GROUPS

BACKGROUND

Field

The present invention is generally directed to dimeric and polymeric fluorescent or colored dyes having rigid spacing groups, and methods for their preparation and use in various analytical methods.

Description of the Related Art

Fluorescent and/or colored dyes are known to be particularly suitable for applications in which a highly sensitive detection reagent is desirable. Dyes that are able to preferentially label a specific ingredient or component in a sample enable the researcher to determine the presence, quantity and/or location of that specific ingredient or component. In addition, specific systems can be monitored with respect to their spatial and temporal distribution in diverse environments.

Fluorescence and colorimetric methods are extremely widespread in chemistry and biology. These methods give useful information on the presence, structure, distance, orientation, complexation and/or location for biomolecules. In addition, time-resolved methods are increasingly used in measurements of dynamics and kinetics. As a result, many strategies for fluorescence or color labeling of biomolecules, such as nucleic acids and protein, have been developed. Since analysis of biomolecules typically occurs in an aqueous environment, the focus has been on development and use of water soluble dyes.

Highly fluorescent or colored dyes are desirable since use of such dyes increases the signal to noise ratio and provides other related benefits. Accordingly, attempts have been made to increase the signal from known fluorescent and/or colored moieties. For example, dimeric and polymeric compounds comprising two or more fluorescent and/or colored moieties have been prepared in anticipation that such compounds would result in brighter dyes. However, as a result of intramolecular fluorescence quenching, the known dimeric and polymeric dyes have not achieved the desired increase in brightness.

There is thus a need in the art for water soluble dyes having an increased molar brightness. Ideally, such dyes and biomarkers should be intensely colored or fluorescent and should be available in a variety of colors and fluorescent wavelengths. The present invention fulfills this need and provides further related advantages.

BRIEF SUMMARY

In brief, embodiments of the present invention are generally directed to compounds useful as water soluble, fluorescent and/or colored dyes and/or probes that enable visual detection of analyte molecules, such as biomolecules, as well as reagents for their preparation. Methods for visually detecting analyte molecules using the dyes are also described.

Embodiments of the presently disclosed dyes include two or more fluorescent and/or colored moieties covalently linked by a rigid linker ("A"). In contrast to previous reports of dimeric and/or polymeric dyes, the present dyes are significantly brighter than the corresponding monomelic dye compound. While, not wishing to be bound by theory, it is believed that the rigid linker moiety provides sufficient spatial separation between the fluorescent and/or colored moieties such that intramolecular fluorescence quenching is reduced and/or eliminated.

The water soluble, fluorescent or colored dyes of embodiments of the invention are intensely colored and/or fluorescent and can be readily observed by visual inspection or other means. In some embodiments the compounds may be observed without prior illumination or chemical or enzymatic activation. By appropriate selection of the dye, as described herein, visually detectable analyte molecules of a variety of colors may be obtained.

In one embodiment, compounds having the following structure (I) are provided:

(I)

or a stereoisomer, tautomer or salt thereof, wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , L ¹ , L ² , L ³ , L ⁴ , L ⁵ A, M, m and n are as defined herein. Compounds of structure (I) find utility in a number of applications, including use as fluorescent and/or colored dyes in various analytical methods.

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

In still other embodiments, the present disclosure provides a method for visually detecting an analyte molecule, comprising:

(a) providing a compound of (I); and

(b) detecting the compound by its visible properties.

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

(a) admixing a compound of structure (I) with one or more biomolecules; and

(b) detecting the compound by its visible properties.

Other embodiments are directed to a composition comprising a compound of structure (I) and one or more biomolecules. Use of such compositions in analytical methods for detection of the one or more biomolecules is also provided.

In some other different embodiments is provided a compound of structure (II):

(Π)

or a stereoisomer, salt or tautomer thereof, wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , L ^la , L ² , L ³ , L ⁴ , L ⁵ , A, G, m and n are as defined herein. Compounds of structure (II) find utility in a number of applications, including use as intermediates for preparation of fluorescent and/or colored dyes of structure (I).

In yet other embodiments a method for labeling an analyte molecule is provided, the method comprising:

(a) admixing a compound of structure (II), wherein R ² or R ³ 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) reacting the conjugate with a compound of formula M-L ^lb -G', thereby forming at least one covalent bond by reaction of G and G', wherein R ² , R ³ , Q, G and M-L ^lb -G' are as defined herein.

In some different embodiments another method for labeling an analyte molecule is provided, the method comprising:

(a) admixing a compound of structure (II), wherein R ² or R ³ is Q or a linker comprising a covalent bond to Q, with a compound of formula M-L ^lb -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 molecule, thereby forming a conjugate of the product of step (A) and the analyte molecule wherein R ² , R ³ , Q, G and M-L ^lb -G' are as defined herein.

In more different embodiments, a method for preparing a compound of structure (I) is provided, the method comprising admixing a compound of structure (II) with a compound of formula M-L ^lb -G', thereby forming at least one covalent bond by reaction of G and G', wherein G and M-L ^lb -G' are as defined herein. These and other aspects of the invention will be apparent upon reference to the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, identical reference numbers identify similar elements. The sizes and relative positions of elements in the figures are not necessarily drawn to scale and some of these elements are arbitrarily enlarged and positioned to improve figure legibility. Further, the particular shapes of the elements as drawn are not intended to convey any information regarding the actual shape of the particular elements, and have been solely selected for ease of recognition in the figures.

Figure 1 provides UV absorbance spectra for comparative dye compounds.

Figure 2 provides fluorescence emmision spectra for comparative dye compounds.

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, one skilled in the art will understand that the invention may be practiced without these details.

Unless the context requires otherwise, throughout the present specification and claims, the word "comprise" and variations thereof, such as,

"comprises" and "comprising" are to be construed in an open, inclusive sense, that is, as

"including, but not limited to".

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present 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, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. "Amino" refers to the - H ₂ group.

"Carboxy" refers to the -C0 ₂ H group.

"Cyano" refers to the -CN group.

"Formyl" refers to the -C(=0)H group.

"Hydroxy" or "hydroxyl" refers to the -OH group.

"Imino" refers to the = H group.

"Nitro" refers to the -N0 ₂ group.

"Oxo" refers to the =0 substituent group.

"Sulfhydryl" refers to the -SH group.

"Thioxo" refers to the =S group.

"Alkyl" refers to a straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to twelve carbon atoms (Ci-Ci ₂ alkyl), one to eight carbon atoms (Ci-C ₈ alkyl) or one to six carbon atoms (Ci-C ₆ alkyl), and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, ^-propyl, 1-methylethyl (z ^' so-propyl), «-butyl, «-pentyl, 1, 1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Unless stated otherwise specifically in the specification, alkyl groups are optionally substituted.

"Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing no unsaturation, and having from one to twelve carbon atoms, e.g., methylene, ethylene, propylene, «-butylene, ethenylene, propenylene, «-butenylene, propynylene, «-butynylene, and the like. 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 points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, alkylene is optionally substituted.

"Alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing at least one carbon-carbon double bond and having from two to twelve carbon atoms, e.g., ethenylene, propenylene, «-butenylene, and the like. 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 within the chain. Unless stated otherwise specifically in the specification, alkenylene is optionally substituted.

"Alkynylene" or "alkynylene chain" refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing at least one carbon-carbon triple bond and having from two to twelve carbon atoms, e.g., ethenylene, propenylene,

«-butenylene, and the like. 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 within the chain. Unless stated otherwise specifically in the specification, alkynylene is optionally substituted.

"Alkylether" refers to any alkyl group as defined above, wherein at least one carbon-carbon bond is replaced with a carbon-oxygen bond. The carbon-oxygen bond may be on the terminal end (as in an alkoxy group) or the carbon oxygen bond may be internal (i.e., C-O-C). Alkylethers include at least one carbon oxygen bond, but may include more than one. For example, polyethylene glycol (PEG) is included within the meaning of alkylether. Unless stated otherwise specifically in the specification, an alkylether group is optionally substituted. For example, in some embodiments an alkylether is substituted with an alcohol or -OP(=R _a )(Rb)R _c , wherein each of R _a , Rb and R _c is as defined for compounds of structure (I).

"Alkoxy" refers to a group of the formula -OR _a where R _a is an alkyl group as defined above containing one to twelve carbon atoms. Unless stated otherwise specifically in the specification, an alkoxy group is optionally substituted.

"Heteroalkylene" refers to an alkylene group, as defined above, comprising at least one heteroatom (e.g., N, O, P or S) within the alkylene chain or at a terminus of the alkylene chain. In some embodiments, the heteroatom is within the alkylene chain (i.e., the heteroalkylene comprises at least one carbon-heteroatom- carbon bond). In other embodiments, the heteroatom is at a terminus of the alkylene and thus serves to join the alkylene to the remainder of the molecule (e.g., M1-H-B-M2, where Ml and M2 are portions of the molecule, H is a heteroatom and B is an alkylene). Unless stated otherwise specifically in the specification, a heteroalkylene group is optionally substituted. An exemplary heteroalkylene linking group is illustrated below:

"C linker"

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

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

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

"Heteroatomic" in reference to a "heteroatomic linker" refers to a linker group consisting of one or more heteroatom. Exemplary heteroatomic linkers include single atoms selected from the group consisting of O, N, P and S, and multiple heteroatoms for example a linker having the formula -P(0 ^" )(=0)0- or -OP(0 ^" )(=0)0- and multimers and combinations thereof.

