Water-soluble polymer dyes containing pendant chromophores

Abstract

Water-soluble light-harvesting multichromophores having pendant chromophore groups are provided. The light-harvesting multichromophores have a polymer backbone including non-conjugated repeat units and a plurality of pendant donor chromophore groups linked to the non-conjugated repeat units of the polymer backbone. The pendant chromophore groups can be BODIPY groups substituted with one or more water-soluble groups. Polymeric tandem dyes based on the subject multichromophores are provided, further including an acceptor fluorophore linked to the non-conjugated repeat units of the polymer backbone and configured in energy-accepting proximity to the pendant donor chromophore groups. Labeled specific binding members comprising the subject polymeric tandem dyes are also provided. Methods of evaluating samples for target analytes and methods of labeling target molecules in which the subject polymeric tandem dyes find use are provided. Systems and kits for practicing the subject methods are also provided.

Description

[Background technology]

[0001] Fluorescent dyes are compounds that, when irradiated with light of a wavelength they absorb, (usually) emit light of a different wavelength. Fluorescent dyes find use in a variety of applications in biochemistry, biology, and medicine, such as in diagnostic kits, microscopy, or drug screening. Fluorescent dyes are characterized by several parameters that allow users to select a suitable dye according to their desired purpose.

[0002] The parameters of interest include the maximum excitation wavelength, maximum emission wavelength, Stokes shift, annihilation coefficient, fluorescence quantum yield, and fluorescence lifetime. Dyes may be selected according to the intended application, for example, to allow the penetration of excitation radiation into a biological sample, minimize background fluorescence, and / or achieve a high signal-to-noise ratio.

[0003] Molecular recognition involves the specific binding of two molecules. Molecules with binding specificity to target biomolecules find use in a variety of research and diagnostic applications, including analyte labeling and separation, flow cytometry, insight hybridization, enzyme-linked immunosorbent assay (ELISA), Western blotting, magnetic cell separation, and chromatography. Target biomolecules can be detected by labeling with fluorescent dyes. [Overview of the Initiative]

[0004] Water-soluble light-harvesting multichromophores having multiple pendant chromophore groups are provided. The light-harvesting multichromophore has a polymer backbone containing non-conjugated repeating units and multiple pendant donor chromophore groups independently linked to each non-conjugated repeating unit of the polymer backbone. The pendant chromophore groups can be BODIPY groups substituted with one or more water-soluble groups. Polymer tandem dyes based on the multichromophores of the subject are also provided, which further include acceptor fluorophores linked to the non-conjugated repeating units of the polymer backbone and configured in a vicinity that accepts energy to at least one pendant donor chromophore group of the light-harvesting multichromophore. Labeled specific binding members containing the polymer tandem dyes of the subject are also provided. Methods for evaluating a sample for the presence of a target analyte and methods for labeling target molecules for which the polymer tandem dye of the subject finds use are also provided. Systems and kits for practicing the methods of the subject are also provided. [Brief explanation of the drawing]

[0005] Please understand that the drawings below are for illustrative purposes only. They are not intended to limit the scope of this instruction in any way.

[0006] [Figure 1] The general structure of an exemplary polymer tandem dye in which the "dye" is an acceptor fluorophore is shown. [Figure 2] Normalized absorbance spectra for exemplary multichromophores and emission spectra for a series of polymer tandem dyes containing multichromophores are shown. [Figure 3] Exemplary multichromophores and emission spectra of a series of polymer tandem dyes, including multichromophores, are shown, all with an absorbance of 0.04 OD. [Figure 4] The structure of an exemplary polymer tandem dye having a peptide backbone is shown. D is the BODIPY donor pendant group, A is the acceptor dye, and the biolinker is a linker containing chemoselective functional groups for attaching the tandem dye to a biomolecule. [Figure 5A] The absorbance and emission spectra of the BODIPY donor pendant groups used to prepare the multichromophore shown in Figure 4 are presented. [Figure 5B] The absorbance and emission spectra of the acceptor dyes used to prepare the multichromophore shown in Figure 4 are presented. [Figure 5C] Figure 4 shows the absorbance and emission spectra of an exemplary polymer tandem dye. [Figure 6A] This illustrates homoenergy transfer between pendant-donor chromophores, which preferentially leads to continuous, reversible energy transfer between equal chromophores rather than luminescence from a single chromophore. This process can result in significantly lower self-quench and quantum yield than those observed for a single isolated chromophore. [Figure 6B] This primarily illustrates heteroenergy transfer, which leads to unidirectional energy transfer between different chromophores. Energy transfer to secondary chromophores preferentially leads to luminescence, limited by the quantum yield of the acceptor and single-donor chromophores. [Figure 7A] A synthetic scheme for the preparation of exemplary multichromophores having linked BODIPY dyes ("dyes") is illustrated using click polymerization. "Dyes" refers to donor dyes such as BODIPY. [Figure 7B] A synthetic scheme for the preparation of exemplary multichromophores having polymer tandem dyes is illustrated using click polymerization. "Donor" refers to a donor dye such as BODIPY, and "acceptor" refers to an acceptor fluorophore. [Figure 8] Figure 7A shows the absorbance and emission spectra of an exemplary multichromophore containing linked pendant BODIPY dyes. [Figure 9A] This shows the absorbance of a series of exemplary polymer tandem dyes, including linked pendant BODIPY donor dyes and various acceptor dyes with different maximum emission wavelengths. [Figure 9B]The emission spectra of a series of exemplary polymer tandem dyes, including linked pendant BODIPY donor dyes and various acceptor dyes with different maximum emission wavelengths, are shown. [Modes for carrying out the invention]

[0007] definition Before describing exemplary embodiments in more detail, the following definitions are provided to illustrate and specify the meaning and scope of terms used in the description.

[0008] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs. Nevertheless, for clarity and ease of reference, certain terms are defined below.

[0009] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context otherwise explicitly indicates otherwise. For example, the term “dye” refers to one or more dyes, i.e., a single dye and multiple dyes. It should be further noted that the claims may be drafted to exclude optional elements. Thus, this statement is intended to function as an antecedent for the use of exclusive terms such as “simply” and “only” in relation to the enumeration of elements of the claims or the use of “negative” restrictions.

[0010] As used herein, the terms “chemoselective functional group” and “chemoselective tag” are used interchangeably and, in some cases, refer to a functional group that, after the activation of one of the functional groups of any choice, can selectively react with another compatible functional group to form a covalent bond. The chemoselective functional groups in question include, but are not limited to, thiols and maleimides, or iodoacetamides, amines and carboxylic acids, or their activated esters, as well as, for example, azide and alkyne groups (e.g., cyclooctin group), tetrazine, transcyclooctene, dienes and dieneophils, as well as azides, sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, and groups that can react with each other via click chemistry, such as hydroxyls, hydrazides, hydrazinos, aldehydes, ketones, azides, alkynes, phosphines, and epoxides.

[0011] As used herein, the term “sample” refers to a material or mixture of materials containing one or more analytes of interest, in some cases in liquid form. In some embodiments, the term is used most broadly to refer to any plant, animal or bacterial material containing cells or producing cellular metabolites, e.g., individuals (including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, tears, saliva, and tissue sections), or tissues or fluids isolated from in vitro cell culture components, as well as samples from the environment. The term “sample” may also refer to a “biological sample.” As used herein, the term “biological sample” refers to an entire organism or a subset of its tissues, cells, or component parts (e.g., body fluids including, but not limited to, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, sheep's blood, urine, vaginal fluid, and semen). "Biological sample" can also refer to a whole organism or a subset of its tissues, cells, or component parts, or homogenates, lysates, or extracts prepared from fractions or parts thereof, including, but not limited to, serum, cerebrospinal fluid, lymph, external skin sections, respiratory tract, intestinal tract, and urinary tract, tears, saliva, milk, blood cells, tumors, and organs. In certain embodiments, the sample is removed from an animal or plant. A biological sample may include cells. The term "cell" in its conventional sense means a cell having at least a nucleus and a cell membrane, and eukaryotes and prokaryotes. The term is used to refer to the basic structural units of both living organisms. In certain embodiments, cells include prokaryotic cells, such as those derived from bacteria. In other embodiments, cells include eukaryotic cells, for example, cells obtained from biological samples from animals, plants, or fungi.

[0012] The terms “support-bound” and “bound to support” are used interchangeably and refer to a portion (e.g., a specific binding member) that is covalently or non-covalently bound to the support of interest. Covalent bonding can occur through a chemical reaction between two compatible functional groups (e.g., two chemoselective functional groups, such as electrophilic and nucleophilic) to form a covalent bond between two portions of interest (e.g., the support and the specific binding member). In some cases, non-covalent bonding can occur through specific binding between two portions of interest (e.g., a hapten and an antibody, or two affinity portions such as a biotin moiety and streptavidin). In certain specific cases, non-covalent bonding can occur through absorption into a substrate.

[0013] 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 exceeding 50 amino acids in length. The terms “polypeptide” and “protein” are used interchangeably herein. The term “polypeptide” includes polymers of encoded and unencoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backchain in which the conventional backchain is replaced with a non-spontaneously occurring or synthetic backchain. Polypeptides can be of any convenient length, for example, two or more amino acids, for example four or more amino acids, ten or more amino acids, twenty or more amino acids, fifty or more amino acids, one hundred or more amino acids, three hundred or more amino acids, for example up to 500 or one thousand or more amino acids. A “peptide” can be two or more amino acids, for example four or more amino acids, ten or more amino acids, twenty or more amino acids, for example up to 50 amino acids. In some embodiments, peptides are 5 to 30 amino acids long.

[0014] As used herein, the term “isolated” means a portion of a subject that, prior to purification, is at least 60%, at least 75%, at least 90%, at least 95%, at least 98%, and even at least 99% free from the other components to which it relates.

[0015] "Multiple" includes at least two members. In certain cases, multiple may have five or more members, for example, six or more, seven or more, eight or more, nine or more, ten or more, twenty or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, eighty or more, ninety or more, one hundred or more, three hundred or more, one thousand or more, three thousand or more, one hundred thousand or more, one hundred thousand or more, one hundred thousand or more, one hundred thousand or more, or one hundred thousand or more.

[0016] A numerical range includes the numerical values ​​that define the range.

[0017] The term "specific binding" refers to the ability of a capture agent (or the first member of a specific binding pair) to preferentially bind to a particular analyte (or the second member of a specific binding pair) present, for example, in a homogeneous mixture of different analytes. In some cases, specific binding interactions distinguish desirable and undesirable analytes in a sample with a specificity of 10 times or more compared to undesirable analytes, such as 100 times or more, or 1000 times or more. In some cases, the affinity between the capture agent and the analyte when they specifically bind to each other within the capture agent / analyte complex is at least 10 -8 M, at least 10 -9 M, for example, up to 10 -10 It is M.

[0018] "Affinity" refers to the strength of the bond, and an increase in binding affinity correlates with a lower Kd.

[0019] The methods described herein include a plurality of steps. Each step may be performed after a predetermined time has elapsed between steps, as necessary. Thus, the time between performing 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, or 60 minutes or more, including 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 for a few minutes, for example, up to an incubation or waiting period after the completion of the previous step, such as an overnight waiting period.

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

[0021] As used herein, the term “separate” refers to the physical separation of two elements (e.g., by size or affinity), as well as the decomposition of one element while leaving the other element intact.

[0022] The term “linker” or “link” refers to a linking portion that connects two groups and has a back chain of no more than 100 atoms in length. A linker or link may be a covalent bond connecting two groups or a chain of 1 to 100 atoms in length, e.g., a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20 or more carbon atoms, and the linker may be linear, branched, cyclic, or single-atom. In some cases, a linker is a branched linker, referring to a linking portion that connects three or more groups. In certain particular cases, one, two, three, four, or five or more carbon atoms in the linker back chain may be optionally substituted with sulfur, nitrogen, or oxygen heteroatoms. In some cases, the linker back chain contains linking functional groups such as ethers, thioethers, aminos, amides, sulfonamides, carbamates, thiocarbamates, ureas, thioureas, esters, thioesters, or imines. The bonds between the main chain atoms may be saturated or unsaturated, and in some cases, one, two, or three or fewer unsaturated bonds may be present in the linker main chain. The linker may contain one or more substituents having, for example, alkyl, aryl, or alkenyl groups. The linker may, but is not limited to, polyethylene glycol, ethers, thioethers, tertiary amines, and alkyl groups that may be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethyl (t-butyl), etc. The linker main chain may contain cyclic groups, for example, cyclic groups with two or more atoms of the cyclic group, for example, two, three, or four atoms included in the main chain, such as aryl, heterocyclic, or cycloalkyl groups. The linker may be cleavable or incleavable.

[0023] The terms "polyethylene oxide," "PEO," "polyethylene glycol," and "PEG" are used interchangeably, and the formula is (CH2-CH2-O-). n- Refers to a polymer group comprising a chain described by or its derivatives. 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. The peg polymer group may be of any convenient length, but is not limited thereto, and may include a variety of terminal groups and / or further substituents, including alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amide terminals and / or substituents. Peg groups suitable for use in the subject's multichromophore include those described by S. Zalipsky in “Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6(2), 150-165; and by Zhu et al in “Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy”, Chem. Rev., 2012, 112(8), pp 4687-4735.

[0024] The term "alkyl" refers to a saturated branched or linear monovalent hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of a parent alkane, either by itself or as part of another substituent. The alkyl groups in question 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-methylpropan-1-yl or 2-methylpropan-2-yl). In some embodiments, the alkyl group contains 1 to 20 carbon atoms. In some embodiments, the alkyl group contains 1 to 10 carbon atoms. In certain embodiments, the lower alkyl group contains 1 to 6 carbon atoms, such as 1 to 4 carbon atoms. This term includes, as an example, linear and branched hydrocarbyl groups, such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0025] The term "substituted alkyl" refers to a group in which one or more carbon atoms in the alkyl chain are optionally -O-, -N-, -S-, or -S(O). n-(where n is 0 to 2), -NR- (where R is hydrogen or alkyl), etc., substituted with heteroatoms, and alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketone, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R b refers to an alkyl group as defined herein having 1 to 5 substituents selected from the group consisting of 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.

[0026] "Alkoxy" refers to an -O-alkyl group, 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, etc. The term "alkoxy" also refers to alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-groups, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0027] The term "substituted alkoxy" refers to substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- groups, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0028] "Alkynyl" refers to a linear or branched monovalent hydrocarbyl group having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and at least 1, preferably 1 to 2 triple-bonded unsaturated moieties. Examples of such alkynyl groups include acetylenyl (-C≡CH) and propargyl (-CH2C≡CH).

[0029] The term "substituted alkynyl" refers to an alkynyl group as defined herein having one to five substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminosiloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylyl, thioaryloxy, thioheteroaryloxy, thioheteroaryloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl, or having one to three substituents.

[0030] "Amino" refers to the -NH2 group. The term "substituted amino" refers to a -NRR group in which 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, provided that at least one R is not hydrogen.

[0031] The term "aryl," either in 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 in an aromatic ring system. The aryl groups in question include, but are not limited to, groups derived from acetantrylene, acenaphthylene, acephenantrylene, anthracene, azulene, benzene, chrysene, coronene, fluorantene, fluorene, hexacene, hexaphene, hexalene, asindacene, s-indacene, indan, indene, naphthalene, octane, octafene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthalene, picene, pleiaden, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, etc. 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.

[0032] Unless otherwise restricted by the definition of aryl substituents, "substituted aryl" refers to an aryl group substituted with 1 to 5 substituents selected from 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, aryloxy, azide, 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, -SO2-aryl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl, or an aryl group substituted with 1 to 3 substituents.

[0033] "Heteroaryl" refers to a monovalent heteroaromatic radical induced by removing one hydrogen atom from a single atom of a heteroaromatic ring system, either as a heteroaryl itself or as part of another substituent. The heteroaryl groups in question are not limited to those listed above, but include groups derived from acridine, arsindol, carbazole, β-carbolin, chroman, chromane, cinolin, furan, imidazole, indazole, indole, indoline, indidine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenantriazole, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, quinazoline, quinoline, quinolidine, quinoxaline, tetrazoline, thiadiazole, thiazoline, triazole, benzole, thiophene, triazole, xanthene, and benzole. In certain embodiments, the heteroaryl group is a 5- to 20-membered heteroaryl. In certain embodiments, the heteroaryl group is a 5- to 10-membered heteroaryl. In certain embodiments, the heteroaryl group is a group derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole, and pyrazine.

[0034] "Heterocyclic," "heterocyclic formula," "heterocycloalkyl," and "heterocyclyl" refer to saturated or unsaturated groups having a monocyclic or multiple fused rings, including fused-bridged ring systems and spirocyclic systems, and having 3 to 20 ring atoms 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 may be cycloalkyl, aryl, or heteroaryl, provided that the attachment site is via a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety.

[0035] Examples of heterocyclic and heteroaryl compounds include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indidine, isoindole, indole, dihydroindole, indazole, purine, quinoridine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carborin, phenanthridine, acridine, phenanthroline, isothiazole, phena Examples include din, isoxazole, 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, morpholinil, thiomorpholinil (also called thiamorpholinil), 1,1-dioxothiomorpholinil, piperidinil, pyrrolidine, and tetrahydrofuranyl.

[0036] Unless otherwise restricted by the definition of substituents, "substituted heteroaryl" refers to a heteroaryl group substituted with 1 to 5 substituents selected from 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, azide, 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, -SO2-aryl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl, or a heteroaryl group substituted with 1 to 3 substituents.

[0037] The terms "alkalyl" or "aralkyl" as defined herein refer to alkylene, substituted alkylene, and aryl groups, and to alkylene-aryl and substituted alkylene-aryl groups.

[0038] "Alkylene" refers to a divalent aliphatic hydrocarbyl group having preferably 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, which can be either linear or branched, and optionally -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 -is interrupted by one or more groups selected from the following. This term includes, for example, methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), etc. "Substituted alkylene" refers to an alkylene group having 1 to 3 hydrogens that are replaced by substituents as described for carbon in the definition of "substituted" below.

[0039] "Substituted" refers to a group in which one or more hydrogen atoms are independently substituted with the same or different substituents. The substituents in question are not limited to these, but include alkylenedioxy (such as methylenedioxy), -M, and -R. 60 , -O - ,=O,-OR 60 , -SR 60 , -S - ,=S,-NR 60 R 61 ,=NR 60 , -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2O - -S(O)2OH, -S(O)2R 60 -OS(O)2O - -OS(O)2R 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 It includes, M is a halogen, and R 60 , R 61 , R 62 , and R 63 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 it is bonded, it forms a cycloheteralkyl or substituted cycloheteralkyl ring, R 64 and R 65 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 bonded nitrogen atom, they form a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certain embodiments, the substituents are -M, -R 60 ,=O,-OR 60 , -SR 60 , -S - ,=S,-NR60 R 61 、 =NR 60 、 -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2R 60 、 -OS(O)2O - 、 -OS(O)2R 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[[ID=2\6]] 60 、 -C(O)NR 60 R 61 、 -C(O)O - 、 -NR 62 C(O)NR 60 R 61 includes. In certain embodiments, the substituent is -M, -R 60 [[ID=)1]]、 =O, -OR 60 、 -SR 60 、 -NR 60 R 61 、 -CF3, -CN, -NO2, -S(O)2R 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 - includes. In certain embodiments, the substituent is -M, -R 60 、 =O, -OR 60 、 -SR 60 、 -NR 60 R 61 、 -CF3, -CN, -NO2, -S(O)2R 60 、 -OP(O)(OR 60 )(OR[[ID=8\4]] 61 )、 -C(O)R 60 、 -C(O)OR 60 、 -C(O)O - includes, R 60 、 R61 , and R 62 As stated above, for example, a substituent may have one, two, or three substituents selected from methylenedioxy substituents, halogen atoms, (1-4C)alkyl groups, and (1-4C)alkoxy groups. When the substituted group is an aryl or heteroaryl group, the substituent (as described herein, for example) may be called an "aryl substituent."

[0040] "Sulfonylamino" is -NR 21 SO2R 22 It refers to the base, and in the formula, R 21 and R 22 R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic. 21 and R 22 These are optionally linked together with the atoms to which they are bonded to form heterocyclic or substituted heterocyclic groups, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic groups are as defined herein.

[0041] It should be understood that polymers achieved by defining substituents having further substituents themselves (for example, substituted aryls that are themselves substituted with a substituted aryl group and have a substituted aryl group as a substituent that is further substituted with a substituted aryl group) are not intended to be included herein. In such cases, the maximum number of such substitutions is 3. For example, the sequential substitution of substituted aryl groups specifically intended herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0042] Unless otherwise specified, the naming of substituents not explicitly defined herein is achieved by naming the terminal portion of the functional group followed by the functional group adjacent to the attachment point. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0043] With respect to any of the groups disclosed herein that include one or more substituents, it is understood that such groups do not include any substitutions or substitution patterns that are sterically impossible and / or synthetically impossible. In addition, the compounds of the subject matter include all stereochemical isomers resulting from the substitutions of these compounds.

[0044] Other definitions of terms may appear throughout this specification.

[0045] As summarized above, water-soluble light-harvesting multichromophores having multiple pendant chromophore groups are provided. The light-harvesting multichromophore has a polymer backbone containing non-conjugated repeating units and multiple pendant donor chromophore groups, each independently linked to the non-conjugated repeating units of the polymer backbone. The pendant chromophore groups may be BODIPY groups substituted with one or more water-soluble groups. Polymer tandem dyes based on the multichromophores of the subject are also provided, which further include acceptor fluorophores linked to the non-conjugated repeating units of the polymer backbone and configured in a vicinity that accepts energy to at least one pendant donor chromophore group of the light-harvesting multichromophore. Labeled specific binding members containing the polymer tandem dyes of the subject are also provided. Methods for evaluating a sample for the presence of a target analyte and methods for labeling target molecules for which the polymer tandem dye of the subject finds use are also provided. Systems and kits for practicing the methods of the subject are also provided.

[0046] Before the present invention is described in more detail, it should be understood that the present invention is not limited to the specific embodiments described and is therefore naturally subject to change. Furthermore, since the scope of the present invention is limited only by the appended claims, it should also be understood that the terms used herein are merely for describing specific embodiments and are not intended to limit them.

[0047] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that each intermediate value up to one-tenth of the lower limit unit between the upper and lower limits of that range and any other stated or intermediate value within that stated range is included within the scope of the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, subject to any specifically excluded restrictions within the stated range, and are also included within the invention. If a stated range includes one or both of the restrictions, the range excluding either or both of those included restrictions is also included within the invention.

[0048] In this specification, the term “approximately” is used to indicate a specific range before a number. In this specification, the term “approximately” is used to provide literal support for the exact number it precedes, as well as for any number that is close to or approximates the number it precedes. When determining whether a number is close to or approximates a specifically enumerated number, a close or approximate unenumerated number may, in the context in which it is presented, provide a substantial equivalent of the specifically enumerated number.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Any methods and materials similar or equivalent to those described herein may also be used in carrying out or testing the present invention, but representative exemplary methods and materials are described herein.

[0050] All publications and patents cited herein are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe methods and / or materials relating to the cited publications. Any citation of any publication is for its disclosure prior to the filing date and shall not be construed as acknowledging that the present invention does not have prior rights to such publication by prior invention. Furthermore, the publication dates provided may differ from the actual publication dates which may need to be independently verified.

[0051] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context otherwise explicitly indicates. It should also be noted that the claims may be drafted to exclude any element. Therefore, this statement is intended to function as an antecedent for the use of exclusive terms such as “simply,” “only,” etc., in relation to the enumeration of elements of the claims or the use of “negative” restrictions.

[0052] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated from or combined with any of the features of several other embodiments without departing from the scope or spirit of the invention. Any enumerated method may be performed in the order of the enumerated events, or in any other logically possible order.

[0053] Apparatus and methods are described or will be described for grammatical fluidity with functional description, but claims should not necessarily be construed as being limited by constructing “means” or “step” limitations unless expressly formulated under 35 U.S. SC § 112, and should be granted the meaning of the definitions provided by the claims and the full scope of the equivalent under the judicial theory of equivalents, and if the claims are expressly formulated under 35 U.S. SC § 112, it should be explicitly understood that a full statutory equivalent should be granted under 35 U.S. SC § 112.

[0054] In further describing the present invention, the light-harvesting multichromophore containing the acceptor fluorophore and the associated polymer tandem dye will first be described in more detail. Next, the labeled specific binding member containing the polymer tandem dye of the subject will be described. Then, the target methods in which the polymer tandem dye of the subject will find use will be considered. Systems and kits that may be used in practicing the methods of this disclosure will also be described.

[0055] Light-gathering multichromophore As summarized above, the disclosure includes light-collecting multichromophores having a modular scaffold to which pendant light-absorbing chromophore groups are attached. The term “pendant group” refers to a side chain group that is attached to the main chain of the modular scaffold but is not part of the main chain. In contrast to the light-absorbing comonomers of conjugated polymer dyes, the pendant light-absorbing chromophore groups of the subject multichromophores are not pi-conjugated to one another and do not form a delocalized pi-electron system. Rather, the modular scaffold of the subject multichromophores provides a configuration of multiple light-absorbing chromophore groups in a compact area sufficient for efficient energy transfer between chromophores (see, e.g., Figure 6A) and, if present, to acceptor fluorophores (see, e.g., Figure 6B). Combining this configuration of pendant light-absorbing chromophore groups forms a light-collecting multichromophore having an absorption wavelength (e.g., as described herein) to which the optically active chromophore groups absorb light and form an excited state. Therefore, the light-absorbing chromophore groups are configured in a neighboring area that accepts energy from each other, and when present, they can donate energy to the acceptor fluorophore.

[0056] The terms “light-gathering multichromophore” and “polymer dye” are used interchangeably and refer to the polymers of this disclosure having multiple pendant chromophore groups capable of capturing light having a specific maximum absorption wavelength and converting it into light emitted at a longer maximum emission wavelength. The polymers may have a main chain that is saturated or partially unsaturated.

[0057] Further pendant groups, such as acceptor fluorophores, secondary donor chromophore groups, linkers, and chemoselective tags capable of biomolecular conjugation and water solubility, can also be attached to the modular scaffold. In some cases, acceptor fluorophores can be installed in conjunction with two types of donor chromophores (primary and secondary donor chromophores) to provide desired fluorescence emission from the acceptor fluorophores. The number and positioning of acceptor fluorophores relative to the configuration of the pendant donor chromophores can be controlled.

[0058] The specific configuration of the pendant group can be determined and controlled by the arrangement of repeating units of the underlying modular scaffold to which the pendant group is attached. The subject multichromophore can provide a water-soluble light-harvesting multichromophore by including multiple water-soluble groups attached to the scaffold and / or pendant group at any convenient location. The modular scaffold can consist of repeating units that form a polymer backbone having side-chain groups to which the pendant group can be attached. The repeating units can be arranged in various configurations to provide a water-soluble light-harvesting multichromophore with desired spectral properties. The distance and arrangement of sites for covalent bonding between the pendant donor chromophore and the acceptor fluorophore (if present) can be controlled to provide a desired energy transfer process. This can result in desirable high light-harvesting and signal amplification characteristics.

