodiments, L 1b Contains one of the following structures:

[0175]

[0176] in

[0177] a, b, c, d, and e are each independently an integer ranging from 1 to 6. In some embodiments, a, b, c, d, or e is an integer of 1. In some embodiments, a, b, c, d, or e is an integer of 2. In some embodiments, a, b, c, d, or e is an integer of 3. In some embodiments, a, b, c, d, or e is an integer of 4. In some embodiments, a, b, c, d, or e is an integer of 5. In some embodiments, a, b, c, d, or e is an integer of 6. In some specific embodiments, a is an integer of 6 and d is an integer of 4.

[0178] In some embodiments, M of structure (I) 1 -L 1b At least one occurrence of has one of the following structures:

[0179]

[0180]

[0181] In some embodiments, M of structure (I) 1 -L 1b Each occurrence of has one of the following structures:

[0182]

[0183] In some embodiments, L 7 In other embodiments, at least one occurrence of is an optionally substituted heteroalkylene linker. 7 is independently at each occurrence an optionally substituted heteroalkylene. 7 Contains an amide functional group. For example, in some embodiments, L 7 At least one occurrence of has one of the following structures:

[0184]

[0185] In other embodiments, L 7 Each occurrence of has one of the following structures:

[0186]

[0187] In some specific embodiments, L 7 At least one occurrence of has one of the following structures:

[0188]

[0189] In some other specific embodiments, L 7 Each occurrence of has one of the following structures:

[0190]

[0191] In some embodiments, R 3 At least one occurrence of is H.

[0192] In yet other embodiments of compounds of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'), R 5 is independently OH, O- or OR at each occurrence d It should be understood that “OR d ” and “SR d " is intended to refer to the O- and S- - For example, the disodium salt of a phosphate group can be represented as:

[0193]

[0194] Where R d Sodium + ).

[0195] In other embodiments of compounds of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'), R 4In other embodiments of any of structures (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'), R 4 At each occurrence is oxo.

[0196] In other various embodiments, R 1 and R 2 Each is independently OH or -OP (= R a )(R b )R c In some different embodiments, R 1 or R 2 is OH or -OP (=R a )(R b )R c , and R 1 or R 2 The other one of them is Q or a linker comprising a covalent bond to Q.

[0197] In still further embodiments of any of the foregoing compounds of Structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'), R 1 and R 2 Each is independently -OP(=R a )(R b )R c In some of these embodiments, R c It's OL'.

[0198] In other embodiments, R 1 and R 2 Each is independently -OP(=R a )(R b )OL', and L' is an alkylene or heteroalkylene linker to: Q, a targeting moiety, an analyte (e.g., an analyte molecule), a solid support, a solid support residue, a nucleoside, or another compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC').

[0199] In some embodiments, the analyte molecule is a nucleic acid, an amino acid, or a polymer thereof. In some other embodiments, the analyte molecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion.

[0200] In some embodiments, the targeting moiety is an antibody or a cell surface receptor antagonist. In some other embodiments, the solid support is a polymeric bead or a non-polymeric bead.

[0201] The linker L' can be any linker suitable for attaching Q, a targeting moiety, an analyte (e.g., an analyte molecule), a solid support, a solid support residue, a nucleoside, or another compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') to a compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'). Advantageously, certain embodiments include using a selected L' moiety to increase or optimize the water solubility of the compound. In certain embodiments, L' is a heteroalkylene moiety. In some other certain embodiments, L' comprises an alkyleneoxy or phosphodiester moiety, or a combination thereof.

[0202] In certain embodiments, L' has the structure:

[0203]

[0204] in:

[0205] m" and n" are independently integers from 1 to 10;

[0206] R e is H, electron pair or counter ion;

[0207] L" is R e or a direct bond or a bond to: Q, a targeting moiety, an analyte (e.g., an analyte molecule), a solid support, a solid support residue, a nucleoside, or another compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC').

[0208] In some embodiments, m" is an integer from 4 to 10, e.g., 4, 6, or 10. In other embodiments, n" is an integer from 3 to 6, e.g., 3, 4, 5, or 6. In some embodiments, n" is an integer from 18 to 28, e.g., 21 to 23.

[0209] In some other embodiments, L" is an alkylene, alkyleneheterocyclylene, alkyleneheterocyclylenealkylene, alkylenecyclylene, alkylenecyclylenealkylene, heteroalkylene, heteroalkyleneheterocyclylene, heteroalkyleneheterocyclyleneheteroalkylene, heteroalkylenecyclylene, or heteroalkylenecyclyleneheteroalkylene moiety. In some other certain embodiments, L" comprises an alkyleneoxy, a phosphodiester moiety, a sulfhydryl, a disulfide, or a maleimide moiety, or a combination thereof.

[0210] In certain of the foregoing embodiments, the targeting moiety is an antibody or a cell surface receptor antagonist.

[0211] In some embodiments, the antibodies include CD3, CD4, FoxP3, TNF-α, IFN-γ, clone 4S.B3, clone206D, CD8α (D8A8Y) Rabbit mAb, Vimentin (D21H3) 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 αE / CD103, CCR9, or MOPC-21.

[0212] In other more specific embodiments of any of the foregoing compounds of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'), R 1 or R 2 Has one of the following structures:

[0213]

[0214]

[0215] In other more specific embodiments of any of the foregoing compounds of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'), R 1 or R 2 Has one of the following structures:

[0216]

[0217]

[0218] Certain embodiments of compounds of structures (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') can be prepared according to solid phase synthesis methods similar to those known in the art for preparing oligonucleotides. Thus, in some embodiments, L' is a bond to a solid support, a solid support residue, or a nucleoside. Solid supports containing activated deoxythymidine (dT) groups are readily available and can be used in some embodiments as starting materials for preparing compounds of structures (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'). Thus, in some embodiments, R 1 or R 2 Has the following structure:

[0219]

[0220] Those skilled in the art will appreciate that the dT group depicted above is included and is not essential only for ease of synthesis and economic efficiency. Other solid supports may be used and will result in different nucleosides or solid support residues being present on L', or nucleosides or solid support residues may be removed or modified after synthesis.

[0221] In yet other embodiments, Q is independently a moiety comprising a reactive group capable of forming a covalent bond with an analyte molecule or a solid support at each occurrence. In other embodiments, Q is independently a moiety comprising a reactive group capable of forming a covalent bond with a complementary reactive group Q' at each occurrence. For example, in some embodiments, Q' is present on a compound of another structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') (e.g., on R 2 or R 3 position), and Q and Q' comprise complementary reactive groups such that the reaction of a compound of structure (I) and another compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') produces a covalently bound dimer of a compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'). Multimeric compounds of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') can also be prepared in a similar manner and are included within the scope of the embodiments of the present disclosure.

[0222] The type of Q group and the connectivity of the Q group to the rest of the compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') are not limited, provided that Q comprises a moiety with appropriate reactivity for forming the desired bond.

[0223] In certain embodiments, Q is a moiety that is not susceptible to hydrolysis under aqueous conditions, but is sufficiently reactive to form a bond with an analyte molecule or a corresponding group on a solid support (eg, an amine, azide, or alkyne).

[0224] Certain embodiments of compounds of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') comprise a Q group commonly employed in the field of bioconjugation. For example, in some embodiments, Q comprises a nucleophilic reactive group, an electrophilic reactive group, or a cycloaddition reactive group. In some more specific embodiments, Q comprises a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, olefin, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, or maleimide functional group. In some embodiments, the activated ester is an N-succinimidyl ester, an imidoester, or a polyfluorophenyl ester. In other embodiments, the alkyne is an alkyl azide or an acyl azide.

[0225] The Q group can be conveniently provided in a protected form to increase storage stability or other desired properties, and then the protecting group is removed at an appropriate time for conjugation with, for example, a targeting moiety or an analyte. Therefore, the Q group includes a "protected form" of a reactive group, including any of the reactive groups described in the above and following Table 1. A "protected form" of Q refers to a portion with lower reactivity under predetermined reaction conditions relative to Q, but it can be converted to Q under conditions that preferably do not degrade or react with other portions of a compound of structure (I), (IA), (IA'), (IB), (IB'), (IC) or (IC'). Those skilled in the art can derive a suitable protected form of Q based on specific Q and desired end uses and storage conditions. For example, when Q is SH, the protected form of Q includes a disulfide, which can be reduced to show the SH moiety using commonly known techniques and reagents.

[0226] Exemplary Q moieties are provided in Table 1 below.

[0227] Table 1. Exemplary Q fractions

[0228]

[0229]

[0230]

[0231]

[0232] It should be noted that in some embodiments, where Q is SH, the SH moiety will tend to form a disulfide bond with another sulfhydryl group on, for example, another compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'). Thus, some embodiments include compounds of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') in the form of a disulfide dimer, the disulfide bond being derived from the SH Q group.