"Phosphate" refers to the -OP(=0)(R _a )Rb group, wherein R _a is OH, O ^" or OR _c ; and R is OH, O ^" , OR _c , a thiophosphate group or a further phosphate group, wherein R _c is a counter ion (e.g., Na+ and the like).

"Phosphoalkyl" refers to the -OP(=0)(R _a )R _b group, wherein R _a is OH,

O ^" or OR _c ; and R _b is -Oalkyl, wherein R _c is a counter ion (e.g., Na+ and the like).

Unless stated otherwise specifically in the specification, a phosphoalkyl group is optionally substituted. For example, in certain embodiments, the -Oalkyl moiety in a phosphoalkyl group is optionally substituted with one or more of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,

phosphoalkylether or thiophosphoalkylether.

"Phosphoalkylether" refers to the -OP(=0)(R _a )R _b group, wherein R _a is OH, O ^" or OR _c ; and R is -Oalkylether, wherein Rc is a counter ion (e.g., Na+ and the like). Unless stated otherwise specifically in the specification, a phosphoalkylether group is optionally substituted. For example, in certain embodiments, the -Oalkylether moiety in a phosphoalkylether group is optionally substituted with one or more of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl,

thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether.

"Thiophosphate" refers to the -OP(=R _a )(R _b )R _c group, wherein R _a is O or

S, R _b is OH, O ^" , S ^" , OR _d or SR _d ; and R _c is OH, SH, O ^" , S ^" , OR _d , SR _d , a phosphate group or a further thiophosphate group, wherein R _d is a counter ion (e.g., Na+ and the like) and provided that: i) R _a is S; ii) R _b is S ^" or SR _d ; iii)Rc is SH, S ^" or SR _d ; or iv) a combination of i), ii) and/or iii).

"Thiophosphoalkyl" refers to the -OP(=R _a )(R _b )Rc group, wherein R _a is

O or S, R _b is OH, O ^" , S ^" , OR _d or SR _d ; and Rc is -Oalkyl, wherein R _d is a counter ion (e.g., Na+ and the like) and provided that: i) R _a is S; ii) R _b is S ^" or SR _d ; or iii)R _a is S and R _b is S ^" or SR _d . Unless stated otherwise specifically in the specification, a

thiophosphoalkyl group is optionally substituted. For example, in certain embodiments, the -Oalkyl moiety in a thiophosphoalkyl group is optionally substituted with one or more of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether.

"Thiophosphoalkylether" refers to the -OP(=R _a )(R _b )Rc group, wherein R _a is O or S, R _b is OH, O ^" , S ^" , OR _d or SR^ and R _c is -Oalkylether, wherein R _d is a counter ion (e.g., Na+ and the like) and provided that: i) R _a is S; ii) R _b is S ^" or SR _d ; or iii)R _a is S and R _b is S ^" or SR _d . Unless stated otherwise specifically in the specification, a thiophosphoalkylether group is optionally substituted. For example, in certain embodiments, the -Oalkylether moiety in a thiophosphoalkyl group is optionally substituted with one or more of hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether. "Carbocyclic" refers to a stable 3- to 18-membered aromatic or non-aromatic ring comprising 3 to 18 carbon atoms. Unless stated otherwise specifically in the specification, 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 saturated. Non-aromatic carbocyclyl radicals include cycloalkyl, while aromatic carbocyclyl radicals include aryl. Unless stated otherwise specifically in the specification, a carbocyclic group is optionally substituted.

"Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic carbocyclic ring, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, preferably having from three to ten carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond.

Monocyclic cyclocalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptly, and cyclooctyl. Polycyclic cycloalkyls include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo-[2.2.1]heptanyl, and the like. Unless stated otherwise specifically in the specification, a cycloalkyl group is optionally substituted.

"Aryl" refers to a ring system comprising at least one carbocyclic aromatic ring. In some embodiments, an aryl cromprises from 6 to 18 carbon atoms. The aryl ring may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. Aryls include, but are not limited to, aryls 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 stated otherwise specifically in the specification, an aryl group is optionally substituted.

"Heterocyclic" refers to a stable 3- to 18-membered aromatic or non-aromatic ring comprising one to twelve carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclic ring may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and 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 partially or fully saturated. Examples of aromatic heterocyclic rings are listed below in the definition of heteroaryls (i.e., heteroaryl being a subset of heterocyclic). Examples of non-aromatic heterocyclic rings include, but are not limited to, dioxolanyl, thienyl[l,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.

"Heteroaryl" refers to a 5- to 14-membered ring system comprising one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring. For purposes of certain embodiments of this invention, the heteroaryl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quatemized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzthiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[£][l,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl,

benzo[4,6]imidazo[l,2-a]pyridinyl, benzoxazolinonyl, benzimidazolthionyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl,

1 -phenyl- lH-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, pteridinonyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyridinonyl, pyrazinyl, pyrimidinyl, pryrimidinonyl, pyridazinyl, pyrrolyl, pyrido[2,3-<i]pyrimidinonyl, quinazolinyl, quinazolinonyl, quinoxalinyl, quinoxalinonyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, thieno[3,2-<i]pyrimidin-4-onyl, thieno[2,3- <i]pyrimidin-4-onyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). Unless stated otherwise specifically in the specification, a heteroaryl group is optionally substituted.

"Fused" refers to a ring system comprising at least two rings, wherein the two rings share at least one common ring atom, for example two common ring atoms. When the fused ring is a heterocyclyl ring or a heteroaryl ring, the common ring atom(s) may be carbon or nitrogen. Fused rings include bicyclic, tricyclic, tertracyclic, and the like.

The term "substituted" used herein means any of the above groups (e.g., alkyl, alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene,

heteroalkynylene, alkoxy, alkylether, phosphoalkyl, phosphoalkylether,

thiophosphoalkyl, thiophosphoalkylether, carbocyclic, cycloalkyl, aryl, heterocyclic and/or heteroaryl) wherein at least one hydrogen atom (e.g., 1, 2, 3 or all hydrogen atoms) is replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, CI, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. "Substituted" also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, "substituted" includes any of the above groups in which one or more hydrogen atoms are replaced with - R _g R _h , - R _g C(=0)R _h , -OR _g , -SR _g , -SOR _g , -S0 ₂ R _g , -OS0 ₂ R _g , -S0 ₂ OR _g , =NS0 ₂ R _g , and -S0 ₂ R _g R _h . "Substituted also means any of the above groups in which one or more hydrogen atoms are replaced with -C(=0)R _g , -C(=0)OR _g , -CH ₂ S0 ₂ R _g , -CH ₂ S0 ₂ R _g R _h . In the foregoing, R _g and R _h are the same or different and independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N- heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl.

"Substituted" further means any of the above groups in which one or more hydrogen atoms are replaced by 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 addition, each of the foregoing substituents may also be optionally substituted with one or more of the above substituents.

"Conjugation" refers to the overlap of one p-orbital with another p- orbital across an intervening sigma bond. Conjugation may occur in cyclic or acyclic compounds. A "degree of conjugation" refers to the overlap of at least one p-orbital with another p-orbital across an intervening sigma bond. For example, 1, 3-butadine has one degree of conjugation, while benzene and other aromatic compounds typically have multiple degrees of conjugation. Fluorescent and colored compounds typically comprise at least one degree of conjugation.

"Fluorescent" refers to a molecule which is capable of absorbing light of a particular frequency and emitting light of a different frequency. Fluorescence is well- known to those of ordinary skill in the art.

"Colored" refers to a molecule which absorbs light within the colored spectrum (i.e., red, yellow, blue and the like).

A "linker" refers to a contiguous chain of at least one atom, such as carbon, oxygen, nitrogen, sulfur, phosphorous and combinations thereof, which connects a portion of a molecule to another portion of the same molecule or to a different molecule, moiety or solid support (e.g., microparticle). Linkers may connect the molecule via a covalent bond or other means, such as ionic or hydrogen bond interactions.

The term "biomolecule" refers to any of a variety of biological materials, including nucleic acids, carbohydrates, amino acids, polypeptides, glycoproteins, hormones, aptamers and mixtures thereof. More specifically, the term is intended to include, without limitation, RNA, DNA, oligonucleotides, modified or derivatized nucleotides, enzymes, receptors, prions, receptor ligands (including hormones), antibodies, antigens, and toxins, as well as bacteria, viruses, blood cells, and tissue cells. The visually detectable biomolecules of the invention (e.g., compounds of structure (I) having a biomolecule linked thereto) are prepared, as further described herein, by contacting a biomolecule with a compound having a reactive group that enables attachment of the biomolecule to the compound via any available atom or functional group, such as an amino, hydroxy, carboxyl, or sulfhydryl group on the biomolecule.

