The present invention is generally directed to dimeric and polymeric chromophore compounds (e.g., polymer compounds comprising fluorescent dye moieties) having 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 dyes compatible with aqueous systems that can elucidate desired spatial information and biomolecule interactions.

Accordingly, techniques involving resonance energy transfer have been developed to reveal such structural information. Specifically, Förster resonance energy transfer (“FRET”– sometimes also used interchangeably with fluorescence resonance energy transfer) techniques produce information that reliably measures change

biomolecular distances and interactions. Resonance energy transfer techniques are relatively cheap and measurements can be obtained rapidly; however, FRET suffers from several limitations related to the orientation and positioning of chromophores as well as energy transfer masking due to free fluorophores and undesirable pH sensitivity.

There is thus a need in the art for water soluble dyes, especially resonance energy transfer dyes, having an increased molar brightness and/or increased FRET emission signal. 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. In particular, in some embodiments, the compounds of this disclosure are useful because they enable FRET fluorescence emission associated with the same. 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 (i.e., chromophores) covalently linked by a linker (“L4”). In contrast to previous reports of dimeric and/or polymeric dyes, the present dyes are significantly brighter than the corresponding monomeric dye compound and enable FRET absorbance and emission as a result of intramolecular interactions. While, not wishing to be bound by theory, it is believed that particular linkers provide sufficient proximity between the fluorescent and/or colored moieties such that intramolecular FRET is optimized.

The water soluble, fluorescent or colored dyes of embodiments of the invention are intensely colored, enable FRET processes (e.g., absorbance, emission, Stokes shifts) 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 as well as valuable spatial information about target molecules.

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

or a stereoisomer, tautomer or salt thereof, wherein R1, R2, R3, R4, R5, L1, L2, L3, L4, M1, M2, m and n are as defined herein.

Another embodiment provides a compound having the following structure (II):

or a stereoisomer, salt or tautomer thereof, wherein R1, R2, R3, R4, R5, L1, L2, L3, L4, M1, M2, m and n are as defined herein.

Yet another embodiment provides a polymer compound comprising an acceptor chromophore having an acceptor transition dipole moment and being covalently linked to a polymer backbone, and a donor chromophore having a donor transition dipole moment and being covalently linked to the polymer backbone, wherein the polymer compound adopts a confirmation in solution at physiological conditions wherein the effective distance between the acceptor chromophore and the donor

chromophore is less than about 50.0 nm and the acceptor transition dipole and the donor transition dipole are substantially parallel.

The foregoing embodiments describe compounds that find utility in a number of applications, including use as FRET dyes, 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 one of the foregoing compounds 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 one of the foregoing compounds (e.g., a compound of structure (I) or structure (II)); and

(b) detecting the compound by its visible properties.

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

(a) ad-mixing one of the foregoing compounds (e.g., a compound of structure (I) or structure (II)) with one or more biomolecules; and

(b) detecting the compound by its visible properties.

Other embodiments are directed to a composition comprising at least one of the foregoing compounds (e.g., a compound of structure (I) or structure (II)) and one or more biomolecules. Use of such compositions in analytical methods for detection of the one or more analyte (e.g., biomolecules) is also provided.

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 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 shows the emission spectra of Compounds I-1 through I-4, each having various spacer lengths between pendant boron-dipyrromethene (“BODIPY”) and fluorescein-based chromophores.

Figure 2 illustrates the emission spectra of Compounds I-5 through I-8, each having various spacer lengths between pendant Texas Red and fluorescein-based chromophores.

Figure 3 depicts the emission spectra of Compounds I-9 through I-12 to compare donor emission for representative polymer dye compounds.

Figure 4 shows emission spectra of Compounds I-13 through I-19 to compare how changes in the polymer compound affect emission.

Figure 5 shows the emission spectra for Compounds I-20 through I-23 and the effect of spacer length on the same.

Figure 6 depicts the absorbance spectrum of Compound I-24, which has 3 distinct dye moieties pendant to the polymer backbone

Figure 7 displays emission spectra for Compound I-24 for pH values of 7.0 and 9.0.

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 ˗NH2 group.

“Carboxy” refers to the ˗CO2H group.

“Cyano” refers to the ˗CN group.

“Formyl” refers to the ˗C(=O)H group.

“Hydroxy” or“hydroxyl” refers to the ˗OH group.

“Imino” refers to the =NH group.

“Nitro” refers to the ˗NO2 group.

“Oxo” refers to the =O 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 (C1-C12 alkyl), one to eight carbon atoms (C1-C8 alkyl) or one to six carbon atoms (C1-C6 alkyl), and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, 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, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-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, n-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, n-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(=Ra)(Rb)Rc, wherein each of Ra, Rb and Rc is as defined for compounds of structure (I).