[0233] Also included within the scope of certain embodiments are compounds of structure (I) wherein R 1 and R 2 One or both of the compounds of structure (I) contain a bond to another compound of structure (I). 1 and R 2 One or both of them are -OP(=R a )(R b )R c , and Rc is OL', and L' is a linker comprising a covalent bond to another compound of structure (I). Such compounds can be prepared by preparing a first compound of structure (I) having, for example, about 10 "M" moieties (i.e., n=9) and having an appropriate "Q" for reacting with a complementary Q' group on a second compound of structure (I). In this manner, compounds of structure (I) having any number of "M" moieties (e.g., 100 or more) can be prepared without coupling each monomer sequentially.

[0234] The value of m is another variable that can be selected based on the desired fluorescence and / or color intensity. In some embodiments, m is one or more integers at each occurrence. In some embodiments, m is independently an integer of 1 to 10 at each occurrence. In other embodiments, m is independently an integer of 1 to 6 at each occurrence, such as 1, 2, 3, 4, 5 or 6. In some specific embodiments, m is an integer of 1. In some embodiments, m is an integer of 2. In some embodiments, m is an integer of 3. In some embodiments, m is an integer of 4. In some embodiments, m is an integer of 5. In some embodiments, m is an integer of 6.

[0235] Fluorescence intensity can also be adjusted by selecting different n values. In some embodiments, n is an integer of one or more at each occurrence. In certain embodiments, n is an integer of 1 to 100. In other embodiments, n is an integer of 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.

[0236] The value of q is another variable that can be selected based on the desired fluorescence and / or color intensity. In some embodiments, q is an integer of one or more at each occurrence. In some embodiments, q is an integer of 1 to 5 independently at each occurrence. For example, in some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, q is 4. In some embodiments, q is 5.

[0237] The value of w is another variable that can be selected based on the desired fluorescence and / or color intensity. In some embodiments, w is an integer of one or more at each occurrence, provided that when n is an integer of 1, q is an integer greater than w. In some embodiments, w is an integer of 1 to 10. In some embodiments, w is an integer of 1 to 5. In some embodiments, w is an integer of 1. In some embodiments, w is an integer of 2. In some embodiments, w is an integer of 3. In some embodiments, w is an integer of 4. In some embodiments, w is an integer of 5.

[0238] The values ​​of q, w, n, and m are variables that can be selected based on the desired fluorescence and / or color intensity. In some more specific embodiments, q is an integer of 2, w is an integer of 1, n is an integer of 1, and m is an integer of 1. In some other more specific embodiments, q is an integer of 3, w is an integer of 1, n is an integer of 1, and m is an integer of 1. In some other more specific embodiments, q is an integer of 1, w is an integer of 1, n is an integer of 2, and m is an integer of 1 at each occurrence. In some other more specific embodiments, q is an integer of 1 for the first occurrence and an integer of 2 for the second occurrence, w is an integer of 1 for each occurrence, n is an integer of 2, and m is an integer of 1 at each occurrence. In some other more specific embodiments, q is an integer of 2 at each occurrence, w is an integer of 1 at each occurrence, n is an integer of 2, and m is an integer of 1 at each occurrence. In some other more specific embodiments, q is an integer of 1 for the first occurrence and an integer of 2 for the second occurrence, w is an integer of 1 at each occurrence, n is an integer of 2, and m is the integer of 1 at each occurrence.

[0239] M 1 and M 2 Based on the desired optical properties, for example, based on the desired color and / or fluorescence emission wavelength. In some embodiments, M 1 and M 2 is different at each occurrence. For example, in some embodiments, each M 1 and M 2 are different, and choose different M 1 and M 2 The moieties may have absorption and / or emission for use in fluorescence resonance energy transfer (FRET) methods. For example, in such embodiments, different M moieties are selected to form FRET donor-acceptor pairs such that absorption of radiation at one wavelength results in emission of radiation at a different wavelength via the FRET mechanism. In this aspect, M 1 and M 2 Parts form FRET pairs. 1 and M 2 The moieties can be appropriately selected by one of ordinary skill in the art based on the desired end use. 1 and M 2 Some of them include fluorescein and Alexa 594 dye. In some other embodiments, the M used in the FRET method 1 and M 2 Some of them include fluorescein and Alexa In some other embodiments, the M used in the FRET method 1and M 2 Some of them include fluorescein and Alexa 568 dye. In some other embodiments, the M used in the FRET method 1 and M 2 Some of them include fluorescein and Alexa 532 dye. In some other embodiments, the M used in the FRET method 1 and M 2 Some of them include fluorescein and Alexa 546 dye. In some other embodiments, the M used in the FRET method 1 and M 2 Some include Cy3 and Alexa 680 dyes.

[0240] M 1 and M 2 From M 1 and M 2 Any position (i.e., atom) on M can be attached to the rest of the molecule. Those skilled in the art will recognize that 1 and M 2 A means of attachment to the rest of the molecule.

[0241] In some embodiments, M 1 and M 2It is a fluorescent or colored part. Any fluorescent and / or colored part can be used, and those known in the art and typically used in colorimetry, UV, and / or fluorescence determination can be used. Examples of M parts that can be used in various embodiments of the present disclosure include, but are not limited to: xanthene derivatives (e.g., fluorescein, rhodamine, Oregon green (Oregon green), eosin or Texas red (Texas red)); cyanine derivatives (e.g., cyanine, indole carbocyanine, oxacarbocyanine (oxacarboL1cyanine), thiocarbocyanine or merocyanine); squaraine derivatives and ring-substituted squaraine, including Seta, SeTau and Square dyes; naphthalene derivatives (e.g., dansyl and 2-propionyl-6-dimethylaminonaphthalene (prodan) derivatives); coumarin derivatives; oxadiazole derivatives (e.g., pyridyl oxazole, nitrobenzoxadiazole or benzoxadiazole); anthracene derivatives (e.g., anthraquinone, including DRAQ5, DRAQ7 and CyTRAK Orange); pyrene derivatives such as cascade blue; oxazine derivatives (e.g., Nile Red, Nile Blue, Cresyl Violet, Oxazine 170); acridine derivatives (e.g., proflavine, acridine orange, acridine yellow); arylmethine derivatives: auramine, crystal violet, malachite green; and tetrapyrrole derivatives (e.g., porphine, phthalocyanine, or bilirubin). Other exemplary M moieties include: cyanine dyes, xanthate dyes (e.g., Hex, Vic, Nedd, Joe, or Tet); Yakima Yellow; Redmond Red; tamra; Texas Red, and Alexa Red. Dyes such as Alexa 350. Alexa 430. Alexa 488. Alexa 532. Alexa 546. Alexa 555. Alexa 568. Alexa 594. Alexa 633. Alexa 647、Alexa 660、Alexa 680 or Alexa 750.

[0242] The compounds of the present disclosure find utility as fluorescent and / or coloring dyes with high quantum efficiency. This is due in part to the donor moiety (e.g., M 1 ) has an emission spectrum similar to that of the acceptor moiety (e.g., M 2) with an absorption or excitation spectrum of 300 to 900 nm and an emission maximum of 350 to 900 nm. For example, in some embodiments, the FRET donor includes 2,5-diphenyloxazole with an excitation maximum of 311 nm and an emission maximum of 375 nm. In another example, in some embodiments, the FRET donor includes a dansyl fluorophore with an excitation maximum of 333 nm and an emission maximum of 518 nm. In yet another example, in some embodiments, the FRET donor Alexa Fluor has an excitation maximum of 311 nm and an emission maximum of 375 nm. In yet another example, in some embodiments, the FRET donor includes pyrene having an excitation maximum of 340 nm and an emission maximum of 376 nm. In yet another example, in some embodiments, the FRET donor includes coumarin 343 having an excitation maximum of 437 nm and an emission maximum of 477 nm. In yet another example, in some embodiments, the FRET donor includes Alexa Fluor having an excitation maximum of 346 nm and an emission maximum of 442 nm. In yet another example, in some embodiments, the FRET donor comprises a cyanide dye (CY3) having an excitation maximum of 550 nm and an emission maximum of 615 nm. In yet another example, in some embodiments, the FRET donor comprises an Alexa Fluor® dye having an excitation maximum of 430 nm and an emission maximum of 539 nm. In yet another example, in some embodiments, the FRET donor comprises a fluorescein dye (FAM) having an excitation maximum of 495 nm and an emission maximum of 519 nm. In yet another example, in some embodiments, the FRET donor comprises a cyanide dye (CY3) having an excitation maximum of 550 nm and an emission maximum of 615 nm. 555 has an excitation maximum of 555 nm and an emission maximum of 572 nm. In yet another example, in some embodiments, the FRET donor Alexa 568 has an excitation maximum of 578 nm and an emission maximum of 603 nm. In yet another example, in some embodiments, the FRET donor Alexa 633 has an excitation maximum at 630 nm and an emission maximum at 650 nm. In yet another example, in some embodiments, the FRET donor Alexa 647 has an excitation maximum of 650 nm and an emission maximum of 668 nm. In yet another example, in some embodiments, the FRET donor MB800 has an excitation maximum of 774 nm and an emission maximum of 798 nm. In yet another example, in some embodiments, the FRET donor Alexa 800 has an excitation maximum of 801 nm and an emission maximum of 814 nm. In yet another example, in some embodiments, the FRET donor Alexa In yet another example, in some embodiments, the FRET donor CF820 has an excitation maximum of 820 nm and an emission maximum of 830 nm. In yet another example, in some embodiments, the FRET donor CF810 has an excitation maximum of 812 nm and an emission maximum of 826 nm. 820 has an excitation maximum at 820 nm and an emission maximum at 849 nm. In yet another example, in some embodiments, the FRET donor PromoFluor 840 / 840 has an excitation maximum at 838 nm and an emission maximum at 880 nm. In yet another example, in some embodiments, the FRET donor The 860 has an excitation maximum at 852 nm and an emission maximum at 877 nm.

