Ultra-bright dimer or polymer dye having spacing linker group

Abstract

To provide a compound that is useful as fluorescent or coloring dye.SOLUTION: A compound useful as fluorescent or coloring dye is disclosed. The compound has the following structure (I) or a stereoisomer, tautomer or salt thereof. A preparation and use of said compound and a related method are also provided. The present invention generally relates to a dimeric and polymeric fluorescent or coloring dye having rigid spacing group and to a preparation method thereof and a use in various analytical methods.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] (background) (Field) The present invention relates generally to dimeric and polymeric fluorescent or colored dyes having rigid spacing groups, and methods for their preparation and use in various analytical methods. [Background technology]

[0002] Description of Related Art Fluorescent and / or colored dyes are known to be particularly suitable for applications where sensitive detection reagents are desired. Dyes that can preferentially label specific components or constituents in a sample allow researchers to determine the presence, amount and / or location of the specific components or constituents. Furthermore, specific systems can be monitored with respect to their spatial and temporal distribution in various environments.

[0003] Fluorescence and colorimetric methods are extremely widespread in chemistry and biology. These methods provide useful information about the presence, structure, distance, orientation, complexation and / or location of biomolecules. Furthermore, time-resolved methods are increasingly used in measuring dynamics and kinetics. As a result, many strategies for fluorescent or color labeling of biomolecules (e.g., nucleic acids and proteins) have been developed. Since the analysis of biomolecules typically occurs in an aqueous environment, the focus has been on the development and use of water-soluble dyes.

[0004] Highly fluorescent or colored dyes are desirable because the use of such dyes increases the signal-to-noise ratio and provides other related benefits. Thus, attempts have been made to increase the signal from known fluorescent and / or colored moieties. For example, dimeric and polymeric compounds containing two or more fluorescent and / or colored moieties have been prepared in the hope that such compounds will produce brighter dyes. However, as a result of intramolecular fluorescence quenching, known dimeric and polymeric dyes have not achieved the desired increase in brightness.

[0005] Thus, there is a need in the art for water-soluble dyes with increased per molar brightness. Ideally, such dyes and biomarkers should be strongly colored or fluorescent and available in a variety of colors and fluorescent wavelengths. The present invention fulfills this need and provides further related advantages. Summary of the Invention [Means for solving the problem]

[0006] (Brief summary) Briefly, embodiments of the present invention generally relate to compounds useful as water-soluble, fluorescent and / or colored dyes and / or probes that enable visual detection of analyte molecules (e.g., biomolecules), and reagents for their preparation. Methods for visually detecting analyte molecules using the dyes are also described.

[0007] Dye embodiments of the present disclosure include a linker ("L 4 "). In contrast to previous reports of dimeric and / or polymeric dyes, the dyes of the present invention are significantly brighter than their corresponding monomeric dye compounds. Without wishing to be bound by theory, it is believed that this linker moiety provides sufficient spatial distance between the fluorescent and / or colored moieties such that intramolecular fluorescence quenching is reduced and / or eliminated.

[0008] The water-soluble fluorescent or colored dyes of the present embodiments are strongly colored and / or fluorescent and can be easily observed by visual or other means. In some embodiments, the compounds can be observed without prior illumination or chemical or enzymatic activation. As described herein, by appropriate selection of the dye, visually detectable analyte molecules of various colors can be obtained.

[0009] In one embodiment, the compound has the following structure (I): [ka] or a stereoisomer, tautomer, or salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 , L 4 , M, m and n are as defined herein. The compounds of structure (I) find utility in many applications, including use as fluorescent and / or colored dyes in a variety of analytical methods.

[0010] In another embodiment, a method for staining a sample is provided comprising adding to the sample a compound of structure (I) in an amount sufficient to produce an optical response when the sample is illuminated with an appropriate wavelength.

[0011] In yet another embodiment, the present disclosure provides a method for visually detecting an analyte molecule, the method comprising: (a) providing a compound of structure (I); and (b) detecting said compound by its visual properties; Includes:

[0012] Other disclosed methods include a method for visually detecting a biomolecule, the method comprising: (a) combining a compound of structure (I) with one or more biomolecules; and (b) detecting said compound by its visual properties; Includes:

[0013] Another embodiment provides a method for visually detecting an analyte, the method comprising: (a)R 2 or R 3 providing a compound as disclosed herein comprising a linker comprising a covalent bond to a targeting moiety having specificity for said analyte; (b) mixing the compound and the analyte, thereby causing the targeting moiety and the analyte to associate; and (c) detecting said compound by its visual properties; Includes:

[0014] Other embodiments relate to compositions comprising a compound of structure (I) and one or more analyte molecules, such as biomolecules. The use of such compositions in analytical methods for the detection of one or more biomolecules is also provided.

[0015] In some other different embodiments, structure (II): [ka] or a stereoisomer, salt or tautomer thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1a , L 2 , L 3 , L 4, G, m and n are as defined herein. Compounds of structure (II) find utility in many applications, including use as intermediates for preparing fluorescent and / or colored dyes of structure (I).

[0016] In yet another embodiment, a method for labeling an analyte molecule is provided, the method comprising: (a)R 2 or R 3 mixing a compound of structure (II), which is Q or a linker comprising a covalent bond to Q, with the analyte molecule; (b) forming a conjugate of the compound and the analyte molecule; and (c) The above combination with the formula ML 1b - reacting a compound of formula G' with a compound of formula G', thereby forming at least one covalent bond by reaction of G and G'; where R 2 , R 3 , Q, G and M.L. 1b -G' is as defined herein.

[0017] In several different embodiments, another method for labeling an analyte molecule is provided, the method comprising: (a)R 2 or R 3 is a compound of structure (II) which is a linker containing a covalent bond to Q or Q, and a compound of formula ML 1b - mixing a compound of formula G' with a compound of formula G', thereby forming at least one covalent bond by reaction of G and G'; and (b) reacting the product of step (A) with said analyte molecule, thereby forming a conjugate of the product of step (A) and said analyte molecule; where R 2 , R 3 , Q, G and M.L. 1b -G' is as defined herein.

[0018] In yet another embodiment, a method for preparing a compound of structure (I) is provided, the method comprising reacting a compound of structure (II) with a compound of formula ML 1b -G', thereby forming at least one covalent bond by reaction of G and G', wherein G and ML 1b -G' is as defined herein.

[0019] Even more embodiments relate to fluorescent compounds comprising Y fluorescent moieties, M, which have a peak fluorescence emission upon excitation with ultraviolet light of a given wavelength that is at least 85% Y times the peak fluorescence emission of a single M moiety upon excitation with ultraviolet light of the same wavelength, and where Y is an integer equal to or greater than 2.

[0020] These and other aspects of the present invention will become evident upon reference to the following detailed description.

[0021] In the drawings, identical reference numbers identify similar elements. The sizes and relative positions of elements in the drawings are not necessarily drawn to scale, and some of these elements have been arbitrarily enlarged and positioned to improve the legibility of the drawings. Furthermore, the particular shapes of elements as drawn are not intended to convey any information regarding the actual shape of that particular element, but have only been selected for ease of recognition in the drawings. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 provides UV absorbance spectra of representative and comparative compounds containing a triethylene glycol spacer at 5 μm and pH 9.

[0023] [Diagram 2] FIG. 2 shows UV absorbance data for representative and comparative compounds containing a hexaethylene glycol spacer at 5 μm and pH 9.

[0024] [Diagram 3] FIG. 3 shows the fluorescence emission spectra of representative and comparative compounds containing a triethylene glycol spacer at 50 nM and pH 9.

[0025] [Figure 4] FIG. 4 shows the fluorescence emission spectra of representative and comparative compounds containing a hexaethylene glycol spacer at 50 nM and pH 9.

[0026] [Diagram 5] FIG. 5 shows UV absorbance data at 5 μm for representative compounds containing four hexaethylene glycol spacers and two or three fluorescein moieties versus a comparative compound with one fluorescein moiety.

[0027] [Figure 6] FIG. 6 is a graph of fluorescence emission data at 5 μm for representative compounds containing four hexaethylene glycol spacers and two or three fluorescein moieties versus a comparative compound having one fluorescein moiety.

[0028] [Figure 7] FIG. 7 shows the comparative fluorescence responses of exemplary compounds with various m values.

[0029] [Figure 8] FIG. 8 provides data comparing the fluorescence emission of “HEG” compounds where m is 1, 2, or 3 to compound A.

[0030] [Figure 9] FIG. 9 provides UV absorbance data for Compound I-32, Compound I-46 and Compound B.

[0031] [Figure 10] FIG. 10 shows the results of the reaction to trimerize compound I-42, as analyzed by PAGE.

[0032] [Figure 11] FIG. 11 provides data comparing the fluorescent signal of seven compounds in dead and necrotic cell populations.

[0033] [Figure 12] FIG. 12 shows the fluorescence intensity of antibody conjugates of I-51 versus compound G.

[0034] [Figure 13] FIG. 13 shows a comparison of the I-51 conjugate and the Compound G reference antibody.

[0035] [Figure 14] FIG. 14 shows a comparison of UCHT1-I-51, UCHT1-BB515, and UCHT1-FITC.

[0036] [Figure 15] FIG. 15 shows the expression levels of CD3 compared to a MEF standard curve.

[0037] [Figure 16] FIG. 16 shows a comparison of UCHT1-I-16 fractions to FITC.

[0038] [Figure 17] FIG. 17 shows a comparison of the UCHT1-I-16 fraction to the I-56 conjugate.

[0039] [Figure 18] FIG. 18 shows a comparison of the UCHT1-I-51-like analog UCHT1 I-16 with UCHT1 I-56 (10×) and UCHT1 I-53 (6×).

[0040] [Figure 19] FIG. 19 provides data comparing the UCHT1 I-51-like analog UCHT1 I-16 with UCHT1 I-56 (10×) and UCHT1 I-53 (6×).

[0041] [Figure 20] FIG. 20 shows the results of a regression analysis performed on data generated when UCHT1 I-16 and UCHT1 I-49 conjugates were tested, demonstrating equivalence between the conjugations.

[0042] [Figure 21A] Figure 21A shows the correlation between I-16 and I-45 as determined using regression analysis. Figure 21B shows the titration curve overlay and compared to a reference. Figure 21C shows exemplary qualitative data showing background FL and cell morphology comparing compound D and I-45. [Figure 21B] Figure 21A shows the correlation between I-16 and I-45 as determined using regression analysis. Figure 21B shows the titration curve overlay and compared to a reference. Figure 21C shows exemplary qualitative data showing background FL and cell morphology comparing compound D and I-45. [Figure 21C] Figure 21A shows the correlation between I-16 and I-45 as determined using regression analysis. Figure 21B shows the titration curve overlay and compared to a reference. Figure 21C shows exemplary qualitative data showing background FL and cell morphology comparing compound D and I-45.

[0043] [Figure 22] FIG. 22 shows the affinity curves as a histogram along with compound emission detected in the FL1-A channel.

[0044] [Figure 23] FIG. 23A shows a comparison of the fluorescence intensity of off-target non-specific binding of UCHT1-I-21B, UCHT1-I-16, and the reference UCHT1-FITC, and FIG. 23B shows supporting data.

[0045] [Figure 24] FIG. 24 shows the results of a regression analysis applied to the data to examine correlation and relative affinity.

[0046] [Diagram 25] FIG. 25 shows signal to noise data for UCHT1-I-21B, UCHT1-I-51, and UCHT1-FITC.

[0047] [Figure 26A] Figures 26A and 26B provide data comparing UCHT1 Compound G and UCHT1 I-51 in a plasma interference study using PBMCs. Figure 26A shows data resulting from the addition of 0% glycine, and Figure 26B shows data resulting from the addition of 2.5% glycine. [Figure 26B] Figures 26A and 26B provide data comparing UCHT1 Compound G and UCHT1 I-51 in a plasma interference study using PBMCs. Figure 26A shows data resulting from the addition of 0% glycine, and Figure 26B shows data resulting from the addition of 2.5% glycine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] (Detailed Description) In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, it will be understood by one of ordinary skill in the art that the invention may be practiced without these details.

[0049] Unless the context requires otherwise, throughout this specification and claims, the word "comprise" and variations thereof (e.g., "comprises" and "comprising") are to be interpreted in an open-ended, inclusive sense, i.e., "including but not limited to."

[0050] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0051] "Amino" refers to the group -NH2.

[0052] "Carboxy" refers to the group -CO2H.

[0053] "Cyano" refers to the group -CN.

[0054] "Formyl" refers to the group -C(=O)H.

[0055] "Hydroxy" or "hydroxyl" refers to the group --OH.

[0056] "Imino" refers to the group =NH.

[0057] "Nitro" refers to the -NO2 group.

[0058] "Oxo" refers to the =O substituent.

[0059] "Sulfhydryl" refers to the -SH group.

[0060] "Thioxo" refers to the group ═S.

[0061] "Alkyl" means an alkyl group consisting solely of carbon and hydrogen atoms, containing no unsaturation, and having 1 to 12 carbon atoms (C1-C 12"C-C alkyl" refers to a straight or branched hydrocarbon chain radical having 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl) and attached to the remainder of the molecule by a single bond (e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, etc.). Unless otherwise specifically stated in the specification, alkyl groups are optionally substituted.

[0062] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that links the rest of the molecule to a radical group, consists solely of carbon and hydrogen, contains no unsaturation, and has 1 to 12 carbon atoms (e.g., methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc.). The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The point of attachment of the alkylene chain to the rest of the molecule or to the radical group can be through one carbon or any two carbons in the chain. Unless stated otherwise specifically in the specification, alkylene is optionally substituted.

[0063] "Alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain (e.g., ethenylene, propenylene, n-butenylene, etc.) that links the rest of the molecule to a radical group, consists solely of carbon and hydrogen, contains at least one carbon-carbon double bond, and has 2 to 12 carbon atoms. The alkenylene chain is attached to the rest of the molecule through a single bond and to the radical group through 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 group can be through one carbon or any two carbons in the chain. Unless stated otherwise specifically in the specification, alkenylene is optionally substituted.

[0064] "Alkynylene" or "alkynylene chain" refers to a straight or branched divalent hydrocarbon chain (e.g., ethenylene, propenylene, n-butenylene, etc.) that links the rest of the molecule to a radical group, consists solely of carbon and hydrogen, contains at least one carbon-carbon triple bond, and has 2 to 12 carbon atoms. The alkynylene chain is attached to the rest of the molecule through a single bond and to the radical group through 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 group can be through one carbon or any two carbons in the chain. Unless stated otherwise specifically in the specification, alkynylene is optionally substituted.

[0065] "Alkyl ether" refers to any alkyl group, as defined above, where at least one carbon-carbon bond is replaced with a carbon-oxygen bond. The carbon-oxygen bond can be at a terminal portion (as in an alkoxy group) or the carbon-oxygen bond can be internal (i.e., COC). An alkyl ether contains at least one carbon-oxygen bond, but can contain more than one. For example, polyethylene glycol (PEG) is included within the meaning of an alkyl ether. Unless specifically stated otherwise herein, an alkyl ether group is optionally substituted. For example, in some embodiments, an alkyl ether is an alkyl group selected from the group consisting of an alcohol or -OP(=R a )(R b )R c where R a , R b and R c Each of is as defined for compounds of structure (I).

[0066] "Alkoxy" means a group of the formula -OR a where R a refers to a group which is an alkyl group, as defined above, containing 1 to 12 carbon atoms. Unless stated otherwise specifically in the specification, an alkoxy group is optionally substituted.

[0067] "Alkoxyalkyl ether" means a group of the formula -OR a R b (where R a is an alkylene group as defined above containing 1 to 12 carbon atoms, and Rb is an alkyl ether group as defined herein. Unless otherwise specifically stated in the specification, an alkoxyalkyl ether group can be optionally substituted, for example, an alcohol or -OP(=R a )(R b )R c where R a , R b and R c Each of is as defined for compounds of structure (I).

[0068] "Heteroalkyl" refers to an alkyl group (as defined above) that contains at least one heteroatom (e.g., N, O, P, or S) within or at the terminus of the alkyl group. In some embodiments, the heteroatom is within the alkyl group (i.e., a heteroalkyl has at least one carbon-[heteroatom] x -carbon bond, where x is 1, 2, or 3. In other embodiments, the heteroatom is at the end of the alkyl group, and thus serves to attach the alkyl group to the remainder of the molecule (e.g., M1-HA, where M1 is part of the molecule, H is the heteroatom, and A is the alkyl group). Unless otherwise specifically stated herein, heteroalkyl groups are optionally substituted. Exemplary heteroalkyl groups include ethylene oxide (e.g., polyethylene oxide), optionally including a phosphorus-oxygen bond such as a phosphodiester bond.

[0069] "Heteroalkoxy" means a heteroalkoxy group of the formula -OR a (where R a is a heteroalkyl group as defined above containing 1 to 12 carbon atoms. Unless stated otherwise specifically in the specification, a heteroalkoxy group is optionally substituted.

[0070] "Heteroalkylene" refers to an alkylene group as defined above that contains at least one heteroatom (e.g., N, O, P, or S) within the alkylene chain or at the end of the alkylene chain. In some embodiments, the heteroatom is in the alkylene chain (i.e., the heteroalkylene contains at least one carbon-[heteroatom]-carbon bond, where x is 1, 2, or 3). In other embodiments, the heteroatom is at the end of the alkylene, thus serving to link the alkylene to the remainder of the molecule (e.g., M1-HA-M2, where M1 and M2 are part of the molecule, H is a heteroatom, and A is an alkylene). Unless otherwise specifically stated herein, heteroalkylene groups are optionally substituted. Exemplary heteroalkylene groups include ethylene oxide (e.g., polyethylene oxide) and the "C" linking groups illustrated below: [ka] Includes.

[0071] Multimers of the above C-linkers are included in the various embodiments of the heteroalkylene linker.

[0072] "Heteroalkenylene" is a heteroalkylene (as defined above) containing at least one carbon-carbon double bond. Unless stated otherwise specifically in the specification, a heteroalkenylene group is optionally substituted.

[0073] "Heteroalkynylene" is a heteroalkylene containing at least one carbon-carbon triple bond. Unless stated otherwise specifically in the specification, a heteroalkynylene group is optionally substituted.

[0074] "Heteroatom" in reference to a "heteroatom linker" refers to a linker group consisting of one or more heteroatoms. Exemplary heteroatom linkers include an atom selected from the group consisting of O, N, P, and S, as well as multiple heteroatoms (e.g., a linker group of the formula -P(O- )(=O)O- or -OP(O - )(=O)O-- and multimers and combinations thereof.