“Alkoxy” refers to a group of the formula ˗ORa where Ra 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, where x is 1, 2 or 3). 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-A-M2, where M1 and M2 are portions of the molecule, H is a heteroatom and A is an alkylene). Unless stated otherwise specifically in the specification, a heteroalkylene group is optionally substituted. Exemplary

heteroalkylene groups include ethylene oxide (e.g., polyethylene oxide) and the“C,” “HEG,”“TEG,”“PEG 1K” and variations thereof, linking groups illustrated below:

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

In some embodiments,

In some embodiments of the PEG 1K linker, n is 25. Multimers may comprise, for example, the following structure:

wherein x is 0 or an integer greater than 0, for example, x ranges from 0-100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

“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(O-)(=O)O˗ or ˗OP(O-)(=O)O˗ and multimers and combinations thereof.

“Phosphate” refers to the ˗OP(=O)(Ra)Rb group, wherein Ra is OH, O- or ORc; and Rb is OH, O-, ORc, a thiophosphate group or a further phosphate group, wherein Rc is a counter ion (e.g., Na+ and the like).

“Phosphoalkyl” refers to the ˗OP(=O)(Ra)Rb group, wherein Ra is OH, O- or ORc; and Rb is ˗Oalkyl, wherein Rc 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(=O)(Ra)Rb group, wherein Ra is OH, O- or ORc; and Rb 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(=Ra)(Rb)Rc group, wherein Ra is O or S, Rb is OH, O-, S-, ORd or SRd; and Rc is OH, SH, O-, S-, ORd, SRd, a phosphate group or a further thiophosphate group, wherein Rd is a counter ion (e.g., Na+ and the like) and provided that: i) Ra is S; ii) Rb is S- or SRd; iii) Rc is SH, S- or SRd; or iv) a combination of i), ii) and/or iii).

“Thiophosphoalkyl” refers to the ˗OP(=Ra)(Rb)Rc group, wherein Ra is O or S, Rb is OH, O-, S-, ORd or SRd; and Rc is ˗Oalkyl, wherein Rd is a counter ion (e.g., Na+ and the like) and provided that: i) Ra is S; ii) Rb is S- or SRd; or iii)Ra is S and Rb is S- or SRd. 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(=Ra)(Rb)Rc group, wherein Ra is O or S, Rb is OH, O-, S-, ORd or SRd; and Rc is ˗Oalkylether, wherein Rd is a counter ion (e.g., Na+ and the like) and provided that: i) Ra is S; ii) Rb is S- or SRd; or iii)Ra is S and Rb is S- or SRd. 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 comprises 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[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,

imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl,

1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, a heterocyclic group is optionally substituted.

“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 quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzthiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl,

benzo[4,6]imidazo[1,2-a]pyridinyl, benzoxazolinonyl, 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-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl,

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

The suffix“-ene” refers to a particular structural feature (e.g., alkyl, aryl, heteroalkyl) attached to the rest of the molecule through a single bond and to the radical group through a single bond. In other words, the suffix“-ene” refers to a particular structural feature having the description given herein which is a linker between the molecule and a radical group. The points of attachment of the“-ene” chain to the rest of the molecule and to the radical group can be through one atom of or any two atoms within the chain. For example, an alkyleneheteroalkylene refers to a linker comprising a alkylene portion and a heteroalkylene portion.

“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.

“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).

“FRET” refers to Förster resonance energy transfer refers to a physical interaction whereby energy from the excitation of one moiety (e.g., a first chromophore or“donor”) is transferred to an adjacent moiety (e.g., a second chromophore or “acceptor”).“FRET” is sometimes also used interchangeably with fluorescence resonance energy transfer (i.e., when each chromophore is a fluorescent moiety).

Generally, FRET requires that (1) the excitation or absorption spectrum of the acceptor chromophore overlaps with the emission spectrum of the donor chromophore; (2) the transition dipole moments of the acceptor and donor chromophores are substantially parallel (i.e., at about 0° or 180°); and (3) the acceptor and donor chromophores share a spatial proximity (i.e., close to each other). The transfer of energy from the donor to the acceptor occurs through non-radiative dipole-dipole coupling and the distance between the donor chromophore and acceptor chromophore is generally much less than the wavelength(s) of light.

“Donor” or“donor chromophore” refers to a chromophore (e.g., a fluorophore) that is or can be induced into an excited electronic state and may transfer its excitation energy to a nearby acceptor chromophore in a non-radiative fashion through long-range dipole-dipole interactions. Without wishing to be bound by theory, it is thought that the energy transfer occurs because the oscillating dipoles of the respective chromophores have similar resonance frequencies. A donor and acceptor that have these similar resonance frequencies are referred to as a“donor-acceptor pair(s),” which is used interchangeably with“FRET moieties” or“FRET dyes.”