[0243] In some embodiments, a FRET acceptor is provided that has an excitation maximum of 400 to 800 nm and an emission maximum of 500 to 500 nm. For example, in some embodiments, the FRET acceptor 5-carboxyfluorescein (FAM) has 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® with an excitation maximum of 548 nm and an emission maximum of 566 nm. 543. In yet another example, in some embodiments, the FRET acceptor comprises an Alexa Fluor® having an excitation maximum at 532 nm and an emission maximum at 554 nm. 532. In yet another example, in some embodiments, the FRET acceptor comprises an Alexa Fluor® having an excitation maximum at 554 nm and an emission maximum at 570 nm. 546. In yet another example, in some embodiments, the FRET acceptor comprises an Alexa Fluor® having an excitation maximum at 555 nm and an emission maximum at 572 nm. 555. In yet another example, in some embodiments, the FRET acceptor comprises an Alexa Fluor® having an excitation maximum at 578 nm and an emission maximum at 603 nm. 568. In yet another example, in some embodiments, the FRET acceptor comprises an Alexa Fluor® having an excitation maximum at 590 nm and an emission maximum at 617 nm. 594. In yet another example, in some embodiments, the FRET acceptor comprises an Alexa Fluor® having an excitation maximum at 630 nm and an emission maximum at 650 nm. 633. In yet another example, in some embodiments, the FRET acceptor comprises an Alexa Fluor® having an excitation maximum at 663 nm and an emission maximum at 690 nm. 660. In yet another example, in some embodiments, the FRET acceptor comprises an Alexa Fluor® having an excitation maximum at 650 nm and an emission maximum at 668 nm. 647. In yet another example, in some embodiments, the FRET acceptor comprises an Alexa Fluor® having an excitation maximum at 679 nm and an emission maximum at 702 nm. 680. In yet another example, in some embodiments, the FRET acceptor comprises an Alexa Fluor® having an excitation maximum at 756 nm and an emission maximum at 776 nm. 750.

[0244] Embodiments of the present disclosure allow for various combinations of FRET donor / acceptor pairs to enhance brightness as a sensor. For example, in some embodiments, the FRET donor / acceptor pair is 2,5-diphenyloxazole as a FRET donor and Alexa Fluor as a FRET acceptor. 430. In another example, in some embodiments, the FRET donor / acceptor pair is a dansyl fluorophore as a FRET donor and an Alexa Fluor as a FRET acceptor. 543 or Alexa 532. In yet another example, in some embodiments, the FRET donor / acceptor pair is an Alexa Fluor 2000 as the FRET donor. 350 and Alexa Fluor as FRET acceptor 430. In yet another example, in some embodiments, the FRET donor / acceptor pair is pyrene as the FRET donor and Alexa Fluor as the FRET acceptor. 430. 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 as the FRET donor. 430 and Alexa Fluor as FRET acceptors 543. Alexa 532. Alexa 546. Alexa 555. Alexa 568 or Alexa 594. In yet another example, in some embodiments, the FRET donor / acceptor pair is FAM as the FRET donor and Alexa Fluor as the FRET acceptor. 532. Alexa 555. Alexa 546. Alexa 568 or Alexa 594. In yet another example, in some embodiments, the FRET donor / acceptor pair is CY3 as a FRET donor and Alexa Fluor as a FRET acceptor. 532. Alexa 633. In yet another example, in some embodiments, the FRET donor / acceptor pair is an Alexa Fluor 2000 as the FRET donor. 555 and Alexa Fluor as FRET acceptors 633 or Alexa 660. In yet another example, in some embodiments, the FRET donor / acceptor pair is an Alexa Fluor 2000 as the FRET donor. 568 and Alexa Fluor as FRET acceptor 633. Alexa 647、Alexa 660 or Alexa 680. In yet another example, in some embodiments, the FRET donor / acceptor pair is an Alexa Fluor 2000 as the FRET donor. 633 and Alexa Fluor as FRET acceptors 680. In yet another example, in some embodiments, the FRET donor / acceptor pair is an Alexa Fluor 2000 as the FRET donor. 647 and Alexa Fluor as FRET acceptors 680 or Alexa 750.

[0245] In yet other embodiments of any of the foregoing, M 1 and M 2 In some embodiments, M 1 and M 2 Contains six aryl or heteroaryl rings, or combinations thereof. In other embodiments, the rings are fused. For example, in some embodiments, M 1 and M 2 Contains three or more fused rings, four or more fused rings, five or more fused rings, or even six or more fused rings.

[0246] In some embodiments, M 1 or M 2 For example, in some embodiments, M 1 or M2 In other embodiments, M 1 or M 2 In yet other embodiments of the foregoing, M 1 or M 2 In some embodiments, the aryl moiety is polycyclic. In other more specific examples, the aryl moiety is a fused polycyclic aryl moiety, for example, it can include at least 3, at least 4, or even more than 4 aryl rings.

[0247] In other embodiments of any of the foregoing compounds of Structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'), M 1 or M 2 In each occurrence, independently at least one heteroatom is included. For example, in some embodiments, the heteroatom is nitrogen, oxygen, or sulfur.

[0248] In yet more embodiments of any of the foregoing, M 1 or M 2 In some embodiments, the substituent is fluoro, chloro, bromo, iodo, amino, alkylamino, arylamino, hydroxyl, sulfhydryl, alkoxy, aryloxy, phenyl, aryl, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl, carboxyl, sulfonate, amide, or formyl.

[0249] In some even more specific embodiments of the foregoing, M 1 or M 2 is independently at each occurrence a dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, bis-fluorophenyl-BODIPY, acridine, terrylene, hexaphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluoroboron-diaza-indacenyl)phenyl, (bis-fluorophenyl-difluoroboron-diaza-indacenyl)phenyl, biquatphenyl, bibenzothiazole, tribenzothiazole, binaphthyl, bianthraquinone, squarylium, 9,10-ethynylanthracene, or trinaphthyl moiety. In other embodiments, M 1 or M 2 is independently at each occurrence terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthrene, Rubrene, coronene, cyanine, perylene imide, or perylene amide or its derivatives. In still more embodiments, M 1 or M 2is independently at each occurrence a coumarin dye, a resorufin dye, a dipyrromethene boron difluoride dye, a ruthenium bipyridine dye, an energy transfer dye, a thiazole orange dye, a polymethine, or an N-aryl-1,8-naphthalimide dye.

[0250] In yet more embodiments of any of the foregoing, each M 1 or M 2 In still more embodiments, one or more M 1 or M 2 are the same and one or more M 1 or M 2 are different.

[0251] In some embodiments, M 1 or M 2 It is pyrene, perylene, perylene monoimide, 5-carboxyfluorescein (FAM), 6-FAM, 6-FITC, 5-FITC, or a derivative thereof.

[0252] In some embodiments, M 1 and M 2 In one or more occurrences, M independently includes a fused polycyclic aryl or heteroaryl moiety comprising at least four fused rings. 1 or M 1 -L 1b Each occurrence independently has one of the following structures:

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259] In some embodiments, M 2 Each occurrence independently has one of the following structures:

[0260]

[0261]

[0262] Although M containing a carboxylic acid group 1 or M 2 Part of it is in the form of the above anions (CO 2- ), but those skilled in the art will appreciate that this will vary depending on pH, and that the protonated form (CO 2 H) is included in various embodiments.

[0263] In some embodiments, the compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') is selected from Table 2. The compounds in Table 2 were prepared according to the procedures described in the Examples and their identities were confirmed by mass spectrometry.

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270] As used in Table 2 and throughout this disclosure, unless otherwise indicated, R 1 , R 2 , z and n have the definitions provided for compounds of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC').

[0271] As used in Table 2 above and throughout this disclosure, M 1 and M 2 is independently at each occurrence a fluorescent or colored moiety as described above. 1 and M 2 One is the FRET donor, and M 1 and M 2 The other of M is a FRET acceptor. In some embodiments, M 1 It's Alexa 594(AF594) and M 2 is FAM. In some embodiments, M 1 It's Alexa 555(AF555) and M 2 is FAM. In some embodiments, M 1 It's Alexa 568(AF568) and M 2 is FAM. In some embodiments, M 1 It's Alexa 680(AF680) and M 2 It's Cy3.