[0059] As shown in Figure 6A, the configuration of the pendant donor chromophore group can exhibit self-quenching of fluorescence compared to an unquenched isolated donor chromophore group when excited by incident light. Self-quenching means a fluorescence increase of 10% or more, e.g., 20%, 25%, 30%, 40%, or 50% or more, compared to an unquenched isolated donor chromophore group.

[0060] Modular scaffolds can consist of polymer backbones of non-conjugated repeating units having any convenient configuration, such as linear, branched, or dendrimer configurations. The polymer backbone can be a linear polymer. The polymer backbone can be branched. In some cases, a light-harvesting multichromophore comprises multiple pendant donor chromophore groups, each independently linked to a non-conjugated repeating unit of the polymer backbone. The configuration of the pendant groups can be set during or after the synthesis of the polymer backbone. The incorporation of the pendant groups can be achieved in a sequence-specific manner, depending on the specific synthesis method used, via random, block, or stepwise synthesis.

[0061] The term “unit” refers to a structural subunit of a polymer. The term “unit” includes monomers, comonomers, coblocks, repeating units, and so on. A “repeating unit” or “repeat unit” is a subunit of a polymer defined by the minimum number of distinct structural features required for a unit to be considered a monomer, such that when the unit is repeated n times, the resulting structure describes the polymer or its block. In some cases, a polymer may contain two or more distinct repeating units; for example, when the polymer is a multiblock polymer, a random arrangement of units, or a defined sequence, each block may define a distinct repeating unit. Various arrangements of repeating units or blocks are possible, and it is understood that in the descriptive formulas of multichromophores of the subject matter described herein, any convenient linear arrangement of various lengths may be included within the overall polymer structure. A polymer may also be represented by a formula with respect to the molar percentage value of each unit in the polymer, and it is understood that such a formula may represent various arrangements of repeating units, such as random or multiblock polymers, or defined residue sequences. In some cases, the repeating units of a polymer contain a single monomer group. In certain cases, the repeating units of a polymer contain two or more monomer groups, i.e., comonomer groups, for example, two, three, four or more comonomer groups. The term "comonomer" or "comonomer group" refers to a polymer structural unit that can itself be part of the repeating units of the polymer.

[0062] The light-harvesting multichromophore comprises a modular scaffold having a linear polymer backbone of non-conjugated repeating units. The modular scaffold may have a polymer backbone containing a random configuration of non-conjugated repeating units. The modular scaffold may have a polymer backbone containing a block or coblock configuration of non-conjugated repeating units. Alternatively, the modular scaffold may have a polymer backbone containing a specific defined sequence of non-conjugated repeating units, e.g., amino acid residues of a polypeptide sequence. These configurations can be characterized by polymer segments of repeating units (e.g., as described herein), and these segments themselves may be repeated throughout the modular scaffold.

[0063] "Non-conjugated" means that at least a portion of the repeating units contain saturated back-chain groups (e.g., groups with two or more consecutive single covalent bonds) that exclude pi-conjugation or extended delocalization electronic structures along the polymer back-chain from one repeating unit to the next. Even if one repeating unit is not conjugated to an adjacent repeating unit, such a repeating unit may contain one or more isolated unsaturated groups, including unsaturated bonds (e.g., unsaturated bonds of alkenylene or alkynylene groups) and / or aryl or heteroaryl groups, and these groups may be part of the back-chain. In some cases, each repeating unit of the polymer back-chain contains one side chain containing a linked pendant group or a chemoselective tag for linking to a pendant group.

[0064] In some cases, the multichromophore contains a segment of formula (I),

[0065] [ka]

[0066] During the ceremony, Each M 1 and M 2 These are, independently, unsaturated comonomers. Each S 1and S 2 These are independent, non-conjugate spacer units. Each D 1 M 1 A pendant light-absorbing chromophore (for example, as described herein) is connected to it, each Z 1 M 2 It is a chemoselective tag linked to, x is 75 mol% or more, y is less than 25 mol%, and * indicates the attachment of the multichromophore to the polymer backbone.

[0067] In some cases of equation (I), M 1 and S 1 is the first repeating unit (M 1 ~S 1 ) forms M 2 and S 2 This is the second repeating unit of the polymer backbone (M 2 ~S 2 ) forms. The first and second repeating units can be arranged randomly or in a coblock configuration. In the first repeating unit, D 1 D is the first chemoselective tag. 1 M via conjugation to the precursor 1 It can be linked to. In the second iteration unit, Z 1 The group is used to attach a second chemoselective tag (Z) to a pendant group such as a second light-absorbing chromophore, acceptor fluorophore, or (for example, as described herein) a linked biomolecule. 2 ) can be further conjugated to the target molecule via . In a particular case of formula (I), the D of the first repeating unit 1 The pendant base comprises two or more (e.g., two or three) different types of pendant focusing chromophores that together provide a focusing multichromophore system. In certain cases of formula (I), the first repeating unit D 1 The base of all the pendants is the same.

[0068] In some cases of equation (I), x 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 cases of equation (I), y 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, 1 mol% or less.

[0069] Any convenient unsaturated comonomer is, for example, M in the multichromophore of the subject of formula (I). 1 and M 2 They can be used as a base. An unsaturated comonomer means a comonomer having at least one unsaturated covalent bond in the polymer backbone. The unsaturated comonomers in question include, but are not limited to, aryl or heteroaryl comonomers, alkynyl comonomers (e.g., ethynylene) or segments, and alkenyl comonomers (e.g., vinylene) or segments. The aryl or heteroaryl comonomers in question that find use in multichromophores (e.g., of formula (I)) include, but are not limited to, phenyl comonomers, biphenyl comonomers, benzoxazole comonomers, benzothiazole comonomers, polyphenylene comonomers, and condensed tricyclic comonomers, e.g., fluorene comonomers, carbazole comonomers, silole comonomers, and crosslinked biphenyl comonomers. The aryl or heteroaryl comonomers may be further optionally substituted, for example, as described herein. In some cases of formula (I), each M 1 and M 2 It independently comprises one or more groups selected from fluorene, carbazole, silol, biphenylene, and phenylene.

[0070] The light-harvesting multichromophore of formula (I) comprises a polymer backbone of non-conjugated repeating units, with each S 1 and S 2 It is independent of the adjacent M 1 and / or M 2It is a saturated spacer unit that eliminates pi conjugation between comonomers. In some cases, S 1 and S 2 These are independently selected from divalent polyethylene glycol (PEG) and divalent modified PEG groups. Divalent means a PEG or modified peg linker that connects two adjacent comonomers. In certain cases, the PEG or modified PEG is 3 to 100 polyethylene glycol units, such as 6 to 100 units (e.g., 6 to 100 PEGs). 100 ) includes.

[0071] In some cases, the multichromophore contains a segment of equation (II),

[0072] [ka]

[0073] During the ceremony, The polymer backbone of the non-conjugated repeating units is composed of SMs, each of which is an independent non-conjugated comonomer. 1 SM 2 , and SM 3 Contains a copolymer, Each D 1 SM 1 It is a pendant light-absorbing chromophore connected to it, each Z 1 SM 2 It is a chemoselective tag linked to, each Z 2 SM 3 An optional side chain group linked to, x is 50 mol% or more, y+z is less than 50 mol%, and * indicates the attachment of the multichromophore to the polymer backbone.

[0074] Z 2 The side chain may be absent or may be any convenient side chain group, such as a light-absorbing chromophore, chemoselective tag, linker, linked biomolecule, acceptor fluorophore, WSG, etc. In certain cases of formula (II), SM3 is, Z 2 It is a spacer comonomer in which no spacer comonomer exists. In certain cases of formula (II), SM 3 This is the second pendant light-absorbing chromophore, Z 2 It is a comonomer containing a group, and each D 1 and each Z 2 It provides a light-gathering multichromophore system together. In some cases, SM 3 This is the second chemoselective tag (Z 2 ), for example, Z provides selective placement of the target part. 1 It is a comonomer containing a protective functional group or tag orthogonal to it.

[0075] In certain cases of equation (II), 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 cases of equation (II), 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 cases of equation (II), 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 cases of equation (II), z is at least 1 mol% and 10 mol% or less, for example, 5 mol% or less, or even less.

[0076] In a specific case of equation (II), SM 3 It does not exist, i.e., z is 0 mol%. Therefore, the multichromophore can contain the segment of equation (III),

[0077] [ka]

[0078] During the ceremony, The polymer backbone of the non-conjugated repeating units is composed of SMs, each of which is an independent non-conjugated comonomer. 1 and SM 2 Contains a copolymer, Each D 1 SM 1 It is a pendant light-absorbing chromophore connected to it, each Z 1 SM 2 It is a chemoselective tag linked to, x is 75 mol% or more, y is less than 25 mol%, and * indicates the attachment of the multichromophore to the polymer backbone.

[0079] In a particular embodiment of formula (III), SM 1 and SM 2 Each of these is independently a saturated non-conjugated comonomer, for example, a comonomer that provides only a single covalent CC bond. In some embodiments of formula (III), SM 1 and SM 2 Each of these is independently a partially saturated non-conjugated comonomer, for example, a comonomer that provides an isolated double C=C covalent bond in the main chain of a saturated covalent bond. The first and second repeating units of formula (III) (SM 1 and SM 2 The elements can be arranged in a random configuration, a block, a coblock configuration, or a specific sequence. In the first repeating unit, D 1 D is the first chemoselective tag. 1 SM via conjugation to the precursor 1 It can be linked to the second repeating unit (SM 2 ) then, Z 1 The group is used to attach a second chemoselective tag (Z) to a pendant group such as a second light-harvesting chromophore, acceptor fluorophore, or (for example, as described herein) a linked biomolecule. 2 ) can be further conjugated to the target molecule via . In a particular case of formula (III), the D of the first repeating unit 1The pendant group comprises two or more (e.g., two or three) different types of pendant light-absorbing chromophores that together provide a light-collecting multichromophore system. In certain cases of formula (III), the D of the first repeating unit 1 The base of all the pendants is the same.

[0080] In some cases of equation (III), x 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 cases of equation (III), y 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, 1 mol% or less.

[0081] A polymer backbone in a multichromophore of formula (II) or (III) can be provided using any convenient comonomer. Target comonomers that find use in the preparation of fully saturated or partially saturated polymer backbones include, but are not limited to, acrylates, methacrylates, acrylamides, polystyrenes, ROMP (ring-opening metathesis polymerization) monomers, ADMET (acyclic diene metathesis) monomers, cyclic carbonates, monomers derived from polyethylene glycol, and monomers derived from polyethylenemine. Comonomers may optionally be substituted, for example, with chemoselective tags. Comonomers may be polymerized or linked using any convenient chemistry, but are not limited to, alkene polymerization, ring-opening polymerization, radical polymerization, and click chemistry, or conjugation between compatible chemoselective functional groups or tags. The target ADMET monomers are not limited to these, but include those described by Mutlu et al. ("Acyclic diene metathesis: a versatile tool for the construction of defined polymer architectures", Chem. Soc. Rev., 2011, 40, 1404-1445). In some cases, SM 1 SM2 , and / or SM 3 It has the following formula:

[0082] [ka]

[0083] During the ceremony, R 21 is, -L 1 -D 1 or -L 2 -Z 1 D 1 It is a pendant donor chromophore, Z 1 is a chemoselective tag, L 1 and L 2 It is an optional linker, n and m are independent integers between 1 and 6 (for example, 1 or 2). * indicates the attachment of a multichromophore to the polymer backbone.

[0084] The ROMP monomers covered are not limited to these, but include those described by Song et al. ("Scope of the Ring-Opening Metathesis Polymerization (ROMP) Reaction of 1-Substituted Cyclobutenes", J.Am.Chem.Soc., 2010, 132(30), pp 10513-10520). In some cases, SM 1 SM 2 , and / or SM 3 It has one of the following formulas:

[0085] [ka]

[0086] During the ceremony, R 21 is, -L 1 -D 1 or -L 2 -Z1 D 1 It is a pendant donor chromophore, Z 1 is a chemoselective tag, L 1 and L 2 is any linker, X is either CH2 or O. * indicates the attachment of a multichromophore to the polymer backbone.

[0087] In some cases, SM 1 SM 2 , and / or SM 3 This includes one of the following formulas:

[0088] [ka]

[0089] During the ceremony, R 21 is, -L 1 -D 1 or -L 2 -Z 1 And, D 1 It is a pendant donor chromophore, Z 1 It is a chemoselective tag, L 1 and L 2 It is an optional linker, X is O or NR'', R' is H or a lower alkyl group (e.g., methyl). R'' is H, lower alkyl, substituted lower alkyl, and WSG. * indicates a connection to the polymer backbone of a multichromophore. In some cases, the polymer backbone comprises a mixture of polystyrene and acrylate or acrylamide-derived comonomers (e.g., as described herein).

[0090] Multichromophores may have a hydrocarbon backbone prepared using any convenient polymerization method. In some cases, the hydrocarbon backbone is derived from acrylates, acrylamides, or styrene comonomers, or their derivatives. In some cases, the multichromophore is represented by formula (X),

[0091] [ka]

[0092] During the ceremony, Each D 1 It is, independently, a pendant donor chromophore, each Z 1 is a chemoselective tag (as described herein, for example), Each L 1 , L 2 , and L 3 It is, independently, a linker, a, b, and c are the mol% values ​​of each comonomer. d represents the total polymerization or average length of the polymer (for example, d is 2-1000, such as 2-500, 2-200, 2-100, or 2-50), WSG is a water-soluble group (as described herein, for example), G 1 and G 2 Each of these is independently selected from terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, linkers, and linked specific binding members. In some cases of equation (X), c=0. In some cases of equation (X), a>0 and b>0. In some cases of equation (X), 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 cases of equation (X), 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, 1 mol% or less. In some cases of equation (X), a is 65-95 mol%, b is 5-35 mol%, c is 0-30 mol%, and a+b+c=100%. In a particular case of equation (X), L 1 ~L 3 This involves linking to the main chain of a polymer selected from -COO-, -CONR''-, -Ph-, and -O-, where R'' is H, a lower alkyl, a substituted lower alkyl, or a WSG. Such linking is D 1 , Z 1 , and WSG can be used to connect to the polymer backbone.

[0093] In a specific case of equation (X), L 1 ~D 1 It is represented by one of the following:

[0094] [ka]

[0095] In the formula, R 4 is H, a lower alkyl group, a substituted lower alkyl group, or a WSG. In certain cases of formula (X), the WSG is a water-soluble group as described in any one of the embodiments and structures of such a group described herein.

[0096] Multichromophores may have a main chain derived from comonomers linked via click chemistry-conjugated reactions or groups (as described herein, for example). Any convenient divalent comonomer group can be derivatized with terminal chemoselective tags and polymerized via conjugation of compatible chemoselective tags. Figures 7A and 7B illustrate exemplary schemes for polymerization of comonomers having alkyne and azide chemoselective tags via click chemistry. In some cases, the comonomer contains one or more ethylene oxide or ethylene amino groups that constitute part of the main chain of the polymer. Such groups can provide the desired water solubility of the resulting polymer dye. The comonomer may further contain a trivalent unit for linking to a donor or acceptor dye, or a side chain group such as a WSG. In some cases, the comonomer contains a propylene oxide or propylene amino group in the main chain, which is further substituted at the 2-position with a side chain group or substituent. This group may contain a linked chemoselective tag, a linked donor or acceptor dye, or a linked WSG.

[0097] In some cases of equation (II), the multichromophore is of equation (XXI),

[0098] [ka]

[0099] During the ceremony, The polymer backbone of the non-conjugated repeating units is linked via groups T, which are products of click chemistry or chemoselective group-conjugation reactions (e.g., azide-alkyne click chemistry) (SM). 1 SM 2 , and SM 3 Contains a copolymer, SM 3 This optionally includes a concatenated WSG. Each D 1 SM 1 It is a pendant light-absorbing chromophore connected to it, each Z 1 SM 2 It is a chemoselective tag linked to, each Z 2 SM 3 An optional side chain group linked to, x is 50 mol% or more, y+z is less than 50 mol%, * represents the attachment of a multichromophore or terminal group to the polymer backbone, as described herein. In a specific case of formula (XXI), SM 1 SM 2 , and SM 3 This comprises repeating units selected from ethylene oxide, ethylene amino, disubstituted propylene oxide, and disubstituted propylene amino. In some cases of formula (II), each T is a 1,4-substituted 1,2,3-triazole, i.e., the product of an azido-alkyn click chemistry conjugation reaction.

[0100] In some cases of formula (XXII), SM 1 SM 2 , and / or SM 3 It has the following structure:

[0101] [ka]

[0102] During the ceremony, Each X is independently O or NR 31 And R 31 is H, alkyl, substituted alkyl, alkanoyl, or substituted alkanoyl, Each r and s is independently 1 to 6 (e.g., 1, 2, or 3). Each d and e is independently 1 to 12 (for example, 1 to 6 such as 1, 2, 3, 4, 5, or 6). t is either 0 or 1, D 1 It is a pendant donor chromophore, Z 1 is a chemoselective tag (as described herein, for example), WSG is a water-soluble group (as described herein, for example), Each L 1 , L 2 , and L 3 It is, independently, a linker, * indicates connection to 1,4-substituted 1,2,3-triazole (T).

[0103] The above SM 1 ~SM 3 With respect to any of the comonomers from which the structure is derived, it is understood that either an azide or an alkyne group may be available at the end of the comonomer for linking during polymerization. Thus, 1,4-substituted 1,2,3-triazole(T) can exist in one of two possible orientations, as follows:

[0104] [ka]

[0105] The ends of the polymer backbone can contain any convenient end groups, such as azide or alkyne groups, linkers, or linked specific binding moieties.

[0106] Exemplary multichromophore structures and their precursors are shown in Example 3 of the experimental section and in the following structures.

[0107] [ka]

[0108] During the ceremony, G 1 is a terminal group (as described herein, for example), L 1 and L 2 It is, independently, a linker, D 1is a pendant chromophore (for example, as described herein), Each of d, e, and f is independently 1 to 6. n is between 1 and 1000 (for example, 2 to 1000, 2 to 500, 2 to 100, or 2 to 50), Each R 4 1 is selected from alkyl, substituted alkyl, and WSG. A "linker" is a linker that contains an optional chemoselective functional group for, for example, conjugation to a comonomer or biomolecule. In some cases of formula (XXI), L 1 -L 3 This involves linking a polymer selected from -NHCO-alkyl groups to the main chain.

[0109] The cyclic carbonates and protected carbonate monomers of interest that may be adapted for use in the preparation of the polymer backbone of the multichromophore of interest (see, for example, formula (IX) described herein) include, but are not limited to, those described in Barnes et al. in WO2013036532, Cooley et al. (J.Am.Chem.Soc., 131, 45, 1640-3, 2009), and Rothbard et al. in USPatent 7, 169, 814. Such monomers are utilized in polymerization reactions using an initiator and a cyclic carbonate monomer in a suitable supply ratio to provide the polymer backbone. Alternative protected carbonate monomers can be assembled in stepwise synthesis to provide the defined sequence. In some cases of formulas (II) to (III), the polymer backbone has a polycarbonate backbone. Thus, the multichromophore may have formula (IX).

[0110] [ka]

[0111] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, each Z 1 These are independently chemoselective tags, Each L 1 and L 2 It is, independently, a linker, x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2 Each is independently selected from the group consisting of terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, linkers, and linked specific binding members.

[0112] In certain embodiments, the iterative units (SM) of equations (II) to (III) 1 SM 2 , and / or SM 3 The molecules are arranged in a defined linear sequence. Any convenient comonomer that can be polymerized in a defined stepwise manner can be used to construct the multichromophore of formulas (II) to (III). The comonomer may be derived from an amino acid, a peptoid monomer, or a protected or cyclic carbonate monomer.

[0113] In some cases of formulas (II) to (III), the polymer backbone is a polypeptide having a defined sequence of α-amino acid residues and / or β-amino acid residues. Two types of β-amino acids and polypeptides can be found to be used in the polymer backbone of the subject multichromophore, those having a side chain group next to the amine are called β3 peptides / residues, and those having a side chain group next to the carbonyl group are called β2 peptides / residues. In certain embodiments, the multichromophore is of formula (IV),

[0114] [ka]

[0115] During the ceremony, Each D 1These are independently pendant light-absorbing chromophore groups, each Z 1 These are independently chemoselective tags, Each L 1 and L 2 It is, independently, a linker, p1 and q1 are independently either 0 or 1, and p1 + q1 ≤ 1. p2 and q2 are independently 0 or 1, and p2 + q2 ≤ 1. x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2 Each of these is independently selected from terminal groups, polymer segments, light-absorbing (e.g., donor) chromophore groups, acceptor fluorophores, linkers, and linked specific binding members.

[0116] In some embodiments of formula (IV), p1 and p2 are each 0, and q1 and q2 are each 1 (e.g., a β3-amino acid residue). In some embodiments of formula (IV), p1 and p2 are each 1, and q1 and q2 are each 0 (e.g., a β2-amino acid residue). In some cases, p1, p2, q1, and q2 are each 0, and the multichromophore is of formula (V) below,

[0117] [ka]

[0118] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, each Z 1 These are independently chemoselective tags, L 1 and L 2 Each of them is an independent linker, x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2 Each of these is independently selected from terminal groups, polymer segments, light-absorbing (e.g., donor) chromophore groups, acceptor fluorophores, linkers, and linked specific binding members. It is understood that the multichromophore described by formula (V) includes any convenient arrangement of comonomers in a defined linear sequence having a defined mol% ratio of total x and y. In some cases, Z 1 The contained comonomers are spaced apart throughout the entire polymer backbone sequence and are therefore always adjacent on both sides to one or more D1-containing comonomers.

[0119] In certain cases of equation (V), the multichromophore includes the segment of equation (VI),

[0120] [ka]

[0121] During the ceremony, Each D 1 These are independently pendant light-absorbing chromophore groups, each Z 1 These are independently chemoselective tags, Each L 1 and L 2 It is, independently, a linker, n and p are each independent integers between 1 and 20, and in the equation, n + p ≥ 2. m is either 1 or 2.

[0122] In some cases of equation (VI), n and p are each independently between 1 and 10, such as 2 to 20, 3 to 10, or 3 to 6. In some examples of equation (VI), n+p is an integer between 2 and 20, such as 3 to 20, 4 to 20, 5 to 20, 5 to 15, or 5 to 12. In a particular embodiment of equation (VI), m is 1.

[0123] The subject multichromophore may contain multiple segments of formula (VI), each segment containing a comonomer D 1 One isolated Z containing a comonomer adjacent to the block 1 This includes. In some cases, the multichromophore is, for example, 3-20, 4-20, 5-20, 5-15, or 5-12 D 1 Blocks of comonomers containing 2 to 20 D 1 Two isolated Z molecules separated by the block of contained comonomers. 1 To provide a comonomer containing two or more segments of formula (VI) oriented adjacent to each other, the multichromophore comprises a segment of the block copolymer of formula (VII),

[0124] [ka]

[0125] In the formula, each (n) q and each (p) q These are independent integers from 1 to 20, and in the expression, for each segment of q, (n) q +(p) q The value is ≥ 3, and q is an integer between 1 and 100.

[0126] It is understood that the α-amino acid residues in formulas (V) to (VII) can be substituted with β2-amino acid residues or β3-amino acid residues to provide the corresponding polypeptide product. In formulas (V) to (VII), D 1 D is the first chemoselective tag. 1 It can be linked to the amino acid side chain linker L1 via conjugation to the precursor. In equations (V) to (VII), Z 1 The group is used to attach a second chemoselective tag (Z) to a pendant group such as a second light-harvesting chromophore, acceptor fluorophore, or (for example, as described herein) a linked biomolecule. 2) can be further coupled to the target molecule.

[0127] Any suitable amino acid may be adapted for use to provide a polymer backbone to which a pendant group can be covalently bonded. Amino acids may be naturally occurring or unnatural. For example, amino acids such as lysine and ornithine have side-chain amino groups suitable for conjugation with amino-reactive groups such as activated carboxylic acids. Cysteine ​​contains side-chain thiol groups suitable for conjugation with thiol-reactive groups such as maleimide or haloacetyl. Aspartic acid and glutamic acid have side-chain carboxylic acid groups that can be conjugated with nucleophiles such as amino groups. Methods for preparing multichromophores of the subject, including peptide synthesis methods, are described herein.

[0128] In some cases, the polymer backbone of the multichromophore has one or more of the following polypeptide sequence segments: XYXX XXYXX XXXYXXX XXXYXXXX XXXXYXXX XXXXYXXXX XXXXXYXXXXX XXXXXXYXXXXXX XXXXXXXYXXXXXXX XXXXXXXXYXXXXXXXX XXXXXXXXXYXXXXXXXXX Y(X) n Y XY(X) n YX XXY(X) n YXX XXXY(X) n YXXX XXXXY(X) n YXXXX XXXXXY(X) n YXXXXX During the ceremony, Each X is a first amino acid residue having a side-chain-linked first chemoselective tag or a side-chain-linked pendant donor chromophore. Each Y is a second amino acid residue having a side-chain-linked second chemoselective tag or a side-chain-linked pendant acceptor fluorophore. n is an integer between 2 and 20, such as 2-10, 3-10, 4-10, or 5-10. For example, n could be 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0129] In certain embodiments, a third type of amino acid residue (Z) can be incorporated into the polymer backbone, for example, between two of the segments, in addition to one or more polypeptide segments described herein. In certain cases, a single isolated third amino acid residue is incorporated between any two of the segments described herein, for example, (segment 1)-Z-(segment 2). This third amino acid residue can be a spacer residue, or a residue having a chemoselective functional group suitable for the selective placement of the additional portion of the target, such as a second pendant light-absorbing chromophore, a chemoselective tag (e.g., a bioorthogonal click chemist tag), a linker, a linked biomolecule, an acceptor fluorophore, or a WSG. In certain cases, the additional portion of the target is incorporated into the third amino acid residue before preparation.

[0130] In certain cases, the first amino acid residue (X) contains a side-chain amino group, e.g., lysine or ornithine, or a protected version thereof. In the SPPS of the polymer backbone, these side-chain amino groups remain protected (e.g., with a Cbz or Boc protecting group) and can then be orthogonally deprotected to provide conjugation of the first residue to a pendant donor chromophore group. In certain cases, the second amino acid residue contains a side-chain thiol group, e.g., cysteine, or a protected version thereof. Similarly, in the SPPS of the polymer backbone, these side-chain thiol groups remain protected and can then be orthogonally deprotected to provide conjugation of the second residue to a target pendant group, e.g., an acceptor fluorophore.

