Water-soluble polymeric dyes containing pendant chromophores

Abstract

The present invention provides water soluble polymer tandem dyes containing pendant group donor fluorophores. The water-soluble polymer tandem dye has a polypeptide skeleton, a plurality of side group donor fluorophores independently connected with amino acid residues of the polymer skeleton, and a receptor fluorophore connected with the amino acid residues of the polypeptide skeleton. The pendant group donor fluorophore may be a BODIPY group substituted with one or more water soluble groups. The invention also provides a labeled specific binding member based on a water-soluble polymer tandem dye, which further comprises a specific binding member linked to a polymer tandem dye, the specific binding member being an antibody or a binding fragment thereof. Further, the present invention also provides methods of assessing a target analyte in a sample as well as methods of labeling a target molecule in which a polymer tandem dye may be used in the target molecule. The invention also provides systems and kits for carrying out the methods.

Description

[0001] This application is a divisional application of an invention patent application with an application date of March 28, 2019, application number 201980024229.X, and invention name “Water-soluble polymeric dyes containing side-group chromophores”.

[0002] This patent application claims priority to U.S. Provisional Patent Application No. 62 / 650,935, filed on March 30, 2018, and U.S. Provisional Patent Application No. 62 / 715,722, filed on August 7, 2018; the contents of these patents are incorporated herein by reference. Technical Field

[0003] The present invention relates to a water-soluble polymer tandem dye containing a side-donating fluorophore and a labeled specific binding member based on the water-soluble polymer tandem dye. The present invention provides a method for evaluating a target analyte in a sample, a method for labeling a target molecule, and a system and a kit for implementing the target method. Background Art

[0004] Fluorescent dyes are compounds that emit light of (usually) a different wavelength when irradiated with light of the wavelength they absorb. Fluorescent dyes can be used in various biochemical, biological and medical applications, for example, in diagnostic kits, microscopy or drug screening. Fluorescent dyes are characterized by a number of parameters that allow the user to select the appropriate dye according to the intended use. Parameters of interest include maximum excitation wavelength, maximum emission wavelength, Stokes shift, extinction coefficient, fluorescence quantum yield and fluorescence lifetime. For example, the dye can be selected according to the intended application in order to allow the excitation radiation to penetrate the biological sample, minimize background fluorescence and / or obtain a high signal-to-noise ratio.

[0005] Molecular recognition involves the specific binding of two molecules. Molecules with binding specificity for a target biomolecule can be used in a variety of research and diagnostic applications, such as analyte labeling and separation, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell sorting, and chromatography. Target biomolecules can be detected by labeling with fluorescent dyes. Summary of the invention

[0006] The present invention provides a water-soluble light-harvesting multichromophore containing a plurality of side-group chromophores. The light-harvesting multichromophore has a polymer backbone including non-conjugated repeating units and a plurality of side-group donor chromophores independently connected to the non-conjugated repeating units of the polymer backbone. The side-group chromophore may be a BODIPY group substituted with one or more water-soluble groups. The present invention also provides a polymer tandem dye based on a target multichromophore, further comprising an acceptor fluorophore connected to the non-conjugated repeating unit of the polymer backbone, wherein the acceptor fluorophore is configured to approach at least one side-group donor chromophore of the light-harvesting multichromophore upon energy reception. The present invention also provides a labeled specific binding member comprising a target polymer tandem dye. In addition, the present invention also provides a method for evaluating the presence of a target analyte in a sample and a method for labeling a target molecule, wherein the target polymer tandem dye may be used in the target molecule. The present invention also provides a system and a kit for implementing the target method. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] It should be understood that the following drawings are only used for illustrative purposes and are not intended to limit the scope of the teachings of the present invention in any way.

[0008] Figure 1 The general structure of an exemplary polymer tandem dye is depicted, where the "dye" is the acceptor fluorophore.

[0009] Figure 2 Shown are normalized absorption spectra of exemplary multichromophores and emission spectra of a series of polymer tandem dyes including the multichromophores.

[0010] Figure 3 Shown are emission spectra of an exemplary multichromophore and a series of polymer tandem dyes including the multichromophore, all taken at 0.04 OD (absorbance).

[0011] Figure 4 The structure of an exemplary polymeric tandem dye with a peptide backbone is depicted. D is a BODIPY donor side group, A is an acceptor dye, and the biolinker is a linker comprising a chemoselective functional group for attaching the tandem dye to a biomolecule.

[0012] Figure 5A Shown for preparation Figure 4 Absorption and emission spectra of the multichromophore BODIPY donor side groups shown. Figure 5B Shown for preparation Figure 4 Absorption and emission spectra of the multichromophore acceptor dyes are shown. Figure 5C Shows Figure 4 Absorption and emission spectra of exemplary polymer tandem dyes shown.

[0013] Fig. 6A Homogeneous energy transfer processes between side-donor chromophores are elucidated, which tend to result in continuous reversible energy transfer between identical chromophores rather than emission from a single chromophore. Such energy transfer processes may lead to self-quenching and may result in quantum yields significantly lower than those observed in single isolated chromophores. Figure 6B Heterogeneous energy transfer is elucidated, which mainly causes one energy transfer mode between different chromophores. Energy transfer to the secondary chromophore tends to cause emission, which is limited by the quantum yields of the acceptor and the single donor chromophore.

[0014] Figure 7A-7B The invention discloses a method for preparing a BODIPY dye ("dye") ( Fig. 7A ) and polymer tandem dyes ( Figure 7B ) is a schematic diagram of the synthesis of an exemplary multichromophore of ). "Dye" and "donor" may refer to a donor dye, such as BODIPY, while "acceptor" refers to an acceptor fluorophore.

[0015] Figure 8 Absorption and emission spectra are shown for the exemplary multichromophore shown in FIG. 7 , wherein the exemplary multichromophore includes a BODIPY dye attached to a pendant group.

[0016] Figures 9A-9B The absorption of a series of exemplary polymer tandem dyes is shown ( Fig.9A ) and emission spectra ( Fig. 9B ), wherein the exemplary polymer tandem dye comprises a BODIPY donor dye attached to a side group and various acceptor dyes having different maximum emission wavelengths.

[0017] definition

[0018] Before describing exemplary embodiments in more detail, the following definitions are set forth in order to illustrate and define the meaning and scope of the terms used in this specification.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Nevertheless, for clarity and ease of reference, certain terms are defined as follows.

[0020] It must be noted that, as used in this patent and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. For example, the term "dye" refers to one or more dyes, i.e., a single dye and multiple dyes. It should also be noted that claims can be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a pre-conditional basis for the use of special terms such as "solely", "only", etc. in connection with stating claim elements or for the use of a "negative" limitation.

[0021] As used herein, the terms "chemoselective functional group" and "chemoselective tag" are used interchangeably and refer to a functional group that, in some cases, can selectively react with another compatible functional group to form a covalent bond after optionally activating one of the functional groups. Chemoselective functional groups of interest include, but are not limited to: thiols and maleimides or iodoacetamides; amines and carboxylic acids or their activated esters; groups that can react with each other via click chemistry, such as azido and alkynyl groups (e.g., cyclooctynyl); tetrazines, trans-cyclooctenes, dienes, dienophiles, azides, hexavalent sulfur-fluoride exchange chemistry (SuFEX), fluorinated sulfonyls; and hydroxyls, hydrazides, hydrazines, aldehydes, ketones, azidos, alkynes, phosphines, epoxides, and the like.

[0022] The term "sample" as used herein refers to a material or material mixture containing one or more target analytes, which is in a liquid state in some cases. In some embodiments, in a broad sense, "sample" refers to any plant, animal or bacterium containing cells or producing cell metabolites, for example, tissue or liquid separated from an individual (including but not limited to plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or in vitro cell culture components and samples from the environment. The term "sample" may also refer to a "biological sample". The term "biological sample" as used herein refers to a whole organism or a subset of its tissues, cells or components (for example, body fluids, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A "biological sample" may also refer to a homogenate, lysate or extract prepared with a whole organism or a subset of its tissues, cells or components or its fragments or parts (including but not limited to plasma, serum, spinal fluid, lymph, skin outer layer sections, respiratory tract, intestinal and urogenital tract, tears, saliva, milk, blood cells, tumors and organs). In certain embodiments, the sample has been taken from an animal or plant. A biological sample may include a cell. According to conventional meaning, the term "cell" refers to the basic structural unit of eukaryotic and prokaryotic organisms having at least a nucleus and a cell membrane. In certain embodiments, the cell includes a prokaryotic cell, for example, a prokaryotic cell from bacteria. In other embodiments, the cell includes a eukaryotic cell, for example, a cell obtained from a biological sample of an animal, plant, or fungus.

[0023] The terms "support binding" and "connected to a support" are used interchangeably and refer to a portion (e.g., a specific binding member) that is covalently or non-covalently connected to a target support. Covalent connection may involve a chemical reaction of two compatible functional groups (e.g., two chemoselective functional groups, an electrophile and a nucleophile, etc.) to form a covalent bond between two target moieties (e.g., a support and a specific binding member). In some cases, non-covalent connection may involve specific binding between two target moieties (e.g., two affinity moieties, such as a hapten and an antibody or a biotin moiety and streptavidin, etc.). In some cases, non-covalent connection may involve absorption of a substrate.

[0024] The term "polypeptide" refers to a polymeric form of amino acids of any length, including peptides of 2-50 amino acids in length and polypeptides of greater than 50 amino acids in length. The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" includes polymers of coded and non-coded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides with modified peptide backbones, wherein the conventional backbone has been replaced by a non-naturally occurring or synthetic backbone. The polypeptide can have any suitable length, for example, 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 50 or more amino acids, 100 or more amino acids, 300 or more amino acids, such as up to 500 or 1000 or more amino acids. A "peptide" can be 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, such as up to 50 amino acids. In some embodiments, the length of the peptide is between 5 and 30 amino acids.

[0025] As used herein, the term "isolated" refers to a fraction of interest that is at least 60% separated, at least 75% separated, at least 90% separated, at least 95% separated, at least 98% separated, or even 99% separated from other components (i.e., components associated with the fraction of interest prior to purification).

[0026] A "plurality" contains at least 2 members. In some cases, a plurality can have 5 or more members, such as 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 300 or more, 1000 or more, 3000 or more, 10,000 or more, 100,000 or more members.

[0027] Numerical ranges are inclusive of the numbers defining the range.

[0028] The term "specific binding" refers to the ability of a capture agent (or a first member of a specific binding pair) to preferentially bind to a particular analyte (or a second member of a specific binding pair) present in, for example, a homogeneous mixture of different analytes. In some cases, the specific binding interaction will distinguish between desirable and undesirable analytes in a sample, wherein the specificity for the desirable analyte is 10 times or more, such as 100 times or more, or 1000 times or more, higher than that for the undesirable analyte. In some cases, when a capture agent specifically binds to an analyte in the form of a capture agent / analyte complex, the affinity between them is at least 10 -8 M. At least 10 -9 M, for example, up to 10 -10 M.

[0029] "Affinity" refers to the strength of binding, with increased binding affinity being associated with decreased Kd.

[0030] Methods described herein include multiple steps. As required, each step can be performed after a predetermined amount of time between steps. Therefore, the time between performing each step can be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, including 5 hours or more. In certain embodiments, each subsequent step is performed immediately after the previous step is completed. In other embodiments, a step can be performed after incubation or waiting time after the previous step is completed, for example, after a few minutes to overnight waiting time.

[0031] As used herein, the terms "evaluate," "determine," "measure," "assess," and "measure" are used interchangeably and include both quantitative and qualitative measurements.

[0032] As used herein, the term "separation" refers to the physical separation of two elements (eg, separation by size or affinity, etc.) as well as the degradation of one element while the other remains intact.

[0033] The term "linker" or "key" refers to a connecting portion connecting two groups and having a skeleton with a length of 100 or less atoms. A linker or key can be a covalent bond connecting two groups or a chain of 1 to 100 atoms in length, for example, a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms in length, wherein the linker can be a straight chain, branched, cyclic or single atom. In some cases, the linker is a branched linker, i.e., refers to a connecting portion connecting three or more groups. In some cases, one, two, three, four or five or more carbon atoms of the linker skeleton can be optionally substituted by sulfur, nitrogen or oxygen heteroatoms. In some cases, the linker skeleton includes a connecting functional group, for example, ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester or imine. The bonds between the backbone atoms may be saturated bonds or unsaturated bonds; in some cases, the number of unsaturated bonds in the linker backbone does not exceed one, two or three. The linker may include one or more substituents, for example, substituted by an alkyl, aryl or alkenyl group. The linker may include, but is not limited to, polyethylene glycol; ether; thioether; tertiary amine; alkyl, which may be linear or branched, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linker backbone may include a cyclic group, for example, an aryl, heterocyclic or cycloalkyl group, wherein the backbone includes 2 or more atoms of the cyclic group, for example, 2, 3 or 4 atoms. The linker may be cleavable or non-cleavable.