[0272] FAM refers to a moiety having one of the following structures:

[0273]

[0274] AF594 refers to a portion having the following structure:

[0275]

[0276] AF555 refers to a part with the following structure:

[0277]

[0278] AF568 refers to a portion having the following structure:

[0279]

[0280] AF680 refers to a part having one of the following structures:

[0281]

[0282]

[0283] Cy3 refers to a moiety having the following structure:

[0284]

[0285] As used above in Table 2 and throughout this disclosure, dT refers to the following structure:

[0286]

[0287] in:

[0288] R is H or a direct bond.

[0289] The FRET donor-acceptor ratio is another variable that can be selected based on the desired fluorescence and / or color intensity. 1 With the corresponding FRET donor M 2 The ratio is 1:1. In other words, for each FRET donor M 2 , the polymer dye contains a FRET acceptor M 1 In some embodiments, the FRET acceptor M 1 With the corresponding FRET donor M 2 The ratio is 1:2. In other words, for every two FRET donors M 2, the polymer dye contains a FRET acceptor M 1 In some embodiments, the FRET acceptor M 1 With the corresponding FRET donor M 2 The ratio is 1:3. In other words, for every three FRET donors M 2 , the polymer dye contains a FRET acceptor M 1 In some embodiments, the FRET acceptor M 1 With the corresponding FRET donor M 2 The ratio is 2:3. In other words, for every three FRET donors M 2 The polymer dye contains two FRET acceptors M 1 .

[0290] Some embodiments include any of the foregoing compounds, including specific compounds provided in Table 2 conjugated to a targeting moiety such as an antibody. In some embodiments, the antibodies include CD3, CD4, FoxP3, TNF-α, IFN-γ, clone 4S.B3, clone 206D, CD8α (D8A8Y) Rabbit mAb, Vimentin (D21H3) 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 αE / CD103, CCR9, or MOPC-21.

[0291] The present disclosure generally provides compounds with increased fluorescence emission relative to earlier known compounds. Thus, certain embodiments relate to fluorescent compounds comprising Y fluorescent moieties M, wherein the fluorescent compound has a peak fluorescence emission when excited with ultraviolet light of a predetermined wavelength that is at least 85% greater than the peak fluorescence emission of a single M moiety when excited with ultraviolet light of the same wavelength, and wherein Y is an integer of 2 or greater. Fluorescent compounds include compounds that emit a fluorescent signal when excited with light, such as ultraviolet light.

[0292] Also provided are compositions comprising a fluorescent compound of any of structures (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') and an analyte.

[0293] The compounds disclosed herein are "tunable", meaning that by appropriately selecting the variables in any of the foregoing compounds, one skilled in the art can obtain compounds having a desired and / or predetermined molar fluorescence (molar brightness). The tunability of the compounds allows the user to easily obtain compounds having a desired fluorescence and / or color for use in a particular assay or for identifying a particular analyte of interest. Although all variables may have an effect on the molar fluorescence of a compound, M 1 、M 2 , L 4 , L 5 , L 6 The appropriate selection of L, m, n, q, w and z is believed to play an important role in the molar fluorescence of the compound. Therefore, in one embodiment, a method for obtaining a compound with a desired molar fluorescence is provided, the method comprising selecting an M moiety with a known fluorescence, preparing a compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') comprising the M moiety, and selecting a molar fluorescence ratio for L. 4 , L 5 , L 6 , m, n, q, w and z are appropriately varied to obtain the desired molar fluorescence.

[0294] In certain embodiments, molar fluorescence can be expressed as a fold increase or decrease relative to the fluorescence emission of the parent fluorophore (e.g., monomer). In some embodiments, the molar fluorescence of the compounds of the invention is 1.1x, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or even higher relative to the parent fluorophore. Various embodiments include by appropriately selecting L 4 , L 5 , L 6 , m, n, q, w and z are used to prepare compounds having a desired fold increase in fluorescence relative to the parent fluorophore.

[0295] For ease of illustration, the anionic state (e.g., -OPO(OH)O-, -OPO 3 2- ) of various compounds containing phosphorus moieties (e.g., phosphate groups, etc.). Those skilled in the art will readily appreciate that the charge is pH dependent, and that uncharged (e.g., protonated or salts, such as sodium or other cations) forms are also included within the scope of the embodiments of the present disclosure.

[0296] In various other embodiments, compositions comprising any of the aforementioned compounds and one or more analyte molecules (e.g., biomolecules) are provided. In some embodiments, uses of such compositions in analytical methods for detecting the one or more analyte molecules are also provided.

[0297] In yet other embodiments, the compounds can be used in various analytical methods. For example, in certain embodiments, the present disclosure provides a method for staining a sample, the method comprising adding a compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') to the sample, for example, wherein R 1 or R 2 One of them is a linker comprising a covalent bond to an analyte molecule (eg, a biomolecule) or a microparticle, and R 1 or R 2 The other is H, OH, alkyl, alkoxy, alkyl ether or -OP(=R a )(R b )R c , an amount sufficient to produce an optical response when the sample is illuminated at an appropriate wavelength.

[0298] In some embodiments of the foregoing methods, R 1 or R 2 A linker is a linker comprising a covalent bond to an analyte molecule, such as a biomolecule. For example, a nucleic acid, an amino acid, or a polymer thereof (e.g., a polynucleotide or a polypeptide). In still more embodiments, the biomolecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion.

[0299] In yet other embodiments of the foregoing methods, R 1 or R 2 is a linker comprising a covalent bond to a solid support such as a microparticle. For example, in some embodiments, the microparticle is a polymeric bead or a non-polymeric bead.

[0300] In even more embodiments, the optical response is a fluorescent response.

[0301] In other embodiments, the sample comprises cells, and some embodiments further comprise observing the cells by flow cytometry.

[0302] In still more embodiments, the method further comprises distinguishing the fluorescent response from a fluorescent response of a second fluorophore having a detectably different optical property.

[0303] In other embodiments, the present disclosure provides a method for visually detecting an analyte molecule (such as a biomolecule), comprising:

[0304] (a) providing a compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'), wherein R 1 or R 2 One of them is a linker group containing a covalent bond to the analyte molecule, and R1 or R 2 The other is H, OH, alkyl, alkoxy, alkyl ether or -OP(=R a )(R b )R c ;and

[0305] (b) Detecting compounds by their visible properties.

[0306] In some embodiments, the analyte molecule is a nucleic acid, an amino acid, or a polymer thereof (eg, a polynucleotide or a polypeptide). In still more embodiments, the analyte molecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion.

[0307] In other embodiments, a method is provided for visually detecting an analyte molecule (such as a biomolecule), the method comprising:

[0308] (a) admixing any of the foregoing compounds with one or more analyte molecules; and

[0309] (b) Detecting compounds by their visible properties.

[0310] In other embodiments, a method for visually detecting an analyte molecule is provided, the method comprising:

[0311] (a) a compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') (where R 1 or R 2 is Q or a linker comprising a covalent bond to Q) and an analyte molecule;

[0312] (b) forming a conjugate of the compound and the analyte molecule; and

[0313] (c) Detection of the conjugate by its visual properties.

[0314] Other exemplary methods include a method for detecting an analyte, the method comprising:

[0315] (a) providing a compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'), wherein R 1 or R 2 including a linker comprising a covalent bond to a targeting moiety specific for the analyte;

[0316] (b) admixing the compound with the analyte, thereby associating the targeting moiety with the analyte; and

[0317] (c) Detecting the compound, for example by its visible or fluorescent properties.

[0318] In certain embodiments of the foregoing methods, the analyte is a particle, such as a cell, and the method includes the use of flow cytometry. For example, the compound can be provided with a targeting moiety, such as an antibody, for selectively associating with the desired cell, thereby making the cell detectable by any number of techniques, such as visible or fluorescent detection. In some embodiments, the antibody is a polyclonal antibody. In other embodiments, the antibody is a monoclonal antibody. Appropriate antibodies can be selected by one of ordinary skill in the art depending on the desired end use. Exemplary antibodies used in certain embodiments include CD3 (cloneUCHT1), CD4 (cloneOKT4), FoxP3, TNF-α, IFN-γ, clone 4S.B3, clone 206D, CD8α (D8A8Y) Rabbit mAb, Vimentin (D21H3) 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 αE / CD103, CCR9, and MOPC-21.

[0319] In certain embodiments, the conjugation efficiency of the conjugate formed by the compound comprising structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') and the analyte is greater than about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 98.5%, or 99%.

[0320] In yet other embodiments, the present disclosure provides a method for increasing the brightness of a dye, the method comprising:

[0321] (a) providing a dye solution comprising a compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'); and

[0322] (b) allowing the dye solution to age for a certain period of time.

[0323] 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 prior to use.

[0324] The dye solution may include various buffers. In some embodiments, the dye comprises ETOH. In some embodiments, the dye solution comprises BD brilliant. In some embodiments, the dye solution comprises sodium chloride or potassium chloride.