[0131] In some embodiments, the polymer backbone of the multichromophore is derived from one or more of the following polypeptide sequence segments: KCKK (Sequence ID: 1) KKCK (Sequence ID: 2) KKYKK (Sequence ID: 3) KKKYKK (Sequence ID: 4) KKYKKK (Sequence ID: 5) KKKYKKK (Sequence ID: 6) KKKYKKKK (Sequence number: 7) KKKKYKKK (Sequence number: 8) KKKKCKKKK (Sequence ID: 9) KKKKKCKKKKK (Sequence number: 10) KKKKKKCKKKKKK (Sequence ID: 11) KKKKKKKCKKKKKKK(Sequence number: 12) KKKKKKKKCKKKKKKKK (Sequence number: 13) KKKKKKKKKCKKKKKKKKK(Sequence number: 14) KKKKCKKKKKKKKKKKKKKKK(Sequence number: 15) C(K) n C (Sequence ID: 16) KC(K) n CK (Sequence ID: 17) KKC(K) n CKK (Sequence ID: 18) KKKC(K) n CKKK (Sequence ID: 19) KKKKC(K) n CKKKK (Sequence ID: 20) KKKKKC(K) n CKKKKK (Sequence ID: 21) In the formula, each K is a lysine residue, a protected lysine residue, or a lysine residue covalently bonded to a pendant donor chromophore group via a side-chain amino group, n is an integer between 2 and 20 (e.g., 2 to 10, 3 to 10, 4 to 10, or 5 to 10, e.g., n is 2, 3, 4, 5, 6, 7, 8, 9, or 10), and C is a cysteine ​​residue or a protected cysteine ​​residue.

[0132] In some embodiments, the polymer backbone of the multichromophore is derived from one or more of the following polypeptide sequence segments: OCOO (Sequence ID: 22) OOCO (Sequence ID: 23) OOCOO (Sequence ID: 24) OOOCOO (Sequence ID: 25) OOCOOO (Sequence ID: 26) OOOCOOO (Sequence ID: 27) OOOCOOOO (Sequence number: 28) OOOOCOOO (Sequence number: 29) OOOOCOOOO (Sequence number: 30) OOOOOCOOOOO (Sequence ID: 31) OOOOOOCOOOOOO (Sequence number: 32) OOOOOOOCOOOOOOO(array number:33) OOOOOOOOCOOOOOOOO(array:34) OOOOOOOOOCOOOOOOOOO(array number:35) OOOOOOOOOOCOOOOOOOOOO(array:36) C(O) n C (Sequence ID: 37) OC(O)n CO (Sequence ID: 38) OOC(O) n COO (Sequence ID: 39) OOOC(O) n COOO (Sequence ID: 40) OOOOC(O) n COOOO (Sequence ID: 41) OOOOOC(O) n COOOOO (Sequence ID: 42) During the ceremony, O is an ornithine residue covalently bonded to a pendant donor chromophore group via a side-chain amino group, a protected ornithine residue, or an ornithine residue. n is an integer between 2 and 20 (for example, 2 to 10, 3 to 10, 4 to 10, or 5 to 10, for example, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10). C is a cysteine ​​residue or a protected cysteine ​​residue.

[0133] In some cases of formulas (II) to (III), the polymer backbone is a peptoid backbone. Therefore, the multichromophore may have formula (VIII).

[0134] [ka]

[0135] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, each Z 1 These are independently chemoselective tags, Each L 1 and L 2 It is, independently, a linker, x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2Each of these is independently selected from terminal groups, polymer segments, light-absorbing (e.g., donor) chromophore groups, acceptor fluorophores, linkers, and linked specific binding members.

[0136] It is understood that the multichromophore described by formula (VIII) includes any convenient arrangement of comonomers in a defined linear sequence having a defined mol% ratio of total x and y. In some cases, Z 1 The contained comonomers are spaced apart throughout the entire polymer backbone sequence and are therefore always adjacent on both sides to one or more D1-containing comonomers.

[0137] In some examples of equations (IV), (V), (XIII), and (IX), x 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 equations (IV), (V), (XIII), and (IX), y 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, 1 mol% or less.

[0138] pendant chromophore base Any convenient light-absorbing chromophore group can be adapted for use in the multichromophore of the subject. The terms “light-absorbing chromophore group” and “donor chromophore group” are used interchangeably and refer to pendant groups in a multichromophore that absorb light at a specific maximum absorption wavelength and transfer energy to a nearby chromophore or acceptor fluorophore, or convert it into emitted light at a longer maximum emission wavelength.

[0139] BODIPY chromophore group The pendant chromophore group can be a BODIPY group. In some cases of formulas (I) to (IX), each D 1This is independently a BODIPY group. In some cases, the BODIPY group is a pendant donor chromophore group. The term "BODIPY group" refers to the pendant group of a multichromophore containing a chromophore group having the following boron-dipyromethane (BODIPY) core structure.

[0140] [ka]

[0141] In the formula, Q is C or N, and each R is any convenient boron substituent. In some cases, Q is C. In some cases, each R is independently selected from F, OH, H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl, and substituted alkynyl.

[0142] The BODIPY core structure can be linked to the repeating unit of a multichromophore via an optional side-chain linker, or via any convenient position on the core structure. The BODIPY core structure can be further optionally substituted. In certain embodiments, the BODIPY group defines a side-chain group of a comonomer that is part of the repeating unit. Any convenient BODIPY-containing structure can be adapted as the BODIPY group for use in the multichromophore of the subject. The BODIPY-containing structures in question include, but are not limited to, those described in Loudet and Burgess in “BODIPY Dyes and Their Derivatives: Syntheses and Spectroscopic Properties”, Chem. Rev. 2007, 107(11): 4891-4932, Suzuki et al. in US Patent No. 8, 193, 350, Ulrich et al. in US Patent No. 8, 476, 461, and Ulrich et al. in US Patent No. 7, 897, 786, and their disclosures are incorporated herein by reference in their entirety.

[0143] The BODIPY pendant chromophore group can be described by formula (XI),

[0144] [ka]

[0145] During the ceremony, Q is either C or N, R 1 ~R 7 Each of these is independently H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG), and -L. 1 -Z 1 Selected from, or Optionally, R 6 and R 7 , R 2 and R 3 , R 5 and R 6 , R 3 and R 4 , R 4 and R 1 Furthermore, R 5 and R 1 Any one or more substituent pairs selected from the above form a divalent radical together, which is cyclically linked and, together with the carbon atoms to which they are bonded, provides a 5- or 6-membered condensed heterocycle, carbocyclic ring, aryl or heteroaryl ring (e.g., a 5- or 6-membered ring containing carbon atoms and 0 to 3 heteroatoms selected from O, S and N), the ring may be unsubstituted or independently alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and -L 1 -Z 1 They may be further substituted with substituents selected from, L 1 It is a linker, Z1 These are non-conjugated repeating units of the polymer backbone, Y 1 and Y 2 These are independently selected from F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and WSG. Y 1 , Y 2 , and R 1 ~R 7 One of these is linked to a non-conjugated repeating unit of the polymer backbone.

[0146] In equation (XI), substituent Y 1 , Y 2 and R 1 -R 7 It is understood that Q may be selected from groups that do not inhibit the fluorescence of the BODIPY group. In certain cases of formula (XI), Q is C. For any of the BODIPY group formulas described herein, it is understood that a corresponding formula may be included if the atom represented by Q in formula (XI) is a nitrogen atom. In certain embodiments of formula (XI), Y 1 , Y 2 , and R 1 ~R 7 One or more of these include WSG. In certain cases of equation (XI), Y 1 and Y 2 Each includes WSG. In certain cases of equation (XI), -L 1 -Z 1 The linker includes the WSG.

[0147] In equation (XI), substituent pairs R 6 and R 7 And / or R 2 and R 3This can provide a cyclically linked 5- or 6-membered condensed ring that is unsubstituted or substituted. In some cases of formula (XI), the 5- or 6-membered condensed ring is an aryl or heteroaryl ring selected from furan, thiophene, pyrrole, oxazole, isoxazole, thiazole, isothiazole, imidazole, and pyrazole.

[0148] In some cases of formula (XI), the BODIPY group is that of formula (XIIa) or (XIIb),

[0149] [ka]

[0150] During the ceremony, Z 11 and Z 12 These are independently fused 5-membered or 6-membered fused heterocycles, carbon rings, aryl or heteroaryl rings, Each "i" is independently between 0 and 3. Each R 20 R in equation (X) is independent. 2 ~R 7 These are substituents defined for each other.

[0151] In certain embodiments of equations (XIIa) to (XIIb), Z 11 and Z 12 These are independently 5-membered or 6-membered condensed aryl or heteroaryl rings. In some cases, Z 11 and Z 12 The following are independently selected from furan, thiophene, pyrrole, oxazole, isoxazole, thiazole, isothiazole, imidazole, and pyrazole. In some cases of formulas (XIIa) to (XIIb), Z 11 and / or Z 12 is either furan or thiophene. In certain cases of formula (XIIa), R 1 ~R 5 None of them are connected in a ring.

[0152] In some cases of equations (XIa) to (XIIb), Z 11 and Z 12 These are independently selected from the following rings,

[0153] [ka]

[0154] During the ceremony, X is either O or S, Y is O, S, or NR, R is H, alkyl, substituted alkyl, or R of formula (XIIa)~(XIIb). 20 A substituent defined with respect to, R 21 ~R 23 These are independently H and R of equations (XIIa)~(XIIb). 20 A substituent is selected from those defined for the property.

[0155] In certain cases of equations (XIIa) to (XIIb), Z 11 and Z 12 These are independently selected from the following ring, as defined above.

[0156] [ka]

[0157] In a particular case of equation (XIIb), Z 11 and Z 12 These are the same ring. In a particular case, Z 11 and Z 12 This includes different rings.

[0158] In certain embodiments of formula (XIIa), the BODIPY group is of formula (XIIIa) or (XIIIb),

[0159] [ka]

[0160] During the ceremony, X is either O or S, R 16 and R 17 R in equation (I) 6 and R 7 These are substituents defined for each other.

[0161] In certain cases of equation (XIIIa) or (XIIIb), R 1 ~R 5 None of them are connected in a ring.

[0162] In certain embodiments of formulas (XI) and (XIIb), the BODIPY group is of formula (XIVa) or (XIVb),

[0163] [ka]

[0164] During the ceremony, Each X is either O or S, R 12 , R 13 , R 16 , and R 17 R in equation (I) 2 , R 3 , R 6 , and R 7 These are substituents defined for each other.

[0165] In certain embodiments of formulas (XI) to (XIVb), the BOBIPY group is a light-collecting multichromophore (e.g., -L 1 -Z 1 ) contains linked chemoselective functional groups or target molecules. In some cases, R 1 ~R 7 One of them is -L 1 -Z 1 This includes Y. In certain cases, 1 or Y 2 is, -L1 -Z 1 This includes. In certain embodiments of formulas (XI) to (XIVb), R 1 is, -L 1 -Z 1 And in the formula, L 1 It is a linker, Z 1 R is a chemoselective functional group or a light-harvesting multichromophore. In certain embodiments of formulas (XI) to (XIVb), R 4 or R 5 is, -L 1 -Z 1 In certain embodiments of equations (XI), (XIIa), and (XIIIa) to (XIIIb), R 2 or R 7 is, -L 1 -Z 1 In certain embodiments of equations (XI), (XIIa), and (XIIIa) to (XIIIb), R 3 or R 6 is, -L 1 -Z 1 In certain embodiments of equations (XIIa) to (XIIb), R 20 The substituent is -L 1 -Z 1 In certain embodiments of equations (XIIIa) to (XIIIb), R 16 or R 17 is, -L 1 -Z 1 In certain embodiments of equations (XIVa) to (XIVb), R 12 , R 13 , R 16 , or R 17 is, -L 1 -Z 1 That is the case.

[0166] In some cases, R 1 is, -L 1 -Z 1 And L 1 R is a linker having a main chain of 20 atoms or less in length (for example, as described herein). 1 In some cases, L 1The linker is selected from alkyl or substituted alkyl linkers, alkoxy or substituted alkoxy linkers, peg linkers, sulfonamide alkyl or substituted sulfonamide alkyl linkers, amide alkyl or substituted amide alkyl linkers, and alkylamide alkyl or substituted alkylamide alkyl linkers. The linker may be substituted with a WSG such as a peg group. 1 In a particular case, L 1 C1~C 12 Alkyl or substituted alkyl linker, C1-C 12 Alkoxy or substituted alkoxy linkers, C1-C 12 Amidoalkyl or substituted amidoalkyl linkers, as well as C1-C 12 Selected from alkylamide alkyl or substituted alkylamide alkyl linker. 1 In a particular case, Z 1 It is conjugated to the BODIPY group via a carboxylic acid or its active ester.

[0167] In certain cases of equations (XI) to (XIVb), R 1 This includes a repeating unit of a light-harvesting multichromophore or an optionally substituted carbocyclic or heterocyclic group linked to a chemoselective functional group. In certain cases, R 1 The group is an optionally substituted aryl or heteroaryl group, which is linked to the repeating unit of the light-harvesting multichromophore, for example, via a chemoselective functional group. The divalent carbocyclic or heterocyclic groups of interest include, but are not limited to, 1,4-cyclohexyl, 1,3-cyclohexyl, piperidinyl (e.g., 1,4-piperidinyl), piperazinyl (e.g., 1,4-piperazinyl), etc. The divalent aryl or heteroaryl groups of interest include, but are not limited to, 1,4-phenyl, 1,3-phenyl, 2,5-pyridyl, 2,6-pyridyl, 3,5-pyridyl, etc. 1 The divalent carbocyclic or heterocyclic group, or the divalent aryl or heteroaryl group, is, for example, as described in any one of the embodiments herein, L 2 -Z 1 It may also be connected to L2 It is a linking group.

[0168] In some embodiments of equation (XI), Q is C and R 1 ~R 7 Each of these is independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. In certain examples of formula (XI), Y 1 and Y 2 Each contains one or more water-soluble groups (WSGs). In some cases, Y 1 and Y 2 This is an alkynyl substituted with WSG. In some cases, Y 1 and Y 2 Each of these is an alkinyl substituted with a branch WSG. In some examples of equation (XI), Y 1 and Y 2 (Each is -CC-CH2) n -O(CH2CH2O) m -R, where n is 1 to 6, m is 2 to 50 such as 2 to 30, 2 to 20, 6 to 20, 8 to 20, or 10 to 20, and R is H, alkyl, or substituted alkyl (e.g., methyl).

[0169] In some embodiments of formula (XI), the BODIPY pendant donor chromophore group can be described by formula (XIa),

[0170] [ka]

[0171] During the ceremony, * indicates a linkage point to the non-conjugated repeating unit of the polymer backbone. Y 1 and Y 2 Each of these is an alkynyl substituted with one or more WSGs.

[0172] In some cases, Y1 and Y 2 Each of these is an alkynyl substituted with a branch WSG. In some examples of equation (XIa), each R 10 (is -CC-CH2) n -O(CH2CH2O) m -R, where n is 1 to 6, m is 2 to 50 such as 2 to 30, 2 to 20, 6 to 20, 8 to 20, or 10 to 20, and R is H, alkyl, or substituted alkyl (e.g., methyl).

[0173] In certain cases of formula (XI), the BODIPY group is that of formula (XV),

[0174] [ka]

[0175] During the ceremony, L 3 These are covalent bonds, oxo (-O-), alkylenes (e.g., C1-C6-alkylenes), -O-alkylenes, or their substituted versions. R 11 R 1 As defined, Each R 9 is an optional substituent selected from halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, where t is 0 to 4.

[0176] In a particular case of equation (XV), L 3 This is a covalent bond. In some cases of equation (XV), L 3 is an oxo. In a specific case of equation (XV), L 3 This is a covalent bond.

[0177] In a specific case of formula (XV), the BODIPY group is that of formula (XVa),

[0178] [ka]

[0179] During the ceremony, R 11 R 1 As defined, Each R 9 is an optional substituent selected from halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, where t is 0 to 4.

[0180] In some cases of equation (XV)~(XVa), R 11 L 1 -Z 1 (For example, as described herein). In some cases of formulas (XV)~(XVa), R 11 This includes sulfonamide alkyl or substituted sulfonamide alkyl linkers, amide alkyl or substituted amide alkyl linkers, or alkylamide alkyl or substituted alkylamide alkyl linkers. In certain examples of formulas (XV) to (XVa), R 2 , R 4 , R 5 , and R 7 Each of these is independently H, alkyl, or substituted alkyl. In certain examples of formulas (XV)~(XVa), R 3 and R 6 Each of these is independently H, alkyl, or substituted alkyl. In some cases, R 3 and R 6 These are H, respectively. In certain cases of equation (XV)~(XVa), R 2 , R 4 , R 5 and R 7 Each of these is independently a C1-C6 alkyl group or a substituted C1-C6 alkyl group.

[0181] In certain cases of formula (XV), the BODIPY group is formula (XVIa) or (XVIb),

[0182] [ka]

[0183] During the ceremony, * indicates a linkage point to a light-harvesting multichromophore or a repeating unit of a chemoselective functional group. Y 1 and Y 2 The following are independently selected from the group consisting of F, OH, H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl, substituted alkynyl, and WSG.

[0184] In certain cases of formula (XI), the BODIPY group is of formula (XVIIa) or (XVIIb),

[0185] [ka]

[0186] During the ceremony, n is between 0 and 12. Z 1 is a chemoselective functional group or a linked molecule of the target (for example, as described herein).

[0187] In certain cases of equations (XVIIa)~(XVIIb), n is 1~12 or 1~6, for example, 1, 2, 3, 4, 5, or 6. In certain cases of equations (XVIIa)-(XVIIb), R 2 -R 4 and R 5 -R 7 Each is independently selected from H, C1-C6 alkyl, and substituted C1-C6 alkyl. In certain cases of formulas (XVIIa) to (XVIIb), R 2 and R 4 , and / or R 5 and R 7Each is independently selected from C1-C6 alkyl and substituted C1-C6 alkyl. In certain cases of formulas (XVIIa)-(XVIIb), R 3 and / or R 6 is H. In certain cases of equations (XVIIa)~(XVIIb), R 3 and / or R 6 It is an alkyl or substituted alkyl group.

[0188] In a specific case of formula (XIIIa), the BODIPY group is that of formula (XVIII),

[0189] [ka]

[0190] During the ceremony, L 1 It is a linker, Z 1 This is a linked, non-conjugated repeating unit of a polymer backbone (as described herein, for example).

[0191] In a specific case of formula (XVIII), the BODIPY group is that of formula (XVIIIa),

[0192] [ka]

[0193] During the ceremony, L 2 is a linker (for example, L 1 (The linking group component of) Each R 9 is an optional substituent selected from halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, where t is 0 to 4.

[0194] In a specific case of formula (XIVa), the BODIPY group is that of formula (XIX),

[0195] [ka]

[0196] During the ceremony, R 12 and R 13 R in equation (XI) 2 and R 3 A substituent defined with respect to, L 1 It is a linker, Z 1 This is a linked, non-conjugated repeating unit of a polymer backbone (as described herein, for example).

[0197] In a specific case of formula (XIX), the BODIPY group is that of formula (XIXa),

[0198] [ka]

[0199] During the ceremony, L 2 is a linker (for example, L 1 (The linking group component of) Each R 9 is any substituent selected from halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, where t is 0 to 4.

[0200] In any embodiment of formulas (XI) to (XIXa) described herein, the BODIPY group is a specific -L as described in any of the following embodiments. 1 -Z 1 It can include a base.

[0201] L 1This can be a linker (as described herein) having a main chain of 20 atoms or less in length. In some cases, L 1 The linker is selected from alkyl or substituted alkyl linkers, alkoxy or substituted alkoxy linkers, peg linkers, sulfonamide alkyl or substituted sulfonamide alkyl linkers, amide alkyl or substituted amide alkyl linkers, and alkylamide alkyl or substituted alkylamide alkyl linkers. The linker may be substituted with a WSG such as a peg group. In certain cases, L 1 C1~C 12 Alkyl or substituted alkyl linker, C1-C 12 Alkoxy or substituted alkoxy linkers, C1-C 12 Amidoalkyl or substituted amidoalkyl linkers, as well as C1-C 12 The linker L is selected from alkylamide alkyl or substituted alkylamide alkyl linkers. Linker L is selected from several linking components, for example, independently, lower alkylene, substituted lower alkylene, alkenylene (-CH=CH-), substituted alkenylene, alkylylene (-CC-), substituted or unsubstituted amide (e.g., -NRCO- or -CONR-, where R is H, alkyl or substituted alkyl), substituted or unsubstituted sulfonamide (e.g., -NRSO2- or -SO2NR-, where R is H, alkyl or substituted alkyl), oxo (-O-), thio (-S-), ethylene glycol (-OCH2CH2O-), polyethylene glycol (e.g., -(CH2CH2O) n - where n may contain one group or linking group selected from 2-20 (e.g., 2-10 or 2-6, or 2, 3, 4, 5 or 6), arylene, substituted arylene, heteroarylene, substituted heteroarylene, alpha amino acid residue, β-amino acid residue, etc.

[0202] Polymer backbone (Z 1The linked non-conjugated repeating units of ) can be conjugated to the BODIPY group via any convenient chemoselective functional group, e.g., a functional group suitable for conjugation to the molecule of interest having a compatible functional group. Not limited to these, chemoselective functional groups of interest that find use in linking the BODIPY group of interest to the polymer backbone include, but are not limited to, amine groups (e.g., -NH2), carboxylic acids (-CO2H), active esters (e.g., NHS or sulfo-NHS esters), thiols, maleimides, iodoacetamides, hydroxyls, hydrazides, hydrazinos, aldehydes, ketones, azides, alkynes, tetrazines, alkenes, phosphines, and epoxides. In some cases, it is understood that the chemoselective functional group used to link the BODIPY group is a synthetic precursor or protected version of the functional group of interest that can be converted into a reactive functional group that can conjugate the polymer backbone. For example, carboxylic acids are chemoselective functional groups that can bond to amine groups on the molecule of interest. Carboxylic acids can be converted to active esters that couple with amine groups, either in situ or before coupling.

[0203] In some embodiments of equations (XI) to (XIXa), L 1 This includes optionally substituted carbocyclic or heterocyclic groups linked to the non-conjugated repeating units of the polymer backbone. In certain cases, L 1 The group is an optionally substituted aryl or heteroaryl group. The divalent carbocyclic or heterocyclic groups in question are not limited to these, but include 1,4-cyclohexyl, 1,3-cyclohexyl, piperidinyl (e.g., 1,4-piperidinyl), piperazinyl (e.g., 1,4-piperazinyl), etc. The divalent aryl or heteroaryl groups in question are not limited to these, but include 1,4-phenyl, 1,3-phenyl, 2,5-pyridyl, 2,6-pyridyl, 3,5-pyridyl, etc. 1 The divalent carbocyclic or heterocyclic group, or the divalent aryl or heteroaryl group, is, for example, as described in any one of the embodiments herein, L 2 -Z 1 It may also be connected to L 2It is a linking group.

[0204] In some embodiments of equations (I) to (IXa), -L 1 -Z 1 It is described by one of the following structures:

[0205] [ka]

[0206] During the ceremony, R 11 L 2 -Z 1 And, L 2 It is a linker, Z 1 These are non-conjugated repeating units of the polymer backbone, t is between 0 and 4. Each R 9 These are independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, sulfonic acid, and water-soluble group (WSG).

[0207] In a particular embodiment, -L 1 -Z 1 It is described by one of the following structures:

[0208] [ka]

[0209] During the ceremony, L 2 It is a linker, Z 1 These are non-conjugated repeating units of the polymer backbone, t is between 0 and 4. Each R 9These are independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, sulfonic acid, and water-soluble group (WSG).

[0210] In a particular embodiment, -L 1 -Z 1 It is described by one of the following structures:

[0211] [ka]

[0212] During the ceremony, L 2 It is a linker, Z 1 These are non-conjugated repeating units of the polymer backbone, R 32 These are H, alkyl, substituted alkyl, and water-soluble groups (WSG), L 3 The linker is selected from alkylenes (e.g., C1-C6-alkylenes), -O-alkylenes, and their substituted versions. t is between 0 and 4. Each R 9 These are independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, sulfonic acid, and water-soluble group (WSG).

[0213] In any embodiment of formulas (XI) to (XIXa) described herein, the BODIPY group is a specific Y as described in any of the following embodiments. 1 and Y 2 It can include a base. In some cases of equations (XI) to (XIXa), Y 1 It contains a water-soluble group (WSG). In certain cases, Y 1 This is an alkynyl substituted with WSG. 2 Y 1It may be the same or different. In some cases of equations (XI) to (XIXa), Y 2 The following are selected from F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. In some cases, Y 2 This is selected from F, CN, phenyl, and substituted phenyl.

[0214] In some cases of equations (XI) to (XIXa), Y 1 and Y 2 Each contains a water-soluble group (WSG). In a particular case, Y 1 and / or Y 2 is an alkynyl substituted with WSG (as described herein, for example). In certain cases, Y 1 and / or Y 2 This is an alkynyl substituted with polyethylene glycol (PEG) or modified (PEG).

[0215] In a particular case of any embodiment of formulas (I) to (IXa), Y 1 and / or Y 2 This is the formula shown below:

[0216] [ka]

[0217] During the ceremony, s is between 1 and 12. q is between 0 and 50. R 21 is H, alkyl, or substituted alkyl.

[0218] In certain cases, s is 1-6, such as 1, 2, or 3. In some cases, q is 1-50, 1-30, 2-30, 4-30, 6-30, 8-30, 10-30, 10-20, or 11-16. In certain cases, q is 10-50, such as 10-30, 10-20, or 11-16.

[0219] In certain embodiments of formulas (XI) and (XVIIa), the BODIPY group has the following structure:

[0220] [ka]

[0221] During the ceremony, Z 1 These are linked non-conjugated repeat units of the polymer backbone, n is between 0 and 6. Each R, R 2 , R 4 , R 5 , and R 7 These are independently H, C1-C6 alkyl, or substituted C1-C6 alkyl. Each q is between 6 and 20 (for example, between 10 and 20).

[0222] In certain embodiments of formulas (XI) and (XVIIa), the BODIPY group has one of the following structures:

[0223] [ka]

[0224] In certain embodiments of formulas (XV) and (XVa), the BODIPY dye has the following structure:

[0225] [ka]

[0226] During the ceremony, Z 1 These are linked non-conjugated repeat units of the polymer backbone, n is between 0 and 6. R, R 2 , R 4 , R 5 , and R 7 These are independently H, C1-C6 alkyl, or substituted C1-C6 alkyl. Each q is between 6 and 20 (for example, between 10 and 20).

[0227] In certain embodiments of formulas (XV), (XVa), and (XVIa) or (XVIb), the BODIPY dye has one of the following structures:

[0228] [ka]

[0229] In a particular embodiment of formula (XVIII), the BODIPY group has the following structure:

[0230] [ka]

[0231] During the ceremony, Z 1 These are linked non-conjugated repeat units of the polymer backbone, n is between 0 and 6. Each R, R 2 , and R 4 q is independently H, C1-C6 alkyl, or substituted C1-C6 alkyl, and each q is 6-20 (e.g., 10-20).