[0034] The terms "polyethylene oxide", "PEO", "polyethylene glycol" and "PEG" are used interchangeably to refer to a polyethylene glycol having the chemical formula -- (CH2--CH2--O--) n -A polymer group or a derivative thereof of the chain shown in Figure 1. In some embodiments, "n" is 5000 or less, for example, 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, for example, 3 to 15 or 10 to 15. It should be understood that the PEG polymer group can have any suitable length and can include various end groups and / or other substituents, including but not limited to alkyl, aryl, hydroxyl, amino, acyl, acyloxy and amine end groups and / or substituents. PEG groups suitable for use as target multichromophores include those described in the following publications: S. Zalipsky, Functionalized poly(ethylene glycol)s for the preparation of biologically relevant conjugates, Bioconjugate Chemistry, 1995, 6(2), pp. 150-165; and Zhu et al., Water-soluble conjugated polymers for imaging, diagnosis, and therapy, Chemical Reviews, 2012, 112(8), pp. 4687–4735.

[0035] The term "alkyl" (by itself or as part of another substituent) refers to a saturated branched or straight-chain monovalent hydrocarbon radical derived from a single carbon atom of a parent alkane by removing one hydrogen atom. Alkyl groups of interest include, but are not limited to, methyl; ethyl, propyl, e.g., prop-1-yl or prop-2-yl; and butyl, e.g., but-1-yl, but-2-yl, 2-methylprop-1-yl or 2-methylprop-2-yl. In some embodiments, the alkyl group comprises 1 to 20 carbon atoms. In some embodiments, the alkyl group comprises 1 to 10 carbon atoms. In certain embodiments, the lower alkyl group comprises 1 to 6 carbon atoms, e.g., 1 to 4 carbon atoms. For example, the term includes straight-chain and branched hydrocarbon groups such as methyl (CH3), ethyl (CH3CH2), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2), isobutyl ((CH3)2CHCH2), sec-butyl ((CH3)(CH3CH2)CH-), tert-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0036] The term "substituted alkyl" refers to an alkyl group as defined herein in which one or more carbon atoms in the alkyl chain have been optionally replaced by a heteroatom (e.g., O-, N-, S-, -S(O) n -(wherein n is between 0 and 2), -NR-(wherein R is hydrogen or alkyl)), and has 1 to 5 substituents selected from the group, wherein the group consists of the following: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thione, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R b , wherein R' and R" may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocycle.

[0037] "Alkoxy" refers to the group -O-alkyl, where alkyl is as defined herein. For example, alkoxy includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0038] The term "substituted alkoxy" refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, wherein substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0039] "Alkynyl" refers to a linear or branched monovalent hydrocarbon group having 2 to 6 carbon atoms (preferably 2 to 3 carbon atoms) and having at least 1 (preferably 1 to 2) triple bond unsaturated sites. Examples of the alkynyl group include ethynyl (C≡CH) and propargyl (CH2C≡CH).

[0040]

[00136] The term "substituted alkynyl" refers to an alkynyl group, as defined herein, having from 1 to 5 substituents or from 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thione, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0041] "Amino" refers to the group -NH2. The term "substituted amino" refers to the group -NRR, wherein 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.

[0042] "Aryl" (either by itself or as part of another substituent) refers to a monovalent aromatic hydrocarbon radical derived from a single carbon atom in an aromatic ring system after removing one hydrogen atom. Target aryl groups include, but are not limited to, groups derived from the following: aceanthrene, acenaphthylene, acephenanthrene, anthracene, azulene, benzene, lettuce, coronene, fluoranthene, fluorene, hexacene, hexylene, hexalene, asymmetric indacene, symmetric indacene, indane, indene, naphthalene, octacene, octalene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenanthene, phenanthrenes, phenanthrenes, pyrenes, pyranthracenes, rubrocene, triphenylene, trinaphthalene, etc. In certain embodiments, the aryl group includes 6 to 20 carbon atoms. In certain embodiments, the aryl group includes 6 to 12 carbon atoms. Examples of aryl groups are phenyl and naphthyl.

[0043] Unless otherwise limited by the definition of aryl substituents, "substituted aryl" refers to an aryl group substituted with 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of 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, amido, alkaryl, Aryl, aryloxy, azido, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl and trihalomethyl.

[0044] "Heteroaryl" (by itself or as part of another substituent) means a monovalent heteroaryl radical derived by removal of one hydrogen atom from a single atom of a heteroaromatic ring system. Contemplated heteroaryl groups include, but are not limited to, groups derived from acridine, arsinole, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, In certain embodiments, the heteroaryl group is a 5-20 yuan heteroaryl. In certain embodiments, the heteroaryl group is a 5-10 yuan heteroaryl. In certain embodiments, the heteroaryl group is a group derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine.

[0045] "Heterocycle", "heterocyclic", "heterocycloalkyl" and "heterocyclyl" refer to a saturated or unsaturated group having a single ring or multiple fused rings (including fused bridged rings and spiro ring systems) and having from 3 to 20 ring atoms (including 1 to 10 heteroatoms). The ring atoms are selected from the group consisting of nitrogen, sulfur or oxygen, wherein, in a fused ring system, one or more rings can be cycloalkyl, aryl or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide nitrogen oxide, -S(O)- or -SO2- moieties.

[0046] Examples of heterocyclic and heteroaryl groups include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenanthroline ... Thiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also known as thiomorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0047] Unless otherwise limited by the definition of substituents, "substituted heteroaryl" refers to heteroaryl groups substituted with 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of 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, amido, alkaryl, aromatic The invention also includes but is not limited to alkyl, aryl, azido, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl and trihalomethyl.

[0048] The term "alkaryl" or "aralkyl" refers to the groups -alkylene-aryl and substituted alkylene-aryl wherein alkylene, substituted alkylene and aryl are as defined herein.

[0049] "Alkylene" refers to a divalent aliphatic hydrocarbon group preferably having 1 to 6 (more preferably 1 to 3) linear or branched carbon atoms, which is optionally substituted by one or more selected from -O-, -NR- 10 -、NR 10 C(O)-、-C(O)NR 10 -). For example, the term includes 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-), and the like. "Substituted alkylene" refers to an alkylene group having from 1 to 3 hydrogen atoms replaced by substituents as indicated by the description of the carbon atoms in the definition of "substituted" below.

[0050] "Substituted" refers to a group in which one or more hydrogen atoms are independently replaced by the same or different substituents. Substituents of interest include, but are not limited to, alkylenedioxy (e.g., methylenedioxy), M, 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 R61 , wherein M is halogen; R 60 , R 61 , R 62 and R 63 is 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 attached, it forms a cycloheteroalkyl or substituted cycloheteroalkyl ring; and R 64 and R 65 is independently hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally, R 64 and R 65 Together with the nitrogen atom to which it is attached, it forms a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certain embodiments, the substituents include M, R 60 , =O, OR 60 , SR 60 ,S,=S,NR 60 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 60 、C(O)NR 60 R 61 , C(O)O and NR 62 C(O)NR 60 R 61 In certain embodiments, the substituents include M, R 60 , =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 In certain embodiments, the substituents include M, R 60 , =O, OR 60 , SR 60 NR 60 R 61 , CF3, CN, NO2, S(O)2R 60 OP(O)(OR 60 )(OR 61 )、C(O)R 60 、C(O)OR 60 and C(O)O, where R 60 , R 61 and R 62 As defined above. For example, the substituted group may have a methylenedioxy substituent or one, two or three substituents selected from halogen atoms, (1-4C)alkyl groups and (1-4C)alkoxy groups. When the substituted group is an aryl or heteroaryl group, the substituent (e.g., as described herein) may be referred to as an "aryl substituent".

[0051] "Sulfonylamino" refers to the group -NR 21 S02R 22 , where R 21 and R 22 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, heterocycle, and substituted heterocycle, wherein R 21 and R 22 and optionally linked to the atoms to which they are attached to form a heterocyclic or substituted heterocyclic group, and wherein 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 are as defined herein.

[0052] It is to be understood that, among all the substituted groups defined above, polymers obtained by substituents which themselves have further substituents (e.g., a substituted aryl having a substituted aryl group as a substituent, which itself has been substituted by a substituted aryl group, which has been further substituted by a substituted aryl group, etc.) are not suitable for inclusion herein. In such cases, the maximum number of substitutions is three. For example, the continuous substitution results of substituted aryl groups specifically contemplated herein are limited to substituted aryl-(substituted aryl)-substituted aryl.

[0053] Unless otherwise indicated, substituents not expressly defined herein are named by the name of the terminal functional group plus the name of the adjacent functionality toward the point of attachment. For example, the substituent "arylalkoxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0054] For any group containing one or more substituents disclosed in the present invention, it should be understood, of course, that the group does not contain any substitution or substitution pattern that is sterically impractical and / or synthetically infeasible. In addition, the target compound includes all stereochemical isomers produced by the substitution of the compound.

[0055] Additional definitions of terms may appear elsewhere in the specification. DETAILED DESCRIPTION

[0056] As described above, the present invention provides a water-soluble light-harvesting multichromophore containing multiple side-group chromophores. The light-harvesting multichromophore has a polymer backbone including non-conjugated repeating units and multiple side-group donor chromophores independently connected to the non-conjugated repeating units of the polymer backbone. The side-group chromophore can be a BODIPY group substituted with one or more water-soluble groups. The present invention also provides a polymer tandem dye based on a target multichromophore, which further includes an acceptor fluorophore connected to the non-conjugated repeating unit of the polymer backbone, and the acceptor fluorophore is configured to approach at least one side-group donor chromophore of the light-harvesting multichromophore when receiving energy. The present invention also provides a labeled specific binding member including a target polymer tandem dye. In addition, the present invention also provides a method for evaluating the presence of a target analyte in a sample and a method for labeling a target molecule, wherein the target polymer tandem dye can be used in the target molecule. The present invention also provides a system and a kit for implementing the target method.

[0057] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, because there will be differences in actual implementation. It should also be understood that the terms used herein are only used to describe specific embodiments, and are not intended to limit the inventive concept, and the scope of the present invention will only be limited by the appended claims.

[0058] Where a numerical range is provided, it is to be understood that each intermediate value between the upper and lower limits of the range and any other specified value or intermediate value within the range are included within the scope of the present invention. Unless the context clearly specifies otherwise, each intermediate value should be as low as one-tenth of the lower limit unit. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges, and are also included within the present invention, subject to the requirements of any specifically excluded limits within the range. Where the range includes one or two limits, the range excluding any one or two of those included limits is also included within the present invention.

[0059] Certain ranges set forth herein are preceded by the term "about." The term "about" is used herein for the purpose of providing literal support for the exact number that follows, as well as for numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, a close or approximately unrecited number may be a number that is substantially equivalent to the specifically recited number in the context in which it appears.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described below.

[0061] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and the purpose of incorporating by reference into this document is to disclose and describe the methods and / or materials related to the cited publications. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by reason of prior invention. In addition, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0062] It is noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is also noted that claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of special terminology such as "solely," "only," etc. in connection with reciting claim elements or use of a "negative" limitation.

[0063] After reading the present invention, the following will be apparent to those skilled in the art that each individual embodiment described and listed in this patent has layered components and features that can be quickly decomposed or combined with the features of any of the other several embodiments without departing from the scope and spirit of the present invention. Any stated method can be implemented in the order of events stated or in any other order that is logically possible.

[0064] Although the apparatus and method have been or will be described for grammatical fluency and functional description, it should be clearly understood that, except as expressly provided in 35 U.S.C. § 112, the claims shall not be construed as necessarily being limited to "means" or "steps" under any circumstances, but shall be consistent with the meaning and full scope of equivalents set forth in the claims in accordance with the judicial doctrine of equivalents, and when the claims are expressly drafted in accordance with the provisions of 35 U.S.C. § 112, the claims shall be fully consistent with the statutory equivalents in 35 U.S.C. § 112.