[0325] Thus, embodiments of the compounds of the present invention find utility in any number of methods, including, but not limited to: cell counting; cell sorting; biomarker detection; quantification of apoptosis; determination of cell viability; identification of cell surface antigens; determination of total DNA and / or RNA content; identification of specific nucleic acid sequences (e.g., as nucleic acid probes); and diagnosis of diseases, such as blood cancers.

[0326] In addition to the above methods, embodiments of compounds of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') find utility in a variety of disciplines and methods, including but not limited to: imaging in endoscopic procedures for identifying cancerous and other tissues; single cell and / or single molecule analytical methods, such as detection of polynucleotides with little or no amplification; cancer imaging, for example by including a targeting moiety such as an antibody or a sugar or other moiety that preferentially binds to cancer cells in a compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC'); imaging in surgical procedures; binding to histones for identifying various diseases; drug delivery, for example by replacing the M moiety in a compound of structure (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') with an active drug moiety; and / or contrast agents in dental work and other procedures, for example by preferentially binding a compound of structure (I) to various flora and / or organs.

[0327] It is to be understood that any embodiment of the compound of structure (I) as described above and for the compound of structure (I) as described above wherein R 1 , R 2 , R 3 , R 4 , R 5 , L', L 1a , L 1b , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 、M 1 、M 2Any specific selection of the variables R, m, n, q, and w set forth herein may be independently combined with other embodiments and / or variables of the compounds of structure (I) to form embodiments of the present disclosure not specifically set forth above. In addition, any specific R in the description of specific embodiments and / or claims may be independently combined with other embodiments and / or variables of the compounds of structure (I) to form embodiments of the present disclosure not specifically set forth above. 1 , R 2 , R 3 , R 4 , R 5 , L', L 1a , L 1b , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 、M 1 、M 2 Where a list of options is presented for the m, n, q and w variables, it should be understood that each individual option may be deleted from the specific embodiments and / or claims and the remaining list of options will be considered to be within the scope of the present disclosure.

[0328] It is understood that in the present description, combinations of substituents and / or variables of the depicted formulas are permissible only if such contributions result in stable compounds.

[0329] It will also be appreciated by those skilled in the art that in the methods described herein, the functional groups of the intermediate compounds may need to be protected by suitable blocking groups. Such functional groups include hydroxyl, amino, sulfhydryl and carboxylic acid. Suitable blocking groups for hydroxyl include trialkylsilyl or diarylalkylsilyl (e.g., tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable blocking groups for amino, amidino and guanidino include tert-butyloxycarbonyl, benzyloxycarbonyl, etc. Suitable blocking groups for sulfhydryl include-C (O) -R "(wherein R " is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl, etc. Suitable blocking groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Blocking groups can be added or removed according to standard techniques, which are known to those skilled in the art and as described herein. The use of blocking groups is described in detail in Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), the 3rd edition, Wiley. As will be appreciated by those skilled in the art, the protecting group may also be a polymeric resin such as Wang resin, Rink resin or 2-chlorotrityl-chloride resin.

[0330] In addition, all compounds of the present disclosure that exist in free base or acid form can be converted to their salts by methods known to those skilled in the art by treatment with appropriate inorganic or organic bases or acids. Salts of compounds of the present disclosure can be converted to their free base or acid form by standard techniques.

[0331] The following reaction schemes illustrate exemplary methods for preparing compounds of structures (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') of the present disclosure. It should be understood that those skilled in the art can prepare these compounds by similar methods or by combining other methods known to those skilled in the art. It should also be understood that those skilled in the art will be able to prepare other compounds of structures (I), (IA), (IA'), (IB), (IB'), (IC), or (IC') not specifically described below in a similar manner as described below, by using appropriate starting components and modifying the parameters of the synthesis as needed. Typically, starting components can be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc., 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.

[0332] Reaction Scheme I

[0333]

[0334] Reaction Scheme I shows an exemplary method for preparing intermediates useful for preparing compounds of structure (I), wherein R 1 , L 2 , L 3 and M are as defined above, R 2 and R 3 As defined above or a protected variant thereof and L is an optional linker. Referring to Reaction Scheme 1, compounds of structure a can be purchased or prepared by methods well known to those of ordinary skill in the art. Under Suzuki coupling conditions known in the art, the reaction of a with MX (wherein x is a halogen, such as bromine) yields compounds of structure b. Compounds of structure b can be used to prepare compounds of structure (I) as described below.

[0335] Reaction Scheme II

[0336]

[0337] Reaction Scheme II shows an alternative method for preparing intermediates useful for preparing compounds of structure (I). Referring to Reaction Scheme II, wherein R 1 , L 1 , L 2 , L 3 , G and M are as defined above, and R 2 and R 3 As defined above, or a protected variation thereof, a compound of structure c (which can be purchased or prepared by well-known techniques) is reacted with M-G' to obtain a compound of structure d. Here, G and G' represent functional groups with complementary reactivity (i.e., functional groups that react to form a covalent bond). G' can be a side chain of M or part of the structural backbone of M. G and G' can be any number of functional groups described herein, such as alkyne and azide, respectively, amine and activated ester, respectively, or amine and isothiocyanate, respectively, etc.

[0338] The compound of structure (I) can be prepared from one of structures b or d by reacting with a phosphoramidite compound having the following structure (e) under well-known automated DNA synthesis conditions:

[0339]

[0340] wherein L is an optional linking group.

[0341] DNA synthesis methods are well known in the art. Briefly, two alcohol groups (e.g., R in intermediate b or d above) are reacted to form a molecule. 2 and R 3 ) are functionalized with dimethoxytrityl (DMT) and 2-cyanoethyl-N,N-diisopropylaminophosphoramidite groups, respectively. Typically, in the presence of an activator such as tetrazole, the phosphoramidite group is coupled to an alcohol group and then oxidized to the phosphorus atom with iodine. The dimethoxytrityl group can be removed with an acid (e.g., chloroacetic acid) to expose a free alcohol, which can be reacted with the phosphoramidite group. By treating with ammonia, the 2-cyanoethyl can be removed after oligomerization.

[0342] The preparation of phosphoramidites used in oligomerization methods is also well known in the art. For example, a primary alcohol (e.g., R 3 ) can be protected as a DMT group by reaction with DMT-Cl. The secondary alcohol (e.g., R 2 ) is functionalized to a phosphoramidite. Methods for preparing phosphoramidites and their oligomerization are well known in the art and are described in more detail in the Examples.

[0343] Compounds of structure (I) are prepared by oligomerization of intermediates b or d and e according to the well-known phosphoramidite chemistry described above. The desired number of m and n repeating units are incorporated into the molecule by repeating the phosphoramidite coupling the desired number of times.

[0344] In addition, the compounds of the present disclosure can be prepared according to the methods described in PCT Publication Nos. WO 2016 / 183185; WO 2017 / 173355; and WO 2017 / 177065 (each of which is hereby incorporated by reference).

[0345] The efficiency of the FRET process depends in part on the characteristics of the chromophore. Specifically, high-efficiency FRET requires a large overlap between the absorption spectrum of the donor chromophore and the emission spectrum of the acceptor chromophore. In addition, the distance and orientation of the chromophore play an important role. The FRET efficiency is inversely proportional to the sixth power of the distance between the chromophores, and the angle of the transition dipole moment should be substantially aligned to be parallel (i.e., close to 0 ° or 180 °). Therefore, in certain embodiments, the covalent attachment of the first and second chromophores to the polymer backbone is selected so that the distance between the first and second chromophores is minimized and the transition dipole moments are substantially aligned. The efficiency of FRET can be expressed according to the following equation:

[0346]

[0347] Where E FRET is the FRET efficiency, R is the distance between chromophores, and Ro is expressed according to the following equation:

[0348] Ro=(8.8×10 23 JK 2 Q o n -4 ) 1 / 6

[0349] where J is the spectral overlap of the absorbance spectrum of the acceptor with the emission spectrum of the donor, Q o is the donor quantum efficiency, n -4 is the medium index (constant) between the donor and the acceptor, and K 2 is the dipole direction matching.

[0350] Thus, one embodiment provides a polymer compound comprising an acceptor chromophore having an acceptor transition dipole moment and covalently attached to a polymer backbone and a donor chromophore having a donor transition dipole moment and covalently attached to the polymer backbone, wherein the polymer compound is identified in solution under physiological conditions, wherein the effective distance between the acceptor chromophore and the donor chromophore is less than about 50.0 nm and the acceptor transition dipole and the donor transition dipole are substantially parallel.

[0351] In some embodiments, the effective distance between the acceptor chromophore and the donor chromophore is less than about 25.0 nm. In some embodiments, the effective distance between the acceptor chromophore and the donor chromophore is less than about 10.0 nm. In some embodiments, the effective distance between the acceptor chromophore and the donor chromophore is less than about 30.0 nm, less than about 27.0 nm, less than about 22.0 nm, less than about 20.0 nm, less than about 17.0 nm, less than about 15.0 nm, less than about 12.0 nm, less than about 11.0 nm, less than about 9.0 nm, less than about 8.0 nm, less than about 7.0 nm, less than about 6.0 nm, less than about 5.0 nm, less than about 4.0 nm, less than about 3.0 nm, less than about 2.0 nm, or less than about 1.0 nm.