[0232] In a particular embodiment of formula (XVIII), the BODIPY group has one of the following structures:

[0233] [ka]

[0234] In certain embodiments of formulas (XIX) to (XIXa), the BODIPY group has the following structure:

[0235] [ka]

[0236] During the ceremony, Z 1 These are linked non-conjugated repeat units of the polymer backbone, n is between 0 and 6. Each R, and R 13 q is independently H, C1-C6 alkyl, or substituted C1-C6 alkyl, and each q is 6-20 (e.g., 10-20).

[0237] In certain embodiments of formulas (XIX) to (XIXa), the BODIPY group has the following structure.

[0238] [ka]

[0239] In a particular embodiment of formula (XIIIa), the BODIPY group has the following structure:

[0240] [ka]

[0241] In a particular embodiment of formula (XIVa), the BODIPY group has the following structure:

[0242] [ka]

[0243] In a particular embodiment of formula (XIXa), the BODIPY group has one of the following structures:

[0244] [ka]

[0245] Aryl or heteroarylchromophore group The light-absorbing chromophore group may be an aryl or heteroarylchromophore group. The aryl or heteroarylchromophore groups that find use in the multichromophores of the subject (e.g., formulas (I) to (IX)) include, but are not limited to, phenyl, biphenyl, benzoxazole, benzothiazole, polyphenylene, and condensed tricyclic groups such as fluorine, carbazole, silole, biphenyl, and crosslinked biphenyl. The aryl or heteroarylchromophore group may optionally be further substituted with, for example, a water-soluble group and / or an aryl or heteroaryl substituent that imparts the desired light-absorbing properties to the aryl or heteroaryl group. In some cases of formulas (I) to (IX), each D 1 These independently include fused tricyclic aryl or heteroaryl compounds. In some cases of formulas (I) to (IX), each D 1 It independently comprises one or more groups selected from fluorene, carbazole, silole, biphenyl, and crosslinked biphenyl.

[0246] A condensed tricyclic chromophore is a group comprising a tricyclic aromatic group having three fused rings in which two aryl or heteroaryl six-membered rings are fused to a central five- or six-membered carbon or heterocyclic ring. In some cases, the condensed tricyclic group comprises two benzo or pyrido rings fused to a central five- or six-membered carbon or heterocyclic ring. The condensed tricyclic group can be linked to the side chains of comonomers in the polymer backbone via any convenient ring atoms of the fused rings. The central five- or six-membered ring may be a carbon or heterocyclic ring, may be aromatic or partially saturated, and may further contain side chain substituents, e.g., WSGs and / or chemoselective tags or linkers to the comonomer side chains. A crosslinked biphenyl comonomer is a condensed tricyclic group having a biphenyl group, where two phenyl rings are further linked to each other via a central six-membered carbon or heterocyclic ring.

[0247] In certain examples of multichromophores (for example, in formulas (I) to (IX)), the pendant donor chromophore group is a fused tricyclic aryl or heteroaryl having one of the following formulas:

[0248] [ka]

[0249] During the ceremony, * indicates a linkage point to the non-conjugated repeating unit of the polymer backbone. Y is C(R 13 )2, -C(R 13 )2C(R 13 )2-, -C(R 13 )2Si(R 13 )2-, NR 13 , Si(R 13 )2 or Se, Each Z is independently CH, CR, or N. Each R 13These are independently H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amide, substituted amide, aralkyl, substituted aralkyl, peg portion, -L 11 -Z 1 Selected from, L 11 It is a linker, Z 1 is either a non-conjugated repeating unit and a WSG, or any two convenient R 3 The bases are optionally connected in a ring shape. Each R is independently H or one or more substituents (e.g., WSG), and two convenient R groups are optionally linked cyclically.

[0250] In some cases, R and R 13 One of these is linked to a non-conjugated repeating unit of the polymer backbone.

[0251] In certain cases, a fused tricyclic group is described by one of the following structures:

[0252] [ka]

[0253] In the formula, Y and each R are as defined above, and the fused tricyclic group can be linked to the non-conjugated repeating unit of the polymer backbone via Y or R.

[0254] In certain cases, the condensed tricyclic group is such that Y is C(R 3 )2 is fluorene. In some cases the condensed tricyclic group is carbazole, and Y is NR 3 In some cases, the condensed tricyclic group is silole, and Y is Si(R 3 )2. In some cases, the condensed tricyclic group is a crosslinked biphenyl, and Y is -C(R 3 )2C(R 3 )2- or -C(R 3 )2Si(R 3)2-. In some cases, the condensed tricyclic compound is a cross-linked biphenyl, and Y is -CHR 3 CHR 3 - In certain cases of any of the condensed tricyclic groups described herein, each R is independently selected from H, halogen, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. In certain cases, each R is independently selected from H, fluoro, chloro, methoxy, substituted alkoxy, alkyl, and substituted alkyl.

[0255] In certain embodiments of a fused tricyclic group, the group comprises two cyclically linked R substituents to provide a carbocyclic or heterocyclic ring A that is optionally further substituted,

[0256] [ka]

[0257] During the ceremony, Y is C(R 3 )2, -C(R 3 )2C(R 3 )2-, -C(R 3 )2Si(R 3 )2-, NR 3 , Si(R 3 )2 or Se, and each R 3 These are independently H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amide, substituted amide, aralkyl, substituted aralkyl, peg portion, -L 11 -Z 1 And L 11 It is a linker, Z 1 is a chemoselective tag (e.g., a tag containing a chemoselective functional group), and is selected from WSG. Each R is as defined above, and the condensed tricyclic group is R 3 Alternatively, it can be linked to the non-conjugated repeating units of the polymer backbone via R.

[0258] In certain cases, the condensed tricyclic group has the following structure:

[0259] [ka]

[0260] During the ceremony, R 8 ~R 9 Each of these independently consists of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amide, substituted amide, aralkyl, substituted aralkyl, peg moiety, and -L. 11 -Z 1 And, L 11 It is a linker, Z 1 is a chemoselective tag (e.g., a tag containing a chemoselective functional group), and is selected from WSG. Each R is as defined above, and the condensed tricyclic group is Y, R 8 -R 8 Alternatively, Y may be linked to the non-conjugated repeating units of the polymer backbone via R, and in some cases of comonomers, Y is C(R 3 )2.

[0261] water solubilizing group This disclosure includes water-soluble light-harvesting multichromophores having polymer tandem dyes comprising pendant chromophore groups and acceptor fluorophores. The multichromophores can be substituted with multiple water-soluble groups (WSGs). In some cases, the WSGs are pendant groups directly attached to a modular scaffold, for example, as side chains of the polymer backbone. In certain cases, the WSGs are substituents attached to the pendant donor chromophore group or the pendant acceptor fluorophore. In some cases, each of the pendant donor chromophore groups is substituted with one or more WSGs.

[0262] As used herein, the terms “water-soluble group,” “water-soluble group,” and “WSG” are used interchangeably and refer to a group or substituent that, when well solvated in an aqueous environment, e.g., under physiological conditions, confers improved water solubility to the molecule to which it adheres. WSGs can increase the solubility of multichromophores, primarily in aqueous solutions, compared to control multichromophores lacking WSGs. A water-soluble group can be any convenient hydrophilic group that is well solvated in an aqueous environment.

[0263] The water-soluble multichromophores of this disclosure are soluble under aqueous conditions, making them particularly suitable for application in a variety of biological assays. The water-soluble multichromophores and polymer tandem dyes of this subject, as well as their conjugations, may be resistant to undesirable aggregation, providing advantageous fluorescence and spectral properties in a variety of biological assays. Aggregation of dyes is undesirable because it can result in a reduced fluorescence signal, for example, through aggregation-induced quenching of dye fluorescence. The water-soluble multichromophores and polymer tandem dyes of this subject can be used as fluorescence reporters for a variety of biosensors, providing signals of outstanding brightness with a variety of excitation and emission wavelengths for applications such as flow cytometry and imaging.

[0264] Various water-soluble polymer groups can be adapted for use in the WSG of the subject multichromophore. Any convenient water-soluble group (WSG) may be included in the multichromophore described herein to provide increased water solubility. The increase in solubility may vary, but in some cases the increase (compared to a compound without WSG) is more than two times, for example, 5 times, 10 times, 25 times, 50 times, 100 times or more. In some cases the hydrophilic water-soluble group is charged, for example, positively or negatively charged. In certain cases the hydrophilic water-soluble group is a neutral hydrophilic group. In some embodiments the WSG is branched (for example, as described herein). In certain embodiments the WSG is linear. In some embodiments the WSG is a hydrophilic polymer, for example polyethylene glycol, modified PEG, peptide sequence, peptoid, carbohydrate, oxazoline, polyol, dendron, dendritic polyglycerol, cellulose, chitosan, or derivatives thereof. The target water-soluble groups are not limited to these, but include carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfonium, esters, polyethylene glycol (PEG) and modified PEG, hydroxyl, 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, polyethers, -COOM', -SO3M', -PO3M', -NR3 + ,Y′,(CH2CH2O) p R comprises a glycol containing mixtures thereof, where 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), and M' can be a cation counterion or hydrogen, --(CH2CH2O) yy CH2CH2XR yy ,--(CH2CH2O) yy CH2CH2X--, --X(CH2CH2O) yyIt can be CH2CH2--, glycol, and polyethylene glycol, yy is selected from 1 to 1000, and X is O, S, and NR ZZ Selected from, R ZZ and R YY H and C are independent of each other. 1~3 Selected from alkyl groups. In some cases, WSG is (CH2) x (OCH2CH2) y The molecule is OCH3, where each x is an integer between 0 and 20, and each y is an integer between 0 and 50. In some cases, the water-soluble group includes a nonionic polymer (e.g., a peg polymer) substituted at the terminals with an ionic group (e.g., a sulfonate).

[0265] In some embodiments of the formula, the pendant group in question is (CH2) x (OCH2CH2) y It comprises substituents selected from OCH3, where each x is independently an integer from 0 to 20, and each y is independently an integer from 0 to 50, as well as one or more halogens, hydroxyls, C1-C 12 Alkoxy, or (OCH2CH2) z Benzyl is optionally substituted with OCH3, where each z is an independent integer between 0 and 50. In some cases, the substituent is (CH2)3(OCH2CH2) 11 It is OCH3. In some embodiments, one or more of the substituents are (CH2) x (OCH2CH2) y Benzyl substituted with at least one WSG group (e.g., one or two WSG groups) selected from OCH3, where each x is an integer between 0 and 20, and each y is an integer between 0 and 50.

[0266] Multiple WSGs may be incorporated at a single position within the multichromophore of interest via a branched linker. In certain embodiments, the branched linker is an aralkyl substituent, further disubstituted with a water-soluble group. Thus, in some cases, the branched linker group is a substituent of the multichromophore that connects the multichromophore to two or more water-soluble groups. In certain embodiments, the branched linker is an amino acid, for example, a lysine amino acid connected to three groups via an amino group and a carboxylic acid group. In some cases, the incorporation of multiple WSGs via a branched linker imparts a desired solubility to the multichromophore. In some cases, the WSG is a nonionic side chain group that can impart water solubility greater than 50 mg / mL. In some cases, the WSG is a nonionic side chain group that can impart water solubility greater than 100 mg / mL. In some embodiments, the multichromophore comprises substituents selected from the group consisting of alkyl, aralkyl, and heterocyclic groups, each of which is further substituted with a water-soluble group, a hydrophilic polymer group, such as polyethyl glycol (PEG) (e.g., 2 to 20 PEG units).

[0267] The target water-soluble polymers that can be used in WSG include polyethylene glycol (PEG) groups or modified PEG groups. The target water-soluble polymers are not limited to these, but also include polyalkylene oxide-based polymers, such as polyethylene glycol "PEG" (e.g., “Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications”, JM Harris, Ed., Plenum Press, New York, NY (1992); and “Poly(ethylene glycol) Chemistry and Biological Applications”, JM Harris and S. Zalipsky, Eds., ACS (1997), and international patent applications, WO90 / 13540, WO92 / 00748, WO92 / 16555, WO94 / 04193, WO94 / 14758, WO94 / 17039, WO94 / 18247, WO94 / 28937, WO95 / 11924, WO96 / 00080, WO96 / 23794, WO98 / 07713 , WO98 / 41562, WO98 / 48837, WO99 / 30727, WO99 / 32134, WO99 / 33483, WO99 / 53951, WO01 / 26692, WO95 / 13312, WO96 / 21469, WO97 / 03106, WO99 / 45964, and U.S. Patents No. 4,179,337, No. 5,075,046, No. 5,089,261, No. 5, No. 100,992, No. 5,134,192, No. 5,166,309, No. 5,171,264, No. 5,213,891, No. 5,219,564, No. 5,275,838, No. 5, No. 281,698, No. 5,298,643, No. 5,312,808, No. 5,321,095, No. 5,324,844, No. 5,349,001, No. 5,352,756, No. 5, No. 405,877, No. 5,455,027, No. 5,446,090, No. 5,470,829, No. 5,478,805, No. 5,567,422, No. 5,605,976, No. 5, No. 612,460, No. 5,614,549, No. 5,618,528, No. 5,672,662, No. 5,637,749, No. 5,643,575, No. 5,650,388, No. 5,No. 681,567, No. 5,686,110, No. 5,730,990, No. 5,739,208, No. 5,756,593, No. 5,808,096, No. 5,824,778, No. 5,824, No. 784, No. 5,840,900, No. 5,874,500, No. 5,880,131, No. 5,900,461, No. 5,902,588, No. 5,919,442, No. 5,919,455 (See also issues 5,932,462, 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).

[0268] Examples of the target water-soluble polymers include, but are not limited to, polyalkylene oxides, polyamidealkylene oxides, or polyalkylene oxides and their derivatives containing ethylene oxide repeating units of the formula -(CH2-CH2-O)-. Further examples of the polymer of interest include those of the formula -[C(O)-XC(O)-NH-Y-NH]n- or -[NH-Y-NH-C(O)-XC(O)] n - comprises a polyamide having a molecular weight of 1,000 Daltons or more, where X and Y may be the same or different, may be branched or linear, n is a distinct integer from 2 to 100, such as 2 to 50, and either or both of X and Y comprises a biocompatible, substantially non-antigenic, water-soluble repeating unit, which may be linear or branched. Further examples of water-soluble repeating units include ethylene oxide of the formula -(CH2-CH2-O)- or -(O-CH2-CH2)-. The number of such water-soluble repeating units may vary considerably, such as 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 6 to 100, for example, 2 to 50, or 6 to 50. An example of one embodiment is where one or both of X and Y are -((CH2) n1 -(CH2-CH2-O) n2 -(CH2)- or -((CH2)n1 -(O-CH2-CH2) n2 -(CH2) n-1 -) is selected from n1 is 1-6, 1-5, 1-4, or 1-3, and n2 is 2-50, 2-25, 2-15, 2-10, 2-8, or 2-5. A further example of one embodiment is that X is -(CH2-CH2)- and Y is -(CH2-(CH2-CH2-O)3-CH2-CH2-CH2)- or -(CH2-CH2-CH2-(O-CH2-CH2)3-CH2)-.

[0269] The term modified polymer, such as modified PEGs, refers to a water-soluble polymer that is modified or derivatized at either or both ends, for example, to include terminal substituents (e.g., terminal alkyl, substituted alkyl, alkoxy, or substituted alkoxy) and / or terminal linking functional groups suitable for attachment to the polymer's multichromophores (e.g., amino or carboxylic acid groups suitable for attachment via amide bond formation). The water-soluble polymer of the subject can be adapted to include any convenient linking group. In some cases, it is understood that water-soluble polymers may have some dispersibility with respect to polymer length, depending on the method of preparation and / or purification of the polymer initiator. In some cases, water-soluble polymers are monodisperse.

[0270] Water-soluble polymers may contain one or more spacers or linkers. Examples of spacers or linkers include linear or branched moieties containing one or more repeating units, diamino and / or dibasic acid units, natural or unnatural amino acids or their derivatives, and aliphatic moieties containing alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, etc., which may contain, for example, up to 18 carbon atoms or additional polymer chains.

[0271] One or more of the water-soluble polymer moieties, or the spacers or linkers of the polymer moieties when present, may contain polymer chains or units that are biostable or biodegradable. For example, polymers with repeating bonds have varying degrees of stability under physiological conditions, depending on the bond resistance. Polymers with such bonds can be classified by their relative hydrolysis rates under physiological conditions, based on the known hydrolysis rates of low molecular weight analogs, for example, from low stability to high stability, such as polyurethane (-NH-C(O)-O-) > polyorthoester (-OC((OR)(R'))-O-) > polyamide (-C(O)-NH-). Similarly, the linking systems for attaching water-soluble polymers to target molecules may be biostable or biodegradable, ranging from low stability to high stability, for example, carbonates (-OC(O)-O-) > esters (-C(O)-O-) > urethanes (-NH-C(O)-O-) > orthoesters (-OC((OR)(R'))-O-) > amides (-C(O)-NH-). In general, it may be desirable to avoid the use of sulfated polysaccharides depending on the degree of sulfate liberation. In addition, the use of polycarbonates and polyesters may be less desirable. These linkages are provided as examples and are not intended to limit the types of linkages that can be used in polymer chains or linking systems of water-soluble polymers useful for WSG disclosed herein.

[0272] In some cases, WSGs are branched nonionic water-soluble groups (WSGs) that contain linked branching groups and provide further linkage to two or more nonionic water-soluble polymers.

[0273] In some cases, branched nonionic WSGs have one of the following equations:

[0274] [ka]

[0275] During the ceremony, Each B 1 and B 2It is independently a branching element, Each W 1 This is a nonionic water-soluble polymer that independently contains, for example, six or more monomer units. T 3 is an optional linker to a pendant group or repeating unit of a multichromophore, Each p and q is independently 0 or 1, and if present, each T 1 and each T 2 It is, independently, a linker.

[0276] In a particular case, each W 1 These are independently pegs or modified peg polymers. In certain cases, each W 1 These are independently selected from substituted alkyl groups, PEG or modified PEG groups, and WSGs. In a particular case, each W 1 These are independently PEGs or modified PEG polymers of 6 to 30 monomer units, such as 6 to 24 or 10 to 30 monomer units, 10 to 24 or 10 to 20 monomer units, 12 to 24 monomer units, 12 to 20 monomer units, 12 to 16 monomer units, or 16 to 20 monomer units.

[0277] In some cases, branched nonionic WSGs have the following formula:

[0278] [ka]

[0279] During the ceremony, Each B 1 is a branching base, Each W 1 This is a nonionic water-soluble polymer that independently contains, for example, six or more monomer units. T 3 This is an optional linker for condensed 6-5-6 tricyclic comonomers, Each p is independently 0 or 1, and if present, each T 1 It is, independently, a linker.

[0280] In a particular case, each W 1 These are independently pegs or modified peg polymers. In certain cases, each W 1 These are independently selected from substituted alkyl groups, PEG or modified PEG groups, and WSGs. In a particular case, each W 1 These are independently PEGs or modified PEG polymers of 6 to 30 monomer units, such as 6 to 24 or 10 to 30 monomer units, 10 to 24 or 10 to 20 monomer units, 12 to 24, 12 to 20 monomer units, 12 to 16, or 16 to 20 monomer units. In some embodiments of branched nonionic WSGs, B 1 This is a tetrasubstituted aryl group (e.g., 1,3,4,5-phenyl).

[0281] In some embodiments of branched nonionic WSGs (for example, as shown in the formula above), B 1 p is selected from CH, N, C(=O)N, SO2N, trisubstituted aryl groups (e.g., 1,3,5-phenyl), tetrasubstituted aryl groups, and trisubstituted heteroaryl groups. In some embodiments of the branched nonionic WSG, each p is 0. In some embodiments of the branched nonionic WSG, p is 1, and each T 1 is, -(CH2) n -O-, -O-(CH2) n -,-(CH2) n Selected from -, and -O-, where n is 1 to 12, for example, 1 to 6. In some embodiments of branched nonionic WSG, each T 2 and / or T 3 These are, independently, C1-C12 alkyl linkers, such as C1-C6 alkyl linkers, and one or more main chain atoms are arbitrarily substituted with heteroatoms.

[0282] In some embodiments of the subject multichromophore, the pendant donor chromophore group is independently substituted with one or more water-soluble groups (WSGs) selected from the following formulas:

[0283] [ka]

[0284] During the ceremony, T 5 It is an optional linker, Each T 6 It is a linker, R 11 And R are independently H, alkyl, or substituted alkyl. Each s is an integer between 1 and 100 (for example, between 6 and 100 or between 6 and 50).

[0285] In some specific cases, each s is independently 6-30, such as 6-24, 6-20, 11-20, 12-20, 12-18, or 12-16. In some specific cases, each s is independently 6-30, such as 6-24, 8-24, 10-24, 12-24, 13-24, 14-24, 15-22, or 16-20. In some cases, each s is independently 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, each s is independently 7 or more, for example 8, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or more, and in some cases has up to 50 monomer units, for example up to 40, up to 30, or up to 24 monomer units. In some cases, each s is independently 6 to 30 monomer units, for example 6 to 24 or 10 to 30, 10 to 24 or 10 to 20, 12 to 24, 12 to 20, 12 to 16 or 16 to 20 monomer units. In some cases, each s is identical. In some embodiments of the WSG, T 5 and / or T 6 The linkers are, for example, C1-C6 alkyl linkers or C1-C12 alkyl linkers, and one or more main chain atoms are optionally substituted with heteroatoms (e.g., -O-). In some embodiments of the WSG, each R 11 H is H. In some embodiments of the WSG, each R 11 It is methyl.

[0286] It should be understood that hydroxy-terminated peg chains may be used instead of methoxy-terminated peg chains in any of the WSG groups described above. In certain examples of any one of the formulas described herein, one or more of the repeating units or pendant groups are replaced by a WSG which is a dendron selected from one of the following structures:

[0287] [ka]

[0288] In certain examples of any one of the formulas described herein, one or more of the repeating units or pendant groups are replaced by a WSG which is a polyol selected from one of the following structures:

[0289] [ka]

[0290] In certain examples of any one of the formulas described herein, one or more of the repeating units or pendant groups are replaced by WSGs, which are oxazolines having the following structure:

[0291] [ka]

[0292] In certain examples of any one of the formulas described herein, one or more repeating units or pendant groups are replaced by WSGs, which are peptoids selected from one of the following structures:

[0293] [ka]

[0294] Water-soluble groups (WSGs) can impart water solubility exceeding 10 mg / mL to multichromophores or polymer tandem dyes, such as exceeding 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL. In certain cases, branched nonionic water-soluble groups (WSGs) can impart water solubility of 20 mg / mL or more (e.g., aqueous buffer) to multichromophores or polymer tandem dyes, such as 30 mg / mL or more, 40 mg / mL or more, 50 mg / mL or more, 60 mg / mL or more, 70 mg / mL or more, 80 mg / mL or more, 90 mg / mL or more, 100 mg / mL or more, or even more. It is understood that water-soluble multichromophores may form separate water-solvable nanoparticles in an aqueous system under certain conditions. In certain cases, water-solvated nanoparticles are resistant to aggregation and have found use in a variety of biological assays.

[0295] The polymer backbone of a multichromophore can have any convenient length. In some cases, a particular number of monomer repeating units in a multichromophore can fall within the range of, for example, 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 within the range of, for example, 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 cases, the specific number of monomer repeating units or segments in the polymer backbone of a multichromophore can fall within the range of 2 to 1,000, such as 2-500, 2-500, 3-500, 4-500, 5-500, 6-500, 7-500, 8-500, 9-500, 10-500, 10-400, 10-300, 10-200, 10-100, or 20-100 units or segments.

[0296] Multichromophores can have any convenient molecular weight (MW). In some cases, the MW of a multichromophore can be expressed as an average molecular weight. In some cases, polymer dyes have average molecular weights in the range of 500 to 500,000, such as 1,000 to 100,000, 2,000 to 100,000 (e.g., 2,000 to 10,000 or 10,000 to 100,000), or even in the range of 50,000 to 100,000. In some cases, the polymer backbone of a multichromophore is prepared to have specific distinct monomer sequences so that the MW of the multichromophore can be expressed as an exact molecular weight. In some cases, polymer dyes have precise molecular weights in the range of 500 to 500,000, such as an average molecular weight in the range of 1,000 to 100,000, 1,000 to 50,000, 2,000 to 50,000 (e.g., 2,000 to 10,000 or 10,000 to 50,000), or even an average molecular weight in the range of 50,000 to 100,000.

[0297] Polymer tandem dyes As summarized above, the light-harvesting multichromophore of the subject can contain a polymer backbone of non-conjugated repeating units containing multiple pendant-donor chromophore groups, each of which can be linked to one of the repeating units. As described above, the water-soluble light-harvesting multichromophore of the subject enables homoenergy transfer between pendant-donor chromophores, which can result in continuous, reversible energy transfer between equal chromophores, rather than emission from a single chromophore. Thus, while the multichromophore system itself can be fluorescent, via a self-quenching process illustrated in Figure 6A, the multichromophore system can have a significantly lower quantum yield than that observed for a single isolated chromophore.

[0298] Water-soluble light-harvesting multichromophores can transfer energy to linked acceptor fluorophores. See, for example, Figure 6B. Thus, the polymer tandem dye of the subject further comprises acceptor signal fluorophores covalently bonded to the vicinity of the energy-receiving donor water-solvated light-harvesting multichromophore system, i.e., to the vicinity of at least one pendant donor chromophore group. The terms “acceptor fluorophore” and “acceptor chromophore” are used interchangeably herein.

[0299] Acceptor signal fluorophores can be linked to non-conjugated repeating units of the polymer backbone as pendant groups. Excitation of the multichromophore donor can result in energy transfer to the covalently bonded acceptor signal fluorophore and subsequent emission. The number of repeating units of the donor aqueous solvation nucleating multichromophore having linked acceptor signal fluorophore groups can vary, and in some cases, the number ranges from 1 mol% to 50 mol% of the repeating units, for example, 1 mol% to 25 mol%, 2 mol% to 25 mol%, 3 mol% to 25 mol%, 4 mol% to 25 mol%, 5 mol% to 25 mol%, or 10 mol% to 25 mol%.

[0300] Mechanisms for energy transfer between the light-gathering chromophores in a multichromophore system, and from these donor chromophores to the connected acceptor-signal fluorophores, include, for example, resonant energy transfer (e.g., Forster (or fluorescence) resonant energy transfer, Fret), quantum charge exchange (Dexter energy transfer), etc. These energy transfer mechanisms can be over relatively short ranges, i.e., the proximity of the chromophores in a light-gathering multichromophore system to each other and / or to the acceptor fluorophores provides efficient energy transfer.

