Purple excitable tandem dyes and methods of making and using same - Patents.com

Abstract

A purple-excitable tandem dye is provided. The tandem dye of the present invention comprises a non-conjugated polymer backbone, one or more purple-excitable donor fluorophores linked to the non-conjugated polymer backbone, and one or more acceptor fluorophores linked to the non-conjugated polymer backbone, wherein the one or more purple-excitable donor fluorophores and the one or more acceptor fluorophores are in an energy transfer relationship. Also provided are methods of making and using the tandem dye, and kits containing the tandem dye for use in the method embodiments.

Description

[Background technology]

[0001] Fluorescent dyes are compounds that, when illuminated with light of a wavelength that the fluorescent dye absorbs, emit light of a (usually) different wavelength. Fluorescent dyes are used in a variety of applications in biochemistry, biology and medicine, for example in diagnostic kits, microscopy or drug screening. Fluorescent dyes are characterized by a number of parameters that allow the user to select the appropriate dye depending on the desired purpose. Parameters of interest include excitation wavelength maximum, emission wavelength maximum, Stokes shift, extinction coefficient, fluorescence quantum yield and fluorescence lifetime. Dyes can be selected according to the application of interest, for example to allow penetration of excitation radiation into biological samples, to minimize background fluorescence and / or to achieve a high signal-to-noise ratio.

[0002] Molecular recognition involves the specific binding of two molecules. Molecules with binding specificity for target biomolecules are used in a variety of research and diagnostic applications, such as labeling and separation of analytes, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation, and chromatography. Target biomolecules can be detected by labeling with fluorescent dyes.

[0003] Some applications, such as flow cytometry applications, employ combinations or panels of different fluorescently labeled specific binding members, such as antibodies, for the detection of multiple different targets, such as internal or cell surface markers. In such applications, multiple different fluorescent dyes are employed with the same sample, where the multiple different fluorescent dyes are distinguishable from each other in terms of excitation and / or emission maxima. One type of fluorescent dye used in such applications is a tandem fluorescent dye. A tandem fluorescent dye is a compound with two covalently linked different fluorophores, which can be covalently linked to each other directly or through a linking group. One of the fluorophores acts as a donor fluorophore and the other acts as an acceptor fluorophore. The donor and acceptor fluorophores together form a fluorescence resonance energy transfer (FRET) pair. Such a FRET pair behaves as an intrinsic dye with the excitation properties of the donor fluorophore and the emission properties of the acceptor fluorophore. Summary of the Invention [Problem to be solved by the invention]

[0004] The inventors have realized that there is a need for tandem fluorescent dyes in which the donor fluorophore is excited in the violet wavelength range, for example so that it can be excited by commonly available violet lasers.Embodiments of the present invention fulfill this need. [Means for solving the problem]

[0005] A purple-excitable tandem dye is provided. The tandem dye of the present invention comprises a non-conjugated polymer backbone, one or more purple-excitable donor fluorophores linked to the non-conjugated polymer backbone, and one or more acceptor fluorophores linked to the non-conjugated polymer backbone, wherein the one or more purple-excitable donor fluorophores and the one or more acceptor fluorophores are in an energy transfer relationship. Methods of making and using the tandem dye, as well as kits containing the tandem dye for use in embodiments of the method are also provided. [Brief description of the drawings]

[0006] The invention may be best understood from the following detailed description when read in conjunction with the accompanying drawing figures, in which:

[0007] [Figure 1] FIG. 1 is a chart showing donor absorption and emission spectra for three coumarin purple excitable donor fluorophores that may be employed in the tandem dyes of the present embodiments. [Figure 2A] 1 is a chart showing the absorption and emission spectra of seven different violet excitable tandem dyes according to embodiments of the present invention. [Figure 2B] 1 is a chart showing the absorption and emission spectra of seven different violet excitable tandem dyes according to embodiments of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] definition As used herein, the terms "chemoselective functional group" and "chemoselective tag" are used interchangeably and refer to a functional group that can selectively react with another compatible functional group to form a covalent bond, in some cases after optionally activating one of the functional groups.Chemoselective functional groups of interest include, but are not limited to, thiols and maleimides or iodoacetamides, amines and carboxylic acids or their active esters, and groups that can react with each other via click chemistry, such as azides and alkyne groups (e.g., cyclooctyne groups), tetrazines, transcyclooctenes, dienes and dienophiles, and azides, sulfur (VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, and hydroxyls, hydrazides, hydrazinos, aldehydes, ketones, azides, alkynes, phosphines, epoxides, etc.

[0009] As used herein, the term "sample" refers to a material or mixture of materials, in some cases in liquid form, that contains one or more analytes of interest. In some embodiments, the term used in its broadest sense refers to any plant, animal or bacterial material that contains cells or produces cellular metabolic products, such as tissues or fluids isolated from an individual (including but not limited to plasma, serum, cerebrospinal fluid, lymphatic fluid, tears, saliva and tissue sections) or from in vitro cell culture components, as well as samples from the environment. The term "sample" can also refer to a "biological sample". As used herein, the term "biological sample" refers to a whole organism or a subset of its tissues, cells or component parts (e.g., bodily fluids including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). "Biological sample" can also refer to homogenates, lysates or extracts prepared from a whole organism or a subset of its tissues, cells or components, or fractions or portions thereof, including, but not limited to, plasma, serum, spinal fluid, lymphatic fluid, external parts of the skin, respiratory, intestinal and genitourinary tracts, tears, saliva, milk, blood cells, tumors and organs. In certain embodiments, the sample has been removed from an animal or plant. The biological sample may include cells. The term "cell" is used in its conventional sense to refer to the basic structural unit of an organism, both eukaryotic and prokaryotic organisms, having at least a nucleus and a cell membrane. In certain embodiments, the cell includes a prokaryotic cell, such as from a bacterium. In other embodiments, the cell includes a eukaryotic cell, such as a cell obtained from a biological sample from an animal, a plant or a fungus.

[0010] The terms "support-bound" and "linked to a support" are used interchangeably and refer to a moiety (e.g., a specific binding member) that is covalently or non-covalently bound to a support of interest. Covalent binding can include a chemical reaction of two compatible functional groups (e.g., two chemically selective functional groups, such as an electrophile and a nucleophile) to form a covalent bond between two moieties of interest (e.g., a support and a specific binding member). In some cases, non-covalent binding can include a specific bond between two moieties of interest (e.g., two affinity moieties, such as a hapten and an antibody, or a biotin moiety and streptavidin). In certain cases, non-covalent binding can include absorption to a substrate.

[0011] The term "polypeptide" refers to a polymeric form of amino acids of any length, including peptides ranging from 2 to 50 amino acids in length, and polypeptides greater than 50 amino acids in length. The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" includes coded and non-coded amino acids, polymers of amino acids that have been modified or derivatized chemically or biochemically, and polypeptides having modified peptide backbones in which the conventional backbone has been replaced with a non-naturally occurring or synthetic backbone. A polypeptide can be of any convenient length, such as, for example, 2 or more amino acids, such as 4 or more amino acids, such as 10 or more amino acids, such as 20 or more amino acids, such as 50 or more amino acids, such as 100 or more amino acids, such as 300 or more amino acids, such as up to 500 or 1000 or more amino acids. A "peptide" can be 2 or more amino acids, such as 4 or more amino acids, such as 10 or more amino acids, such as 20 or more amino acids, such as up to 50 amino acids. In some embodiments, a peptide is 5 to 30 amino acids in length.

[0012] As used herein, the term "isolated" refers to a moiety of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which it is associated prior to purification.

[0013] A "plurality" includes at least two members. In certain cases, a plurality can have 5 or more, e.g., 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 300 or more, 1000 or more, 3000 or more, 10,000 or more, 100,000 or more members.

[0014] Numeric ranges are inclusive of the numbers defining the range.

[0015] The term "specific binding" refers to a direct association between two molecules due to covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bonding interactions, including, for example, interactions such as salt bridges and water bridges. A specific binding member describes a member of a pair of molecules that has binding specificity for one another. Members of a specific binding pair can be naturally derived or synthetically produced in whole or in part. One member of the pair of molecules has an area or cavity on its surface that specifically binds to, and is thus complementary to, a particular spatial polar organization of the other member of the pair of molecules. Thus, the members of the pair have the property of specifically binding to one another. Examples of pairs of specific binding members are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The specific binding members of a binding pair exhibit high affinity and binding specificity for binding to one another. Typically, the affinity between a pair of specific binding members is greater than 10 -6 M or less, e.g. 10 -7 M or less, and 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, and 10 -15 K below M dThe affinity is characterized by a dissociation constant (KD). "Affinity" refers to the strength of binding, and an increase in binding affinity correlates with a lower KD. In an embodiment, affinity is determined by surface plasmon resonance (SPR), for example, as used by the Biacore system. The affinity of one molecule to another molecule is determined, for example, by measuring the on-rate of the interaction at 25°C. "Affinity" refers to the strength of binding, and an increase in binding affinity correlates with a lower KD. In an embodiment, affinity is determined by surface plasmon resonance (SPR), for example, as used by the Biacore system. The affinity of one molecule to another molecule is determined, for example, by measuring the on-rate of the interaction at 25°C.

[0016] The specific binding member may be proteinaceous. As used herein, the term "proteinaceous" refers to a moiety composed of amino acid residues. The proteinaceous moiety may be a polypeptide. In certain cases, the proteinaceous specific binding member is an antibody. In certain embodiments, the proteinaceous specific binding member is an antibody fragment, e.g., a binding fragment of an antibody that specifically binds to a polymeric dye. As used herein, the terms "antibody" and "antibody molecule" are used interchangeably and refer to a protein consisting of one or more polypeptides substantially encoded by all or part of a recognized immunoglobulin gene. The recognized immunoglobulin genes include, for example, in humans, the kappa (k), lambda (l), and heavy chain loci, which together comprise a myriad of variable region genes and the constant region genes mu (u), delta (d), gamma (g), sigma (e), and alpha (a), which code for the IgM, IgD, IgG, IgE, and IgA isotypes, respectively. An immunoglobulin light or heavy chain variable region consists of a "framework" region (FR) interrupted by three hypervariable regions, also called complementarity determining regions or "CDRs". The extent of the framework region and CDRs has been precisely defined (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., US Department of Health and Human Services, (1991)). The numbering of all antibody amino acid sequences discussed herein conforms to the Kabat system. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. The term antibody is meant to include full length antibodies and may refer to natural antibodies from any organism, modified antibodies, or antibodies that have been recombinantly generated for experimental, therapeutic, or other purposes as described below.

[0017] Antibody fragments of interest include Fab, Fab', F(ab') fragments produced by modification of either whole antibodies or those synthesized de novo using recombinant DNA technology. 2 Antibody fragments include, but are not limited to, Fv, scFv, or other antigen-binding subsequences of antibodies. Antibodies can be monoclonal or polyclonal and can have other specific activities on cells (e.g., antagonist, agonist, neutralizing, inhibitory, or stimulatory antibodies). It is understood that antibodies can have additional conservative amino acid substitutions that do not substantially affect antigen binding or other antibody functions.

[0018] According to certain embodiments, the specific binding member is a Fab fragment, F(ab') 2 In certain embodiments, the specific binding member is an antibody. In some examples, the specific binding member is a murine antibody or binding fragment thereof. In certain examples, the specific binding member is a recombinant antibody or binding fragment thereof.

[0019] The methods described herein may include multiple steps. Each step may be performed after a predetermined time has elapsed between steps, if necessary. Thus, the time between each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, and 5 hours or more. In certain embodiments, each subsequent step is performed immediately after the completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting period after the completion of the previous step, such as a waiting period of several minutes to overnight.

[0020] As used herein, the terms "evaluating," "determining," "measuring," and "assessing" and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

[0021] The term "separating" as used herein refers to the physical separation of two elements (e.g., by size or affinity, etc.), as well as the disintegration of one element while leaving the other intact.

[0022] The term "linker" or "link" refers to a linking moiety that connects two groups and has a backbone that is 100 atoms or less in length. The linker or linkage can be a covalent bond that connects two groups or chains that are 1 to 100 atoms in length, for example, a chain that is 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms in length, and the linker can be linear, branched, cyclic, or a single atom. In some cases, the linker is a branched linker, which refers to a linking moiety that connects three or more groups. In certain cases, one, two, three, four, or five or more carbon atoms of the linker backbone can be optionally substituted with sulfur, nitrogen, or oxygen heteroatoms. In some examples, the linker backbone includes a linking functional group such as an ether, a thioether, an amino, an amide, a sulfonamide, a carbamate, a thiocarbamate, a urea, a thiourea, an ester, a thioester, or an imine. The bonds between the backbone atoms can be saturated or unsaturated, and in some examples, there are no more than 1, 2, or 3 unsaturated bonds in the linker backbone. The linker can include one or more substituents, such as alkyl, aryl, or alkenyl groups. The linker can include, but is not limited to, polyethylene glycol; ether, thioether, tertiary amine, which can be linear or branched, alkyl, such as methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone can include a cyclic group, such as an aryl, heterocyclic, or cycloalkyl group, with two or more atoms of the cyclic group, such as 2 atoms, 3 atoms, or 4 atoms, included in the backbone. The linker can be cleavable or non-cleavable.

[0023] As used herein, the terms "water-solubilizing group", "water-soluble group" and "WSG" are used interchangeably and refer to a group or substituent that is sufficiently solvated in an aqueous environment, e.g., under physiological conditions, and that confers improved water solubility to the molecule to which it is attached. A WSG can increase the solubility of a tandem dye or a component thereof, e.g., a donor or acceptor fluorophore, primarily in aqueous solution, compared to a control tandem dye or component thereof that lacks the WSG. A water-solubilizing group can be any convenient hydrophilic group that is sufficiently solvated in an aqueous environment.

[0024] A variety of water-soluble polymeric groups can be adapted for use in the WSG of the subject dyes. Any convenient water-solubilizing group (WSG) can be included in the dyes described herein to provide increased water solubility. The increase in solubility can vary, but in some instances the increase is 2-fold or more (compared to a compound without the WSG(s)), e.g., 5-fold, 10-fold, 25-fold, 50-fold, 100-fold or more. In some instances, the hydrophilic water-solubilizing group is, for example, positively charged or negatively charged. In certain instances, the hydrophilic water-solubilizing group is a neutral hydrophilic group. In some embodiments, the WSG is branched (e.g., as described herein). In certain instances, the WSG is linear. In some embodiments, the WSG is a hydrophilic polymer, e.g., polyethylene glycol, modified PEG, peptide sequence, peptoid, carbohydrate, oxazoline, polyol, dendron, dendritic polyglycerol, cellulose, chitosan, or derivatives thereof. Water solubilizing groups of interest include, but are not limited to, carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfonium, esters, polyethylene glycols (PEGs) and modified PEGs, hydroxyls, amines, amino acids, ammonium, guanidinium, pyridinium, polyamines and sulfonium, polyalcohols, linear or cyclic sugars, primary, secondary, tertiary, or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols, and polyethers, -COOM', -SO 3 M', -PO 3 M', -NR3 + , Y', (CH 2 CH 2 O) p R and mixtures thereof, Y' can be any halogen, sulfate, sulfonate, or oxygen-containing anion, p can be 1 to 500, each R can independently be H or alkyl (such as methyl), M' can be a cationic counterion or hydrogen, --(CH 2 CH 2 O) yy CH 2 CH 2 XR yy , --(CH 2 CH 2 O) yy CH 2 CH 2 X--, --X(CH 2 CH 2 O) yy CH 2 CH 2 --, glycol, and polyethylene glycol, where yy is selected from 1 to 1000, and X is O, S, or NR ZZ Selected from R ZZ and R YY are independently H and C 1-3 In some cases, WSG is selected from (CH 2 ) x (OCH 2 CH 2 ) y OCH 3 where each x is independently an integer from 0 to 20 and each y is independently an integer from 0 to 50. In some cases, the water-solubilizing group comprises a nonionic polymer (e.g., a PEG polymer) terminally substituted with an ionic group (e.g., a sulfonate).

[0025] In some embodiments of the formula, the pendant group of interest is (CH 2 ) x (OCH 2 CH 2 ) y OCH 3(each x is independently an integer from 0 to 20, and each y is independently an integer from 0 to 50); one or more of halogen, hydroxyl, C 1 ~C 12 Alkoxy, or (OCH 2 CH 2 ) z OCH 3 In some examples, the substituents are selected from benzyl optionally substituted with (CH 2 ) 3 (OCH 2 CH 2 ) 11 OCH 3 In some embodiments, one or more of the substituents is (CH 2 ) x (OCH 2 CH 2 ) y OCH 3 wherein each x is independently an integer from 0 to 20 and each y is independently an integer from 0 to 50.

[0026] Multiple WSGs may be included at a single position in the subject dyes via a branched linker. In certain embodiments, the branched linker is an aralkyl substituent, which is further disubstituted with a water-solubilizing group. Thus, in some cases, the branched linker group is a substituent of the dye that connects the dye to two or more water-solubilizing groups. In certain embodiments, the branched linker is an amino acid, such as a lysine amino acid, which is connected to three groups via an amino group and a carboxylic acid group. In some examples, the incorporation of multiple WSGs via a branched linker confers desirable solubility to the dye. In some examples, the WSG is a non-ionic side group capable of conferring solubility in water of greater than 50 mg / mL. In some examples, the WSG is a non-ionic side group capable of conferring solubility in water of greater than 100 mg / mL. In some embodiments, the dye comprises a substituent(s) selected from the group consisting of alkyl, aralkyl, and heterocyclic groups, each group being further substituted with a water-solubilizing group comprising a hydrophilic polymer group, such as polyethyl glycol (PEG) (e.g., a PEG group of 6 to 24 units).