[0352] In some embodiments, the acceptor chromophore is a fluorescent dye moiety. In certain embodiments, the donor chromophore is a fluorescent dye moiety. In certain related embodiments, both the acceptor chromophore and the donor chromophore are fluorescent dye moieties.

[0353] In some embodiments, the angle between the acceptor transition dipole moment and the donor transition dipole moment ranges from 120° to 180°. For example, in some embodiments, the angle between the acceptor transition dipole moment and the donor transition dipole moment ranges from 125° to 180°, 130° to 180°, 140° to 180°, 150° to 180°, 160° to 180°, 170° to 180°, 172° to 180°, 175° to 180°, or 177° to 180°.

[0354] In certain embodiments, the angle between the acceptor transition dipole moment and the donor transition dipole moment ranges from 0° to 60°. For example, in some embodiments, the angle between the acceptor transition dipole moment and the donor transition dipole moment ranges from 0° to 50°, 0° to 40°, 0° to 30°, 0° to 20°, 0° to 10°, 0° to 8°, 0° to 5°, 0° to 3°, or 0° to 2°.

[0355] In some more specific embodiments, the polymer compound further comprises a first acceptor chromophore covalently linked at the proximal end of the polymer backbone, a second acceptor chromophore covalently linked at the distal end of the polymer backbone, and a donor chromophore covalently linked between the proximal and distal ends of the polymer backbone.

[0356] In certain embodiments, the polymer backbone comprises a phosphate linkage. In some embodiments, the polymer backbone comprises a plurality of phosphate linkages.

[0357] In some related embodiments, the polymer backbone comprises an alkylene oxide linker. In some more specific embodiments, the alkylene oxide is an ethylene oxide. In some specific embodiments, the polymer backbone comprises a HEG linker, a C linker, or a combination thereof.

[0358] In some embodiments, the polymer compound has a molecular weight of less than 20,000 g / mol. In some embodiments, the polymer compound has a molecular weight of less than 19,000 g / mol, 18,500 g / mol, 18,000 g / mol, 17,500 g / mol, 17,000 g / mol, 16,500 g / mol, 16,000 g / mol, 15,500 g / mol, 15,000 g / mol, 14,500 g / mol, 14,000 g / mol, 13,500 g / mol, 13,000 g / mol, 12,500 g / mol, 11,500 g / mol, 11,000 g / mol, 10,500 g / mol, mol, 10,000g / mol, 9,500g / mol, 9,000g / mol, 8,500g / mol, 8,000g / mol, 7,500g / mol, 7,000g / mol, 6,500g / mol, 6,000g / mol, 5,500 A molecular weight of g / mol, 5,000g / mol, 4,500g / mol, 4,000g / mol, 3,500g / mol, 3,000g / mol, 2,500g / mol, 2,000g / mol, 1,500g / mol, or 1,000g / mol.

[0359] In some embodiments, the polymer compound is not a peptide or protein. In some other embodiments, the polymer backbone does not have amide bonds.

[0360] The following examples are provided for purposes of illustration only and not limitation. Example

[0361] General approach

[0362] Mass spectrometry analysis was performed on a Waters / Micromass Quattro micro MS / MS system (in MS mode only) using MassLynx 4.1 acquisition software. The mobile phase for LC / MS on the dye was 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6 mM triethylamine (TEA), pH 8. Phosphoramidites and precursor molecules were also analyzed using a Waters Acquity UHPLC system 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 calculated using the HPLC-MS / MS software on a Waters / Micromass Quattro micro MS / MS system (in MS mode only). Onium cation infusion enhanced ionization was obtained. Excitation and emission curve experiments were recorded on a Cary Eclipse spectrophotometer.

[0363] Unless otherwise noted, all reactions were performed in oven-dried glassware under a nitrogen atmosphere. Commercially available DNA synthesis reagents were purchased from Glen Research (Sterling, VA). Anhydrous pyridine, toluene, dichloromethane, diisopropylethylamine, triethylamine, acetic acid, pyridine, and THF were purchased from Aldrich. All other chemicals were purchased from Aldrich or TCI and used as received without additional purification.

[0364] Example 1

[0365] Synthesis of dyes with alkylene-polyethylene glycol-alkylene spacer groups

[0366] Compounds having an alkylene-polyethyleneoxy-alkylene linker were prepared as follows:

[0367] Oligofluoroside constructs (ie, compounds of structure (I)) were synthesized on an Applied Biosystems 394 DNA / RNA synthesizer at a 1 μmol scale and had either a 3′-phosphate group or a 3′-S 2 -(CH 2 ) 6-OH group or any other group described herein. Synthesis was performed directly on CPG beads or on polystyrene solid supports using standard phosphoramidite chemistry. Oligofluoroglycosides were synthesized in the 3' to 5' direction using standard solid phase DNA methods and coupling employed standard β-cyanoethyl phosphoramidite chemistry. Fluoroglycoside phosphoramidites and spacers (e.g., polyethylene glycol phosphoramidites, propane-diol phosphoramidites, butane-diol phosphoramidites, and hexane-diol phosphoramidites) and linkers (e.g., 5'-amino-modifier phosphoramidites and thiol-modifier S2 phosphoramidites) are dissolved in acetonitrile to prepare a 0.1M solution and added in a continuous 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 activator reagent in acetonitrile, 3) oxidize P(III) with iodine / pyridine / water to form a stable P(v), and 4) cap any unreacted 5'-hydroxyl groups with acetic anhydride / 1-methylimidazole / acetonitrile. The synthesis cycle is repeated until the full-length oligofluoroglycoside construct is assembled. At the end of the chain assembly, the monomethoxytrityl (MMT) or dimethoxytrityl (DMT) is removed with dichloroacetic acid in dichloromethane.

[0368] Compounds were provided on controlled pore glass (CPG) supports at 0.2 umol scale in labeled Eppendorf tubes. 400 μL of 20%-30% NH 4 OH and mix slowly. The open tube is placed at 55 ° C for ~ 5 minutes or until excess gas has been released, and then tightly closed and incubated for 2 hours (+ / - 15min.). The tube is taken out from the heating block and allowed to reach room temperature, then centrifuged at 13,400RPM for 30 seconds to consolidate the supernatant and solid. The supernatant is carefully taken out and placed in a marked tube, and then 150 μL acetonitrile is added to wash the carrier. After the washing solution is added to the tube, it is placed in a CentriVap device at 40 ° C until dry.

[0369] The product was characterized by ESI-MS, UV-absorption and fluorescence spectroscopy.

[0370] Example 2

[0371] General flow cytometry methods

[0372] Unless otherwise noted, the following general procedures were used throughout the following examples:

[0373] Lysis of whole blood:

[0374] Buffered ammonium chloride method. For staining of live cells, ethylenediaminetetraacetate (EDTA) anticoagulated normal human blood was lysed at room temperature (RT) with ammonium chloride solution (ACK), 15 mL blood to 35 mL hemolytic agent super-large (bulk) for 15 min. The cells were washed twice with 50% Hank's balanced salt solution (HBSS) and 50% 1% fetal bovine serum (FBS) 1x Dulbecco's phosphate buffered saline (PBS) with 0.02% sodium azide. The cells were then resuspended in donor plasma to 100 μL / test article / 0.1-1x10e6. The cells in plasma were added to the V for 100 μL 1% bovine serum albumin (BSA) and 1x DPBS with 0.02% sodium azide in a polypropylene 96-well HTS plate. f After incubation at room temperature for 45 min., cells were washed twice with 50% HBSS and 50%-1% FBS 1xDPBS with 0.02% sodium azide.

[0375] Lysis / fixation method. Blood was lysed at room temperature with 1.0mL RBC lysate (ammonium chloride), 100-15mL blood to 35mL hemolytic agent for 15min. The cells were then washed twice with 50% HBSS and 50%-1% FBS1xDPBS with 0.02% sodium azide. The cells were then resuspended in donor plasma to 100 μL / test product / 1x10e6. Pre-diluted antibodies were added to 100 μL 1% BSA and 1x DPBS with 0.02% sodium azide. 100 μL cells were added to 96-well polypropylene HTS plates (a total of 200 μL test product size). After incubation at room temperature for 45min., the cells were washed twice with 50% HBSS and 50% 1% FBS1x DPBS with 0.02% sodium azide.

[0376] Preparation of Antibody Conjugates:

[0377] The antibody conjugate is prepared by reacting a compound of structure (I) comprising a Q moiety having the following structure with the desired antibody:

[0378]

[0379] Thus, the compound and antibody are conjugated by reaction of the S and Q moieties on the antibody to form the following linked structure:

[0380]

[0381] The antibody conjugates are indicated by the antibody name followed by the compound number. For example, UCHT1-I-1 indicates a conjugate formed between the UCHT1 antibody and the compound of structure (I)I-1. If the reference compound number does not include the Q moiety above in Table 1, it is understood that the Q moiety is already provided and the conjugate is prepared from the resulting compound with the Q moiety.