[0301] Under conditions for efficient energy transfer, amplification of emission from a luminescent acceptor fluorophore can occur when the emission from the luminescent acceptor fluorophore is stronger when the incident light ("pump light") is at a wavelength absorbed by the chromophore of a condensing multichromophore and transmitted from there, than when the luminescent acceptor fluorophore is directly excited by the pump light.

[0302] "Efficient" energy transfer means that 10% or more of the energy collected by the donor chromophore is transferred to the acceptor, for example, 20% or more, or 30%, 40%, 50%, etc. "Amplification" means that the signal from the acceptor fluorophore is 1.5 times or more when excited by energy transfer from a donor-focusing multichromophore system 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 polymer tandem dyes, the acceptor fluorophore emission is 1.5 times greater when excited by a multichromophore compared to the direct excitation of the acceptor fluorophore by incident light, for example, 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 compared to when the acceptor fluorophore is directly excited by incident light.

[0303] Any convenient fluorescent dye can be used as an acceptor fluorophore in the polymer tandem dye. The terms “fluorescent dye” and “fluorophore” are used interchangeably herein. Acceptor fluorophore (e.g., each A 1 ) can be small molecule fluorophores. Acceptor fluorophores (for example, each A 1) can be a dye molecule selected from rhodamine, coumarin, xanthene, cyanine, polymethine, pyrene, thiadin, acridine, dipyrometheneboron difluoride, naptalimide, phycobiliprotein, peridinium chlorophyll protein, their conjugates, and combinations thereof. In certain embodiments, acceptor fluorophores (A 1 These are cyanine pigments, xanthene pigments, coumarin pigments, thiadin pigments, or acridine pigments. In some cases, acceptafluorophores (A 1) are selected from DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy 3.5, and diethylaminocoumarin. The fluorescent dyes covered are not limited to these, but include fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodamine, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-chrome, phycoerythrin, PerCP (peridinin chlorophyll-α 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 This includes 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, 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, conjugated forms, and combinations thereof. The lanthanide chelates covered include, but are not limited to, europium chelates, terbium chelates, and samarium chelates.In some embodiments, the polymer tandem dye comprises a multichromophore linked to an acceptor fluorophore selected from Cy 3, Cy 3.5, Cy 5, Cy 5.5, Cy 7, Alexa 488, Alexa 647, and Alexa 700. In certain embodiments, the polymer tandem dye comprises a multichromophore linked to an acceptor fluorophore selected from a dymics dye (e.g., DY 431, DY 485XL, DY 500XL, DY 530, DY 610, DY 633, DY 640, DY 651, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800, or DY 831), biotium CF 555, Cy 3.5, and diethylaminocoumarin.

[0304] In some cases, the acceptor fluorophore is a BODIPY group, for example, any one of the BODIPY groups of formulas (XI) to (XIXa) described herein, or any embodiment thereof. It is understood that any convenient BODIPY group described herein having a suitable absorption and emission profile may be configured as an acceptor fluorophore in a vicinity that accepts energy into a donor water solvation focused multichromophore system, i.e., in a vicinity that accepts energy into at least one compatible pendant donor chromophore group.

[0305] In some embodiments, the selected acceptor fluorophore has a maximum emission wavelength in the range of 300-900 nm, such as 350-850 nm, 350-600 nm, 360-500 nm, 370-500 nm, 380-500 nm, 390-500 nm, or 400-500 nm. Specific examples of the maximum emission values ​​of the signal chromophore in question are not limited to these, but include 395 nm ± 5 nm, 420 nm ± 5 nm, 430 nm ± 5 nm, and 440 nm. Includes ±5nm, 450nm±5nm, 460nm±5nm, 470nm±5nm, 480nm±5nm, 490nm±5nm, 500nm±5nm, 510nm±5nm, 520nm±5nm, 530nm±5nm, 540nm±5nm, 550nm±5nm, 560nm±5nm, 570nm±5nm, 580nm±5nm, 590nm±5nm, 605nm±5nm, 650nm±5nm, 680nm±5nm, 700nm±5nm, and 805nm±5nm.

[0306] The coupled luminescent acceptor fluorophore emission of polymer tandem dyes can have a yield of 0.03 or higher, such as 0.04 or higher, 0.05 or higher, 0.06 or higher, 0.07 or higher, 0.08 or higher, 0.09 or higher, 0.1 or higher, 0.15 or higher, 0.2 or higher, 0.3 or higher, or even higher. In some cases, polymer tandem dyes have yields of 5 × 10⁻⁶. 5 cm -1 M -1 For example, 6 x 10 5 cm -1 M -1 The above 7 x 10 5 cm -1 M -1 The above 8 x 10 5 cm -1 M -1 The above is 9 x 10 5 cm -1 M -1 For example, 1 × 10 6 cm -1 M -1 The above is 1.5 × 10 6 cm -1 M -1 The above is 2 x 10 6 cm -1M -1 The above is 2.5 × 10 6 cm -1 M -1 The above is 3 x 10 6 cm -1 M -1 The above is 4 x 10 6 cm -1 M -1 The above 5 x 10 6 cm -1 M -1 The above 6 x 10 6 cm -1 M -1 The above 7 x 10 6 cm -1 M -1 or more, or 8 x 10 6 cm -1 M -1 It has the above-mentioned annihilation coefficient. In some embodiments, the polymer tandem dye is 5 × 10 5 M -1 cm -1 It has the above molar annihilation coefficient. In a particular embodiment, the polymer tandem dye is 1 × 1 6 M -1 cm -1 It has the above molar annihilation coefficient.

[0307] The polymer tandem dyes of the subject can be isolated and provide fluorescence emission from luminescent signal chromophore dyes that is brighter than the emission possible from such luminescent dyes alone. The coupled luminescent signal chromophore emission of polymer tandem dyes is, for example, 60 mM. -1 cm -1 Above 70mM -1 cm -1 Above 80mM -1 cm -1 Above 90mM -1 cm -1 Above 100mM -1 cm -1 More than 150mM -1 cm -1 Above 200mM -1 cm -1 more than 250mM -1 cm -1 Above 300mM -1 cm-1 50mM or more, or even more. -1 cm -1 It can have a brightness of such magnitude. In certain cases, the linked signaling chromophore emission of polymer tandem dyes has a brightness at least 5 times greater than that of directly excited luminescent dyes, for example, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, at least 300 times, or even greater than that of directly excited luminescent dyes.

[0308] The polymer tandem dyes in question can provide fluorescence emission with a Stokes shift of 100 nm or more, such as 110 nm or more, 120 nm or more, 130 nm or more, 140 nm or more, or 150 nm or more. In some cases, the Stokes shift is 300 nm or less, such as 200 nm or less.

[0309] In some embodiments, the polymer tandem dye comprises one of the water-soluble light-harvesting multichromophores of formulas (I) to (IX), where Z 1 Chemoselective tags are acceptor fluorophore groups (A 1 ) can be replaced by. It is understood that any embodiment of the subject multichromophore of formulas (I) to (IX) can also be carried out with respect to the polymer tandem dyes of this disclosure. In certain examples of formulas (I) to (IX), Z 1 One or more of the groups are conjugated to the acceptor fluorophore precursor, forming an acceptor fluorophore group (A 1 ) can be provided. Therefore, polymer tandem dyes may include a segment of formula (Ia).

[0310] [ka]

[0311] During the ceremony, Each M 1 and M 2These are, independently, unsaturated comonomers (e.g., aryl or heteroaryl comonomers), Each S 1 and S 2 These are independent, non-conjugate spacer units. Each D 1 M 1 A pendant donor chromophore connected to (for example, as described herein), Each A 1 M 2 It is an acceptor fluorophore linked to, x is 75 mol% or more, y is 25 mol% or less.

[0312] The first (M 1 -S 1 ) and the second repeating unit (M 2 -S 2 ) can be arranged in a random or coblock configuration. In a particular case of formula (Ia), the D of the first iteration unit 1 The pendant group comprises two or more (e.g., two or three) different types of pendant light-absorbing chromophores that together provide a light-collecting multichromophore system. In certain cases of formula (Ia), the first repeating unit D 1 The base of all the pendants is the same.

[0313] In some cases of equation (Ia), x 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 cases of equation (Ia), y 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, 1 mol% or less.

[0314] In some cases, polymer tandem dyes contain a segment of formula (IIa),

[0315] [ka]

[0316] During the ceremony, The polymer backbone of the non-conjugated repeating units is composed of SMs, each of which is an independent non-conjugated comonomer. 1 SM 2 , and SM 3 Contains a copolymer, Each D 1 SM 1 It is a pendant donor chromophore connected to, Each A 1 SM 2 It is an acceptor fluorophore linked to, each Z 2 SM 3 An optional side chain group linked to, x is 50 mol% or more, The concentration of y+z is less than 50 mol%.

[0317] Z 2 This may be absent or may be any convenient side chain group, such as a light-absorbing chromophore, chemoselective tag, linker, linked biomolecule, acceptor fluorophore, WSG, etc. In a particular case of formula (IIa), SM 3 is, Z 2 It is a spacer comonomer in which no spacer comonomer exists. In certain cases of formula (IIa), SM 3 This is the second pendant light-absorbing chromophore, Z 2 It is a comonomer containing a group, and each D 1 and each Z 2 It provides a light-gathering multichromophore system together. In some cases, SM 3 This is the second chemoselective tag (Z 2 ), for example, Z provides selective placement of the target part. 1 It is a comonomer containing a protective functional group or tag orthogonal to it.

[0318] In certain cases of equation (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 cases of equation (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 cases of equation (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 cases of equation (IIa), z is at least 1 mol% and 10 mol% or less, for example, 5 mol% or less, or even less.

[0319] In some cases, polymer tandem dyes contain a segment of formula (IIIa),

[0320] [ka]

[0321] During the ceremony, The polymer backbone of the non-conjugated repeating units is composed of SMs, each of which is an independent non-conjugated comonomer. 1 and SM 2 Contains a copolymer, Each D 1 SM 1 It is a pendant donor chromophore connected to, Each A 1 SM 2 It is an acceptor fluorophore linked to, x is 75 mol% or more, y is 25 mol% or less.

[0322] In a particular embodiment of formula (IIIa), SM 1 and SM 2Each of these is independently a saturated non-conjugated comonomer, for example, a comonomer that provides only a single covalent CC bond. In some embodiments of formula (IIIa), SM 1 and SM 2 Each of these is independently a partially saturated non-conjugated comonomer, for example, a comonomer that provides an isolated double C=C covalent bond in the main chain of a saturated covalent bond. The first and second repeating units (SM) of formula (IIIa) 1 and SM 2 ) can be arranged in a random configuration, block, or coblock configuration, or in a specific sequence. In a particular case of formula (IIIa), SM 1 D 1 The pendant group comprises two or more (e.g., two or three) different types of pendant light-absorbing chromophores that together provide a light-collecting multichromophore system. In certain cases of formula (IIIa), the first repeating unit D 1 The base of all the pendants is the same.

[0323] In some cases of equation (IIIa), x 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 cases of equation (IIIa), y 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, 1 mol% or less.

[0324] In certain cases, the polymer tandem dye is given by formula (IVa),

[0325] [ka]

[0326] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, Each A 1It is independently an acceptor fluorophore, Each L 1 and L 2 It is, independently, a linker, p1 and q1 are independently 0 or 1, and p1 + q1 ≤ 1. p2 and q2 are independently 0 or 1, and p2 + q2 ≤ 1. x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2 Each of these is independently selected from terminal groups, polymer segments, light-absorbing (e.g., donor) chromophore groups, acceptor fluorophores, linkers, and linked specific binding members.

[0327] In some embodiments of formula (IVa), p1 and p2 are each 0, and q1 and q2 are each 1 (e.g., a β3-amino acid residue). In some embodiments of formula (IVa), p1 and p2 are each 1, and q1 and q2 are each 0 (e.g., a β2-amino acid residue). In some cases, p1, p2, q1, and q2 are each 0, and the polymer tandem dye is of the following formula (Va):

[0328] [ka]

[0329] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, Each A 1 It is independently an acceptor fluorophore, L 1 and L 2 Each of them is an independent linker, x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2Each of these is independently selected from terminal groups, polymer segments, light-absorbing (e.g., donor) chromophore groups, linkers, and linked specific binding members.

[0330] It is understood that the multichromophore described by formula (Va) includes any convenient arrangement of comonomers in a defined linear sequence having a defined mol% ratio of total x and y. In some cases, A 1 The contained comonomers are spaced apart throughout the entire polymer backbone sequence and are therefore always adjacent on both sides to one or more D1-containing comonomers.

[0331] In certain cases of formula (Va), the polymer tandem dye contains a segment of formula (VIa),

[0332] [ka]

[0333] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, Each A 1 It is, independently, an acceptchromophore, Each L 1 and L 2 It is, independently, a linker, n and p are each independent integers between 1 and 20, and in the equation, n + p ≥ 2. m is either 1 or 2. In some cases of equation (VIa), n and p are independently between 1 and 10, such as 2 to 20, 3 to 10, or 3 to 6.

[0334] In some examples of equation (VIa), n+p is an integer between 2 and 20, such as 3 to 20, 4 to 20, 5 to 20, 5 to 15, or 5 to 12. In a particular embodiment of equation (VIa), m is 1.

[0335] The polymer tandem dye of the subject may contain multiple segments of formula (VIa), each segment containing a comonomer D 1 One isolated A containing a comonomer adjacent to the block 1 This includes. In some cases, the multichromophore is, for example, 3-20, 4-20, 5-20, 5-15, or 5-12 D 1 Blocks of comonomers containing 2 to 20 D 1 Two isolated A molecules separated by the block of contained coprimordia. 1 To provide a comonomer containing two or more segments of formula (VIa) oriented adjacent to each other, the polymer tandem dye comprises a segment q of the block copolymer and is of formula (VIIa),

[0336] [ka]

[0337] In the formula, each (n) q and each (p) q These are independent integers from 1 to 20, and in the expression, for each segment of q, (n) q +(p) q The value is ≥ 3, and q is an integer between 1 and 100.

[0338] In a particular embodiment, the polymer tandem dye has formula (VIIIa),

[0339] [ka]

[0340] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, Each A 1 It is, independently, an acceptchromophore, Each L 1 and L 2It is, independently, a linker, x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2 Each of these is independently selected from terminal groups, polymer segments, donor chromophore groups, linkers, and linked specific binding members.

[0341] In a particular embodiment, the polymer tandem dye has formula (IXa),

[0342] [ka]

[0343] During the ceremony, Each D 1 It is independently a pendant BODIPY donor chromophore, Each A 1 It is independently an acceptor fluorophore, Each L 1 and L 2 It is, independently, a linker, x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2 Each is independently selected from the group consisting of terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, linkers, and linked specific binding members.

[0344] In some examples of formulas (IVa), (Va), (XIIIa), and (IXa), x 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 formulas (IV), (V), (XIII), and (IX), y 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, 1 mol% or less.

[0345] In a particular embodiment, the polymer tandem dye has formula (Xa),

[0346] [ka]

[0347] During the ceremony, Each D 1 It is, independently, a pendant donor chromophore, Each A 1 It is independently an acceptor fluorophore, Each L 1 , L 2 , and L 3 It is, independently, a linker, a, b, and c are the mol% values ​​of each comonomer. d represents the total polymerization or average length of the polymer (for example, d is 2-1000, such as 2-500, 2-200, 2-100, or 2-50), WSG is a water-soluble group (as described herein, for example), G 1 and G 2 Each is independently selected from the group consisting of terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, linkers, and linked specific binding members.

[0348] In some cases of formula (Xa), c = 0. In some cases of formula (Xa), a > 0 and b > 0. In some examples of formula (Xa), a is, for example, 80 mol% or more, such as 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, for example, 20 mol% or less, such as 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, 1 mol% or less. In some cases of formula (Xa), a is 65 - 95 mol%, b is 5 - 35 mol%, c is 0 - 30 mol%, and a + b + c = 100%.

[0349] In a certain case of formula (Xa), each D 1 is a linked BODIPY group (for example, as described herein). In some cases, each acceptor dye A 1 is selected from DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, iFluor dyes 350, 405, 488, 514, 532, 594, 660, 680, 700, 710, 790, Tide Fluor dyes 5WS, 7WS, 8ws, ICG, BODIPY-based dyes, Biotium CF 555, Cy 3.5, and diethylaminocoumarin. In a certain case of formula (Xa), L 1 ~D 1 is selected from the following,

[0350]

Chemical formula

[0351] wherein R 4 is H, lower alkyl, substituted lower alkyl, and WSG.

[0352] Instead, in some instances of formula (Xa), the donor dye and acceptor dye D 1 and A 1 pairs may be selected from Dyomics dyes DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, iFluor dyes 350, 405, 488, 514, 532, 594, 660, 680, 700, 710, 790, Tide Fluor dyes 5WS, 7WS, 8ws, ICG, BODIPY-based dyes, Biotium CF 555, Cy 3.5, and diethylaminocoumarin. In certain instances of formula (Xa), the WSG is a solubilizing group as described in any one of the embodiments and structures of such groups described herein.

[0353] In certain instances, the polymeric tandem dye is derived from a multi-chromophore of formula (XXI), and the chemo-selective tag Z 2 of SM 1 is conjugated to an acceptor fluorophore. Thus, exemplary polymeric tandem dye structures are shown in Example 3 of the experimental section and in the following structures,

[0354]

Chemical formula

[0355] wherein, G 1 is a terminal group (e.g., as described herein), L 1 and L 2 are independently linkers, D 1 is a pendant chromophore (e.g., as described herein), each A 1 is an acceptor fluorophore,​Each of d, e, f, g, and h is independently 1 to 6. Each n and m is independently between 1 and 1000 (e.g., 2 to 1000, 2 to 500, 2 to 100, or 2 to 50), Each R 4 1 is selected from alkyl, substituted alkyl, and WSG. A "linker" is a linker that contains an optional chemoselective functional group, for example, for conjugation to a comonomer or biomolecule.

[0356] In a particular case, each D 1 is a BODIPY chromophore group (as described herein, for example). In some cases, polymer tandem dyes derived from formula (XXI) are described by the following general structure:

[0357] [ka]

[0358] Each n and m is independently between 1 and 1000 (e.g., 2 to 1000, 2 to 500, 2 to 100, or 2 to 50).

[0359] In some cases, the donor chromophore is a BODIPY chromophore (e.g., as described herein), and the acceptor dyes are DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, iFluor dyes 350, 405, 488, 514, 532, 594, 660, 680, 700, 710, 790, Tide Fluor dyes 5WS, 7WS, 8ws, ICG, BODIPY-based dyes, Biotium CF 555, Cy 3.5 and diethylaminocoumarin are selected.

[0360] Labeled specific binding member Aspects of this disclosure include labeled specific binding members. A labeled specific binding member is a conjugation of a polymer dye of the subject (e.g., those described herein) to the specific binding member. Either the polymer dye or polymer tandem dye described herein may be conjugated to a specific binding member. The specific binding member and the polymer dye may be conjugated (e.g., covalently bonded) to each other at any convenient position on the two molecules via an optional linker. In some embodiments, the labeled specific binding member is agglomeration-resistant. As used herein, “agglomeration-resistant” means a labeled specific binding member that can form a homogeneous aqueous composition without agglomerating at concentrations of 1 mg / ml or higher in the aqueous buffer of the subject, for example, 2 mg / ml or higher, 3 mg / ml or higher, 4 mg / ml or higher, 5 mg / ml or higher, 6 mg / ml or higher, 7 mg / ml or higher, 8 mg / ml or higher, 9 mg / ml or higher, 10 mg / ml or higher, or many more of the labeled specific binding members.

[0361] In certain embodiments, the labeled specific binding member includes a water-solvated polymer dye having a deep ultraviolet excitation spectrum and containing segments of π-conjugated comonomers and conjugated modification repeat units, as well as a specific binding member covalently bonded to a multichromophore.

[0362] As used herein, the term “specific binding member” refers to a pair of members of a molecule that have binding specificity to each other. One member of the pair of molecules may have a cavity that specifically binds to an area on its surface, or to an area on the surface of the other member of the pair of molecules, or to a cavity within it. Thus, the pair of members have the property of specifically binding to each other to form a binding complex. In some embodiments, the affinity between specific binding members in the binding complex is, for example, 10 -8 10 including M or below -7 M or less, for example, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13M or less, 10 -14 M or less, 10 -15 10 including M or below -6 K below M d Characterized by the (dissociation constant). In some embodiments, the specific binding member binds specifically with high affinity. High affinity means that the binding member binds specifically with 10 × 10 -9 For example, 1 × 10 -9 Below, 3 x 10 -10 M or less, 1×10 -10 M or less, 3×10 -11 M or less, 1×10 -11 M or less, 3×10 -12 M or less, or 1 × 10 -12 M or below, apparent K d This means that it specifically binds with apparent affinity characterized by [the specified characteristic].

[0363] The specific binding member may be proteinaceous. As used herein, the term “proteinaceous” refers to a portion consisting of amino acid residues. The proteinaceous portion 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 polymer dye. As used herein, the terms “antibody” and “antibody molecule” are interchangeable 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 gene, for example in humans, includes kappa(k), lambda(I), and the heavy chain gene Ioci, which together comprise a multitude of variable region genes, each containing constant region genes mu(u), delta(d), gamma(g), sigma(e), and α(a) encoding IgM, IgD, IgG, IgE, and IgA isotypes, respectively. The variable region of an immunoglobulin light or heavy chain 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 is precisely defined (see “Sequences of Proteins of Immunological Interest,” E. Kabat et al., USD Department of Health and Human Services, (1991)). The numbering of all antibody amino acid sequences considered herein conforms to the Kabat system. The sequences of the framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody, i.e., the combined framework region of the constituent light and heavy chains, plays a role in positioning and aligning the CDRs. The CDRs are primarily involved in the binding of the antigen to the epitope. The term antibody means to include full-length antibodies and may refer to naturally occurring antibodies of any organism, genetically engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic, or other purposes, as further defined below.

[0364] The antibody fragments of interest include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of antibodies produced by modification of whole antibodies or novel antibodies synthesized using recombinant DNA technology. The antibodies may be monoclonal or polyclonal and may possess other specific activities on cells (e.g., antagonist, agonist, neutralizing, inhibitory, or stimulating antibody). It is understood that antibodies may have additional conserved amino acid substitutions that do not substantially affect antigen binding or other antibody functions.

[0365] In certain embodiments, the specific binding member is a Fab fragment, an F(ab')2 fragment, an scFv, a diabody, or a triabody. In certain embodiments, the specific binding member is an antibody. In some cases, the specific binding member is a mouse antibody or its binding fragment. In certain cases, the specific binding member is a recombinant antibody or its binding fragment.

[0366] In some embodiments, the labeled specific binding member includes a water-solvated light-harvesting multichromophore (e.g., as described herein), a signal chromophore covalently bonded to the energy-accepting vicinity of the multichromophore (e.g., as described herein), and a specific binding member covalently bonded to the multichromophore. Some examples of the labeled specific binding member include a multichromophore of any of the formulas described herein, where G 1 and G 2 Each is independently selected from the group consisting of a terminal group (e.g., terminal group), a linker, and a linked specific binding member, G 1 and G 2 At least one of them is a linked specific binding member.

[0367] In certain embodiments of the formulas described herein, G 1 and / or G 2This refers to linkers such as linkers containing functional groups suitable for conjugation to specific bonding sites. Multichromophore G 1 and / or G 2 The linker located at this position is a side chain of the multichromophore (for example, Z 2 It is understood that any other linker may be selected to be orthogonal to any chemoselective tag (e.g., as described herein) that may be present in the ). In certain embodiments, the amino functional group or its derivatives is G 1 and / or G 2 The carboxylic acid functional group or its derivative is included in Z 2 It is included in certain embodiments. In certain embodiments, the carboxylic acid functional group or its derivative is G 1 and / or G 2 It contains an amino functional group or its derivative, Z 2 It is included in.

[0368] In some embodiments of the formulas described herein, G 1 and G 2 At least one of them is -L 3 -Z 4 And L 3 is a linker (as described herein, for example), and Z 4 is a specific binding member (for example, as described herein). In some embodiments of the formulas described herein, G 1 and G 2 At least one of them is -L 3 -Z 3 And L 3 is a linker (as described herein, for example), and Z 3is a chemoselective tag (for example, as described herein). Any convenient chemoselective tag and conjugation chemistry can be adapted for use in the multichromophore of the subject. The chemoselective tags of interest include, but are not limited to, amines, active esters, maleimides, thiols, sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, Diers-Alder cycloaddition click reagents, and click chemists, tetrazine, transcyclooctene, aldehydes, alkoxylamines, alkynes, cyclooctine, azides, etc. In some cases, Z 3 The active ester is selected from the group consisting of carboxylic acids, active esters (e.g., N-hydroxysuccinimidyl ester (NHS) or sulfo-NHS), aminos, maleimides, iodoacetyls, and thiols.

[0369] The biomolecules in question are not limited to these, but include polypeptides, polynucleotides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs thereof, and combinations thereof. In certain cases, Z 4 It is an antibody. In some cases, Z 4 This is an antibody fragment or its conjugated derivative. In some cases, the antibody fragment or its conjugated derivative is selected from the group consisting of Fab fragments, F(ab')2 fragments, scFv, diabodies, and triabodies.

[0370] How to use As summarized above, aspects of this disclosure include methods for evaluating a sample for the presence of a target analyte. Aspects of the method include contacting the sample with a polymer dye conjugate that specifically binds to the target analyte to produce a labeled composition contact sample. As used herein, the terms “polymer dye conjugate” and “labeled specific binding member” are interchangeable. Thus, the polymer dye conjugate may include (i) a water-solvable polymer dye (e.g., those described herein) and (ii) a specific binding member (e.g., those described herein). In some cases, the polymer dye conjugate further includes a signal chromophore covalently bonded near a multichromophore of the polymer dye that accepts energy.

[0371] A labeled composition contact sample can be generated by contacting the sample with a polymer dye conjugate that specifically binds to the target analyte using any convenient method. In some cases, the sample is contacted with the polymer dye conjugate under conditions that, if present, the specific binding member specifically binds to the target analyte. For specific binding of the specific binding member of the conjugate to the target analyte, a suitable solution that maintains the biological activity of the components of the sample and the specific binding member may be used. The solution may be a balanced salt solution, such as physiological saline, PBS, or Hank's balanced salt solution, conveniently supplemented with fetal bovine serum, human platelet lysate, or other factors, in combination with a buffer that is acceptable at low concentrations, such as 5–25 mM. Convenient buffers include HEPES, phosphate buffer, and lactate buffer. Various media are commercially available and may be used according to the properties of the target analyte, including dMEM, HBSS, dPBS, RPMI, and Iscove's medium, which may also be supplemented with fetal calf serum or human platelet lysate. The final components of the solution may be selected according to the components of the sample contained.