[0027] Water-soluble polymers of interest that can be utilized in the WSG include polyethylene glycol (PEG) groups or modified PEG groups. Water-soluble polymers of interest include, but are not limited to, polyalkylene oxide-based polymers such as polyethylene glycol "PEG" (see, e.g., "Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications", J.M. Harris, Ed., Plenum Press, New York, NY (1992); and "Poly(ethylene glycol) Chemistry and Biological Applications", J.M. Harris and S. Zalipsky, Eds., ACS (1997); WO 90 / 13540, WO 92 / 00748, WO 92 / 16555, WO 94 / 04193, WO 94 / 14758, WO 94 / 17039, WO 94 / 18247, WO 94 / 28937, WO 95 / 11924, WO 96 / 00080, WO 96 / 23794, WO 98 / 07713, WO 98 / 41562, WO 98 / 48837, WO 99 / 307 27, WO 99 / 32134, WO 99 / 33483, WO 99 / 53951, WO 01 / 26692, WO 95 / 13312, WO 96 / 21469, WO 97 / 03106, WO 99 / 45964, and U.S. Pat. Nos. 4,179,337; 5,075,046; 5,089,261; 5,100,992; 5,134,192; 5,166,309; 5,171,264; 5,213,891;U.S. Patent No. 5,219,564; U.S. Patent No. 5,275,838; U.S. Patent No. 5,281,698; U.S. Patent No. 5,298,643; U.S. Patent No. 5,312,808; U.S. Patent No. 5,321,095; U.S. Patent No. 5,324,844; U.S. Patent No. 5,349,001; U.S. Patent No. 5,352,756; U.S. Patent No. 5,405,877; U.S. Patent No. 5,455,027; U.S. Patent No. 5,446,090; U.S. Patent No. 5,470,829; U.S. Patent No. US Patent No. 5,478,805; US Patent No. 5,567,422; US Patent No. 5,605,976; US Patent No. 5,612,460; US Patent No. 5,614,549; US Patent No. 5,618,528; US Patent No. 5,672,662; US Patent No. 5,637,749; US Patent No. 5,643,575; US Patent No. 5,650,388; US Patent No. 5,681,567; US Patent No. 5,686,110; US Patent No. 5,730,990; US No. 5,739,208; U.S. Pat. No. 5,756,593; U.S. Pat. No. 5,808,096; U.S. Pat. No. 5,824,778; U.S. Pat. No. 5,824,784; U.S. Pat. No. 5,840,900; U.S. Pat. No. 5,874,500; U.S. Pat. No. 5,880,131; U.S. Pat. No. 5,900,461; U.S. Pat. No. 5,902,588; U.S. Pat. No. 5,919,442; U.S. Pat. No. 5,919,455; U.S. Pat. No. 5,932,462; U.S. Pat. (See U.S. Patent Nos. 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011,042; 6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; and 6,214,966).

[0028] Examples of water-soluble polymers of interest include those of the formula -(CH 2 -CH 2 Examples of polymers of interest include, but are not limited to, those containing polyalkylene oxides, polyamide alkylene oxides, or derivatives thereof, including polyalkylene oxides and polyamide alkylene oxides with ethylene oxide repeat units of the formula -[C(O)-XC(O)-NH-Y-NH]n- or [NH-Y-NH-C(O)-XC(O)] n -, wherein X and Y are divalent radicals which may be the same or different and may be branched or linear, and n is a discrete integer between 2 and 100, e.g., between 2 and 50, and either or both of X and Y comprise biocompatible, substantially non-antigenic water-soluble repeating units which may be linear or branched. Further examples of water-soluble repeating units include those of the formula -(CH 2 -CH 2 -O)- or (O-CH 2 -CH 2 The number of such water-soluble repeating units can vary significantly, with the number of such units being 2-500, 2-400, 2-300, 2-200, 2-100, 6-100, for example 2-50 or 6-50. An example embodiment is where one or both of X and Y are -((CH 2 ) n1 -(CH 2 -CH 2 -O) n2 -(CH 2 )- or ((CH 2 ) n1 -(O-CH 2 -CH 2 ) n2 -(CH 2 ) n-1 -), n1 is 1 to 6, 1 to 5, 1 to 4, or 1 to 3, and n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2 to 8, or 2 to 5. A further example of the embodiment is where X is -(CH 2 -CH 2)- and Y is -(CH 2 -(CH 2 -CH 2 -O) 3 -CH 2 -CH 2 -CH 2 )- or -(CH 2 -CH 2 -CH 2 -(O-CH 2 -CH 2 ) 3 -CH 2 )- is what it is.

[0029] The term modified polymer, such as modified PEG, refers to a water-soluble polymer that has been modified or derivatized at either or both termini to include, for example, terminal substituents (e.g., terminal alkyl, substituted alkyl, alkoxy, or substituted alkoxy, etc.) and / or terminal linking functional groups (e.g., amino or carboxylic acid groups suitable for attachment via amide bond formation) suitable for attaching the polymer to a molecule of interest (e.g., to a light-harvesting chromophore via a branching group). The subject water-soluble polymers can be adapted to include any convenient linking group. It is understood that in some cases, the water-soluble polymer can include some dispersity over the length of the polymer, depending on the method of preparation and / or purification of the polymer starting material. In some examples, the water-soluble polymer is monodisperse.

[0030] The water soluble polymer can include one or more spacers or linkers. Examples of spacers or linkers include linear or branched chain moieties comprising one or more repeat units employed in the water soluble polymer, diamino and / or diacid units, natural or unnatural amino acids or derivatives thereof, and aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, etc., and can contain, for example, up to 18 carbon atoms, or even additional polymer chains.

[0031] The water-soluble polymer moiety, or one or more of the spacers or linkers of the polymer moiety, if present, may comprise polymer chains or units that are biostable or biodegradable. For example, polymers with repeating linkages have varying degrees of stability under physiological conditions, depending on bond lability. Polymers with such linkages can be classified by their relative rate of hydrolysis under physiological conditions, for example, based on known hydrolysis rates of low molecular weight analogues, for example, from less stable to more stable, e.g., polyurethane (-NH-C(O)-O-)>polyorthoester (-OC((OR)(R'))-O-)>polyamide (-C(O)-NH-). Similarly, linking systems attaching the water-soluble polymers to the targeting molecules can be biostable or biodegradable, for example, from lesser to more stable: carbonate (-OC(O)-O-)>ester (-C(O)-O-)>urethane (-NH-C(O)-O-)>orthoester (-OC((OR)(R'))-O-)>amide (-C(O)-NH-). In general, it may be desirable to avoid the use of sulfated polysaccharides, depending on the lability of the sulfate group. Additionally, the use of polycarbonates and polyesters may be less desirable. These linkages are provided by way of example and are not intended to limit the types of linkages that may be employed in the polymer chains or linking systems of the water-soluble polymers useful in the WSGs disclosed herein.

[0032] The water-soluble group (WSG) can provide the subject dye or polymer tandem dye with a solubility in water of greater than 10 mg / mL, greater than 20 mg / mL, greater than 30 mg / mL, greater than 40 mg / mL, greater than 50 mg / mL, greater than 60 mg / mL, greater than 70 mg / mL, greater than 80 mg / mL, greater than 90 mg / mL, or greater than 100 mg / mL. In certain cases, the branched non-ionic water-soluble group (WSG) can provide the subject dye or polymer tandem dye with a solubility in water (e.g., aqueous buffer) of greater than 20 mg / mL, such as greater than 30 mg / mL, greater than 40 mg / mL, greater than 50 mg / mL, greater than 60 mg / mL, greater than 70 mg / mL, greater than 80 mg / mL, greater than 90 mg / mL, greater than 100 mg / mL, or greater. It is understood that the water-soluble dipyrromethene dye can form discrete water-solvated nanoparticles in aqueous systems under certain conditions. In certain cases, water-solvated nanoparticles are resistant to aggregation and are used in various biological assays.

[0033] The terms "polyethylene oxide", "PEO", "polyethylene glycol" and "PEG" are used interchangeably and represent a group of the formula --(CH 2 ----O--) n- or derivatives thereof. In some embodiments, "n" is 5000 or less, e.g., 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, e.g., 3 to 15, or 10 to 15. It is understood that the PEG polymer group may be of any convenient length and may include a variety of end groups and / or further substituents, including, but not limited to, alkyl, aryl, hydroxyl, amino, acyl, acyloxy, amide end groups and / or substituents. PEG groups that may be adapted for use in the subject multichromophores include those PEGs described by S. Zalipsky “Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6(2), 150-165; and Zhu et al “Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy”, Chem. Rev., 2012, 112(8), pp 4687-4735.

[0034] The term "alkyl" by itself or as part of another substituent refers to a saturated branched or straight chain monovalent hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of a parent alkane. Alkyl groups of interest include, but are not limited to, methyl, ethyl, propyl, such as propan-1-yl or propan-2-yl, and butyl, such as butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, or 2-methyl-propan-2-yl. In some embodiments, an alkyl group comprises 1-20 carbon atoms. In some embodiments, an alkyl group comprises 1-10 carbon atoms. In certain embodiments, a lower alkyl group comprises 1-6 carbon atoms, such as 1-4 carbon atoms. The term is by way of example only, such as methyl (CH 3 -), ethyl (CH 3 CH 2 -), n-propyl (CH 3 CH2 CH 2 -), isopropyl ((CH 3 ) 2 CH-), n-Butyl (CH 3 CH 2 CH 2 CH 2 -), isobutyl ((CH 3 ) 2 CHCH 2 -), sec-butyl ((CH 3 )(CH 3 CH 2 )CH-), t-butyl ((CH 3 ) 3 C-), n-pentyl (CH 3 CH 2 CH 2 CH 2 CH 2 -), and neopentyl (CH 3 ) 3 CCH 2 -), and other linear and branched hydrocarbyl groups.

[0035] The term "substituted alkyl" refers to an alkyl group, as defined herein, wherein one or more carbon atoms in the alkyl chain is not substituted with any of -O-, -N-, -S-, -S(O), n -(n is 0 to 2), -NR-(R is hydrogen or alkyl), and optionally substituted with a heteroatom such as alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -Alkyl, -SO 2 -Aryl, -SO 2-heteroaryl, and NR a R b wherein R′ and R″ may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocycle.

[0036] "Alkoxy" refers to the group -O-alkyl, where alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0037] The term "substituted alkoxy" refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0038] "Alkenyl" refers to a monoradical, branched or straight chain, cyclic or acyclic hydrocarbonyl group with a carbon-carbon double bond. Examples of alkenyl groups include ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, and tetracosenyl. In some examples, alkenyl groups have 1 to 24 carbon atoms, such as 1 to 18 carbon atoms or 1 to 12 carbon atoms. The term "lower alkenyl" refers to an alkyl group having 1 to 6 carbon atoms.

[0039] "Alkynyl" or "alkyne" refers to a straight or branched chain monovalent hydrocarbyl group having from 2 to 6 carbon atoms, preferably from 2 to 3 carbon atoms, and having at least 1, and preferably 1 to 2, sites of triple bond unsaturation. Examples of such alkynyl groups are acetylenyl (-C≡CH), and propargyl (-CH 2 C≡CH).

[0040] The term "substituted alkynyl" or "substituted alkyne" refers to any of the following: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -Alkyl, -SO 2 -substituted alkyl, -SO 2 -aryl, and SO 2 -heteroaryl, or alkynyl groups as defined herein having 1 to 5 substituents selected from 1 to 3 substituents.

[0041] "Heterocyclyl" refers to a monoradical, cyclic group that contains a heteroatom (e.g., O, S, N) as a ring atom and is not aromatic (i.e., distinguishing heterocyclyl groups from heteroaryl groups). Examples of heterocyclyl groups include piperidinyl, tetrahydrofuranyl, dihydrofuranyl, and thiocanyl.

[0042] "Amino" is the group -NH 2The term "substituted amino" refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, and where at least one R is not hydrogen.

[0043] "Aryl" by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of an aromatic ring system. Aryl groups of interest include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and the like. In certain embodiments, the aryl group contains 6 to 20 carbon atoms. In certain embodiments, the aryl group contains 6 to 12 carbon atoms. Examples of aryl groups are phenyl and naphthyl.

[0044] Unless otherwise constrained by the definition of the aryl substituent, "substituted aryl" includes any of the following: acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -Alkyl, -SO 2 -substituted alkyl, -SO 2 -Aryl, -SO 2 -heteroaryl, and trihalomethyl, or an aryl group substituted with 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of aryl, heteroaryl, and trihalomethyl.

[0045] "Heteroaryl" by itself or as part of another substituent refers to a monovalent heteroaromatic radical derived by removing one hydrogen atom from a single atom of a heteroaromatic ring system. Heteroaryl groups of interest include, but are not limited to, acridine, arsindol, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, triazole, benzotriazole, thiophene, triazole, xanthene, benzodioxole, and the like. In certain embodiments, the heteroaryl group is a 5-20 membered heteroaryl. In certain embodiments, the heteroaryl group is a 5-10 membered heteroaryl. In certain embodiments, the heteroaryl group is derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole, and pyrazine.

[0046] "Heterocycle", "heterocyclic", "heterocycloalkyl" and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple fused rings, including fused bridged and spiro ring systems, having 3 to 20 ring atoms and containing 1 to 10 heteroatoms. These ring atoms are selected from the group consisting of nitrogen, sulfur, or oxygen, and in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, and the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of a heterocycle group can be N-oxide, -S(O)-, or -SO 2 - optionally oxidized to give the moiety.

[0047] Examples of heterocycles and heteroaryls include azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, furan, and the like. These include, but are not limited to, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also called thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0048] "Substituted heteroaryl" means, unless otherwise constrained by the definition of the substituents, acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -Alkyl, -SO 2 -substituted alkyl, -SO 2 -Aryl, -SO 2-heteroaryl, and trihalomethyl.

[0049] The terms "alkaryl" or "aralkyl" refer to the groups -alkylene-aryl and substituted alkylene-aryl, where alkylene, substituted alkylene, and aryl are defined herein.

[0050] "Alkylene" refers to either a straight or branched chain, -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 -, and the like. This term refers to a divalent aliphatic hydrocarbyl group having preferably 1 to 6, more preferably 1 to 3, carbon atoms, optionally interrupted by one or more groups selected from methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), n-propylene (-CH 2 CH 2 CH 2 -), iso-propylene (-CH 2 CH(CH 3 )-), (-C(CH 3 ) 2 CH 2 CH 2 -), (-C(CH 3 ) 2 CH 2 C(O)-), (-C(CH 3 ) 2 CH 2 C(O)NH-), (-CH(CH 3 )CH 2 "Substituted alkylene" refers to an alkylene group having 1 to 3 hydrogens replaced with a substituent as described for carbon in the definition of "substituted" below.

[0051] "Substituted" refers to a group in which one or more hydrogen atoms are independently replaced with the same or different substituent(s). Substituents of interest include alkylenedioxy (such as methylenedioxy), -M, -R 60, -O - , =O, -OR 60 , -SR 60 , -S - , =S, -NR 60 R 61 , =NR 60 , -CF 3 , -CN, -OCN, -SCN, -NO, -NO 2 , =N 2 , -N 3 , -S(O) 2 O - , -S(O) 2 OH, -S(O) 2 R 60 , -OS(O) 2 O - , -OS(O) 2 R 60 , -P(O)(O - ) 2 , -P(O)(OR 60 )(O - ), -OP(O)(OR 60 )(OR 61 ), -C(O)R 60 , -C(S)R 60 , -C(O)OR 60 , -C(O)NR 60 R 61 , -C(O)O - , -C(S)OR 60 , -NR 62 C(O)NR 60 R 61 , -NR 62 C(S)NR 60 R 61 , -NR 62 C(NR 63 )NR 60 R 61 and -C(NR 62 )NR 60 R 61 M is a halogen; R 60 , R 61 , R 62 and R 63are independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, or optionally R 60 and R 61 together with the nitrogen atom to which they are attached form a cycloheteroalkyl or substituted cycloheteroalkyl ring; R 64 and R 65 are independently hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, or optionally R 64 and R 65 together with the nitrogen atom to which they are attached form a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certain embodiments, the substituents are -M, -R 60 , =O, -OR 60 , -SR 60 , -S - , =S, -NR 60 R 61 , =NR 60 , -CF 3 , -CN, -OCN, -SCN, -NO, -NO 2 , =N 2 , -N 3 , -S(O) 2 R 60 , -OS(O) 2 O - , -OS(O) 2 R 60 , -P(O)(O - ) 2 , -P(O)(OR 60 )(O - ), -OP(O)(OR 60 )(OR 61 ), -C(O)R 60 , -C(S)R 60 , -C(O)OR 60 , -C(O)NR 60 R 61 , -C(O)O - , -NR 62 C(O)NR60 R 61 In certain embodiments, the substituents include -M, -R 60 , =O, -OR 60 , -SR 60 , -NR 60 R 61 , -CF 3 , -CN, -NO 2 , -S(O) 2 R 60 , -P(O)(OR 60 )(O - ), -OP(O)(OR 60 )(OR 61 ), -C(O)R 60 , -C(O)OR 60 , -C(O)NR 60 R 61 , -C(O)O - In certain embodiments, the substituents include -M, -R 60 , =O, -OR 60 , -SR 60 , -NR 60 R 61 , -CF 3 , -CN, -NO 2 , -S(O) 2 R 60 , -OP(O)(OR 60 )(OR 61 ), -C(O)R 60 , -C(O)OR 60 , -C(O)O - Including R 60 , R 61 and R 62 is as defined above. For example, the substituent may bear a methylenedioxy substituent, or one, two or three substituents selected from a halogen atom, a (1-4C) alkyl group and a (1-4C) alkoxy group. When the substituted group is an aryl or heteroaryl group, the substituent(s), such as those described herein, may be referred to as "aryl substituent(s)".

[0052] In all of the above defined substituents, it is understood that polymers obtained by defining the substituents with further substituents on themselves (e.g., substituted aryl having a substituted aryl group as a substituent, which is itself substituted with a substituted aryl group, which is further substituted with a substituted aryl group, etc.) are not intended to be included herein. In such cases, the maximum number of such substitutions is 3. For example, the tandem substitution of substituted aryl groups specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0053] "Acyl" refers to a group of the formula -C(O)R, where R is alkyl, alkenyl, or alkynyl. For example, an acetyl group has the formula -C(O)CH 3 has.

[0054] "Halo" and "halogen" refer to chloro, bromo, fluoro, and iodo groups.

[0055] "Carboxyl", "carboxy", and "carboxylate" are -CO 2 H group and its salts.

[0056] "Sulfonyl" is -SO 2 R refers to the group R, where R is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, and substituted versions thereof. An exemplary sulfonyl group is -SO 2 CH 3 and -SO 2 (C 6 H 5 ).

[0057] Unless otherwise specified, a reference to an atom is meant to include all isotopes of that atom. For example, a reference to H is 1 H, 2 H (i.e. D) and 3 H (i.e. T) is meant to include C. 12 C and all isotopes of carbon ( 13C, etc. Additionally, any group listed includes all stereoisomers of that group.

[0058] Unless otherwise indicated, the nomenclature of substituents not explicitly defined herein is accomplished by naming the terminal portion of the functional group followed by the adjacent functional group toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0059] With respect to any of the groups disclosed herein that contain one or more substituents, it is of course understood that such groups do not contain any substitutions or substitution patterns that are sterically impractical and / or synthetically impractical. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0060] Detailed Description A purple-excitable tandem dye is provided. The tandem dye of the present invention comprises a non-conjugated polymer backbone, one or more purple-excitable donor fluorophores linked to the non-conjugated polymer backbone, and one or more acceptor fluorophores linked to the non-conjugated polymer backbone, wherein the one or more purple-excitable donor fluorophores and the one or more acceptor fluorophores are in an energy transfer relationship. Methods of making and using the tandem dye, as well as kits containing the tandem dye for use in the method embodiments, are also provided.