A "reactive group" is a moiety capable of reacting with a second reactive groups (e.g., a "complementary reactive group") to form one or more covalent bonds, for example by a displacement, oxidation, reduction, addition or cycloaddition reaction. Exemplary reactive groups are provided in Table 1, and include for example, nucleophiles, electrophiles, dienes, dienophiles, aldehyde, oxime, hydrazone, alkyne, amine, azide, acylazide, acylhalide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imidoester, activated ester, ketone, α,β-unsaturated carbonyl, alkene, maleimide, a-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin, thiirane and the like.

The terms "visible" and "visually detectable" are used herein to refer to substances that are observable by visual inspection, without prior illumination, or chemical or enzymatic activation. Such visually detectable substances absorb and emit light in a region of the spectrum ranging from about 300 to about 900 nm. Preferably, such substances are intensely colored, preferably having a molar extinction coefficient of at least about 40,000, more preferably at least about 50,000, still more preferably at least about 60,000, yet still more preferably at least about 70,000, and most preferably at least about 80,000 M^cm ^"1 . The compounds of the invention may be detected by observation with the naked eye, or with the aid of an optically based detection device, including, without limitation, absorption spectrophotometers, transmission light microscopes, digital cameras and scanners. Visually detectable substances are not limited to those which emit and/or absorb light in the visible spectrum. Substances which 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 emitting and/or absorbing in other regions of the electromagnetic spectrum are also included with the scope of "visually detectable" substances.

For purposes of embodiments of the invention, the term "photostable visible dye" refers to a chemical moiety that is visually detectable, as defined hereinabove, and is not significantly altered or decomposed upon exposure to light. Preferably, the photostable visible dye does not exhibit significant bleaching or decomposition after being exposed to light for at least one hour. More preferably, the visible dye is stable after exposure to light for at least 12 hours, still more preferably at least 24 hours, still yet more preferably at least one week, and most preferably at least one month. Nonlimiting examples of photostable visible dyes suitable for use in the compounds and methods of the invention include azo dyes, thioindigo dyes,

quinacridone pigments, dioxazine, phthalocyanine, perinone, diketopyrrolopyrrole, quinophthalone, and truarycarbonium.

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 analogously. In some preferred embodiments, a derivative (e.g., perylene, pyrene, anthracene or naphthalene derivative) is an imide, bisimide or hydrazamimide derivative of perylene, anthracene, naphthalene, or pyrene.

The visually detectable molecules of various embodiments of the invention are useful for a wide variety of analytical applications, such as biochemical and biomedical applications, in which there is a need to determine the presence, location, or quantity of a particular analyte (e.g., biomolecule). In another aspect, therefore, the invention provides a method for visually detecting a biomolecule, comprising: (a) providing a biological system with a visually detectable biomolecule comprising the compound of structure (I) linked to a biomolecule; and (b) detecting the biomolecule by its visible properties. For purposes of the invention, the phrase "detecting the biomolecule by its visible properties" means that the biomolecule, without illumination or chemical or enzymatic activation, is observed with the naked eye, or with the aid of a optically based detection device, including, without limitation, absorption spectrophotometers, transmission light microscopes, digital cameras and scanners. A densitometer may be used to quantify the amount of visually detectable biomolecule present. For example, the relative quantity of the biomolecule in two samples can be determined by measuring relative optical density. If the stoichiometry of dye molecules per biomolecule is known, and the extinction coefficient of the dye molecule is known, then the absolute concentration of the biomolecule can also be determined from a measurement of optical density. As used herein, the term

"biological system" is used to refer to any solution or mixture comprising one or more biomolecules in addition to the visually detectable biomolecule. Nonlimiting examples of such biological systems include cells, cell extracts, tissue samples, electrophoretic gels, assay mixtures, and hybridization reaction mixtures.

"Solid support" refers to any solid substrate known in the art for solid- phase support of molecules, for example a "microparticle" refers to any of a number of small particles useful for attachment to compounds of the invention, including, but not limited to, glass beads, magnetic beads, polymeric beads, nonpolymeric beads, and the like. In certain embodiments, a microparticle comprises polystyrene beads.

"Base pairing moiety" refers to a heterocyclic moiety capable of hybridizing with a complementary heterocyclic moiety via 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 such adenosine, guanosine, thymidine, cytosine and uridine and analogues thereof.

Embodiments of the invention disclosed herein are also meant to encompass all compounds of structure (I) or (II) being isotopically-labelled by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as ² H, ³ H, ^U C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ 0, ¹⁷ 0, ¹⁸ 0, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ³⁶ C1, ¹²³ I, and ¹²⁵ I, respectively.

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

"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.

"Optional" or "optionally" means that the subsequently described event or circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. 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 having no substitution.

"Salt" includes both acid and base addition salts.

"Acid addition salt" refers to those salts which are formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid,

naphthalene-l,5-disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, ^-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

"Base addition salt" refers to those salts which are prepared from addition of an inorganic base or an organic base to the 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, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol,

dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, tri ethanolamine, tromethamine, purines, piperazine, piperidine,

N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

Crystallizations may produce a solvate of the compounds described herein. Embodiments of the present invention include all solvates of the described compounds. As used herein, the term "solvate" refers to an aggregate that comprises one or more molecules of a compound of the invention with 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 a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms. The compounds of the invention may be true solvates, while in other cases the compounds of the invention may merely retain adventitious water or another solvent or be a mixture of water plus some adventitious solvent.

Embodiments of the compounds of the invention (e.g., compounds of structure I or II), or their salts, tautomers or solvates may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids. 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 (5)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

A "stereoisomer" refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof and includes "enantiomers", which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another.

A "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 said compounds. Various tautomeric forms of the compounds are easily derivable by those of ordinary skill in the art.

The chemical naming protocol and structure diagrams used herein are a modified form of the I.U.P.A.C. nomenclature system, using the ACD/Name Version 9.07 software program and/or ChemDraw Ultra Version 11.0 software naming program (CambridgeSoft). Common names familiar to one of ordinary skill in the art are also used.

As noted above, in one embodiment of the present invention, compounds useful as fluorescent and/or colored dyes in various analytical methods are provided. In other embodiments, compounds useful as synthetic intermediates for preparation of compounds useful as fluorescent and/or colored dyes are provided. In general terms, embodiments of the present invention are directed to dimers and higher polymers of fluorescent and/or colored moieties. The fluorescent and or colored moieties are linked by one or more rigid linkers (i.e., moiety "A"). Without wishing to be bound by theory, it is believed 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 having a high molar "brightness" (e.g., high fluorescence emission).

Accordingly, in some embodiments the compounds have the following structure (A):

(A)

wherein L is a linker with sufficient rigidity to maintain spatial separation between one or more (e.g., each) M group so that intramolecular quenching is reduced or eliminated,

1 2 3 1 2 3

and R , R R V, L , V and n are as defined for structure (I).

In other embodiments is provided a compound having the following structure (I):

or a stereoisomer, salt or tautomer thereof, wherein:

A is, at each occurrence, independently a moiety comprising one or more, fused, carbocyclic or heterocyclic ring system;

M is, at each occurrence, independently a moiety comprising two or more carbon-carbon double bonds and at least one degree of conjugation;

L ¹ is at each occurrence, independently either: i) an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker; or ii) a linker comprising a functional group capable of formation by reaction of two complementary reactive groups; L ² , L ³ , L ⁴ and L ⁵ are, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene linker or heteroatomic linker;

R ¹ is, at each occurrence, independently H, alkyl or alkoxy; R ² and R ³ are each independently H, OH, SH, alkyl, alkoxy, alkylether,

-OP(=R _a )(Rb)Rc, 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 is OH, SH, O ^" , S ^" , OR _d or SR _d ; R _c is OH, SH, O ^" , S ^" , OR _d , SR _d , alkyl, alkoxy, alkylether , alkoxyalkylether, phosphate, thiophosphate,

phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether; and R _d is a counter ion;

R ⁴ is, at each occurrence, independently OH, SH, O ^" , S ^" , OR _d or SR _d ; R ⁵ is, at each occurrence, independently oxo, thioxo or absent;

Q is, at each occurrence, independently a moiety comprising a reactive group capable of forming a covalent bond with an analyte molecule, a solid support or a complementary reactive group Q';

m is, at each occurrence, independently an integer of zero or greater, provided that at least one occurrence of m is an integer of one or greater; and

n is an integer of one or greater.

The various linkers and substituents (e.g., M, A, Q, R ¹ , R ² , R ³ , R ^c L ¹ , L ² , L ³ , L ⁴ and L ⁵ ) in the compound of structure (I) are optionally substituted with one more substituent. For example, in some embodiments the optional substituent is selected to optimize the water solubility or other property of the compound of structure (I). In certain embodiments, each alkyl, alkoxy, alkylether , alkoxyalkylether, phosphoalkyl, thiophosphoalkyl, phosphoalkylether and thiophosphoalkylether in the compound of structure (I) is optionally substituted with one more substituent selected from the group consisting of hydroxyl, alkoxy, alkylether , alkoxyalkylether, sulfhydryl, amino, alkylamino, carboxyl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether and thiophosphoalkylether. In some embodiments, A is at each occurrence, independently a moiety comprising one or more, fused, aryl or heteroaryl ring system. In different

embodiments, A is at each occurrence, independently a moiety comprising one or more, fused, bicyclic or tricyclic, aryl or heteroaryl ring system.