[0075] "Phosphate" means -OP(=O)(R a )R b A group, where R a OH, O - OR c and R b OH, O - , OR c , a thiophosphate group or a further phosphate group, where R c is the counter ion (e.g., Na + etc.)

[0076] "Phosphoalkyl" means -OP(=O)(R a )R b A group, where R a OH, O - OR c and R b is -Oalkyl, where R c is the counter ion (e.g., Na + and the like). Unless otherwise specifically stated herein, phosphoalkyl groups are optionally substituted. For example, in certain embodiments, the -Oalkyl portion of a phosphoalkyl group is hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether, thiophosphoalkylether, or -OP(=R a )(R b )R c where R is optionally substituted with one or more of a , R b and R c Each of is as defined for compounds of structure (I).

[0077] "Phosphoalkyl ether" means -OP(=O)(R a )Rb A group, where R a OH, O - OR c and R b is an -O alkyl ether, where R c is the counter ion (e.g., Na + and the like). Unless otherwise specifically stated herein, a phosphoalkyl ether group is optionally substituted. For example, in certain embodiments, the -O alkyl ether portion in a phosphoalkyl ether group is hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether, or -OP(=R a )(R b )R c where R is optionally substituted with one or more of a , R b and R c Each of is as defined for compounds of structure (I).

[0078] "Thiophosphate" is -OP(=R a )(R b )R c A group, where R a is O or S; R b OH, O - , S - , OR d or S.R. d and R c OH, SH, O - , S - , OR d , S.R. d , a phosphate group or a further thiophosphate group, where R d is the counter ion (e.g., Na + etc.), with the proviso that: i) R a is S; ii) R b is S - or S.R. d and iii) R c , SH, S -or S.R. d or iv) a combination of i), ii) and / or iii).

[0079] "Thiophosphoalkyl" means -OP(=R a )(R b )R c A group, where R a is O or S; R b OH, O - , S - , OR d or S.R. d and R c is -Oalkyl, where R d is the counter ion (e.g., Na + etc.), with the proviso that: i) R a is S or 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 herein, a thiophosphoalkyl group is optionally substituted. For example, in certain embodiments, the -Oalkyl portion of the thiophosphoalkyl group is hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether, thiophosphoalkylether, or -OP(=R a )(R b )R c where R is optionally substituted with one or more of a , R b and R c Each of is as defined for compounds of structure (I).

[0080] "Thiophosphoalkyl ether" means -OP(=R a )(R b )R c A group, where R ais O or S, and R b OH, O - , S - , OR d or S.R. d and R c is an -O alkyl ether, where R d is the counter ion (e.g., Na + etc.), with the proviso that: i) R a is S or 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 herein, a thiophosphoalkyl ether group is optionally substituted. For example, in certain embodiments, the -O alkyl ether portion of the thiophosphoalkyl group is hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether, or -OP(=R a )(R b )R c where R is optionally substituted with one or more of a , R b and R c Each of is as defined for compounds of structure (I).

[0081] "Carbocyclic" refers to a stable 3- to 18-membered aromatic or non-aromatic ring containing 3 to 18 carbon atoms. Unless otherwise specifically stated herein, a carbocyclic ring may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system which may include fused or bridged ring systems and may be partially or fully unsaturated. Non-aromatic carbocyclyl radicals include cycloalkyl, while aromatic carbocyclyl radicals include aryl. Unless otherwise specifically stated herein, a carbocyclic group is optionally substituted.

[0082] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic carbocyclic ring, which may include fused or bridged ring systems, has 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, and is saturated or unsaturated and attached to the remainder of the molecule by a single bond. Monocyclic cyclocalkyls 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 herein, cycloalkyl groups are optionally substituted.

[0083] "Aryl" refers to a ring system containing at least one carbocyclic aromatic ring. In some embodiments, the aryl contains 6 to 18 carbon atoms. The aryl ring can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems. Aryl includes, but is not limited to, aryl derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless otherwise specifically stated herein, aryl groups are optionally substituted.

[0084] "Heterocyclic" refers to a stable 3-18 membered aromatic or non-aromatic ring containing 1-12 carbon atoms and 1-6 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless otherwise specifically stated herein, the heterocyclic ring may include fused or bridged ring systems, the nitrogen, carbon or sulfur atoms in the heterocyclic ring may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclic ring may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system which may be partially or fully unsaturated. Examples of aromatic heterocyclic rings are listed below in the definition of heteroaryl (i.e., heteroaryl is a subset of heterocyclic). Examples of non-aromatic heterocyclic rings include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, a heterocyclic group is optionally substituted.

[0085] "Heteroaryl" refers to a 5-14 membered ring system containing 1-13 carbon atoms, 1-6 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring. For purposes of certain embodiments of the present invention, the heteroaryl radical can include fused or bridged ring systems; the nitrogen, carbon or sulfur atoms in the heteroaryl radical can be optionally oxidized; and the nitrogen atom can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system which can be optionally quaternized.Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzthiazolyl, benzoindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzo Furanyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, benzoxazolinonyl, benzimidazolethionyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizyl nyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, pteridinonyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyridinonyl, pyrazinyl, pyrimidinyl, pyri pryrimidinonyl, pyridazinyl, pyrrolyl, pyrido[2,3-d]pyrimidinonyl, quinazolinyl, quinazolinonyl, quinoxalinyl, quinoxalinonyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, thieno[3,2-d]pyrimidin-4-onyl, thieno[2,3-d]pyrimidin-4-onyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwise specifically in the specification, heteroaryl groups are optionally substituted.

[0086] "Fused" refers to a ring system that contains at least two rings, where the two rings share at least one common ring atom, e.g., two common ring atoms. When the fused ring is a heterocyclyl or heteroaryl ring, the common ring atom can be carbon or nitrogen. Fused rings include bicyclic, tricyclic, tetracyclic, etc.

[0087] As used herein, the term "substituted" refers to any of the above groups (e.g., alkyl, alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, alkoxy, alkyl ether, alkoxyalkyl ether, heteroalkyl, heteroalkoxy, phosphoalkyl, phosphoalkyl ether, thiophosphoalkyl, thiophosphoalkyl ether, carbocyclic, cycloalkyl, aryl, heterocyclic, and / or heteroaryl) in which at least one hydrogen atom (e.g., 1, 2, 3, or all of the hydrogen atoms) has been replaced with a non-hydrogen atom (e.g., including, but not limited to, F, Cl, "Substituted" also means that one or more hydrogen atoms are replaced by a bond to a heteroatom (e.g., a halogen atom such as , Br, and I; an oxygen atom in groups such as hydroxyl, alkoxy, and ester groups; a sulfur atom in groups such as thiol, thioalkyl, sulfone, sulfonyl, and sulfoxide groups; a nitrogen atom in groups such as amine, amide, alkylamine, dialkylamine, arylamine, alkylarylamine, diarylamine, N-oxide, imide, and enamine; a silicon atom in groups such as trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, and triarylsilyl groups; and other heteroatoms in various other groups). "Substituted" also means that one or more hydrogen atoms are replaced by a higher bond (e.g., a double bond or a triple bond) to a heteroatom (e.g., an oxygen atom in groups such as oxo, carbonyl, carboxyl, and ester groups; and a nitrogen atom in groups such as imine, oxime, hydrazone, and nitrile). For example, "substituted" means that one or more hydrogen atoms are replaced by a bond to -NR g Rh , -NR g C(=O)R h , -NR g C(=O)NR g R h , -NR g C(=O)OR h , -NR g SO2R h , -OC(=O)NR g R h , -OR g , -SR g , -SOR g , -SO2R g , -OSO2R g , -SO2OR g , =NSO2R g , and -SO2NR g R h "Substituted" also includes any of the above groups replaced with one or more hydrogen atoms -C(=O)R. g , -C(=O)OR g , -C(=O)NR g R h , -CH2SO2R g , -CH2SO2NR g R h In the above, R g and R hare 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 refers to any of the above groups in which one or more hydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl, and / or heteroarylalkyl group. In some embodiments, the optional substituents are -OP(=R a )(R b )R c where R a , R b and R c Each of the is as defined for compounds of structure (I). Additionally, each of the foregoing substituents may also be optionally substituted with one or more of the above-described substituents.

[0088] "Conjugation" refers to the overlap of one p orbital with another p orbital across an intervening sigma bond. 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-butadiene has a degree of conjugation of one, while benzene and other aromatic compounds typically have multiple degrees of conjugation. Fluorescent and colored compounds typically contain a degree of conjugation of at least one.

[0089] "Fluorescent" refers to a molecule that can absorb light of a particular frequency and emit light of a different frequency. Fluorescence is well known to those of skill in the art.

[0090] "Colored" refers to molecules that absorb light within the colored spectrum (i.e., red, yellow, blue, etc.).

[0091] "Linker" refers to a continuous chain of at least one atom (e.g., carbon, oxygen, nitrogen, sulfur, phosphorus, and combinations thereof) that connects one portion of a molecule to another portion of the same molecule or to a different molecule, moiety, or solid support (e.g., a microparticle). A linker may connect the molecules via a covalent bond or other means (e.g., ionic or hydrogen bonding interactions).

[0092] The term "biomolecule" refers to any of a variety of biological substances, including nucleic acids, carbohydrates, amino acids, polypeptides, glycoproteins, hormones, aptamers, and mixtures thereof. More specifically, the term is intended to include, but is not limited to, RNA, DNA, oligonucleotides, modified or derivatized nucleotides, enzymes, receptors, prions, receptor ligands (including hormones), antibodies, antigens, and toxins, as well as bacteria, viruses, blood cells, and tissue cells. The visually detectable biomolecules of the present invention (e.g., compounds of structure (I) to which a biomolecule is linked) are prepared by contacting a biomolecule with a compound (which has a reactive group that allows the binding of the biomolecule to the compound via any available atom or functional group, such as an amino, hydroxyl, carboxyl, or sulfhydryl group on the biomolecule), as further described herein.

[0093] A "reactive group" is a moiety that can react with a second reactive group (e.g., a "complementary reactive group") to form one or more covalent bonds, for example, by substitution, oxidation, reduction, addition, or cycloaddition reactions. Exemplary reactive groups are provided in Table 1 and include, for example, nucleophilic groups, electrophilic groups, 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, alkenes, maleimides, α-haloimides, epoxides, aziridines, tetrazines, tetrazoles, phosphines, biotins, thiiranes, and the like.

[0094] The terms "visual" and "visually detectable" are used herein to refer to substances that are observable by the human eye without prior illumination or chemical or enzymatic activation. Such visually detectable substances absorb and emit light in a region of the spectrum ranging from about 300 to about 900 nm. Preferably, such substances are strongly colored, and have a chromaticity of preferably at least about 40,000, more preferably at least about 50,000, even more preferably at least about 60,000, even more preferably at least about 70,000, and most preferably at least about 80,000 M. -1 cm -1The compounds of the present invention can be detected by observation with the naked eye or with the aid of optical-based detection devices, including but not limited to, absorption spectrophotometers, transmission light 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 10 nm to about 400 nm), infrared (IR) region (about 700 nm to about 1 mm), and substances that emit and / or absorb in other regions of the electromagnetic spectrum are also included within the scope of "visually detectable" substances.

[0095] For the purposes of the present embodiment, the term "photostable visible dye" refers to a chemical moiety that is visually detectable and does not change or decompose significantly when exposed to light, as defined herein above. Preferably, the photostable visible dye does not exhibit significant bleaching or decomposition after exposure to light for at least 1 hour. More preferably, the visual dye is stable after exposure to light for at least 12 hours, even more preferably at least 24 hours, even more preferably at least 1 week, and most preferably at least 1 month. Non-limiting examples of photostable visual dyes suitable for use in the compounds and methods of the present invention include azo dyes, thioindigo dyes, quinacridone pigments, dioxazines, phthalocyanines, perinones, diketopyrrolopyrroles, quinophthalones, and trueycarboniums.

[0096] As used herein, the term "perylene derivative" is intended to include any substituted perylene that is visually detectable. However, the term is not intended to include perylene itself. The terms "anthracene derivative", "naphthalene derivative", and "pyrene derivative" are used similarly. In some preferred embodiments, the derivative (e.g., a derivative of perylene, pyrene, anthracene, or naphthalene) is an imide, bisimide, or hydrazamimide derivative of perylene, anthracene, naphthalene, or pyrene.

[0097] The visually detectable molecules of various embodiments of the present invention are useful in a wide variety of analytical applications (e.g., biochemical and biomedical applications) in which the presence, location, or amount of a particular analyte (e.g., biomolecule) needs to be determined. Thus, in another aspect, the present invention provides a method for visually detecting a biomolecule, comprising: (a) providing a biological system with a visually detectable biomolecule comprising a compound of structure (I) linked to a biomolecule; and (b) detecting the biomolecule by its visual properties. For the purposes of the present invention, the phrase "detecting a biomolecule by its visual properties" means that the biomolecule is observed with the naked eye or with the aid of an optical-based detection device (including, but not limited to, an absorption spectrophotometer, a transmitted light microscope, a digital camera, and a scanner) without illumination or chemical or enzymatic activation. A densitometer can be used to quantify the amount of visually detectable biomolecule present. For example, the relative amounts of the biomolecules in two samples can be determined by measuring the relative optical density. If the stoichiometry of dye molecules per biomolecule is known and the extinction coefficient of the dye molecule is known, the absolute concentration of the biomolecule can also be determined from the measurement of optical density.As used herein, the term "biological system" is used to refer to any solution or mixture that contains one or more biomolecules in addition to visually detectable biomolecules.Non-limiting examples of such biological systems include cells, cell extracts, tissue samples, electrophoretic gels, assay mixtures, and hybridization reaction mixtures.

[0098] "Solid support" refers to any solid substrate known in the art for solid phase support of molecules, for example, "microparticle" refers to any of a number of small particles useful for binding to the compounds of the invention, including, but not limited to, glass beads, magnetic beads, polymeric beads, non-polymeric beads, etc. In certain embodiments, the microparticle comprises a polystyrene bead.

[0099] "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 obtained based on the structure of the solid support and the group that links the molecule to the solid support.

[0100] A "targeting moiety" is a moiety that selectively binds or associates with a particular target (e.g., an analyte molecule). By "selectively" binds or associates, it is meant that the targeting moiety preferentially associates or binds with the desired target compared to other targets. In some embodiments, the compounds disclosed herein include a linkage to a targeting moiety for the purpose of selectively binding or associating the compound with the analyte of interest (i.e., the target of the targeting moiety), thus 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, and the like. In some embodiments, the targeting moiety is a moiety 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 cellular structure), such as an antibody, thus allowing detection of the cell of interest. Small molecules that selectively bind or associate with the desired analyte are also contemplated as targeting moieties in certain embodiments. Those skilled in the art will recognize other analytes and corresponding targeting moieties that are useful in various embodiments.

[0101] "Base pairing moiety" refers to a heterocyclic moiety that can hybridize with a complementary heterocyclic moiety through hydrogen bonds (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 (e.g., adenosine, guanosine, thymidine, cytosine and uridine and their analogs).

[0102] The embodiments of the invention disclosed herein are also meant to encompass all compounds of structure (I) or (II) that are isotopically labeled by replacing one or more atoms with an atom having a different atomic mass or mass number. Examples of isotopes that may be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine (e.g., isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, respectively). 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I, and 125 I) can be mentioned.

[0103] Isotopically labeled compounds of structure (I) or (II) may generally be prepared by conventional techniques known to those of skill in the art, or by processes analogous to those described below and in the Examples below, using the appropriate isotopically labeled reagent in place of the unlabeled reagent previously used.

[0104] "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.

[0105] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when said event or circumstance occurs and instances when it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and that the description includes both substituted alkyl groups and alkyl groups that have no substitution.

[0106] The term "salt" includes both acid addition salts and base addition salts.

[0107] "Acid addition salts" include salts of inorganic acids (e.g., but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like) and organic acids (e.g., 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, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, glycer ... and the like.

[0108] "Base addition salt" refers to a salt prepared from the addition of an inorganic or 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 salts, 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 (e.g., 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, and the like). Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0109] Crystallization may produce solvates of the compounds described herein. The embodiments of the present invention encompass all solvates of the compounds described. As used herein, the term "solvate" refers to an aggregate that contains one or more molecules of the compounds of the present invention and one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present invention may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., as well as their corresponding solvated forms. The compounds of the present invention may be true solvates, while in other cases, the compounds of the present invention may only retain adventitious water or another solvent, or may be a mixture of water and some adventitious solvent.

[0110] The compound embodiments of the present invention (e.g., compounds of structure I or II) or their salts, tautomers or solvates may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined in terms of absolute stereochemistry as (R)- or (S)-, or with respect to amino acids as (D)- or (L)-. The embodiments of the present invention are meant 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 separated using conventional techniques (e.g., chromatography and fractional crystallization). Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from appropriate optically pure precursors, or separation of racemic mixtures (or racemic mixtures of salts or derivatives) using, for example, chiral high pressure liquid chromatography (HPLC). The compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise specified, are intended to include both E and Z geometric isomers, as well as all tautomeric forms.

[0111] "Stereoisomers" refer to compounds composed of the same atoms linked by the same bonds but having different three-dimensional structures that are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0112] "Tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present invention includes tautomers of any of the above compounds. Various tautomeric forms of the above compounds can be easily derived by one skilled in the art.

[0113] The chemical naming protocols and structure diagrams used herein are modifications of the IUPAC nomenclature 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 skilled in the art are also used.

[0114] As stated above, in one embodiment of the present invention, compounds are provided that are useful as fluorescent and / or colored dyes in various analytical methods. In other embodiments, compounds are provided that are useful as synthetic intermediates for the preparation of compounds that are useful as fluorescent and / or colored dyes. In general terms, embodiments of the present invention relate to dimers and higher order polymers of fluorescent and / or colored moieties. The fluorescent and / or colored moieties are linked by a linking moiety. Without wishing to be bound by theory, it is believed that the linker helps to maintain sufficient spatial distance between the fluorescent and / or colored moieties such that intramolecular quenching is reduced or eliminated, thus resulting in a dye compound with higher molar "brightness" (e.g., high fluorescence emission).

[0115] Thus, in some embodiments, the compound has the following structure (A): [ka] where L is a linker sufficient to maintain spatial separation between one or more (e.g., each) M groups such that intramolecular quenching is reduced or eliminated, and R 1 , R 2 , R 3 , L 1 , L 2 , L 3 and n is as defined for structure (I). In some embodiments of structure (A), L is a linker comprising one or more ethylene glycol or polyethylene glycol moieties.