[0061] Before describing the exemplary embodiments in more detail, the following definitions are set forth to illustrate and define the meaning and scope of terms used in the description.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Additionally, for clarity and ease of reference, certain terms are defined below. Additionally, although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described herein.

[0063] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any otherwise stated or intervening value in that stated range, is included in the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included in the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0064] A range is presented herein, and the numerical values ​​are preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that the term precedes, as well as a number that is close to or approximately the number that the term precedes. In determining whether a number is close to or approximately equal to a specifically stated number, the near or approximate unstated number may be a number that, in the context in which the specifically stated number is presented, results in a substantial equivalence to the specifically stated number.

[0065] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0066] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a prelude to the use of exclusive terminology such as "sole," "only," and the like, or the use of a "negative" limitation in relation to the recitation of claim elements.

[0067] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein comprises separate components and features which may be readily separated from, or combined with, any of the features of the other multiple embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0068] Although the apparatus and methods have been described or will be described for grammatical fluidity with functional descriptions, the claims should not be construed as necessarily limited in any way by "means" or "step" configuration limitations unless expressly recited under 35 U.S.C. 112, but should be accorded the full scope of meaning and equivalents of the definitions given by the claims under the judicial theory of equivalents, with the express understanding that if a claim is expressly recited under 35 U.S.C. 112, then the full statutory equivalents under 35 U.S.C. 112 should be accorded.

[0069] In further describing various embodiments of the present invention, the purple excitable tandem dyes will be discussed in more detail first, followed by a discussion of methods of using and making the purple excitable tandem dyes, as well as a discussion of kits containing the conjugates.

[0070] Purple-excitable tandem dye As summarized above, the present disclosure provides violet-excitable fluorescent tandem dye conjugates. A violet-excitable tandem dye is a dye that can be excited by violet light, such as that provided by a violet laser. In some examples, the violet-excitable tandem dye is excitable by light having a wavelength in the range of 350-450 nm. The excitation maxima of the violet-excitable tandem dyes of the present invention can vary, and in some embodiments, range from 375-425 nm, such as 390-410 nm, such as 400-410 nm. Thus, the violet-excited tandem dyes of the present invention may be excited by a laser producing light having a wavelength in the range of 375-425 nm, e.g., 390-410 nm, e.g., 400-410 nm, examples of such lasers include, but are not limited to, violet laser diodes (e.g., as described in Telford et al., "Violet laser diode in flow cytometry: an update", Cytometry A, (November 1, 2006) 69(11):1153-60).

[0071] As summarized above, the purple excitable tandem dye of the present invention comprises a non-conjugated polymer backbone, one or more purple excitable donor fluorophores linked to the non-conjugated polymer backbone, and one or more acceptor fluorophores linked to the non-conjugated polymer backbone, wherein the one or more purple excitable donor fluorophores and the one or more acceptor fluorophores are in an energy transfer relationship. Each of these components of the purple excitable dye will now be considered in more detail.

[0072] Purple-excitable donor fluorophores As summarized above, the tandem dyes of the present invention include one or more violet excitable donor fluorophores. The violet excitable donor fluorophore is a fluorophore that absorbs light in the violet wavelength range, e.g., 350-450 nm, e.g., 375-425 nm. In certain cases, the violet excitable donor fluorophore has an absorption maximum in the range of 375-425 nm, e.g., 390-410 nm, e.g., 400-410 nm, e.g., 400 nm, 401 nm, 402 nm, 403 nm, 404 nm, 405 nm, 406 nm, 407 nm, 408 nm, 409 nm, and 410 nm. In certain embodiments, the violet excitable donor fluorophore exhibits a narrow absorption spectrum. A narrow absorption spectrum is one that has an absorption maximum peak with a full width at half maximum (FWHM) of 35 nm or less, e.g., 25 nm or less, 20 nm or less, 15 nm or less, 12 nm or less, or 10 nm or less, as measured, for example, in ethanol or phosphate buffered saline (PBS). The donor violet-excitable fluorophores of embodiments of the present invention can vary. Types of donor violet-excitable fluorophores that can be found in the tandem dyes of the present invention include, but are not limited to, coumarin fluorophores, pyrene fluorophores, triphenyl fluorophores, and the like.

[0073] In some examples, the violet excitable donor fluorophore is a coumarin fluorophore. A coumarin fluorophore is a fluorophore having a coumarin core, which can be described as a benzene molecule with two adjacent hydrogen atoms replaced with a lactone-like chain -=--O-, forming a second six-membered heterocycle sharing two carbons with the benzene ring. The coumarin core can be represented by the following structure:

[0074] [ka]

[0075] Coumarin donor fluorophores of interest include coumarin derivatives. By "coumarin" derivatives is meant molecules having a coumarin core, where one or more of the coumarin core atoms are substituted or conjugated to additional moieties. In some examples, the coumarin fluorophores used as violet excitable donor fluorophores in embodiments of the present invention are described by the following formula:

[0076] [ka]

[0077] R 1 OR 1 ' or NR 2 'R 3 ', where R 1 ', R 2 ' and R 3 ' is independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, water solubilizing group (WSG), or carboxyalkyl, where alkyl is optionally substituted with hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl or a water solubilizing group (WSG), or R 1 ' and R 2 , or R 1 ' and R 3together form a 5-, 6- or 7-membered ring selected from aryl, heterocyclic, heteroaryl, and heteroaromatic; or R 2 ', R 2 , R 3 ' and R 3 together independently form at least one 5-, 6-, or 7-membered ring selected from aryl, heterocyclic, heteroaryl, and heteroaromatic; R 2 , R 3 and R 4 are independently selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, carboxyalkyl, aryl, heterocyclic, heteroaryl, and heteroaromatic, where alkyl is optionally substituted with hydroxyl, amino, carboxyl, carboxylic ester, amide, carbamate, or aminoalkyl, any of which groups can be further substituted, for example, with WSG; R 5 and R 6 are independently selected from hydrogen, halogen, alkyl, alkoxy, keto, carboxylate, carboxylate ester, carbamate, amide, amino, alkylamino, benzimidazolyl, polyether, alkylthio, cyano, nitro, sulfonyl, pyrimidine, purine, any of which groups may be further substituted, for example, with WSG.

[0078] In some examples, the coumarin donor fluorophore of the tandem dyes of the present invention is a coumarin donor fluorophore having one of the following formulas:

[0079] [ka]

[0080] [ka]

[0081] [ka]

[0082] [ka]

[0083] [ka]

[0084] [ka]

[0085] [ka]

[0086] [ka]

[0087] In some examples, the purple excitable donor fluorophore is a pyrene donor fluorophore. Pyrene donor fluorophores are fluorophores that contain polycyclic aromatic hydrocarbons (PAHs) with four fused benzene rings, resulting in a planar aromatic system. The pyrene core may be represented by the following structure:

[0088] [ka]

[0089] Pyrene donor fluorophores of interest include pyrene derivatives. By "pyrene" is meant a molecule having four fused benzene rings of pyrene, one or more of the pyrene carbon atoms being substituted or conjugated to further moieties. In some examples, the pyrene donor fluorophore present in the tandem dye of the present invention is described by the following formula:

[0090] [ka]

[0091] (a)R i may be a chemically reactive functional group such as, but not limited to, an active ester, an active carbonate, an acyl azide, an acid halide, or other reactive group, including, for example, acrylamides, alkyl and aryl azides, alkynes and constrained cyclic alkynes, anhydrides, halides, sulfonate esters, amines, alcohols, carboxylic acids, haloacetamides, isothiocyanates and isocyanates, or conjugates of such reactive groups and attachment moieties, such as R short alkyl chains, or cyclic rings with or without heteroatoms such as nitrogen, oxygen, sulfur, phosphorus, which rings may be substituted or unsubstituted; (b)R 2 is a hydrogen atom, or a short alkyl chain, or a cyclic ring with or without heteroatoms such as nitrogen, oxygen, sulfur, phosphorus, or water solubilizing groups (WSG).

[0092] Examples of pyrene donor fluorophores according to embodiments of the present invention include, but are not limited to:

[0093] [ka]

[0094] In some examples, the pyrene donor fluorophore present in the tandem dyes of the invention is described by the formula:

[0095] [ka]

[0096] (a)R 1may be a chemically reactive functional group such as, but not limited to, an active ester, an active carbonate, an acyl azide, an acid halide, or other reactive group, including, for example, acrylamides, alkyl and aryl azides, alkynes and constrained cyclic alkynes, anhydrides, halides, sulfonate esters, amines, alcohols, carboxylic acids, haloacetamides, isothiocyanates and isocyanates, or conjugates of such reactive groups with additional moieties, such as R short alkyl chains, or cyclic rings with or without heteroatoms such as nitrogen, oxygen, sulfur, phosphorus, which rings may be substituted or unsubstituted; (b)R 2 and R 3 is a hydrogen atom, or a short alkyl chain, or a cyclic ring with or without heteroatoms such as nitrogen, oxygen, sulfur, phosphorus, or water solubilizing groups (WSG).

[0097] In some examples, the pyrene donor fluorophore of the tandem dyes of the invention is a pyrene donor fluorophore having the formula:

[0098] [ka]

[0099] In some examples, the violet excitable donor fluorophore is a triphenyl donor fluorophore. Triphenyl donor fluorophores of interest include donor fluorophores having a triphenyl core, i.e., described by the following formula:

[0100] [ka]

[0101] In some examples, the triphenyl donor fluorophore is a triarylmethane dye of formula (VI).

[0102] [ka]

[0103] In formula (VI), Same or different R 33 , R 34 , R 35 and R 36 represents a hydrogen atom or a group selected from alkyl, optionally substituted aryl and optionally substituted arylalkyl, and a water-solubilizing group; Same or different R 37 , R 38 , R 39 , R 40 , R 41 , R 42 , R 43 and R 44 is a hydrogen atom or alkyl; alkoxy, alkylthio; (di)(alkyl)amino; hydroxyl, mercapto, -nitro, -nitroso; R 0 -C(X)-X'-, R 0 -X'-C(X)- or R 0 represents a group selected from -X'-C(X)-X"-; R 0 represents a hydrogen atom or an alkyl or aryl group or carboxyl, X, X' and X" which may be the same or different represent an oxygen or sulfur atom or an alkyl group or NR, R represents a hydrogen atom or an alkyl group; or two adjacent groups R 42 and R 41 , or R 43 and R 42 , or R 44 and R 43 together form a fused benzo group.

[0104] In the same example, the triphenyl donor fluorophore of the tandem dye of the invention is a pyrene donor fluorophore having the formula:

[0105] [ka]

[0106] If desired, the purple excitable donor fluorophore present in the tandem dyes of the present embodiments may be substituted with one or more water-solubilizing groups (WSG), for example as defined above.

[0107] Acceptor Fluorophore As summarized above, the tandem dyes of the present invention include one or more acceptor fluorophores. In some examples, the tandem dyes include a single acceptor fluorophore. In other examples, the tandem dyes include two or more acceptor fluorophores. Any convenient fluorescent dye may be utilized in the polymeric tandem dyes as the acceptor fluorophore. The acceptor fluorophore (e.g., each A) can be a small molecule fluorophore. The acceptor fluorophore (e.g., each A) can be a dye molecule selected from rhodamine, perylene, diimide, coumarin, xanthene, cyanine, polymethine, pyrene, thiazine, acridine, dipyrrometheneboron difluoride, naphthalimide, phycobiliprotein, peridinium chlorophyll protein, conjugates thereof, and combinations thereof. In certain embodiments, the acceptor fluorophore (A) is a cyanine dye, a xanthene dye, a coumarin dye, a thiazine dye, or an acridine dye. In some examples, the acceptor fluorophore (A) is selected from DY431, DY485XL, DY500XL, DY610, DY640, DY654, DY682, DY700, DY701, DY704, DY730, DY731, DY732, DY734, DY752, DY778, DY782, DY800, DY831, Biotium CF555, Cy3.5, and diethylaminocoumarin. Fluorescent dyes of interest include fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, cascade blue, cascade yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinin chlorophyll-a protein), and PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and 6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br.sub.2, BODIPY530 / 550, BODIPY558 / 568, BODIPY564 / 570, BODIPY576 / 589, BODIPY581 / 591, BODIPY630 / 650, BODIPY650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, conjugates thereof, and combinations thereof. Lanthanide chelates of interest include, but are not limited to, europium chelates, terbium chelates, and samarium chelates. In some embodiments, the polymeric tandem dye comprises multiple chromophores linked to an acceptor fluorophore selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa647, and Alexa700. In certain embodiments, the polymeric tandem dye is a Dyomics dye (such as DY431, DY485XL, DY500XL, DY530, DY610, DY633, DY640, DY651, DY654, DY682, DY700, DY701, DY704, DY730, DY731, DY732, DY734, DY752, DY754, DY778, DY782, DY800, or DY831), Biotium CF555, Cy3.5, and diethylaminocoumarin. In certain cases, the acceptor fluorophore (A) is fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, cascade blue, cascade yellow, coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®, Cy-Chrome, DyLight350, DyLight405, DyLight488, DyLight549, DyLight594, DyLight633, DyLight649, DyLight680, DyLight750, DyLight800, phycoerythrin, PerCP (peridinin chlorophyll-a protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and 6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa The fluorine-containing amine is selected from Fluor® 680, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY® FL, BODIPY® FL-Br2, BODIPY® 530 / 550, BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 576 / 589, BODIPY® 581 / 591, BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, conjugates thereof, and combinations thereof.

[0108] If desired, the acceptor fluorophore present in the tandem dyes of the present embodiments may be substituted with one or more water-solubilizing groups (WSG), for example as defined above.

[0109] Polymer Backbone As summarized above, the tandem fluorescent dyes of the present invention include a polymer backbone. In some examples, the polymer backbone is composed of non-conjugated repeat units having any convenient configuration, such as a linear, branched, or dendrimer configuration. The polymer backbone can be a linear polymer. The polymer backbone can be branched. In some examples, the dye conjugate includes multiple pendant donor chromophore groups, each independently linked to a non-conjugated repeat unit of the polymer backbone. The configuration of the pendant groups can be attached during or after the synthesis of the polymer backbone. Incorporation of the pendant groups can be achieved in a random configuration, a block configuration, or in a sequence-specific manner via stepwise synthesis, depending on the particular method of synthesis utilized.

[0110] The term "unit" refers to a structural subunit of a polymer. The term unit is meant to include monomers, comonomers, coblocks, repeat units, and the like. A "repeating unit" or "repeat unit" is a subunit of a polymer defined by the minimum number of distinct structural features required for the unit to be considered a monomer, such that when the unit is repeated n times, the resulting structure describes a polymer or a block thereof. In some cases, for example, when the polymer is a multiblock polymer, a random arrangement or a defined sequence of units, the polymer may include two or more different repeat units, each block may define a distinct repeat unit. It is understood that various arrangements of repeat units or blocks are possible, and in the written formulas of the polymer backbones described herein, any convenient linear sequence of various lengths can be included within the structure of the overall polymer. It is understood that the polymer may also be represented by a formula in terms of the mole percentage values ​​of each unit in the polymer, and such formulas may represent various arrangements of repeat units, such as random or multiblock polymers or defined sequences of residues. In some cases, the repeat unit of the polymer includes a single monomer group. In certain instances, the repeat unit of the polymer includes more than one monomer group, i.e., comonomer groups (e.g., two, three, four or more comonomer groups). The term "comonomer" or "comonomer group" refers to a structural unit of a polymer that may itself be part of the repeat unit of the polymer.

[0111] The backbone of the tandem dye can have any convenient length. In some cases, the specific number of monomer repeat units or segments of the chromophore can be in the range of 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000, or 2 to 1,000 units or segments, or such as 5 to 100,000, 10 to 100,000, 100 to 100,000, 200 to 100,000, or 500 to 50,000 units or segments. In some examples, the specific number of monomer repeat units or segments of the backbone can be in the range of 2 to 1,000, e.g., 2 to 500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to 100, 9 to 100, or 10 to 100 units or segments. In certain cases, the specific number of monomer repeat units or segments of the backbone can be in the range of 2 to 500, e.g., 2 to 400, 2 to 300, 2 to 200, or 2 to 100 units or segments. In certain cases, the specific number of monomer repeat units or segments of the backbone can be in the range of 2 to 100, e.g., 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, or 2 to 30 units or segments of repeat monomer units.

[0112] The polymer backbone may have a random configuration of non-conjugated repeat units. The polymer backbone may include a block or conblock configuration of non-conjugated repeat units. Alternatively, the polymer backbone may include specific defined sequences of non-conjugated repeat units, e.g., amino acid residues of a polypeptide sequence. These configurations may be characterized by polymer segments of repeat units (e.g., as described herein), which themselves may be repeated throughout the modular scaffold.

[0113] "Non-conjugated" means that at least a portion of the repeat units contain saturated backbone groups (e.g., groups with two or more consecutive single covalent bonds), which excludes π-conjugation or extended delocalized electronic structures along the polymer backbone from one repeat unit to the next. Even if one repeat unit cannot be conjugated to an adjacent repeat unit, it is understood that such a repeat unit can contain one or more isolated unsaturated groups, including unsaturated bonds (e.g., alkenylene or alkynylene groups) and / or aryl or heteroaryl groups, which can be part of the backbone. In some cases, each repeat unit of the polymer backbone contains one side chain that includes a linking pendant group or a chemoselective tag for linking to a pendant group.

[0114] In certain embodiments of the tandem dye, the polymer backbone is a linear polymer. In certain cases, the linear polymer is selected from a peptide, a peptoid, a hydrocarbon polymer, and a PEG polymer. In certain cases, the linear polymer is a peptide. In certain cases, the linear polymer is a peptoid. In certain other cases, the polymer is a hydrocarbon polymer. In certain other cases, the polymer is a PEG polymer. Further details regarding polymer backbones that may be employed in embodiments of the invention can be found in International Application No. PCT / US2019 / 024662, published as WO 2019 / 191482, and International Application No. PCT / US2020 / 019510, published as WO 2020 / 222894, the disclosures of which are incorporated herein by reference.

[0115] In certain instances, the tandem dyes comprise a linear peptide backbone of 2-100 amino acids, e.g., 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, or 2-30 amino acids. In some cases, the linear peptide backbone comprises two or more amino acids, e.g., 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, up to 100 amino acids. In certain cases, the tandem dyes comprise a linear peptide backbone of 5-30 amino acids, e.g., 5-25, 5-20, 5-15, or 5-10 amino acids.

[0116] If desired, the polymer backbone present in the tandem dyes of the present embodiments may be substituted with one or more water-solubilizing groups (WSG), for example as defined above.