In other more specific embodiments, A is, at each occurrence, independently a fused, carbocyclic or heterocyclic ring system having one of the following structures:

carbocyclic or heterocyclic ring; and

L ⁶ is a direct bond or a linker.

In yet other embodiments A, at each occurrence, independently has of the following structures:

The optional linker L ¹ can be used as a point of attachment of the M moiety to the remainder of the compound. For example, in some embodiments a synthetic precursor to the 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, for example methods referred to as "click chemistry." For this purpose any reaction which 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 the copper catalyzed reaction of an azide and alkyne to form a triazole (Huisgen 1, 3-dipolar cycloaddition), reaction of a diene and dienophile (Diels-Alder), strain-promoted alkyne-nitrone cycloaddition, reaction of a strained alkene with an azide, tetrazine or tetrazole, alkene and azide [3+2] cycloaddition, alkene and tetrazine inverse-demand Diels-Alder, alkene and tetrazole photoreaction and various displacement reactions, such as displacement of a leaving group by nucleophilic attack on an electrophilic atom. In some embodiments the reaction to form L ¹ may be performed in an aqueous environment.

Accordingly, in some embodiments L ¹ is at each occurrence a linker comprising a functional group capable of formation by reaction of two complementary reactive groups, for example a functional group which is the product of one of the foregoing "click" reactions. In various embodiments, for at least one occurrence of L ¹ , the functional group can be formed by reaction of an aldehyde, oxime, hydrazone, alkyne, amine, azide, acylazide, acylhalide, nitrile, nitrone, sulfhydryl, disulfide, sulfonyl halide, isothiocyanate, imidoester, activated ester, ketone, α,β-unsaturated carbonyl, alkene, maleimide, a-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin or thiirane functional group with a complementary reactive group.

In other embodiments, for at least one occurrence of L ¹ , the functional group can be formed by reaction of an alkyne and an azide.

In more embodiments, for at least one occurrence of L ¹ , the functional group comprises an alkene, ester, amide, thioester, disulfide, carbocyclic, heterocyclic or heteroaryl group. In some more specific embodiments, for at least one occurrence of L ¹ , L ¹ is a linker comprising a triazolyl functional group.

In still other embodiments, for at least one occurrence of L ¹ , ΐ Μ has the following structure:

wherein L ^a and L are each independently optional linkers.

In different embodiments, for at least one occurrence of L ¹ , L^M has the following structure:

wherein L ^a and L are each independently optional linkers.

In various embodiments of the foregoing, L ^la or L ^lb , or both, is absent. In other embodiments, L ^la or L ^lb , or both, is present.

In some embodiments L ^la and L ^lb , when present, are each independently alkylene or heteroalkylene. For example, in some embodiments L ^la and L ^lb , when present, independently have one of the following structures:

In still other different embodiments of structure (I), L ¹ is at each occurrence, independently an optional alkylene or heteroalkylene linker.

In more embodiments, L ² , L ³ , L ⁴ and L ⁵ are, at each occurrence, independently Ci-C ₆ alkylene, C2-C ₆ alkenylene or C2-C ₆ alkynylene. For example, in some embodiments the compound has the following structure (IA):

(IA)

wherein:

⁵ and x ⁶ are, at each occurrence, independently an integer from 0 to 6.

In certain embodiments of the compound of structure (IA), x ³ and x ⁴ are both 2 at each occurrence. In other embodiments, x ¹ , x ² , x ⁵ and x ⁶ are each 1 at each occurrence. In some more specific embodiments of the compound of structure (IA), L ¹ , at each occurrence, independently comprises a triazolyl functional group. In other embodiments of the compound of structure (IA), L ¹ , at each occurrence, independently an optional alkylene or heteroalkylene linker.

In still other embodiments of any of the compounds of structure (I) or

(IA), R ⁴ is, at each occurrence, independently OH, O ^" or OR _d . It is understood that "OR _d " and "SR _d " are intended to refer to O ^" and S ^" associated with a cation. For example, the disodium salt of a phosphate group may be represented as:

where R _a is sodium (Na ⁺ ).

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

In some different embodiments of any of the foregoing compounds, R ¹ is

H.

In other various embodiments, R ² and R ³ are each independently OH or

-OP(=R _a )(Rb)Rc. In some different embodiments, R ² or R ³ is OH or -OP(=R _a )(Rb)Rc, and the other of R ² or R ³ is Q or a linker comprising a covalent bond to Q.

In still other embodiments, Q is, at each occurrence, independently a moiety comprising a reactive group capable of forming a covalent bond with an analyte molecule or a solid support. In other embodiments, Q is, at each occurrence, independently a moiety comprising a reactive group capable of forming a covalent bond with a complementary reactive group Q'. For example, in some embodiments, Q' is present on a further compound of structure (I) (e.g., in the R ² or R ³ position), and Q and Q' comprise complementary reactive groups such that reaction of the compound of structure (I) and the further compound of structure (I) results in covalently bound dimer of the compound of structure (I). Multimer compounds of structure (I) can also be prepared in an analogous manner and are included within the scope of embodiments of the invention. The type of Q group and connectivity of the Q group to the remainder of the compound of structure (I) is not limited, provided that Q comprises a moiety having appropriate reactivity for forming the desired bond.

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

Certain embodiments of compounds of structure (I) comprises Q groups commonly employed in the field of bioconjugation. For example in some

embodiments, Q comprises a nucleophilic reactive group, an electrophilic reactive group or a cycloaddition reactive group. In some more specific embodiments, Q comprises a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, a-haloamide, biotin, amino or maleimide functional group. In some embodiments, the activated ester is an N- succinimide ester, imidoester or polyflourophenyl ester. In other embodiments, the alkyne is an alkyl azide or acyl azide.

Exemplary Q moieties are provided in Table I below.

Table 1. Exemplary Q Moieties

 Structure Class

Activated Ester

Activated Ester

Sulfonyl halide

X = halo

Maleimide

Maleimide

0

a-haloimide

X = halo Structure Class

Disulfide

0

Phosphine

0 Ph

-[-N ₃ Azide

Alkyne

Biotin

Diene

Alkene/ di enophil e Alkene/ di enophil e

EWG = eletron withdrawing

group Structure Class

-NH ₂ Amino

It should be noted that in some embodiments, wherein Q is SH, the SH moiety will tend to form disulfide bonds with another sulfhydryl group on another compound of structure (I). Accordingly, some embodiments include compounds of structure (I), which are in the form of disulfide dimers, the disulfide bond being derived from SH Q groups.

In some other embodiments, one of R ² or R ³ is OH or -OP(=R _a )(R _b )R _c , and the other of R ² or R ³ is a linker comprising a covalent bond to an analyte molecule or a linker comprising a covalent bond to a solid support. For example, in some embodiments the analyte molecule is a nucleic acid, amino acid or a polymer thereof. In other embodiments, the analyte molecule is an enzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer or prion. In still different embodiments, the solid support is a polymeric bead or nonpolymeric bead.

The value for m is another variable that can be selected based on the desired fluorescence and/or color intensity. In some embodiments, m is, at each occurrence, independently an integer of 2 or more. In some embodiments, m is, at each occurrence, independently an integer from 1 to 10, 3 to 10 or 3 to 6. In other embodiments, m is, at each occurrence, independently an integer from 1 to 5, for example 1, 2, 3, 4 or 5. In other embodiments, m is, at each occurrence, independently an integer from 5 to 10, for example 5, 6, 7, 8, 9 or 10.

The fluorescence intensity can also be tuned by selection of different values of n. In certain embodiments, n is an integer from 1 to 100. In other embodiments, n is an integer from 1 to 10. In some embodiments, n is 1.

M is selected based on the desired optical properties, for example based on a desired color and/or fluorescence emission wavelength. In some embodiments, M is the same at each occurrence; however, it is important to note that each occurrence of M need not be an identical M, and certain embodiments include compounds wherein M is not the same at 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 emissions for use in fluorescence resonance energy transfer (FRET) methods. For example, in such 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 ordinary 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.

M may be attached to the remainder of the molecule from any position (i.e., atom) on M. One of skill in the art will recognize means for attaching M to the remainder of molecule. Exemplary methods include the "click" reactions described herein.

In some embodiments, M is a fluorescent or colored moiety. Any fluorescent and/or colored moiety may be used, for examples those known in the art and typically employed in colorimetric, UV, and/or fluorescent assays may be used.

Examples of M moieties which are 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., pyridyloxazole, nitrobenzoxadiazole or benzoxadiazole); Anthracene derivatives (e.g., anthraquinones, including DRAQ5, DRAQ7 and

CyTRAK Orange); Pyrene derivatives such as cascade blue; Oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 170); Acridine derivatives (e.g., proflavin, acridine orange, acridine yellow); Arylmethine derivatives: auramine, crystal violet, malachite green; and Tetrapyrrole derivatives (e.g., porphin, 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. In still other embodiments of any of the foregoing, M comprises three or more aryl or heteroaryl rings, or combinations thereof, for example four or more aryl or heteroaryl rings, or combinations thereof, or even five or more aryl or heteroaryl rings, or combinations thereof. In some embodiments, M comprises six aryl or heteroaryl rings, or combinations 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.