[0372] The temperature at which specific binding of a conjugated specific binding member to the target analyte occurs may vary and may be in the range of 5°C to 50°C, for example, 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, for example, 20°C, 25°C, 30°C, 35°C, or 37°C (for example, as described above). In some cases, the temperature at which specific binding occurs is selected to match the biological activity of the specific binding member and / or the target analyte. In certain cases, the temperature is 25°C, 30°C, 35°C, or 37°C. In certain cases, the specific binding member is an antibody or a fragment thereof, and the temperature at which specific binding occurs is room temperature (for example, 25°C), 30°C, 35°C, or 37°C. The incubation time for any convenient specific binding may be selected to allow the formation of a desired amount of binding complex, and in some cases may be 1 minute or longer, such as 2 minutes or longer, 10 minutes or longer, 30 minutes or longer, 1 hour or longer, 2 hours or longer, or even 6 hours or longer.

[0373] Any convenient specific binding member may be utilized within the conjugate. The specific binding members of interest include, but are not limited to, agents that specifically bind to cell surface proteins of various cell types, including, but are not limited to, stem cells, e.g., pluripotent stem cells, hematopoietic stem cells, T cells, T regulatory cells, dendritic cells, B cells, e.g., 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 erythrocytes. Target cells of interest include cells having convenient cell surface markers or antigens that can be captured or labeled by the convenient affinity agent or its conjugate. In some embodiments, 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. In the methods of interest, any convenient cell surface protein or cell marker may be targeted for specific binding to polymer dye conjugates. In some embodiments, the target cells include cell surface markers selected from cell receptors and cell surface antigens. In some cases, the target cells may include 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 α / β, T cell receptor γ / Δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71.

[0374] Any convenient target may be selected for evaluation using the method of the subject. Targets of interest include, but are not limited to, nucleic acids such as RNA, DNA, PNA, CNA, HNA, LNA, or ANA molecules; proteins such as fusion proteins; modified proteins such as phosphorylated proteins, glycosylated proteins, ubiquitinated proteins, SUMOylated proteins, or acetylated proteins; or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins, and drug molecules. As used herein, the term “target protein” refers to all members of the target family and fragments thereof. Target proteins may be any protein of interest, including, but are not limited to, hormones, growth factors, receptors, enzymes, cytokines, bone induction factors, colony-stimulating factors, and immunoglobulins, such as therapeutic or diagnostic targets. The term “target protein” is intended to include recombinant and synthetic molecules that can be prepared using any convenient recombinant expression method, or can be prepared using any convenient synthesis method, or can be commercially available. In some embodiments, polymer dye conjugation includes antibodies or antibody fragments. Any convenient target analytes that specifically bind to the antibody or antibody fragment of interest may be targeted in the method of the subject.

[0375] In some embodiments, the target analyte is associated with cells. In certain cases, the target analyte is a cell surface marker. In certain cases, the cell surface marker is selected from the group consisting of cell receptors and cell surface antigens. In some cases, the target analyte is an intracellular target, and the method further includes lysing the cells.

[0376] In some embodiments, the sample may include a heterogeneous cell population from which target cells are isolated. In some cases, the sample includes peripheral whole blood, peripheral whole blood from which red blood cells have been lysed prior to cell isolation, cord blood, bone marrow, density gradient purified peripheral blood mononuclear cells, or homogenized tissue. In some cases, the sample includes hematopoietic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow, or umbilical cord blood. In certain embodiments, the sample includes tumor cells in peripheral blood. In certain cases, the sample is a sample containing (or suspected to contain) viral cells (e.g., HIV).

[0377] Labeled specific binding members are found to be used in the methods of the subject, for example, to label target cells, particles, targets, or analytes with polymer dyes or polymer tandem dyes. For example, labeled specific binding members are found to be used in labeled cells processed by a flow cytometer (e.g., detected, analyzed, and / or sorted). Labeled specific binding members may include, for example, antibodies 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 the cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression, and / or presentation. Labeled specific binding members can be used in any application that includes (or may include) antibody-mediated labeling of cells, particles, or analytes.

[0378] In some cases of this method, the labeled specific binding member includes a multichromophore described herein (for example, by any one of formulas (I) to (IX)). In certain cases, G 1 and G 2 Each is independently selected from the group consisting of terminal groups, polymer segments, linkers, chemoselective tags, and linked specific binding members, G 1 and G 2 At least one of them is a linked specific binding member.

[0379] Embodiments of the method include assaying a labeled composition-contacted sample for the presence of a polymer dye-conjugated target analyte-binding complex to evaluate whether a target analyte is present in the sample. When the sample comes into contact with the polymer dye conjugate, any convenient method can be used to assay the labeled composition-contacted sample for the presence of the polymer dye-conjugated target analyte-binding complex. The polymer dye-conjugated target analyte-binding complex, if present, is a binding complex formed upon the specific binding of the conjugate's specific binding member to the target analyte. Assaying a sample in contact with the labeled composition may include detecting a fluorescent signal from the binding complex, if present. In some cases, the assay includes a separation step, if present, to separate the target analyte from the sample. Various methods can be used to separate the target analyte from the sample, for example, via immobilization on a support. The assay methods covered include, but are not limited to, any convenient methods and assay formats targeting pairs of specific binding members, such as avidin-biotin or hapten-anti-hapten antibody. The methods and assay formats that may be adapted for use with the composition in question include, but are not limited to, flow cytometry, insight hybridization, enzyme-linked immunosorbent assays (ELISA), Western blot analysis, magnetic cell separation assays, and fluorochrome purification chromatography.

[0380] In certain embodiments, the method further includes contacting a sample with a second specific binding member that specifically binds a target analyte. In certain cases, the second specific binding member is a support binding. Any convenient support may be used to immobilize the components of the method of the subject (e.g., the second specific binding member). In certain cases, the support is a particle, such as a magnetic particle. In some cases, the second specific binding member and the polymer dye conjugate form a sandwich complex that, if present, can be isolated and detected using any convenient method. In some embodiments, the method further includes analyzing the polymer dye-conjugated target analyte-bound complex, i.e., the fluorescently labeled target analyte, by flow cytometry. The assay for the presence of the polymer dye-conjugated target analyte-bound complex may provide assay results (e.g., qualitative or quantitative assay data) that can be used to assess whether the target analyte is present in the sample.

[0381] Any convenient support can be used in the subject method to immobilize any convenient components of the method, such as labeled specific binding members, targets, or secondary specific binding members. The support in question includes, but is not limited to, solid substrates, and the substrate can have a variety of configurations, such as sheets, beads, or other structures like well-equipped plates, beads, polymers, particles, fibrous meshes, hydrogels, porous matrices, pins, microarray surfaces, and chromatographic supports. In some cases, the support is selected from the group consisting of particles, planar solid substrates, fibrous meshes, hydrogels, porous matrices, pins, microarray surfaces, and chromatographic supports. The support can be incorporated into a system that provides cell isolation assisted by any convenient method, such as a manually operated syringe, centrifuge, or automated liquid handling system. In some cases, the support finds use in automated liquid handling systems for high-throughput cell isolation, such as flow cytometers.

[0382] In some embodiments of the method, the separation step includes applying an external magnetic field to fix the magnetic particles. Any convenient magnet can be used as the source of the external magnetic field (e.g., a magnetic field gradient). In some cases, the external magnetic field is generated by a magnetic source, for example, a permanent magnet or an electromagnet. In some cases, fixing the magnetic particles means that the magnetic particles accumulate on the surface closest to the magnetic field gradient source, i.e., near the magnet.

[0383] The separation may further include one or more optional washing steps to remove unbound material from the sample from the support. Any convenient washing method, for example, washing the immobilized support with a biocompatible buffer that preserves the specific binding interaction between the polymer dye and the specific binding member, may be used. The separation of unbound material from the sample from the support and optional washing provide an enriched population of target cells from which undesirable cells and materials can be removed.

[0384] In certain embodiments, the method further includes detecting a labeled target. Detection of the labeled target may include exciting a multichromophore with one or more lasers and then detecting fluorescence emission from the polymer tandem dye using one or more optical detectors. Detection of the labeled target can be performed using any convenient instruments and methods, including but not limited to flow cytometry, FACS systems, fluorescence microscopes, fluorescence, emission, ultraviolet, and / or visible light detection using plate readers, high-performance liquid chromatography (HPLC), and mass spectrometry. When using fluorescently labeled components in the methods and compositions of the present disclosure, it is recognized that different types of fluorescence detection systems can be used to practice the methods of the subject. In some cases, high-throughput screening can be performed, for example, using systems with 96 or more wells of microtiter plates. For example, various methods for performing assays on fluorescent materials can be used, 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; and Turro, NJ, Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Col, Inc. (1978), pp.296-361.

[0385] 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 the excitation optics. The excitation optics excite the sample with the excitation radiation. In response, a fluorescently labeled target in the sample emits radiation having a different wavelength from the excitation wavelength. The collection optics then collect the emission from the sample. The device may include a temperature controller to maintain the sample at a specific temperature while the sample is being scanned. In certain cases, a multi-axis translation stage moves a microtiter plate holding multiple samples to position different wells to be exposed. The multi-axis translation stage, temperature controller, autofocus function, and electronics associated with imaging and data acquisition can be managed by a properly programmed digital computer. The computer can also convert the data collected during the assay into another format for presentation.

[0386] In some embodiments, a method for evaluating a sample for the presence of a target analyte further includes detecting fluorescence in a flow cytometer. In some embodiments, a method for evaluating a sample for the presence of a target analyte further includes imaging the sample in contact with the labeled composition using a fluorescence microscope. Fluorescence microscopy imaging can be used to identify polymer dye-conjugated-target analyte bound complexes in the contacted sample to assess the presence or absence of the target analyte. Microscopy techniques that find use in the methods of the subject include laser scanning confocal microscopy.

[0387] Also provided is a method of labeling a target molecule. The subject polymeric dyes find use in various methods of labeling, separation, detection, and / or analysis. In some embodiments, the method includes contacting a target molecule with a polymeric dye (e.g., as described herein) to produce a labeled target molecule, where the polymeric dye includes a conjugation tag that covalently binds to the target molecule. In some cases, the polymeric dye further includes a signal chromophore covalently bound in an energy-accepting proximity to the multi-chromophore of the polymeric dye. In some examples of the method, the polymeric dye member includes a multi-chromophore according to any one of formulas (I)-(IX) (e.g., as described herein), where G 1 and G 2 one of which is a terminal group and G 1 and G 2 the other of which is a conjugation tag.

[0388] As used herein, the term “conjugation tag” refers to a group that includes a chemoselective functional group (e.g., as described herein) that can covalently bind to a compatible functional group of a target molecule after optional activation and / or deprotection. Any convenient conjugation tag can be utilized in the subject polymeric dyes to conjugate the dye to a target molecule of interest. In some embodiments, the conjugation tag includes a terminal functional group selected from amino, carboxylic acid or its derivatives, thiol, hydroxyl, hydrazine, hydrazide, azide, alkyne, and protein-reactive groups (e.g., amino-reactive, thiol-reactive, hydroxyl-reactive, imidazolyl-reactive, or guanidinyl-reactive).

[0389] Any convenient methods and reagents may be adapted for use in the labeling methods of the subject to covalently bond a conjugated tag to a target molecule. Methods for labeling the subject include, but are not limited to, those described by Hermanson, Bioconjugate Techniques, Third edition, Academic Press, 2013. The contact step may be performed in aqueous solution. In some cases, the conjugated tag contains an amino functional group and the target molecule contains an activated ester functional group such as an NHS ester or sulfo-NHS ester, or vice versa. In certain cases, the conjugated tag contains a maleimide functional group and the target molecule contains a thiol functional group, or vice versa. In certain cases, the conjugated tag contains an alkyne (e.g., cyclooctin group) functional group and the target molecule contains an azide functional group that can be conjugated via click chemistry, or vice versa.

[0390] Any convenient target molecule can be selected for labeling using the subject method. Target molecules of interest include, but are not limited to, nucleic acids such as RNA, DNA, PNA, CNA, HNA, LNA, or ANA molecules; proteins such as fusion proteins; modified proteins such as phosphorylated proteins, glycosylated proteins, ubiquitinated proteins, SUMOylated proteins, or acetylated proteins; or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins, and drug molecules. As used herein, the term “target protein” refers to all members of the target family and fragments thereof. Target proteins can be any protein of interest, including, but are not limited to, hormones, growth factors, receptors, enzymes, cytokines, bone-inducing factors, colony-stimulating factors, and immunoglobulins, such as therapeutic or diagnostic targets. The term “target protein” is intended to include recombinant and synthetic molecules that can be prepared using any convenient recombinant expression method, or that can be prepared using any convenient synthesis method, or that can be commercially available. In some embodiments, the target molecule is a specific binding member (e.g., as described herein). In certain specific cases, the specific binding member is an antibody. In some cases, the specific binding member is an antibody fragment or its bound derivative. In some cases, the antibody fragment or its bound derivative is selected from the group consisting of Fab fragments, F(ab')2 fragments, scFv, diabodies, and triabodies.

[0391] In some cases, the method includes a separation step in which the labeled target molecule is separated from the reaction mixture, for example, an excess of reagent or an unlabeled target. Various methods can be used to separate the target from the sample, for example, by immobilization on a support, precipitation, chromatography, etc.

[0392] In some cases, the method further includes detecting and / or analyzing a labeled target molecule. In some cases, the method further includes fluorescence detection of the labeled target molecule. Any convenient method may be used in conjunction with the method and composition of the subject to detect and / or analyze the labeled target molecule. Analytical methods of the target of the subject that find use in the method of the subject include, but are not limited to, flow cytometry, fluorescence microscopy, insight hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assay, and fluorochrome purification chromatography. Detection methods of the subject include, but are not limited to, fluorescence spectroscopy, fluorescence microscopy, nucleic acid sequencing, fluorescence insight hybridization (FISH), protein mass spectrometry, and flow cytometry.

[0393] Detection can be achieved directly or indirectly via polymer dyes or polymer tandem dyes, or by secondary detection systems. The latter may be based on one or a combination of several different principles, including, but not limited to, antibody-labeled anti-species antibodies, as well as 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). Suitable reporter molecules may be those known in the fields of immunocytochemistry, molecular biology, optics, fluorescence, and electron microscopy, cellular immunophenotyping, cell sorting, flow cytometry, cell visualization, detection, enumeration, and / or signal output quantification. Two or more antibodies of specific and / or nonspecific nature can be labeled and used simultaneously or sequentially to enhance target detection, identification, and / or analysis.

[0394] Preparation method Aspects of this disclosure include methods for preparing multichromophores and polymer tandem dyes of the subject. One advantage of multichromophores of the subject is the modularity of the underlying scaffold. In some cases, the modular scaffold is a linear polymer having a defined sequence of repeating units. The tandem chromophores used can be selected from a wide range of dyes that can be covalently bonded to the polymer backbone either before or after polymer synthesis.

[0395] In some embodiments, the polymer backbone of the subject's multichromophore (see, for example, formula (IX) described herein) may be prepared using cyclic carbonates or protected carbonate monomers. The methods and comonomers described are, but are not limited to, those described by Barnes et al. in WO2013036532, Cooley et al. (J.Am.Chem.Soc., 131, 45, 1640-3, 2009), and Rothbard et al. in US Patent 7, 169, 814. Such monomers can be utilized in polymerization reactions using initiators and cyclic carbonate monomers in preferred supply ratios to provide a polymer backbone. Alternative protected carbonate monomers can be assembled in stepwise synthesis to provide a defined sequence.

[0396] The comonomers in question can be linked using compatible chemoselective functional groups and chemistry. In some cases, comonomers are linked via copper-catalyzed azide-alkyne cycloaddition, strain-enhanced azide-alkyne cycloaddition, alkene azide[3+2] cycloaddition, and reverse demand Diers-Alder and Staudinger ligation of alkenes and tetrazines (see, e.g., Kolb et al., Angew Chem Int Ed Engl. 40:2004-2021, 2001). Thus, the linkage of comonomers can be achieved through the conjugation of compatible chemoselective functional group pairs, such as alkyne / azide, tetrazine / alkene, azide / alkene, and phosphine / azide. Non-limiting examples of azide-alkynecycloaddition reactions include copper-catalyzed azide-alkynecycloaddition (CuAAC) reactions and ruthenium-catalyzed azide-alkynecycloaddition (RuAAC) reactions. CuAACs act over a wide temperature range, are insensitive to aqueous conditions and pH ranges from 4 to 12, and are resistant to a wide range of functional groups (see Himo et al, J Am Chem Soc. 127:210-216, 2005). Active Cu(I) catalysts can be generated from Cu(I) or Cu(II) salts, for example, using sodium ascorbate as a reducing agent. This reaction forms 1,4-substituted products. RuAAC can catalyze the cycloaddition of azides to terminal alkynes, utilizing the pentamethylcyclopentadienylruthenium chloride [Cp*RuCl] complex to locally selectively yield 1,5-disubstituted 1,2,3-triazoles (see Rasmussen et al., Org. Lett. 9:5337-5339, 2007). Furthermore, in contrast to CuAAC, RuAAC can also be used with internal alkynes to provide fully substituted 1,2,3-triazoles.

[0397] In some embodiments, the polymer backbone of the subject multichromophore is a polypeptide. Any convenient peptide synthesis method can be used to prepare such polymer backbone of the subject multichromophore. In some cases, solid-phase peptide synthesis (SPPS) methods are used to prepare the polymer backbone of the subject multichromophore via a defined stepwise synthesis. Conventional protecting group strategies provide deprotection and binding of target amino acid residues within a defined sequence, while the target side-chain functional groups can be orthogonally protected. In some cases, the Fmoc / tert-butyl method is used. In some cases, the Boc / benzyl method is used. Such protecting group strategies can also be adapted for use in placing target pendant groups on the side chains of specific amino acid residues and / or at the polypeptide terminus. In certain cases, one or more of the pendant groups may be placed in the protected amino acid monomer initiating material. In some cases, the pendant groups are placed in the multichromophore after SPPS of the polymer backbone has been performed. By using orthogonal protecting groups, different groups of interest can be independently positioned on the polypeptide backbone, for example, at the N-terminus and C-terminus, and / or at each different type of amino acid residue. Polypeptides may contain β2-amino acid residues, β3-amino acid residues, α-amino acid residues, or mixtures thereof.

[0398] In some embodiments, a method for preparing a light-harvesting multichromophore (for example, as described herein) involves synthesizing a protected polypeptide having a defined amino acid sequence consisting of blocks of first amino acid residues separated by a single second amino acid residue. As described herein, the preparation of a defined sequence of amino acid residues can provide a desired configuration of a pendant donor chromophore and acceptor fluorophore along the polypeptide. Once the placement of the pendant group is achieved after polypeptide synthesis, the chemoselective functional groups of the amino acid side chains along the polypeptide backbone are selectively conjugated to the pendant group. In a particular case, the light-harvesting multichromophore is a polypeptide having a defined sequence, where each block of first amino acid residues comprises at least two residues, each of which contains a protected first chemoselective side chain group, and each of which contains a protected second chemoselective side chain group.

[0399] Alternatively, the placement of pendant donor chromophore groups can be achieved by utilizing protected amino acid construction blocks that already contain chromophore groups as part of the side chain. It is understood that pendant chromophore groups can first be placed on at least the side chain of a defined polypeptide sequence to generate light-harvesting multichromophore groups. Then, various additional pendant groups, including acceptor fluorophores, can be sequentially placed via selective conjugation to chemoselective tags attached to specific residues of the sequence.

[0400] In some cases, the method further comprises coupling a reactive acceptor fluorophore moiety to a deprotected second chemoselective side chain group of a second amino acid residue to generate a pendant acceptor fluorophore.

[0401] In certain cases of this method, the defined amino acid sequence includes one or more amino acid sequence segments selected from the following: XYXX XXYXX XXXYXXX XXXYXXXX XXXXYXXX XXXXYXXXX XXXXXYXXXXX XXXXXXYXXXXXX XXXXXXXYXXXXXXX XXXXXXXXYXXXXXXXX XXXXXXXXXYXXXXXXXXX Y(X) n Y XY(X) n YX XXY(X) n YXX XXXY(X) n YXXX XXXXY(X) n YXXXX XXXXXY(X) n YXXXXX Each X is a first amino acid residue having a first chemoselective functional group or a protected version thereof, and each Y is a second amino acid residue having a second chemoselective functional group or a protected version thereof.

[0402] In certain embodiments of the above sequences, each X is a lysine or ornithine residue having a side-chain amino group that can be selectively covalently bonded to a pendant donor chromophore group, or a protected version thereof, and each Y is a cysteine ​​residue or a protected cysteine ​​residue having a side-chain thiol group that can be selectively covalently N-linked to a pendant acceptor fluorophore.

[0403] Polypeptide sequences containing two or more of the above-described sequence segments may, optionally, be prepared by separation by an additional third amino acid residue (e.g., if Z is the third residue, (segment 1)-Z-(segment 2)). This third amino acid residue can be a spacer residue (e.g., without a side chain or tag), or a residue having a chemoselective functional group suitable for the selective placement of the additional portion of interest, such as a second pendant light-absorbing chromophore, a chemoselective tag (e.g., a bioorthogonal click chemist tag), a linker, a linked biomolecule, an acceptor fluorophore, or a WSG. In certain cases, the additional portion of interest is incorporated into the third amino acid residue before polypeptide synthesis.

[0404] Similarly, various parts can be positioned at the N-terminus and / or C-terminus of a polypeptide during or after peptide synthesis. In certain cases, the method further comprises deprotecting the N-terminus of a protected polypeptide and coupling a G1 group (e.g., as described herein) to the N-terminus of the N-terminally deprotected polypeptide. G1 can be any convenient terminal group (e.g., alkanoyl, a capping group such as acetyl), a donor chromophore group, a linker with a specific chemoselective tag, or the biomolecule of interest.

[0405] In some cases, the method further includes placing a G2 group at the C-terminus of the polypeptide. This can be achieved in various ways, for example, during SPPS where the C-terminal G2 group is placed between the solid support and the first amino acid residue of the sequence, during cleavage of the polypeptide from the solid support, or after synthesis where a moiety of interest (e.g., a specific binding member) or a particular chemoselective tag targeting them can be bound to the C-terminus of the polypeptide. In some cases, natural chemical ligation methods can be used to prepare C-terminal thioester polypeptides suitable for binding to polypeptide fragments or moieties of interest.

[0406] Any of the polypeptide multichromophores described herein can be prepared according to the subject method, for example, the polypeptides of formulas (IV) to (VII) described herein and their synthetic precursors.

[0407] Summary of various methods available for synthesizing the polypeptide multichromophore of the subject can be found in Steward et al., in “Solid Phase Peptide Synthesis”, WH Freeman Co., San Francisco, 1969; Bodanszky et al., in “Peptide Synthesis”, John Wiley & Sons, Second Edition, 1976 and Meienhofer, in “Hormonal Proteins and Peptides”, Vol.2, p.46, Academic Press (New York), 1983; and Kent, Ann. Rev. Biochem., 57, 957, 1988, for solid phase peptide synthesis, and Schroder et al., in “The Peptides”, Vol.1, Academic Press (New York), 1965 for solution synthesis. While not limited to these, any convenient protecting strategy may be used, such as Fmoc solid phase peptide synthesis and Boc solid phase peptide synthesis strategies. In Boc solid-phase peptide synthesis, the Boc-amino protecting group is used at the amino terminus, and benzyl or other convenient protecting groups may be used to protect the side-chain functional group. In Fmoc solid-phase peptide synthesis, the Fmoc-amino protecting group is used at the amino terminus, and tert-butyl or other convenient protecting groups may be used to protect the side-chain functional group. Convenient protecting groups that may be used in such synthetic methods are described in the above references and in McOmic in “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.

[0408] system Aspects of the present invention further include systems for use in practicing the subjective methods and compositions. A sample analysis system may include a sample field or flow channel filled with a sample and a labeled specific binding member. In some embodiments, the system is a flow cytometer, comprising a flow cytometer including a flow path, 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, the system for analyzing a sample is a fluorescence microscope system, comprising a fluorescence microscope including a sample field, and a composition placed in the sample field, the composition comprising a sample and a labeled specific binding member (e.g., as described herein).

[0409] In some cases of the system, the labeled specific binding member includes a water-solvated light-gathering multichromophore (e.g., as described herein) and a specific binding member that specifically binds to the target analyte covalently bonded to the multichromophore. In some cases, the labeled specific binding member further includes a signal chromophore covalently bonded to the multichromophore of the polymer dye near an energy acceptor. In some cases of the system of the subject, the labeled specific binding member, the multichromophore, is described by any one of formulas (I) to (IX) (e.g., as described herein). 1 and G 2 Each is independently selected from the group consisting of terminal groups, polymer-conjugated segments, linkers, and linked specific binding members, G 1 and G 2 At least one of them is a linked specific binding member.

[0410] In certain embodiments of this system, the composition further includes a second specific binding member that is bound to a support and specifically binds to the target analyte. In some cases, the support includes magnetic particles. Thus, in certain specific cases, the system may also include a controllable external paramagnetic field configured to be applied to the assay area of ​​the flow channel.

[0411] The sample may contain cells. In some cases, the sample is a biological sample containing cells. In some cases, the sample contains a labeled specific binding member that is specifically bound to the target cells. In certain cases, the target analyte specifically bound by the specific binding member is a cell surface marker. In certain cases, the cell surface marker is selected from a group consisting of cell receptors and cell surface antigens.

[0412] In certain embodiments, the system may also include a light source configured to direct light into the assay region of the flow channel or sample field of view. The system may also include a detector configured to receive a signal from the assay region of the flow channel or sample field of view, the signal being provided by a fluorescent composition. Optionally, the sample analysis system may include one or more additional detectors and / or light sources for the detection of one or more additional signals.

[0413] In certain embodiments, the system may further include a computer-based system configured to detect the presence of a fluorescent signal. “Computer-based system” means 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 will readily understand that any one of the currently available computer-based systems is suitable for use in the system of the subject. The data storage means may include any product containing a record of the information, as described above, or memory access means capable of accessing such product.

[0414] "Recording" data, programming, or other information onto a computer-readable medium refers to the process of storing information using any such method known in the art. Any convenient data storage structure can be selected based on the means used to access the stored information. Various data processor programs and formats can be used for storage, such as word processing text files and database formats.

[0415] "Processor" refers to any combination of hardware and / or software that performs the necessary functions. For example, any processor as used herein may be a programmable digital microprocessor, available in the form of, for example, an electronic controller, a mainframe, a server, or a personal computer (desktop or portable). If the processor is programmable, appropriate programming may be communicated to the processor from a remote location or pre-stored in a computer program product (e.g., portable or fixed computer-readable storage medium, whether magnetic, optical, or solid-state). For example, a magnetic medium or optical disk may carry programming that can be read by a suitable reader communicating with each processor at its corresponding station.

[0416] For example, in addition to the sensor device and signal processing module as described above, the system of the present invention may include several additional components, such as a data output device such as a monitor and / or speaker, a data input device such as an interface port or keyboard, a fluid processing component, and a power supply.