[0065] In further describing the present invention, light-harvesting multichromophores and related polymer tandem dyes including acceptor fluorophores are first described in more detail. Labeled specific binding members including target polymer tandem dyes are next described. Then, methods of interest in which the target polymer tandem dyes are used are reviewed. In addition, systems and kits that can be used to implement the methods described in the present invention are described.

[0066] Light-harvesting multichromophores

[0067] As described above, the present invention includes light-harvesting multichromophores having modular scaffolds to which pendant light-absorbing chromophores are attached. The term "pendant" refers to side chain groups that are attached to the backbone of the modular scaffold but are not part of the backbone. In contrast to the light-absorbing comonomers of conjugated polymer dyes, the pendant light-absorbing chromophores of the subject multichromophores do not undergo π-conjugation with each other nor form delocalized π-electron systems. The modular scaffolds of the subject multichromophores are more in the form of chromophores (see, e.g., Fig. 6A ) and between the chromophore and the acceptor fluorophore (if any, see, e.g., Figure 6B ) provide a configuration with multiple light-absorbing chromophores within a compact region for efficient energy transfer between the fluorophores. This configuration of pendant light-absorbing chromophores together forms a light-harvesting multichromophore with an absorption wavelength (e.g., as described herein) at which the optically active chromophores absorb light to become excited. Thus, the light-absorbing chromophores are configured to be close to each other upon energy reception and, in the presence of an acceptor fluorophore, are able to provide energy to the acceptor fluorophore.

[0068] The terms "light-harvesting multichromophore" and "polymeric dye" are used interchangeably and refer to polymers of the present invention having multiple pendant chromophores that can capture light having a specific maximum absorption wavelength and convert it into emitted light having a longer maximum emission wavelength. The polymers may have a saturated or partially unsaturated backbone.

[0069] In addition, side groups (e.g., acceptor fluorophores, secondary donor chromophores, linkers and chemoselective tags that enable biomolecule binding) and water solubilizing groups can also be attached to the modular scaffold. In some cases, an acceptor fluorophore can be placed with two donor chromophores (primary and secondary donor chromophores) to provide the desired fluorescence emission from the acceptor fluorophore. The number and position of the acceptor fluorophores can be controlled for the configuration of the side group donor chromophores.

[0070] The specific configuration of the side groups can be determined and controlled by arranging the repeating units of the basic modular scaffold to which the side groups are attached. The target multichromophore may include multiple water-solubilizing groups attached to the scaffold and / or the side groups at any suitable position to provide a water-soluble light-harvesting multichromophore. The modular scaffold may be composed of repeating units that form a polymer backbone with side chain groups to which the side groups may be attached. The repeating units may be arranged in various configurations to provide water-soluble light-harvesting multichromophores with desired spectral properties. The distance and arrangement between the covalent attachment sites of the side group donor chromophore and the acceptor fluorophore (if any) may be controlled to complete the desired energy transfer process. This can achieve desirable high light capture and obtain signal amplification properties.

[0071] like Fig. 6A As shown, the side-donor chromophore of the configuration may exhibit fluorescence self-quenching relative to the unquenched separated donor chromophore under the excitation of incident light. Self-quenching refers to fluorescence quenching of 10% or more, for example, 20% or more, 25% or more, 30% or more, 40% or more, or 50% or more relative to the unquenched separated donor chromophore.

[0072] The modular scaffold can be composed of a polymer backbone comprising non-conjugated repeating units having any suitable configuration, for example, a linear, branched or dendritic configuration. The polymer backbone can be a linear polymer. The polymer backbone can be branched. In some cases, the light-harvesting multichromophore comprises a plurality of side-donor chromophores independently attached to the non-conjugated repeating units of the polymer backbone. The side groups of the configuration can be inserted during or after the synthesis of the polymer backbone. Depending on the specific synthesis method used, the side groups can be introduced in a random configuration, a block configuration or in a specific sequence during a stepwise synthesis process.

[0073] The term "unit" refers to a structural subunit of a polymer. The term "unit" is intended to include monomers, comonomers, coblocks, repeating units, and the like. "Repeat unit" refers to a subunit of a polymer that is defined by the minimum number of different structural features required to be considered a unit of a monomer, so that when the unit is repeated n times, the resulting structure describes the polymer or its block. In some cases, the polymer may include two or more different repeating units, for example, when the polymer is a multi-block polymer, a random arrangement unit, or a defined sequence unit, each block may define a different repeating unit. It should be understood that a variety of arrangements of repeating units or blocks may occur, and in the illustrated formula of the target multi-chromophore described herein, any suitable linear arrangement of various lengths may be included in the structure of the entire polymer. It should be understood that the polymer may also be represented by a formula in terms of the mol% value of each unit, and the formula may represent various arrangements of repeating units, for example, a random or multi-block polymer or a residue of a defined sequence. In some cases, the repeating unit of the polymer includes a single monomer group. In some cases, the repeating unit of the polymer includes 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 structural unit of a polymer, which itself can be part of a repeating unit of a polymer.

[0074] The light-harvesting multichromophore comprises a modular scaffold having a linear polymer backbone comprising non-conjugated repeating units. The modular scaffold may have a polymer backbone comprising non-conjugated repeating units in a random configuration. The modular scaffold may have a polymer backbone comprising non-conjugated repeating units in a block or co-block configuration. In addition, the modular scaffold may have a polymer backbone comprising non-conjugated repeating units of a specific defined sequence (e.g., amino acid residues of a polypeptide sequence). These configurations are characterized by polymer segments of repeating units (e.g., as described herein), wherein the segments themselves may be repeated throughout the modular scaffold.

[0075] "Non-conjugated" means that at least a portion of the repeating units include saturated backbone groups (e.g., groups having two or more consecutive single covalent bonds) that prevent π-conjugated structures or extended delocalized electronic structures from one repeating unit to the next along the polymer backbone. It should be understood that even though a repeating unit may not be conjugated to an adjacent repeating unit, the repeating unit may include one or more isolated unsaturated groups, including an unsaturated bond (e.g., an alkenylene group or an alkynylene group) and / or an aryl or heteroaryl group, which may be part of the backbone. In some cases, each repeating unit of the polymer backbone includes a side chain, which includes a side group that has been attached or a chemoselective tag for attachment to a side group. In some cases, the multichromophore includes a segment as shown in formula (I):

[0076]

[0077] in:

[0078] Each M 1 and M 2 are independently unsaturated comonomers;

[0079] Each S 1 and S 2 are all independently non-conjugated spacer units;

[0080] Each D 1 are independently 1 an attached pendant light absorbing chromophore (e.g., as described herein);

[0081] Each Z 1 are independently 2 attached chemoselective tags;

[0082] x is 75 mol% or more; and

[0083] y is 25 mol% or less, wherein * is the point of attachment to the polymer backbone of the multichromophore.

[0084] In some cases of Formula (I), M 1 and S 1 The first repeating unit (M) forming the polymer backbone 1 -S 1 ), and M 2 and S 2 The second repeating unit (M) of the polymer backbone is formed. 2 -S 2 ). The first and second repeating units may be arranged in a random configuration or a co-block configuration. In the first repeating unit, D 1By conjugating a first chemoselective tag to D 1 M 1 In the second repeating unit, Z 1 The group can be further chemoselective by a second tag (Z 2 ) is conjugated to a molecule of interest to insert a side group, for example, a second light absorbing chromophore, an acceptor fluorophore, or a linked biomolecule (e.g., as described herein). In certain instances of Formula (I), D of the first repeating unit 1 The side groups include two or more (eg, two or three) different types of side-group light-harvesting chromophores, which together constitute a light-harvesting multi-chromophore system. In certain cases of formula (I), D of the first repeating unit 1 The side groups are all identical.

[0085] In some cases of Formula (I), x is 80 mol% or more, e.g., 85 mol% or more, 90 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more, or 99 mol% or more. In some cases of Formula (I), y is 20 mol% or less, e.g., 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, 1 mol% or less.

[0086] Any suitable unsaturated comonomer may be used as M in the target polychromophore (e.g., the polychromophore of formula (I)). 1 and M 2 Group. An unsaturated comonomer refers to a comonomer having at least one unsaturated covalent bond in the polymer backbone. The intended unsaturated comonomers include, but are not limited to, aromatic or heteroaromatic comonomers, alkynyl comonomers (e.g., ethynyl) or segments and alkenyl comonomers (e.g., vinylene) or segments. The intended aromatic or heteroaromatic comonomers used in the multichromophore (e.g., the multichromophore shown in formula (I)) include, but are not limited to, phenyl comonomers, biphenyl comonomers, benzoxazole comonomers, benzothiazole comonomers, polyphenylene comonomers and fused tricyclic comonomers, for example, fluorene comonomers, carbazole comonomers, silole comonomers and bridged biphenyl comonomers. The aromatic or heteroaromatic comonomers may optionally be further substituted, for example, as described herein. In some cases of formula (I), each M 1 and M 2 Independently include one or more groups selected from fluorene, carbazole, silole, biphenylene and phenylene.

[0087] The light-harvesting multichromophore represented by formula (I) comprises a polymer backbone comprising non-conjugated repeating units, wherein each S 1and S 2 is independently a saturated spacer unit, which does not include adjacent M 1 and / or M 2 π conjugated structure between comonomers. In some cases, S 1 and S 2 In some cases, the PEG or modified PEG comprises 3 to 100 polyethylene glycol units, for example, 6 to 100 units (e.g., PEG6 to PEG 100 ).

[0088] In some cases, the multichromophore includes a segment of formula (II):

[0089]

[0090] in:

[0091] The polymer backbone containing non-conjugated repeating units comprises SM 1 , SM 2 and SM 3 comonomers, each of which is independently a non-conjugated comonomer;

[0092] Each D 1 Both are independently 1 a light-absorbing chromophore attached to a side group;

[0093] Each Z 1 Both are independently 2 attached chemoselective tags;

[0094] Each Z 2 Both are related to SM 3 attached optional side chain groups;

[0095] x is 50 mol% or more; and

[0096] y+z is 50 mol % or less, wherein * is the point of attachment to the polymer backbone of the multichromophore.

[0097] Z 2 It may be absent or may be any suitable side chain group, for example, a light absorbing chromophore, a chemical selective tag, a linker, a linked biomolecule, an acceptor fluorophore, WSG, etc. In certain cases of formula (II), SM 3 is a spacer comonomer, wherein Z 2 In certain cases of formula (II), SM 3 Yes, including Z 2A comonomer of a group (i.e., a second side-group light-absorbing chromophore), wherein each D 1 and each Z 2 Together they form a light-harvesting multichromophore system. In some cases, SM 3 is a second chemoselective tag (Z 2 ) comonomers, for example, protected functional groups or with Z 1 Orthogonal tags that can be selectively placed into the intended part.

[0098] In some cases of formula (II), x is 60mol% or more, for example, 65mol% or more, 70mol% or more, 75mol% or more, 80mol% or more, 85mol% or more, 90mol% or more, 95mol% or more, even higher. In some cases of formula (II), y+z is 40mol% or less, for example, 30mol% or less, 25mol% or less, 20mol% or less, 15mol% or less, 10mol% or less, 5mol% or less, even lower. In some cases of formula (II), y is at least 1mol% and no more than 25mol%, for example, 20mol% or less, 15mol% or less, 10mol% or less, 5mol% or less, even lower. In some cases of formula (II), z is at least 1mol% and no more than 10mol%, for example, 5mol% or less, even lower.

[0099] In certain cases of Formula (II), SM 3 does not exist, i.e. z is 0 mol%. Therefore, the multichromophore may include a segment as shown in formula (III):

[0100]

[0101] in:

[0102] The polymer backbone containing non-conjugated repeating units comprises SM 1 and SM 2 comonomers, each of which is independently a non-conjugated comonomer;

[0103] Each D 1 Both are independently 1 a light-absorbing chromophore attached to a side group;

[0104] Each Z 1 Both are independently 2 attached chemoselective tags;

[0105] x is 75 mol% or more; and

[0106] y is 25 mol% or less, wherein * is the point of attachment to the polymer backbone of the multichromophore.