[0382] Dilution of the conjugate:

[0383] The antibody is brought to room temperature. The antibody conjugate is diluted to a concentration in the range of 0.1-540nM (8.0 micrograms or less / test product) in cell staining buffer (1X DPBS, 1% BSA, 0.02% sodium azide). In certain embodiments, the serial dilutions of each sample start with 269nM antibody in cell staining buffer, and the antibody dilutions are kept in dark until use. In other experiments, the dilutions start with 4.0 μg antibody / test product size, wherein the test product size ranges from 100-200 μL. Titers are performed with two or four times dilution to generate binding curves. In some cases, 8.0 or 2.0 μg / test product size is used in the first hole in the dilution series.

[0384] Flow cytometry with conjugates:

[0385] After physical characterization, the activity and functionality of the conjugates (antibody binding affinity and brightness of the dye) were tested and compared with reference antibody staining. The quality of resolution was then determined by brightness compared to an autofluorescence negative control and other nonspecific binding using a flow cytometer. Whole blood screening is the most conventional method for testing conjugates. As new constructs were formed, connectivity studies were performed.

[0386] Perform free dye flow cytometry:

[0387] After molecular and physical characterization, the potential affinity of the dye to cells compared with the reference dye staining is also tested. Since the dye also has the potential to act as a cell probe and bind to cellular materials, the dye can usually be screened for blood at high concentrations (>100nM- to -10,000nM) to determine specific characteristics. The brightness and linearity compared with the automatic fluorescent negative control and other dye controls after dilution are then evaluated using a flow cytometer to identify expected or unexpected off-target binding.

[0388] Flow cytometry workflow: Cultivate cells and observe visual signs of metabolic stress for dye screening or off-target binding (data not shown), or use fresh healthy cells for conjugate screening. Cells are counted regularly to check cell density (1x 10e5 and 1x 10e6 viable cells / mL). Antibody conjugates are diluted (preferably in plates or tubes) in staining buffer (DPBS, 0.1% BSA, 0.02% sodium azide) before harvesting cells. Cells with a viability range of 80%-85% are used. Cells are washed twice by centrifugation and washing cells with buffer to remove pH indicators, and cells are blocked with Ig and other proteins contained in FBS. Cell density is adjusted to the size of the test article in the staining buffer. Cell plates are inoculated, one test article / well, or dye (pre-diluted) is applied to cells in the plate. Then, cells are incubated at 23°C for 45min. Cells are washed twice by centrifugation and washing cells with washing buffer, and then the plate is aspirated. Cells are resuspended in acquisition buffer. 5000 intact cells were collected by flow cytometry.

[0389] Fluorescence of the dye was detected by flow cytometry using a 488 nM blue laser line and a 525 / 50 bandpass filter to detect peak emission (521 nM).At least 1500 intact cells were collected by flow cytometry, with a target of 3000-5000 intact cells, and analyzed to identify live cells present in the cell preparation.

[0390] Data analysis methods:

[0391] Descriptive Statistics. The EC-800 software allows the user to collect a number of statistics for each sample acquisition. The mean or median fluorescence intensity (MFI) in the FL1-A channel is used to measure the brightness of the antibody-dye reagent when it is interrogated by flow cytometry and when examining the noise. Other statistics are evaluated to determine the dye characteristics and overall quality of the reagent, including the median signal-to-noise ratio and absolute fluorescence (median or geometric mean).

[0392] Histogram. Flow cytometry events are size gated relative to side scatter (cell volume relative to cell granularity) in the forward direction. Those cells are then gated by fluorescence emission of mean fluorescence intensity (MFI) at 515nm. The collected data are presented as a two-parameter histogram plotted as the number of events on the y-axis relative to fluorescence intensity (which is represented on the x-axis on a logarithmic scale). The data can be summarized by affinity curves or histograms of relative fluorescence intensity.

[0393] Binding curves. MFI was chosen because it is the best parameter to measure the brightness of an antibody-dye reagent when it is interrogated by FCM, which can be expressed as a geometric mean, median, or average, and represents an absolute fluorescence measurement. For comparison, in cases where noise can be highly characterized, the signal-to-noise ratio is reported as MFI, S / N.

[0394] Bivariate, biparameter histograms. In some cases, to examine qualitative output, FCM events were not gated and data were presented as cell granularity (SSC) versus dye fluorescence. This approach allows for an overall assessment of all populations recovered in whole blood.

[0395] Example 3

[0396] Flow cytometric analysis of compounds I-1 and I-2 were conjugated with CD4 (clone OKT4) antibodies and eluted in 1x d-PBS (phosphate buffered saline). The dyes used included FAM and Alexa 594 (AF594). Whole blood was stained with 0.5 ug of the antibody conjugate and screened on a spectrometer.

[0397] Example 4

[0398] Preparation of phosphoramidites and compounds

[0399] Exemplary compounds were prepared using standard solid phase oligonucleotide synthesis protocols and fluorescein-containing phosphoramidites having the following structures:

[0400]

[0401] It was purchased from ChemGenes (Cat. # CLP-9780).

[0402] An exemplary linker (L) was coupled to a phosphoramidite having the structure 6 ) is included in the compound:

[0403]

[0404] It is also commercially available.

[0405] The exemplary linker (L) is coupled to a phosphoramidite having one of the following structures: 7 / L 1b ) is included in the compound:

[0406]

[0407] It is also commercially available.

[0408] Other exemplary compounds were prepared using phosphoramidites prepared according to the following scheme:

[0409]

[0410] Final deprotection produces the desired Fx moiety. Other commercially available phosphoramidite reagents are used as appropriate to set up the various moieties of the compound. The Q moiety having the following structure:

[0411]

[0412] Set up by reaction with a free sulfhydryl group:

[0413]

[0414] Other Q moieties are arranged in a similar manner according to the knowledge of one of ordinary skill in the art.

[0415] The various embodiments described above can be combined to provide additional embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the application data sheet, including U.S. Provisional Patent Application No. 63 / 352,563 filed on June 15, 2022, are incorporated herein by reference in their entirety. If necessary, the aspects of the embodiment can be modified to provide yet another embodiment using the concepts of various patents, applications, and publications. These and other changes can be made to the embodiment according to the above detailed description. Generally, in the following claims, the terms used should not be interpreted as limiting the claims to the specific embodiments disclosed in this specification and the claims, but should be interpreted as including all possible embodiments and the full equivalent range of such claims being authorized. Therefore, the claims are not limited by this disclosure.

Claims

1. A compound or a stereoisomer, salt or tautomer thereof, wherein the compound has the following structure (I): in: M 1 and M 2 is independently a chromophore at each occurrence, provided that M 1 is a FRET acceptor and M 2 is the corresponding FRET donor, and M 1 and M 2 Forming a FRET pair; L 1a is independently at each occurrence a heteroalkylene or heteroarylene linker; L 1b , L 2 , L 3 , L 5 , L 6 and L 7 is independently at each occurrence an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene or heteroalkynylene linker; L 4 Has one of the following structures at each occurrence: in: z is an integer from 1 to 100; and * indicates bonds to adjacent phosphorus atoms; R 1 and R 2 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a )(R b )R c , Q, or a protected form thereof, or L'; R 3 is independently at each occurrence H, alkyl or alkoxy; R 4 is, independently at each occurrence, OH, SH, O - , S-, OR d or SR d ; R 5 is independently at each occurrence oxo, thio or absent; R a is O or S; R b OH, SH, O-, S-, OR d or SR d ; R c OH, SH, O-, S-, OR d , OL', SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, alkyl phosphate, thiophosphate alkyl, phosphate alkyl ether, or thiophosphate alkyl ether; R d is the counter ion; Q is independently at each occurrence a moiety comprising a reactive group, or a protected form thereof, capable of forming a covalent bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q′; L' is independently at each occurrence a linker comprising a covalent bond to Q, a linker comprising a covalent bond to a targeting 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 another compound of structure (I); m, at each occurrence, is an integer of one or greater; q is an integer of one or greater at each occurrence; w is, in at least one occurrence, an integer of one or greater, provided that when n is an integer of 1, q is an integer greater than w; and n is an integer of one or more.

2. The compound according to claim 1, wherein L 1a and R is independently at each occurrence an optionally substituted 5-7 membered heteroarylene linker.

3. The compound according to claim 1 or 2, wherein L 1a Has one of the following structures:

4. The compound according to any one of claims 1 to 3, or a stereoisomer, salt or tautomer thereof, wherein the compound has one of the following structures (IA) or (IA'):

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

6. The compound according to any one of claims 1 to 5, or a stereoisomer, salt or tautomer thereof, wherein the compound has one of the following structures (IB) or (IB'):

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

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

9. The compound according to any one of claims 1 to 8, wherein L 3 At least one occurrence of is alkylene.

10. The compound according to any one of claims 1 to 9, wherein L 3 Each occurrence of is an alkylene group.

11. The compound according to any one of claims 7 to 10, or a stereoisomer, salt or tautomer thereof, wherein the compound has one of the following structures (IC) or (IC'): where y 1 ,y 2 and 3 is independently an integer from 1 to 6 at each occurrence.