“Acceptor” or“acceptor chromophore” refers to a chromophore (e.g., a fluorophore) to which excitation energy from a donor chromophore is transferred via a non-radiative transfer through long-range dipole-dipole interaction.

“Stoke’s shift” refers to a difference between positions (e.g.,

wavelengths) of the band maxima of absorption and emission spectra of an electronic transition (e.g., from excited state to non-excited state, or vice versa). In some embodiments, the compounds have a Stoke’s shift greater than 25 nm, greater than 30

nm, greater than 35 nm, greater than 40 nm, greater than 45 nm, greater than 50 nm, greater than 55 nm, greater than 60 nm, greater than 65 nm, greater than 70 nm, greater than 75 nm, greater than 80 nm, greater than 85 nm, greater than 90 nm, greater than 95 nm, greater than 100 nm, greater than 110 nm, greater than 120 nm, greater than 130 nm, greater than 140 nm, greater than 150 nm, greater than 160 nm, greater than 170 nm, greater than 180 nm, greater than 190 nm, or greater than 200 nm.

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.

“Physiological conditions” refers to a solution or medium having a temperature ranging from about 20 to 40°C, an atmospheric pressure of about 1 atm (101 kPa or 14.7 psi), a pH of about 6 to 8, a glucose concentration of about 1 to 20 mM, atmospheric oxygen concentration, and/or earth gravity.“Physiological conditions” includes a solution or medium having a subset of these properties (e.g., having a temperature ranging from 20 to 40°C and a pH of about 6 to 8). Such conditions may also include buffer components or systems including, but not limited to phosphate, bicarbonate, hemoglobin and/or protein.

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 structures (I) or (II) 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 group (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, ^ ^ ^-unsaturated carbonyl, alkene, maleimide, ^-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin, thiirane and the like.


“Bio-conjugation” or“bio-conjugate” and related variations refer to a chemical reaction strategy for forming a stable covalent bond between two molecules. The term“bio-conjugation” is generally used when one of the molecules is a biomolecule (e.g., an antibody), but can be used to describe forming a covalent bond with a non-biomolecule (e.g., a polymeric resin). The product or compound resulting from such a reaction strategy is a“conjugate,”“bio-conjugate” or a grammatical equivalent.

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-1cm-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. Non-limiting 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 polymer compounds 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, spatial interaction 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 the embodiments disclosed herein (e.g., structure (I) or structure (II)) 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. Non-limiting examples of such biological systems include cells, cell extracts, tissue samples, electrophoretic gels, assay mixtures, and hybridization reaction mixtures.

“Solid support” or“solid support residue” 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, non-polymeric 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 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 31P, 32P, 35S, 18F, 36Cl, 123I, and 125I, 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; such a description includes instances where the event or circumstance occurs and instances where 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-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,

propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, 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, triethanolamine, 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 stereocenters 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 (S)-, 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 non-superimposable 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 FRET, 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 FRET, fluorescent and/or colored dyes are provided. In general terms, embodiments of the present invention are directed to dimers and higher polymers of FRET, fluorescent and/or colored moieties. The FRET, fluorescent and/or colored moieties are linked by a linking moiety. Without wishing to be bound by theory, it is believed the linker helps to maintain sufficient spatial distance/proximity between the donor-acceptor pair(s) such that intramolecular quenching is reduced or eliminated, while maintain sufficient proximity to facilitate the non-radiative transfer of energy.

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

wherein L is a linker sufficient to maintain spatial separation between one or more (e.g., each) M group so that intramolecular quenching is reduced or eliminated, and R1, R2, R3, L1, L2, L3 and n are as defined for structure (I) or structure (II).

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

or a stereoisomer, salt or tautomer thereof, wherein:

M1 and M2 are, at each occurrence, independently a chromophore, provided that at least one occurrence of M1 and M2 combine to form a FRET donor-acceptor pair;

L1 is, at each occurrence, an optional linker;

L2 and L3 are, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker;

L4 is, at each occurrence, independently an alkylene or heteroalkylene linker;

R1 is, at each occurrence, independently H, alkyl or alkoxy; R2 and R3 are each independently H, OH, SH, alkyl, alkoxy, alkylether, heteroalkyl, ˗OP(=Ra)(Rb)Rc, Q, or a protected form thereof, or L;

R4 is, at each occurrence, independently OH, SH, O-, S-, ORd or SRd; R5 is, at each occurrence, independently oxo, thioxo or absent;

Ra is O or S;

Rb is OH, SH, O-, S-, ORd or SRd;

Rc is OH, SH, O-, S-, ORd, OL, SRd, alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether;

Rd is a counter ion;

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

L is, at each occurrence, independently a linker comprising a covalent bond to Q, a linker comprising a covalent bond to a targeting moiety, a linker comprising a covalent bond to an analyte molecule, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, a linker comprising a covalent bond to a nucleoside or a linker comprising a covalent bond to a further compound of structure (I);

m is, at each occurrence, independently an integer of zero or greater; and n is an integer of one or greater.