[0107] In certain embodiments of Formula (III), SM 1 and SM 2 Each 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 is independently a partially saturated non-conjugated comonomer, for example, a comonomer that provides isolated double C=C covalent bonds in a saturated covalent bond-containing backbone. The first and second repeating units (SM 1 and SM 2 ) can be arranged in a random configuration, a block configuration, a co-block configuration, or in a specific sequence. In the first repeating unit, D 1 By conjugating a first chemoselective tag to D 1 SM 1 In the second repeating unit (SM 2 ), Z 1 The group can be further chemoselective by a second tag (Z 2 ) is conjugated to a molecule of interest to insert a side group, for example, a second light-harvesting chromophore, an acceptor fluorophore, or a linked biomolecule (e.g., as described herein). In certain instances of formula (III), D of the first repeating unit 1 The side groups include two or more (eg, two or three) different types of side group light absorbing chromophores, which together constitute a light-harvesting multichromophore system. In certain cases of formula (III), D of the first repeating unit 1 The side groups are all identical.

[0108] In some cases of formula (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 formula (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, or 1 mol% or less.

[0109] Any suitable comonomer can be used as the polymer backbone in the multichromophore shown in formula (II) or formula (III). Target comonomers that can be used to prepare fully saturated or partially saturated polymer backbones include, but are not limited to, comonomers derived from 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 polyethyleneimine. The comonomers may optionally be substituted with, for example, chemically selective tags. The comonomers may be polymerized or linked by any suitable chemical action, including, but not limited to, olefin polymerization, ring-opening polymerization, free radical polymerization, and click chemistry reactions or conjugation between compatible chemically selective functional groups or tags. Target ADMET monomers include, but are not limited to, monomers described in the following publications: Mutlu et al., Acyclic diene metathesis: a versatile tool for constructing determined polymer structures, Chemical Society Reviews, 2011, 40, pp. 1404-1445. In some cases, SM 1 , SM 2 and / or SM 3 The structural formula is as follows:

[0110]

[0111] in:

[0112] R 21 Yes – L 1 -D 1 or –L 2 -Z 1 , where D 1 is the side-donating chromophore, Z 1 is a chemically selective tag, while L 1 and L 2 is an optional linker;

[0113] n and m are independently integers between 1 and 6 (e.g., 1 or 2); and

[0114] * is the point of attachment to the polymer backbone of the multichromophore.

[0115] ROMP monomers of interest include, but are not limited to, monomers described in the following publications: Song et al., Scope of Ring-Opening Metathesis Polymerization (ROMP) of 1-Substituted Cyclobutenes, Journal of the American Chemical Society, 2010, 132(30), pp. 10513–10520. In some cases, SM 1 , SM 2 and / or SM 3 The structural formula is shown in any of the following:

[0116]

[0117] in:

[0118] R 21 Yes – L 1 -D 1 or –L 2 -Z 1 , where D 1 is the side-donating chromophore, Z 1 is a chemically selective tag, while L 1 and L 2 is an optional linker;

[0119] X is CH2 or O; and

[0120] * is the point of attachment to the polymer backbone of the multichromophore.

[0121] In some cases, SM 1 , SM 2 and / or SM 3 Including any of the following structural formulas:

[0122]

[0123] in:

[0124] R 21 Yes – L 1 -D 1 or –L 2 -Z 1 ;

[0125] D 1 It is a side-donating chromophore;

[0126] Z 1 It is a chemical selective tag;

[0127] L 1 and L 2 is an optional linker;

[0128] X is O or NR";

[0129] R' is H or lower alkyl (e.g., methyl);

[0130] R" is H, lower alkyl, substituted lower alkyl, and WSG; and

[0131] * is the point of attachment to the polymer backbone of the multichromophore. In some cases, the polymer backbone comprises a mixture of polystyrene and acrylate or acrylamide derived comonomers (eg, as described herein).

[0132] The multichromophore may have a hydrocarbon backbone prepared by any suitable polymerization method. In some cases, the hydrocarbon backbone is derived from an acrylate, acrylamide or styrene comonomer or a derivative thereof. In some cases, the multichromophore has a structural formula as shown in formula (X):

[0133]

[0134] in:

[0135] Each D 1 Each is independently a side-group donor chromophore;

[0136] Each Z 1 are all chemoselective tags (e.g., as described herein);

[0137] Each L 1 , L 2 and L 3 are independently linkers;

[0138] a, b and c are the mol% values ​​of each comonomer;

[0139] d represents the total polymerization or average length of the polymer (e.g., d is 2-1000, such as 2-500, 2-200, 2-100 or 2-50);

[0140] WSG is a water solubilizing group (e.g., as described herein); and

[0141] G 1 and G 2 Each is independently selected from the group consisting of an end group, a polymer segment, a donor chromophore, an acceptor fluorophore, a linker, and a linked specific binding member.

[0142] In some cases of formula (X), c=0. In some cases of formula (X), a>0 and b>0. In some cases of formula (X), a is 80mol% or more, for example, 85mol% or more, 90mol% or more, 95mol% or more, 96mol% or more, 97mol% or more, 98mol% or more or 99mol% or more. In some cases of formula (X), b is 20mol% or less, for example, 15mol% or less, 10mol% or less, 5mol% or less, 4mol% or less, 3mol% or less, 2mol% or less, 1mol% or less. In some cases of formula (X), a is 65-95mol%, b is 5-35mol%, and c is 0-30mol%, wherein a+b+c=100%. In some cases of formula (X), L 1 -L 3The invention comprises a bond connecting a polymer backbone selected from the group consisting of: -COO-, -CONR"-, -Ph-, -O-, wherein R" is H, lower alkyl, substituted lower alkyl, or WSG. The bond can be used to connect D 1 , Z 1 and WSG are attached to the polymer backbone.

[0143] In certain cases of formula (X), L 1 -D 1 The structural formula is shown in any of the following:

[0144]

[0145] Where R 4 is H, lower alkyl, substituted lower alkyl or WSG. In certain instances of formula (X), WSG is a water-solubilizing group as described in any of the Examples, the structure of which is as described herein.

[0146] The multichromophore may have a backbone derived from comonomers connected by bonds or groups derived from a click chemistry conjugation reaction (e.g., as described herein). Any suitable divalent comonomer group may be derivatized with a terminal chemoselective tag and polymerization may be achieved by conjugation of a compatible chemoselective tag. Fig. 7A and 7B Exemplary schematic diagrams of comonomer polymerization with alkyne and azide chemoselective tags by click chemistry are illustrated. In some cases, the comonomer includes one or more ethylene oxide or ethylene amino groups constituting a part of the polymer backbone. The group can provide the desired water solubility for the resulting polydye. The comonomer may further include a trivalent unit for being connected to a side chain group (e.g., a donor or acceptor dye or WSG). In some cases, the comonomer includes an propylene oxide or propylene amino group in the backbone, which is further substituted by a side chain group or a substituent at the 2-position. The group may include a connected chemoselective tag, a connected donor or acceptor dye, or a connected WSG.

[0147] In some cases of formula (II), the multichromophore has a structural formula as shown in formula (XXI):

[0148]

[0149] in:

[0150] The polymer backbone of the non-conjugated repeating unit comprises SM 1 , SM 2 and SM 3comonomers, each of which is linked via a group T that is the product of a click chemistry reaction or a chemoselective group conjugation reaction (e.g., an azide-alkyne click chemistry reaction);

[0151] SM 3 Optionally including a connected WSG;

[0152] Each D 1 Both are independently 1 a light-absorbing chromophore attached to a side group;

[0153] Each Z 1 Both are independently 2 attached chemoselective tags;

[0154] Each Z 2 Both are related to SM 3 attached optional side chain groups;

[0155] x is 50 mol% or more; and

[0156] y+z is 50 mol % or less, wherein * is the point of attachment to the polymer backbone of the multichromophore or end group (e.g., as described herein). In certain instances of Formula (XXI), SM 1 , SM 2 and SM 3 The compound comprises repeating units selected from ethylene oxide, ethylene amino, 2-substituted propylene oxide and 2-substituted propylene amino. In some cases of formula (II), each T is a 1,4-substituted 1,2,3-triazole, i.e., the product of azide-alkyne click chemistry conjugation reaction.

[0157] In some cases of Formula (XXII), SM 1 , SM 2 and SM 3 The structure is as follows:

[0158]

[0159] in:

[0160] Each X is independently O or NR 31 , where R 31 is H, alkyl, substituted alkyl, alkanoyl or substituted alkanoyl;

[0161] Each r and s is independently 1-6 (e.g., 1, 2 or 3);

[0162] each d and e is independently 1-12 (e.g., 1-6, such as 1, 2, 3, 4, 5 or 6);

[0163] t is 0 or 1;

[0164] D 1 It is a side-donating chromophore;

[0165] Z 1 is a chemoselective tag (e.g., as described herein);

[0166] WSG is a water solubilizing group (e.g., as described herein);

[0167] Each L 1 , L 2 and L 3 are independently linkers; and

[0168] * is the point of attachment to the 1,4-substituted 1,2,3-triazole (T).

[0169] It should be understood that for the derivation of the above SM 1 -SM 3 Any comonomer of the structure may use an azide or alkyne group at the comonomer terminus for attachment during polymerization. Thus, a 1,4-substituted 1,2,3-triazole (T) may exist in either of the following two possible orientations:

[0170]

[0171] The polymer backbone termini may include any suitable terminal groups, for example, azide or alkyne groups, linkers, or attached specific binding moieties.

[0172] An exemplary multi-chromophore structure and its precursor are detailed in Example 3 of the Experimental Section, and its structure is as follows:

[0173]

[0174] in:

[0175] G 1 is a terminal group (e.g., as described herein);

[0176] L 1 and L 2 independently a linker;

[0177] D 1 is a pendant chromophore (e.g., as described herein);

[0178] Each of d, e and f is independently 1-6;

[0179] n is 1-1000 (e.g., 2-1000, 2-500, 2-100, or 2-50);

[0180] Each R 41 are selected from alkyl, substituted alkyl and WSG; and

[0181] A "linker" is a linker that includes an optional chemoselective functional group (e.g., a functional group for conjugation with a comonomer or biomolecule). In some cases of Formula (XXI), L 1 –L 3 Contains bonds to the polymer backbone selected from -NHCO-alkyl groups.

[0182] Cyclic carbonate and protected carbonate monomers suitable for preparing the polymer backbone of the target multichromophore (see, for example, Formula (IX) described herein) include, but are not limited to, monomers described in the following publications: Barnes et al., WO2013036532; Cooley et al., Journal of the American Chemical Society, 131, 45, 1640-3, 2009; and Rothbard et al., U.S. Patent No. 7,169,814. The monomers are added to the polymerization reaction with an initiator and a suitable cyclic carbonate monomer feed ratio to provide a polymer backbone. In addition, protected carbonate monomers can be assembled in a stepwise synthesis process to provide a defined sequence. In some cases of Formulas (II)-(III), the polymer backbone has a polycarbonate backbone. Therefore, the structural formula of the multichromophore is shown in Formula (IX):

[0183]

[0184] in:

[0185] Each D 1 are all independently side-donating chromophores;

[0186] Each Z 1 Each is independently a chemoselective tag;

[0187] Each L 1 and L 2 are all independently linkers;

[0188] x is 75 mol% or more;

[0189] y is 25 mol% or less; and

[0190] G 1 and G 2 Each is independently selected from the group consisting of: an end group, a polymer segment, a donor chromophore, an acceptor fluorophore, a linker, and an attached specific binding member.

[0191] In certain embodiments, the repeating units (SM) represented by formula (II)-(III)1 , SM 2 and / or SM 3 ) are arranged in a defined linear sequence. Any suitable comonomer that can be polymerized in a defined stepwise manner can be used to construct the multichromophore shown in formula (II)-(III). The comonomer can be derived from an amino acid, a peptoid monomer, or a protected or cyclic carbonate monomer.

[0192] In some cases of formula (II)-(III), the polymer backbone is a polypeptide having a defined sequence of α-amino acid residues and / or β-amino acid residues. The following two types of β-amino acids and polypeptides can be used in the polymer backbone of the target multichromophore: the type with side chain groups close to amines is called β3-peptide / residue; and the type with side chain groups close to carbonyl groups is called β2-peptide / residue. In certain embodiments, the structural formula of the multichromophore is shown in formula (IV):

[0193]

[0194] in:

[0195] Each D 1 Each is independently a side-group light-absorbing chromophore;

[0196] Each Z 1 Each is independently a chemoselective tag;

[0197] Each L 1 and L 2 are all independently linkers;

[0198] p1 and q1 are independently 0 or 1, where p1+q1≤1;

[0199] p2 and q2 are independently 0 or 1, where p1+q1≤1;

[0200] x is 75 mol% or more;

[0201] y is 25 mol% or less; and

[0202] G 1 and G 2 Each is independently selected from a terminal group, a polymer segment, a light absorbing (eg, donor) chromophore, an acceptor fluorophore, a linker, and a linked specific binding member.