[0116] In another embodiment, the following structure (I): [ka] or a stereoisomer, salt, or tautomer thereof, wherein: M, in each occurrence, is independently a moiety that contains two or more carbon-carbon double bonds and at least one degree of conjugation; L 1 is, independently at each occurrence, either i) an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker; or ii) a linker that contains a functional group that can be formed by the reaction of two complementary reactive groups; L 2 and L 3 is independently at each occurrence an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker; L 4 is, at each occurrence, independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker greater than 3 atoms in length, where the heteroatoms in said heteroalkylene, heteroalkenylene, and heteroalkynylene linkers are selected from O, N, and S; R 1 is, at each occurrence, independently H, alkyl, or alkoxy; R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a )(R b )R c , Q or L'; R 4 is, in each occurrence, independently, OH, SH, O - , S - , OR d or S.R. d and; R5 is, at each occurrence, independently oxo, thioxo, or absent; R a is O or S; R b OH, SH, O - , S - , OR d or S.R. d and; R c OH, SH, O - , S - , OR d , O.L.', S.R. d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counter ion; Q, in each occurrence, is independently a moiety that contains a reactive group or a protected analog thereof that is capable of forming a covalent bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group, Q'; L', in each occurrence, is independently 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 moiety, a linker comprising a covalent bond to a nucleoside, or a linker comprising a covalent bond to an additional compound of structure (I); m, in each occurrence, is independently 0 or a greater integer, provided that at least one occurrence of m is an integer 1 or greater; and n is an integer equal to or greater than 1.

[0117] In different embodiments of the compounds of structure (I): M, in each occurrence, is independently a moiety that contains two or more carbon-carbon double bonds and at least one degree of conjugation; L 1is, independently at each occurrence, either i) an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker; or ii) a linker that contains a functional group that can be formed by the reaction of two complementary reactive groups; L 2 and L 3 is independently at each occurrence an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker; L 4 is, at each occurrence, independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker greater than 3 atoms in length, where the heteroatoms in said heteroalkylene, heteroalkenylene, and heteroalkynylene linkers are selected from O, N, and S; R 1 is, at each occurrence, independently H, alkyl, or alkoxy; R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, -OP(=R a )(R b )R c , Q, a linker comprising a covalent bond to Q, a linker comprising a covalent bond to an analyte molecule, a linker comprising a covalent bond to a solid support or a linker comprising a covalent bond to a further compound of structure (I), wherein: R a is O or S; R b OH, SH, O - , S - , OR d or S.R. d ;R c OH, SH, O - , S - , OR d , S.R. d , alkyl, alkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; and Rd is the counter ion; R 4 is, in each occurrence, independently, OH, SH, O - , S - , OR d or S.R. d and; R 5 is, at each occurrence, independently oxo, thioxo, or absent; Q, in each occurrence, is independently a moiety that contains a reactive group that can form a covalent bond with an analyte molecule, a solid support, or a complementary reactive group, Q'; m, in each occurrence, is independently 0 or a greater integer, provided that at least one occurrence of m is an integer 1 or greater; and n is an integer equal to or greater than 1.

[0118] The various linkers and substituents (e.g., M, Q, R 1 , R 2 , R 3 , R c , L 1 , L 2 , L 3 and L 4 ) is optionally substituted with one or more substituents. For example, in some embodiments, the optional substituents are selected to optimize the water solubility or other properties of the compounds of structure (I). In certain embodiments, each alkyl, alkoxy, alkyl ether, alkoxyalkyl ether, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, and thiophosphoalkyl ether in a 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, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, and thiophosphoalkyl ether. In certain embodiments, the optional substituent is -OP(=R a )(Rb )R c where R a , R b and R c is as defined for compounds of structure (I).

[0119] In some embodiments, L 1 is, at each occurrence, independently, an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker. 1 is, in each occurrence, independently, a linker that contains two complementary reactive groups, e.g., a functional group that can be formed by reaction of Q groups.

[0120] In some embodiments, L 4 is, at each occurrence, independently a heteroalkylene linker. In other more specific embodiments, L 4 is, at each occurrence, independently an alkylene oxide linker. For example, in some embodiments, L 4 is polyethylene oxide, and the compound has the following structure (IA): [ka] where z is an integer from 2 to 100. In some embodiments of (IA), z is an integer from 2 to 30, e.g., about 20 to 25, or about 23. In some embodiments, z is an integer from 2 to 10, e.g., 3 to 6. In some embodiments, z is 3. In some embodiments, z is 4. In some embodiments, z is 5. In some embodiments, z is 6.

[0121] Optional Linker L 1can be used as the point of attachment of the M moiety to the remainder of the compound. For example, in some embodiments, a synthetic precursor to a compound of structure (I) is prepared, and the M moiety is attached to the synthetic precursor using any number of facile methods known in the art (e.g., methods referred to as "click chemistry"). To this end, any reaction that is rapid and substantially irreversible can be used to attach M to the synthetic precursor to form a compound of structure (I). Exemplary reactions include copper-catalyzed reactions of azides and alkynes to form triazoles (Husgen 1,3-dipolar cycloaddition), reactions of dienes and dienophiles (Diels-Alder), strain-promoted alkyne-nitrone cycloaddition, reactions of strained alkenes with azides, tetrazines or tetrazoles, [3+2] cycloadditions of alkenes and azides, alkene and tetrazine inverse electron demand Diels-Alder, alkene and tetrazole photochemical reactions, and various substitution reactions (e.g., displacement of a leaving group by nucleophilic attack on an electrophilic atom). Exemplary substitution reactions include reactions of amines with the following: activated esters; N-hydroxysuccinimide esters; isocyanates; isothioscyanates, and the like. In some embodiments, L 1 The reaction to form may be carried out in an aqueous environment.

[0122] Thus, in some embodiments, L 1 is a linker that, in each occurrence, contains 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. In various embodiments, L 1For at least one occurrence of, the functional group may be formed by reaction of an aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imido ester, activated ester (e.g., N-hydroxysuccinimide ester), ketone, α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin, or thiirane functional group with a complementary reactive group, for example, reaction of an amine with an N-hydroxysuccinimide ester or an isothiocyanate.

[0123] In another embodiment, L 1 For at least one occurrence of L, the functional group may be formed by the reaction of an alkyne and an azide. In other embodiments, for at least one occurrence of L, the functional group may be formed by the reaction of an amine (e.g., a primary amine) and an N-hydroxysuccinimide ester or an isothiocyanate.

[0124] In more embodiments, L 1 For at least one occurrence of L, the functional group comprises an alkene, ester, amide, thioester, disulfide, carboxylic acid group, heterocyclic or heteroaryl group. In more embodiments, for at least one occurrence of L, the functional group comprises an alkene, ester, amide, thioester, thiourea, disulfide, carbocyclic, heterocyclic or heteroaryl group. In other embodiments, the functional group comprises an amide or thiourea. In some more specific embodiments, L 1 For at least one occurrence of L 1 is a linker that includes a triazolyl functional group, while in other embodiments, for at least one occurrence of L1, L1 is a linker that includes an amide or thiourea functional group.

[0125] In yet another embodiment, L 1For at least one occurrence of L 1 -M has the following structure: [ka] where L 1a and L 1b are each independently an optional linker.

[0126] In a different embodiment, L 1 For at least one occurrence of L 1 -M has the following structure: [ka] where L 1a and L 1b are each independently an optional linker.

[0127] In various embodiments of the foregoing, L 1a Or L 1b , or both are absent. 1a Or L 1b , or both are present.

[0128] In some embodiments, L 1a and L 1b Each occurrence, when present, is independently alkylene or heteroalkylene. For example, in some embodiments, L 1a and L 1b may, if present, independently have the structure:

[0129] [ka] has one of the following:

[0130] In yet another different embodiment of structure (I), L 1 is, at each occurrence, independently, an optional alkylene or heteroalkylene linker. In certain embodiments, L 1has the following structure: [ka] has one of the following:

[0131] In more embodiments, L 2 and L 3 is, at each occurrence, independently, C1-C6 alkylene, C2-C6 alkenylene, or C2-C6 alkynylene. For example, in some embodiments, the compound has the following structure (IB): [ka] where: x 1 , x 2 , x 3 and x 4 is, in each occurrence, independently an integer from 0 to 6; and z is an integer of 2 to 100, for example, an integer of 3 to 6.

[0132] In certain embodiments of the compound of structure (IB), x 1 , x 2 , x 3 or x 4 At least one occurrence of x is 1. 1 , x 2 , x 3 and x 4 In another embodiment, x 1 and x 3 In some embodiments, x 2 and x 4 In still other embodiments, x 1 and x 3 are 0 in each occurrence, and x 2 and x 4 are each 1 in each occurrence.

[0133] In some more specific embodiments of the compound of structure (IB), L 1 In some other specific embodiments of the compound of structure (IB), L 1 In another embodiment of the compound of structure (IB), L 1 is independently at each occurrence an optional alkylene or heteroalkylene linker.

[0134] In still other embodiments of any of the compounds of structure (I), R 4 is, in each occurrence, independently, OH, O - OR d "OR d " and "SR d " is the O associated with the cation - and S - For example, the disodium salt of a phosphate group is [ka] where R d is sodium (Na + ).

[0135] In other embodiments of any of the compounds of structure (I), R 5 is oxo at each occurrence.

[0136] In some different embodiments of any of the aforementioned compounds, R 1 is H.

[0137] In various other embodiments, R 2 and R 3 are each independently OH or -OP(=R a )(R b )R c In some different embodiments, R 2 or R 3is OH or -OP(=R a )(R b )R c and R 2 or R 3 The other is a linker that comprises a covalent bond to Q or Q.

[0138] In yet another embodiment of any of the foregoing compounds of structure (I), R 2 and R 3 are each independently -OP(=R a )(R b )R c In some of these embodiments, R c is OL'.

[0139] In other embodiments, R 2 and R 3 are each independently -OP(=R a )(R b ) OL', where 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 moiety, a nucleoside, or an additional compound of structure (I).

[0140] The linker L' can be any linker suitable for linking Q, a targeting moiety, an analyte (e.g., an analyte molecule), a solid support, a solid support residue, a nucleoside, or an additional compound of structure (I) to the compound of structure (I). Advantageously, certain embodiments include the use of an L' moiety selected to increase or optimize the water solubility of the compound. In certain embodiments, L' is a heteroalkylene moiety. In some other certain embodiments, L' includes an alkylene oxide or a phosphodiester moiety, or a combination thereof.

[0141] In certain embodiments, L' has the structure: [ka] where: m″ and n″ are independently integers from 1 to 10; R e is H, an electron pair or a counterion; L” is R e Or it is a direct bond or it is a linkage to Q, a targeting moiety, an analyte (eg, an analyte molecule), a solid support, a solid support moiety, a nucleoside or a further compound of structure (I).

[0142] In some embodiments, m" is an integer from 4 to 10, such as 4, 6, or 10. In other embodiments, n" is an integer from 3 to 6, such as 3, 4, 5, or 6.

[0143] In some other embodiments, L" is an alkylene or heteroalkylene moiety. In some other certain embodiments, L" comprises an alkylene oxide, a phosphodiester moiety, a sulfhydryl, a disulfide, or a maleimide moiety, or a combination thereof.

[0144] In certain of the foregoing embodiments, the targeting moiety is an antibody or a cell surface receptor antagonist.

[0145] In other more specific embodiments of any of the foregoing compounds of structure (I), R 2 or R 3 has the following structure: [ka] [ka] has one of the following:

[0146] Certain embodiments of the compounds of structure (I) can be prepared according to solid phase synthesis methods similar to those known in the art for the preparation of oligonucleotides. Thus, in some embodiments, L' is a linkage 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 the preparation of compounds of structure (I). Thus, in some embodiments, R 2 or R 3 has the following structure:

[0147] [ka] has.

[0148] Those skilled in the art will appreciate that the dT group shown above is included for ease of synthesis and economic efficiency only, and is not required. Other solid supports may be used, resulting in different nucleosides or solid support moieties present in L', or the nucleoside or solid support moiety may be removed or modified after synthesis.

[0149] In still other embodiments, Q, at each occurrence, is a moiety that independently comprises a reactive group capable of forming a covalent bond with an analyte molecule or a solid support. In other embodiments, Q, at each occurrence, is a moiety that independently comprises a reactive group that can form a covalent bond with a complementary reactive group Q'. For example, in some embodiments, Q' is a moiety that independently comprises a reactive group capable of forming a covalent bond with a complementary reactive group Q' on an additional compound of structure (I) (e.g., R 2 Rank or R 3 In the embodiment, Q and Q' are present at positions (A, B, C, C, D, E, E, F ...

[0150] There are no limitations on the type of Q group and its attachment to the remainder of the compound of structure (I), provided that Q has the appropriate reactivity to form the desired bond.

[0151] In certain embodiments, Q is a moiety that is not susceptible to hydrolysis under aqueous conditions, yet is sufficiently reactive to form a bond with a corresponding group (eg, an amine, azide, or alkyne) on an analyte molecule or a solid support.

[0152] Certain embodiments of the compound of structure (I) include a Q group that is commonly used in the field of bioconjugation reactions. For example, in some embodiments, Q includes a nucleophilic reactive group, an electrophilic reactive group, or a cycloaddition reactive group. In some more specific embodiments, Q includes a sulfhydryl, a disulfide, an activated ester, an isothiocyanate, an azide, an alkyne, an alkene, a diene, a dienophile, an acid halide, a sulfonyl halide, a phosphine, an α-haloamide, a biotin, an amino, or a maleimide functional group. In some embodiments, the activated ester is an N-succinimide ester, an imido ester, or a polyfluorophenyl ester. In other embodiments, the alkyne is an alkyl azide or an acyl azide.

[0153] Q group can be conveniently provided in a protected form to enhance storage stability or other desired properties, and the protecting group can then be removed at the appropriate time, for example, for conjugation with a targeting moiety or analyte. Thus, Q group includes "protected forms" of reactive groups, including any of the reactive groups described above or in Table 1 below. A "protected form" of Q refers to a moiety that has a lower reactivity compared to Q under a given reaction condition, but preferably does not degrade or react with other moieties of the compound of structure (I), and can be converted to Q under such conditions. Those skilled in the art can derive the appropriate protected form of Q based on the specific Q and the desired end use and storage conditions. For example, if Q is SH, the protected form of Q includes a disulfide that can be reduced to reveal an SH moiety using commonly known techniques and reagents.

[0154] Exemplary Q moieties are provided in Table I below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0155] It should be noted that in some embodiments where Q is SH, this SH moiety will tend to form a disulfide bond with another sulfhydryl group on another compound, for example, of structure (I). Thus, some embodiments include compounds of structure (I) that are in the form of a disulfide dimer, in which the disulfide bond is derived from the SH Q group.

[0156] Also included within the scope of certain embodiments are 2 and R 3is a compound of structure (I) in which one or both of R 2 and R 3 One or both of the following must be -OP(=R a )(R b )R c where Rc is OL', and L' is a linker comprising a covalent bond to an additional compound of structure (I). Such compounds can be prepared, for example, by preparing a first compound of structure (I) having about 10 "M" moieties (i.e., n=9) and a "Q" suitable for reaction 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 the need to sequentially couple each monomer. An exemplary embodiment of such a compound of structure (I) is the following structure (I'): [ka] where: R 1 , R 2 , R 3 , R 4 , R 5 , L 1 , L 2 , L 3 , L 4 each occurrence of M, m, and n is independently as defined for compounds of structure (I); L″ is a linker containing a functional group resulting from reaction of a Q moiety with the corresponding Q′ moiety; and α is an integer greater than 1, for example, 1 to 100, or 1 to 10.

[0157] Exemplary compounds of structure (I') are provided in Example 5. Other compounds of structure (I') can be derived by one of skill in the art, for example, by dimerizing or polymerizing the compounds of structure (I) provided herein.

[0158] In other embodiments, the Q moiety is conveniently masked (e.g., protected) as a disulfide moiety, which can then be reduced to provide an activated Q moiety for attachment to a desired analyte molecule or targeting moiety. For example, the Q moiety has the following structure: [ka] where R is an optionally substituted alkyl group. For example, in some embodiments, Q can be masked as a disulfide having the following structure: [ka] where n is an integer from 1 to 10, e.g., 6.

[0159] In some other embodiments, R 2 or R 3 One of the groups is OH or -OP(=R a )(R b )R c and R 2 or R 3 The other of the two is a linker that includes a covalent bond to the analyte molecule or a linker that includes a covalent bond to the solid support. For example, in some embodiments, the analyte molecule is a nucleic acid, an amino acid, or a polymer thereof. In other embodiments, the analyte molecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion. In yet different embodiments, the solid support is a polymeric bead or a non-polymeric bead.

[0160] The value for m is another variable that can be selected based on the desired fluorescence and / or color intensity. In some embodiments, m, at each occurrence, is independently an integer from 1 to 10. In other embodiments, m, at each occurrence, is independently an integer from 1 to 5, e.g., 1, 2, 3, 4, or 5.

[0161] In other embodiments, m, at each occurrence, is independently an integer greater than 2 and z is an integer from 3 to 10; e.g., in some embodiments, m, at each occurrence, is independently an integer greater than 2 (e.g., 3, 4, 5, or 6) and z is an integer from 3 to 6.

[0162] The fluorescence intensity can also be tuned by selection of various values ​​of n. In certain embodiments, n is an integer between 1 and 100. In other embodiments, n is an integer between 1 and 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.

[0163] M is selected based on the desired optical properties, for example, based on the desired color and / or fluorescence emission wavelength. In some embodiments, M is the same in each occurrence; however, it is important to note that each occurrence of M does not have to be the same M, and certain embodiments include compounds in which M is not the same in each occurrence. For example, in some embodiments, each M is not the same, and the different M moieties are selected to have absorbance and / or emission for use in fluorescence resonance energy transfer (FRET) methods. For example, in some embodiments, the different M moieties are selected such that absorbance of radiation at one wavelength causes emission of radiation at a different wavelength by a FRET mechanism. Exemplary M moieties can be appropriately selected by one of skill in the art based on the desired end use. Exemplary M moieties for FRET methods include fluorescein and 5-TAMRA (5-carboxytetramethylrhodamine, succinimidyl ester) dyes.

[0164] M can be attached to the remainder of the molecule from any position (i.e., atom) on M. Those of skill in the art will recognize means of attaching M to the remainder of the molecule. Exemplary methods include the "click" reaction described herein.

[0165] In some embodiments, M is a fluorescent or colored moiety. Any fluorescent and / or colored moiety may be used, such as those known in the art and typically used in colorimetric, UV, and / or fluorescent assays. Examples of M moieties useful in various embodiments of the invention include, but are not limited to, xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, or Texas Red); cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, or merocyanine); squaraine derivatives and ring-substituted squaraines, including Seta, SeTau, and Square dyes; naphthalene derivatives (e.g., dansyl and prodan derivatives); coumarin derivatives; oxadiazole derivatives (e.g., pyridyloxazo anthracene derivatives (e.g., anthraquinone (including DRAQ5, DRAQ7, and CyTRAK orange)); pyrene derivatives (e.g., 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 fluor® dyes.