In some embodiments, M is cyclic. For example, in some embodiments M is carbocyclic. In other embodiment, 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 multicyclic. In other more specific examples, the aryl moiety is a fused-multicyclic aryl moiety, for example which may comprise at least 3, at least 4, or even more than 4 aryl rings.

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

In still more embodiments of any of the foregoing, M, at each occurrence, independently comprises 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, amide, or formyl group.

In some even more specific embodiments of the foregoing, M, at each occurrence, independently is a dimethylaminostilbene, quinacridone, fluorophenyl- dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine, terrylene, sexiphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl, squaraine, squarylium, 9, 10- ethynyl anthracene or ter-naphthyl moiety. In other embodiments, M is, at each occurrence, 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 is, at each occurrence, independently a coumarin dye, resorufin dye, dipyrrometheneboron difluoride dye, ruthenium bipyridyl dye, energy transfer dye, thiazole orange dye, polymethine or N-aryl-l,8-naphthalimide dye.

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

In some embodiments, M is pyrene, perylene, perylene monoimide or 6- FAM or derivative thereof. In some other embodiments, M has one of the following structures:

In some specific embodiments, the compound is a compound selected from Table 2: Table 2. Exem lary Compounds of Structure I

As used in Table 2, and throughout the application, F, E and Y refer to fluorescein, perylene and pyrene moieties, respectively, and have the following structures:

The presently disclosed dye compounds are "tunable," meaning that by proper selection of the variables in any of the foregoing compounds, one of skill in the art can arrive at a compound having a desired and/or predetermined molar fluorescence (molar brightness). The tunability of the compounds allows the user to easily arrive at compounds having the desired fluorescence and/or color for use in a particular assay or for identifying a specific analyte of interest. Although all variables may have an effect on the molar fluorescence of the compounds, proper selection of M, A, m and n is believed to play an important role in the molar fluorescence of the compounds.

Accordingly, in one embodiment is provided a method for obtaining a compound having a desired molar fluorescence, the method comprising selecting an M moiety having a known fluorescence, preparing a compound of structure (I) comprising the M moiety, and selecting the appropriate variables for A, m and n to arrive at the desired molar fluorescence.

Molar fluorescence in certain embodiments 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 present compounds is l . lx, 1.5x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x lOx or even higher relative to the parent fluorophore. Various embodiments include preparing compounds having the desired fold increase in fluorescence relative to the parent fluorophore by proper selection of A, m and n.

For ease of illustration, various compounds comprising phosphorous moieties (e.g., phosphate and the like) are depicted in the anionic state (e.g.,

-OPO(OH)0 ^" , -ΟΡΟ3 ^2" )· One of skill in the art will readily understand that the charge is dependent on pH and the uncharged (e.g., protonated or salt, such as sodium or other cation) forms are also included in the scope of embodiments of the invention.

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, use of such compositions in analytical methods for detection of the one or more analyte molecules are also provided.

In still other embodiments, the compounds are useful in various analytical methods. For example, in certain embodiments the disclosure provides a method of staining a sample, the method comprising adding to said sample a compound of structure (I), for example wherein one of R ² or R ³ is a linker comprising a covalent bond to an analyte molecule (e.g., biomolecule) or microparticle, and the other of R ² or R ³ is H, OH, alkyl, alkoxy, alkylether or -OP(=R _a )(R _b )R _c , in an amount sufficient to produce an optical response when said sample is illuminated at an appropriate wavelength.

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

In yet other embodiments of the foregoing method, R ² is a linker comprising a covalent linkage to a solid support such as a microparticle. For example, in some embodiments the microparticle is a polymeric bead or nonpolymeric bead.

In even more embodiments, said optical response is a fluorescent response.

In other embodiments, said sample comprises cells, and some embodiments further comprise observing said cells by flow cytometry.

In still more embodiments, the method further comprises distinguishing the fluorescence response from that of a second fluorophore having detectably different optical properties.

In other embodiments, the disclosure provides a method for visually detecting an analyte molecule, such as a biomolecule, comprising:

(a) providing a compound of structure (I), for example, wherein one of R ² or R ³ is a linker comprising a covalent bond to the analyte molecule, and the other of R ² or R ³ is H, OH, alkyl, alkoxy, alkylether or -OP(=R _a )(R _b )R _c ; and

(b) detecting the compound by its visible properties.

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

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

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

(b) detecting the compound by its visible properties.

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

(a) admixing the compound of claim 1, wherein R ² or R ³ 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) detecting the conjugate by its visible properties.

In some other different embodiments, the compounds of structure (I) can be used in various for analysis of cells. For example, by use of flow cytometry, the compounds can be used to discriminate between live and dead cells, evaluate the health of cells (e.g., necrosis vs. early apoptitic vs. late apoptitic vs. live cell), tracking ploidy and mitosis during the cell cycle and determining various states of cell proliferation. While not wishing to be bound by theory, it is believed that embodiments of the compounds of structure (I) preferentially bind to postively charged moieties.

Accordingly, in some embodiments the compounds may be used in methods for determining the presence of non-intact cells, for example nectrotic cells. For example, the presence of nectrotic cells can be determined by admixing a sample containing cells with a compound of structure (I) and analyzing the mixture by flow cytometry. The compound of structure (I) binds to nectrotic cells, and thus there presence is detectable under flow cytometry conditions. In contrast to other staining reagents which require an amine reactive group to bind to nectrotic cells, embodiments of the staining methods of employing compounds of structure (I) do not require a protein-free incubation buffer, and thus the methods are more efficient to perform than related known methods.

In various other embodiments, the compounds can be used in related methods for determing the presence of positively charged moieties in intact or non- intact cells, apoptitic bodies, depolarized membranes and/or permealized membranes.

In addition to the above methods, embodiments of the compounds of structure (I) find utility in various disciplines and methods, including but not limited to: imaging in endoscopy procedures for identification of cancerous and other tissues; identification of necrotic tissue by preferential binding of the compounds to dead cells; single-cell and/or single molecule analytical methods, for example detection of polynucleotides with little or no amplification; cancer imaging, for example by conjugating a compound of structure (I) to an antibody or sugar or other moiety that preferentially binds cancer cells; imaging in surgical procedures; binding of histones for identification of various diseases; drug delivery, for example by replacing the M moiety in a compound of structure (I) with an active drug moiety; and/or contrast agents in dental work and other procedures, for example by preferential binding of the compound of structure (I) to various flora and/or organisms.

It is understood that any embodiment of the compounds of structure (I), as set forth above, and any specific choice set forth herein for a R ¹ , R ² , R ³ , R ⁴ , R ⁵ , L ¹ , L ² , L ³ , L ⁴ , L ⁵ , A, M, m and/or n variable in the compounds of structure (I), 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 inventions not specifically set forth above. In addition, in the event that a list of choices is listed for any particular R ¹ , R ² , R ³ , R ⁴ , R ⁵ , L ¹ , L ² , L ³ , L ⁴ , L ⁵ , A, M, m and/or n variable in a particular embodiment and/or claim, it is understood that each individual choice may be deleted from the particular embodiment and/or claim and that the remaining list of choices will be considered to be within the scope of the invention.

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

It will also be appreciated by those skilled in the art that in the process 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 (for example, 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(0)-R" (where R" is alkyl, aryl or arylalkyl), /?-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin. Furthermore, all compounds of the invention which exist in free base or acid form can be converted to their salts by treatment with the appropriate inorganic or organic base or acid by methods known to one skilled in the art. Salts of the compounds of the invention can be converted to their free base or acid form by standard techniques.

The following Reaction Schemes illustrate exemplary methods of making compounds of this invention. It is understood that one skilled in the art may be able to make these compounds by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art would be able to make, in a similar manner as described below, other compounds of structure (I) not specifically illustrated below by using the appropriate starting components and modifying the parameters of the synthesis as needed. In general, starting components may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described in this invention.

Reaction Scheme I

a

b Reaction Scheme I illustrates an exemplary method for preparing an intermediate useful for preparation of compounds of structure (I), where R ¹ , L ² , L ³ and M are as defined above, R ² and R ³ are as defined above or are protected variants thereof and L is an optional linker. Referring to Reaction Scheme 1, compounds of structure a can be purchased or prepared by methods well-known to those of ordinary skill in the art. Reaction of a with M-X, where x is a halogen such as bromo, under Suzuki coupling conditions known in the art results in compounds of structure b. Compounds of structure b can be used for preparation of compounds of structure (I) as described below.

Reaction Scheme II

Reaction Scheme II illustrates an alternative method for preparation of intermediates useful for preparation of compounds of structure (I). Referring to

1 1 2 3 2 3 reaction Scheme II, where R , L , L , L , G and M are as defined above, and R and R are as defined above or are protected variants thereof, a compound of structure c, which can be purchased or prepared by well-known techniques, is reacted with M-G' to yield compounds of structure d. Here, G and G' represent functional groups having complementary reactivity (i.e., functional groups which react to form a covalent bond).

G' may be pendant to M or a part of the structural backbone of M. G may be any number of functional groups described herein, such as alkyne.

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

(e)

wherein A is as defined herein and each L is independently an optional linker.