[0203] 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 structured as shown in formula (V):

[0204]

[0205] in:

[0206] Each D 1 are all independently side-donating chromophores;

[0207] Each Z 1 Each is independently a chemoselective tag;

[0208] L 1 and L 2 each independently a linker;

[0209] x is 75 mol% or more;

[0210] y is 25 mol% or less; and

[0211] G 1 and G 2 Each is independently selected from an end group, a polymer segment, a light absorbing (e.g., donor) chromophore, an acceptor fluorophore, a linker, and a linked specific binding member. It should be understood that the multi-chromophore represented by formula (V) includes any suitable comonomer arranged in a defined linear sequence, which has x and y totaling to a defined mol% ratio. In some cases, the Z-containing 1 The comonomers of are spaced throughout the polymer backbone sequence and are therefore always flanked on both sides by one or more comonomers containing D1.

[0212] In certain instances of formula (V), the multichromophore comprises a segment as shown in formula (VI):

[0213]

[0214] in:

[0215] Each D 1 Each is independently a side-group light-absorbing chromophore;

[0216] Each Z 1 Each is independently a chemoselective tag;

[0217] Each L 1 and L 2 are all independently linkers;

[0218] n and p are each independently an integer between 1 and 20, wherein n+p≥2; and

[0219] m is 1 or 2.

[0220] In some instances of Formula (VI), n and p are each independently 1 to 10, e.g., 2 to 20, 3 to 10, or 3 to 6. In some instances of Formula (VI), n+p is an integer between 2 to 20, e.g., 3 to 20, 4 to 20, 5 to 20, 5 to 15, or 5 to 12. In certain embodiments of Formula (VI), m is 1.

[0221] The target multichromophore may include multiple segments of formula (VI), wherein each segment includes a separate Z-containing 1 The comonomer is flanked by D 1 In some cases, the multichromophore comprises two or more segments of formula (VI) oriented adjacent to each other to provide two separate Z-containing 1 The comonomer contains 2-20 D 1 Blocks of comonomers (e.g., containing 3 to 20, 4 to 20, 5 to 20, 5 to 15, or 5 to 12 D 1 Therefore, in certain embodiments, the multi-chromophore comprises a q segment of a block copolymer, and its structural formula is shown in formula (VII):

[0222]

[0223] Where: Each (n) q and each (p) q are independently integers between 1 and 20, wherein for each q segment, (n) q +(p) q ≥3; and q is an integer between 1 and 100.

[0224] It should be understood that the α-amino acid residues shown in formulas (V)-(VII) can be replaced by β2-amino acid residues or β3-amino acid residues to obtain the corresponding polypeptide products. 1 By conjugating a first chemoselective tag to D 1 The precursor is connected to the amino acid side chain linker L1. In formula (V)-(VII), Z 1 The group can be further chemoselective by a second tag (Z 2) can be conjugated to a molecule of interest to introduce a side group, for example, a second light-harvesting chromophore, an acceptor fluorophore, or an attached biomolecule (eg, as described herein).

[0225] Any suitable amino acid can be used to form a polymer backbone to which side groups can be covalently attached. The amino acids can be naturally occurring or non-naturally occurring. For example, amino acids (e.g., lysine, ornithine) have side chain amino groups suitable for conjugation with amino-reactive groups (e.g., activated carboxylic acids). Cysteine ​​includes side chain thiol groups suitable for conjugation with thiol-reactive groups (e.g., maleimide or haloacetyl). Aspartic acid and glutamic acid have side chain carboxylic acid groups that can be conjugated with nucleophilic groups (e.g., amino groups). The present invention describes methods for preparing target multichromophores, including peptide synthesis methods.

[0226] In some cases, the multichromophore polymer backbone has one or more of the following polypeptide sequence segments:

[0227]

[0228]

[0229] in:

[0230] Each X is a first amino acid residue having a side chain-linked first chemoselective tag or a side chain-linked side group donor chromophore;

[0231] Each Y is a second amino acid residue having a side chain-linked second chemoselective tag or a side chain-linked side group acceptor fluorophore; and

[0232] 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.

[0233] In certain embodiments, in addition to one or more polypeptide segments described herein, a third amino acid residue (Z) may be incorporated into the polymer backbone, for example, introduced between two segments. In some cases, a single isolated third amino acid residue is introduced between any two segments described herein, for example, (segment 1)-Z-(segment 2). The third amino acid residue may be a spacer residue, or a residue having a chemoselective functional group suitable for selective placement of other target portions, for example, a second side-group absorbing chromophore, a chemoselective tag (e.g., a bioorthogonal click chemistry tag), a linker, a connected biomolecule, a receptor fluorophore, WSG, etc. In some cases, the other target portions are introduced into the third amino acid residue prior to preparation.

[0234] In some cases, the first amino acid residue (X) includes a side chain amino group, such as lysine or ornithine or a protected form thereof. During polymer backbone SPPS, the side chain amino group can remain protected (e.g., protected with a Cbz or Boc protecting group), followed by orthogonal deprotection to conjugate the first residue with a side group donor chromophore. In some cases, the second amino acid residue includes a side chain thiol group, such as cysteine ​​or a protected form thereof. Similarly, during polymer backbone SPPS, the side chain thiol group can remain protected, followed by orthogonal deprotection to conjugate the second residue with a side group of interest (e.g., an acceptor fluorophore).

[0235] In some embodiments, the multichromophore polymer backbone has or is derived from one or more of the following polypeptide sequence segments:

[0236]

[0237] wherein: each K is a lysine residue, a protected lysine residue, or a lysine residue covalently linked to a side-donor chromophore 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.

[0238] In some embodiments, the multichromophore polymer backbone has or is derived from one or more of the following polypeptide sequence segments:

[0239]

[0240] in:

[0241] O is an ornithine residue, a protected ornithine residue, or an ornithine residue covalently linked to a pendant donor chromophore 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.

[0242] In some cases of formula (II)-(III), the polymer backbone is a peptoid backbone. Therefore, the structural formula of the multi-chromophore is shown in formula (VIII):

[0243]

[0244] in

[0245] Each D 1 are all independently side-donating chromophores;

[0246] Each Z 1 Each is independently a chemoselective tag;

[0247] Each L 1 and L 2 are independently linkers;

[0248] x is 75 mol% or more;

[0249] y is 25 mol% or less; and

[0250] G 1 and G 2 Each is independently selected from an end group, a polymer segment, a light absorbing (e.g., donor) chromophore, an acceptor fluorophore, a linker, and a linked specific binding member. It should be understood that the multi-chromophore represented by formula (VIII) may include any suitable comonomer arranged in a defined linear sequence, which has x and y totaling to a defined mol% ratio. In some cases, the polychromophore containing Z 1 The comonomers of are spaced throughout the polymer backbone sequence and are therefore always flanked on both sides by one or more comonomers containing D1.

[0251] In some cases of formula (IV), (V), (XIII) and (IX), x is 80 mol% or more, e.g., 85 mol% or more, 90 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more or 99 mol% or more. In some cases of formula (IV), (V), (XIII) and (IX), y is 20 mol% or less, e.g., 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, 1 mol% or less.

[0252] Pendant chromophore

[0253] Any suitable light absorbing chromophore may be used in the subject polychromophores. The terms "light absorbing chromophore" and "donor chromophore" are used interchangeably and refer to side groups of a polychromophore that are capable of absorbing light at a specific maximum absorption wavelength and transferring the energy to an adjacent chromophore or an acceptor chromophore or converting it into emitted light having a longer maximum emission wavelength.

[0254] BODIPY Chromophore

[0255] The pendant chromophore group can be a BODIPY group. In some cases of Formulas (I)-(IX), D 1is independently a BODIPY group. In some cases, the BODIPY group is a pendant donor chromophore. The term "BODIPY group" refers to a polychromophore pendant group, which includes a chromophore having the following boron-dipyrromethene (BODIPY) core structure:

[0256]

[0257] wherein Q is C or N; each R is any suitable 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.

[0258] The BODIPY core structure can be linked to the repeating unit of the multichromophore via an optional side chain linker at any suitable position of 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 suitable BODIPY-containing structure can be used as a BODIPY group in the target multichromophore. The BODIPY-containing structure includes, but is not limited to, the BODIPY dyes and their derivatives described in the following publications: Loudet and Burgess, BODIPY dyes and their derivatives: synthesis and spectral properties, Chemical Reviews, 2007, 107 (11): pp. 4891-4932; Suzuki et al., U.S. Pat. No. 8,193,350; Ulrich et al., U.S. Pat. No. 8,476,461; Ulrich et al., U.S. Pat. No. 7,897,786, the contents of which are incorporated herein by reference in their entirety.

[0259] The structural formula of the BODIPY side chromophore group can be shown as formula (XI):

[0260]

[0261] in:

[0262] Q is C or N;

[0263] R 1 -R 7 are each independently selected from H, 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 -Z 1 ,or

[0264] Optionally, selected from 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 and R 5 and R 1 Any one or more pairs of substituents of which together form a divalent radical and are connected to the carbon atoms to which they are attached to form a cyclic structure, together forming a 5- or 6-membered fused heterocyclic ring, carbocyclic ring, aryl ring or heteroaryl ring (e.g., a 5- or 6-membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), wherein the ring may be unsubstituted or further substituted with substituents independently selected from the following groups: alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water solubilizing group (WSG) and -L 1 -Z 1 ;

[0265] L 1 is a linker;

[0266] Z 1 is a non-conjugated repeating unit of the polymer backbone; and

[0267] Y 1 and Y 2 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;

[0268] where Y 1 , Y 2 and R 1 -R 7 One of the substituents Y is connected to the non-conjugated repeating unit of the polymer backbone. 1 , Y 2 and R 1 -R 7 Can be selected from groups that do not inhibit the fluorescence emission of the BODIPY group. In certain cases of formula (XI), Q is C. It should be understood that for any BODPIY group formula described herein, corresponding formulas can be included, wherein 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 -R7 In certain cases of Formula (XI), Y 1 and Y 2 Each includes WSG. In certain instances of Formula (XI), –L 1 -Z 1 The linker includes WSG.

[0269] In formula (XI), the substituents to R 6 and R 7 and / or R 2 and R 3 Can be connected into a ring structure to form a 5 or 6-membered fused ring, the ring is unsubstituted or substituted. In some cases of formula (XI), the 5 or 6-membered fused ring is an aryl or heteroaryl ring selected from furan, thiophene, pyrrole, oxazole, isoxazole, thiazole, isothiazole, imidazole and pyrazole. In some cases of formula (XI), the structural formula of the BODIPY group is shown in formula (XIIa) or formula (XIIb):

[0270]

[0271] in:

[0272] Z 11 and Z 12 is independently a fused 5- or 6-membered heterocyclic, carbocyclic, aryl or heteroaryl ring;

[0273] Each "i" is independently 0-3; and

[0274] Each R 20 are independently for R in formula (X) 2 -R 7 The substituents are defined.

[0275] In certain embodiments of Formulas (XIIa)-(XIIb), Z 11 and Z 12 is independently a 5- or 6-membered fused aryl or heteroaryl ring. 11 and Z 12 is independently selected from furan, thiophene, pyrrole, oxazole, isoxazole, thiazole, isothiazole, imidazole and pyrazole. In some cases of Formulas (XIIa)-(XIIb), Z 11 and / or Z 12 is furan or thiophene. In certain cases of formula (XIIa), R 1 -R 5 In some cases of Formulas (XIa)-(XIIb), Z 11 and Z 12 Independently selected from the following rings:

[0276]

[0277] in:

[0278] X is O or S;

[0279] Y is O, S or NR, wherein R is H, alkyl, substituted alkyl or for R in formula (XIIa)-(XIIb) 20 substituents as defined; and

[0280] R 21 -R 23 are independently selected from H and for R in formula (XIIa)-(XIIb) 20 In certain cases of Formulas (XIIa)-(XIIb), Z 11 and Z 12 Independently selected from the following rings:

[0281] As mentioned above.

[0282] In certain cases of Formula (XIIb), Z 11 and / or Z 12 are the same ring. In some cases, Z 11 and Z 12 Includes different rings.