[0117] Further aspects of purple excitable tandem dyes The violet-excitable polymeric fluorescent tandem dye of the present invention may vary. In some examples, the polymeric fluorescent tandem dye of the present invention comprises a polymer backbone; a plurality of violet-excitable pendant donor fluorophores, each independently linked to a repeat unit of the polymer backbone; and one or more pendant acceptor fluorophores linked to a repeat unit of the polymer backbone, the violet-excitable pendant donor fluorophores and the pendant acceptor fluorophores being in an energy transfer relationship. Thus, the polymeric tandem dye of the present invention comprises one or more pendant donor fluorophores and one or more pendant acceptor fluorophores configured in energy transfer proximity to the one or more pendant donor fluorophores, e.g., both are linked to a common polymer backbone. In some embodiments, there are multiple pendant donor fluorophores and configured in energy transfer proximity to the pendant acceptor fluorophore(s), and in some examples, the multiple pendant donor fluorophores range from 2 to 20, e.g., 2 to 15, e.g., 2 to 10. The term "pendant group" refers to a side group that is connected to the backbone but is not part of the backbone itself. In embodiments of polymer tandem dyes, the donor fluorophore can transfer energy to the linked acceptor fluorophore. Thus, the subject tandem dyes include a linked acceptor signaling fluorophore in energy-accepting proximity to the donor fluorophore system, i.e., in energy-accepting proximity to at least one linked donor fluorophore. The particular configuration of the pendant group can be determined and controlled by the arrangement of the repeating units of the underlying polymer backbone (also referred to herein as the "modular scaffold" to which the pendant group is attached). The tandem dyes can include multiple water-solubilizing groups attached to the scaffold and / or the pendant groups at any convenient location to provide a water-soluble polymer dye. The polymer backbone, i.e., the modular scaffold, can be composed of repeating units that form a polymer backbone with side groups to which the pendant groups can be attached.The repeat units can be arranged in a variety of configurations to provide tandem dyes with desired spectral properties. The distance and location between the sites for covalent attachment of the pendant donor fluorophore and acceptor fluorophore(s), if present, can be controlled to provide the desired energy transfer process.

[0118] As mentioned above, if desired, the polymer backbone and / or the pendant fluorophores (i.e., donor and acceptor fluorophores) may include one or more water-solubilizing groups (WSGs). In some cases, the WSGs are pendant groups attached directly to the polymer backbone, e.g., as side chains of the polymer backbone. In certain cases, the WSGs are substituents attached to the pendant donor fluorophore or the pendant acceptor fluorophore. In some examples, each of the pendant donor fluorophore groups is substituted with one or more WSGs. As used herein, the terms "water-solubilizing group", "water-soluble group" and WSGs are used interchangeably and refer to a group or substituent that is sufficiently solvated in an aqueous environment, e.g., under physiological conditions, and that confers improved water solubility to the molecule to which it is attached. The WSGs can increase the solubility of a given polymer tandem dye, primarily in aqueous solutions, compared to a control dye lacking the WSG. The water-solubilizing group can be any convenient hydrophilic group that is sufficiently solvated in an aqueous environment. The water-soluble tandem dyes of the present disclosure have solubility under aqueous conditions that make them particularly suitable for application in a variety of biological assays. A variety of water-soluble polymeric groups can be adapted for use with the subject dyes in WSGs. Any convenient water-solubilizing group (WSG) can be included in the dyes described herein to enhance water solubility, for example as described above.

[0119] As summarized above, the polymeric fluorescent tandem dyes of the present invention include a plurality of pendant donor fluorophores and one or more pendant acceptor fluorophores. In some examples, the number of donor fluorophores exceeds the number of acceptor fluorophores. In certain embodiments of the subject tandem dyes, the ratio of donor fluorophores to acceptor fluorophores is selected from 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 20:2. In certain cases, the ratio of donor fluorophores to acceptor fluorophores is 5:1. In certain cases, the ratio of donor fluorophores to acceptor fluorophores is 6:1. In certain cases, the ratio of donor fluorophores to acceptor fluorophores is 7:1. In certain cases, the ratio of donor fluorophores to acceptor fluorophores is 8:1. In certain cases, the ratio of donor fluorophores to acceptor fluorophores is 9:1. In certain cases, the ratio of donor fluorophores to acceptor fluorophores is 10:1.

[0120] As described above, in the polymeric fluorescent tandem dyes of the present invention, the pendant donor fluorophore and the pendant acceptor fluorophore are in an energy transfer relationship. Thus, in embodiments of the present invention, excitation of the donor can result in energy transfer to and emission from the covalently linked acceptor signaling fluorophore. Mechanisms for energy transfer between the donor chromophore and the linked acceptor signaling fluorophore include, for example, resonance energy transfer (e.g., Forster (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), and the like. These energy transfer mechanisms can be relatively short-range, i.e., the proximity of the chromophores of the light-harvesting multichromophore system to each other and / or to the acceptor fluorophore provides efficient energy transfer. Under conditions for efficient energy transfer, amplification of emission from the acceptor fluorophore can occur when the emission from the emitting acceptor fluorophore is stronger when the incident light ("pump light") is at a wavelength that is absorbed by and transferred from the chromophore of the light-harvesting chromophore than when the emitting acceptor fluorophore is directly excited by the pump light. "Efficient" energy transfer means 10% or more, such as 20% or more, or 30% or more, 40% or more, 50% or more, of the energy recovered by the donor chromophore. "Amplification" means that when excited by energy transfer from the donor light-harvesting chromophore system, the signal from the acceptor fluorophore is 1.5 times or more compared to direct excitation of the acceptor fluorophore with incident light of equivalent intensity. The signal can be measured using any convenient method. In some cases, a signal of 1.5 times or more refers to the intensity of the emitted light. In certain cases, a signal of 1.5 times or more refers to an increased signal-to-noise ratio. In certain embodiments of the tandem dye, the acceptor fluorophore emission, upon excitation at the chromophore, is 1.5 times or more, e.g., 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 8 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, or even greater than compared to direct excitation of the acceptor fluorophore with incident light.

[0121] The tandem dyes of the present embodiments can be of any convenient molecular weight (MW). In some cases, the MW of the tandem dye can be expressed as an average molecular weight. In some examples, the tandem dye has an average molecular weight in the range of 500-500,000, such as 1,000-100,000, 2,000-100,000, 10,000-100,000, or even 50,000-100,000 Daltons. In some examples, the polymeric fluorescent dye has a molecular weight in the range of 5-75 kDa, such as 10-50 kDa, such as 15-45 kDa.

[0122] As summarized above, the tandem dye of the present invention is a violet-excitable tandem dye. Thus, a violet-excitable tandem dye is a dye that can be excited by violet light, such as that provided by a violet laser. In some examples, the violet-excitable tandem dye is excitable by light having a wavelength in the range of 350-450 nm. The excitation maximum of the violet-excitable tandem dye of the present invention can vary, and in some embodiments, is in the range of 375-425 nm, such as 390-410 nm, such as 400-410 nm.

[0123] In some examples, the tandem dye exhibits an effective Stokes shift of 100 nm or more, e.g., 110 nm or more, 120 nm or more, 130 nm or more, 140 nm or more, 150 nm or more, 160 nm or more, 170 nm or more, 180 nm or more, 190 nm or more, 200 nm or more, 250 nm or more, when the light-harvesting chromophore is directly excited with incident light. In some cases, the effective Stokes shift of the tandem dye is up to about 300 nm, e.g., 100-300 nm, 100-250 nm, or 100-200 nm.

[0124] The emission of the polymeric tandem dye can have a quantum yield of 0.03 or more, for example, 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.3 or more, or more. In some examples, the polymeric tandem dye has a quantum yield of 5×10 5 cm -1 M -1 Or more, for example, 6×10 5 cm -1 M -1 That's it, 7 x 10 5 cm -1 M -1 That's it, 8 x 10 5 cm -1 M -1 That's it, 9 x 10 5 cm -1 M -1 Or more, for example, 1×10 6 cm -1 M -1 , 1.5×10 6 cm -1 M -1 That's it, 2 x 10 6 cm -1 M -1 That's it, 2.5 x 10 6 cm -1 M -1 That's it, 3 x 10 6 cm -1 M -1 That's it, 4 x 10 6 cm -1 M -1 That's it, 5 x 10 6 cm -1 M -1 That's it, 6 x 10 6 cm -1 M -1 That's it, 7 x 10 6 cm -1 M -1 or more, or 8 x 10 6 cm -1 M -1 In some embodiments, the polymeric tandem dye has an extinction coefficient of 5×10 5 M -1 cm -1 In certain embodiments, the tandem dye has a molar extinction coefficient of 1×106 M -1 cm -1 It has a molar extinction coefficient of or greater.

[0125] In embodiments, the subject tandem dyes provide fluorescent emission from an acceptor fluorophore that is brighter than the emission possible from such a fluorescent dye alone. -1 cm -1 More than 60mM, for example -1 cm -1 Above 70mM -1 cm -1 Above 80mM -1 cm -1 Above 90mM -1 cm -1 More than 100mM -1 cm -1 More than 150mM -1 cm -1 More than 200mM -1 cm -1 more than 250mM -1 cm -1 More than 300mM -1 cm -1 In certain examples, the emission of the tandem dye is at least 5 times greater than the brightness of the directly excited acceptor fluorophore, at least 10 times greater, at least 20 times greater, at least 30 times greater, at least 50 times greater, at least 100 times greater, at least 300 times greater, or even greater than the brightness of the directly excited acceptor fluorophore.

[0126] In addition to the attributes discussed above, the tandem dyes of the present embodiments may have one or more additional desirable spectral properties, such as a particular emission maximum wavelength, extinction coefficient, quantum yield, or the like.

[0127] The different emission profiles depend on various factors such as the selected comonomers, linking groups, substituents and linked acceptor fluorophores of which the tandem dyes are composed, etc. In some embodiments, the tandem dyes have an emission maximum wavelength in the range of 425-900 nm, e.g., 450 nm-900 nm, 475 nm-900 nm, or 500 nm-900 nm.

[0128] In some embodiments, the polymeric tandem dye can include a segment of formula (Ia).

[0129] [ka]

[0130] Each M 1 and M 2 are independently unsaturated comonomers (e.g., amino acid residues); Each S 1 and S 2 are independently optional non-conjugated spacer units, Each D 1 is independent, M 1 a pendant violet excitable donor fluorophore (e.g., as described herein) linked to Each A 1 is independent, M 2 is an acceptor fluorophore linked to x is 75 mol % or more; y is 25 mol % or less.

[0131] The first repeat unit (M 1 -S 1 ) and the second repeating unit (M 2 -S 2 ) may be arranged in a random or coblock configuration. In certain cases of formula (Ia), D of the first repeat unit 1 The pendant group includes two or more (e.g., two or three) distinct types of pendant light-absorbing chromophores, which together provide a light-harvesting multichromophore system. In a particular example of formula (Ia), D of the first repeat unit1 All of the pendant groups are the same.

[0132] In some examples of Formula (Ia), x is 80 mol% or more, e.g., 85 mol% or more, 90 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more, or 99 mol% or more. In some examples of Formula (Ia), y is 20 mol% or less, e.g., 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, or 1 mol% or less.

[0133] In some examples, the polymeric tandem dye comprises a segment of formula (IIa).

[0134] [ka]

[0135] The polymer backbone of each non-conjugated repeat unit is independently a non-conjugated comonomer, SM 1 , S.M. 2 and S.M. 3 Equipped with accessories, Each D 1 is independent, SM 1 a pendant violet excitable donor fluorophore (e.g., as described herein) linked to Each A 1 is independent, SM 2 is an acceptor fluorophore linked to each Z 2 is S.M. 3 is an optional side chain group linked to x is 50 mol % or more; y+z is 50 mol % or less.

[0136] Z 2can be absent or any convenient side chain group, such as a light absorbing chromophore, a chemoselectivity tag, a linker, a linking biomolecule, an acceptor fluorophore, a WSG (e.g., as described in International Application No. PCT / US2019 / 024662, published as WO 2019 / 191482, the disclosure of which is incorporated herein by reference). In the particular case of formula (IIa), SM 3 is Z 2 is an absent spacer comonomer. In a particular example of formula (IIa), SM 3 is the second pendant light-absorbing chromophore, Z 2 Each D 1 And each Z 2 Together they provide a light-harvesting multichromophore system. In some cases, SM 3 is the second chemical selection tag (Z 2 ), e.g., a protected functional group, or Z allowing the selective attachment of a moiety of interest. 1 It is a comonomer containing a tag orthogonal to.

[0137] In certain cases of formula (IIa), x is 60 mol% or more, for example 65 mol% or more, 70 mol% or more, 75 mol% or more, 80 mol% or more, 85 mol% or more, 90 mol% or more, 95 mol% or more, or even more. In certain examples of formula (IIa), y+z is 40 mol% or less, for example 30 mol% or less, 25 mol% or less, 20 mol% or less, 15 mol% or less, 10 mol% or less, 5 mol% or less, or even less. In certain examples of formula (IIa), y is at least 1 mol% and 25 mol% or less, for example 20 mol% or less, 15 mol% or less, 10 mol% or less, 5 mol% or less, or even less. In certain examples of formula (IIa), z is at least 1 mol% and 10 mol% or less, for example 5 mol% or less, or even less.

[0138] In some examples, the polymeric tandem dye comprises a segment of formula (IIIa).

[0139] [ka]

[0140] The polymer backbone of each non-conjugated repeat unit is independently a non-conjugated comonomer, SM 1 and S.M. 2 Equipped with accessories, Each D 1 is independent, SM 1 a pendant violet excitable donor fluorophore (e.g., as described herein) linked to Each A 1 is independent, SM 2 is an acceptor fluorophore linked to x is 75 mol % or more; y is 25 mol % or less.

[0141] In certain embodiments of Formula (IIIa), SM 1 and S.M. 2 are each independently saturated non-conjugated comonomers, e.g., comonomers that provide only a single covalent C-C bond. In some embodiments of formula (IIIa), SM 1 and S.M. 2 are each independently a partially saturated non-conjugated comonomer, e.g., a comonomer that provides an isolated double C=C covalent bond in a backbone of saturated covalent bonds. The first and second repeat units (SM 1 and S.M. 2 ) may be arranged in a random configuration, in a block or coblock configuration, or in a specific sequence. In the specific case of formula (IIIa), SM 1 D 1 The pendant groups include two or more (e.g., two or three) different types of pendant light-absorbing chromophores that together provide a light-harvesting multichromophore system. In a particular example of formula (IIIa), D of the first repeat unit 1 All of the pendant groups are the same.

[0142] In some examples of Formula (IIIa), x is 80 mol% or more, e.g., 85 mol% or more, 90 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more, or 99 mol% or more. In some examples of Formula (IIIa), y is 20 mol% or less, e.g., 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, or 1 mol% or less.

[0143] In a particular example, the polymeric tandem dye is a polymeric tandem dye of formula (IVa):

[0144] [ka]

[0145] Each D 1 are independently a pendant violet excitable donor fluorophore (e.g., as described herein), Each A 1 are independently acceptor fluorophores, Each L 1 and L 2 is a separate linker, p 1 and q 1 is independently 0 or 1, p 1 +q 1 ≦1, p 2 and q 2 is independently 0 or 1, p 1 +q 1 ≦1, x is 75 mol % or more; y is 25 mol % or less, G 1 and G 2 are each independently selected from an end group, a polymer segment, a light absorbing (eg, donor) chromophore group, an acceptor fluorophore, a linker, and a linker to a specific binding member, eg, as described above.

[0146] In some embodiments of formula (IVa), p 1 and p2 are 0, and q 1 and q 2 is 1 (e.g., a β3-amino acid residue). In some embodiments of formula (IVa), p 1 and p 2 are 1, and q 1 and q 2 are each 0 (e.g., β2-amino acid residues). In some cases, p 1 , p 2 , q 1 and q 2 are each 0, and the polymer tandem dye has the following formula (Va):

[0147] [ka]

[0148] Each D 1 are independently a pendant violet excitable donor fluorophore (e.g., as described herein), Each A 1 are independently acceptor fluorophores, L 1 and L 2 are each independently linkers, x is 75 mol % or more; y is 25 mol % or less, G 1 and G 2 are each independently selected from an end group, a polymer segment, a light absorbing (e.g., donor) chromophore group, a linker, and a linker to a specific binding member, such as those described above. It is understood that the tandem dyes described by formula (Va) include any convenient arrangement of the comonomers in a defined linear sequence, which collectively has a defined mole percent ratio of x and y. In some cases, the A containing comonomers 1 are spaced throughout the sequence of the polymer backbone, thus forming one or more D 1 They are always adjacent to each other on both sides.

[0149] In a particular example of formula (Va), the polymeric tandem dye comprises a segment of formula (VIa).

[0150] [ka]

[0151] Each D 1 are independently a pendant violet excitable donor fluorophore (e.g., as described herein), Each A 1 are independently acceptor chromophores, Each L 1 and L 3 is a separate linker, n and p each independently represent an integer of 1 to 20, and n+p≧2; m is 1 or 2.

[0152] In some cases of Formula (VIa), n and p are each independently 1 to 10, such as 2 to 20, 3 to 10, or 3 to 6. In some examples of Formula (VIa), n+p is an integer from 2 to 20, such as 3 to 20, 4 to 20, 5 to 20, 5 to 15, or 5 to 12. In certain embodiments of Formula (VIa), m is 1.

[0153] The subject polymeric tandem dyes can include multiple segments of formula (VIa), each segment containing a comonomer D 1 One isolated A containing comonomers flanked by blocks of 1 In some cases, the multichromophore comprises two or more segments of formula (VIa) arranged next to each other, and has 2 to 20 D containing comonomers. 1 D blocks of 3 to 20, 4 to 20, 5 to 20, 5 to 15 or 5 to 12, e.g. 1 Two isolated A-containing comonomers separated by a block of 1 Thus, in certain embodiments, the polymeric tandem dye comprises q segments of a block copolymer and is a polymeric tandem dye of formula (VIIa):

[0154] [ka]

[0155] each(n) q and each (p) q is independently an integer from 1 to 20, and for each of the q segments, (n) q +(p) q ≧3, and q is an integer from 1 to 100.

[0156] In certain embodiments, the polymeric tandem dye has the formula (VIIIa):

[0157] [ka]

[0158] Each D 1 are independently a pendant violet excitable donor fluorophore (e.g., as described herein), Each A 1 are independently acceptor chromophores, Each L 1 and L 2 is a separate linker, x is 75 mol % or more; y is 25 mol % or less, G 1 and G 2 are each independently selected from an end group, a polymer segment, a donor chromophore group, a linker, and a linker to a specific binding member, such as those described above.