[0166] In still other embodiments of any of the foregoing, M comprises three or more aryl or heteroaryl rings, or a combination thereof, for example, four or more aryl or heteroaryl rings, or a combination thereof, or even five or more aryl or heteroaryl rings, or a combination thereof. In some embodiments, M comprises six aryl or heteroaryl rings, or a combination thereof. In further embodiments, the rings are fused. For example, in some embodiments, M comprises three or more fused rings, four or more fused rings, five or more fused rings, or even six or more fused rings.

[0167] In some embodiments, M is cyclic. For example, in some embodiments, M is carbocyclic. In other embodiments, M is heterocyclic. In still other embodiments of the foregoing, M, at each occurrence, independently comprises an aryl moiety. In some of these embodiments, the aryl moiety is polycyclic. In other more specific embodiments, the aryl moiety is a fused polycyclic aryl moiety, for example, which may comprise at least three, at least four, or even more than four aryl rings.

[0168] In other embodiments of any of the foregoing compounds of structure (I), (IA), (IB) or (I'), M, at each occurrence, independently comprises at least one heteroatom. For example, in some embodiments, the heteroatom is nitrogen, oxygen or sulfur.

[0169] In still more embodiments of any of the foregoing, M, at each occurrence, independently includes at least one substituent, for example, in some embodiments, the substituent is a fluoro, chloro, bromo, iodo, amino, alkylamino, arylamino, hydroxy, sulfhydryl, alkoxy, aryloxy, phenyl, aryl, methyl, ethyl, propyl, butyl, isopropyl, t-butyl, carboxy, sulfonate, amido, or formyl group.

[0170] In still more specific embodiments of some of the foregoing, M, at each occurrence, is independently dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine, terylene, sexiphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl, squaraine, squarylium, 9,10-ethynylanthracene, or ter-naphthyl moiety. In other embodiments, M, at each occurrence, is independently p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, or perylene amide, or a derivative thereof. In still more embodiments, M at each occurrence is independently a coumarin dye, a resorufin dye, a dipyrrometheneboron difluoride dye, a ruthenium bipyridyl dye, an energy transfer dye, a thiazole orange dye, a polymethine, or an N-aryl-1,8-naphthalimide dye.

[0171] In still more embodiments of any of the foregoing, M is the same at each occurrence. In other embodiments, each M is different. In still more embodiments, one or more M are the same, or one or more M are different.

[0172] In some embodiments, M is pyrene, perylene, perylene monoimide, or 6-FAM or a derivative thereof. In some other embodiments, M has the following structure: [ka] has one of the following:

[0173] The M moiety containing the carboxylic acid group is shown above in anionic form (CO2 - ), but one of skill in the art will appreciate that this varies depending on pH, and that the protonated form (COH) is included in various embodiments.

[0174] In some specific embodiments, the compound is a compound 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. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Table 2-16] [Table 2-17] [Table 2-18] [Table 2-19] [Table 2-20] [Table 2-21] [Table 2-22] [Table 2-23] [Table 2-24] * TBD=To be determined

[0175] As used in Table 2 and throughout this application, R 2 , R3 , m, n and L′ have the definitions provided for the compounds of structure (I) unless otherwise indicated, and F, F′ and F″ are each independently selected from the following structures: [ka] The term "fluorescein moiety" refers to a fluorescein moiety having the formula: "dT" has the following structure: [ka] This refers to.

[0176] Some embodiments include any of the aforementioned compounds, including the specific compounds provided in Table 2, conjugated to a targeting moiety (eg, an antibody).

[0177] The present disclosure generally provides compounds with increased fluorescence emission relative to prior known compounds. Thus, certain embodiments relate to fluorescent compounds comprising Y fluorescent moieties M, where the fluorescent compound has a peak fluorescence emission upon excitation with a given wavelength of ultraviolet light that is at least 85% Y times the peak fluorescence emission of a single M moiety upon excitation with the same wavelength of ultraviolet light, where Y is an integer of 2 or greater. Fluorescent compounds include compounds that emit a fluorescent signal upon excitation with light, such as ultraviolet light.

[0178] In some embodiments, the fluorescent compound has a peak fluorescence emission that is at least Y times 90%, Y times 95%, Y times 97%, or Y times 99% of the peak fluorescence emission of an M moiety.

[0179] In some embodiments, Y is an integer from 2 to 100, for example, 2 to 10.

[0180] In some embodiments, the Y M moieties are independently selected from the following structures: [ka] where [ka] indicates the point of attachment to the fluorescent compound.

[0181] In other embodiments, one M moiety independently has the structure: [ka] has one of the following:

[0182] In more specific embodiments, the fluorescent compounds independently have the following structure: [ka] wherein Y M moieties have one of the following structures: [ka] indicates a point of attachment to the fluorescent compound, and the one M moiety has the structure: [ka] has.

[0183] In other embodiments, the peak fluorescent emission is at a wavelength ranging from about 500 to about 550 nm.

[0184] In even more embodiments, the fluorescent compound includes at least one ethylene oxide moiety.

[0185] A composition comprising a fluorescent compound according to any one of the claims and an analyte is also provided.

[0186] The compounds of the present disclosure are "tunable", meaning that by appropriately selecting the variables in any of the aforementioned compounds, one of skill in the art can arrive at a compound with a desired and / or predetermined molar fluorescence (molar brightness). The tunability of the compounds allows the user to easily arrive at a compound with a desired fluorescence and / or color for use in a particular assay or to identify a particular analyte of interest. While all variables can have an effect on the molar fluorescence of the compound, the M, L, and 4 It is believed that the appropriate selection of m and n plays an important role in the molar fluorescence of the compound. Thus, in one embodiment, a method for obtaining a compound having a desired molar fluorescence is provided, the method comprising the steps of selecting an M moiety having known fluorescence, preparing a compound of structure (I) comprising the M moiety, and 4 , m and n to achieve the desired molar fluorescence.

[0187] In certain embodiments, molar fluorescence can be expressed in terms of the 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 are 4 By appropriate selection of , m and n, compounds having a desired fold increase in fluorescence relative to the parent fluorophore are prepared.

[0188] For ease of illustration, various compounds that contain phosphorus moieties (e.g., phosphate, etc.) may be represented in an anionic state (e.g., -OPO(OH)O - , -OPO3 2- ) whose charge is dependent on pH. One of skill in the art will readily appreciate that uncharged (e.g., protonated or salt (e.g., sodium or other cation) forms are also within the scope of embodiments of the present invention.

[0189] Compositions comprising any of the foregoing compounds and one or more analyte molecules (e.g., biomolecules) are provided in various other embodiments. In some embodiments, the use of such compositions in analytical methods for the detection of one or more analyte molecules is also provided.

[0190] In yet other embodiments, the compounds are useful in a variety of analytical methods. For example, in certain embodiments, the disclosure provides a method for staining a sample, the method comprising administering to the sample a compound of structure (I) (e.g., where R 2 or R 3 is a linker that includes a covalent bond to an analyte molecule (e.g., a biomolecule) or a microparticle, and R 2 or R 3 The other of the two is H, OH, alkyl, alkoxy, alkyl ether or -OP(=R a )(R b )R c The method includes adding a sufficient amount of a dye, such as dimethylformamide, to the sample to produce an optical response when the sample is illuminated with an appropriate wavelength.

[0191] In some embodiments of the aforementioned methods, R 2 is a linker that includes a covalent bond to an analyte molecule (e.g., a biomolecule), such as a nucleic acid, an amino acid, or a polymer thereof (e.g., a polynucleotide or a polypeptide). In even more embodiments, the biomolecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion.

[0192] In still other embodiments of the aforementioned methods, R 2 , is a linker that comprises a covalent bond to a solid support (e.g., a microparticle). For example, in some embodiments, the microparticle is a polymeric or non-polymeric bead.

[0193] In even more embodiments, the optical response is a fluorescent response.

[0194] In other embodiments, the sample comprises cells, and some embodiments further comprise observing the cells by flow cytometry.

[0195] In even more embodiments, the method further comprises distinguishing the fluorescent response from that of a second fluorophore having detectably different optical properties.

[0196] In other embodiments, the present disclosure provides a method for visually detecting an analyte molecule (e.g., a biomolecule), the method comprising: (a) a compound of structure (I), e.g., where R 2 or R 3 is a linker comprising a covalent bond to the analyte molecule, and R 2 or R 3 The other of the two is H, OH, alkyl, alkoxy, alkyl ether or -OP(=R a )(R b )R c and (b) detecting said compound by its visual properties; Includes:

[0197] In some embodiments, the analyte molecule is a nucleic acid, an amino acid, or a polymer thereof (e.g., a polynucleotide or a polypeptide). In even more embodiments, the analyte molecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion.

[0198] In another embodiment, a method for visually detecting an analyte molecule (e.g., a biomolecule) is provided, the method comprising: (a) mixing any of the foregoing compounds with one or more analyte molecules; and (b) detecting said compound by its visual properties; Includes:

[0199] In another embodiment, a method for visually detecting an analyte molecule is provided, the method comprising: (a)R 2 or R 3 is Q or a linker comprising a covalent bond to Q, mixing the compound of claim 1 with the analyte molecule; (b) forming a conjugate of the compound and the analyte molecule; and (c) detecting the conjugate by its visual properties; Includes:

[0200] Other exemplary methods include a method for detecting an analyte, the method comprising: (a)R 2 or R 3 providing a compound of structure (I) comprising a linker comprising a covalent bond to a targeting moiety having specificity for an analyte; (b) mixing the compound and the analyte, thereby causing the targeting moiety and the analyte to associate; and (c) detecting the compound, e.g., by its visual or fluorescent properties; Includes:

[0201] In certain embodiments of the above method, the analyte is a particle (e.g., a cell), and the method includes the use of flow cytometry. For example, the compound is provided with a targeting moiety (e.g., an antibody) to selectively associate with the desired cell, thus rendering the cell detectable by any number of techniques (e.g., visual or fluorescent detection). The appropriate antibody can be selected by those skilled in the art depending on the desired end use. Exemplary antibodies for use in certain embodiments include UCHT1 and MOPC-21.

[0202] Embodiments of the compounds of the present invention thus find utility in any number of methods, including, but not limited to, cell counting; cell sorting; biomarker detection; quantitating apoptosis; determining cell viability; identifying cell surface antigens; determining total DNA and / or RNA content; identifying specific nucleic acid sequences (e.g., as nucleic acid probes); and diagnosing disease (e.g., blood cancers).

[0203] In addition to the methods described above, embodiments of the compounds of structure (I) find utility in a variety of disciplines and methods, including, but not limited to, imaging in endoscopic procedures for the identification of cancerous and other tissues; single cell and / or single molecule analytical methods, e.g., detection of polynucleotides with little or no amplification; imaging of cancer, e.g., by including in the compounds of structure (I) a targeting moiety such as an antibody or sugar or other moiety that preferentially binds to cancer cells; imaging in surgical procedures; histone binding for the identification of various diseases; drug delivery, e.g., by replacing the M moiety in the compounds of structure (I) with an active drug moiety; and / or as contrast agents in dental work and other procedures, e.g., by preferential binding of the compounds of structure (I) to various flora and / or organisms.

[0204] In any embodiment of the compound of structure (I) (as shown above), as well as in the compound of structure (I), R 1 , R 2 , R 3 , R 4 , R 5 , L', L 1 , L 2 , L 3 , L 4 It is understood that any specific selection set forth herein for the variables R, M, m and / or n (as set forth above) may be independently combined with other embodiments and / or variables of the compounds of structure (I) to form embodiments of the invention not specifically set forth above. Additionally, the list of options may be combined with any particular R, M, m and / or n variables in a particular embodiment and / or claim. 1, R 2 , R 3 , R 4 , R 5 , L', L 1 , L 2 , L 3 , L 4 , M, m and / or n variables, it is understood that each individual selection may be deleted from the particular embodiment and / or claim and that the recitation of the remaining selections is considered to be within the scope of the invention.

[0205] It is understood that combinations of substituents and / or variables of the depicted formulae herein are permissible only if such contributions result in stable compounds.

[0206] It will also be appreciated by those skilled in the art that in the processes described herein, the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (wherein R" is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups can be added or removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art will recognize, the protecting group can also be a polymer resin, such as a Wang resin, a Rink resin, or a 2-chlorotrityl-chloride resin.

[0207] Additionally, all compounds of the invention that exist in free base or acid form can be converted to their salts by treatment with an appropriate inorganic or organic base or acid by methods known to those skilled in the art. Salts of compounds of the invention can be converted to their free base or free acid forms by standard techniques.

[0208] The following reaction schemes illustrate exemplary methods for making the compounds of the present invention. It is understood that those skilled in the art may be capable of making these compounds by similar methods or by combining other methods known to those skilled in the art. It is also understood that those skilled in the art may make other compounds of structure (I) that are not specifically illustrated below by using appropriate starting components in a similar manner to those described below and, if necessary, modifying the parameters of synthesis. In general, starting components can be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc., or can be 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 can be prepared as described in the present invention. Reaction Scheme I [ka]

[0209] Reaction Scheme I illustrates the reaction of a compound of structure (I), where R 1 , L 2 , L 3 and M is as defined above, R 2 and R 3 FIG. 1 illustrates an exemplary method for preparing intermediates useful for the preparation of (I) (wherein x is 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 skilled in the art. Reaction with MX (wherein x is a halogen, such as bromo) under Suzuki coupling conditions known in the art gives compounds of structure b. Compounds of structure b can be used for the preparation of compounds of structure (I) as described below. Reaction Scheme II [ka]

[0210] Reaction Scheme II illustrates an alternative method for preparing intermediates useful in the preparation of compounds of structure (I). 1 , L 1 , L 2 , L 3 , G and M are as defined above, and R 2 and R 3 With reference to M-G', which is as defined above or a protected variant thereof, a compound of structure c, which can be purchased or prepared by well-known techniques, is reacted with M-G' to provide a compound of structure d, where G and G' represent functional groups with complementary reactivity (i.e., functional groups that react to form a covalent bond). G' can be pendant to M or can be part of the structural backbone of M. G and G' can be any number of functional groups described herein, such as an alkyne and azide, respectively, an amine and activated ester, respectively, or an amine and isothiocyanate, respectively, etc.

[0211] Compounds of structure (I) can be synthesized by synthesis of the following structure (e): [ka] wherein A is as defined herein and each L is independently an optional linker.

[0212] Methods for DNA synthesis are well known in the art. Briefly, two alcohol groups (e.g., R 2 and R 3) are functionalized with a dimethoxytrityl (DMT) group and a 2-cyanoethyl-N,N-diisopropylamino phosphoramidite group, respectively. The phosphoramidite group is typically coupled to an alcohol group in the presence of an activator such as tetrazole, followed by oxidation of 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. The 2-cyanoethyl group can be removed after oligomerization by treatment with aqueous ammonia.

[0213] The preparation of phosphoramidites used in the above oligomerization methods is also well known in the art. For example, primary alcohols (e.g., R 3 ) can be protected as a DMT group by reaction with DMT-Cl. A secondary alcohol (e.g., R 2 ) is functionalized as a phosphoramidite by reaction with a suitable reagent such as 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. Methods for the preparation of phosphoramidites and their oligomerization are well known in the art and are described in more detail in the Examples.

[0214] 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 repeating units m and n are incorporated into the molecule by repeating phosphoramidite coupling a desired number of times. It is understood that compounds of structure (II) can be prepared by similar methods, as described below.

[0215] In various other embodiments, compounds useful for the preparation of compounds of structure (I) are provided. These compounds can be prepared as described above in the form of monomers, dimers and / or oligomers, and then the M moiety can be covalently attached to the compound via any number of synthetic methodologies (e.g., the "click" reaction described above) to form a compound of structure (I). Thus, in various embodiments, the following compounds of structure (II): [ka] or a stereoisomer, salt, or tautomer thereof, wherein: G, in each occurrence, is independently a moiety that contains a reactive group or a protected analog thereof that is capable of forming a covalent bond with a complementary reactive group; L 1a , L 2 and L 3 is independently at each occurrence an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker; L 4 is, at each occurrence, independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker greater than 3 atoms in length, where the heteroatoms in said heteroalkylene, heteroalkenylene, and heteroalkynylene linkers are selected from O, N, and S; R 1 is, at each occurrence, independently H, alkyl, or alkoxy; R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a )(R b )R c , Q or L'; R 4 is, in each occurrence, independently, OH, SH, O - , S - , OR d or S.R. d and; R 5 is, at each occurrence, independently oxo, thioxo, or absent; R a is O or S; R b OH, SH, O - , S - , OR d or S.R. d and; R c OH, SH, O - , S - , OR d , O.L.', S.R. d , alkyl, alkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether; R d is the counter ion; Q, in each occurrence, is independently a moiety that contains a reactive group or a protected analog thereof that is capable of forming a covalent bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group, Q'; L', in each occurrence, is independently 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 moiety, a linker comprising a covalent bond to a nucleoside, or a linker comprising a covalent bond to an additional compound of structure (II); m, in each occurrence, is independently 0 or a greater integer, provided that at least one occurrence of m is an integer 1 or greater; and n is an integer equal to or greater than 1.

[0216] In another embodiment of structure (II): G, in each occurrence, is independently a moiety that contains a reactive group that is capable of forming a covalent bond with a complementary reactive group; L 1a , L 2 and L 3is independently at each occurrence an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker; L 4 is, at each occurrence, independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker greater than 3 atoms in length, where the heteroatoms in said heteroalkylene, heteroalkenylene, and heteroalkynylene linkers are selected from O, N, and S; R 1 is, at each occurrence, independently H, alkyl, or alkoxy; R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, -OP(=R a )(R b )R c , Q, a linker comprising a covalent bond to Q, a linker comprising a covalent bond to an analyte molecule, a linker comprising a covalent bond to a solid support or a linker comprising a covalent bond to a further compound of structure (II), wherein: R a is O or S; R b OH, SH, O - , S - , OR d or S.R. d ;R c OH, SH, O - , S - , OR d , S.R. d , alkyl, alkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; and R d is the counter ion; R 4 is, in each occurrence, independently, OH, SH, O - , S - , OR d or S.R. d and; R 5is, at each occurrence, independently oxo, thioxo, or absent; Q, in each occurrence, is independently a moiety that contains a reactive group that can form a covalent bond with an analyte molecule, a solid support, or a complementary reactive group, Q'; m, in each occurrence, is independently 0 or a greater integer, provided that at least one occurrence of m is an integer 1 or greater; and n is an integer equal to or greater than 1.