[0283] In certain embodiments of formula (XIIa), the structural formula of the BODIPY group is as shown in formula (XIIIa) or formula (XIIIb):

[0284]

[0285]

[0286] in:

[0287] X is O or S; and

[0288] R 16 and R 17 is for R in formula (I) 6 and R 7 The substituents are defined.

[0289] In certain cases of Formula (XIIIa) or (XIIIb), R 1 -R 5 None of them are cyclic linking groups.

[0290] In certain embodiments of formula (XI) or (XIIb), the structural formula of the BODIPY group is as shown in formula (XIVa) or (XIVb):

[0291]

[0292] in:

[0293] each X is O or S; and

[0294] R 12 , R 13 , R 16 and R 17 is for R in formula (I) 2 , R 3 , R 6 and R 7 The substituents are defined.

[0295] In certain embodiments of Formulae (XI)-(XIVb), the BOBIPY group includes an attached chemoselective functional group or molecule of interest, such as a light-harvesting multichromophore (e.g., -L 1 -Z 1 ). In some cases, R 1 -R 7 One of the included –L 1 -Z 1 In some cases, Y 1 or Y 2 Includes –L 1 -Z 1 In certain embodiments of Formulae (XI)-(XIVb), R 1 Yes – L 1 -Z 1 , where L 1 is the linker, Z 1 is a chemoselective functional group or a light-harvesting multichromophore. In certain embodiments of Formulas (XI)-(XIVb), R 4 or R 5 Yes – L 1 -Z 1 In certain embodiments of Formulae (XI), (XIIa) and (XIIIa)-(XIIIb), R 2 or R 7 Yes – L 1 -Z 1 In certain embodiments of Formulae (XI), (XIIa) and (XIIIa)-(XIIIb), R 3 or R 6 Yes – L 1 -Z 1 In certain embodiments of Formulae (XIIa)-(XIIb), R 20 The substituent is –L 1 -Z 1In certain embodiments of Formula (XIIIa)-(XIIIb), R 16 or R 17 Yes – L 1 -Z 1 In certain embodiments of Formulas (XIVa)-(XIVb), R 12 , R 13 , R 16 or R 17 Yes – L 1 -Z 1 .

[0296] In some cases, R 1 Yes-L 1 -Z 1 , where L 1 is a linker (e.g., as described herein) having a backbone of 20 or fewer atoms in length. 1 In some cases, L 1 is selected from alkyl or substituted alkyl linkers, alkoxy or substituted alkoxy linkers, PEG linkers, sulfonamido-alkyl or substituted sulfonamido-alkyl linkers, amino-alkyl or substituted amino-alkyl linkers, and alkyl-amino-alkyl or substituted alkyl-amino-alkyl linkers. The linker may be substituted with WSG (e.g., a PEG group). In R 1 In some cases, L 1 Selected from C1-C 12 Alkyl or substituted alkyl linker, C1-C 12 Alkoxy or substituted alkoxy linker, C1-C 12 Amine-alkyl or substituted amine-alkyl linker and C1-C 12 Alkyl-amino-alkyl or substituted alkyl-amino-alkyl linker. 1 In some cases, Z 1 Conjugation to the BODIPY group is via a carboxylic acid or its activated ester.

[0297] In certain cases of Formulas (XI)-(XIVb), R 1 includes an optionally substituted carbocyclic or heterocyclic group attached to the repeating unit or chemoselective functional group of the light-harvesting multichromophore. 1is an optionally substituted aryl or heteroaryl group connected to the repeating unit of the light-harvesting multichromophore (e.g., connected 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-pyridinyl, 2,6-pyridinyl, 3,5-pyridinyl, etc. R 1 The divalent carbocyclic or heterocyclic group or divalent aromatic or heteroaromatic group may be combined with -L 2 -Z 1 Connection, where L 2 is a linking group, for example, a linking group described in any of the Examples herein.

[0298] In some embodiments of Formula (XI), Q is C; R 1 -R 7 are each 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 instances of Formula (XI), Y 1 and Y 2 In some cases, Y 1 and Y 2 are all alkynyl substituted with WSG. In some cases, Y 1 and Y 2 are all alkynyl substituted with branched WSG. In some cases of formula (XI), Y 1 and Y 2 All are –CC-CH2) n -O(CH2CH2O) m -R, wherein n is 1 to 6, m is 2 to 50 (eg, 2 to 30, 2 to 20, 6 to 20, 8 to 20, or 10 to 20); R is H, alkyl, or substituted alkyl (eg, methyl).

[0299] In some embodiments of formula (XI), the structural formula of the BODIPY side-donor chromophore group may be as shown in formula (XIa):

[0300]

[0301] in:

[0302] * is the bonding point of the non-conjugated repeating unit of the polymer backbone;

[0303] Y 1 and Y 2are alkynyl substituted with one or more WSG. 1 and Y 2 are all alkynyl substituted with branched WSG. In some cases of Formula (XIa), each R 10 All are –CC-CH2) n -O(CH2CH2O) m -R, wherein n is 1 to 6, m is 2 to 50 (eg, 2 to 30, 2 to 20, 6 to 20, 8 to 20, or 10 to 20); R is H, alkyl, or substituted alkyl (eg, methyl).

[0304] In certain cases of formula (XI), the BODIPY group has a structural formula as shown in formula (XV):

[0305]

[0306] in:

[0307] L 3 is a covalent bond, oxo (—O—), alkylene (e.g., C1-C6-alkylene), —O-alkylene, or a substituted form thereof;

[0308] R 11 It is for R 1 the defined group;

[0309] Each R 9 are all optional substituents selected from halogen, hydroxy, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and t is 0-4. In certain cases of Formula (XV), L 3 is a covalent bond. In some cases of Formula (XV), L 3 In certain cases of Formula (XV), L 3 It is a covalent bond.

[0310] In certain cases of formula (XV), the structural formula of the BODIPY group is shown in formula (XVa):

[0311]

[0312] Where: R 11 It is for R 1 The defined group; each R 9 is an optional substituent selected from halogen, hydroxy, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and t is 0-4. In some cases of Formulas (XV)-(XVa), R 11 YesL1 -Z 1 (e.g., as described herein). In some cases of Formulas (XV)-(XVa), R 11 In some cases of Formula (XV)-(XVa), R 2 , R 4 , R 5 and R 7 are each independently H, alkyl or substituted alkyl. In certain cases of Formulas (XV)-(XVa), R 3 and R 6 Each is independently H, alkyl or substituted alkyl. 3 and R 6 are H. In certain cases of Formulas (XV)-(XVa), R 2 , R 4 , R 5 and R 7 Each is independently a C1-C6 alkyl group or a substituted C1-C6 alkyl group.

[0313] In certain cases of formula (XV), the structural formula of the BODIPY group is as shown in formula (XVIa) or formula (XVIb):

[0314]

[0315] in:

[0316] * is a bonding point to a repeating unit or a chemoselective functional group of the light-harvesting multichromophore; and

[0317] Y 1 and Y 2 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.

[0318] In certain cases of formula (XI), the structural formula of the BODIPY group is as shown in formula (XVIIa) or formula (XVIIb):

[0319]

[0320] Where: n is 0-12, Z 1is a chemoselective functional group (e.g., as described herein) or a target linker molecule. In certain instances of Formulas (XVIIa)-(XVIIb), n is 1-12 or 1-6, e.g., 1, 2, 3, 4, 5 or 6. In certain instances of Formulas (XVIIa)-(XVIIb), R 2 -R 4 and R 5 -R 7 are each independently selected from H, C1-C6 alkyl and substituted C1-C6 alkyl. In certain cases of Formula (XVIIa)-(XVIIb), R 2 and R 4 and / or R 5 and R 7 are each independently selected from C1-C6 alkyl and substituted C1-C6 alkyl. In certain cases of Formula (XVIIa)-(XVIIb), R 3 and / or R 6 is H. In certain instances of Formulas (XVIIa)-(XVIIb), R 3 and / or R 6 It is an alkyl group or a substituted alkyl group.

[0321] In certain cases of formula (XIIIa), the structural formula of the BODIPY group is shown in formula (XVIII):

[0322]

[0323] Where: L 1 is the linker, Z 1 is a non-conjugated repeating unit of the polymer backbone (e.g., as described herein). In certain instances of Formula (XVIII), the BODIPY group has a structural formula as shown in Formula (XVIIIa):

[0324]

[0325] Where: L 2 is a linker (e.g., L 1 The linking group component) of 9 are all optional substituents selected from halogen, hydroxy, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and t is 0-4.

[0326] In certain cases of formula (XIVa), the BODIPY group has a structural formula as shown in formula (XIX):

[0327]

[0328] Where: R 12 and R 13 is for R in formula (XI) 2 and R 3 The substituents defined; L 1 is the linker, Z 1 is a non-conjugated repeating unit of the polymer backbone (e.g., as described herein). In certain instances of Formula (XIX), the BODIPY group has a structural formula as shown in Formula (XIXa):

[0329]

[0330] Where: L 2 is a linker (e.g., L 1 The linking group component); each R 9 are all optional substituents selected from halogen, hydroxy, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and t is 0-4.

[0331] In any of the embodiments of Formula (XI)-(XIXa) described herein, the BODIPY group may include a specific -L as described in any of the following embodiments: 1 -Z 1 Group.

[0332] L 1 L may be a linker (e.g., as described herein) having a backbone of 20 or fewer atoms in length. 1 is selected from alkyl or substituted alkyl linkers, alkoxy or substituted alkoxy linkers, PEG linkers, sulfonamido-alkyl or substituted sulfonamido-alkyl linkers, amino-alkyl or substituted amino-alkyl linkers, and alkyl-amino-alkyl or substituted alkyl-amino-alkyl linkers. The linker may be substituted with WSG (e.g., a PEG group). In some cases, L 1 Selected from C1-C 12 Alkyl or substituted alkyl linker, C1-C 12 Alkoxy or substituted alkoxy linker, C1-C 12 Amine-alkyl or substituted amine-alkyl linker and C1-C 12Alkyl-amino-alkyl or substituted alkyl-amino-alkyl linker. The linker L may include several connected components, for example, one or more linking groups independently selected from the following: lower alkylene, substituted lower alkylene, alkenylene (-CH=CH-), substituted alkenylene, alkynylene (-CC-), substituted or unsubstituted amine (e.g., -NRCO- or -CONR-, wherein R is H, alkyl or substituted alkyl), substituted or unsubstituted sulfonamide (e.g., -NRSO2- or -SO2NR-, wherein R is H, alkyl or substituted alkyl), oxo (-O-), thio (-S-), ethylene glycol (-OCH2CH2O-), polyethylene glycol (e.g., -(CH2CH2O) n -, wherein n is 2-20, e.g., 2-10 or 2-6, or 2, 3, 4, 5 or 6), arylene, substituted arylene, heteroarylene, substituted heteroarylene, α-amino acid residue, β-amino acid residue, and the like.

[0333] The polymer backbone (Z 1 ) can be conjugated to the BODIPY group by any suitable chemoselective functional group (e.g., a functional group suitable for conjugation with a target molecule having a compatible functional group). The target chemoselective functional group that can be used to connect the BODIPY group to the polymer backbone includes, but is not limited to, an amine group (e.g., -NH2), a carboxylic acid (-CO2H), an activated ester (e.g., NHS or sulfo-NHS ester), a thiol, a maleimide, an iodoacetamide, a hydroxyl group, a hydrazide group, a hydrazine group, an aldehyde, a ketone, an azide group, an alkyne, a tetrazine, an olefin, a phosphine, and an epoxide. It should be understood that in some cases, the chemoselective functional group used to connect the BODIPY group is a synthetic precursor or a protected form of the target functional group, which can be converted into a reactive functional group that can be conjugated to the polymer backbone. For example, a carboxylic acid is a chemoselective functional group that can be coupled to an amine group on the target molecule. The carboxylic acid can be converted to an activated ester that is coupled to an amine group in situ or before coupling.