In some embodiments, at least one occurrence of L4 is heteroalkylene. In more specific embodiments, the heteroalkylene comprises alkylene oxide. In some related embodiments, the heteroalkylene comprises ethylene oxide.

In embodiments, at least one occurrence of L4 is alkylene. In some more specific embodiments, at least one alkylene is ethylene. In some embodiments, the alkylene is ethylene at each occurrence.

In some embodiments, L1 is, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker. In some embodiments, L1 is a linker comprising a functional group capable of formation by reaction of two complementary reactive groups (e.g., an azide and an alkyne). In some embodiments, L1 is, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, alkyleneheteroarylenealkylene,

alkyleneheterocyclylenealkylene, alkylenecarbocyclylenealkylene,

heteroalkyleneheteroarylenealkylene, heteroalkyleneheterocyclylenealkylene, heteroalkylenecarbocyclylenealkylene, heteroalkyleneheteroaryleneheteroalkylene, heteroalkyleneheterocyclyleneheteroalkylene,

heteroalkylenecarbocyclyleneheteroalkylene, alkyleneheteroaryleneheteroalkylene, alkyleneheterocyclyleneheteroalkylene, alkylenecarbocyclyleneheteroalkylene, heteroarylene, heterocyclylene, carbocyclylene, alkyleneheteroarylene,

alkyleneheterocyclylene, heteroarylenealkylene, alkylenecarbocyclylene,

carbocyclylenealkylene, heteroalkyleneheteroarylene, heteroalkyleneheterocyclylene, heteroaryleneheteroalkylene, heteroalkylenecarbocyclylene,

carbocyclyleneheteroalkylene or heteroatomic linker. In some embodiments, L1 is optionally substituted.

In some embodiments, the compound has the following structure (IA):

wherein:

z is, at each occurrence, independently an integer from 1 to 100; and m1, m2 and m3 are, at each occurrence, independently an integer from 0 to 6.

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

.

wherein:
z is, at each occurrence, independently an integer from 1 to 100.

In a different embodiment, the compound has the following structure (IC):

wherein:

z is, at each occurrence, independently an integer from 1 to 100; and x1, x2, x3 and x4 are, at each occurrence, independently an integer from 0 to 6.

In certain related embodiments, z is an integer from 3 to 6 at one or more occurrences. In some specific embodiments, x1 and x3 are each 0 at each occurrence, and x2 and x4 are each 1 at each occurrence. In some embodiments, x1, x2, x3 and x4 are each 1 at each occurrence. In one particular embodiment, L4 has the following structure:

wherein:

p is, at each occurrence, independently an integer from 0 to 6; and y is, at each occurrence, independently an integer from 1 to 100.

In some embodiments, R3 comprises the following structure:

wherein:

p is, at each occurrence, independently an integer from 0 to 6; and y is, at each occurrence, independently an integer from 1 to 100.

In certain embodiments, the compound has the following structure (ID):

.

In some related embodiments, the compound has the following structure (IE):

Another embodiment provides a compound having the following structure (II):

or a stereoisomer, salt or tautomer thereof, wherein:

M1 and M2 are, at each occurrence, independently a chromophore, provided that at least one occurrence of M1 and M2 combine to form a FRET acceptor-donor pair;

L1 is at each occurrence, an optional linker;

L2 and L3 are, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker;

L4 is, at each occurrence, independently an alkylene or heteroalkylene linker;

R1 is, at each occurrence, independently H, alkyl or alkoxy;

R2 and R3 are each independently H, OH, SH, alkyl, alkoxy, alkylether, heteroalkyl, ˗OP(=Ra)(Rb)Rc, Q, or a protected form thereof, or L;

R4 is, at each occurrence, independently OH, SH, O-, S-, ORd or SRd;

R5 is, at each occurrence, independently oxo, thioxo or absent;

Ra is O or S;

Rb is OH, SH, O-, S-, ORd or SRd;

Rc is OH, SH, O-, S-, ORd, OL, SRd, alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether;

Rd is a counter ion;

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

L is, at each occurrence, independently a linker comprising a covalent bond to Q, a linker comprising a covalent bond to a targeting moiety, a linker comprising a covalent bond to an analyte molecule, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, a linker comprising a covalent bond to a nucleoside or a linker comprising a covalent bond to a further compound of structure (II);

m is, at each occurrence, independently an integer of zero to two or greater than 6; and

n is an integer of one or greater.