[0417] In certain embodiments, the system includes a flow cytometer. Suitable flow cytometry systems and methods for analyzing samples are, but are not limited to, 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 (pt 1): 17-28; Linden, et al., Semin Throm 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 This includes what is described in Syst.24(3):203-255, and these disclosures are incorporated herein by reference.In certain cases, the target flow cytometry system is the BD Biosciences FACSCanto® flow cytometer, BD Biosciences FACSVantage®, BD Biosciences FACSort®, BD Biosciences FACSCount®, BD Biosciences FACScan®, and the BD Biosciences FACSCalibur® system, BD Biosciences Accuri® system, BD Biosciences FACSCanto® system, BD Biosciences FACSCelesta® system, BD Biosciences FACSLyric® system, BD Biosciences FACSVerse® system, BD Biosciences FACSymphony® system, BD Biosciences LSRFortessa® system, BD Biosciences Influx® cell sorter, BD Biosciences FACSJazz® cell sorter, and BD Biosciences FACSAria® cell sorter. Includes FACSMelody® cell sorting machine, etc.

[0418] In a particular embodiment, the subject system is as specified in U.S. Patents No. 3,960,449, No. 4,347,935, No. 4,667,830, No. 4,704,891, No. 4,770,992, No. 5,030,002, No. 5,040,890, No. 5,047,321, No. 5,245,318, No. 5,317,162, No. 5,464,581, No. 5,483,469, No. 5,602,039, No. 5,620,842, No. 5,627,040, No. 5,643,796, No. 5,700,692, and No. 6,372. A flow cytometer system incorporating one or more components of the flow cytometers described in Nos. 506, 6,809,804, 6,813,017, 6,821,740, 7,129,505, 7,201,875, 7,544,326, 8,140,300, 8,233,146, 8,753,573, 8,975,595, 9,092,034, 9,095,494, and 9,097,640, the disclosures of which are incorporated herein by reference.

[0419] Other systems may find use in practicing the subject method. In certain embodiments, the system may be a fluorometer or microscope packed with a sample having a fluorescent composition of any of the embodiments considered herein. The fluorometer or microscope may include a light source configured to direct light into an assay region of the flow channel or sample field. The fluorometer or microscope may also include a detector configured to receive a signal from the assay region of the flow channel or sample field, the signal provided by the fluorescent composition.

[0420] kit Aspects of the present invention further include kits for use in practicing the subject methods and compositions. The compositions of the present invention may be included in the kit as reagents, either as initiating materials or as provided for use in the methods described above, for example.

[0421] The kit may include a polymer dye comprising a water-solvable light-gathering multichromophore (e.g., as described herein) and a container. Any convenient container can be used, such as a tube, bottle, or well, box, bag, or insulating container in a multiwell strip or plate. The kit of the subject may further include one or more components selected from polymer tandem dyes, fluorophores, specific binding members, specific binding member conjugates, support-binding specific binding members, cells, support, biocompatible aqueous elution buffer, and instructions for use. In some embodiments of the kit, the multichromophore is covalently bound to a specific binding member. In some cases, the specific binding member is an antibody. In certain specific cases, the specific binding member is an antibody fragment or its bound derivative. In certain specific cases, the antibody fragment or its bound derivative is selected from the group consisting of Fab fragments, F(ab')2 fragments, scFv, diabodies, and triabodies.

[0422] In certain embodiments, the kit finds use in evaluating a sample for the presence of a target analyte, such as an intracellular target. Therefore, in some cases, the kit includes one or more components suitable for cell lysis. One or more additional components of the kit may be provided in separate containers (e.g., separate tubes, bottles, or wells in a multiwell strip or plate).

[0423] In certain embodiments, the kit further comprises reagents for performing a flow cytometry assay. The reagents in question, though not limited to these, include buffers for reconstitution and dilution, buffers for contacting multichromophore cell samples, washing buffers, control cells, control beads, fluorescent beads for flow cytometer calibration, and combinations thereof. The kit may also include one or more cell fixation reagents, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination thereof or buffers. Furthermore, the kit may include cell permeability reagents, such as methanol, acetone, or washing agents, such as Triton, NP-40, saponin, Tween 20, digitonin, leucoperme, or any combination thereof or buffers. Other protein transport inhibitors, cell fixation reagents, and cell permeability reagents well known to those skilled in the art are within the scope of the kits in question.

[0424] The kit composition may be provided in a liquid composition such as any suitable buffer. Alternatively, the kit composition 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 a particular embodiment, the kit may include aliquots of the composition provided in a separate container (e.g., a separate tube, bottle, or well in a multiwell strip or plate).

[0425] In addition, one or more components may be combined into a single container, such as a glass or plastic vial, tube, or bottle. In certain cases, the kit may further include a container (e.g., a box, bag, insulating container, bottle, tube, etc.) in which all components (and their separate containers) reside. The kit may be separate from the kit container or attached to the kit container and may further include printed information on the kit, the components of the kit, and / or instructions for using the kit.

[0426] In addition to the components described above, the subject kit may further include instructions for practicing the subject method. These instructions may be present in the subject kit in various forms, and one or more of them may be present in the kit. One possible form in which these instructions may be present is as information printed on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, the kit's packaging, accompanying documentation, etc. Yet another means is a computer-readable medium on which the information is recorded, such as a diskette, CD, DVD, portable flash drive, etc. Yet another possible form is a website address that can be used via the internet to access the information at a deleted site. Any convenient means may be present in the kit.

[0427] Utility The polymer dyes, compositions, methods, and systems described herein can be found 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 such as cytometry, microscopy, immunoassays (e.g., competitive or non-competitive), evaluation of free analytes, and evaluation of receptor-binding ligands. The compositions, systems, and methods described herein, though not limited to these, may be useful for the analysis of any of several samples, including biological fluids, cell culture samples, and tissue samples. In certain embodiments, the compositions, systems, and methods described herein can be found in methods where the analyte is detected in a sample, using fluorescent labeling, if present, such as in fluorescence-activated cell sorting or analysis, immunoassays, or immunostaining. In certain cases, the compositions and methods find use in applications where evaluation of the sample for the presence of a target analyte is important.

[0428] In some cases, the methods and compositions find use in any assay format where the detection and / or analysis of a target from a sample is important, including, but not limited to, flow cytometry, fluorescence microscopy, insight hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assay, and fluorochrome purification chromatography. In certain specific cases, the methods and compositions find use in any application where the fluorescent labeling of the target molecule is important. The compositions of the subject matter may be adapted for use in any convenient application where pairs of specific binding members find use, such as biotin-streptavidin and hapten-anti-hapten antibodies. [Examples]

[0429] Example 1 The synthesis of the polymer dyes in question can be achieved by the Suzuki coupling method. Other methods, such as CH-linked arylation or Stille coupling, can also be used to construct and polymerize repeating units containing aryl or heteroaryl comonomers with saturated peg comonomers.

[0430] The formulas for tandem dyes based on multichromophore A (MC) are shown in Figure 1. A series of tandem dyes having the following core structures with different acceptor fluorophores ("dyes") were prepared.

[0431] [ka]

[0432] The spectral properties of exemplary polymer tandem dyes were evaluated as shown in Figures 2 and 3.

[0433] [Table 1]

[0434] Example 2 As shown in Figure 4, the sequences of the lysine and cysteine ​​amino acid residues KCKK were prepared using a solid-phase peptide synthesis method. A carboxylic acid-substituted BODIPY group was conjugated to the N-terminus of the peptide and the side-chain amino group of the lysine residue via amide bond coupling. A maleimide-substituted acceptor fluorophore (dye 7) was conjugated to the cysteine ​​residue using maleimidethiol coupling chemistry. The peptide was prepared with a C-terminal linker suitable for conjugation to biomolecules, such as proteins.

[0435] The spectral properties of the BODIPY-based tandem dyes in Figure 4 were characterized. Figure 5A shows the absorbance and emission spectra of the BODIPY donor pendant group. Figure 5B shows the absorbance and emission spectra of the acceptor dye (dye 7). Figure 5C shows the absorbance and emission spectra of the exemplary polymer tandem dye in Figure 4. For the emission spectrum of the scaffold-type chromophore, the primary chromophore is excited. With respect to this system, donor emission is almost completely quenched, and the majority of the emission is from the acceptor fluorophore. The quantum yield of the polymer tandem dye is comparable to that of the acceptor fluorophore (dye 7), suggesting that the tandem dye exhibits an efficient energy transfer process. The fluorescence of this exemplary tandem dye was amplified approximately twice as much as the fluorescence of the acceptor fluorophore alone (dye 7).

[0436] Polypeptides providing donor-to-acceptor ratios of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 20:2 are prepared and characterized using the methods described above.

[0437] Example 3 Water-soluble fluorescent polymer dyes can be prepared by click polymerization under mild conditions. These polymer dyes can covalently bond to biorecognition units (biomolecules) and function as fluorescent probes for targets. When a secondary dye is incorporated as an acceptor, a polymer tandem dye is formed. By adjusting the color of the acceptor dye, polymer tandem dyes can be prepared that have the same excitation wavelength (see Figure 9A) but a range of different alternative emission wavelengths (see Figure 9B).

[0438] A pegged BODIPY dye with a desired narrow absorbance is selected as the fluorophore pendant dye. The pendant fluorophore is chemically bonded to a water-soluble ethylene glycol ether monomer terminated with a diazide, dialkyne, or azide / alkyne. The pegged polymer chain with the attached BODIPY dye can be prepared using a Cu(I)-catalyzed 1,3-dipolar adduct of alkyne to azide with good MW (e.g., as described herein). When only one type of fluorophore dye is bonded as the pendant group, the resulting polymer exhibits fluorescence as a simple fluorescent polymer dye (see Figure 8). When a second acceptor fluorophore is attached, a polymer chain with two fluorophore pendants is achieved. One fluorophore with a higher energy level can function as an energy donor, and the second fluorophore with a lower energy level can function as an energy acceptor. By mixing suitable donor / acceptor pairs and ratios, polymer tandem dyes with desired emission wavelengths can be obtained.

[0439] The following polymer dyes are prepared by click polymerization using the comonomers and methods described by the synthesis schemes in Figures 7A and 7B, where n and m can both represent the average length of the polymer and the relative ratio of the comonomers, and it is understood that n and m can be selected as desired by controlling the parameters of the polymerization reaction using any convenient method. Depending on the polymerization method, it is understood that the n and m repeating units may be present in random or block configurations. In addition, the following polymer structures can be further conjugated to any convenient target molecule, e.g., biomolecules, using click chemistry via terminal azide or alkyne groups. In some cases, n and m are each independently selected from 1 to 1000, such as 1 to 500, 1 to 200, 1 to 100, 2 to 100, or 5 to 100.

[0440] [ka]

[0441] Figure 8 shows the absorbance and emission spectra of base polymer dyes, including the pendant BODIPY dye with the general structure shown above. Other exemplary polymer dyes, in which R is a linked donor dye (e.g., the BODIPY dye described above) and which contain side chain -NH2 groups suitable for conjugation (e.g., via amide bonds and linkers to acceptor dyes), are as follows:

[0442] [ka]

[0443] An exemplary polymer tandem dye structure is shown below, where R is a conjugated donor dye (e.g., the BODIPY dye described above), and includes a conjugated acceptor dye of choice (e.g., DY-754 or DY-704, which are suitable for conjugation (e.g., conjugation to the acceptor dye)), and DY refers to a diomics dye having a maximum emission wavelength of, for example, 754 nm or 704 nm, respectively. By selecting a target acceptor dye that at least partially overlaps with the emission spectrum of the base polymer (see Figure 8), polymer tandem dyes with various emission wavelengths can be generated. See, for example, Figures 9A-9B.

[0444] [ka]

[0445] Additional Embodiments Notwithstanding the attached claims, the disclosure described herein is also meant by the following note:

[0446] Note 1. A water-soluble light-collecting multichromophore, A polymer backbone containing non-conjugated repeating units, Multiple pendant donor chromophore groups, each independently linked to a non-conjugated repeating unit of the polymer backbone, Including a light-gathering multichromophore. Note 2. Each pendant donor chromophore group is replaced with a water-soluble group, as described in Note 1, for the light-collecting multichromophore. Note 3. The polymer main chain is a linear polymer, a light-harvesting multichromophore as described in Note 1. Note 4. The pendant donor chromophore groups are configured in close proximity to each other, and are light-gathering multichromophores as described in Note 1. Note 5. The pendant donor chromophore group is a light-collecting multichromophore as described in Note 4, which exhibits fluorescence quenching relative to an isolated, unquenched donor chromophore group when excited by incident light.

[0447] Note 6. The pendant donor chromophore group is a light-harvesting multichromophore as described in any one of Notes 1 to 5, selected from a condensed tricyclic aryl group or heteroaryl group and a BODIPY group. Note 7. The pendant donor chromophore group is a light-gathering multichromophore as described in Note 6, selected from fluorene, carbazole, and silole. Note 8. A pendant donor chromophore group is a fused tricyclic aryl group or heteroaryl group having one of the following formulas:

[0448] [ka]

[0449] During the ceremony, * indicates a linkage point to the non-conjugated repeating unit of the polymer backbone. Y is C(R 13 )2, -C(R 13 )2C(R 13 )2-, -C(R 13 )2Si(R 13 )2-, NR 13 , Si(R 13 )2 or Se, Each Z is independently CH, CR, or N. Each R 13 These are independently H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amide, substituted amide, aralkyl, substituted aralkyl, peg moiety, WSG, and -L 11 -Z 1 Selected from, L 11 It is a linker, Z 1 It is either a non-conjugated repeating unit, or any two convenient R' groups are optionally linked in a ring. Each R is independently H or one or more substituents, and two convenient R groups are optionally linked cyclically. R and R 13One of these is a light-harvesting multichromophore as described in Appendix 6 or 7, which is linked to a non-conjugated repeating unit of the polymer backbone. Note 9. The pendant donor chromophore group is a BODIPY group, a light-gathering multichromophore as described in any one of Notes 1 to 6.

[0450] Note 10. The BODIPY group is represented by the following formula:

[0451] [ka]

[0452] During the ceremony, R 1 ~R 7 Each of these is independently H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG), and -L. 1 -Z 1 Selected from, or Optionally, R 6 and R 7 , R 2 and R 3 , R 5 and R 6 , R 3 and R 4 , R 4 and R 1 Furthermore, R 5 and R 1 Any one or more substituent pairs selected from the above form a divalent radical together, which is cyclically linked and, together with the carbon atoms to which they are bonded, provides a 5- or 6-membered condensed heterocycle, carbocyclic ring, aryl or heteroaryl ring (e.g., a 5- or 6-membered ring containing carbon atoms and 0 to 3 heteroatoms selected from O, S and N), the ring may be unsubstituted or independently alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and -L1 -Z 1 They may be further substituted with substituents selected from, L 1 It is a linker, Z 1 These are non-conjugated repeating units of the polymer backbone, Y 1 and Y 2 These are independently selected from F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and WSG. Y 1 , Y 2 , and R 1 ~R 7 One of these is a light-harvesting multichromophore, as described in Appendix 9, which is linked to a non-conjugated repeating unit of the polymer backbone.

[0453] Note 11.R 1 The light-harvesting multichromophore described in Appendix 10 is an optionally substituted aryl or heteroaryl linked to the non-conjugated repeating units of the polymer backbone. Note 12.Y 1 and Y 2 Each of these is a light-harvesting multichromophore as described in Appendix 10 or 11, containing a water-soluble group (WSG). Note 13. The pendant donor chromophore group is described by the following structure:

[0454] [ka]

[0455] During the ceremony, * indicates a linkage point to the non-conjugated repeating unit of the polymer backbone. Y 1 and Y 2 Each of these is an alkynyl substituted with WSG, a light-collecting multichromophore as described in any one of appendices 10 to 12. Note 14. The pendant donor chromophore group is described by the following structure:

[0456] [ka]

[0457] During the ceremony, R 11 L 1 -Z 1 (a linked, non-conjugated repeating unit of the polymer backbone), Each R 9 The light-harvesting multichromophore described in Appendix 13, wherein is an optional substituent selected from halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, and t is 0 to 4. Note 15. The pendant donor chromophore group is independently substituted with one or more water-soluble groups (WSGs) selected from the following formulas:

[0458] [ka]

[0459] During the ceremony, T 5 It is an optional linker, Each T 6 It is a linker, R 11 And R are independently H, alkyl, or substituted alkyl. Each s is an integer from 1 to 50, a light-gathering multichromophore as described in one of the appendices 1 to 14.

[0460] Note 16. The following expressions include segments:

[0461] [ka]

[0462] During the ceremony, Each M 1 and M 2 These are independently aryl or heteroaryl comonomers, Each S 1 and S 2 These are independent, non-conjugate spacer units. Each D 1 M 1 It is a pendant donor chromophore connected to, each Z 1 M 2 It is a chemoselective tag linked to, x is 75 mol% or more, y is 25 mol% or less, and is a light-collecting multichromophore as described in any one of the appendices 1 to 15. Note 17. M of the polymer backbone 1 -S 1 and M 2 -S 2 The repeating unit is a light-gathering multichromophore as described in Appendix 16, having a random configuration. Appendix 18.Each M 1 and M 2 It independently comprises one or more groups selected from fluorene, carbazole, silol, biphenylene, and phenylene. Each S 1 and S 2 The light-harvesting multichromophore described in Appendix 16 or 17 is independently a saturated spacer unit selected from divalent polyethylene glycol (PEG) and divalent modified PEG groups. Note 19. The following expressions include segments:

[0463] [ka]

[0464] During the ceremony, The polymer backbone of the non-conjugated repeating units is SM, where each unit is independently a saturated non-conjugated comonomer. 1 and SM 2 Contains a copolymer, Each D1 SM 1 It is a pendant donor chromophore connected to, each Z 1 SM 2 It is a chemoselective tag linked to, x is 75 mol% or more, y is 25 mol% or less, and is a light-collecting multichromophore as described in any one of the appendices 1 to 15. Note 20. SM of polymer backbone 1 and SM 2 The repeating unit is a light-gathering multichromophore as described in Appendix 19, having a random configuration.

[0465] Note 21.SM 1 and SM 2 The light-harvesting multichromophore according to Appendix 19 or 20 is a comonomer derived from acrylate, methacrylate, acrylamide, polystyrene, ROMP monomer, ADMET monomer, or cyclic carbonate. Note 22.SM 1 and SM 2 The following formula is selected:

[0466] [ka]

[0467] During the ceremony, R 21 is, -L 1 -D 1 or -L 2 -Z 1 And, D 1 M 1 It is a pendant donor chromophore connected to, Z 1 M 2 It is a chemoselective tag linked to, L 1 and L 2 It is an optional linker, X is O or NR'', R 22 is H or a lower alkyl group, R'' is H or a lower alkyl, a substituted lower alkyl, and a WSG. * indicates a light-harvesting multichromophore as described in Appendix 21, which is connected to the polymer main chain.

[0468] Note 23. The multichromophore is of formula (XXI),

[0469] [ka]

[0470] During the ceremony, The polymer backbone of the non-conjugated repeating units is linked via groups T, which are products of click chemistry or chemoselective group-conjugation reactions, forming SMs. 1 SM 2 , and SM 3 Contains a copolymer, SM 3 This optionally includes a concatenated WSG. Each D 1 SM 1 It is a pendant light-absorbing chromophore connected to it, each Z 1 SM 2 It is a chemoselective tag linked to, x is 50 mol% or more, y+z is 50 mol% or less, and * is a multichromophore or terminal group connected to the polymer backbone, as described in Appendix 19. Note 24.SM 1 SM 2 , and SM 3 It includes the following structure:

[0471] [ka]

[0472] During the ceremony, Each X is independently O or NR 31 And R 31 is H, alkyl, substituted alkyl, alkanoyl, or substituted alkanoyl, Each r and s is independently 1 to 6 (e.g., 1, 2, or 3). Each d and e is independently 1 to 12 (for example, 1 to 6 such as 1, 2, 3, 4, 5, or 6). t is either 0 or 1, D 1 It is a pendant donor chromophore, Z 1 is a chemoselective tag (as described herein, for example), WSG is a water-soluble group (as described herein, for example), Each L 1 , L 2 , and L 3 It is, independently, a linker, * indicates a connection to a 1,4-substituted 1,2,3-triazole(T) having one of the following structures,

[0473] [ka]

[0474] Alternatively, terminal group G (as described herein, for example) 1 Or G 2 This is the light-gathering multichromophore described in Appendix 23. Note 25. The repeating units of the polymer backbone are light-harvesting multichromophores as described in Note 19, having a defined linear arrangement.

[0475] Note 26.SM 1 and SM 2 The light-harvesting multichromophore described in Appendix 25 is a comonomer derived from an amino acid, peptoid monomer, protected carbonate monomer, or cyclic carbonate monomer. Note 27. A light-harvesting multichromophore according to any one of Notes 19, 25, and 26, wherein the polymer backbone is a polypeptide having a defined sequence of α-amino acid residues and / or β-amino acid residues. Note 28. The multichromophore is defined by the following formula:

[0476] [ka]

[0477] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, each Z 1 These are independently chemoselective tags, Each L 1 and L 2 It is, independently, a linker, p1 and q1 are independently either 0 or 1, and p1 + q1 ≤ 1. p2 and q2 are independently 0 or 1, and p2 + q2 ≤ 1. x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2 Each of the following is independently selected from terminal groups, polymer segments, donor chromophore groups, acceptor fluorophore groups, linkers, and linked specific binding members, as described in Appendix 19 and any one of 25-27. Note 29. A light-gathering multichromophore as described in Note 28, where p1 and p2 are each 0, and q1 and q2 are each 1. Note 30. A light-gathering multichromophore as described in Note 28, where p1 and p2 are each 1, and q1 and q2 are each 0.

[0478] Note 31. p1, p2, q1, and q2 are each 0, and the multichromophore is given by the following equation:

[0479] [ka]

[0480] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, each Z 1 These are independently chemoselective tags, L 1 and L 2 Each of them is an independent linker, x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2 Each of these is independently selected from terminal groups, polymer segments, donor chromophore groups, acceptor fluorophore groups, linkers, and linked specific binding members, as described in Appendix 28. Note 32. The following expressions include segments:

[0481] [ka]

[0482] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, each Z 1 These are independently chemoselective tags, Each L 1 and L 2 It is, independently, a linker, n and p are each independent integers between 1 and 20, and in the equation, n + p ≥ 2. A light-gathering multichromophore as described in any one of appendices 27, 28, and 31, wherein m is 1 or 2. Note 33. The light-harvesting multichromophore contains a segment of the copolymer q and has the following formula:

[0483] [ka]

[0484] During the ceremony, each(n) q and each (p) q These are independent integers from 1 to 20, and in the expression, for each segment of q, (n) q +(p) q ≥ 3, q is an integer between 1 and 100, as described in Appendix 32, for a light-gathering multichromophore. Note 34. The polymer backbone contains one or more amino acid sequences selected from the following: XYXX XXYXX XXXYXXX XXXYXXXX XXXXYXXX XXXXYXXXX XXXXXYXXXXX XXXXXXYXXXXXX XXXXXXXYXXXXXXX XXXXXXXXYXXXXXXXX XXXXXXXXXYXXXXXXXXX Y(X) n Y XY(X) n YX XXY(X) n YXX XXXY(X) n YXXX XXXXY(X) n YXXXX XXXXXY(X) n YXXXXX During the ceremony, Each X is a lysine or ornithine residue covalently N-linked to a pendant donor chromophore group. Each Y is a cysteine ​​residue or a protective cysteine ​​residue, as described in any one of the appendices 19, 25-28, and 31-33, for a light-harvesting multichromophore.

[0485] Note 35. The light-gathering multichromophore has the following formula:

[0486] [ka]

[0487] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, each Z 1 These are independently chemoselective tags, Each L 1 and L 2 It is, independently, a linker, x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2 Each of the following is independently selected from terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, linkers, and linked specific binding members: a light-harvesting multichromophore according to any one of Appendices 19, 25, and 26.

[0488] Note 36. The light-gathering multichromophore has the following formula:

[0489] [ka]

[0490] During the ceremony, Each D 1 It is, independently, a pendant donor chromophore, each Z 1 These are independently chemoselective tags, Each L 1 and L 2 It is, independently, a linker, x is 75 mol% or more, y is 25 mol% or less. G 1 and G2 Each of these is independently selected from the group consisting of terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, linkers, and linked specific binding members, as described in any one of Appendices 19-21 and 26. Note 37. The pendant donor chromophore group is a BODIPY group, a light-gathering multichromophore as described in any one of Notes 16-36. Note 38. Polymer tandem dyes, It is a light-gathering multichromophore, Polymer backbone containing non-conjugated repeating units, and A light-harvesting multichromophore comprising multiple pendant donor chromophore groups, each independently linked to a non-conjugated repeating unit of the polymer backbone, An acceptor fluorophore is linked to the non-conjugated repeating units of the polymer backbone and configured in the vicinity of at least one pendant donor chromophore group of the light-harvesting multichromophore to accept energy. A polymer tandem dye containing [a specific ingredient / method]. Note 39. The polymer tandem dye described in Note 38, wherein each pendant donor chromophore group is replaced with a water-soluble group. Note 40. The polymer tandem dye described in Note 38 has a linear polymer main chain.

[0491] Note 41. The pendant donor chromophore groups are arranged in a proximity that transmits energy to each other, as described in Note 38, in the polymer tandem dye. Note 42. A polymer tandem dye as described in Note 36, having a Stokes shift of 100 nm or more. Note 43. The pendant donor chromophore group is selected from a fused tricyclic aryl group, a fused tricyclic heteroaryl group, and a BODIPY group, as described in any one of Notes 36 to 40, for the polymer tandem dye. Note 44. The pendant donor chromophore group is selected from optionally substituted fluorene, carbazole, and silole groups, as described in Note 43, for the polymer tandem dye. Note 45. A pendant donor chromophore group is a fused tricyclic aryl group or heteroaryl group having one of the following formulas:

[0492] [ka]

[0493] During the ceremony, * indicates a linkage point to the non-conjugated repeating unit of the polymer backbone. Y is C(R 13 )2, -C(R 13 )2C(R 13 )2-, -C(R 13 )2Si(R 13 )2-, NR 13 , Si(R 13 )2 or Se, Each Z is independently CH, CR, or N. Each R 13 These are independently H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amide, substituted amide, aralkyl, substituted aralkyl, peg moiety, WSG, and -L 11 -Z 1 Selected from, L 11 It is a linker, Z 1 is either a non-conjugated repeating unit or any two convenient R units. 3 The bases are optionally connected in a ring shape. Each R is independently H or one or more substituents, and two convenient R groups are optionally linked cyclically. R and R 13 One of these is a polymer tandem dye as described in Appendix 43 or 44, which is linked to a non-conjugated repeating unit of the polymer backbone.