[0334] In some embodiments of Formulae (XI)-(XIXa), L 1 Includes an optionally substituted carbocyclic or heterocyclic group attached to the non-conjugated repeating unit of the polymer backbone. In some cases, L 1 is an optionally substituted aryl or heteroaryl. The intended divalent carbocyclic or heterocyclic group includes, but is not limited to, 1,4-cyclohexyl, 1,3-cyclohexyl, piperidinyl (e.g., 1,4-piperidinyl), piperazinyl (e.g., 1,4-piperazinyl), etc. The intended divalent aryl or heteroaryl group includes, but is not limited to, 1,4-phenyl, 1,3-phenyl, 2,5-pyridinyl, 2,6-pyridinyl, 3,5-pyridinyl, etc. L 1The divalent carbocyclic or heterocyclic group or divalent aromatic or heteroaromatic group may be combined with -L 2 -Z 1 Connection, where L 2 is a linker, for example, a linker described in any of the Examples herein. In some instances of Formulas (I)-(IXa), -L 1 -Z 1 is structured as either:

[0335]

[0336] in:

[0337] R 11 YesL 2 -Z 1 ;

[0338] L 2 is the linker, Z 1 is a non-conjugated repeating unit of the polymer backbone;

[0339] t is 0-4; and each R 9 is independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, sulfonic acid, and a water solubilizing group (WSG). In certain embodiments, -L 1 -Z 1 is structured as either:

[0340]

[0341] in:

[0342] L 2 is the linker, Z 1 is a non-conjugated repeating unit of the polymer backbone;

[0343] t is 0-4; and each R 9 is independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, sulfonic acid, and a water solubilizing group (WSG). In certain embodiments, -L 1 -Z 1 is structured as either:

[0344]

[0345] in:

[0346] L 2 is the linker, Z 1 is a non-conjugated repeating unit of the polymer backbone;

[0347] R 32 is H, alkyl, substituted alkyl, and water solubilizing group (WSG);

[0348] L 3 is a linker selected from alkylene (e.g., C1-C6-alkylene), -O-alkylene, and substituted forms thereof;

[0349] t is 0-4; and each R 9 Independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, sulfonic acid and water solubilizing group (WSG).

[0350] In any of the embodiments of formula (XI)-(XIXa) described herein, the BODIPY group may include the specific Y described in any of the following embodiments: 1 and Y 2 In some cases of Formulas (XI)-(XIXa), Y 1 In some cases, Y 1 is an alkynyl group substituted with WSG. 2 Can be used with Y 1 In some cases of Formulas (XI)-(XIXa), Y 2 is selected from the group consisting of 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 is selected from the group consisting of F, CN, phenyl and substituted phenyl.

[0351] In some cases of Formulas (XI)-(XIXa), Y 1 and Y 2 In some cases, Y 1 and / or Y 2 is an alkynyl substituted with WSG (e.g., as described herein). 1 and / or Y 2 is an alkynyl substituted with polyethylene glycol (PEG) or a modified (PEG). In certain cases described in any one of the embodiments of Formula (I)-(IXa), Y 1 and / or Y 2 The structural formula is as follows:

[0352]

[0353] in:

[0354] s is 1 to 12;

[0355] q is 0 to 50; and

[0356] R 21 In some cases, s is 1 to 6, e.g., 1, 2, or 3. In some cases, q is 1 to 50, e.g., 1 to 30, 2 to 30, 4 to 30, 6 to 30, 8 to 30, 10 to 30, 10 to 20, or 11 to 16. In some cases, q is 10 to 50, e.g., 10 to 30, 10 to 20, or 11 to 16.

[0357] In certain embodiments of Formula (XI) and (XVIIa), the structure of the BODIPY group is as follows:

[0358]

[0359] Where Z 1 is a linked non-conjugated repeating unit of the polymer backbone; n is 0-6; each R, R 2 , R 4 , R 5 and R 7 are independently H, C1-C6 alkyl or substituted C1-C6 alkyl; and each q is 6-20 (e.g., 10-20). In certain embodiments of formula (XI) and (XVIIa), the structure of the BODIPY group is shown in any of the following:

[0360] In certain embodiments of Formula (XV) and (XVa), the BODIPY dye has the following structure:

[0361]

[0362] Where Z 1 is a connected non-conjugated repeating unit of the polymer backbone; n is 0-6; R, R 2 , R 4 , R 5 and R 7 is independently H, C1-C6 alkyl or substituted C1-C6 alkyl; and each q is 6-20 (e.g., 10-20). In certain embodiments of formula (XV), (XVa) and (XVIa) or (XVIb), the structure of the BODIPY dye is shown in any of the following:

[0363]

[0364] In certain embodiments of Formula (XVIII), the structure of the BODIPY group is as follows:

[0365]

[0366] Where Z 1 is a linked non-conjugated repeating unit of the polymer backbone; n is 0-6; each R, R 2 and R 4 are independently H, C1-C6 alkyl or substituted C1-C6 alkyl; and each q is 6-20 (e.g., 10-20). In certain embodiments of formula (XVIII), the structure of the BODIPY group is shown in any of the following:

[0367]

[0368] In certain embodiments of Formula (XIX)-(XIXa), the structure of the BODIPY group is as follows:

[0369]

[0370] Where Z 1 is a linked non-conjugated repeating unit of the polymer backbone; n is 0-6; each R and R 13 are independently H, C1-C6 alkyl or substituted C1-C6 alkyl; and each q is 6-20 (e.g., 10-20). In certain embodiments of formula (XIX)-(XIXa), the structure of the BODIPY group is as follows:

[0371]

[0372] In certain embodiments of Formula (XIIIa), the structure of the BODIPY group is as follows:

[0373]

[0374] In certain embodiments of Formula (XIVa), the structure of the BODIPY group is as follows:

[0375]

[0376] In certain embodiments of Formula (XIXa), the structure of the BODIPY group is as shown in any one of the following:

[0377]

[0378]

[0379] Aryl or heteroaryl chromophore

[0380] The light absorbing chromophore may be an aryl or heteroaryl chromophore. The aryl or heteroaryl chromophore of interest in the polychromophores (e.g., polychromophores of formulas (I)-(IX)) include, but are not limited to, phenyl, biphenyl, benzoxazole, benzothiazole, polyphenylene, and fused tricyclic groups, such as fluorene, carbazole, silole, biphenyl, and bridged biphenyl. The aryl or heteroaryl chromophore may optionally be further substituted with aryl or heteroaryl substituents such as water solubilizing groups and / or aryl or heteroaryl substituents that impart the desired light absorbing properties to the aryl or heteroaryl groups. In some cases of formulas (I)-(IX), each D 1 independently includes fused tricyclic aryl or heteroaryl. In some cases of Formulas (I)-(IX), each D 1 Independently include one or more groups selected from fluorene, carbazole, silole, biphenyl and bridged biphenyl.

[0381] The fused tricyclic chromophoric group is a group including a tricyclic aromatic group (having three fused rings in the configuration), wherein two aryl or heteroaryl 6-rings are fused to a central 5 or 6-ring carbocycle or heterocycle. In some cases, the fused tricyclic group includes two benzo or pyrido rings fused to a central 5 or 6-ring carbocycle or heterocycle. The fused tricyclic group can be connected to the side chain of the comonomer in the polymer backbone by any suitable ring atom on the fused ring. The central 5 or 6-ring can be an aromatic or partially saturated carbocycle or heterocycle, and can further include a side chain substituent, for example, WSG and / or a linker connecting a chemical selective tag or a comonomer side chain. The bridged biphenyl comonomer is a fused tricyclic group with a biphenyl group, wherein two benzene rings are further connected to each other by a central 6-ring carbocycle or heterocycle.

[0382] In certain cases of multichromophores (eg, in formulas (I)-(IX)), the pendant donor chromophore is a fused tricyclic aryl or heteroaryl group having the following structural formulas:

[0383]

[0384] in:

[0385] * is the bonding point of the non-conjugated repeating unit of the polymer backbone;

[0386] 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;

[0387] Each Z is independently CH, CR or N;

[0388] Each R 13 are independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amine, substituted amine, aralkyl, substituted aralkyl, PEG moiety, -L 11 -Z 1 , where L 11 is the linker, Z 1 is a non-conjugated repeating unit and WSG, or any two suitable R 3 The groups are optionally linked to form a ring structure; and

[0389] Each R is independently H or one or more substituents (e.g., WSG), and any two suitable R groups are optionally linked to form a ring structure. 13 One of the two is connected to the non-conjugated repeating unit of the polymer backbone.

[0390] In some cases, the structure of the fused tricyclic group is as shown in any of the following:

[0391]

[0392] wherein Y and each R are as defined above; the fused tricyclic group may be linked to the non-conjugated repeating unit of the polymer backbone via Y or R.

[0393] In certain cases, the fused tricyclic group is fluorene, wherein Y is C(R 3 )2. In some cases, the fused tricyclic group is carbazole, wherein Y is NR 3 In some cases, the fused tricyclic group is a silole, wherein Y is Si(R 3 )2. In some cases, the fused tricyclic group is a bridged biphenyl, wherein Y is -C(R 3 )2C(R 3 )2-or-C(R 3 )2Si(R 3 )2-. In some cases, the fused tricyclic ring is a bridged biphenyl, wherein Y is -CHR 3 CHR 3 -. In certain cases of any fused tricyclic group 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.

[0394] In certain embodiments of the fused tricyclic group, the group includes two R substituents connected to form a cyclic structure to provide a carbocyclic or heterocyclic ring A which is optionally further substituted:

[0395]

[0396] Where 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 are independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amine, substituted amine, aralkyl, substituted aralkyl, PEG moiety, -L 11 -Z 1 , where L 11 is the linker, Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG; each R is as defined above; and the fused tricyclic group can be connected by R 3 Or R is connected to the non-conjugated repeating unit of the polymer backbone. In some cases, the structure of the fused tricyclic group is as follows:

[0397]

[0398] Where R 8 -R 9 are each independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amine, substituted amine, aralkyl, substituted aralkyl, PEG moiety, -L 11 -Z 1 , where L 11 is the linker, Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG; Y and each R are as defined above; and the fused tricyclic group can be connected by Y, R 8 -R 8 or R is linked to the non-conjugated repeating unit of the polymer backbone. In some cases of the comonomer, Y is C(R 3 )2.

[0399] Water-solubilizing groups

[0400] The present invention includes water-soluble light-harvesting multichromophores with side-group chromophores and polymer tandem dyes including acceptor fluorophores. The multichromophores can be substituted with multiple water-solubilizing groups (WSG). In some cases, WSG is a side group directly connected to a modular scaffold, for example, as a side chain of a polymer backbone. In some cases, WSG is a substituent attached to a side-group donor chromophore or a side-group acceptor fluorophore. In some cases, each side-group donor chromophore is substituted with one or more WSGs.

[0401] As used herein, the terms "water-solubilizing group", "water-soluble group" and WSG are used interchangeably and refer to a group or substituent that is effectively solvated in an aqueous environment (e.g., under physiological conditions) and can increase the water solubility of the molecule to which it is attached. WSG can increase the solubility of the multichromophore in a predominantly aqueous solution compared to a control multichromophore without WSG. The water-soluble group can be any suitable hydrophilic group that is effectively solvated in an aqueous environment.

[0402] The water-soluble multichromophores of the present invention are soluble in aqueous environments, which makes them particularly suitable for various biological assays. The subject water-soluble multichromophores and polymer tandem dyes and their conjugates can resist undesirable aggregation, which can produce favorable fluorescence and spectral properties in various biological assays. Dye aggregation is undesirable because it can result in a reduction in fluorescence signal, for example, polymerization-induced dye fluorescence quenching. The subject water-soluble multichromophores and polymer tandem dyes can be used as fluorescent reporters for various biosensors and provide exceptionally bright signals and a range of excitation and emission wavelength options suitable for various applications (e.g., flow cytometry and imaging).