DNA synthesis methods are well-known in the art. Briefly, two alcohol groups, for example R ² and R ³ in intermediates b or d above, are functionalized with a dimethoxytrityl (DMT) group and a 2-cyanoethyl-N,N-diisopropylamino

phosphoramidite group, respectively. The phosphoramidite group is coupled to an alcohol group, typically in the presence of an activator such as tetrazole, followed by oxidation of the phosphorous atom with iodine. The dimethoxytrityl group can be removed with acid (e.g., chloroacetic acid) to expose the free alcohol, which can be reacted with a phosphoramidite group. The 2-cyanoethyl group can be removed after oligomerization by treatment with aqueous ammonia.

Preparation of the phosphoramidites used in the oligomerization methods is also well-known in the art. For example, a primary alcohol (e.g., R ³ ) can be protected as a DMT group by reaction with DMT-C1. A secondary alcohol (e.g., R ² ) is then functionalized as a phosphoramidite by reaction with an appropriate reagent such as 2-cyanoethyl Ν,Ν-dissopropylchlorophosphoramidite. Methods for preparation of phosphoramidites and their oligomerization are well-known in the art and described in more detail in the examples.

Compounds of structure (I) are prepared by oligomerization of intermediates b or d and e according to the well-known phophoramidite chemistry described above. The desired number of m and n repeating units is incorporated into the molecule by repeating the phosphoramidite coupling the desired number of times. It will be appreciated that compounds of structure (II) as, described below, can be prepared by analogous methods.

In various other embodiments, compounds useful for preparation of the compound of structure (I) are provided. The compounds can be prepared above in monomer, dimer and/or oligomeric form and then the M moiety covalently attached to the compound via any number of synthetic methodologies (e.g., the "click" reactions described above) to form a compound of structure (I). Accordingly, in various embodiments a compound is provided having the following structure (II):

(Π)

or a stereoisomer, salt or tautomer thereof, wherein: A is, at each occurrence, independently a moiety comprising one or more, fused, carbocyclic or heterocyclic ring system;

G is, at each occurrence, independently a moiety comprising a reactive group capable of forming a covalent bond with a complementary reactive group;

L ^la , L ² , L ³ , L ⁴ and L ⁵ are, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker;

R ¹ is, at each occurrence, independently H, alkyl or alkoxy;

R ² and R ³ are each independently H, OH, SH, alkyl, alkoxy, alkylether, -OP(=R _a )(Rb)Rc, 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 is OH, SH, O ^" , S ^" , OR _d or SR _d ; R _c is OH, SH, O ^" , S ^" , OR _d , SR _d , alkyl, alkoxy, alkylether , alkoxyalkylether, phosphate, thiophosphate,

phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether; and R _d is a counter ion;

R ⁴ is, at each occurrence, independently OH, SH, O ^" , S ^" , OR _d or SR _d ;

R ⁵ is, at each occurrence, independently oxo, thioxo or absent;

Q is, at each occurrence, independently a moiety comprising a reactive group capable of forming a covalent bond with an analyte molecule, a solid support or a complementary reactive group Q';

m is, at each occurrence, independently an integer of zero or greater, provided that at least one occurrence of m is an integer of one or greater; and

n is an integer of one or greater.

The G moiety in the compound of structure (II) can be selected from any moiety comprising a group having the appropriate reactivity group for forming a covalent bond with a complementary group on an M moiety. In exemplary

embodiments, the G moiety can be selected from any of the Q moieties described herein, including those specific examples provided in Table 1. In some embodiments, G comprises, at each occurrence, independently a moiety suitable for reactions including: the copper catalyzed reaction of an azide and alkyne to form a triazole (Huisgen 1, 3-dipolar cycloaddition), reaction of a diene and dienophile (Di els- Alder), strain-promoted alkyne-nitrone cycloaddition, reaction of a strained alkene with an azide, tetrazine or tetrazole, alkene and azide [3+2] cycloaddition, alkene and tetrazine inverse-demand Diels-Alder, alkene and tetrazole photoreaction and various displacement reactions, such as displacement of a leaving group by nucleophilic attack on an electrophilic atom.

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

In other embodiments, G comprises, at each occurrence, independently an alkyne or an azide group. In different embodiments, G comprises, at each occurrence, independently a reactive group capable of forming a functional group comprising an alkene, ester, amide, thioester, disulfide, carbocyclic, heterocyclic or heteroaryl group, upon reaction with the complementary reactive group. For example, in some embodiment the heteroaryl is triazolyl.

In various other embodiments of the compound of structure (II), L ² , L ³ , L ⁴ and L ⁵ are, at each occurrence, independently Ci-C ₆ alkylene, C ₂ -C ₆ alkenylene or C ₂ -C ₆ alkynylene.

In other embodiments, the compound has the following structure (IIA):

(IIA)

wherein:

⁵ and x ⁶ are, at each occurrence, independently an intege from 0 to 6. In other embodiments of structure (II), each L ^la is absent. In other embodiments, each L ^la is present, for example L ^la is, at each occurrence, independently heteroalkylene. In c cture:

In other of any of the foregoing embodiments of compound (II), G is, at each occurrence, independently ^ or ^ ³ .

In various embodiments of the compound of structure (Ila), x ³ and x ⁴ are both 2 at each occurrence. In other embodiments, x ¹ , x ² , x ⁵ and x ⁶ are each 1 at each occurrence.

In some other embodiments of the compound of structure (II) or (Ila), A is at each occurrence, independently a moiety comprising one or more, fused, aryl or heteroaryl ring system. In different embodiments, A is at each occurrence,

independently a moiety comprising one or more, fused, bicyclic or tricyclic, aryl or heteroaryl ring system.

In other more specific embodiments of the compound of structure (II) or (Ila), A is, at each occurrence, independently a fused, carbocyclic or heterocyclic ring system having one of the following structures:

wherein:

a ¹ , a ² and a ³ are, at each occurrence, independently a 5, 6 or 7-membered carbocyclic or heterocyclic ring; and

L ⁶ is a direct bond or a linker.

In yet other embodiments of the compound of structure (II) or (Ila), A, at each occurrence, independently has one of the following structures:

In other embodiments, R ⁴ is, at each occurrence, independently OH, O or OR _d , and in different embodiments R ⁵ is, at each occurrence, oxo.

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

In other various embodiments of the compounds of structure (II) or (Ila), R ² and R ³ are each independently OH or -OP(=R _a )(Rb)Rc In some different embodiments, R ² or R ³ is OH or -OP(=R _a )(R )R _c , and the other of R ² or R ³ is Q or a linker comprising a covalent bond to Q.

In still other embodiments of compounds of structure (II) or (Ila), Q is, at each occurrence, independently a moiety comprising a reactive group capable of forming a covalent bond with an analyte molecule or a solid support. In other embodiments, Q is, at each occurrence, independently a moiety comprising a reactive group capable of forming a covalent bond with a complementary reactive group Q'. For example, in some embodiments, Q' is present on a further compound of structure (II) or (Ila) (e.g., in the R ² or R ³ position), and Q and Q' comprise complementary reactive groups such that reaction of the compound of structure (II) or (Ila) and the further compound of structure (II) or (Ila) results in covalently bound dimer of the compound of structure (II) or (Ila). Multimer compounds of structure (II) of (Ila) can also be prepared in an analogous manner and are included within the scope of embodiments of the invention.

The type of Q group and connectivity of the Q group to the remainder of the compound of structure (II) or (Ila) is not limited, provided that Q comprises a moiety having appropriate reactivity for forming the desired bond. In certain embodiments of compounds of structure (II) or (Ha), the Q is a moiety which is not susceptible to hydrolysis under aqueous conditions, but is sufficiently reactive to form a bond with a corresponding group on an analyte molecule or solid support (e.g., an amine, azide or alkyne).

Certain embodiments of compounds of structure (II) or (Ila) comprises Q groups commonly employed in the field of bioconjugation. For example in some embodiments, Q comprises a nucleophilic reactive group, an electrophilic reactive group or a cycloaddition reactive group. In some more specific embodiments, Q comprises a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, a-haloamide, biotin, amino or maleimide functional group. In some embodiments, the activated ester is an N- succinimide ester, imidoester or polyflourophenyl ester. In other embodiments, the alkyne is an alkyl azide or acyl azide.

Exemplary Q moieties for compounds of structure (II) or (Ila) are provided in Table I above.

As with compounds of structure (I) or (la), in some embodiments of compounds of structure (II) or (Ila), wherein Q is SH, the SH moiety will tend to form disulfide bonds with another sulfhydryl group on another compound of structure (II) or (Ila). Accordingly, some embodiments include compounds of structure (II) or (Ila), which are in the form of disulfide dimers, the disulfide bond being derived from SH Q groups.

In some other embodiments of compounds of structure (II) or (Ila), one of R ² or R ³ is OH or -OP(=R _a )(Rb)Rc, and the other of R ² or R ³ is a linker comprising a covalent bond to an analyte molecule or a linker comprising a covalent bond to a solid support. For example, in some embodiments the analyte molecule is a nucleic acid, amino acid or a polymer thereof. In other embodiments, the analyte molecule is an enzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer or prion. In still different embodiments, the solid support is a polymeric bead or nonpolymeric bead.