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

13. The compound according to claim 12, wherein L 1b At least one occurrence of comprises a functional group formed by the reaction of an alkyne and an azide.

14. The compound according to claim 13, wherein L 1b At least one occurrence of is a linker comprising a triazole functional group.

15. The compound according to any one of claims 1 to 14, wherein L 1b -M 1 At least one occurrence of contains the following structure: Where L c and L d Each is independently an optional linker.

16. The compound according to claim 15, wherein L c or L d , or both do not exist.

17. The compound according to claim 15, wherein L c or L d , or both exist.

18. The compound according to claim 17, wherein When present, L c and L d Each is independently alkylene or heteroalkylene.

19. The compound according to claim 17, wherein L c and L d Independently has one of the following structures:

20. The compound according to any one of claims 1 to 19, wherein L 1b Contains one of the following structures: in a, b, c, d and e are each independently an integer ranging from 1-6.

21. The compound according to any one of claims 1 to 20, wherein M 1 -L 1b At least one occurrence of has one of the following structures:

22. The compound of any one of claims 1 to 21, wherein M 1 -L 1b Each occurrence of has one of the following structures:

23. A compound according to any one of claims 1 to 22, wherein L 7 At least one occurrence of is an optionally substituted heteroalkylene linker.

24. A compound according to any one of claims 1 to 23, wherein L 7 Each occurrence is independently optionally substituted heteroalkylene.

25. The compound of any one of claims 1 to 24, wherein L 7 Contains amide functionality.

26. A compound according to any one of claims 1 to 25, wherein L 7 At least one occurrence of has one of the following structures:

27. A compound according to any one of claims 1 to 26, wherein L 7 Each occurrence of has one of the following structures:

28. A compound according to any one of claims 1 to 27, wherein L 7 At least one occurrence of has one of the following structures:

29. A compound according to any one of claims 1 to 28, wherein L 7 Each occurrence of has one of the following structures:

30. A compound according to any one of claims 1 to 29, wherein R 3 At least one occurrence of is H.

31. A compound as described in any one of claims 1 to 30, wherein R 5 is independently OH, O- or OR at each occurrence d .

32. A compound as described in any one of claims 1 to 31, wherein R 4 At each occurrence is oxo.

33. A compound as described in any one of claims 1 to 32, wherein R 1 and R 2 Each is independently OH or -OP (= R a )(R b )R c .

34. A compound as described in any one of claims 1 to 32, wherein R 1 or R 2 One of them is OH or -OP (=R a )(R b )R c , and R 1 or R 2 The other one of them is Q or a linker comprising a covalent bond to Q.

35. A compound as described in any one of claims 1 to 32, wherein R 1 and R 2 Each is independently -OP(=R a )(R b )R c .

36. A compound as described in any one of claims 33 to 35, wherein R c It's OL'.

37. The compound of claim 36, wherein L' is a heteroalkylene linker to Q, a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside, or another compound of structure (I).

38. The compound of claim 37, wherein The analyte molecules are nucleic acids, amino acids or polymers thereof.

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

40. The compound of claim 37, wherein The targeting moiety is an antibody or a cell surface receptor antagonist.

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

42. The compound of claim 37, wherein L' comprises an alkyleneoxy or phosphodiester moiety, or a combination thereof.

43. The compound of claim 42, wherein L' has the following structure: in: m" and n" are independently integers from 1 to 10; R e is H, electron pair or counter ion; L” is R e or a direct bond or a bond to: Q, a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside or another compound of structure (I).

44. A compound as described in any one of claims 1 to 43, wherein R 1 or R 2 Has one of the following structures:

45. A compound as described in any one of claims 1 to 44, wherein R 1 or R 2 Has the following structure:

46. ​​A compound as described in any one of claims 1 to 45, wherein Q comprises a nucleophilic reactive group, an electrophilic reactive group, or a cycloaddition reactive group.

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

48. The compound of claim 47, wherein The activated ester is an N-succinimidyl ester, an imidic acid ester or a polyfluorophenyl ester.

49. The compound of claim 47, wherein The azide is an alkyl azide or an acyl azide.

50. A compound as described in any one of claims 1 to 49, wherein Q is a moiety selected from Table 1.

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

52. A compound as described in any one of claims 1 to 51, wherein M 1 and M 2 In one or more occurrences are independently fluorescent or colored.

53. The compound of claim 52, wherein M 1 and M 2 It's fluorescent.

54. A compound as described in any one of claims 1 to 53, wherein M 1 and M 2 [0063] In one or more occurrences independently includes fused polycyclic aryl or heteroaryl moieties comprising at least four fused rings.

55. A compound as described in any one of claims 1 to 54, wherein M 1 or M 1 -L 1b Each occurrence independently has one of the following structures:

56. A compound as described in any one of claims 1 to 54, wherein M 2 Each occurrence independently has one of the following structures:

57. A compound as described in any one of claims 1 to 56, wherein For each FRET donor M 2 , the polymer dye comprises a FRET acceptor M 1 .

58. A compound as described in any one of claims 1 to 56, wherein For every two FRET donors M 2 , the polymer dye comprises a FRET acceptor M 1 .

59. A compound as described in any one of claims 1 to 56, wherein For each of the three FRET donors M 2 , the polymer dye comprises a FRET acceptor M 1 .

60. The compound of any one of claims 1 to 56, wherein For each of the three FRET donors M 2 The polymer dye comprises two FRET acceptors M 1 .

61. A compound as described in any one of claims 1 to 60, wherein n is an integer from 1 to 100.

62. A compound as described in any one of claims 1 to 61, wherein n is an integer of 1 to 10.

63. A compound as described in any one of claims 1 to 62, wherein q is an integer from 1 to 10.

64. A compound as described in any one of claims 1 to 63, wherein q is an integer from 1 to 5.

65. A compound as described in any one of claims 1 to 64, wherein q is an integer of 1, w is an integer of 1, and n is an integer of 2.

66. A compound as described in any one of claims 1 to 64, wherein q is an integer of 2, w is an integer of 1, and n is an integer of 1.

67. A compound as described in any one of claims 1 to 64, wherein q is an integer of 3, w is an integer of 1, and n is an integer of 1.

68. A compound as described in any one of claims 1 to 64, wherein q is an integer of 2, w is an integer of 1, and n is an integer of 2.

69. A compound as described in any one of claims 1 to 68, wherein m is an integer of 1-6.

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

71. The compound of claim 11, wherein y 1 ,y 2 and 3 Each is 1 at each occurrence.

72. A compound selected from Table 2.

73. A method of staining a sample comprising adding to the sample a compound as described in any one of claims 1-72 in an amount sufficient to produce an optical response when the sample is illuminated at an appropriate wavelength.

74. The method of claim 73, wherein: The optical response is a fluorescent response.

75. The method of claim 73 or 74, wherein: The sample comprises cells.

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

77. The method of claim 76, further comprising distinguishing the fluorescent response from a fluorescent response of a second fluorophore having a detectably different optical property.

78. A method for visually detecting an analyte molecule, the method comprising: (a) providing a compound as described in any one of claims 1 to 72, wherein R 1 or R 2 is a linker comprising a covalent bond to the analyte molecule; and (b) detecting the compound by its visual properties.

79. A method for visually detecting an analyte molecule, the method comprising: (a) a compound as described in any one of claims 1 to 72 (wherein R 1 or R 2 is Q or a linker comprising a covalent bond to Q) is admixed with the analyte molecule; (b) forming a conjugate of said compound and said analyte molecule; and (c) detecting the conjugate by its visual properties.

80. A method for visually detecting an analyte, the method comprising: (a) providing a compound as described in any one of claims 1 to 72, wherein R 1 or R 2 comprising a linker comprising a covalent bond to a targeting moiety specific for said analyte; (b) admixing the compound with the analyte, thereby associating the targeting moiety with the analyte; and (c) detecting the compound by its visual properties.

81. The method of claim 80, 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) 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 αE / CD103, CCR9, and MOPC-21.

82. A method for increasing the brightness of a dye, the method comprising: (a) providing a dye solution comprising a compound as described in any one of claims 1 to 72; and (b) aging the dye solution for a certain period of time.

83. The method of claim 82, wherein: Aging the dye solution comprises storing the dye solution for at least one week.

84. The method of claim 83, wherein: Aging the solution included storing the dye solution for three weeks.

85. The method of any one of claims 82-84, wherein: The dye solution contains ETOH.

86. The method of any one of claims 82-85, wherein The dye solution contained BD Brightener.

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

88. A composition comprising a compound as described in any one of claims 1-72 and one or more analyte molecules.

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

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