[0217] The G moiety in the compound of structure (II) can be selected from any moiety that contains a group having a suitable reactive group for forming a covalent bond with a complementary group on the M moiety. In exemplary embodiments, the G moiety can be selected from any of the Q moieties described herein, including the specific examples provided in Table 1. In some embodiments, G, at each occurrence, independently contains a moiety suitable for reactions including copper-catalyzed reactions of azides and alkynes to form triazoles (Husgen 1,3-dipolar cycloaddition reaction), reactions of dienes and dienophiles (Diels-Alder), strain-promoted alkyne-nitrone cycloaddition, reactions of strained alkenes with azides, tetrazines or tetrazoles, [3+2] cycloadditions of alkenes and azides, alkene and tetrazine inverse electron demand Diels-Alder, alkene and tetrazole photochemical reactions, and various substitution reactions (e.g., displacement of a leaving group by nucleophilic attack on an electrophilic atom).

[0218] In some embodiments, G, at each occurrence, is independently a moiety that contains an aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imido ester, activated ester, ketone, α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin, or thiirane functional group.

[0219] In other embodiments, G, at each occurrence, independently, comprises an alkyne or azide group. In other embodiments, G, at each occurrence, independently, comprises an amino, isothiocyanate, or activated ester group. In different embodiments, G, at each occurrence, independently, comprises a reactive group that can form a functional group, including an alkene, an ester, an amide, a thioester, a disulfide, a carboxylic acid group, a heterocyclic, or a heteroaryl group, upon reaction with a complementary reactive group. For example, in some embodiments, heteroaryl is triazolyl.

[0220] In various other embodiments of the compound of structure (II), L 2 and L 3 is, at each occurrence, independently, C1-C6 alkylene, C2-C6 alkenylene, or C2-C6 alkynylene.

[0221] In other embodiments, the compound has the following structure (IIA): [ka] where: x 1 , x 2 , x 3 and x 4 is independently, in each occurrence, an integer from 0 to 6.

[0222] In another embodiment of structure (II), each L 1a In other embodiments, each L 1a exists, for example, L 1a is, independently at each occurrence, heteroalkylene. In certain embodiments, L 1a has the following structure: [ka] has.

[0223] In any other of the foregoing embodiments of Compound (II), G, at each occurrence, is independently [ka] It is.

[0224] In various embodiments of the compound of structure (IIA), x 1 , x 2 , x 3 or x 4 At least one occurrence of x is 1. 1 , x 2 , x 3 and x 4 In another embodiment, x 1 and x 3 In some embodiments, x 2 and x 4 In still other embodiments, x 1 and x 3 are 0 in each occurrence, and x 2 and x 4 are each 1 in each occurrence.

[0225] In some other embodiments of the compounds of structure (II) or (IIA), L 4 is, at each occurrence, independently a heteroalkylene linker. In other more specific embodiments, L 4 is, at each occurrence, independently an alkylene oxide linker. For example, in some embodiments, L 4 is polyethylene oxide, the compound having the following structure (IB): [ka] where z is an integer from 2 to 100, for example, an integer from 3 to 6.

[0226] In other embodiments, R 4 is, in each occurrence, independently, OH, O - OR dIn a different embodiment, R 5 is oxo at each occurrence.

[0227] In some different embodiments of any of the foregoing compounds of structure (II) or (IIa), R 1 is H.

[0228] In other various embodiments of the compound of structure (II), R 2 and R 3 are each independently OH or -OP(=R a )(R b )R c In some different embodiments, R 2 or R 3 is OH or -OP(=R a )(R b )R c and R 2 or R 3 The other is a linker that comprises a covalent bond to Q or Q.

[0229] In yet another embodiment of any of the foregoing compounds of structure (II), R 2 and R 3 are each independently -OP(=R a )(R b )R c In some of these embodiments, R c is OL'.

[0230] In another embodiment of structure (II), R 2 and R 3 are each independently -OP(=R a )(R b ) OL', where L' is a heteroalkylene linker to Q, a targeting moiety, an analyte (eg, an analyte molecule), a solid support, a solid support moiety, a nucleoside, or a further compound of structure (II).

[0231] The linker L' can be any linker suitable for linking Q, a targeting moiety, an analyte (e.g., an analyte molecule), a solid support, a solid support residue, a nucleoside, or an additional compound of structure (II) to the compound of structure (II). Advantageously, certain embodiments include the use of an L' moiety selected to increase or optimize the water solubility of the compound. In some certain embodiments, L' comprises an alkylene oxide or a phosphodiester moiety, or a combination thereof.

[0232] In certain embodiments, L' has the structure: [ka] where: m″ and n″ are independently integers from 1 to 10; R e is H, an electron pair or a counterion; L” is R e or is a direct bond or is a linkage to Q, a targeting moiety, an analyte (eg, an analyte molecule), a solid support, a solid support moiety, a nucleoside, or a further compound of structure (II).

[0233] In certain of the foregoing embodiments, the targeting moiety is an antibody or a cell surface receptor antagonist.

[0234] In other more specific embodiments of any of the foregoing compounds of structure (II), R 2 or R 3 has the following structure: [ka] [ka] has one of the following:

[0235] Certain embodiments of the compounds of structure (II) can be prepared according to solid phase synthesis methods similar to those known in the art for the preparation of oligonucleotides. Thus, in some embodiments, L' is a linkage 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 the preparation of compounds of structure (II). Thus, in some embodiments, R 2 or R 3 has the following structure:

[0236] [ka] has.

[0237] In yet other embodiments of the compound of structure (II), Q, at each occurrence, is a moiety that contains a reactive group that can independently form a covalent bond with an analyte molecule or a solid support. In other embodiments, Q, at each occurrence, is a moiety that contains a reactive group that can independently form a covalent bond with a complementary reactive group, Q'. For example, in some embodiments, Q' is a moiety that contains a reactive group that can independently form a covalent bond with a complementary reactive group, Q', on an additional compound of structure (II) (e.g., R 2 Rank or R 3 In the present invention, Q and Q' are present at positions (positions 1 and 2) and Q and Q' contain complementary reactive groups such that reaction of a compound of structure (II) with an additional compound of structure (II) results in a covalently linked dimer of a compound of structure (II). Multimeric compounds of structure (II) can also be prepared in an analogous manner and are included within the scope of embodiments of the present invention.

[0238] The type of Q group and its attachability to the remainder of the compound of structure (II) are not limited, provided that Q contains a moiety with appropriate reactivity to form the desired bond.

[0239] In certain embodiments of the compound of structure (II), Q is a moiety that is not susceptible to hydrolysis under aqueous conditions, but is sufficiently reactive to form a bond with a corresponding group (e.g., an amine, azide, or alkyne) on an analyte molecule or a solid support.

[0240] Certain embodiments of the compound of structure (II) include Q groups commonly used in the field of bioconjugation reactions. For example, in some embodiments, Q includes a nucleophilic reactive group, an electrophilic reactive group, or a cycloaddition reactive group. In some more specific embodiments, Q includes a sulfhydryl, a disulfide, an activated ester, an isothiocyanate, an azide, an alkyne, an alkene, a diene, a dienophile, an acid halide, a sulfonyl halide, a phosphine, an α-haloamide, a biotin, an amino, or a maleimide functional group. In some embodiments, the activated ester is an N-succinimide ester, an imido ester, or a polyfluorophenyl ester. In other embodiments, the alkyne is an alkyl azide or an acyl azide.

[0241] Exemplary Q moieties for compounds of structure (II) are provided in Table I above.

[0242] Similar to compounds of structure (I), in some embodiments of compounds of structure (II) where Q is SH, the SH moiety tends to form a disulfide bond with another sulfhydryl group on another compound of structure (II), and thus some embodiments include compounds of structure (II) in the form of a disulfide dimer, the disulfide bond being derived from the SH Q group.

[0243] In some other embodiments of the compound of structure (II), R 2 or R 3 One of the groups is OH or -OP(=R a )(R b )R c and R 2 or R 3The other of the linkers is a linker that includes a covalent bond to the analyte, a linker that includes a covalent bond to the targeting moiety, or a linker that includes a covalent bond to the solid support. For example, in some embodiments, the analyte molecule is a nucleic acid, an amino acid, or a polymer thereof. In other embodiments, the analyte is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion. In some embodiments, the targeting moiety is an antibody or a cell surface receptor antagonist. In yet another embodiment, the solid support is a polymeric bead or a non-polymeric bead.

[0244] In other embodiments of the compound of structure (II), m, at each occurrence, is independently an integer from 1 to 10. For example, in some embodiments, m, at each occurrence, is independently an integer from 1 to 5, e.g., 1, 2, 3, 4, or 5. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5.

[0245] In still different embodiments of the compound of structure (II), n is an integer from 1 to 100. For example, in some embodiments, n is an integer from 1 to 10. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

[0246] In other different embodiments, the compound of structure (II) is selected from Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10]

[0247] In various embodiments, G in the compounds of Table 3 is alkynyl (e.g., ethynyl). In other embodiments, G in the compounds of Table 3 is azide. In other embodiments, G in the compounds of Table 3 is amino (NH2). In other embodiments, G in the compounds of Table 3 is isothiocyanate. In other embodiments, G in the compounds of Table 3 is an activated ester (e.g., an ester of N-hydroxysuccinimide).

[0248] The compounds of structure (II) can be used in a variety of methods, for example, in embodiments, a method is provided for labeling an analyte (e.g., an analyte molecule) or targeting moiety, the method comprising: (a)R 2 or R 3 mixing the analyte molecule with any of the compounds described in structure (I), where Q is Q or a linker containing a covalent bond to Q; (b) forming a conjugate of the compound and the analyte molecule; and (c) The above combination with the formula ML 1b - reacting a compound of formula (I) with a compound of formula (I) of formula (II) to form at least one covalent bond by reaction of at least one G and at least one G; where: M is a moiety containing two or more carbon-carbon double bonds and at least one degree of conjugation; L 1b is an optional alkylene, heteroalkylene or heteroatom linker; and G' is a complementary reactive group to G. A different embodiment is a method for labeling an analyte, e.g., an analyte molecule or a targeting moiety, the method comprising: (a)R 2 or R 3 is Q or a linker comprising a covalent bond to Q; and any of the compounds of structure (II) disclosed herein, 1b - mixing a compound of formula G' with a compound of formula G', thereby forming at least one covalent bond by reaction of G and G'; and (b) reacting the product of step (A) with the analyte or targeting moiety, thereby forming a conjugate of the product of step (A) and the analyte molecule; where: M is a moiety containing two or more carbon-carbon double bonds and at least one degree of conjugation; L 1b is an optional alkylene, heteroalkylene or heteroatom linker; and G' is a linker with complementary reactivity to G.

[0249] Additionally, as noted above, compounds of structure (II) are useful for preparing compounds of structure (I). Thus, in one embodiment, a method for preparing a compound of structure (I) is provided, the method comprising reacting a compound of structure (II) with a compound of formula ML 1b- mixing a compound of formula G' with a compound of formula G', thereby forming at least one covalent bond by reaction of G and G', wherein: M is a moiety containing two or more carbon-carbon double bonds and at least one degree of conjugation; L 1b is an optional alkylene, heteroalkylene or heteroatom linker; and G' is a linker with complementary reactivity to G.

[0250] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0251] General method Mass spectrometry was performed on a Waters / Micromass Quattro micro MS / MS system (in MS-only mode) using MassLynx 4.1 acquisition software. The mobile phase used for LC / MS for the dyes was 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6 mM triethylamine (TEA), pH 8. The phosphoramidites and precursor molecules were also analyzed using a Waters Acquity UHPLC system equipped with a 2.1 mm x 50 mm Acquity BEH-C18 column held at 45°C using an acetonitrile / water mobile phase gradient. The molecular weights of the monomer intermediates were obtained using tropylium cation infusion enhanced ionization on a Waters / Micromass Quattro micro MS / MS system (in MS-only mode). Excitation and emission profile experiments were recorded on a Cary Eclipse spectrophotometer.

[0252] All reactions were carried out in oven-dried glassware under a nitrogen atmosphere unless otherwise stated. Commercial DNA synthesis reagents were purchased from Glen Research (Sterling, VA). Anhydrous pyridine, toluene, dichloromethane, diisopropylethylamine, triethylamine, acetic acid, pyridine, and THF were purchased from Aldrich. All other chemicals were purchased from Aldrich or TCI and used as is without further purification.

[0253] Example 1 Synthesis of dyes with ethylene glycol spacers Compounds with ethylene oxide linkers were prepared as follows:

[0254] Oligofluoroside constructs (i.e., compounds of structure (I)) were synthesized on an Applied Biosystems 394 DNA / RNA synthesizer at 1 μmol scale, and had either a 3′-phosphate group or a 3′-S2-(CH2)6-OH group or other groups as described herein. Synthesis was performed directly on CPG beads or polystyrene solid support using standard phosphoramidite chemistry. The oligofluorosides were synthesized in the 3′ to 5′ direction using standard solid-phase DNA methods and coupling using standard β-cyanoethyl phosphoramidite chemistry. Fluoroside phosphoramidites and spacers (e.g., hexaethyloxy-glycol phosphoramidite, triethyloxy-glycol phosphoramidite, polyethylene glycol phosphoramidite) and linkers (e.g., 5'-amino-modifier phosphoramidite and thiol-modifier S2 phosphoramidite) were dissolved in acetonitrile to make a 0.1 M solution and added in sequential order using the following synthesis cycle: 1) removal of the 5'-dimethoxytrityl protecting group with dichloroacetic acid in dichloromethane, 2) coupling to the next phosphoramidite with an activator in acetonitrile, 3) oxidation of P(III) to form stable P(v) with iodine / pyridine / water, and 4) capping of any unreacted 5'-hydroxyl groups with acetic anhydride / 1-methylimidizole / acetonitrile. The synthesis cycle was repeated until the full-length oligofluoroside construct was assembled. At the end of chain assembly, the monomethoxytrityl (MMT) or dimethoxytrityl (DMT) groups were removed with dichloroacetic acid in dichloromethane.

[0255] The compounds were provided on a controlled-pore glass (CPG) support at 0.2 μmol scale in labeled Eppendorf tubes. 400 μL of 20-30% NH4OH was added and mixed gently. The open tubes were placed at 55°C for approximately 5 minutes or until excess gas was released, then the tubes were tightly closed and incubated for 2 hours (± 15 minutes). The tubes were removed from the heating block and allowed to reach room temperature, followed by centrifugation at 13,400 RPM for 30 seconds to solidify the supernatant and solids. The supernatant was carefully removed and placed in a labeled tube, then 150 μL acetonitrile was added to wash the support. After the washes were added to the tubes, they were placed in a CentriVap instrument at 40°C until dry.

[0256] The product was characterized by ESI-MS (see Table 2), UV absorbance, and fluorescence spectroscopy.

[0257] Example 2 Spectral testing of compounds

[0258] The dried compounds were reconstituted in 150 μL of 0.1M Na2CO3 buffer to make approximately 1 mM stocks. The concentrated stocks were diluted 50× in 0.1×PBS and analyzed on a NanoDrop UV spectrometer to obtain absorbance traces. The absorbance traces were calculated based on the extinction coefficient (75,000 M for each FAM unit). -1 cm -1 ) and Beer's Law to determine the actual concentration of the stock.

[0259] From the calculated stock concentrations, approximately 4 mL of 5 μM solutions were made in 0.1 M Na2CO3, pH 9, and analyzed in a 1 x 1 cm quartz cuvette on a Cary 60 UV spectrometer using a spectral range of 300 nm to 700 nm, and total absorbance was measured for the group. From these 5 μM solutions, second dilutions were made at either 50 nM or 25 nM (also in 0.1 M Na2CO3, pH 9) for spectral analysis on a Cary Eclipse Fluorometer. Excitation was set at 494 nm, and emission spectra were collected from 499 nm to 700 nm.

[0260] Figures 1 and 2 provide the UV absorbance of a representative compound of structure (I) and a comparative compound ("Compound A"). As can be seen in Figures 1 and 2, the UV extinction coefficient of a representative compound of structure (I) containing two fluorescein moieties is approximately twice that of Compound A. [ka]

[0261] The fluorescence emission spectra of representative compounds of structure (I) were also determined and compared to the emission spectrum of compound A. As shown by the data in Figures 3 and 4, the fluorescence emission of representative compounds of structure (I) is higher than that of compound A, and the emission increases as the number of triethylene glycol or hexaethylene glycol units increases. Without wishing to be bound by theory, this unexpected increase in fluorescence emission is believed to be due to the fact that L 4 This is believed to be related to the reduced internal quenching associated with the spatial distance provided by

[0262] Compounds I-10 and I-11 were tested to determine the effect of the number of M moieties on the UV absorbance and fluorescence emission of the compounds. Figure 5 provides data comparing the UV absorbance of compounds I-10 and I-11 to a comparison compound with one M moiety ("Compound B") at 5 μm. At 5 uM, compound B (which contains one FAM unit) absorbs at 0.43 AU, while compound I-10 (3 FAM units) absorbs at 1.17 AU and compound I-11 (5 FAM units) absorbs at 2.00 AU. [ka]

[0263] The fluorescence emission spectra of compounds I-10, I-11 and B at 25 nM are shown in Figure 6. Rather than quenching (such that the spatially closer FAM units are quenched), compounds I-10 and I-11 showed 2.5x and 4.3x increased emission responses, respectively, compared to the values ​​for compound B.

[0264] Example 3 Comparative fluorescence response

[0265] R 2 and R 3 Compounds "HEG", "TEG", "C2", "C3", "C4" and "C6", in which m is as defined for compound I-3 and m varies from 1 to 9, were prepared and their fluorescence emission spectra were determined. The results are shown in FIG. 7. The data show that compounds according to embodiments of the present invention (i.e., HEG and TEG) have increased fluorescence emission despite having fewer repeat spacer moieties (i.e., lower values ​​of m) relative to the other dye compounds.

[0266] 8 provides data comparing the fluorescence emission of "HEG" compound (where m is 1, 2, or 3) to compound A (50 nM, pH=9). The data shows an increase in the fluorescence emission of HEG relative to compound A when m is greater than 2. [ka] [ka]

[0267] Example 4 Preparation of Representative Compounds

[0268] Compound I-29, compound I-32 and representative analogs were prepared, 4 Compounds with long linkers (approximately 1,000 daltons PEG) have shorter L 4 Compounds with linkers but multiple repeats (i.e., m is greater than 1) were tested to determine whether they would have similar properties. Figure 9 provides UV absorbance data for Compound I-60, Compound I-46, and Compound B. The data show that compounds with long L 4 4 shows that the compound with the linker has a similar UV absorbance to that of the compound with multiple repeats of the shorter linker, and both compounds have increased absorbance relative to the control compound B.