The various linkers and substituents (e.g., M1, M2, Q, R1, R2, R3, Rc L1, L2, L3, and L4) in the compound of structures (I) and (II) 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 compounds of structures (I) or (II). In certain embodiments, each alkyl, alkoxy, alkylether , alkoxyalkylether, phosphoalkyl, thiophosphoalkyl, phosphoalkylether and

thiophosphoalkylether in the compounds of structures (I) and (II) are 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.

The optional linker L1 can be used as a point of attachment of the M1 and M2 moieties to the remainder of the compound. For example, in some embodiments a synthetic precursor to the compound of structure (I) or structure (II) is prepared, and the M1 and/or M2 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 M1 or M2 or both to the synthetic precursor to form a compound of structure (I) or structure (II). 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 L1 may be performed in an aqueous environment.

Accordingly, in some embodiments L1 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 L1, 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, ^ ^ ^-unsaturated carbonyl, alkene, maleimide, ^-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 L1, the functional group can be formed by reaction of an alkyne and an azide.

In more embodiments, for at least one occurrence of L1, 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 L1, L1 is a linker comprising a triazolyl functional group.

In still other embodiments, for at least one occurrence of L1, L1-M has the following structure:

wherein L1a and L1b are each independently optional linkers.

In different embodiments, for at least one occurrence of L1, L1-M has the following structure:

wherein L1a and L1b are each independently optional linkers.

In various embodiments of the foregoing, L1a or L1b, or both, is absent. In other embodiments, L1a or L1b, or both, is present.

In some embodiments L1a and L1b, when present, are each independently alkylene or heteroalkylene. For example, in some embodiments L1a and L1b, when present, independently have one of the following structures:

In still other different embodiments, L1 is at each occurrence, independently an optional alkylene or heteroalkylene linker.

In more embodiments, L2 and L3 are, at each occurrence, independently C1-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene.

In some embodiments, at least one occurrence of L1 has one of the following structures:

wherein
a, b, and c are each independently an integer ranging from 1-6.

In some embodiments, each occurrence of L1 has one of the following structures:

wherein

a, b, and c are each independently an integer ranging from 1-6.

In some embodiments, at least one occurrence of L1 has one of the following structures:

.

In some embodiments, each occurrence of L1 has one of the following structures:

In still other embodiments, R4 is, at each occurrence, independently OH, O- or ORd. It is understood that“ORd” and“SRd” 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 Ra is sodium (Na+).

In other embodiments, R5 is, at each occurrence, oxo.

In some different embodiments of any of the foregoing compounds, R1 is H.

In other various embodiments, R2 and R3 are each independently OH or ˗OP(=Ra)(Rb)Rc. In some different embodiments, R2 or R3 is OH or ˗OP(=Ra)(Rb)Rc, and the other of R2 or R3 is Q or a linker comprising a covalent bond to Q. In some embodiments, R2 and R3 are each independently -OP(=Ra)(Rb)Rc. In some specific embodiments, Rc is OL . In some of those embodiments, L is a heteroalkylene linker to: Q, a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside or a further compound of structure (I) or a further compound of structure (II). In some embodiments, L comprises an alkylene oxide or phosphodiester moiety, or combinations thereof. In certain embodiments, L has the following structure:

wherein:

m² and n² are independently an integer from 1 to 10;

Re is H, an electron pair or a counter ion;

L² is Re or a direct bond or linkage to: Q, a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside or a further compound of structure (I) or a further compound of structure (II).

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) or structure (II) (e.g., in the R2 or R3 position), and Q and Q comprise complementary reactive groups such that reaction of the compound of structure (I) or structure (II) and the further compound of structure (I) or structure (II) results in covalently bound dimer of the compound of structure (I) or structure (II). Multimer compounds of structure (I) or structure (II) 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) or structure (II) is not particularly limited, provided that Q comprises a moiety having appropriate reactivity for forming the desired bond.

In certain embodiments, 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) and/or structure (II) comprise Q groups commonly employed in the field of bio-conjugation. 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, ^-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. In some embodiments, Q comprises a maleimide functional group.