[0159] In certain embodiments, the polymeric tandem dye has the formula (IXa):

[0160] [ka]

[0161] Each D 1are independently a pendant violet excitable donor fluorophore (e.g., as described herein), Each A 1 are independently acceptor fluorophores, Each L 1 and L 2 is a separate linker, x is 75 mol % or more; y is 25 mol % or less, G 1 and G 2 are each independently selected from the group consisting of an end group, a polymer segment, a donor chromophore group, an acceptor fluorophore, a linker, and a linker to a specific binding member, such as those described above.

[0162] In some examples of Formula (IVa), Formula (Va), Formula (XIIa), and Formula (IXa), x is 80 mol% or more, e.g., 85 mol% or more, 90 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more, or 99 mol% or more. In some examples of Formula (IV), Formula (V), Formula (XIII), and Formula (IX), y is 20 mol% or less, e.g., 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, or 1 mol% or less.

[0163] In certain embodiments, the polymeric tandem dye has the formula (Xa):

[0164] [ka]

[0165] Each D 1 are independently a pendant violet excitable donor fluorophore (e.g., as described herein), Each A 1 are independently acceptor fluorophores, Each L 1 , L 2 and L 3 is a separate linker, a, b and c are mole percent values ​​for each comonomer; d represents the total length or average length of the polymer (e.g., d is 2 to 1000, e.g., 2 to 500, 2 to 200, 2 to 100, or 2 to 50); WSG is a water solubilizing group (e.g., as described in International Application No. PCT / US2019 / 024662, published as WO 2019 / 191482, the disclosure of which is incorporated herein by reference); G 1 and G 2 are each independently selected from the group consisting of an end group, a polymer segment, a donor chromophore group, an acceptor fluorophore, a linker, and a linker to a specific binding member as described above.

[0166] In some examples of formula (Xa), c=0. In some examples of formula (Xa), a>0 and b>0. In some examples of formula (Xa), a is 80 mol% or more, for example, 85 mol% or more, 90 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more, or 99 mol% or more. In some examples of formula (Xa), b is 20 mol% or less, for example, 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, or 1 mol% or less. In some examples of formula (Xa), a is 65-95 mol%, b is 5-35 mol%, c is 0-30 mol%, and a+b+c=100%.

[0167] Any convenient end group (e.g., G 1 and G 2 ) may be utilized at the terminus of a tandem fluorescent dye. As used herein, the terms "end group" and "terminal group" are used interchangeably to refer to a group located at the end of the polymeric structure of a tandem dye, for example as described herein. 1 Group and G 2 Groups include, but are not limited to, H, chemoselective groups, linkers (which may or may not be activated), and linkers that couple to specific binding members, e.g., as described above. 1 Position and / or G2 It is understood that the linker at position G may be selected to be orthogonal to any other linker, including a chemoselectivity tag (e.g., as described herein), that may be present in the side chain of the tandem dye. In certain embodiments, the amino functionality or a derivative thereof is 1 and / or G 2 In certain embodiments, the carboxylic acid functionality or a derivative thereof is included in G 1 and / or G 2 is included.

[0168] In some embodiments of the formulas described herein, G 1 and G 2 At least one of the -L 6 -Z 4 And L 6 is a linker (e.g., as described herein), and Z 4 is a specific binding member (e.g., as described herein). In some embodiments of the formulas described herein, G 1 and G 2 At least one of the -L 6 -Z 3 And L 6 is a linker (e.g., as described herein), and Z 3 is a chemoselectivity tag (e.g., as described herein). Any convenient chemoselectivity tag and conjugation chemistry can be adapted for use in the subject light-harvesting chromophores. Chemoselectivity tags of interest include, but are not limited to, amines, active esters, maleimides, thiols, sulfur (VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, Diels-Alder cycloaddition click reagents and click chemistry, tetrazines, transcyclooctene, aldehydes, alkoxylamines, alkynes, cyclooctynes, azides, and the like. In some examples, Z 3 is selected from the group consisting of carboxylic acid, active ester (e.g., N-hydroxysuccinimidyl ester (NHS) or sulfo-NHS), amino, maleimide, iodoacetyl, and thiol. In certain embodiments of the formulas described herein, G1 and G 2 At least one of the following is described by the following structure: *-Ar-LZ Ar is a π-conjugated aryl group, L is a linker, and Z is a chemoselective tag or specific binding member. In some cases, the LZ group can be attached towards the terminal comonomer. In certain embodiments of the formulas described herein, G 1 and G 2 At least one of the following is described by the following structure:

[0169] [ka]

[0170] q is 0 or an integer from 1 to 12, L is an optional linker, Z is a chemoselective tag or specific binding member. Further details regarding WSG groups that may be found in the tandem dyes of the present embodiments can be found in International Application No. PCT / US2019 / 024662, published as WO 2019 / 191482, the disclosure of which is incorporated herein by reference.

[0171] An exemplary violet excitable polymeric fluorescent tandem dye according to an embodiment of the present invention has the following structure:

[0172] [ka]

[0173] As can be seen, the purple excitable polymeric tandem dye described above comprises 16 purple excitable donor coumarin fluorophores (8 donors on each side) adjacent to a single acceptor fluorophore, and the purple excitable polymeric tandem dye is conjugated to an antibody for specific binding to a target analyte of interest. The purple excitable coumarin donor fluorophores are substituted with PEG water-solubilizing groups, and the peptide backbone comprises a water-solubilizing PEG spacer between the peptide and the end-linked antibody.

[0174] method Aspects of the invention include methods of evaluating a sample for the presence of a target analyte. In aspects of the method, a sample is contacted with a labeled specific binding member that specifically binds the target analyte to generate an assay composition comprising a labeled specific binding member-contacted sample. The labeled specific binding member employed in embodiments of the method of the invention includes a specific binding member conjugated to a purple excitable polymeric fluorescent tandem dye, for example as described above. In the following sections, the target analyte may be a target molecule of interest or a reagent, for example a primary antibody bound to a target molecule, depending on whether the labeled specific binding member is employed as a primary label or a secondary label. Any convenient method may be used to contact the sample with the labeled specific binding member that specifically binds the target analyte to generate the assay composition. In some examples, the sample is contacted with the labeled specific binding member under conditions that allow the labeled specific binding member to specifically bind to the target analyte, if present. A suitable medium that maintains the biological activity of the sample and the components of the single domain antibody may be used to allow the labeled specific binding member to specifically bind to the target analyte. The medium may be a balanced salt solution, such as normal saline, PBS, Hank's balanced salt solution, etc., conveniently supplemented with fetal bovine serum, human platelet lysate or other factors, along with an acceptable buffer at a low concentration, such as 5-25 mM. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. In some cases, a variety of media, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., supplemented with fetal bovine serum or human platelet lysate, are commercially available and may be used according to the nature of the target analyte. The final components of the medium, which may be a solution, may be selected depending on the components of the sample involved. The temperature at which specific binding of the labeled specific binding member to the target analyte occurs may vary, and in some instances may be in the range of 5° C. to 50° C., such as 10° C. to 40° C., 15° C. to 40° C., 20° C. to 40° C., such as 20° C., 25° C., 30° C., 35° C. or 37° C. (e.g., as described above). In some instances, the temperature at which specific binding occurs is selected to be compatible with the biological activity of the specific binding member and / or the target analyte, hi particular instances, the temperature is 25°C, 30°C, 35°C or 37°C.In certain cases, the temperature at which specific binding occurs is room temperature (e.g., 25° C.), 30° C., 35° C., or 37° C. Any convenient incubation time for specific binding can be selected to allow for the formation of a desired amount of binding complex, and in some instances can be 1 minute (min) or more, such as 2 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, or even 6 hours or more.

[0175] Any convenient specific binding member may be utilized for the labeled specific binding member employed in the methods of the present invention. Specific binding members of interest include, but are not limited to, those specific binding members that specifically bind to cell surface proteins of various cell types, including, but not limited to, stem cells, such as pluripotent stem cells, hematopoietic stem cells, T cells, T regulatory cells, dendritic cells, B cells, such as memory B cells, antigen-specific B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest include cells that have a convenient cell surface marker or antigen that can be captured by a convenient specific binding member conjugate. In some embodiments, the target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood. Any convenient cell surface protein or cell marker may be targeted for specific binding to the conjugates employed in the subject methods, hi some embodiments, the target cell comprises a cell surface marker selected from a cell receptor and a cell surface antigen. In some cases, the target cell may comprise cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71.

[0176] Any convenient target may be selected for evaluation using the subject method. Targets of interest include, but are not limited to, nucleic acids such as RNA, DNA, PNA, CNA, HNA, LNA or ANA molecules, fusion proteins, modified proteins, such as proteins that are phosphorylated, glycosylated, ubiquitinated, sumoylated or acetylated, or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins and drug molecules. As used herein, the term "target protein" refers to all members of a target family, and fragments thereof. Target proteins can be any protein of interest, such as therapeutic or diagnostic targets, including, but not limited to, hormones, growth factors, transcription factors, receptors, enzymes, cytokines, osteoinductive factors, colony stimulating factors and immunoglobulins. The term "target protein" is intended to include recombinant and synthetic molecules that can be prepared using any convenient recombinant expression method or using any convenient synthetic method, or purchased commercially. In some embodiments, the polymer-dye conjugate comprises an antibody or an antibody fragment. Any convenient target analyte that specifically binds to the antibody or antibody fragment of interest can be targeted in the subject methods.

[0177] In some embodiments, the target analyte is associated with a cell. In certain instances, the target analyte is a cell surface marker of the cell. In certain cases, the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen. In some instances, the target analyte is an intracellular target, and the method further comprises treating the cell to allow the labeled specific binding member to access the intracellular target, for example, by permeabilizing or lysing the cell. Thus, the labeled specific binding member employed in the method of the invention may target a cell surface or intracellular antigen. Alternatively, the labeled specific binding member employed in the method of the invention may target a primary antibody that then specifically binds to the target cell surface or intracellular antigen.

[0178] In some embodiments, the sample may comprise a heterogeneous cell population from which target cells are isolated. In some examples, the sample comprises peripheral whole blood, peripheral whole blood from which red blood cells have been lysed prior to cell isolation, umbilical cord blood, bone marrow, density gradient purified peripheral blood mononuclear cells, or homogenized tissue. In some cases, the sample comprises hematopoietic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow, or umbilical cord blood. In certain embodiments, the sample comprises tumor cells in peripheral blood. In certain examples, the sample is a sample that contains (or is suspected of containing) viral cells (e.g., HIV).

[0179] Labeled specific binding members are used in the subject methods, for example, to label target cells, particles, targets, or analytes with polymer tandem fluorescent dyes. For example, labeled specific binding members are used to label cells that are processed (e.g., detected, analyzed, and / or sorted) in a flow cytometer. Labeled specific binding members can include specific binding members, for example, antibodies or binding fragments thereof, that specifically bind to cell surface proteins of various cell types (e.g., as described herein). Labeled specific binding members can be used to investigate various biological (e.g., cellular) properties or processes, such as cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression and / or presentation, etc. Labeled specific binding members can be used in any application that includes (or may include) antibody-mediated labeling of cells, particles, or analytes.

[0180] In aspects of the method, the assay composition, i.e., the labeled specific binding member contacted sample, is assayed for the presence of a labeled specific binding member-target analyte binding complex to assess whether the target analyte is present in the sample. Once the sample is contacted with the labeled specific binding member, any convenient method may be utilized in assaying the resulting assay composition for the presence of a labeled specific binding member-target analyte binding complex. The labeled specific binding member-target analyte binding complex is a binding complex generated upon specific binding of the labeled specific binding member to the target analyte, if present (or a primary binding member, e.g., a primary antibody to the target antigen, depending on the embodiment). When assaying the assay composition, a fluorescent signal may be detected from the binding complex, if present. In some cases, the assay includes a separation step in which the target analyte, if present, is separated from the sample. A variety of methods may be utilized to separate the target analyte from the sample, for example, by immobilization on a carrier. Assay methods of interest include, but are not limited to, any convenient methods and assay formats of interest, in which a specific binding member pair, such as avidin-biotin or hapten-antihapten antibody, is used. Methods and assay formats of interest that can be adapted for use with the subject compositions include, but are not limited to, flow cytometry, in-situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography.

[0181] In certain embodiments, the method further comprises contacting the sample with a second specific binding member that specifically binds to the target analyte. In certain examples, the second specific binding member is carrier-bound. Any convenient carrier may be utilized to immobilize the components of the subject method (e.g., the second specific binding member). In certain examples, the carrier is a particle, such as a magnetic particle. In some examples, the second specific binding member and the polymer-dye conjugate form a sandwich complex that can be isolated and detected, if present, using any convenient method. In some embodiments, the method further comprises flow cytometrically analyzing the polymer-dye conjugate-target analyte binding complex, i.e., the fluorescently labeled target analyte. Assaying for the presence of the labeled specific binding member-target analyte binding complex may provide an assay result (e.g., qualitative or quantitative assay data) that can be used to assess whether the target analyte is present in the sample.

[0182] Any convenient carrier may be utilized in the subject method to immobilize any convenient component of the method, such as labeled specific binding members, targets, secondary specific binding members, etc. Carriers of interest include, but are not limited to, solid substrates, which may have a variety of configurations, such as, for example, sheets, beads, or other structures, such as wells, beads, polymers, particles, fibrous meshes, hydrogels, porous matrices, pins, microarray surfaces, chromatography supports, etc. In some examples, the carrier is selected from the group consisting of particles, planar solid substrates, fibrous meshes, hydrogels, porous matrices, pins, microarray surfaces, and chromatography supports. The carrier may be incorporated into a system that provides cell isolation assisted by any convenient method, such as a manually operated syringe, a centrifuge, or an automated liquid handling system. In some cases, the carrier is used in an automated liquid handling system for high throughput isolation of cells, such as a flow cytometer.

[0183] In some embodiments of the method, the separation step includes applying an external magnetic field to immobilize the magnetic particles. Any convenient magnet can be used as the source of the external magnetic field (e.g., magnetic field gradient). In some cases, the external magnetic field is generated by a magnetic source, for example by a permanent magnet or an electromagnet. In some cases, immobilization of the magnetic particles means that the magnetic particles accumulate near the surface closest to the magnetic field gradient source, i.e., the magnet.

[0184] The separation step may further include one or more optional washing steps to remove unbound material of the sample from the carrier. Any convenient washing method may be used, such as washing the immobilized carrier with a biocompatible buffer that maintains the specific binding interaction between the polymer dye and the specific binding member. Separation and optional washing of the unbound material of the sample from the carrier, from which undesired cells and materials may be removed, results in an enriched population of target cells.

[0185] In certain embodiments, the method detects the labeled target analyte. In detecting the labeled target analyte, the polymeric fluorescent tandem dye may be excited with one or more lasers, and then the fluorescent emission from the polymeric fluorescent tandem dye may be detected using one or more optical detectors. The detection of the labeled target may be performed using any convenient instrument and method, including but not limited to flow cytometry, FACS system, fluorescence microscopy; fluorescence, luminescence, ultraviolet, and / or visible light detection using a plate reader; high performance liquid chromatography (HPLC); and mass spectrometry. When using fluorescent labeling components in the methods and compositions of the present disclosure, it is recognized that the subject method may be performed using different types of fluorescence detection systems. In some cases, high throughput screening may be performed, for example, a system using 96 well or more microtiter plates. Various methods of performing assays on fluorescent materials are available, such as those described in Lakowicz, JR, Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, DL & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, NJ, Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Col, Inc. (1978), pp. 296-361.

[0186] Fluorescence in a sample can be measured using a fluorometer. In some cases, excitation radiation from an excitation source having a first wavelength passes through excitation optics. The excitation optics causes the excitation radiation to excite the sample. In response, fluorescently labeled targets in the sample emit radiation having a wavelength different from the excitation wavelength. The collection optics then collects the emission from the sample. The device can include a temperature controller to maintain the sample at a particular temperature while the sample is being scanned. In a particular example, a multi-axis translation stage moves a microtiter plate holding multiple samples to expose and position different wells. The multi-axis translation stage, temperature controller, autofocus function, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer. The computer can also convert data collected during the assay into another format for presentation.

[0187] In some embodiments, the method of evaluating a sample for the presence of a target analyte further comprises detecting the fluorescence with a flow cytometer. In some embodiments, the method of evaluating a sample for the presence of a target analyte further comprises imaging the sample contacted with the labeling composition using fluorescence microscopy. Fluorescence microscopy imaging can be used to identify polymer-dye conjugate-target analyte binding complexes in the contacted sample and assess whether the target analyte is present. Microscopy of interest for use in the subject methods includes laser scanning confocal microscopy.

[0188] Also provided is a method of making a violet-excitable polymeric fluorescent tandem dye, e.g., as described herein. In some embodiments, the method contacts a specific binding member with a violet-excitable polymeric fluorescent tandem dye (e.g., as described herein) to produce a specific binding member / polymeric fluorescent tandem dye conjugate, where the polymeric fluorescent tandem dye comprises a conjugation tag that covalently links the dye to the specific binding member. The term "conjugation tag" refers to a group that comprises a chemoselective functional group (e.g., as described herein) that can be covalently linked to a compatible functional group of a specific binding member after optional activation and / or deprotection. Any convenient conjugation tag may be utilized in the subject polymeric dyes to conjugate the polymeric fluorescent tandem dye to a specific binding member, e.g., an antibody or fragment thereof of interest. In some embodiments, the conjugation tag comprises a terminal functional group selected from amino, carboxylic acid or derivatives thereof, thiol, hydroxyl, hydrazine, hydrazide, azide, alkyne, and protein-reactive groups (e.g., amino-reactive, thiol-reactive, hydroxyl-reactive, imidazolyl-reactive, or guanidinyl-reactive). Any convenient methods and reagents can be adapted for use in the subject methods to covalently attach the conjugation tag to a specific binding member. Methods of interest for labeling the target include, but are not limited to, those methods and reagents described in Hermanson, Bioconjugate Techniques, Third edition, Academic Press, 2013. The contacting step may be carried out in an aqueous solution. In some examples, the conjugation tag comprises an amino functional group and the target molecule comprises an active ester functional group, such as an NHS ester or a sulfo-NHS ester, or vice versa. In certain examples, the conjugation tag comprises a maleimide functional group and the target molecule comprises a thiol functional group, or vice versa. In certain instances, the conjugated tag comprises an alkyne (e.g., a cyclooctyne group) functional group and the target molecule comprises an azide functional group, or vice versa, and can be conjugated via click chemistry. In some cases, the method includes a separation step in which the product labeled specific binding member is separated from the reaction mixture, e.g., excess reagents or unlabeled specific binding member.To separate the target from the sample, various methods may be used, for example by immobilization on a support, precipitation, chromatography, etc.