[0269] Example 5 Preparation of 99-mer dyes

[0270] Compound I-42, which has 33 fluorescein moieties, was prepared using standard solid-phase oligonucleotide techniques as described herein. I-42 (represented by "A" in the scheme below) was trimerized to form a 99-mer dye as shown and described below. [ka]

[0271] In a 200 μL polypropylene tube, sodium phosphate buffer (3.5 μL, 100 mM, pH=6.5) and a solution of I-42 bis-disulfide (5.5 μL, 0.18 mM in water) were placed. To this, a solution of tris(2-carboxyethyl)phosphine (TCEP, 1.0 μL, 10 mM in water) was added. The tube was capped, vortexed, and allowed to incubate at room temperature for 2 hours. The mixture was desalted through a micro Zeba Spin desalting column (Pierce, Cat. No. 89877). The desalted solution was treated with sodium phosphate buffer (2.0 μL, 500 mM, pH=7.2) and a solution of bismaleimidoethane in DMSO (BMOE, 1.0 μL, 0.25 mM) and incubated overnight at room temperature. The reaction mixture was diluted with water (100 μL) and analyzed by PAGE ( FIG. 10 , Invitrogen EC6875, 10% TBE-urea gel, 180 V constant, electrophoresis stopped upon completion of separation of the highest MW species and visualized by UV illumination (365 nm)).

[0272] Other oligomeric dyes having any desired number of dye moieties are prepared in a similar manner.

[0273] Example 6 Common Flow Cytometry Methods

[0274] Unless otherwise indicated, the following general procedures were used throughout the following examples.

[0275] Lysis of whole blood: Buffered Ammonium Chloride Method. To stain live cells, ethylenediaminetetraacetic acid (EDTA) anticoagulated normal human blood was lysed en bloc with ammonium chloride solution (ACK), 15 mL blood to 35 mL lysis solution, for 15 min at room temperature (RT). 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 (DPBS) with 0.02% sodium azide. The cells were then resuspended in donor plasma to 100 μL / test / 0.1-1x10e6. Cells in plasma were transferred to a polypropylene 96-well HTS plate with 100 μL of 1% bovine serum albumin (BSA), and 1x DPBS with 0.02% sodium azide. f After incubation for 45 min at RT, the cells were washed twice with 50%-1% FBS 1×DPBS containing 50% HBSS and 0.02% sodium azide.

[0276] Lysis / fixation method. Blood was lysed with 1.0 mL RBC lysis solution (ammonium chloride), 100-15 mL blood to 35 mL lysis solution, for 15 min at RT. The cells were then washed twice with 50%-1% FBS 1xDPBS with 50% HBSS and 0.02% sodium azide. The cells were then resuspended to 100 μL / test / 1x10e6 in donor plasma. Pre-diluted antibodies were added in 100 μL of 1% BSA, and 1xDPBS with 0.02% sodium azide. 100 μL cells were added to a 96-well polypropylene HTS plate (total test size of 200 μL). After incubation at RT for 45 min, the cells were washed twice with 50% HBSS, and 50% 1% FBS 1xDPBS with 0.02% sodium azide.

[0277] Preparation of antibody conjugates: The antibody conjugate may have the following structure: [ka] The compound and antibody were then conjugated by reaction of the S on the antibody with the Q moiety to form the following linking structure: [ka]

[0278] Antibody conjugates are designated by the antibody name followed by a compound number. For example, UCHT1-I-45 refers to a conjugate formed between compound I-45 and UCHT1 antibody. If the referenced compound number does not include a Q moiety as described above in Table 2, it is understood that the Q moiety was introduced and the conjugate was prepared from the resulting compound with the Q moiety.

[0279] Conjugate Dilution: Antibodies were brought to RT. The antibody conjugates were diluted in cell staining buffer (1x DPBS, 1% BSA, 0.02% sodium azide) to concentrations ranging from 0.1 to 540 nM (8.0 μg or less per test). In some instances, serial dilutions of each sample started at 269 nM antibody in cell staining buffer and the antibody dilutions were kept protected from light until use. In other experiments, dilutions started at 4.0 μg antibody / test size, with test sizes ranging from 100 to 200 μL. Titrations were performed at 2- or 4-fold dilutions to generate binding curves. In some cases, 8.0 μg or 2.0 μg / test size was used in the first well in the dilution series.

[0280] Flow cytometry with conjugates: After physical characterization, the conjugates were tested for activity and functionality (antibody binding affinity and dye brightness) and compared to reference antibody staining. The quality of separation was then determined by examining brightness in comparison to autofluorescence negative controls and other nonspecific binding using a flow cytometer. Extensive studies of mouse IgG1,k isotype control MOPC-21 conjugates were not included when testing I-45 because MOPC-21 nonspecific binding was characterized during testing of UCHT1-Compound C and UCHT1-I-45 in previous studies. The I-45 conjugates were tested against Jurkat T cells, Ramos B cells, and heterogeneous populations of leukocytes in human blood or peripheral blood mononuclear cells (PBMCs), and using polystyrene goat anti-mouse Ig coated beads. Whole blood screening was the most conventional for testing UCHT1 I-45 and its analogs. Crosslinking studies were performed when new constructs were formed. Additional flow cytometry methods were used when testing conjugates (UCHT1-I-56, I-48, I-49, I-16, and I-21B) and in most studies were compared to a Sony Biotechnology antibody conjugate reference (UCHT1-FITC) and previously characterized key cross-linking references (e.g., UCHT1-I-45, UCHT1-I-49). [ka]

[0281] Performing free dye flow cytometry: After molecular and physical characterization, the dyes were also tested for potential affinity to cells in comparison to reference dye staining. Since the dyes have the potential to act as cell probes and bind to cellular material, the dyes can be broadly screened against blood at high concentrations (>100 nM to 10,000 nM) to confirm specific properties. Predicted or unexpected off-target binding was then qualified by assessing brightness and linearity upon dilution using a flow cytometer in comparison to autofluorescence negative controls, and other dye controls. A study of Compound D (Compound C free dye, but not functionalized), a positive control for bright off-target binding of the dye, has been previously characterized when in conjugate form. The I-45 dye was tested against a heterogeneous population of leukocytes in human blood when cells were treated with lysis and fixation solutions, and when blood was laid down, or when applied to a PBMC monocyte population. Cross-linking studies ranking affinity (Compound D, I-45, I-49, and I-16) were conducted for dye lot comparison, including dyes from the very early studies when characterizing Compound D. [ka]

[0282] Flow cytometry workflow: Cells were cultured and observed for visual signs of metabolic stress for dye screening or off-target binding (data not shown) or fresh healthy cells were used for conjugate screening. Cells were counted periodically to check cell density (1x10e5 and 1x10e6 viable cells / mL). Antibody conjugates were diluted (preferably in plates or tubes) before harvesting cells in staining buffer (DPBS, 0.1% BSA, 0.02% sodium azide). Cells with a viability range of 80-85% were used. The cells were washed twice by centrifuging and washing with buffer to remove pH indicator and blocked cells with Ig and other proteins contained in FBS. Cell density was adjusted to test size in staining buffer. The cells were plated (one test per well) or dye (pre-diluted) was applied to cells in the plate. The cells were then incubated for 45 minutes at 23°C. The cells were washed twice by centrifuging and washing with washing buffer and then the plate was aspirated. The cells were resuspended in acquisition buffer. 5000 intact cells were acquired by flow cytometry.

[0283] The fluorescence of the dye was detected by flow cytometry with a 488 nM blue laser line at peak emission (521 nM) detected using a 525 / 50 bandpass filter. At least 1500 intact cells (acquisition goal was 3000-5000 intact cells) were acquired and analyzed by flow cytometry to identify viable cells present in the cell preparation.

[0284] Data Analysis Methodology: Descriptive statistics. The EC-800 software allows the user to collect a number of statistical data regarding each sample acquisition. The mean or median fluorescence intensity (MFI) in the FL1-A channel was used to measure the brightness of the antibody-dye reagent when interrogated by flow cytometry and to consider noise. Other statistics were assessed to determine the dye characteristics and overall quality of the reagent, including median signal-to-noise and absolute fluorescence (median or geometric mean).

[0285] Histograms. Flow cytometry events were gated by size for forward vs. side scatter (cell volume vs. cell granularity). The cells were then gated by fluorescence emission at 515 nm for mean fluorescence intensity (MFI). The collected data are presented as dual parameter histograms plotted as number of events on the y-axis against fluorescence intensity (represented on a logarithmic scale on the x-axis). The data can be summarized by affinity curves, or histograms of relative fluorescence intensity.

[0286] Binding curve. MFI was selected because it is the parameter that best measures the brightness of the antibody-dye reagent when interrogated by FCM, which can be expressed as the geometric mean, median, or average, and can represent the absolute fluorescence measurement. For comparison, when the noise can be highly characterized, the signal-to-noise ratio is reported as MFI, S / N. In Example 7, Example 8, and Example 14, the MFI of UCHT1-Compound C conjugate against concentration is shown to show the binding curve of the reagent.

[0287] Bivariate dual parameter histograms. In some cases, FCM events were not gated to consider a qualitative output, and data are expressed by cell granularity (SSC) versus dye fluorescence. This method allows for a global assessment of all populations recovered in whole blood.

[0288] Example 7 Evaluation of dyes for non-specific and off-target binding using necrotic and apoptotic populations of heat-stressed Jurkat T cells

[0289] Jurkat cells were cultured according to the instructions provided by the American Type Culture Collection (ATCC), harvested alive, heat stressed, washed 2-3 times, and stained with conjugated antibodies. Staining was performed by applying the cells to pre-diluted dye and pre-diluted conjugated antibodies, incubating, washing, and then acquiring by flow cytometry. The dead and necrotic cell populations (approximately 10% of acquired cells) were evaluated for fluorescent signal. The results are shown in Figure 11. As shown in Figure 11, fluorescence is observed to be higher in 10x Compound D free dye compared to 10x I-47, 5x Compound E, 5x I-44, 3x Compound F, and 3x I-43. [ka]

[0290] Example 8 Evaluation of conjugates for fluorescence intensity: UCHT1-Compound G vs. UCHT1-I-51

[0291] Live Jurkat cells were cultured according to the instructions provided by ATCC, harvested, and then collected at approximately 225 RCF for 6 minutes in a temperature-controlled centrifuge set at 23°C. The supernatant was removed. The cells were then washed twice in cell suspension buffer (1x DPBS without calcium and magnesium, 1.0% FBS, 0.02% sodium azide, pH 7.2). After the second wash, the cells were centrifuged, the supernatant was removed, and the cells (approximately 5x10 5 Viable cells / sample) were resuspended to the test size (final volume 100 μL). Cells were incubated with the antibody-dye conjugate solution for 45 min at RT. After incubation, samples were washed twice and then suspended in acquisition buffer.

[0292] Data was acquired and evaluated on a SONY EC-800 FCM and plotted relative fluorescence geometric mean versus nM of antibody protein as shown in Figure 12. As can be seen, MOPC-21-Compound G has some non-specific binding, but both conjugates are 4-5 times brighter than the FITC reference. This example also shows that MOPC21-I-51 shows reduced non-specific binding (at these dye on label (DOL) levels) compared to the UCHT1-Compound G conjugate. [ka]

[0293] Example 9 Assessment of CD3 expression in heterogeneous cell samples and peripheral whole blood cells (specificity and resolution)

[0294] Whole blood was collected from normal donors into EDTA stabilized sample tubes for transport and short term storage. Blood was lysed with ACK, 15mL blood to 35mL lysis solution for 15 minutes at RT. The cells were washed twice with 50% Hanks Balanced Salt Solution (HBSS) and 50%-1% FBS 1xDPBS with 0.02% sodium azide. The cells were resuspended in donor plasma to 100μL / test / 1x10e6. Antibodies were pre-diluted in 100μL 1% BSA and 1xDPBS with 0.02% sodium azide and added to 100μL cells in polypropylene 96-well HTS plates (total 200μL test size). The cells were incubated for 45 min at RT, washed twice with 50% HBSS and 50%-1% FBS 1xDPBS containing 0.02% sodium azide, and resuspended in 1% FBS 1xDPBS containing 0.02% sodium azide.

[0295] Figure 13 shows a comparison of I-51 conjugate and Compound G reference antibody. In Figure 13, the cell morphology (SSC-Lin) is shown in a dual parameter histogram with dye emission detected in the FL1-A channel. This shows the non-specific binding (NSB) of Compound G conjugate to a heterogeneous population of cells (mainly neutrophils and monocytes). On the other hand, I-51 conjugate shows no NSB. The NSB of UCHT1-Compound G in lysed whole blood effectively reduces the antibody available for binding to CD3.

[0296] Figure 14 shows a comparison of UCHT1-I-51, UCHT1 BB515, and UCHT1-FITC. UCHT1-I-51 is 6 times brighter than UCHT1-FITC.

[0297] Example 10

[0298] CD3 expression levels compared to a standard curve of equivalent fluorophores (MEFs)

[0299] Using lysed whole blood, high antigen density CD3 expression was visualized by fluorescence intensity results and compared with 6-peak (or 8-peak) bead fluorescence output (Sony Biotech, Catalog No. AE700520) to estimate MEF value.UCHT1-FITC (used as reference), UCHT1-Compound G, and UCHT1-I-51, as well as UCHT1-BB515 (used as additional reference conjugate) were compared in the same experiment using standard substances.6-peak beads consist of a mixture of 6 different fluorescence intensity 3.8 micron beads, which were used to verify the linearity and sensitivity of the instrument and estimate MEF when run in parallel in a given protocol.

[0300] Figure 15 shows the expression levels of CD3 compared to the MEF standard curve. As can be seen, I-51 is approximately 6-fold brighter than the reference, and Compound G is approximately 2-fold brighter. The concentration range shown is 133 nM and below. In comparison, note that Figure 13 shows that non-specific binding of UCHT1-Compound G in lysed whole blood effectively reduced the antibody available for binding to CD3.

[0301] Example 11 Comparison of UCHT1 I-16 fraction to FITC and I-56 conjugates

[0302] Beads were pretreated (vortexed and sonicated), washed, and bead numbers were calibrated to 2xC dilutions as determined in preliminary experiments to optimize acquisition and target a linear saturation curve. Beads were incubated with antibody conjugates, washed, and then acquired by flow cytometry. BSA solution (0.1% in 1xDPBS) was used for bead dilution, washing, and acquisition. The antibody was prediluted in 1% BSA staining buffer starting at 4.0 μg in a 200 μL volume in the first well in a 96-well polypropylene plate, then serially diluted 100 μL in each subsequent well with at least 8 dilutions (2-fold). Thoroughly vortexed beads (at 2xC) were then added, and 100 μL of beads were added to 100 μL of antibody in each well. The beads were incubated for 20 min at RT, washed, and acquired by flow cytometry.

[0303] The results are shown in Figure 16. UCHT1-I-16 at DOL 3.0 was nearly equal to the theoretical maximum. Similar experiments were performed to highlight the difference in affinity curves between UCTH1-I-16 and the longer tethered cross-linked reference UCHT1-I-56, as shown in Figure 17.

[0304] Example 12 UCHT1 I-16, a UCHT1 I-51-like analogue, compared with UCHT1 I-56 (10×) and UCHT1 I-53 (6×).

[0305] Peripheral WBCs were treated with lysis buffer (Buffered ACK) for 20 minutes at 25°C with gentle shaking, then centrifuged to remove the lysis buffer. The cells were washed once with HBSS (pH 7.2), then with 1x HBSS (pH 7.2) containing 0.5% FBS and 0.02% sodium azide, and resuspended in staining buffer (1x DPBS, 1% BSA, 0.02% sodium azide (Ph 7.2)). Cells were then applied to pre-diluted conjugated antibodies and incubated for 40 minutes at 23-25°C, protected from light.

[0306] FIG. 18 shows a comparison of the UCHT1-I-51-like analog UCHT1-I-16 with UCHT1-I-56 (10×) and UCHT1-I-53 (6×).

[0307] Example 13 UCHT1 I-16, a UCHT1 I-51-like analogue, compared with UCHT1 I-56 (10×) and UCHT1 I-53 (6×).

[0308] The antibodies were evaluated for specific binding and fluorescence resolution by flow cytometry. Jurkat cells were cultured and harvested live according to the instructions provided by ATCC. Staining was performed when cells were applied to pre-diluted conjugated antibodies, incubated for 20-40 min, washed, and then acquired by flow cytometry. Its UCHT1-I-51-like analog, UCHT1-I-16, was compared with UCHT1-I-56 (10x) and UCHT1-I-53 (6x). The results are shown in Figure 19.

[0309] Example 14 Comparison of UCHT1 binding ability by regression analysis

[0310] Cells were isolated from peripheral whole blood and frozen in freezing buffer. Cells were thawed, settled, treated with autologous plasma to block FcRs and mimic the whole blood environment, washed 2-3 times, and then stained with conjugates as if using whole blood. Beads were pre-calibrated to optimize acquisition and target saturation in antibody staining. Beads were incubated with antibody conjugates, washed, and then acquired by flow cytometry.

[0311] Regression analysis was performed on the data generated when UCHT1-I-16 and UCHT1-I-49 were tested to demonstrate equivalence between the conjugates, and the results are shown in FIG.

[0312] Example 15 I-49 and I-16 affinity testing of raw dyes in whole blood

[0313] To assess background in three populations, monocytes, granulocytes, and lymphocytes, the method of staining, lysing, fixing, and washing whole blood was used to screen the dyes in equivalence tests in the presence of excess dye. Granulocytes present in whole blood were selected as the primary target for analysis. Lymphocytes and monocytes were also studied, but the data are not shown in the regression graph.

[0314] Stock dye was used in excess (starting at 10,000 nM for initial titrations) without the presence of antibody conjugate to also highlight and qualify non-specific binding differences between the two nearly identical constructs. Peripheral WBCs were treated with lysis buffer (buffered ACK) for 20 minutes at 25° C. with slow shaking, centrifuged, and lysis buffer removed. Red blood cell lysis and fixation solution was applied to the dye and cells, and the cells were then washed once with HBSS (pH 7.2), then with 1×HBSS (pH 7.2) containing 0.5% FBS and 0.02% sodium azide, then resuspended in staining buffer (1×DPBS, 1% BSA, 0.02% sodium azide (pH 7.2)).