CLAIMS

1. A compound having the following structure (I):

or a stereoisomer, salt or tautomer thereof, wherein:

M1 and M2 are, at each occurrence, independently a chromophore, provided that at least one of M1 and M2 is a FRET donor, and another one of M1 and M2 is a corresponding FRET acceptor;

L1 is, at each occurrence, an optional linker;

L2 and L3 are, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker;

L4 is, at each occurrence, independently an alkylene or heteroalkylene linker;

R1 is, at each occurrence, independently H, alkyl or alkoxy; R2 and R3 are each independently H, OH, SH, alkyl, alkoxy, alkylether, heteroalkyl, ˗OP(=Ra)(Rb)Rc, Q, or a protected form thereof, or L;

R4 is, at each occurrence, independently OH, SH, O-, S-, ORd or SRd; R5 is, at each occurrence, independently oxo, thioxo or absent;

Ra is O or S;

Rb is OH, SH, O-, S-, ORd or SRd;

Rc is OH, SH, O-, S-, ORd, OL, SRd, alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether;

Rd is a counter ion;

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

L is, at each occurrence, independently a linker comprising a covalent bond to Q, a linker comprising a covalent bond to a targeting moiety, a linker comprising a covalent bond to an analyte molecule, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, a linker comprising a covalent bond to a nucleoside or a linker comprising a covalent bond to a further compound of structure (I);

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

provided that at least one occurrence of L4 is heteroalkylene or at least one occurrence of m is 0 when M1 and M2 are selected from fluorescein, pyrene and perylene chromophores.

2. The compound of claim 1, wherein at least one occurrence of L4 is heteroalkylene.

3. The compound of claim 2, wherein the heteroalkylene comprises alkylene oxide.

4. The compound of any one of claims 2-3, wherein the

heteroalkylene comprises ethylene oxide.

5. The compound of any one of claims 1-4, wherein at least one occurrence of L4 is alkylene.

6. The compound of claim 5, wherein at least one alkylene is ethylene.

7. The compound of claim 5, wherein the alkylene is ethylene at each occurrence.

8. The compound of claims 1-7, wherein the compound has the following structure (IA):

wherein:

z is, at each occurrence, independently an integer from 1 to 100; and m1, m2 and m3 are, at each occurrence, independently an integer from 0 to 6.

9. The compound of any one of claims 1-4, wherein the compound has the following structure (IB):

wherein:

z is, at each occurrence, independently an integer from 1 to 100.

10. The compound of any one of claims 1-4 or 9, wherein the compound has the following structure (IC):

wherein:

x1, x2, x3 and x4 are, at each occurrence, independently an integer from 0 to 6.

11. The compound of any one of claims 8-10, wherein z is an integer from 3 to 6 at one or more occurrences.

12. The compound of any one of claims 10-11, wherein x1 and x3 are each 0 at each occurrence, and x2 and x4 are each 1 at each occurrence.

13. The compound of any one of claims 10-11, wherein x1, x2, x3 and x4 are each 1 at each occurrence.

14. The compound of claim 8, wherein the compound has the following structure (ID):

.

15. The compound of claim 1, wherein the compound has the following structure (IE):

16. A compound having the following structure (II):

or a stereoisomer, salt or tautomer thereof, wherein:

M1 and M2 are, at each occurrence, independently a chromophore, provided that at least one occurrence of M1 and M2 combine to form a FRET acceptor-donor pair;

L1 is at each occurrence, an optional linker;

L2 and L3 are, at each occurrence, independently an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene or heteroatomic linker;

L4 is, at each occurrence, independently an alkylene or heteroalkylene linker;

R1 is, at each occurrence, independently H, alkyl or alkoxy;

R2 and R3 are each independently H, OH, SH, alkyl, alkoxy, alkylether, heteroalkyl, ˗OP(=Ra)(Rb)Rc, Q, or a protected form thereof, or L;

R4 is, at each occurrence, independently OH, SH, O-, S-, ORd or SRd; R5 is, at each occurrence, independently oxo, thioxo or absent;

Ra is O or S;

Rb is OH, SH, O-, S-, ORd or SRd;

Rc is OH, SH, O-, S-, ORd, OL, SRd, alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether;

Rd is a counter ion;

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

L is, at each occurrence, independently a linker comprising a covalent bond to Q, a linker comprising a covalent bond to a targeting moiety, a linker comprising a covalent bond to an analyte molecule, a linker comprising a covalent bond to a solid support, a linker comprising a covalent bond to a solid support residue, a linker comprising a covalent bond to a nucleoside or a linker comprising a covalent bond to a further compound of structure (II);

m is, at each occurrence, independently an integer of zero to two or greater than 6; and

n is an integer of one or greater.

17. The compound of any one of claims 1-16, wherein L1 is at each occurrence a linker comprising a functional group capable of formation by reaction of two complementary reactive groups.

18. The compound of claim 17, wherein for at least one occurrence of L1, 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, ^ ^ ^-unsaturated carbonyl, alkene, maleimide, ^-haloimide, epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin or thiirane functional group with a complementary reactive group.

19. The compound of claim 17, wherein for at least one occurrence of L1, the functional group can be formed by reaction of an alkyne and an azide.

20. The compound of claim 17, wherein for at least one occurrence of L1, the functional group comprises an alkene, ester, amide, thioester, disulfide, carbocyclic, heterocyclic or heteroaryl group.