[0189] In some examples, the method further comprises detecting and / or analyzing the product-labeled specific binding member. In some examples, the method further comprises fluorescently detecting the labeled specific binding member. Any convenient method may be used to detect and / or analyze the specific binding member in conjunction with the subject methods and compositions. Methods for analyzing targets of interest used in the subject methods include, but are not limited to, flow cytometry, fluorescent microscopy, in-situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assay, and fluorescent dye purification chromatography. Methods for detecting targets of interest include, but are not limited to, fluorescent spectroscopy, fluorescent microscopy, nucleic acid sequencing, fluorescent in-situ hybridization (FISH), protein mass spectrometry, flow cytometry, and the like.

[0190] Detection can be achieved directly via the polymer tandem dye or indirectly by a secondary detection system. The secondary detection system can be based on any one or combination of several different principles, including but not limited to antibody-labeled anti-species antibodies and other forms of immunological or non-immunological bridging and signal amplification systems (e.g., biotin-streptavidin technology, protein-A and protein-G mediated technology, or nucleic acid probes / anti-nucleic acid probes, etc.). Suitable reporter molecules can be reporter molecules known in the fields of immunocytochemistry, molecular biology, light, fluorescence, and electron microscopy, cell immunophenotyping, cell sorting, flow cytometry, cell visualization, detection, counting, and / or signal output quantification. Two or more antibodies of specific and / or non-specific nature may be labeled and used simultaneously or sequentially to enhance target detection, identification, and / or analysis.

[0191] system Aspects of the invention further include systems for use in carrying out the subject methods and compositions. A sample analysis system can include a sample field or flow channel filled with a sample and a labeled specific binding member of the invention as described above. In some embodiments, the system is a flow cytometry system including a flow cytometer including a flow path and a composition in the flow path, the composition comprising a sample and a labeled specific binding member (e.g., as described herein). In some embodiments, a system for analyzing a sample is a fluorescent microscope system, including a fluorescent microscope with a sample field and a composition disposed in the sample field, the composition comprising a sample and a labeled specific binding member (e.g., as described herein).

[0192] In certain embodiments of the system, the composition further comprises a second specific binding member that is carrier-bound and specifically binds to the target analyte. In some cases, the carrier comprises magnetic particles. Thus, in certain examples, the system may also include a controllable external paramagnetic field configured to be applied to the assay region of the flow channel.

[0193] The sample may include cells. In some examples, the sample is a cell-containing biological sample. In some examples, the sample includes a labeled specific binding member that specifically binds to a target cell. In certain examples, the target analyte that is specifically bound by the specific binding member is a cell surface marker of the cell. In certain cases, the cell surface marker is selected from a cell receptor and a cell surface antigen.

[0194] In certain aspects, the system may further include a light source configured to direct light to the assay region or sample field of the flow channel. The system may include a detector configured to receive a signal from the assay region or sample field of the flow channel, the signal being provided by the fluorescent composition. Optionally, the sample analysis system may further include one or more additional detectors and / or light sources for detection of one or more additional signals.

[0195] In certain embodiments, the system may further include a computer-based system configured to detect the presence of a fluorescent signal. "Computer-based system" refers to the hardware means, software means, and data storage means used to analyze the information of the present invention. The minimum hardware of the computer-based system of the present invention includes a central processing unit (CPU), input means, output means, and data storage means. Those skilled in the art can easily understand that any one of the currently available computer-based systems is suitable for use in the subject system. The data storage means may include any product that contains a record of the present information as described above, or a memory access means that can access such a product.

[0196] "Recording" data, programming, or other information on a computer readable medium refers to a process for storing information using any method as known in the art. Any convenient data storage structure may be selected based on the means used to access the stored information. A variety of data processor programs and formats may be used for storage, for example, word processing text files, database formats, and the like.

[0197] A "processor" refers to any combination of hardware and / or software that performs the functions required of a processor. For example, any processor herein may be a programmable digital microprocessor, available, for example, in the form of an electronic controller, a mainframe, a server, or a personal computer (desktop or portable). If the processor is programmable, the appropriate programming may be transmitted to the processor from a remote location or may be stored in advance in a computer program product (such as a portable or fixed computer-readable storage medium, whether magnetic, optical, or solid-state device-based). For example, a magnetic medium or optical disk may carry the programming and may be read by a suitable reader that communicates with each processor at its corresponding station.

[0198] In addition to sensor devices and signal processing modules, such as those described above, the systems of the present invention may include a number of additional components, such as data output devices, e.g., monitors and / or speakers, data input devices, e.g., interface ports, keyboards, etc., fluid handling components, power sources, etc.

[0199] In some embodiments, the system comprises a flow cytometer. Suitable flow cytometry systems include, but are not limited to, those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo et al. (2012) Ann Clin Biochem. Jan; 49(pt1): 17-28; Linden et al., Semin Thromb Hemost. 2004 Oct; 30(5): 502-11; Alison et al. J Pathol, 2010 Dec; 222(4): 335-344; and Herbig et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255, the disclosures of which are incorporated herein by reference.In particular examples, flow cytometry systems of interest include BD Biosciences FACSCanto™ flow cytometers, BD Biosciences FACSCanto™ II flow cytometers, BD Accuri™ flow cytometers, BD Accuri™ C6 Plus flow cytometers, BD Biosciences FACSCelesta™ flow cytometers, BD Biosciences FACSLyric™ flow cytometers, BD Biosciences FACSVerse™ flow cytometers, BD Biosciences FACSymphony™ flow cytometers, BD Biosciences LSRFortessa™ flow cytometers, BD Biosciences LSRFortessa™ X-20 flow cytometers, BD Biosciences FACSPresto™ flow cytometers, BD Biosciences FACSVia™ flow cytometers, and BD Biosciences FACSCalibur™ cell sorters, BD Biosciences FACSCount™ cell sorters, BD Biosciences FACSLyric™ cell sorters, BD Cell sorters and cell sorters used in cell sorting include the BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ Fusion cell sorter, and BD Biosciences FACSMelody™ cell sorter, BD Biosciences FACSymphony™ S6 cell sorter, and the like.

[0200] In some embodiments, the subject systems are disclosed in U.S. Pat. Nos. 10,663,476; 10,620,111; 10,613,017; 10,605,713; 10,585,031; 10,578,542; 10,578,469; 10,481,074; 10,302,545; US Patent No. 10,145,793; US Patent No. 10,113,967; US Patent No. 10,006,852; US Patent No. 9,952,076; US Patent No. 9,933,341; US ​​Patent No. 9,726,527; US Patent No. 9,453,789; US Patent No. 9,200,334; US Patent No. 9,097,640; US Patent No. 9,095,494; US Patent No. 9,092 ,034; U.S. Patent No. 8,975,595; U.S. Patent No. 8,753,573; U.S. Patent No. 8,233,146; U.S. Patent No. 8,140,300; U.S. Patent No. 7,544,326; U.S. Patent No. 7,201,875; U.S. Patent No. 7,129,505; U.S. Patent No. 6,821,740; U.S. Patent No. 6,813,017; U.S. Patent No. 6,809,804; U.S. Patent No. Nos. 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; 4,987,086; and 4,498,766, the entire disclosures of which are incorporated herein by reference.

[0201] In certain examples, the flow cytometry system of the present invention may be any of the methods described in Diebold et al., Nature Photonics Vol. 7(10); 806-810 (2013) and U.S. Pat. Nos. 9,423,353; 9,784,661; 9,983,132; 10,006,852; 10,078,045; 10,036,699; 10,222,316; 10,288,546; 10,324,019; 10,408,758; 10,451,538; U.S. Pat. Nos. 5,811,521, 5,811,521, 5,811,521, and 5,911,521, the disclosures of which are incorporated herein by reference. No. 10,620,111; and U.S. Patent Application Publication No. 2017 / 0133857; U.S. Patent Application Publication No. 2017 / 0328826; U.S. Patent Application Publication No. 2017 / 0350803; U.S. Patent Application Publication No. 2018 / 0275042; U.S. Patent Application Publication No. 2019 / 0376895; and U.S. Patent Application Publication No. 2019 / 0376894, configured to image particles in a flow stream by Fluorescence Imaging using Radio Frequency Tag Emission (FIRE).

[0202] Other systems may be used in practicing the subject methods. In certain aspects, the system may be a fluorometer or microscope loaded with a sample having the fluorescent composition of any of the embodiments described herein. The fluorometer or microscope may include a light source configured to direct light to an assay region or sample field of view of the flow channel. The fluorometer or microscope may also include a detector configured to receive a signal from the assay region or field of view of the flow channel, the signal being provided by the fluorescent composition.

[0203] kit Aspects of the invention further include kits for use in carrying out the subject methods and compositions. The compositions of the invention can be included as reagents in the kit as starting materials or can be included in the kit as reagents for use in the methods described above, for example. The kits can include, for example, labeled specific binding members and containers as described above. Any convenient container can be utilized, such as tubes, bottles, or wells in multi-well strips or plates, boxes, bags, insulated containers, etc. The subject kits can further include one or more components selected from primer specific binding members for a given target analyte, carrier-bound specific binding members, cells, carriers, biocompatible aqueous elution buffers, controls (positive and / or negative), etc., and instructions for use, as appropriate. A given kit can include reagents suitable for detection of a single target analyte, or multiple reagents suitable for detection of two or more different target analytes, for example when a given kit is configured for multiplex detection applications.

[0204] In certain embodiments, the kit is used in evaluating a sample for the presence of a target analyte, such as an intracellular target. Thus, in some examples, the kit includes one or more components suitable for permeabilizing or lysing cells. One or more additional components of the kit may be provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0205] In some embodiments, the kit further comprises a reagent for performing a flow cytometry assay. Reagents of interest include, but are not limited to, buffers for reconstitution and dilution, buffers for contacting chromophores with cell samples, wash buffers, control cells, control beads, fluorescent beads for flow cytometer calibration, and combinations thereof. The kit may further comprise one or more cell fixation reagents, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination or buffer thereof. Additionally, the kit may comprise a cell permeabilization reagent, such as methanol, acetone, or detergent (e.g., triton, NP-40, saponin, tween20, digitonin, leucoperm, or any combination or buffer thereof). Other protein transport inhibitors, cell fixation reagents, and cell permeabilization reagents familiar to those skilled in the art are within the scope of the subject kit.

[0206] The compositions of the kit may be provided in a liquid composition, such as any suitable buffer. Alternatively, the compositions of the kit may be provided in a dry composition (e.g., lyophilized), and the kit may optionally include one or more buffers for reconstituting the dry composition. In certain aspects, the kit may include aliquots of the compositions provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0207] In addition, one or more components may be combined in a single container, such as a glass or plastic vial, tube, or bottle. In certain instances, the kit may further include a container (e.g., a box, bag, insulated container, bottle, tube, etc.) in which all of the components (and their separate containers) reside. The kit may further include packaging, separate from or attached to the kit container, printed with information about the kit, the components of the kit, and / or instructions for using the kit.

[0208] In addition to the above components, the subject kits may further include instructions for carrying out the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as information printed on a suitable medium or substrate, such as a sheet or sheets of paper on which the information is printed, the kit packaging, a package insert, and the like. Yet another means may be a computer readable medium, such as a diskette, CD, DVD, portable flash drive, and the like, on which the information is stored. Yet another means may be present is a website address that may be used via the Internet to access the information at the destination site. Any convenient means may be present in the kit.

[0209] usefulness The purple excitable tandem dyes, and labeled specific binding members, compositions, methods, and systems comprising purple excitable tandem dyes, as described herein, may be used in a variety of applications, including diagnostic and research applications, where labeling, detection, and / or analysis of a target of interest is desirable. Such applications include methods of cytometry, microscopy, immunoassays (e.g., competitive or noncompetitive), free analyte assessment, receptor-bound ligand assessment, and the like. The compositions, systems, and methods described herein may be useful in the analysis of any of a number of samples, including, but not limited to, biological fluids, cell culture samples, and tissue samples. In certain aspects, the compositions, systems, and methods described herein may be used in methods to detect an analyte in a sample, if present, using fluorescent labels, for example, in fluorescence-activated cell sorting or analysis, immunoassays, immunostaining, and the like. In certain examples, the compositions and methods are used in applications where it is of interest to evaluate a sample for the presence of a target analyte.

[0210] In some cases, the methods and compositions are used in any assay format in which detection and / or analysis of a target from a sample is of interest, including, but not limited to, flow cytometry, fluorescence microscopy, in-situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography. In particular examples, the methods and compositions are used in any application in which fluorescent labeling of a target molecule is of interest. The subject compositions can be adapted for use in any convenient application in which pairs of specific binding members are used, such as biotin-streptavidin and hapten-antihapten antibodies.

[0211] The following examples are offered by way of illustration and not by way of limitation. [Example] I. Synthesis of Water-Soluble Purple-Excitable Coumarin Donor Fluorophore C, Water-Soluble Purple-Excitable Coumarin Donor Fluorophore G, and Water-Soluble Purple-Excitable Coumarin Donor Fluorophore H A. Synthesis of Compound C

[0212] [ka]

[0213] Compound 1C:N 2 POCl in DMF (45 mL) at 0 °C for 0.5 h under atmospheric pressure 3 Reaction A of (40.82 mg, 266.23 μmol, 24.74 μL, 0.1 equiv.) of 3-[bis[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]phenol (3.18 g, 2.66 mmol, 1 equiv.)) in DMF (40 mL) was added to the stirred solution in reaction B. The resulting mixture was stirred at 25° C. for 16 h. The reaction mixture was cooled to 30° C. and cooled to 30° C. for 1 h ... 2 CO 3 The mixture was acidified to pH = 7 with N 2The mixture was stirred at 55° C. under atmospheric pressure for 3 h. The mixture was diluted with DCM (200 mL) and H 2 The mixture was extracted with 20 mL of 100% H2O and the combined organic phase was washed with brine (100 mL) and anhydrous Na2O3 (100 mL). 2 SO 4 The mixture was dried at 40° C. and concentrated to give a crude residue. The residue was used in the next step without purification. Compound 4-[bis[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]-2-hydroxy-benzaldehyde (compound 1C) (1.83 g, 1.18 mmol, 44.31% yield, 78.8% purity) was obtained as a pale black oil.

[0214] [ka]

[0215] Compound 2C: To a solution of 2-(1H-benzimidazol-2-yl)acetonitrile (2 g, 12.72 mmol, 1 equiv.) in DCM (100 mL) was added DBU (2.32 g, 15.27 mmol, 2.30 mL, 1.2 equiv.) and ethyl 4-bromobutanoate (3.72 g, 19.09 mmol, 2.74 mL, 1.5 equiv.). The mixture was heated at 50 °C with N 2 The mixture was stirred under reduced pressure for 16 hours. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography to give ethyl 4-[2-(cyanomethyl)benzimidazol-1-yl]butanoate (compound 2C) (1.33 g, 4.52 mmol, 35.52% yield, 92.2% purity) as a dark oil.

[0216] [ka]

[0217] Compound 3C: A solution of compound 1C (0.2 g, 163.61 μmol, 1 equiv.) and compound 2C (66.58 mg, 245.41 μmol, 1.5 equiv.) in EtOH (4 mL) was added with N 2 Piperidine (27.86 mg, 327.21 μmol, 32.31 μL, 2 equiv) was added under atmospheric pressure. Some yellow solid precipitated from the mixture. The suspension was stirred at 55° C. for 16 h. The mixture was dissolved in DCM (50 mL) and washed with water (20 mL) and brine (10 mL), and the organic layer was washed with Na 2 SO 4 The crude product was dissolved in 2% HCl (2 mL) at 100 °C for 2 h, dried over N 2 The mixture was cooled to room temperature and then dissolved in DCM (100 mL), washed with water (10 mL) and brine (10 mL), and the organic layer was diluted with Na 2 SO 4 The mixture was dried at 40° C. for 1 hour, filtered, and concentrated under reduced pressure to give a residue: ethyl 4-[2-[7-bis[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]-2-oxo-chromen-3-yl]benzimidazol-1-yl]butanoate (compound 3C) (0.25 g, 155.75 μmol, 95.20% yield, 92% purity) as a yellow oil.

[0218] [ka]

[0219] Compound C:N 2 To a solution of compound 3C (0.05 g, 33.86 μmol, 1 equiv.) in HCl (2.04 g, 5.60 mmol, 2 mL, 10% purity, 165.25 equiv.) at 100 °C for 1 h under atmospheric pressure. The mixture was dissolved in DCM (50 mL) and the organic layer was washed with Na 2 SO 4The mixture was dried at 40° C., filtered, and concentrated under reduced pressure to obtain a residue. The resulting residue was purified by preparative HPLC. Compound 4-[2-[7-bis[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]-2-oxo-chromen-3-yl]benzimidazol-1-yl]butanoic acid (compound C) (0.75 g, 509.95 μmol, 56.83% yield, 98.5% purity) was obtained as a yellow oil.

[0220] B. Synthesis of Compound G

[0221] [ka]

[0222] Compound 1G: 1,3-Difluoro-2,4-dimethoxy-benzene (3 g, 17.23 mmol, 1 equiv.) was diluted in DCM (114 mL) and diluted with BBr 3 (1M, 111.11 mL, 6.45 equiv.) was added via cannula at 20° C. The resulting clear light brown solution was stirred at 20° C. for 40 h. 2 The reactions were quenched by slow addition to a suspension of ice in 20O (400 mL), resulting in the formation of a precipitate. The resulting suspension was stirred at room temperature until the precipitate dissolved. The organic layer was decanted and the aqueous layers from both reactions were combined and purified by HCl. 2 Cl 2 (3×50 mL) until nearly colorless. 2 Cl 2 The layers were discarded and the aqueous layer was extracted with MTBE (3×300 mL). The combined ether layers were dried (MgSO 4 ), filtered, and concentrated under reduced pressure to give a light gray semi-solid. Crystallization was initiated by eluting with CH until a precipitate (10 mL) formed. 2 Cl 2This was followed by removal of the solvent under reduced pressure until a free-flowing light gray powder was obtained. 2,4-Difluorobenzene-1,3-diol (compound 1G) (2.5 g, 15.40 mmol, 89.40% yield, 90% purity) was obtained as a light gray solid.