[0315] The relative MFI data were compared by concentration-matched regression analysis to show the level of agreement and / or similarity between the raw I-45 and I-16. In this test, I-45 and I-16 are found to have approximately equivalent levels of background. Using the same data from the dye screen, the I-45 analogs (I-49 and I-16) are overlaid on each other when examining the entire titration curve and the controls. The I-45 analogs that fall between the MFI of the controls included for reference were Compound D (10x), as well as two analogs Compound D (analogs i and ii) with longer alkylene spacer groups. I-49 has a slightly higher background than I-16, while the I-16 background is very similar to I-45. The 10x fluorophore construct is dimmer in nonspecific binding than the 6x, indicating the effectiveness of backbone adjustments to fit additional fluorophores while reducing the nonspecific binding properties of the 10x molecule. The results are shown in Figures 21A-21C. Figure 21A shows the correlation between I-16 and I-45, Figure 21B shows the titration curve overlay and comparison with the reference, and Figure 21C shows exemplary qualitative data showing background FL and cell morphology comparing Compound D and I-45.

[0316] Example 16 Comparison of UCHT1-I-21B and UCHT1-I-16 in blood cells fixed and stored for 72 hours

[0317] Whole blood was collected from normal donors into EDTA stabilized sample tubes for transport and short-term storage. The blood was treated with lysis agents either before or after staining with antibodies. The cells were lysed with ACK, 15mL blood to 35mL lysis solution for 15 minutes at RT, then washed twice with 50% HBSS, and 50% 1% FBS 1xDPBS with 0.02% sodium azide. The cells were resuspended in donor plasma to 100μL / test / 1x10e6. Antibodies pre-diluted in 100μL 1% BSA and 1xDPBS with 0.02% sodium azide were added to 100μL cells, which were then added to a 96-well HTS polypropylene plate (total 200μL test size). After incubating the cells for 45 min at RT, the cells were washed twice with 50% HBSS + 50% 1% FBS 1xDPBS containing 0.02% sodium azide. The cells were then resuspended in 1% FBS 1xDPBS containing 0.02% sodium azide. The cells were washed once more, fixed in 2% paraformaldehyde at 200 μL / well, washed once with 1xDPBS, stored at 2-8°C for 72 h, washed once more with 1xDPBS, and then acquired with 0.1% BSA in 1xDPBS.

[0318] The resolution of the conjugates was compared with the reference UCHT1-FITC and with the theoretical brightness for a DOL of 3.0. The new construct UCHT1-I-21B best matches the theoretical when the DOL is 3.0 in this method, and is 7 times brighter than UCHT1-FITC. As shown in Figure 22, the affinity curves are shown as histograms with the compound emission detected in the FL1-A channel.

[0319] The evaluation of non-specific binding was completed by measuring the fluorescence of granulocytes. Figure 23 shows a comparison of the fluorescence intensity of off-target non-specific binding of UCHT1-I-21B, UCHT1-I-16, and the reference UCHT1-FITC. All fractions and replicates of UCHT1-I-21B show lower background than other constructs included in the study and the FITC reference. Regression analysis was applied to the data to examine correlation and relative affinity, as shown in Figure 24, and it was determined that UCHT1-I-21B does not have a linear relationship with UCHT1-I-16.

[0320] Example 17 UCHT1 I-21B using the Jurkat cell model and simple two-point titration

[0321] Similar to Example 16, UCHT1 I-21B was tested in a simple two-point titer of 2.0 micrograms and 0.125 micrograms of antibody per test. Jurkat cells were cultured according to the instructions provided by ATCC, either harvested live or heat stressed, washed 2-3 times, and then stained with conjugated antibody. Staining was performed when cells were applied to pre-diluted conjugated antibody, incubated, washed, and then acquired by flow cytometry.

[0322] As shown in Figure 25, UCHT1-I-21B shows higher affinity at lower concentrations compared to UCHT1-I-51, as expected. The actual signal to noise exceeds the theoretical one with a C below saturation of 0.125 micrograms / test, and is superior to UCHT1-I-51.

[0323] Example 18 Comparison of UCHT1 Compound G and UCHT1 I-51 in plasma interference studies using PBMCs

[0324] PBMCs and autologous plasma were previously isolated from peripheral whole blood and then frozen in freezing medium. Cells were thawed, briefly rested, washed two or three times, and then stained with conjugated antibodies as if freshly isolated from whole blood, with autologous plasma, or with HBSS present during antibody staining.

[0325] UCHT1-Compound G interacted with monocytes to form fluorescent events in the presence of activated and inactivated donor plasma, as well as in the presence of the addition of 2.5% glycine. Figure 26A shows data resulting from the addition of 0% glycine, and Figure 26B shows data resulting from the addition of 2.5% glycine. Glycine, a zwitterionic amino acid, plays a role in immunoassays as a proxy for affinity to natural polyamines, amide bonds with natural polyamines, or blocking by natural polyamines, thus exaggerating or blocking the effects of other reagents in live cell staining systems. Plasma reintroduced into PBMCs is either (1) activated with complement to platelets present at normal levels, or (2) inactivated by heating (both complement and other factors of the plasma), filtration, and centrifugation to remove most of the platelets. The inactivated plasma functions better as a blocking agent, while the activated plasma is expected to have high interference.

[0326] This study mimics the range of possible effects in whole blood when blood is transferred and plasma is present, remains, diluted, or washed away. Compound C and I-45 show clear differences in behavior as expected. This supports the reduction in background binding and the clear improvement in activity due to structural modifications to I-45. In general, it is observed that UCHT1-I-51 background is limited in comparison with compound G, while glycine, when present, slightly enhances the background staining of I-51 and suppresses the background of UCHT1-compound G, especially in inactivated plasma and neat controls. Overall, UCHT1-compound G has a higher monocyte background.

[0327] Example 19 Preparation of phosphoramidites and compounds

[0328] Exemplary compounds were synthesized using standard solid phase oligonucleotide synthesis protocols and the following structures: [ka] (This was purchased from ChemGenes (catalog number CLP-9780). The compound was prepared using a fluorescein-containing phosphoramidite having the formula:

[0329] Exemplary linkers (L 4 ) into the following structure: [ka] (Also available commercially) was included in the compound by coupling with a phosphoramidite having the formula:

[0330] Other exemplary compounds are shown in the following scheme: [ka] The compound was prepared using phosphoramidites prepared according to the procedure described above.

[0331] Final deprotection yields the desired F″ moiety. Other commercially available phosphoramidite reagents were used as appropriate to introduce various moieties of the compound. The following structures: [ka] A Q moiety having [ka] with a free sulfhydryl. Other Q moieties are similarly introduced according to the knowledge of one of skill in the art.

[0332] Representative embodiments include, but are not limited to, the following:

[0333] Embodiment 1. A compound having the following structure (I): [ka] or a stereoisomer, salt or tautomer thereof, wherein: M, in each occurrence, is independently a moiety that contains two or more carbon-carbon double bonds and at least one degree of conjugation; L 1 is, independently at each occurrence, either i) an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker; or ii) a linker that contains a functional group that can be formed by the reaction of two complementary reactive groups; L 2 and L 3 is independently at each occurrence an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker; L 4 is, at each occurrence, independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker greater than 3 atoms in length, where the heteroatoms in said heteroalkylene, heteroalkenylene, and heteroalkynylene linkers are selected from O, N, and S; R 1 is, at each occurrence, independently H, alkyl, or alkoxy; R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a )(R b )R c , Q or a protected form thereof, or L'; R 4 is, in each occurrence, independently, OH, SH, O - , S- , OR d or S.R. d and; R 5 is, at each occurrence, independently oxo, thioxo, or absent; R a is O or S; R b OH, SH, O - , S - , OR d or S.R. d and; R c OH, SH, O - , S - , OR d , O.L.', S.R. d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counter ion; Q, in each occurrence, is independently a moiety that contains a reactive group or a protected form thereof that is capable of forming a covalent bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group, Q'; L', in each occurrence, is independently 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 moiety, a linker comprising a covalent bond to a nucleoside, or a linker comprising a covalent bond to an additional compound of structure (I); m, in each occurrence, is independently 0 or a greater integer, provided that at least one occurrence of m is an integer 1 or greater; and n is an integer equal to or greater than 1; The compound, or a stereoisomer, salt or tautomer thereof.

[0334] Embodiment 2. L 4

[0036] In one embodiment, R is an alkyl group, and R is an aryl group.

[0335] Embodiment 3. L 4 The compound of embodiment 2, wherein, at each occurrence, is independently an alkylene oxide linker.

[0336] Embodiment 4. L 4 is polyethylene oxide, said compound having the following structure (IA): [ka] wherein z is an integer from 2 to 100.

[0337] Embodiment 5. The compound of embodiment 4, wherein z is an integer from 3 to 6.

[0338] Embodiment 6. L 1 The compound of any one of embodiments 1-5, wherein, in each occurrence, is a linker that includes a functional group that can be formed by reaction of two complementary reactive groups.

[0339] Embodiment 7. L 1 7. The compound of embodiment 6, wherein for at least one occurrence of, the functional group can be formed by reaction of an aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imido ester, activated ester, ketone, α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin, or thiirane functional group with a complementary reactive group.

[0340] Embodiment 8. L 1 For at least one occurrence of, the functional group can be formed by reaction of an alkyne and an azide.

[0341] Embodiment 9. L 1 For at least one occurrence of, the functional group comprises an alkene, ester, amide, thioester, thiourea, disulfide, carboxylic acid, heterocyclic, or heteroaryl group.

[0342] Embodiment 10. L 1 For at least one occurrence of L 1 The compound of embodiment 6, wherein is a linker comprising a triazolyl functional group.

[0343] Embodiment 11. L 1 For at least one occurrence of L 1 -M has the following structure: [ka] where L 1a and L 1b The compound of embodiment 6, wherein each is independently an optional linker.

[0344] Embodiment 12. L 1 For at least one occurrence of L 1 -M has the following structure: [ka] where L 1a and L 1b The compound of embodiment 6, wherein each is independently an optional linker.

[0345] Embodiment 13. L 1a Or L 1b 13. The compound of any one of embodiments 11-12, wherein, or both are absent.

[0346] Embodiment 14. L 1a Or L 1b 13. The compound of any one of embodiments 11-12, wherein:

[0347] Embodiment 15. L1a and L 1b The compound of embodiment 14, wherein, if present, each is independently alkylene or heteroalkylene.

[0348] Embodiment 16. L 1a and L 1b may, if present, independently have the structure: [ka] The compound of embodiment 14, having one of the following:

[0349] Embodiment 17. L 1 The compound of any one of embodiments 1-5, wherein, at each occurrence, is independently an optional alkylene or heteroalkylene linker.

[0350] Embodiment 18. L 2 and L 3 The compound of any one of embodiments 1-17, wherein, at each occurrence, is independently C1-C6 alkylene, C2-C6 alkenylene, or C2-C6 alkynylene.

[0351] Embodiment 19. The compound has the following structure (IB): [ka] where: x 1 , x 2 , x 3 and x 4 is independently, in each occurrence, an integer from 0 to 6; and z is an integer from 2 to 100; A compound according to any one of embodiments 1 to 18.

[0352] Embodiment 20. x 1 , x 2 , x 3 or x 4 wherein at least one occurrence of is 1.

[0353] Embodiment 21.x 1 , x 2 , x 3 and x 4 The compound of embodiment 19 or 20, wherein, in each occurrence,

[0354] Embodiment 22. L 1 The compound of any one of embodiments 19-21, wherein, at each occurrence, independently comprises a triazolyl functional group.

[0355] Embodiment 23. L 1 The compound of any one of embodiments 19-21, wherein, at each occurrence, is independently an optional alkylene or heteroalkylene linker.

[0356] Embodiment 24. R 4 is, in each occurrence, independently, OH, O - OR d The compound of any one of embodiments 1-23, wherein

[0357] Embodiment 25. R 5 The compound of any one of embodiments 1-24, wherein, at each occurrence, is oxo.

[0358] Embodiment 26. R 1 The compound of any one of embodiments 1-25, wherein, at each occurrence, is H.

[0359] Embodiment 27. R 2 and R 3 are each independently OH or -OP(=R a )(R b )R c The compound of any one of embodiments 1-26, wherein

[0360] Embodiment 28. R 2 or R 3 One of the groups is OH or -OP(=R a )(Rb )R c and R 2 or R 3 The compound of any one of embodiments 1-26, wherein the other of is Q or a linker comprising a covalent bond to Q.

[0361] Embodiment 29. R 2 and R 3 are each independently -OP(=R a )(R b )R c The compound of any one of embodiments 1-26, wherein

[0362] Embodiment 30. R c is OL'.

[0363] Embodiment 31. The compound of embodiment 30, wherein L' is a heteroalkylene linker to Q, a targeting moiety, an analyte molecule, a solid support, a solid support moiety, a nucleoside, or a further compound of structure (I).

[0364] Embodiment 32. The compound of embodiment 31, wherein L' comprises an alkylene oxide or phosphodiester moiety, or a combination thereof.

[0365] Embodiment 33. L' has the structure: [ka] where: m″ and n″ are independently integers from 1 to 10; R e is H, an electron pair or a counterion; L” is R e or a direct bond or a linkage to Q, a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside or a further compound of structure (I); The compound of embodiment 32.

[0366] Embodiment 34. The compound of embodiments 29-33, wherein the targeting moiety is an antibody or a cell surface receptor antagonist.

[0367] Embodiment 35. R 2 or R 3 has the following structure: [ka] [ka] The compound of any one of embodiments 29-34, having one of:

[0368] Embodiment 36. R 2 or R 3 has the following structure: [ka] The compound of any one of embodiments 29-35, having the formula:

[0369] Embodiment 37. The compound of any one of embodiments 1-26 or 28-33, wherein Q comprises a nucleophilic reactive group, an electrophilic reactive group, or a cycloaddition reactive group.

[0370] Embodiment 38. The compound of embodiment 37, wherein Q comprises a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, or maleimide functional group.

[0371] Embodiment 39. The compound of embodiment 38, wherein the activated ester is an N-succinimide ester, an imido ester, or a polyfluorophenyl ester.

[0372] Embodiment 40. The compound of embodiment 38, wherein the alkyne is an alkyl azide or an acyl azide.

[0373] Embodiment 41. The compound of any one of embodiments 1-33, wherein Q comprises a moiety selected from Table 1.

[0374] Embodiment 42. R 2 or R 3 One of the groups is OH or -OP(=R a )(R b )R c and R 2 or R 3 The compound according to any one of embodiments 1 to 26, wherein the other of the linkers is a linker comprising a covalent bond to an analyte molecule, a linker comprising a covalent bond to a targeting moiety, or a linker comprising a covalent bond to a solid support.

[0375] Embodiment 43. The compound of embodiment 42, wherein the analyte molecule is a nucleic acid, an amino acid, or a polymer thereof.

[0376] Embodiment 44. The compound of embodiment 42, wherein the analyte molecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion.

[0377] Embodiment 45. The compound of embodiment 42, wherein the targeting moiety is an antibody or a cell surface receptor antagonist.

[0378] Embodiment 46. The compound of embodiment 42, wherein the solid support is a polymeric bead or a non-polymeric bead.

[0379] Embodiment 47. The compound of any one of embodiments 1 to 46, wherein m, in each occurrence, is independently an integer from 1 to 10.

[0380] Embodiment 48. The compound of any one of embodiments 1 to 46, wherein m, in each occurrence, is independently an integer from 1 to 5.

[0381] Embodiment 49. The compound of any one of embodiments 1 to 48, wherein n is an integer from 1 to 100.

[0382] Embodiment 50. The compound of any one of embodiments 1-48, wherein n is an integer from 1 to 10.

[0383] Embodiment 51. A compound of any one of embodiments 1-50, wherein M, at each occurrence, is independently a moiety containing four or more aryl or heteroaryl rings, or a combination thereof.

[0384] Embodiment 52. The compound of any one of embodiments 1 to 51, wherein M, in each occurrence, is independently fluorescent or colored.

[0385] Embodiment 53. The compound of embodiment 52, wherein M is fluorescent.

[0386] Embodiment 54. A compound of any one of embodiments 1-53, wherein M, in each occurrence, independently comprises a fused polycyclic aryl moiety comprising at least four fused rings.

[0387] Embodiment 55. The compound of any one of embodiments 1-54, wherein M, at each occurrence, is independently a dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine, terylene, sexiphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl, squaraine, squarylium, 9,10-ethynylanthracene, or ter-naphthyl moiety.

[0388] Embodiment 56. The compound of any one of embodiments 1-54, wherein M, in each occurrence, is independently p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, or perylene amide, or a derivative thereof.

[0389] Embodiment 57. The compound of any one of embodiments 1-54, wherein M, in each occurrence, is independently a coumarin dye, a resorufin dye, a dipyrrometheneboron difluoride dye, a ruthenium bipyridyl dye, an energy transfer dye, a thiazole orange dye, a polymethine, or an N-aryl-1,8-naphthalimide dye.

[0390] Embodiment 58. The compound of any one of embodiments 1-54, wherein M, in each occurrence, is independently pyrene, perylene, perylene monoimide, or 6-FAM, or a derivative thereof.

[0391] Embodiment 59. M, in each occurrence, independently has the structure: [ka] The compound of any one of embodiments 1-54, having one of:

[0392] Embodiment 60. A compound selected from Table 2.

[0393] Embodiment 61. A method for staining a sample, comprising adding to the sample a compound according to any one of embodiments 1 to 60 in an amount sufficient to produce an optical response when the sample is illuminated with an appropriate wavelength.

[0394]

[0046] Embodiment 62. The method of embodiment 61, wherein the optical response is a fluorescent response.

[0395]

[0046] Embodiment 63. The method of any one of embodiments 61-62, wherein the sample comprises cells.

[0396] Embodiment 64. The method of embodiment 63, further comprising observing the cells by flow cytometry.

[0397] Embodiment 65. The method of embodiment 62, further comprising distinguishing said fluorescent response from a fluorescent response of a second fluorophore having detectably different optical properties.

[0398] Embodiment 66. A method for visually detecting an analyte molecule, the method comprising: (a)R 2 or R 3 providing the compound of embodiment 1, wherein said linker comprises a covalent bond to said analyte molecule; and (b) detecting the compound by its visual properties; The method includes:

[0399] Embodiment 67. A method for visually detecting an analyte molecule, the method comprising: (a)R 2 or R 3 is Q or a linker comprising a covalent bond to Q, mixing the analyte molecule with a compound of embodiment 1, (b) forming a conjugate of the compound and the analyte molecule; and (c) detecting the conjugate by its visual properties; The method includes:

[0400] Embodiment 68. A method for visually detecting an analyte, the method comprising: (a)R 2 or R 3 providing a compound according to any one of embodiments 1 to 36, comprising a linker comprising a covalent bond to a targeting moiety having specificity for said analyte molecule; (b) mixing the compound and the analyte, thereby causing the targeting moiety and the analyte to associate; and (c) detecting the compound by its visual properties; The method includes:

[0401] Embodiment 69. A composition comprising a compound according to any one of embodiments 1 to 60 and one or more analyte molecules.