21. The compound of claim 17, wherein for at least one occurrence of L1, L1 is a linker comprising a triazolyl functional group.

22. The compound of claim 17, wherein for at least one occurrence of L1, L1-M has the following structure:

wherein L1a and L1b are each independently optional linkers.

23. The compound of claim 17, wherein for at least one occurrence of L1, L1-M has the following structure:

wherein L1a and L1b are each independently optional linkers.

24. The compound of any one of claims 22-23, wherein L1a or L1b, or both, is absent.

25. The compound of any one of claims 22-23, wherein L1a or L1b, or both, is present.

26. The compound of claim 25, wherein L1a and L1b, when present, are each independently alkylene or heteroalkylene.

27. The compound of claim 25, wherein L1a and L1b, when present, independently have one of the following structures:

28. The compound of any one of claims 1-16, wherein L1 is at each occurrence, independently an optional alkylene or heteroalkylene linker.

29. The compound of any one of claims 1-28, wherein L2 and L3 are, at each occurrence, independently C1-C6 alkylene, C2-C6 alkenylene or C2-C6 alkynylene.

30. The compound of any one of claims 1-16 or 28, wherein L1 has one of the following structures:

wherein

a, b, and c are each independently an integer ranging from 1-6.

31. The compound of any one of claims 1-30, wherein R4 is, at each occurrence, independently OH, O- or ORd.

32. The compound of any one of claims 1-31, wherein R5 is, at each occurrence, oxo.

33. The compound of any one of claims 1-32, wherein R1 is, at each occurrence, H.

34. The compound of any one of claims 1-33, wherein one of R2 or R3 is OH or -OP(=Ra)(Rb)Rc, and the other of R2 or R3 is Q or a linker comprising a covalent bond to Q.

35. The compound of any one of claims 1-33, wherein R2 and R3 are each independently -OP(=Ra)(Rb)Rc.

36. The compound of claim 35, wherein Rc is OL .

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

38. The compound of claim 37, wherein L comprises an alkylene oxide or phosphodiester moiety, or combinations thereof.

39. The compound of any one of claims 36-38, wherein L has the following structure:

,

wherein:

m² and n² are independently an integer from 1 to 10;

Re is H, an electron pair or a counter ion;

L² is Re or a direct bond or linkage to: Q, a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside or a further compound of structure (I) or a further compound of structure (II).

40. The compound of any one of claims 1-39, wherein Q comprises a nucleophilic reactive group, an electrophilic reactive group or a cycloaddition reactive group.

41. The compound of any one of claims 1-40, wherein Q comprises a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, ^-haloamide, biotin, amino or maleimide functional group.

42. The compound of any one of claims 1-41, wherein Q comprises a maleimide functional group.

43. The compound of claim 41, wherein the azide is an alkyl azide or acyl azide.

44. The compound of claim 41, wherein the activated ester is an N-succinimide ester, imidoester or polyflourophenyl ester.

45. The compound of any one of claims 1-44, wherein Q has one of the following structures:

wherein each X is independently a halogen.

46. The compound of any one of claims 37 or 40, wherein the targeting moiety is an antibody or cell surface receptor antagonist.

47. The compound of any one of claims 1-33, wherein one of R2 or R3 is OH or -OP(=Ra)(Rb)Rc, and the other of R2 or R3 is a linker comprising a covalent bond to an analyte molecule or a linker comprising a covalent bond to a solid support.

48. The compound of claim 47, wherein the analyte molecule is a nucleic acid, amino acid or a polymer thereof.

49. The compound of claim 47, wherein the analyte molecule is an enzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer or prion.

50. The compound of claim 47, wherein the solid support is a polymeric bead or non-polymeric bead.

51. The compound of any one of claims 1-33, wherein R2 or R3 has one of the following structures:

52. The compound of any one of claims 1-51, wherein m is, at each occurrence, independently an integer from 1 to 10.

53. The compound of any one of claims 1-51, wherein m is, at each occurrence, independently an integer from 1 to 5.

54. The compound of any one of claims 1-53, wherein n is an integer from 1 to 100.

55. The compound of any one of claims 1-53, wherein n is an integer from 1 to 10.

56. The compound of any one of claims 1-55, wherein M1 or M2, or both are, at each occurrence, independently a moiety comprising four or more aryl or heteroaryl rings, or combinations thereof.

57. The compound of any one of claims 1-56, wherein M1 or M2, or both are, at each occurrence, independently fluorescent or colored.

58. The compound of claim 57, wherein M1 or M2, or both are fluorescent.

59. The compound of any one of claims 1-58, wherein M1 or M2, or both, at each occurrence, independently comprises a fused-multicyclic aryl moiety comprising at least four fused rings.