[0223] [ka]

[0224] Compound 2G: TFA (46.15 g, 404.71 mmol, 29.97 mL, 23.65 equiv) was placed in a 100 mL flask and cooled to 0° C. HMTA (4.80 g, 34.23 mmol, 6.40 mL, 2 equiv) was added slowly to the cold TFA with stirring at a rate that maintained the reaction temperature around 0° C. Compound 1G (2.5 g, 17.11 mmol, 1 equiv) was then added in one portion as a solid. The flask was equipped with a reflux condenser and N 2 The mixture was refluxed at about 90° C. for 24 h, during which time the solution slowly turned brown. The mixture was cooled to room temperature. The reaction was quenched with 20% aqueous H2SO4 (14 mL) at room temperature and stirred at room temperature for an additional 4 h. The mixture was poured into ice H2O (100 mL) and extracted with EtOAc (3×50 mL). The combined organic layers were washed with brine (1×100 mL) and then concentrated directly under reduced pressure to give a deep orange semi-solid. The residue was purified by flash silica gel chromatography to give 3,5-difluoro-2,4-dihydroxy-benzaldehyde (compound 2G) (1.26 g, 7.13 mmol, 41.69% yield, 98.57% purity) as a yellow solid.

[0225] [ka]

[0226] Compound 3G: Compound 2G (1.26 g, 7.24 mmol, 1 equiv.) and diethyl malonate (4.50 g, 28.08 mmol, 4.24 mL, 3.88 equiv.) were placed in a flask followed by the addition of MsOH (89.5 mL) at 20 °C with vigorous stirring. The reaction mixture was immediately protected from light with foil and flushed with N2. The mixture was stirred for 16 h. The mixture was worked up by pouring onto ice water (50 mL) and extracting with EtOAc (3 x 50 mL). The combined organic layers were washed with H2O (3 x 50 mL) and brine (1 x 50 mL). The organic layers were dried (Na 2 SO 4 The residue was redissolved in MeCN (6.5 mL) in a flask, flushed with N2, and then heated until all solids were dissolved. The solution was cooled slowly to room temperature, followed by chilling at 4° C. for 1 h. Yellow crystals formed and were collected by vacuum filtration and washed with MTBE (3×4.5 mL). The product was a fluffy yellow powder. Ethyl 6,8-difluoro-7-hydroxy-2-oxo-chromene-3-carboxylate (compound 3G) (1.2 g, 4.31 mmol, 59.54% yield, 97.02% purity) was obtained as a pale yellow solid.

[0227] [ka]

[0228] Compound 4G: In a flask, NaOH (799.39 mg, 19.99 mmol, 54 equiv.) was dissolved in HO (10 mL), MeOH (2 mL) was added to the flask, followed by compound 3G (0.1 g, 370.12 μmol, 1 equiv.). The reaction mixture was immediately protected from light and stirred at 20° C. for 1 h. The reaction solution was acidified with concentrated HCl (50 mL) to near colorlessness and extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (1×50 mL), dried (MgSO), filtered, concentrated under reduced pressure, and protected from light. 6,8-Difluoro-7-hydroxy-2-oxo-chromene-3-carboxylic acid (compound 4G) (70 mg, 287.45 μmol, 77.66% yield, 99.43% purity) was obtained as a pale yellow solid.

[0229] [ka]

[0230] Compound 5G: To a solution of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (20 g, 22.45 mmol, 1 equiv.) in DMF (240 mL) was added (1,3-dioxoisoindolin-2-yl)potassium (4.99 g, 26.93 mmol, 1.2 equiv.). The reaction mixture was then stirred at 100° C. under nitrogen for 12 h to give a colorless solution. The reaction mixture was extracted with DCM (100 mL x 3) and water (200 mL) and the organic layer was washed with water (200 mL x 3). The separated organic layer was dried over Na2SO4, filtered and evaporated to give a residue. Compound 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]isoindoline-1,3-dione (compound 5G) (8 g, 8.82 mmol, 39.28% yield, 95.43% purity) was obtained as a pale yellow oil.

[0231] [ka]

[0232] Compound 6G: To a mixture of solid compound 5G (8 g, 9.24 mmol, 1 equiv.) and EtOH (160 mL) was added hydrazine; hydrate (1.76 g, 35.10 mmol, 1.71 mL, 3.8 equiv.) in one charge. The mixture was heated to 40° C. for 4 h. The reaction mixture was cooled to 20° C., then filtered, the filter cake was washed with DCM (100 mL×2), and the filter was evaporated to give a residue, which was dissolved in DCM (50 mL), filtered, the filter cake was washed with DCM (50 mL×2), and the filter was evaporated to give the desired product. The residue was used directly in the next step without further purification. Compound 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanamine (compound 6G) ​​(6.6 g, crude) was obtained as a pale yellow oil.

[0233] [ka]

[0234] Compound 7G: DMF (20 mL) and H 2To a solution of compound 6G (5 g, 6.79 mmol, 1 equiv.) in 0 (15 mL) was added benzyl prop-2-enoate (1.10 g, 6.79 mmol, 1 equiv.). The reaction mixture was then stirred under nitrogen at 20° C. for 16 h to give a colorless solution. The reaction mixture was cooled to 20° C., then filtered, the filter cake was washed with DCM (20 mL×2), the filter was evaporated to give a residue, the residue was dissolved in DCM (30 mL), filtered, the filter cake was washed with DCM (20 mL×2), the filter was evaporated to give the desired product. The crude product was purified by reverse phase HPLC (0.075% TFA condition) to give product 3g. The 3g residue was purified by preparative HPLC. Compound benzyl 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]propanoate (compound 7G) (2 g, 2.00 mmol, 29.50% yield, 90% purity) was obtained as a colorless oil.

[0235] [ka]

[0236] Compound 8G: To a solution of compound 4G (50 mg, 206.50 μmol, 1 eq.) in DMF (2 mL), HATU (102.07 mg, 268.45 μmol, 1.3 eq.) and DIEA (106.75 mg, 825.99 μmol, 143.87 μL, 4 eq.) were added. The reaction mixture was stirred under nitrogen at 20° C. for 0.5 h. Compound 7G (222.54 mg, 247.80 μmol, 1.2 eq.) was then added to the above solution and stirred under nitrogen at 20° C. for 2 h to obtain a green solution. The reaction mixture was poured into water (10 mL), extracted with DCM (20 mL×3), and the combined organic phase was washed with water (20 mL) and saturated NaCl (20 mL), then anhydrous NaCl was added. 2 SO 4The mixture was dried at 40° C. and concentrated to give a green residue. The residue was used directly in the next step without further purification. Compound benzyl 3-[(6,8-difluoro-7-hydroxy-2-oxo-chromene-3-carbonyl)-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]propanoate (compound 8G) (200 mg, crude) was obtained as a pale yellow oil.

[0237] [ka]

[0238] Compound G: In a flask, LiOH.HO (6.42 mg, 152.94 μmol, 2 equiv.) was dissolved in MeOH (1 mL), and HO (0.2 mL) was added to the flask, followed by compound 8G (0.08 g, 76.47 μmol, 1 equiv.). The reaction mixture was immediately protected from light and stirred at 20 °C for 1 h. The reaction solution was acidified with concentrated HCl (5 mL) to make it nearly colorless, and extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (1 × 50 mL), dried (MgSO4), filtered, concentrated under reduced pressure, and protected from light. The residue was purified by preparative HPLC (column: Boston Green ODS 150 × 30 mm × 5 μm; mobile phase: [water (0.075% TFA)-ACN]; B%: 15%-45%, 9 min). Compound 3-[(6,8-difluoro-7-hydroxy-2-oxo-chromene-3-carbonyl)-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]propanoic acid (compound G) (40 mg, 38.72 μmol, 50.63% yield, 99.9% purity) was obtained as a colorless oil.

[0239] C. Synthesis of Compound H

[0240] [ka]

[0241] Compound 1H:H 2 3-Benzyloxyaniline (20 g, 100.38 mmol, 1 equiv.), 2-bromoethanol (50.17 g, 401.51 mmol, 28.51 mL, 4 equiv.), CaCO in O (200 mL). 3 (20.09 g, 200.75 mmol, 2 equiv.), KI (1.67 g, 10.04 mmol, 0.1 equiv.). The mixture was stirred at 100° C. for 16 h. The mixture was extracted with ethyl acetate (3×200 mL) and the combined organic layers were washed with brine (100 mL) and diluted with Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate=1:1) to obtain the compound 2-[3-benzyloxy-N-(2-hydroxyethyl)anilino]ethanol (30 g, 89.79 mmol, 89.45% yield, 86% purity) as a yellow oil.

[0242] [ka]

[0243] Compound 2H: A solution of compound 1H (4 g, 13.92 mmol, 1 equiv.) in THF (320 mL) was added with N 2NaH (5.57 g, 139.20 mmol, 60% purity, 10 equiv.) was added under atmosphere. The mixture was stirred at 70° C. for 1 h. The mixture was then cooled to 25° C. and a solution of 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (22.41 g, 33.41 mmol, 2.4 equiv.) was added dropwise. The mixture was stirred at 70° C. for an additional 16 h. The mixture was quenched with water (200 mL) and diluted with CH 2 Cl 2 (600 mL), and the combined organic layers were washed with brine (200 mL), and then 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2 , dichloromethane:methanol=10:1) to give compound 2H, 3-benzyloxy-N,N-bis[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]aniline (22 g, 13.87 mmol, 99.66% yield, 81% purity) as a yellow oil, which was characterized by LCMS and HPLC.

[0244] [ka]

[0245] Compound 3H: To a solution of compound 2H (22 g, 17.13 mmol, 1 equiv) in TFA (200 mL), the mixture was stirred at 90 °C for 16 h. The reaction mixture was acidified with NaHCO to pH = 7, filtered, and the cake was washed with HO (200 mL) and DCM (400 mL), then concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2The mixture was purified by elution with 10:1 dichloromethane:methanol. Compound 3-[bis[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]phenol (compound 3H) (13 g, 8.49 mmol, 49.57% yield, 78% purity) was obtained as a dark brown oil and was characterized by LCMS and HPLC.

[0246] [ka]

[0247] Compound 4H: POCl in DMF (130 mL) 3 (1.67 g, 10.88 mmol, 1.01 mL, 1 equiv.) was dissolved in N 2 The mixture was stirred at 0° C. under atmospheric pressure for 0.5 h. Compound 3H (13 g, 10.88 mmol, 1 equiv.) in DMF (96 mL) was added to the stirred solution. The resulting mixture was stirred at 25° C. for 16 h. The reaction mixture was diluted with Na 2 CO 3 The mixture was acidified to pH = 7 with N 2 The mixture was stirred at 55° C. under atmospheric pressure for 3 h. The mixture was diluted with DCM (500 mL) and H 2 The mixture was extracted with 200 mL of ethyl acetate and the combined organic phase was washed with brine (300 mL) and anhydrous NaCl. 2 SO 4 The mixture was dried at 40° C. and concentrated to give a crude residue. Compound 4-[bis[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]-2-hydroxy-benzaldehyde (compound 4H) (11.5 g, crude) was obtained as a dark brown oil, which was characterized by LCMS and HPLC, as well as HNMR.

[0248] [ka]

[0249] Compound 5H: To a solution of compound 4H (2 g, 1.64 mmol, 1 equiv) and diethyl propanedionate (786.13 mg, 4.91 mmol, 741.63 μL, 3 equiv) in EtOH (10 mL) was added piperidine (278.62 mg, 3.27 mmol, 323.15 μL, 2 equiv) and AcOH (314.99 mg, 5.25 mmol, 299.99 μL, 3.21 equiv). The mixture was stirred at 55° C. for 16 h. The mixture was quenched with water (100 mL) and purified by HCl. 2 Cl 2 (150 mL), and the combined organic layers were washed with brine (50 mL) and Na 2 SO 4 The mixture was dried at 40° C., filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, dichloromethane:methanol=10:1). The residue was purified by preparative HPLC (column: Welch Xtimate C18 150×30 mm×5 μm; mobile phase: [water (FA)-ACN]; B%: 20%-50%, 7.7 min). Ethyl 7-[bis[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]-2-oxo-chromene-3-carboxylate (compound 5H) (350 mg, 260.14 μmol, 26.38% yield, 98% purity) was obtained as a yellow oil and was confirmed by LCMS and HPLC.

[0250] [ka]

[0251] Compound H: Compound 5H (300 mg, 227.53 μmol, 1 equiv.) was stirred in 1M HCl (1M, 227.53 μL, 1 equiv.) at 100° C. for 1 h. The mixture was dissolved in DCM (50 mL) and the organic layer was extracted with Na 2 SO 4The mixture was dried over 1000 ml, filtered and concentrated under reduced pressure to give a residue. The residue was purified by reverse phase column (column: 12g C18 reverse phase column; mobile phase: [water-ACN]; B%: 0%-60%, 30 min). Compound 7-[bis[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]amino]-2-oxo-chromene-3-carboxylic acid (compound H) (73.3 mg, 55.10 μmol, 24.22% yield, 97% purity) was obtained as a brown oil and was confirmed by LCMS and HPLC and 1H NMR.

[0252] II. Characterization of Compound C, Compound G, and Compound H A.UV-Vis spectroscopy A UV-2600 spectrophotometer (double beam, single monochromator Shimadzu Scientific Instruments) was set to collect data from 900 nm to 250 nm with a slit width of 1.0 nm in 1 nm increments. A stock solution of donor C dissolved in 1x phosphate buffered saline (PBS) was prepared and 2 mL was transferred to a sample cuvette [1 cm polymethyl methacrylate cuvette]. 2 mL of 1x PBS was then transferred to a second cuvette. The cuvette containing 1x PBS was placed in the blank cuvette position in the UV-2600 and the dye sample cuvette was placed in the sample cuvette position. The instrument absorbance was autozeroed at 900 nm and UV-Vis spectra were collected from 900 nm to 250 nm. The same protocol was performed with donors G and H.

[0253] B. Fluorescence Spectroscopy A small amount of donor C stock solution in 1x PBS was diluted to 25 mA.U. in a total volume of 2 mL of 1x PBS. This solution was transferred to a sample cuvette [1 cm polymethyl methacrylate cuvette] and placed in the sample compartment of the fluorometer. The excitation wavelength was 375 nm and the emission scan range was 400 nm to 900 nm. For signal detection, both the fluorescence signal (S1) and the instrument noise (R1) were recorded. A compensation file applied to the raw output signal converted the S1 and R1 signals into corrected fluorescence (S1c) and corrected noise (R1c). The corrected fluorescence S1c was then normalized with respect to the corrected noise to obtain the final reported fluorescence emission of the sample. The same protocol was performed with donor G and donor H. The results are shown in Figure 1.

[0254] III. Synthesis of tandem dyes bearing purple excitable donor fluorophores A summary of some of the various methods available for synthesizing tandem dyes with purple excitable donor fluorophores can be found in, for solid phase peptide synthesis, Steward et al., "Solid Phase Peptide Synthesis", WH Freeman Co., San Francisco, 1969; Bodanszky et al., "Peptide Synthesis", John Wiley & Sons, Second Edition, 1976 and Meienhofer, "Hormonal Proteins and Peptides", Vol. 2, p. 46, Academic Press (New York), 1983; and Kent, Ann. Rev. Biochem., 57, 957, 1988, and for solution synthesis, Schroder et al., "The Peptides", Vol. 1, Academic Press (New York), 1965. Any convenient protecting group strategy may be used, including, but not limited to, Fmoc solid phase peptide synthesis and Boc solid phase peptide synthesis strategies. In Boc solid phase peptide synthesis, a Boc amino protecting group is used at the amino terminus, and benzyl, or benzyl-based or other convenient protecting groups may be used for protection of side chain functional groups. In Fmoc solid phase peptide synthesis, an Fmoc amino protecting group is used at the amino terminus, and tert-butyl, or benzyl-based or other convenient protecting groups may be used for protection of side chain functional groups. Convenient protecting groups that may be used in such synthesis methods are described in the above references and in McOmic, "Protective Groups in Organic Chemistry", Plenum Press, New York, 1973; and Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 4th Edition, 2006.Tandem dyes with a purple excitable donor fluorophore and a different acceptor fluorophore were prepared: Tandem 570 (with an acceptor provided for emission at 570 nm), Tandem 600 (with an acceptor provided for emission at 600 nm), Tandem 650 (with an acceptor provided for emission at 650 nm), Tandem 712 (with an acceptor provided for emission at 712 nm), Tandem 750 (with an acceptor provided for emission at 750 nm), Tandem 780 (with an acceptor provided for emission at 780 nm), and Tandem 850 (with an acceptor provided for emission at 850 nm).

[0255] IV. Sample Tandem Dye Characterization A.UV-Vis spectroscopy A UV-2600 spectrophotometer (double beam, single monochromator Shimadzu Scientific Instruments) was set to collect data from 900 nm to 250 nm with a slit width of 1.0 nm in 1 nm increments. A stock solution of the tandem dye prepared in III. above (i.e., Tandem 570, Tandem 600, Tandem 650, Tandem 712, Tandem 750, Tandem 780, or Tandem 850) dissolved in 1x phosphate-buffered saline (PBS) was prepared and 2 mL was transferred to a sample cuvette [1 cm polymethyl methacrylate cuvette]. 2 mL of 1x PBS was then transferred to a second cuvette. The cuvette containing 1x PBS was placed in the blank cuvette position within the UV-2600, while the tandem dye sample cuvette was placed in the sample cuvette position. The instrument absorbance was autozeroed at 900 nm and UV-Vis spectra were collected from 900 nm to 250 nm.

[0256] B. Fluorescence Spectroscopy A small amount of tandem dye (i.e., Tandem 570, Tandem 600, Tandem 650, Tandem 712, Tandem 750, Tandem 780, or Tandem 850) stock solution prepared in III. above in 1x PBS was diluted to 25 mA.U. in a total volume of 2 mL of 1x PBS. This solution was transferred to a sample cuvette [1 cm polymethyl methacrylate cuvette] and placed in the sample compartment of the fluorometer. The excitation wavelength was 405 nm, and the emission scan range was 415 nm to 900 nm. For signal detection, both the fluorescence signal (S1) and the instrument noise (R1) were recorded. A compensation file applied to the raw output signal converted the S1 and R1 signals into corrected fluorescence (S1c) and corrected noise (R1c). The corrected fluorescence S1c was then normalized with respect to the corrected noise to obtain the final reported fluorescence emission of the sample. The results are shown in Figure 2.

[0257] Notwithstanding the scope of the appended claims, the present disclosure is also defined by the following appended claims.

[0258] Appendix 1. A non-conjugated polymer backbone; one or more violet-excitable donor fluorophores linked to a non-conjugated polymer backbone; one or more acceptor fluorophores linked to a non-conjugated polymer backbone; Including, Tandem dyes, in which one or more violet excitable donor fluorophores and one or more acceptor fluorophores are in an energy transfer relationship.

[0259] Clause 2. The tandem dye of clause 1, wherein the one or more violet-excitable donor fluorophores are selected to have an absorption maximum in the range of 375-425 nm.

[0260] Clause 3. The tandem dye of clause 1 or 2, wherein the one or more violet-excitable donor fluorophores are selected from the group consisting of coumarin fluorophores, pyrene fluorophores and triphenyl fluorophores.