In other embodiments of compounds of structure (II) or (Ila), m is, at each occurrence, independently an integer from 1 to 10. For example, in some embodiments m is, at each occurrence, independently an integer from 1 to 5. In yet different embodiments of compounds of structure (II) or (Ila) n is an integer from 1 to 100. For example, in some embodiments n is an integer from 1 to 10.

In other different embodiments, the compound of structure (II) is selected from Table 3.

Table 3. Exem lary Compounds of Structure (II)

The compounds of structure (II) or (Ila) can be used in various methods, for example in embodiments is provided a method for labeling an analyte molecule, the method comprising:

(a) admixing any of the described compounds of structure (I), wherein R ² or R ³ 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) reacting the conjugate with a compound of formula M- L ^lb -G', thereby forming at least one covalent bond by reaction of at least one G and at least one G',

wherein:

M is a moiety comprising two or more carbon-carbon double bonds and at least one degree of conjugation;

L ^lb is an optional alkylene, heteroalkylene or heteroatomic linker; and G' is a reactive group complementary to G.

A different embodiment is a method for labeling an analyte molecule, the method comprising:

(a) admixing any of the compounds of structure (II) disclosed herein, wherein R ² or R ³ is Q or a linker comprising a covalent bond to Q, with a compound of formula M-L ^lb -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 molecule, thereby forming a conjugate of the product of step (A) and the analyte molecule,

wherein:

M is a moiety comprising two or more carbon-carbon double bonds and at least one degree of conjugation;

L ^lb is an optional alkylene, heteroalkylene or heteroatomic linker; and G' is a reactive group complementary to G.

Further, as noted above, the compound of structure (II) are useful for preparation of compounds of structure (I). Accordingly, in one embodiment is provided a method for preparing a compound of structure (I), the method comprising admixing a compound of structure (II) with a compound of formula M-L ^lb -G', thereby forming at least one covalent bond by reaction of G and G', wherein:

M is a moiety comprising two or more carbon-carbon double bonds and at least one degree of conjugation;

L ^lb is an optional alkylene, heteroalkylene or heteroatomic linker; and

G' is a reactive group complementary to G.

The following examples are provided for purposes of illustration, not limitation.

EXAMPLES

General Methods

1H MR spectra were obtained on a JEOL 400 MHz spectrometer. 1H spectra were referenced against TMS. Reverse phase HPLC dye analysis was performed using a Waters Acquity UHPLC system with a 2.1mm x 50mm Acquity BEH-C18 column held at 45°C. Mass spectral analysis was performed on a

Waters/Micromass Quattro micro MS/MS system (in MS only mode) using MassLynx 4.1 acquisition software. Mobile phase used for LC/MS on dyes was 100 mM l, l,l,3,3,3-hexafluoro-2-propanol (HFIP), 8.6 mM triethylamine (TEA), pH 8.

Phosphoramidites and precursor molecules were also analyzed using a Waters Acquity UHPLC system with a 2.1mm x 50mm Acquity BEH-C18 column held at 45°C, employing an acetonitrile/water mobile phase gradient. Molecular weights for 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 profiles experiments were recorded on a Cary Eclipse spectra photometer.

All reactions were carried out in oven dried glassware under a nitrogen atmosphere unless otherwise stated. Commercially available DNA synthesis reagents were purchased from Glen Research (Sterling, VA). Anhydrous pyridine, toluene, dichloromethane, diisopropylethyl amine, triethylamine, acetic acid, pyridine, and THF were purchased from Aldrich. All other chemicals were purchase from Aldrich or TCI and were used as is with no additional purification.

All oligonucleotide dyes were synthesized on an ABI 394 DNA synthesizer using standard protocols for the phosphoramidite based coupling approach. The chain assembly cycle for the synthesis of oligonucleotide phosphoramidates was the following: (i) detritylation, 3% trichloroacetic acid in dichloromethane, 1 min; (ii) coupling, 0.1 M phosphoramidite and 0.45 M tetrazole in acetonitrile, 10 min; (iii) capping, 0.5 M acetic anhydride in THF/lutidine, 1/1, v/v 15 s; (iv) oxidation, 0.1 M iodine in THF/pyri dine/water, 10/10/1, v/v/v, 30 s.

Chemical steps within the cycle were followed by acetonitrile washing and flushing with dry argon for 0.2-0.4 min. Cleavage from the support and removal of base and phosphoramidate protecting groups was achieved by treatment with ammonia for 1 hour at room temperature. Oligonucleotide dyes were then analyzed by reverse phase HPLC as described above. EXAMPLE 1

In a 250mL round bottomed flask with stirring bar was placed pyromellitic anhydride (l .Og, 4.59mmol) and dioxane (50mL). To this was added ethanolamine (622 \L, 11.5mmol) and diisopropylethylamine (4.0mL, 23mmol). The flask was equipped with a reflux condenser, placed in an oil bath and heated to reflux overnight. The mixture was allowed to cool. The solids were filtered and retained for later. The filtrate was condensed and partitioned between ethyl acetate and water. The ethyl acetate layer was retained and the aqueous layer was extracted two additional times with ethyl acetate. The organic layers were combined, dried over sodium sulfate, filtered and concentrated to a solid. The solids from extraction and the earlier filtration were found to be identical by TLC and combined to afford the final material. (870mg)

LC/MS of product showed m/z 305 associated with the

largest peak. Overall purity was -79%.

In a lOOmL round bottomed flask with stirring bar was placed the diol (850mg, 2.8 mmol) in pyridine (15mL). The mixture was cooled on ice under a stream of nitrogen. To this was added 4,4 dimethoxytrityl chloride (237mg, 0.7mmol). The mixture was stirred at 4 °C overnight. Methanol (2mL) was added, and the mixture stirred for 15min before concentrating to a paste on the rotovap. The residue was partitioned between a saturated solution of sodium bicarbonate and toluene. Retained toluene layer and extracted aqueous layer two additional times with dichloromethane. The organic layers were combined, dried with sodium sulfate and concentrated. Final purification was accomplished with silica gel chromatography

(dichloromethane/methanol gradient) to afford the final product as a solid (372mg).

In a lOOmL round bottomed flask with stirring bar was placed the

monoprotected DMTr alcohol (150mg, 0.24mmol) in dry

dichloromethane (25mL) with diisopropylethylamine (215μL,

1.23mmol). 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (110

\L, 0.49mmol) was added and the mixture stirred for 30min at which

point TLC indicated the reaction was complete. The mixture was

partitioned between dichloromethane and saturated sodium bicarbonate.

The organic layer was retained, dried over sodium sulfate and

concentrated to a yellow oil which was used directly on the DNA

synthesizer.

In a 250mL round bottomed flask with stirring bar was placed 3, 3', 4,4'- biphenyltetracarboxylic dianhydride (1.2g, 4.1mmol) and dioxane (80mL).

Ethanolamine (616 \L, 10.2mmol) and diisopropylethylamine (3.5mL) were added, the flask was equipped with a reflux condenser and the mixture heated to reflux overnight. The reaction was cooled and added to a stirring beaker of water (1500mL) to effect precipitation. The solids were collected by filtration and dried under vacuum (0.74g)

In a 50mL round bottomed flask with stir bar was placed the diol (700mg, 1.8 mmol) in pyridine (9mL). The mixture was cooled on ice under nitrogen. To this was added 4,4 dimethoxytrityl chloride (468mg, 1.4mmol). The mixture was stirred at 4 °C overnight. Methanol (2mL) was added, and the mixture stirred for lOmin before concentrating to a paste on the rotovap. Final purification was accomplished with silica gel chromatography (dichloromethane/methanol gradient) to afford the final product as a solid (15 lmg).

Overall purity is -89%. M/Z 773.8 is consistent with product + tropylium.

In a lOOmL round bottomed flask with stirring bar was placed the monoprotected DMTr alcohol (132mg, 0.19mmol) in dry dichloromethane (25mL) with diisopropylethylamine (168 \L, 0.74mmol). 2-cyanoethyl-N,N- diisopropylchlorophosphoramidite (86 μΐ., 0.38mmol) was added and the mixture stirred for 30min at which point TLC indicated the reaction was complete. The mixture was partitioned between dichloromethane and saturated sodium bicarbonate. The organic layer was retained, dried over sodium sulfate and concentrated to an oil which was used directly on the DNA synthesizer.

In a 250mL round bottomed flask with stirring bar was placed 4,4'- Oxydiphthalic anhydride (1.2g, 3.9mmol) and dioxane (90mL). Ethanolamine (584 \L, 9.7mmol) and diisopropylethylamine (3.4mL) were added, the flask was equipped with a reflux condenser and the mixture heated to reflux overnight. The reaction was cooled and concentrated on a rotovap to a paste which was partitioned between ethyl acetate and water. The organic layer was retained and the aqueous layer was extracted and additional time with ethyl acetate. The organic layers were combined, dried over sodium sulfate and concentrated to the final solid product (1.33g)

Overall purity is -83%. Main peak at 1.16 min. Calculated MW is 396.4. MW found is 396.2.