[0494] Note 46. The pendant donor chromophore group is a BODIPY group, as described in Note 43, for the polymer tandem dye. Note 47. The BODIPY group is represented by the following formula:

[0495] [ka]

[0496] During the ceremony, R 1 ~R 7 Each of these is independently H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG), and -L. 1 -Z 1 Selected from, or Optionally, R 6 and R 7 , R 2 and R 3 , R 5 and R 6 , R 3 and R 4 , R 4 and R 1 Furthermore, R 5 and R 1 Any one or more substituent pairs selected from the above form a divalent radical together, which is cyclically linked and, together with the carbon atoms to which they are bonded, provides a 5- or 6-membered condensed heterocycle, carbocyclic ring, aryl or heteroaryl ring (e.g., a 5- or 6-membered ring containing a carbon atom and 0 to 3 heteroatoms selected from O, S and N), the ring may be unsubstituted or independently alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and -L 1 -Z 1 They may be further substituted with substituents selected from, L 1 It is a linker, Z 1 These are non-conjugated repeating units of the polymer backbone, Y 1 and Y 2These are independently selected from F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and WSG. Y 1 , Y 2 , and R 1 ~R 7 One of these is the polymer tandem dye described in Appendix 46, which is linked to the non-conjugated repeating units of the polymer backbone. Note 48.R 1 The polymer tandem dye as described in Appendix 47, wherein is an optionally substituted aryl or heteroaryl linked to the non-conjugated repeating units of the polymer backbone. Note 49.Y 1 and Y 2 Each of these is a polymer tandem dye as described in Appendix 47 or 48, each containing a water-soluble group (WSG). Note 50. The pendant donor chromophore group is described by the following structure:

[0497] [ka]

[0498] During the ceremony, * indicates a linkage point to the non-conjugated repeating unit of the polymer backbone. Y 1 and Y 2 Each of the following polymer tandem dyes is an alkynyl substituted with WSG, as described in any one of the appendices 47-49.

[0499] Note 51. The pendant donor chromophore group is described by the following structure:

[0500] [ka]

[0501] During the ceremony, R 11 L 1-Z 1 (a linked, non-conjugated repeating unit of the polymer backbone), Each R 9 The polymer tandem dye as described in Appendix 50, wherein is an optional substituent selected from halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, and t is 0 to 4. Note 52. The pendant donor chromophore group is independently substituted with one or more water-soluble groups (WSGs) selected from the following formulas:

[0502] [ka]

[0503] During the ceremony, T 5 It is an optional linker, Each T 6 It is a linker, R 11 And R are independently H, alkyl, or substituted alkyl. Each s is an integer from 1 to 50, a polymer tandem dye as described in any one of appendices 38 to 51. Note 53. The following expressions include segments:

[0504] [ka]

[0505] During the ceremony, Each M 1 and M 2 These are independently aryl or heteroaryl comonomers, Each S 1 and S 2 These are independent, non-conjugate spacer units. Each D 1 M 1 It is a pendant donor chromophore connected to, Each A1 M 2 It is an acceptor fluorophore linked to, x is 75 mol% or more, y is a polymer tandem dye as described in any one of the appendices 38 to 52, wherein y is 25 mol% or less. Note 54. M of the polymer main chain 1 -S 1 and M 2 -S 2 The repeating units are polymer tandem dyes as described in Appendix 53, having a random configuration. Appendix 55.Each M 1 and M 2 It independently comprises one or more groups selected from fluorene, carbazole, silol, biphenylene, and phenylene. Each S 1 and S 2 The polymer tandem dye according to Appendix 53 or 54, wherein is independently a saturated spacer unit selected from divalent polyethylene glycol (PEG) and divalent modified PEG groups.

[0506] Note 56. The following expressions include segments:

[0507] [ka]

[0508] During the ceremony, The polymer backbone of the non-conjugated repeating units is composed of SMs, each of which is an independent non-conjugated comonomer. 1 and SM 2 Contains a copolymer, Each D 1 SM 1 It is a pendant donor chromophore connected to, Each A 1 SM 2 It is an acceptor fluorophore linked to, x is 75 mol% or more, y is a polymer tandem dye as described in any one of the appendices 38 to 52, wherein y is 25 mol% or less. Note 57. The polymer tandem dye described in Note 56, wherein the repeating units of the polymer main chain have a random configuration. Note 58.SM 1 and SM 2 The polymer tandem dye according to Appendix 56 or 57, wherein the comonomer is derived from an acrylate, methacrylate, acrylamide, polystyrene, ROMP monomer, ADMET monomer, or cyclic carbonate. Note 59.SM 1 and SM 2 The following formula is selected:

[0509] [ka]

[0510] During the ceremony, R 21 is, -L 1 -D 1 or -L 2 -Z 1 And, D 1 M 1 It is a pendant donor chromophore connected to, Z 1 M 2 It is a chemoselective tag linked to, L 1 and L 2 It is an optional linker, X is O or NR'', R' is H or a lower alkyl group. R'' is H or a lower alkyl, a substituted lower alkyl, and a WSG. * indicates a polymer tandem dye as described in Appendix 58, which is a link to the polymer main chain. Note 60. The multichromophore is of formula (XXI),

[0511] [ka]

[0512] During the ceremony, The polymer backbone of the non-conjugated repeating units is linked via groups T, which are products of click chemistry or chemoselective group-conjugation reactions, forming SMs. 1 SM 2 , and SM 3 Contains a copolymer, SM 3 This optionally includes a concatenated WSG. Each D 1 SM 1 It is a pendant light-absorbing chromophore connected to it, Each A 1 SM 2 It is an acceptor fluorophore linked to, x is 50 mol% or more, The polymer tandem dye described in Appendix 56, wherein y+z is 50 mol% or less, and * is a multichromophore or terminal group connection to the polymer backbone.

[0513] Note 61.SM 1 SM 2 , and SM 3 It includes the following structure:

[0514] [ka]

[0515] During the ceremony, Each X is independently O or NR 31 And R 31 is H, alkyl, substituted alkyl, alkanoyl, or substituted alkanoyl, Each r and s is independently 1 to 6 (e.g., 1, 2, or 3). Each d and e is independently 1 to 12 (for example, 1 to 6 such as 1, 2, 3, 4, 5, or 6). t is either 0 or 1, Each L 1 , L2 , and L 3 It is, independently, a linker, * is a polymer tandem dye as described in Appendix 60, wherein the linkage is to a 1,4-substituted 1,2,3-triazole(T) having one of the following structures.

[0516] [ka]

[0517] Note 62. The polymer tandem dye as described in Note 56, wherein the repeating units of the polymer backbone have a defined linear arrangement. Note 63.SM 1 and SM 2 The polymer tandem dye described in Appendix 62 is a comonomer derived from an amino acid, peptoid monomer, protective carbonate monomer, or cyclic carbonate monomer. Note 64. The polymer tandem dye according to any one of Notes 56, 62, and 63, wherein the polymer backbone is a polypeptide having a defined sequence of α-amino acid residues and / or β-amino acid residues. Note 65. The multichromophore is defined by the following formula:

[0518] [ka]

[0519] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, Each A 1 It is independently an acceptor fluorophore, Each L 1 and L 2 It is, independently, a linker, p1 and q1 are independently either 0 or 1, and p1 + q1 ≤ 1. p2 and q2 are independently 0 or 1, and p1 + q1 ≤ 1. x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2 A polymer tandem dye according to any one of Appendices 56 and 62-64, wherein each is independently selected from terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, linkers, and linked specific binding members.

[0520] Note 66. The polymer tandem dye as described in Note 65, wherein p1 and p2 are each 0, and q1 and q2 are each 1. Note 67. The polymer tandem dye as described in Note 65, wherein p1 and p2 are each 1, and q1 and q2 are each 0. Note 68. p1, p2, q1, and q2 are each 0, and the multichromophore is given by the following formula:

[0521] [ka]

[0522] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, Each A 1 It is independently an acceptor fluorophore, L 1 and L 2 Each of them is an independent linker, x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2 The polymer tandem dye as described in Appendix 65, wherein each is independently selected from terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, linkers, and linked specific binding members. Note 69. The following expressions include segments:

[0523] [ka]

[0524] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, Each A 1 It is independently an acceptor fluorophore, Each L 1 and L 2 It is, independently, a linker, n and p are each independent integers between 1 and 20, and in the equation, n + p ≥ 2. A polymer tandem dye as described in any one of appendices 64, 65, and 66, wherein m is 1 or 2. Note 70. The multichromophore contains the q segment of the copolymer and has the following formula:

[0525] [ka]

[0526] During the ceremony, each(n) q and each (p) q These are independent integers from 1 to 20, and in the expression, for each segment of q, (n) q +(p) q ≥ 3, q is an integer between 1 and 100, and is a polymer tandem dye as described in Appendix 69.

[0527] Note 71. The polymer backbone contains an amino acid sequence selected from the following: XYXX XXYXX XXXYXXX XXXYXXXX XXXXYXXX XXXXYXXXX XXXXXYXXXXX XXXXXXYXXXXXX XXXXXXXYXXXXXXX XXXXXXXXYXXXXXXXX XXXXXXXXXYXXXXXXXXX Y(X) n Y XY(X) n YX XXY(X) n YXX XXXY(X) n YXXX XXXXY(X) n YXXXX XXXXXY(X) n YXXXXX During the ceremony, Each X is a lysine or ornithine residue covalently N-linked to a pendant donor chromophore group. Each Y is a cysteine ​​residue covalently bonded to a pendant acceptor fluorophore group, as described in any one of appendices 56, 62-65, and 68-70, for a polymer tandem dye. Note 72. A multichromophore has the following formula:

[0528] [ka]

[0529] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, Each A 1 It is, independently, an acceptchromophore, Each L 1 and L 2 It is, independently, a linker, x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2A polymer tandem dye according to any one of appendices 56, 62, and 63, wherein each is independently selected from terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, linkers, and linked specific binding members. Note 73. A multichromophore has the following formula:

[0530] [ka]

[0531] During the ceremony, Each D 1 It is, independently, a pendant donor chromophore, Each A 1 It is independently an acceptor fluorophore, Each L 1 and L 2 It is, independently, a linker, x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2 A polymer tandem dye according to any one of Appendices 56-58 and 63, wherein each is independently selected from the group consisting of terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, linkers, and linked specific binding members. Note 74. The pendant donor chromophore group is a BODIPY group, as described in any one of Notes 53 to 73. Note 75. Acceptor fluorophores (e.g., each A 1 ) is a small molecule fluorophore, a polymer tandem dye as described in any one of appendices 38 to 74.

[0532] Note 76. Acceptor fluorophores (e.g., each A 1) is a polymer tandem dye as described in any one of Appendix 38 to 75, selected from cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethine, pyrene, dipyrometheneboron difluoride, naptalimide, thiadin dyes, and acridine dyes. Note 77. A labeled specific binding member, A polymer backbone containing non-conjugated repeating units, and a light-harvesting multichromophore containing a plurality of pendant donor chromophore groups independently linked to each non-conjugated repeating unit of the polymer backbone, and A polymer tandem dye comprising an acceptor fluorophore linked to a non-conjugated repeating unit of the polymer backbone and configured in the vicinity of at least one pendant donor chromophore group of a light-harvesting multichromophore that accepts energy, A specific binding member linked to a polymer tandem dye and A labeled specific binding member, including a labeled specific binding member. Note 78. The specific binding member is the antibody, or the labeled specific binding member as described in Note 77. Note 79. The specific binding member is a labeled specific binding member as described in Note 77, which is an antibody fragment or a binding derivative thereof. Note 80. The antibody fragment or its conjugated derivative is a labeled specific binding member as described in Note 79, selected from the group consisting of Fab fragment, F(ab')2 fragment, scFv, diabody, and triabody.

[0533] Note 81. The acceptor fluorophore is a labeled specific binding member as described in any one of Notes 77-80, selected from cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethine, pyrene, dipyrometheneboron difluoride, naptalimide, thiadin dyes, and acridine dyes. Note 82. The polymer tandem dye is a polymer tandem dye as described in any one of Notes 39 to 76, and is a labeled specific binding member as described in any one of Notes 77 to 81. Note 83. The following formula is available:

[0534] [ka]

[0535] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, Each A 1 It is independently an acceptor fluorophore, Each L 1 and L 2 It is, independently, a linker, p1 and q1 are independently either 0 or 1, and p1 + q1 ≤ 1. p2 and q2 are independently 0 or 1, and p1 + q1 ≤ 1. x is 75 mol% or more, y is 25 mol% or less. G 1 These are terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, or linkers. G 2 This is a linked specific binding member, which is a labeled specific binding member as described in any one of appendices 77 to 81. Note 84. p1, p2, q1, and q2 are each 0, and the multichromophore is given by the following formula:

[0536] [ka]

[0537] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, Each A 1 It is independently an acceptor fluorophore, L 1 and L 2 Each of them is an independent linker, x is 75 mol% or more, and y is 25 mol% or less. G 1 These are terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, or linkers. G 2 The labeled specific binding member described in Appendix 83 is a linked specific binding member. Appendix 85. A method for evaluating a sample for the presence of a target analyte, (a) A labeled specific binding member that specifically binds to a target analyte is brought into contact with the sample to produce a labeled composition contact sample, wherein the labeled specific binding member is (i) A polymer tandem dye as described in any one of Appendix 38 to 76, and (ii) A polymer tandem dye comprising a specific binding member linked to it, (b) To assess the presence of a labeled specific binding member-target analyte binding complex, the sample in contact with the labeled composition is assayed to evaluate whether the target analyte is present in the sample. Methods that include...

[0538] Note 86. The method according to Note 85, wherein the acceptor fluorophore of the polymer tandem dye is selected from cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethine, pyrene, dipyrometheneboron difluoride, naptalimide, thiadin dyes, and acridine dyes. Appendix 87. The method according to Appendix 85 or 86, further comprising bringing the sample into contact with a second specific binding member that binds to a support and specifically binds to a target analyte. Appendix 88. The support is the method described in Appendix 87, comprising magnetic particles. Note 89. The target analyte is a cell-related method described in any one of the methods described in Notes 85-88. Note 90. The target analyte is a cell surface marker, as described in Note 89.

[0539] Note 91. The cell surface marker is selected from the group consisting of cell receptors and cell surface antigens, as described in Note 90. Note 92. The target analyte is an intracellular target, and the method further comprises lysing cells, as described in Note 89. Appendix 93. The method is the method according to any one of the appendices 85 to 92, further comprising flow cytometry analysis of a fluorescently labeled target analyte. Appendix 94. A method for labeling a target molecule, This involves contacting a target molecule with a polymer tandem dye to produce a labeled target molecule. The polymer tandem dye is a polymer tandem dye as described in any one of appendices 38 to 76, and the method includes a conjugated tag that covalently binds to a target molecule. Note 95. Polymer tandem dyes have the following formula:

[0540] [ka]

[0541] During the ceremony, Each D 1 It is independently a pendant donor chromophore group, Each A 1 It is independently an acceptor fluorophore, Each L 1 and L 2 It is, independently, a linker, p1 and q1 are independently either 0 or 1, and p1 + q1 ≤ 1. p2 and q2 are independently 0 or 1, and p1 + q1 ≤ 1. x is 75 mol% or more, y is 25 mol% or less. G 1 These are terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, or linkers. G 2 The linker is a conjugate tag, as described in Appendix 94.

[0542] Appendix 96. A method for preparing a light-gathering multichromophore, a) To synthesize a protected polypeptide having a defined amino acid sequence consisting of a block of first amino acid residues separated by the single appearance of a second amino acid residue, Each block of the first amino acid residues contains at least two residues. Each of the first amino acid residues contains a protected first chemoselective side chain group. Each of the second amino acid residues contains a protected second chemoselective side chain group, and is synthesized as follows: b) Deprotecting a protected polypeptide to produce a deprotected polypeptide, wherein the first and second chemoselective side chain groups are deprotected. c) The reactive donor chromophore moiety is bonded to the deprotected first chemoselective side chain group of the first amino acid residue to generate a pendant donor chromophore group. Methods that include... The method according to Appendix 96, further comprising, in succession to step c), coupling the reactive acceptor fluorophore moiety to a deprotected second chemoselective side chain group of a second amino acid residue to generate a pendant acceptor fluorophore. The method according to Appendix 96, further comprising, after step a), deprotecting the N-terminus of the protected polypeptide and attaching a G1 group to the N-terminus of the N-terminally deprotected polypeptide, wherein G1 is a terminal group (e.g., a capping group), a donor chromophore group, or a linker. Appendix 99. The method according to any one of the appendices 96 to 98, further comprising attaching a specific binding member to the C-terminus of the polypeptide after step c). Note 100. The light-gathering multichromophore is as follows:

[0543] [ka]

[0544] During the ceremony, D 1 This is a pendant donor chromophore group, Z 1 is a second chemoselective side chain group, Each L 1 and L 2 It is, independently, a linker, p1 and q1 are independently either 0 or 1, and p1 + q1 ≤ 1. p2 and q2 are independently 0 or 1, and p1 + q1 ≤ 1. x is 75 mol% or more, y is 25 mol% or less. G 1 and G 2 The method according to any one of the appendices 96 to 99, wherein each is independently selected from terminal groups, polymer segments, donor chromophore groups, acceptor fluorophores, linkers, and linked specific binding members.

[0545] Note 101. The light-gathering multichromophore is as follows:

[0546] [ka]

[0547] During the ceremony, each(n) q and each (p) q These are independent integers from 1 to 20, and in the expression, for each segment of q, (n) q +(p) q ≥ 3, q is an integer between 1 and 100, and the method is one of the methods described in appendices 96 through 100. Note 102. The first amino acid residue was independently selected from lysine and ornithine. The second amino acid residue is cysteine, according to any one of the methods described in appendices 96-101. Note 103. The defined amino acid sequence includes an amino acid sequence selected from the following: XYXX XXYXX XXXYXXX XXXYXXXX XXXXYXXX XXXXYXXXX XXXXXYXXXXX XXXXXXYXXXXXX XXXXXXXYXXXXXXX XXXXXXXXYXXXXXXXX XXXXXXXXXYXXXXXXXXX Y(X) n Y XY(X) n YX XXY(X) n YXX XXXY(X) n YXXX XXXXY(X) n YXXXX XXXXXY(X) n YXXXXX During the ceremony, Each X is a lysine or ornithine residue covalently N-linked to a pendant donor chromophore group. The method according to any one of the appendices 96 to 102, wherein each Y is a cysteine ​​residue or a protected cysteine ​​residue.

[0548] Although the above invention has been described in some detail as an example and illustration for the sake of clear understanding, it will be readily apparent to those skilled in the art that certain modifications and alterations may be made in light of the teachings of the invention without departing from the spirit or scope of the appended claims.

[0549] Therefore, the foregoing is merely illustrative of the principles of the present invention. Those skilled in the art will understand that various arrangements embodying the principles of the present invention and falling within its spirit and scope can be devised, even if not expressly described or shown herein. Furthermore, all examples and conditional language enumerated herein are primarily intended to help the reader understand the principles of the present invention and the concepts to which the inventors contribute to further the art, and should be construed as not being limited to such specifically enumerated examples and conditions. Moreover, all descriptions herein enumerating the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both their structural and functional equivalents. In addition, such equivalents are intended to include both currently known equivalents and those to be developed in the future, regardless of their structure, i.e., any elements developed to perform the same function. Furthermore, nothing disclosed herein is intended to be for the public only, whether such disclosure is expressly enumerated in the claims or not.

[0550] 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 are embodied by the appended claims. In the claims, 35 U.SC § 112(f) or 35 U.SC § 112(6) is expressly defined as being invoked for limitation in the claims only when the exact phrase “means for” or the exact phrase “steps for” is enumerated at the beginning of such limitation in the claims, and if such exact phrase is not used in limitation in the claims, 35 U.SC § 112(f) or 35 U.SC § 112(6) is not invoked.

[0551] Cross-reference of related applications In accordance with 35 U.S. SC §119(e), this application claims priority to the filing dates of U.S. Provisional Patent Application No. 62 / 650,935, filed on 30 March 2018, and U.S. Provisional Patent Application No. 62 / 715,722, filed on 7 August 2018, the disclosures of those applications being incorporated herein by reference.

Claims

1. A water-soluble polymer tandem dye, A polymer backbone comprising non-conjugated repeating units derived from an amino acid, peptoid monomer, protective carbonate monomer, or cyclic carbonate monomer, and a light-harvesting multichromophore comprising a plurality of pendant donor chromophore groups independently linked to each of the non-conjugated repeating units of the polymer backbone, An acceptor fluorophore is linked to the non-conjugated repeating unit of the polymer main chain and configured in the vicinity of at least one pendant donor chromophore group of the light-harvesting multichromophore to accept energy. A water-soluble polymer tandem dye containing [the specified ingredient].

2. Each of the aforementioned pendant donor chromophore groups is substituted with a water-soluble group. The aforementioned pendant donor chromophore groups are configured in a proximity that transmits energy to each other, and / or The aforementioned pendant donor chromophore group is selected from a fused tricyclic aryl group, a fused tricyclic heteroaryl group, and a BODIPY group. The water-soluble polymer tandem dye according to claim 1.

3. The aforementioned pendant donor chromophore group is a BODIPY group represented by the following formula: 【Chemistry 1】 During the ceremony, R １ ~R ７ Each of these is independently H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG), and -L. １ - Selected from, or Optionally, R ６ and R ７ , R ２ and R ３ , R ５ and R ６ , R ３ and R ４ , R ４ and R １ as well as R ５ and R １ One or more pairs of substituents selected from form a divalent radical together, are linked cyclically, and together with the carbon atoms to which they are attached provide a 5- or 6-membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring, which ring may be unsubstituted or independently alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, a water solubilizing group (WSG) and -L １ - and may be further substituted with substituents selected from, L １ This is a linker that is linked to the non-conjugated repeating units of the polymer main chain, Y １ and Y ２ These are independently F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, WSG and -L １ - Selected from, Y １ , Y ２ , and R １ ~R ７ One of them is -L １ - is, The water-soluble polymer tandem dye according to claim 1 or 2.

4. The aforementioned pendant donor chromophore group is described by the following structure: 【Chemistry 2】 During the ceremony, * indicates the linkage point of the polymer main chain to the non-conjugated repeating unit, Y １ and Y ２ These are each alkynnyls substituted with WSG. The water-soluble polymer tandem dye according to claim 3.

5. The aforementioned pendant donor chromophore group is described by the following structure: 【Transformation 3】 During the ceremony, R １１ L2 is, L2 is a linker connected to the non-conjugated repeating units of the polymer main chain, Each R ９ is an optional substituent selected from halogens, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, where t is 0 to 4. The water-soluble polymer tandem dye according to claim 4.

6. The aforementioned pendant donor chromophore group is independently substituted with one or more water-soluble groups (WSGs) selected from the following formulas: 【Chemistry 4】 During the ceremony, T ５ It is an optional linker, Each T ６ It is a linker, R １１ And R are independently H, alkyl, or substituted alkyl. Each s is an integer between 1 and 50. The water-soluble polymer tandem dye according to claim 1.

7. The light-gathering multichromophore includes the segment of the following formula: 【Transformation 5】 During the ceremony, The polymer backbone of the non-conjugated repeating units is SM, where each unit is independently a saturated non-conjugated comonomer. １ and SM ２ Contains a copolymer, Each D １ SM is independent. １ It is a pendant donor chromophore connected to, Each Z １ SM is independent. ２ It is a chemoselective tag linked to, x is 75 mol% or more, y is 25 mol% or less. The water-soluble polymer tandem dye according to claim 1.

8. The aforementioned light-gathering multichromophore is defined by the following formula: 【Transformation 6】 During the ceremony, Each D １ It is independently a pendant donor chromophore group, Each Z １ It is independently an acceptor fluorophore, Each L １ and L ２ It is, independently, a linker, p １ and q １ These are independently 0 or 1, and p １ +q １ ≤ 1, p ２ and q ２ These are independently 0 or 1, and p ２ +q ２ ≤ 1, x is 75 mol% or more, y is 25 mol% or less, G １ and G ２ Each is independently selected from terminal groups, polymer segments, donor chromophore groups, acceptor fluorophore groups, linkers, and linked specific binding members. The water-soluble polymer tandem dye according to claim 7.

9. p １ , p ２ , q １ , and q ２ Each of these is 0, and the light-gathering multichromophore is given by the following formula: 【Transformation 7】 During the ceremony, Each D １ It is independently a pendant donor chromophore group, Each Z １ These are independently chemoselective tags, L １ and L ２ Each of them is an independent linker, x is 75 mol% or more, y is 25 mol% or less, G １ and G ２ Each is independently selected from terminal groups, polymer segments, donor chromophore groups, acceptor fluorophore groups, linkers, and linked specific binding members. The water-soluble polymer tandem dye according to claim 8.

10. The light-gathering multichromophore includes the segment of the following formula: 【Transformation 8】 During the ceremony, Each D １ It is independently a pendant donor chromophore group, Each Z １ These are independently chemoselective tags, Each L １ and L ２ It is, independently, a linker, n and p are each independent integers between 1 and 20, and in the equation, n + p ≥ 2. m is either 1 or 2. The water-soluble polymer tandem dye according to claim 9.

11. The light-collecting multichromophore comprises a q segment of the copolymer and has the following formula: 【Chemistry 9】 During the ceremony, each (n) ｑ and each (p) ｑ These are independent integers from 1 to 20, and in the expression, for each segment of q, (n) ｑ + (p) ｑ ≥ 3, q is an integer between 1 and 100. The water-soluble polymer tandem dye according to claim 10.

12. The polymer backbone comprises one or more amino acid sequences selected from the following: XYXX XXYXX XXXYXXX XXXYXXXX XXXXYXXX XXXXYXXXX XXXXXYXXXXXX XXXXXXXYXXXXXX XXXXXXXXXYXXXXXXXX XXXXXXXXXYXXXXXXXX XXXXXXXXXXXYXXXXXXXXXX Y(X) ｎ Y XY(X) ｎ YX XXY(X) ｎ YXX XXXY(X) ｎ YXXX XXXXY(X) ｎ YXXXX XXXXXY(X) ｎ YXXXXX During the ceremony, n is an integer between 2 and 20. Each X is a lysine or ornithine residue covalently N-linked to a pendant donor chromophore group. Each Y is a cysteine ​​residue or a protected cysteine ​​residue. The water-soluble polymer tandem dye according to claim 1.

13. A water-soluble labeled specific binding member, A polymer backbone comprising non-conjugated repeating units derived from amino acids, peptoid monomers, protected carbonate monomers, or cyclic carbonate monomers, and a light-harvesting multichromophore comprising a plurality of pendant donor chromophore groups independently linked to the non-conjugated repeating units of the polymer backbone, and An acceptor fluorophore is linked to the non-conjugated repeating unit of the polymer main chain and configured in the vicinity of at least one pendant donor chromophore group of the light-harvesting multichromophore to accept energy. A water-soluble polymer tandem dye containing, A specific binding member linked to the polymer tandem dye and including, A water-soluble, labeled, specific binding member.

14. The water-soluble labeled specific binding member according to claim 13, wherein the specific binding member linked to the polymer tandem dye is an antibody, an antibody fragment, or a bound derivative thereof.

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