60. The compound of any one of claims 1-59, wherein M1 or M2 or both are, at each occurrence, independently 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-ethynylanthracene or ter-naphthyl moiety.

61. The compound of any one of claims 1-59, wherein M1 or M2 or both are, at each occurrence, independently p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, or perylene amide or derivative thereof.

62. The compound of any one of claims 1-59, wherein M1 or M2 or both are, at each occurrence, independently a coumarin dye, resorufin dye,

dipyrrometheneboron difluoride dye, ruthenium bipyridyl dye, thiazole orange dye, polymethine or N-aryl-1,8-naphthalimide dye.

63. The compound of any one of claims 1-59, wherein M1 and M2 are, at each occurrence, independently boron-dipyrromethene, rhodamine, cyanine, pyrene, perylene, perylene monoimide or 6-FAM or a derivative thereof.

64. The compound of any one of claims 1-63, wherein M1 and M2 at each occurrence, independently have one of the following structures:

65. The compound of any one of claims 1-64, wherein the compound is selected from Table 2.

66. The compound of any one of claims 16-65, wherein the compound is selected from Table 3.

67. A polymer compound comprising:

an acceptor chromophore having an acceptor transition dipole moment and being covalently linked to a polymer backbone; and

a donor chromophore having a donor transition dipole moment and being covalently linked to the polymer backbone;

wherein

the polymer compound adopts a confirmation in solution at

physiological conditions wherein the effective distance between the acceptor chromophore and the donor chromophore is less than about 50.0 nm and the acceptor transition dipole and the donor transition dipole are substantially parallel.

68. The polymer compound of claim 67, wherein the effective distance between the acceptor chromophore and the donor chromophore is less than about 25.0 nm.

69. The polymer compound of any one of claims 67-68, wherein the effective distance between the acceptor chromophore and the donor chromophore is less than about 10.0 nm.

70. The polymer compound of any one of claims 67-69, wherein the acceptor chromophore is a fluorescent dye moiety.

71. The polymer compound of any one of claims 67-70, wherein the donor chromophore is a fluorescent dye moiety.

72. The polymer compound of any one of claims 67-71, wherein the acceptor chromophore and the donor chromophore are both fluorescent dye moieties.

73. The polymer compound of any one of claims 67-72, wherein the angle between the acceptor transition dipole moment and the donor transition dipole moment ranges from 120° to 180°.

74. The polymer compound of any one of claims 67-73, wherein the angle between the acceptor transition dipole moment and the donor transition dipole moment ranges from 0° to 60°.

75. The polymer compound of any one of claims 67-74, wherein the polymer compound further comprises:

a first acceptor chromophore is covalently linked at a proximal end of the polymer backbone;

a second acceptor chromophore is covalently linked at a distal end of the polymer backbone; and

a donor chromophore is covalently linked between the proximal and distal ends of the polymer backbone.

76. The polymer compound of any one of claims 67-75, wherein the polymer backbone comprises a phosphate linker.

77. The polymer compound of any one of claims 67-76, wherein the polymer backbone comprises an alkylene oxide linker.

78. The polymer compound of claim 77, wherein the alkylene oxide is ethylene oxide.

79. The polymer compound of any one of claims 67-78, wherein the polymer compound has a molecular weight less than about 20,000 g/mol.

80. The polymer compound of any one of claims 67-79, wherein the polymer compound has a molecular weight less than about 17,000 g/mol.

81. The polymer compound of any one of claims 67-80, wherein the polymer compound has a molecular weight less than about 15,000 g/mol.

82. A method of staining a sample, comprising adding to said sample the compound of any one of claims 1-81 in an amount sufficient to produce an optical response when said sample is illuminated at an appropriate wavelength.

83. The method of claim 82, wherein said optical response is a fluorescent response.

84. The method of any one of claims 82-83, wherein said sample comprises cells.

85. The method of claim 84, further comprising observing said cells by flow cytometry.

86. The method of claim 83, further comprising distinguishing the fluorescence response from that of a second fluorophore having detectably different optical properties.

87. A method for visually detecting an analyte molecule, the method comprising:

(a) providing the polymer compound of any one of claims 1-81, wherein the polymer compound comprises a covalent bond to the analyte molecule; and

(b) detecting the polymer compound by its visible properties.

88. A method for visually detecting an analyte molecule, the method comprising:

(a) ad-mixing the polymer compound of any one of claims 1-81, wherein the polymer compound comprises a covalent bond to Q selected from Table 1, with the analyte molecule;

(b) forming a bio-conjugate of the polymer compound and the analyte molecule; and

(c) detecting the bio-conjugate by its visible properties.

89. A composition comprising the compound of any one of claims 1-81 and one or more analyte molecules.

90. Use of the composition of claim 89 in an analytical method for detection of the one or more analyte molecules.