[0402] Embodiment 70. Use of the composition of embodiment 69 in an analytical method for the detection of one or more analyte molecules.

[0403] Embodiment 71. A compound having the following structure (II): [ka] or a stereoisomer, salt or tautomer thereof, wherein: G, in each occurrence, is independently a moiety that contains a reactive group or a protected analog thereof that is capable of forming a covalent bond with a complementary reactive group; L 1a , L 2 and L 3 is independently at each occurrence an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker; L 4 is, at each occurrence, independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker greater than 3 atoms in length, where the heteroatoms in said heteroalkylene, heteroalkenylene, and heteroalkynylene linkers are selected from O, N, and S; R 1 is, at each occurrence, independently H, alkyl, or alkoxy; R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a )(R b )R c , Q or L'; R4 is, in each occurrence, independently, OH, SH, O - , S - , OR d or S.R. d and; R 5 is, at each occurrence, independently oxo, thioxo, or absent; R a is O or S; R b OH, SH, O - , S - , OR d or S.R. d and; R c OH, SH, O - , S - , OR d , O.L.', S.R. d , alkyl, alkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether; R d is the counter ion; Q, in each occurrence, is independently a moiety that contains a reactive group or a protected analog thereof that is capable of forming a covalent bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group, Q'; L', in each occurrence, is independently 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 moiety, a linker comprising a covalent bond to a nucleoside, or a linker comprising a covalent bond to an additional compound of structure (II); m, in each occurrence, is independently 0 or a greater integer, provided that at least one occurrence of m is an integer 1 or greater; and n is an integer equal to or greater than 1; The compound, or a stereoisomer, salt or tautomer thereof.

[0404] Embodiment 72. The compound of embodiment 71, wherein G, in each occurrence, independently comprises an aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imidoester, activated ester, ketone, α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin, or thiirane functional group.

[0405] Embodiment 73. The compound of embodiment 71, wherein G, at each occurrence, independently comprises an alkyne or azide group.

[0406] Embodiment 74. The compound of embodiment 71, wherein G, in each occurrence, independently comprises a reactive group that can form, upon reaction with said complementary reactive group, a functional group that comprises an alkene, an ester, an amide, a thioester, a disulfide, a carboxylic acid group, a heterocyclic or a heteroaryl group.

[0407] Embodiment 75. The compound of embodiment 74, wherein the heteroaryl is triazolyl.

[0408] Embodiment 76. L 2 and L 3 The compound of any one of embodiments 71-75, wherein, at each occurrence, is independently C1-C6 alkylene, C2-C6 alkenylene, or C2-C6 alkynylene.

[0409] Embodiment 77. The compound has the following structure (IIA): [ka] where: x 1 , x 2 , x 3 and x 4 is independently, at each occurrence, an integer from 0 to 6; The compound according to embodiment 71.

[0410] Embodiment 78. Each L 1a The compound of any one of embodiments 71-77, wherein is absent.

[0411] Embodiment 79. Each L 1a The compound of any one of embodiments 71-77, wherein is present.

[0412] Embodiment 80. L 1a The compound of embodiment 79, wherein, at each occurrence, is independently heteroalkylene.

[0413] Embodiment 81. L 1a has the following structure: [ka] The compound of embodiment 80, having the formula:

[0414] Embodiment 82. G, in each occurrence, is independently [ka] The compound of embodiment 81, wherein

[0415] 83. 1 , x 2 , x 3 or x 4 The compound of any one of embodiments 77-82, wherein at least one occurrence of is 1.

[0416] 84. 1 , x 2 , x 3 and x 4 The compound of any one of embodiments 77-83, wherein, in each occurrence, each is 1.

[0417] Embodiment 85. L 4 The compound according to any one of embodiments 71 to 84, wherein is as defined in any one of embodiments 2 to 5.

[0418] Embodiment 86. R 4 is, in each occurrence, independently, OH, O - OR d The compound of any one of embodiments 71-85, wherein

[0419] Embodiment 87. R 5 The compound of any one of embodiments 71-86, wherein, at each occurrence, is oxo.

[0420] Embodiment 88. R 1 The compound of any one of embodiments 71-87, wherein

[0421] Embodiment 89. R 2 and R 3 are each independently OH or -OP(=R a )(R b )R c The compound of any one of embodiments 71-88, wherein

[0422] Embodiment 90. R 2 or R 3 One of the groups is OH or -OP(=R a )(R b )R c and R 2 or R 3 The compound of any one of embodiments 71-88, wherein the other of is Q or a linker comprising a covalent bond to Q.

[0423] Embodiment 91. R 2 and R 3 are each independently -OP(=R a )(R b )R c The compound of any one of embodiments 71-88, wherein

[0424] Embodiment 92. R c The compound of embodiment 91, wherein:

[0425] Embodiment 93. The compound of embodiment 92, wherein L' is a heteroalkylene linker to Q, a targeting moiety, an analyte molecule, a solid support, a solid support moiety, a nucleoside, or a further compound of structure (I).

[0426] Embodiment 94. The compound of embodiment 93, wherein L' comprises an alkylene oxide or phosphodiester moiety, or a combination thereof.

[0427] Embodiment 95. L' has the following structure: [ka] where: m″ and n″ are independently integers from 1 to 10; R e is H, an electron pair or a counterion; L” is R e or a direct bond or a linkage to Q, a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside, or a further compound of structure (I).

[0428] Embodiment 96. The compound of embodiments 91-95, wherein the targeting moiety is an antibody or a cell surface receptor antagonist.

[0429] Embodiment 97. R 2 or R 3 has the following structure: [ka] [ka] The compound of any one of embodiments 91-96, having one of:

[0430] Embodiment 98. R 2 or R 3 has the following structure: [ka] The compound of any one of embodiments 91-97, having the formula:

[0431] Embodiment 99. The compound of any one of embodiments 71-98, wherein Q comprises a nucleophilic reactive group, an electrophilic reactive group, or a cycloaddition reactive group.

[0432] Embodiment 100. The compound of embodiment 99, wherein Q comprises a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, or maleimide functional group.

[0433] Embodiment 101. The compound of embodiment 100, wherein the activated ester is an N-succinimide ester, an imido ester, or a polyfluorophenyl ester.

[0434] Embodiment 102. The compound of embodiment 100, wherein the alkyne is an alkyl azide or an acyl azide.

[0435] Embodiment 103. The compound of any one of embodiments 71-98, wherein Q is a moiety selected from Table 1.

[0436] Embodiment 104. R 2 or R 3 One of the groups is OH or -OP(=R a )(R b )R c and R 2 or R 3 The compound according to any one of embodiments 61 to 88, wherein the other of the linkers is a linker comprising a covalent bond to an analyte molecule, a linker comprising a covalent bond to a targeting moiety, or a linker comprising a covalent bond to a solid support.

[0437] Embodiment 105. The compound of embodiment 104, wherein the analyte molecule is a nucleic acid, an amino acid, or a polymer thereof.

[0438] Embodiment 106. The compound of embodiment 104, wherein the analyte molecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer or a prion.

[0439] Embodiment 107. The compound of embodiment 104, wherein the targeting moiety is an antibody or a cell surface receptor antagonist.

[0440] Embodiment 108. The compound of embodiment 104, wherein the solid support is a polymeric bead or a non-polymeric bead.

[0441] Embodiment 109. The compound of any one of embodiments 71-108, wherein m, in each occurrence, is independently an integer from 1 to 10.

[0442] Embodiment 110. The compound of any one of embodiments 71-108, wherein m, in each occurrence, is independently an integer from 1 to 5.

[0443] Embodiment 111. The compound of any one of embodiments 71-110, wherein n is an integer from 1 to 100.

[0444] Embodiment 112. The compound of any one of embodiments 71-110, wherein n is an integer from 1 to 10.

[0445] Embodiment 113. A compound selected from Table 3.

[0446] Embodiment 114. A method for labeling an analyte molecule or a targeting moiety, the method comprising: (a)R 2 or R 3 mixing the compound of embodiment 71, wherein Q is a linker comprising a covalent bond to Q, with the analyte molecule or the targeting moiety; (b) forming a conjugate of the compound and the analyte molecule or the targeting moiety; and (c) reacting said conjugate with a compound of formula M-L1b-G′, thereby forming at least one covalent bond by reaction of at least one G and at least one G′; where: M is a moiety containing two or more carbon-carbon double bonds and at least one degree of conjugation; L 1b is an optional alkylene, heteroalkylene or heteroatom linker; and G' is a complementary reactive group to G; method.

[0447] Embodiment 115. A method for labeling an analyte molecule or a targeting moiety, the method comprising: (a)R 2 or R 3 is a linker comprising a covalent bond to Q or Q; and 1b - mixing a compound of formula G' with a compound of formula G', thereby forming at least one covalent bond by reaction of G and G'; and (b) reacting the product of step (A) with said analyte molecule or targeting moiety, thereby forming a conjugate of the product of step (A) and said analyte molecule or targeting moiety, wherein: M is a moiety containing two or more carbon-carbon double bonds and at least one degree of conjugation; L 1b is an optional alkylene, heteroalkylene or heteroatom linker; and G' is a complementary reactive group to G; method.

[0448] Embodiment 116. A method for preparing a compound according to embodiment 1, comprising mixing a compound according to embodiment 71 with a compound of formula M-L1b-G′, thereby forming at least one covalent bond by reaction of G and G′, wherein: M is a moiety containing two or more carbon-carbon double bonds and at least one degree of conjugation; L 1b is an optional alkylene, heteroalkylene or heteroatom linker; and G' is a complementary reactive group to G; method.

[0449] Embodiment 117. A fluorescent compound comprising Y fluorescent moieties, M, wherein the fluorescent compound has a peak fluorescence emission upon excitation with ultraviolet light of a given wavelength that is at least 85% Y times the peak fluorescence emission of a single M moiety upon excitation with ultraviolet light of the same wavelength, where Y is an integer of 2 or greater.

[0450] Embodiment 118. The fluorescent compound of embodiment 117 having a peak fluorescence emission that is at least 90% Y times the peak fluorescence emission of one M moiety.

[0451] Embodiment 119. The fluorescent compound of embodiment 117 having a peak fluorescence emission that is at least 95% Y times the peak fluorescence emission of one M moiety.

[0452] Embodiment 120. The fluorescent compound of embodiment 117 having a peak fluorescence emission that is at least 97% Y times the peak fluorescence emission of one M moiety.

[0453] Embodiment 121. The fluorescent compound of embodiment 117 having a peak fluorescence emission that is at least 99% Y times the peak fluorescence emission of one M moiety.

[0454] Embodiment 122. The fluorescent compound of any one of embodiments 117-121, wherein Y is an integer from 2 to 100.

[0455] Embodiment 123. The fluorescent compound of any one of embodiments 117-121, wherein Y is an integer from 2 to 10.

[0456] Embodiment 124. The Y M moieties independently have the structure: [ka] where [ka] 124. The fluorescent compound of any one of embodiments 117-123, wherein indicates the point of attachment to the fluorescent compound.

[0457] Embodiment 125. The one M moiety independently has the structure: [ka] 125. The fluorescent compound according to any one of embodiments 117 to 124, having one of the following:

[0458] Embodiment 126. The fluorescent compound independently has the following structure: [ka] wherein Y M moieties have one of [ka] indicates a point of attachment to the fluorescent compound, and the one M moiety has the structure: [ka] 124. The fluorescent compound according to any one of embodiments 117 to 123, having the formula:

[0459] Embodiment 127. The fluorescent compound of any one of embodiments 117-126, wherein the peak fluorescent emission is at a wavelength ranging from about 500 to about 550 nm.

[0460] Embodiment 128. The fluorescent compound of any one of embodiments 117 to 127, wherein the fluorescent compound comprises at least one ethylene oxide moiety.

[0461] Embodiment 129. A composition comprising the fluorescent compound of any one of embodiments 117 to 128 and an analyte.

[0462] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification are incorporated by reference in their entirety herein to the extent not inconsistent with this description.

[0463] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

1. The following structure (II): (II) or a stereoisomer, salt or tautomer thereof, wherein: G, in each occurrence, is independently a moiety that contains a reactive group or a protected analog thereof that is capable of forming a covalent bond with a complementary reactive group; L 1a , L 2 and L 3 is, at each occurrence, independently, any alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or heteroatom linker; L 4 is, at each occurrence, independently a heteroalkylene, heteroalkenylene, or heteroalkynylene linker greater than 3 atoms in length, where the heteroatoms in said heteroalkylene, heteroalkenylene, and heteroalkynylene linkers are selected from O, N, and S; R 1 is, at each occurrence, independently H, alkyl, or alkoxy; R 2 and R 3 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a ) (R b ) R c , Q or L′; R 4 is, in each occurrence, independently, OH, SH, O - , S - , OR d or S.R. d and R 5 is, at each occurrence, independently oxo, thioxo, or absent; R a is O or S; R b OH, SH, O - , S - , OR d or S.R. d and R c OH, SH, O - , S - , OR d , O.L.', S.R. d , alkyl, alkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether or thiophosphoalkyl ether; R d is a counter ion; Q, in each occurrence, is independently a moiety that contains a reactive group or a protected analog thereof that is capable of forming a covalent bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q'; L', at each occurrence, is independently a linker that includes a covalent bond to Q, a linker that includes a covalent bond to a targeting moiety, a linker that includes a covalent bond to an analyte molecule, a linker that includes a covalent bond to a solid support, a linker that includes a covalent bond to a solid support moiety, a linker that includes a covalent bond to a nucleoside, or a linker that includes a covalent bond to an additional compound of structure (II); m, in each occurrence, is independently 0 or greater, provided that at least one occurrence of m is an integer equal to 1 or greater; n is an integer equal to or greater than 1; The compound, or a stereoisomer, salt or tautomer thereof.

2. 2. The compound of claim 1, wherein G, in each occurrence, independently comprises an aldehyde, oxime, hydrazone, alkyne, amine, azide, acyl azide, acyl halide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imido ester, activated ester, ketone, α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin, or thiirane functional group.

3. 2. The compound of claim 1, wherein G, in each occurrence, independently comprises a reactive group that can form a functional group comprising an alkene, an ester, an amide, a thioester, a disulfide, a carboxylic acid group, a heterocyclic or a heteroaryl group upon reaction with a complementary reactive group.

4. The compound of claim 3 , wherein the heteroaryl is triazolyl.

5. The compound has the following structure (IIA): where: x 1 , x 2 , x 3 and x 4 The compound of claim 1 , wherein, at each occurrence, is independently an integer from 0 to 6.

6. L 1a has the following structure:

2. The compound of claim 1 having the formula:

7. G, in each occurrence, independently represents 2. The compound of claim 1,

8. R 4 is, in each occurrence, independently, OH, O - OR d and R 5 The compound of claim 1 , wherein, at each occurrence, is oxo.

9. R 1 The compound of claim 1 , wherein

10. R 2 and R 3 are each independently OH or -OP(=R a ) (R b ) R c 2. The compound of claim 1 ,

11. R c The compound of claim 10, wherein is OL'.

12. 12. The compound of claim 11, wherein L' is a heteroalkylene linker to Q, a targeting moiety, an analyte molecule, a solid support, a solid support moiety, a nucleoside, or a further compound of structure (II).

13. 12. The compound of claim 11, wherein L' comprises an alkylene oxide or a phosphodiester moiety, or a combination thereof.

14. L' has the following structure: where: m″ and n″ are independently integers from 1 to 10; R e is H, an electron pair or a counter ion; L” is R e or a direct bond or a linkage to Q, a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside or a further compound of structure (II).

15. The compound of claim 12 , wherein the targeting moiety is an antibody or a cell surface receptor antagonist.

16. R 2 Or R 3 has the following structure: 【Chemistry 1-1】 【Chemistry 1-2】 The compound of claim 1 having one of the following:

17. 2. The compound of claim 1, wherein Q comprises a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, or maleimide functional group.

18. Q has the following structure: 【Chemistry 2-1】 【Chemistry 2-2】 The compound of claim 1 having one of the following:

19. 2. The compound of claim 1, wherein m, in each occurrence, is independently an integer from 1 to 10.

20. The compound according to claim 1, wherein n is an integer from 1 to 100.

21. The compound has the following structure: 【Chemistry 3-1】 【Chemistry 3-2】 【Chemistry 3-3】 【Chemistry 3-4】 【Chemistry 3-5】 【Chemistry 3-6】 【Chemistry 3-7】 【Chemistry 3-8】 【Chemistry 3-9】 【Chemistry 3-10】 【Chemistry 3-11】 The compound of claim 1 having one of the following:

22. 22. The compound of claim 21, wherein G is an azide, amino, isothiocyanate, or an ester of N-hydroxysuccinimide.

23. 1. A method for labeling an analyte molecule or a targeting moiety, the method comprising: (a) R 2 Or R 3 is a linker comprising a covalent bond to Q or Q, mixing the compound of claim 1 with the analyte molecule or the targeting moiety; (b) forming a conjugate of the compound and the analyte molecule or the targeting moiety; and (c) The conjugate and the formula ML 1b -G' with a compound of formula (I) to form at least one covalent bond by reaction of at least one G and at least one G'; where: M is a moiety containing two or more carbon-carbon double bonds and at least one degree of conjugation; L 1b is any alkylene, heteroalkylene or heteroatom linker; and G' is a complementary reactive group to G; method.

24. 1. A method for labeling an analyte molecule or a targeting moiety, the method comprising: (a) R 2 Or R 3 is Q or a linker containing a covalent bond to Q; and 1b - mixing a compound of formula G' with a compound of formula G', thereby forming at least one covalent bond by reaction of G and G'; and (b) reacting the product of step (A) with said analyte molecule or targeting moiety, thereby forming a conjugate of the product of step (A) and said analyte molecule or targeting moiety, wherein: M is a moiety containing two or more carbon-carbon double bonds and at least one degree of conjugation; L 1b is any alkylene, heteroalkylene or heteroatom linker; and G' is a complementary reactive group to G; method.

25. A method for preparing a compound according to claim 1, comprising the steps of: 1b -G', thereby forming at least one covalent bond by reaction of G and G', wherein: M is a moiety containing two or more carbon-carbon double bonds and at least one degree of conjugation; L 1b is any alkylene, heteroalkylene or heteroatom linker; and G' is a complementary reactive group to G; method.

Images