In a 20mL round bottomed flask with stir bar was placed the diol (700mg, 1.8 mmol) in pyridine (9mL). The mixture was cooled on ice under nitrogen. To this was added 4,4 dimethoxytrityl chloride (449mg, 1.3mmol). The mixture was stirred at 4 °C overnight. Methanol (2mL) was added, and the mixture stirred for 15min before concentrating to a paste on the rotovap. Final purification was accomplished with silica gel chromatography (dichloromethane/methanol gradient) to afford the final product as a solid (600mg).

Overall purity is -82%. Predicted MW is 698.7. MW found is 788.5, which is consistent with product + tropylium.

In a lOOmL round bottomed flask with stirring bar was placed the monoprotected DMTr alcohol (lOOmg, 0.14mmol) in dry dichloromethane (25mL) with diisopropylethylamine (125 \L, 0.71mmol). 2-cyanoethyl-N,N- diisopropylchlorophosphoramidite (64 \L, 0.28mmol) was added and the mixture stirred for 30min at which point TLC indicated the reaction was complete. The mixture was partitioned between dichloromethane and saturated sodium bicarbonate. The organic layer was retained, dried over sodium sulfate and concentrated to a yellow oil which was used directly in solid phase oligonucleotide synthesis.

In a 250mL round bottomed flask with stirring bar was placed 4,4'-(4,4'-

Isopropylidinediphenoxy)bis(phthalic anhydride) (1.5g, 2.9mmol) and dioxane (50mL). Ethanolamine (435 \L, 7.2mmol) and diisopropylethylamine (2.5mL) were added, the flask was equipped with a reflux condenser and the mixture heated to reflux overnight. The reaction was cooled and concentrated on a rotovap to a paste which was partitioned between dichloromethane and water. The organic layer was retained and the aqueous layer was extracted two additional times with dichloromethane. The organic layers were combined, dried over sodium sulfate and concentrated to the final solid product (400mg).

In a 20mL round bottomed flask with stir bar was placed the diol

(400mg, 0.7 mmol) in pyridine (3mL). The mixture was cooled on ice under nitrogen. To this was added 4,4 dimethoxytrityl chloride (168mg, 0.5mmol). The mixture was stirred at 4 °C overnight. Methanol (lmL) was added, and the mixture stirred for lOmin before concentrating to a paste on the rotovap. Final purification was

accomplished with silica gel chromatography (dichloromethane/methanol gradient) to afford the final product as a solid. Overall purity is -36%. M/Z at 1000 is consistent with mass + tro lium)

In a 250mL round bottomed flask with stir bar was placed pyromellitic dianhydride (0.75g, 3.4mmol) and dioxane (80mL). Serine Methyl ester

(1.2g, 7.6mmol) and diisopropylethylamine (4.8mL) were added, the flask was equipped with a reflux condenser and the mixture heated to reflux overnight. The reaction was cooled and concentrated on a rotovap to a paste which was partitioned between ethyl acetate and citric acid (500mM). The organic layer was retained and the aqueous layer was extracted and two additional times with ethyl acetate. The organic layers were combined, dried over sodium sulfate and concentrated to the final oil (1.4g).

Calculated MW is 420.33. MW found is 420.1.

In a lOOmL round bottomed flask with stir bar was placed the diol (1.4g, 3.5 mmol) in pyridine (17mL). The mixture was cooled on ice under nitrogen. To this was added 4,4 dimethoxytrityl chloride (883mg, 2.6mmol). The mixture was stirred at 4 °C overnight. Methanol (5mL) was added, and the mixture stirred for lOmin before concentrating to a paste on the rotovap. The residue was partitioned between a saturated solution of sodium bicarbonate and ethyl acetate. Retained organic layer and extracted aqueous layer two additional times with ethyl acetate. The organic layers were combined, dried with sodium sulfate and concentrated. Final purification was accomplished with silica gel chromatography (dichloromethane/ethyl acetate gradient) to afford the final product as an oil (1.7g). MW at 812 consistent with mass plus tropyllium.

EXAMPLE 2

SYNTHESIS OF OLIGOMER DYES

Oligomer dyes were synthesized on an Applied Biosy stems 394

DNA/RNA synthesizer or on GE AKTA 10 OligoPilot on either 1 μιηοΐ or 10 μιηοΐ scales and possessed a 3 '-phosphate group. Dyes were synthesized directly on CPG beads or on polystyrene solid support. The dyes were synthesized in the 3' to 5' direction by standard solid phase DNA methods. Coupling methods employed standard β-cyanoethyl phosphoramidite chemistry conditions. Different number of repeating units were incorporated by repeating the synthesis cycle the desired number of times with an appropriate phosphoramidite. All phosphoramidite monomers were dissolved in acetonitrile /dichloromethane (0.1 M solutions), and were added in successive order using the following synthesis cycles: 1) removal of the 5'-dimethoxytrityl protecting group with dichloroacetic acid in toluene, 2) coupling of the next phosphoramidite with activator reagent in acetonitrile, 3) oxidation with iodine/pyridine/water, and 4) capping with acetic anhydride/l-methylimidizole/acetonitrile. The synthesis cycle was repeated until the 5' Oligofloroside was assembled. At the end of the chain assembly, the monomethoxytrityl (MMT) group or dimthoxytrityl (DMT) group was removed with dichloroacetic acid in dichloromethane or dichloroacetic acid in toluene.

The dyes were cleaved from the solid support and deprotected as follows: A 1 mL micropipettor was used to add 450μΙ. of concentrated H ₄ OH to -25 mg of reacted CPG solid support in a 1.5 mL Eppendorf tube. The slurry was mixed briefly using a Vortex mixer and allowed to settle before placing (open) on a 55°C heating block until gas formation (and bubbling) started to diminish, at which point the tube was tightly closed. Heat treatment was for 2 hours (+/- 15 minutes) and tubes were then removed to cool to room temperature. The tube and its contents were spun in a centrifuge at its maximum speed (13400 rpm) for 1 minute, and then the supernatant was removed with a glass pipette and placed into a second, labeled, 1.5mL Eppendorf tube, taking care not to include the support. The support was washed and spun-down 2x with ~150μΕ of acetonitrile to help maximize dye removal, and the washings were carefully removed from support and added to the labeled secondary tubes. Clarified supernatant was dried completely in a CentriVap concentrator at 40°C to remove H ₄ OH.

EXAMPLE 3

CHARACTERIZATION OF OLIGOMER DYES lmL of deionized water was added to the dried dye sequence prepared according to Example 2 to re-constitute and establish a concentrated stock of -0.3 to 1.0 mM (determined later). 2μΙ. aliquots of each dye construct were analyzed by HPLC- MS to determine identity and relative purity using 45°C heated ultra-high performance 2.1mm x 50mm C18 column (1.7μπι) with 150mM HFIP/TEA (pH9) mobile phase, and methanol as organic elution component. Gradient was from 1-100% over 10 minutes. Electrospray ionization was used (in negative mode) to determine the molecular weights of the dye sequences and help to characterize impurities.

A sample was taken from a concentrated stock using a micropipettor and diluted appropriately in 0. IX PBS (lOx to lOOx) to be within linear range of the NanoDrop UV-vis spetrophotomer (Thermo Scientific). A blank measurement was performed on the NanoDrop using 0.1X PBS, and then the absorbance of the diluted dye sequence at an appropriate wavelength was recorded. Extinction coefficients (ε) were determined by the total number of fluors (M moieties) in the dye construct, using 75,000 M ^"1 cm ^"1 for each fluorescein (F; read at 494nm); 34,500 for each pyrene (Y; read at 343nm); and 40,000 for each perylene (E; read at 440nm) present in the sequence. Spacers and linkers are presumed to have no effect on ε.

With concentration determined, the dye stock was diluted in the NaP0 ₄ (0.1 M at pH 7.5) and NaC0 ₃ (0.1 M at pH 9.0) buffers to make solutions of 2μΜ (or 5μΜ, whatever works with the linear range of the instrument) at a final volume of -3.5 mL. These solutions were scanned by UV/Vis, and then used them to make a second dilution in the appropriate buffer for reading on the fluorimeter, in the range of 10 - 50nM. The necessary concentration will vary depending upon the identity of the M moiety.

Using a 1 cm quartz cuvette, the absorbance of the 2μΜ sample was determined, scanning from 300nm to 700nm. Scan speed was set to medium.

Using a 1cm quartz cuvette and a Cary Eclipse spectrometer, the emission of the 25nM sample was read using an appropriate excitation wavelength (494nm for above dye) and scanning from 499nm to 700nm. Scan speed was set to medium.

The UV absorbance (Figure 1) and fluorescence emission (Figure 2) spectra of compounds I-l, 1-2, and 1-3 were compared to a "monomeric" fluorescein compound (compound A). As expected, the UV absorbance is approximately 2 times greater for compounds I-l, 1-2 and 1-3, relative to Compound A. Further , it was found that when m=l (Compound I-l), the emission was only slightly higher than the emission for compound A. However, when m = 2 or more (Compounds 1-2 and 1-3), the emission is approximately twice the amount relative to Compound A, indicating little or no quenching of the chromophore. Accordingly, the rigid linker A appears to prevent quenching of the fluorophores, especially as the number of A moieties increases (i.e., as m increases).

Compound A

All of the 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 herein by reference, in their entirety to the extent not inconsistent with the present description.

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 deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.