[0261] Clause 4. The tandem dye of clause 3, wherein the one or more violet-excitable donor fluorophores are coumarin fluorophores.

[0262] Clause 5. The tandem dye of any one of clauses 1-4, wherein one or more violet excitable donor fluorophores are attached to a water-solubilizing group.

[0263] Applicant's claim 6. The tandem dye of Applicant's claim 5, wherein the water-solubilizing group comprises a non-ionic water-solubilizing group.

[0264] Applicant's claim 7. The tandem dye of Applicant's claim 6, wherein the non-ionic water-solubilizing group comprises a polyalkylene glycol.

[0265] Applicant's claim 8. The tandem dye of Applicant's claim 7, wherein the polyalkylene glycol comprises polyethylene glycol.

[0266] Appendix 9. The tandem dye of any one of appendices 1 to 8, wherein one or more acceptor fluorophores have an emission maximum in the range of 500 to 875 nm.

[0267] Clause 10. The tandem dye of any one of clauses 1-9, wherein the one or more acceptor fluorophores are selected from the group consisting of coumarin dyes, cyanine dyes, rhodamine dyes, xanthene dyes, polymethines, dipyrromethene boron difluorides, naphthalimides, thiazine dyes, and acridine dyes.

[0268] Addendum 11. The tandem dye of any one of Addendums 1 to 10, wherein the number of donor fluorophores exceeds the number of acceptor fluorophores.

[0269] Applicant's claim 12. The tandem dye of Applicant's claim 11, wherein the number of donor fluorophores is in the range of 2 to 20.

[0270] Addendum 13. The tandem dye of any one of Addendums 1 to 12, comprising a single acceptor fluorophore.

[0271] Appendix 14. The tandem dye of any one of appendices 1 to 13, which is linked to a biomolecule.

[0272] Applicant's claim 15. The tandem dye of Applicant's claim 14, wherein the biomolecule is selected from the group consisting of carbohydrates, lipids, nucleic acids, and proteins.

[0273] Addendum 16. The tandem dye of Addendum 15, wherein the biomolecule is a specific binding member.

[0274] Addendum 17. The tandem dye of Addendum 16, wherein the specific binding member is an antibody or a binding fragment or derivative thereof.

[0275] Appendix 18. The tandem dye of any one of appendices 1 to 13, which is linked to a chemical selectivity group.

[0276] Appendix 19. The tandem dye of any one of appendices 1 to 18, wherein the non-conjugated polymer backbone comprises a peptide.

[0277] Appendix 20. The tandem dye of appendix 19, wherein the peptide comprises 5 to 100 amino acid residues.

[0278] Appendix 21. A method for evaluating a sample for the presence of a target analyte, comprising: (a) contacting the sample with a labeled specific binding member that specifically binds a target analyte to produce a labeled sample; (b) assaying the labeled composition for the presence of a labeled specific binding member-target analyte binding complex to assess whether the target analyte is present in the sample; The labeled specific binding member is a non-conjugated polymer backbone; one or more violet-excitable donor fluorophores linked to a non-conjugated polymer backbone; one or more acceptor fluorophores linked to a non-conjugated polymer backbone; a specific binding member conjugated to a tandem dye comprising A method wherein one or more violet excitable donor fluorophores and one or more acceptor fluorophores are in an energy transfer relationship.

[0279] Addendum 22. The method of Addendum 21, wherein the one or more violet-excitable donor fluorophores are selected to have an absorption maxima in the range of 375 to 425 nm.

[0280] Addendum 23. The method of any one of Addendum 21 to 22, wherein the one or more violet-excitable donor fluorophores are selected from the group consisting of coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores.

[0281] Clause 24. The method of clause 23, wherein the one or more violet-excitable donor fluorophores are coumarin fluorophores.

[0282] Clause 25. The method of any one of clauses 21-24, wherein one or more violet excitable donor fluorophores are attached to a water-solubilizing group.

[0283] Applicant's claim 26. The method of Applicant's claim 25, wherein the water-solubilizing group comprises a non-ionic water-solubilizing group.

[0284] Applicant's claim 27. The method of claim 26, wherein the non-ionic water-solubilizing group comprises a polyalkylene glycol.

[0285] Addendum 28. The method of Addendum 27, wherein the polyalkylene glycol comprises polyethylene glycol.

[0286] Addendum 29. The method of any one of Addendums 21 to 28, wherein one or more acceptor fluorophores have an emission maximum in the range of 500 to 875 nm.

[0287] Addendum 30. The method of any one of Addendums 21 to 29, wherein the one or more acceptor fluorophores are selected from the group consisting of coumarin dyes, cyanine dyes, rhodamine dyes, xanthene dyes, polymethines, dipyrromethene boron difluorides, naphthalimides, thiazine dyes, and acridine dyes.

[0288] Addendum 31. The method of any one of Addendums 21 to 30, wherein the number of the one or more violet-excitable donor fluorophores exceeds the number of the one or more acceptor fluorophores.

[0289] Addendum 32. The method of Addendum 31, wherein the number of the one or more violet-excitable donor fluorophores is in the range of 2 to 20.

[0290] Addendum 33. The method of any one of Addendums 21 to 32, wherein the tandem dye comprises a single acceptor fluorophore.

[0291] Addendum 34. The method of Addendum 33, wherein the specific binding member is an antibody or a binding fragment or derivative thereof.

[0292] Applicant's 35. The method of any one of Applicants' 21 to 34, wherein the polymer backbone comprises a peptide.

[0293] Appendix 36. The method of Appendix 35, wherein the peptide comprises 2 to 100 amino acid residues.

[0294] Clause 37. The method of any one of clauses 21-36, wherein the sample is contacted with a second specific binding member that is carrier-bound and specifically binds to the target analyte.

[0295] Addendum 38. The method of Addendum 37, wherein the carrier comprises magnetic particles.

[0296] Addendum 39. The method of any one of Addendums 21-38, wherein the target analyte is associated with a cell.

[0297] Clause 40. The method of clause 39, wherein the target analyte is a cell surface marker of a cell.

[0298] Appendix 41. The method of Appendix 40, wherein the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0299] Addendum 42. The method of any one of Addendums 21-39, wherein the target analyte is an intracellular target.

[0300] Addendum 43. The method of Addendum 42, wherein the cells are permeabilized or lysed.

[0301] Addendum 44. The method of any one of Addendums 21 to 43, wherein, in the assay, the labeled sample is analyzed flow cytometrically.

[0302] Addendum 45. A method for synthesizing a fluorescently labeled tandem dye, comprising: forming a precursor non-conjugated polymer backbone comprising first, second and third types of residues having first, second and third orthogonal reactive side chains, respectively; conjugating a purple-excitable donor fluorophore to any first reactive side chain of the precursor non-conjugated polymer backbone, conjugating an acceptor fluorophore to any second reactive side chain of the precursor non-conjugated polymer backbone, and conjugating a biomolecule to any third reactive side chain of the precursor non-conjugated polymer backbone; A method for producing a fluorescently labeled polymer tandem dye.

[0303] Clause 46. The method of clause 45, wherein the one or more violet-excitable donor fluorophores are selected to have an absorption maxima in the range of 375 to 425 nm.

[0304] Clause 47. The method of clause 45 or 46, wherein the one or more violet-excitable donor fluorophores are selected from the group consisting of coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores.

[0305] Clause 48. The method of clause 47, wherein the one or more violet-excitable donor fluorophores are coumarin fluorophores.

[0306] Clause 49. The method of any one of clauses 45-48, wherein one or more violet excitable donor fluorophores are attached to a water-solubilizing group.

[0307] Clause 50. The method of clause 49, wherein the water-solubilizing group comprises a non-ionic water-solubilizing group.

[0308] Addendum 51. The method of Addendum 40, wherein the non-ionic water-solubilizing group comprises a polyalkylene glycol.

[0309] Addendum 52. The method of Addendum 51, wherein the polyalkylene glycol comprises polyethylene glycol.

[0310] Addendum 53. The method of any one of Addendums 45 to 52, wherein one or more acceptor fluorophores have an emission maximum in the range of 500 to 875 nm.

[0311] Addendum 54. The method of any one of Addendums 45 to 53, wherein the one or more acceptor fluorophores are selected from the group consisting of coumarin dyes, cyanine dyes, rhodamine dyes, xanthene dyes, polymethines, dipyrromethene boron difluorides, naphthalimides, thiazine dyes, and acridine dyes.

[0312] Addendum 55. The method of any one of Addendums 45 to 54, wherein the number of violet-excitable donor fluorophores exceeds the number of acceptor fluorophores.

[0313] Addendum 56. The method of Addendum 55, wherein the number of violet-excitable donor fluorophores is in the range of 2 to 20.

[0314] Addendum 57. The method of any one of Addendums 45 to 56, wherein the tandem dye comprises a single acceptor fluorophore.

[0315] Addendum 58. The method of any one of Addendums 45 to 57, wherein the biomolecule is selected from the group consisting of carbohydrates, lipids, nucleic acids, and proteins.

[0316] Addendum 59. The method of Addendum 58, wherein the biological molecule is a specific binding member.

[0317] Addendum 60. The method of Addendum 59, wherein the specific binding member is an antibody or a binding fragment or derivative thereof.

[0318] Addendum 61. The method of any one of Addendums 45 to 60, wherein the non-conjugated polymer backbone comprises a peptide.

[0319] Addendum 62. The method of Addendum 61, wherein the peptide comprises 5 to 100 amino acid residues.

[0320] Addendum 63. Tandem dyes, comprising: a non-conjugated polymer backbone; one or more violet-excitable donor fluorophores linked to a non-conjugated polymer backbone; one or more acceptor fluorophores linked to a non-conjugated polymer backbone; Including, The kit, wherein one or more violet excitable donor fluorophores and one or more acceptor fluorophores are in an energy transfer relationship.

[0321] Item 64. The kit of item 63, wherein the one or more violet-excitable donor fluorophores are selected to have an absorption maxima in the range of 375 to 425 nm.

[0322] Addendum 65. The kit of any one of Addendum 63 to 64, wherein the one or more violet-excitable donor fluorophores are selected from the group consisting of coumarin fluorophores, pyrene fluorophores, and triphenyl fluorophores.

[0323] Addendum 66. The kit of Addendum 65, wherein the one or more violet-excitable donor fluorophores are coumarin fluorophores.

[0324] Addendum 67. The kit of any one of Addendums 63-66, wherein one or more purple-excitable donor fluorophores are attached to a water-solubilizing group.

[0325] Addendum 68. The kit of Addendum 67, wherein the water-solubilizing group comprises a non-ionic water-solubilizing group.

[0326] Addendum 69. The kit of Addendum 68, wherein the non-ionic water-solubilizing group comprises a polyalkylene glycol.

[0327] Addendum 70. The kit of Addendum 69, wherein the polyalkylene glycol comprises polyethylene glycol.

[0328] Addendum 71. The kit of any one of Addendums 63-70, wherein one or more acceptor fluorophores have an emission maximum in the range of 500 to 875 nm.

[0329] Addendum 72. The kit of any one of Addendums 63-71, wherein the one or more acceptor fluorophores are selected from the group consisting of coumarin dyes, cyanine dyes, rhodamine dyes, xanthene dyes, polymethines, dipyrromethene boron difluorides, naphthalimides, thiazine dyes, and acridine dyes.

[0330] Addendum 73. The kit of any one of Addendums 63-72, wherein the number of the one or more violet-excitable donor fluorophores exceeds the number of the one or more acceptor fluorophores.

[0331] Item 74. The kit of item 73, wherein the number of the one or more violet-excitable donor fluorophores is in the range of 2 to 20.

[0332] Addendum 75. The kit of any one of Addendums 63 to 74, wherein the tandem dye comprises a single acceptor fluorophore.

[0333] Addendum 76. The kit of any one of Addendums 63 to 75, wherein the tandem dye is linked to a biomolecule.

[0334] Addendum 77. The kit of Addendum 76, wherein the biological molecule is selected from the group consisting of carbohydrates, lipids, nucleic acids, and proteins.

[0335] Addendum 78. The kit of Addendum 76, wherein the biological molecule is a specific binding member.

[0336] Addendum 79. The kit of Addendum 78, wherein the specific binding member is an antibody or a binding fragment or derivative thereof.

[0337] Addendum 80. The kit according to any one of Addendums 63 to 75, wherein the tandem dye is linked to a chemically selective group.

[0338] Addendum 81. The kit of any one of Addendums 63 to 80, wherein the non-conjugated polymer backbone comprises a peptide.

[0339] Item 82. The kit of item 81, wherein the peptide comprises 5 to 100 amino acid residues.

[0340] In at least some of the above-described embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment, unless such substitution is technically infeasible. It will be understood by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

[0341] In general, those skilled in the art will understand that the terms used herein, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "including" should be interpreted as "including but not limited to," etc.). It will be further understood by those skilled in the art that if a specific number of recitations of an introduced claim are intended, such intent will be explicitly set forth in the claim, and that in the absence of such recitation, no such intent exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce the recitations of the claim. However, the use of such phrases should not be construed as meaning that the introduction of a claim recitation by the indefinite article "a" or "an" limits any particular claim that includes such an introduced claim recitation to embodiments that include only one such recitation, even if that claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"), and the same applies to the use of a definite article used to introduce a claim recitation. Moreover, even if a specific number of introduced claim recitations is explicitly recited, one of skill in the art will recognize that such recitations should be interpreted to mean at least the recited number (e.g., the bare recitation "two recitations" without any other qualifier means at least two recitations, or more than two recitations). Moreover, in those instances where a conventional expression similar to "at least one of A, B, and C, etc." is used, such structure is generally intended in the sense that one of ordinary skill in the art would understand the conventional expression (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.).In those instances where a conventional expression similar to "at least one of A, B, or C, etc." is used, such a structure is generally intended in the sense that one of ordinary skill in the art would understand the conventional expression (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having only A, only B, only C, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). It will be further understood by those of ordinary skill in the art that virtually any alternative word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0342] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is thereby described in terms of any individual members or subgroups of members of the Markush group.

[0343] As will be understood by those skilled in the art, all ranges disclosed herein encompass all possible subranges and combinations of subranges for any and all purposes, including in terms of providing a written description. Any recited range can be readily recognized as fully describing and allowing the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third, etc. As will also be understood by those skilled in the art, all language, such as "up to," "at least," "greater than," "less than," etc., refers to a range that includes the recited numbers and can then be broken down into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0344] Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0345] Thus, the above merely describes the principles of the invention. It will be appreciated that those skilled in the art can devise various configurations that embody the principles of the invention and fall within its spirit and scope, although not expressly described or shown herein. Furthermore, all examples and conditional language described herein are intended primarily to aid the reader in understanding the principles of the invention and the concepts the inventors have contributed to furthering the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein describing the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any developed elements that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be dedicated to the public, regardless of whether such disclosure is expressly set forth in the claims.

[0346] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied in the appended claims. In the claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) are expressly defined to apply to a limitation of a claim only if the exact phrase "means for" or the exact phrase "step for" appears at the beginning of such limitation, and if such exact phrase is not used in a limitation in a claim, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) shall not apply.

[0347] CROSS-REFERENCE TO RELATED APPLICATIONS Pursuant to 35 U.S.C. §119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 322,120, filed March 21, 2022, the disclosure of which is incorporated herein by reference in its entirety.

Claims

1. a non-conjugated polymer backbone; one or more violet-excitable donor fluorophores linked to the non-conjugated polymer backbone; one or more acceptor fluorophores linked to the non-conjugated polymer backbone; Including, The one or more violet excitable donor fluorophores and the one or more acceptor fluorophores are in an energy transfer relationship, a tandem dye.

2. 2. The tandem dye of claim 1, wherein the one or more violet excitable donor fluorophores are selected to have an absorption maxima in the range of 375-425 nm.

3. 3. The tandem dye of claim 1 or 2, wherein the one or more violet excitable donor fluorophores are selected from the group consisting of coumarin fluorophores, pyrene fluorophores and triphenyl fluorophores.

4. The tandem dye of claim 3 , wherein the one or more violet excitable donor fluorophores are coumarin fluorophores.

5. The tandem dye of any one of claims 1 to 4, wherein the one or more violet excitable donor fluorophores are attached to a water-solubilizing group.

6. The tandem dye of claim 5 , wherein the water-solubilizing group comprises a non-ionic water-solubilizing group.

7. The tandem dye of claim 6 , wherein the non-ionic water-solubilizing group comprises a polyalkylene glycol.

8. The tandem dye of claim 7 , wherein the polyalkylene glycol comprises polyethylene glycol.

9. The tandem dye of any one of claims 1 to 8, wherein the one or more acceptor fluorophores have an emission maximum in the range of 500 to 875 nm.

10. 10. The tandem dye of claim 1, wherein the one or more acceptor fluorophores are selected from the group consisting of coumarin dyes, cyanine dyes, rhodamine dyes, xanthene dyes, polymethines, dipyrrometheneboron difluorides, naphthalimides, thiazine dyes, and acridine dyes.

11. The tandem dye of any one of claims 1 to 10, wherein the number of said one or more violet excitable donor fluorophores exceeds the number of said one or more acceptor fluorophores.

12. The tandem dye of any one of claims 1 to 11, wherein the tandem dye is linked to a biomolecule.

13. 13. The tandem dye of claim 12, wherein the biomolecule is selected from the group consisting of carbohydrates, lipids, nucleic acids, and proteins.

14. 1. A method for evaluating a sample for the presence of a target analyte, comprising: (a) contacting the sample with a labeled specific binding member that specifically binds a target analyte to produce a labeled sample; (b) assaying the labeled composition for the presence of a labeled specific binding member-target analyte binding complex to assess whether the target analyte is present in the sample; The labeled specific binding member comprises: a non-conjugated polymer backbone; one or more violet-excitable donor fluorophores linked to the non-conjugated polymer backbone; one or more acceptor fluorophores linked to the non-conjugated polymer backbone; a specific binding member conjugated to a tandem dye comprising The method, wherein the one or more violet excitable donor fluorophores and the one or more acceptor fluorophores are in an energy transfer relationship.

15. 1. A method for synthesizing a fluorescently labeled tandem dye, comprising: forming a precursor non-conjugated polymer backbone comprising first, second and third types of residues having first, second and third orthogonal reactive side chains, respectively; conjugating a violet-excitable donor fluorophore to any first reactive side chain of said precursor non-conjugated polymer backbone, conjugating an acceptor fluorophore to any second reactive side chain of said precursor non-conjugated polymer backbone, and conjugating a biomolecule to any third reactive side chain of said precursor non-conjugated polymer backbone; A method for producing a fluorescently labeled polymeric tandem dye.

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