[0403] Various water-soluble polymer groups can be used in the WSG of the target multi-chromophore. Any suitable water-solubilizing group (WSG) can be included in the multi-chromophore described herein to improve water solubility. Although the increase in solubility may vary, in some cases, the increase (compared to the compound without WSG) can reach 2 times or more, for example, 5 times, 10 times, 25 times, 50 times, 100 times or more. In some cases, the hydrophilic water-solubilizing group is charged, for example, positively charged or negatively charged. In some cases, the hydrophilic water-solubilizing group is a neutral hydrophilic group. In some embodiments, WSG is branched (for example, as described herein). In some cases, WSG is linear. In some embodiments, WSG is a hydrophilic polymer, for example, polyethylene glycol, modified PEG, peptide sequence, peptoid, carbohydrate, oxazoline, polyol, dendron, dendritic polyglycerol, cellulose, chitosan or its derivatives. The water-solubilizing groups of interest include, but are not limited to, carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfoniums, esters, polyethylene glycol (PEG) and modified PEGs, hydroxyls, amines, amino acids, ammonium groups, guanidine groups, pyridinium salts, polyamines and sulfoniums, polyols, linear or cyclic sugars, primary, secondary, tertiary or quaternary amines and polyamines, phosphonates, phosphinates, ascorbates, glycols including polyethers, -COOM', -SO3M', -PO3M', -NR3 + 、Y′、(CH2CH2O) p R and mixtures thereof, wherein Y′ can be any halogen, sulfate, sulfonate or oxyanion; p can be from 1 to 500; each R can independently be H or an alkyl group (e.g., methyl); and M′ can be a cationic counterion or hydrogen, --(CH2CH2O) yy CH2CH2XR yy 、--(CH2CH2O) yy CH2CH2X--、--X(CH2CH2O) yy CH2CH2--, ethylene glycol and polyethylene glycol, wherein yy is selected from 1 to 1000; X is selected from O, S and NR ZZ ; and R ZZ and R YY Independently selected from H and C 1-3 In some cases, WSG is (CH2) x (OCH2CH2) y OCH3, wherein each x is independently an integer between 0 and 20, and each y is independently an integer between 0 and 50. In some cases, the water-solubilizing group includes a nonionic polymer (e.g., a PEG polymer) substituted at the terminal end with an ionic group (e.g., a sulfonate group).

[0404] In some embodiments of the formula, the side groups of interest include those selected from (CH2) x (OCH2CH2) y OCH3 (wherein each x is independently an integer between 0 and 20, and each y is independently an integer between 0 and 50) and optionally substituted by one or more halogen, hydroxyl, C1-C 12 Alkoxy or (OCH2CH2) z OCH3 (wherein each z is independently an integer between 0 and 50) substituted benzyl. In some cases, the substituent is (CH2)3(OCH2CH2) 11 In some embodiments, one or more substituents are benzyl substituted with at least one WSG group (e.g., one or two WSG groups) selected from (CH2) x (OCH2CH2) y OCH3, wherein each x is independently an integer between 0 and 20, and each y is independently an integer between 0 and 50.

[0405] Multiple WSGs can be included in a single position of the target multichromophore via a branched linker. In certain embodiments, the branched linker is an aralkyl substituent, which is further disubstituted by a water-solubilizing group. Therefore, in some cases, the branched linker group is a substituent of the multichromophore that connects the multichromophore to two or more water-solubilizing groups. In certain embodiments, the branched linker is an amino acid, for example, a lysine amino acid, which is connected to three groups via an amino group and a carboxylic acid group. In some cases, after multiple WSGs are introduced via a branched linker, the multichromophore can be made to reach the desired solubility. In some cases, the WSG is a nonionic side chain group that can make the solubility in water exceed 50 mg / mL. In some cases, the WSG is a nonionic side chain group that can make the solubility in water exceed 100 mg / mL. In some embodiments, the multichromophore includes substituents selected from the group consisting of alkyl, aralkyl, and heterocyclic groups, each of which is further substituted with a hydrophilic polymer group including a water-solubilizing group, such as polyethylene glycol (PEG) (e.g., containing 2 to 20 units of a PEG group).

[0406] The water-soluble polymers of interest that can be used in WSG include polyethylene glycol (PEG) groups or modified PEG groups. The water-soluble polymers of interest include, but are not limited to, polyalkylene oxide-based polymers, such as polyethylene glycol (PEG) (see, e.g., Poly(ethylene glycol) Chemicals: Biotechnology and Biomedical Applications, JM Harris, ed., Plenum Press, New York, NY (1992); Poly(ethylene glycol) Chemistry and Biological Applications, JM Harris and S.Zalipsky, editor, ACS (1997); International Patent Applications: WO 90 / 13540, WO 92 / 00748, WO 92 / 16555, WO 94 / 04193, WO 94 / 14758, WO 94 / 17039, WO 94 / 18247, WO 94 / 28937, WO 95 / 11924, WO 96 / 00080, WO 96 / 23794, WO 98 / 07713, WO 98 / 41562, WO 98 / 48837, WO 99 / 30727, WO 99 / 32134, WO 99 / 33483, WO 99 / 53951, WO 01 / 26692, WO 95 / 13312, WO 96 / 21469, WO 97 / 03106, WO 99 / 45964; and U.S. Patents numbered as follows: 4,179,337; 5,075,046; 5,089,261; 5,100,992; 5,134,192; 5,166,309; 5,171,264; 5,213,891; 5,219,564; 5,275,838; 5,281,698; 5,298,643; 5,312,808; 5,321,095; 5,324,844; 5,349,001; 5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662; 5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110; 5,730,990; 5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,442; 5,919,455; 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; 6,214,966).

[0407] Examples of the target water-soluble polymer include, but are not limited to, water-soluble polymers containing polyalkylene oxides, polyamide alkylene oxides or derivatives thereof, including polyalkylene oxides and polyamide alkylene oxides containing ethylene oxide repeating units represented by the formula (CH2-CH2-O)-. Further examples of the target polymer include water-soluble polymers of the formula -[C(O)-XC(O)-NH-Y-NH]n- or -[NH-Y-NH-C(O)-XC(O)] n -a polyamide having a molecular weight greater than 1,000 Daltons, wherein X and Y are divalent radicals, which may be the same or different, and may be branched or linear, and n is a discrete integer between 2 and 100, for example, 2 to 50; and one or both of X and Y contain biocompatible, substantially non-antigenic, water-soluble repeating units, which may be branched or linear. Further examples of water-soluble repeating units include ethylene oxide of the formula -(CH2-CH2-O)- or -(O-CH2-CH2)-. The number of water-soluble repeating units may vary greatly, wherein the number of units ranges from 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 an embodiment is one in which one or both of X and Y are selected from the following: -((CH2) n1 -(CH2-CH2-O) n2 -(CH2)- or -((CH2) n1 -(O-CH2-CH2) n2 -(CH2) n-1 -), wherein n1 is 1 to 6, 1 to 5, 1 to 4 or 1 to 3; n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2 to 8 or 2 to 5. A further example of an embodiment is wherein X is -(CH2-CH2)-; Y is -(CH2-(CH2-CH2-O)3-CH2-CH2-CH2)- or -(CH2-CH2-CH2-(O-CH2-CH2)3-CH2)-.

[0408] The term "modified polymer" (e.g., modified PEG) refers to a water-soluble polymer that has been modified or derivatized at either or both ends, for example, including terminal substituents (e.g., terminal alkyl, substituted alkyl, alkoxy or substituted alkoxy, etc.) and / or terminally attached functional groups (e.g., amino or carboxylic acid groups suitable for attachment via amide bond formation) that are suitable for attaching the polymer to a multichromophore (e.g., via a branching group). The target water-soluble polymer is suitable for including any suitable linking group. It should be understood that in some cases, based on the preparation and / or purification methods of the polymer starting material, the water-soluble polymer may have a certain dispersity relative to the polymer length. In some cases, the water-soluble polymer is monodisperse.

[0409] The water-soluble polymer may include one or more spacers or linkers. Examples of spacers or linkers include linear or branched moieties (which include one or more repeating units used in water-soluble polymers, diamino and / or diacid units, natural or non-natural amino acids or derivatives thereof) and aliphatic moieties (which include alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, etc., which may include, for example, up to 18 carbon atoms or even other polymer chains).

[0410] The water-soluble polymer portion or one or more spacers or linkers of the polymer portion (if any) may include a biostable or biodegradable polymer chain or unit. For example, based on the instability of the bond, polymers with repeating bonds have varying degrees of stability under physiological conditions. Polymers with the bonds can be classified according to their relative hydrolysis rates under physiological conditions based on the known hydrolysis rates of low molecular weight analogs, for example, from less stable to more stable, for example, polyurethane (-NH-C(O)-O)> polyorthoester (-OC((OR)(R'))-O-)> polyamide (-C(O)-NH). Similarly, the bonding system that attaches the water-soluble polymer to the target molecule can be biostable or biodegradable, for example, from less stable to more stable: carbonate (-OC(O)-O)> ester (-C(O)-O)> urethane (-NH-C(O)-O-)> orthoester (-OC((OR)(R'))-O-)> amide (-C(O)-NH-). In general, sulfated polysaccharides may need to be avoided due to the instability of sulfate groups. In addition, polycarbonates and polyesters may be less desirable. The bonds are provided as examples and are not intended to limit the types of bonds in the polymer chains or bonding systems of the water-soluble polymers that can be used in the WSG disclosed herein.

[0411] In some cases, WSG is a branched nonionic water-soluble group (WSG), which comprises a connected branched group and can be further bonded to two, three or more nonionic water-soluble polymers. In some cases, the structural formula of the branched nonionic WSG is shown in any of the following:

[0412]

[0413] in:

[0414] Each B 1 and B 2 are all independently branched linkers;

[0415] Each W 1 Each is independently a nonionic water-soluble polymer, for example, comprising 6 or more monomer units;

[0416] T 3 is an optional linker that connects a side group or a repeating unit of the multichromophore; and

[0417] Each p and q are independently 0 or 1, where each T 1 and each T 2 (if any) are independently linkers. In some cases, each W 1 Each W is independently PEG or a modified PEG polymer. 1 W are independently selected from substituted alkyl, PEG or modified PEG groups and WSG. In some cases, each W 1 Each is independently a PEG or modified PEG polymer containing 6-30 monomer units, for example, 6-24 or 10-30, 10-24 or 10-20, 12-24, 12-20, 12-16 or 16-20 monomer units.

[0418] In some cases, the structural formula of the branched nonionic WSG is as follows:

[0419]

[0420] in:

[0421] Each B 1 All are branched groups;

[0422] Each W 1 Each is independently a nonionic water-soluble polymer, for example, comprising 6 or more monomer units;

[0423] T 3 is an optional linker connecting the fused 6-5-6 tricyclic comonomer; and

[0424] Each p is independently 0 or 1, where each T 1 (if any) are independently linkers. In some cases, each W 1 Each W is independently PEG or a modified PEG polymer. 1 W are independently selected from substituted alkyl, PEG or modified PEG groups and WSG. In some cases, each W 1 Each is independently a PEG or modified PEG polymer containing 6-30 monomer units, for example, 6-24 or 10-30, 10-24 or 10-20, 12-24, 12-20, 12-16 or 16-20 monomer units. In some embodiments of the branched nonionic WSG, B 1 is a tetrasubstituted aryl group (eg, 1,3,4,5-phenyl).

[0425] In some embodiments of the branched nonionic WSG (e.g., as shown in the above formula), B 1 is selected from CH, N, C(=O)N, SO2N, a trisubstituted aryl group (e.g., 1,3,5-phenyl), a tetrasubstituted aryl group, and a trisubstituted heteroaryl group. In some embodiments of the branched nonionic WSG, each p is 0. In some embodiments of the branched nonionic WSG, p is 1; each T 1 Selected from –(CH2) n -O–, –O-(CH2) n –, –(CH2) n – and –O–, wherein n is 1 to 12, for example, 1 to 6. In some embodiments of the branched nonionic WSG, each T 2 and / or T 3 Each is independently a C1-C12-alkyl linker, for example, a C1-C6-alkyl linker, wherein one or more backbone atoms are optionally substituted by a heteroatom.

[0426] In some embodiments of the subject multichromophores, the pendant donor chromophore is substituted with one or more water solubilizing groups (WSG) independently selected from the following formula:

[0427]

[0428]

[0429] in:

[0430] T 5 is an optional linker;

[0431] Each T 6 All are linkers;

[0432] R 11 and R are independently H, alkyl or substituted alkyl; and

[0433] Each s is an integer between 1 and 100 (eg, 6 to 100 or 6 to 50).

[0434] In certain cases, each s is independently 6 to 30, e.g., 6 to 24, 6 to 20, 11 to 20, 12 to 20, 12 to 18, or 12 to 16. In certain cases, each s is independently 6 to 30, e.g., 6 to 24, 8 to 24, 10 to 24, 12 to 24, 13 to 24, 14 to 24, 15 to 22, or 16 to 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 greater, e.g., 8, 9 or greater, 10 or greater, 11 or greater, 12 or greater, 13 or greater, 14 or greater, 15 or greater, or even greater, and in some cases has up to 50 monomeric units, e.g., up to 40, up to 30, or up to 24 monomeric units. In some cases, each s is independently 6-30 monomeric units, e.g., 6-24 or 10-30, 10-24 or 10-20, 12-24, 12-20, 12-16, or 16-20 monomeric units. In some cases, each s is the same. In some embodiments of the WSG, T 5 and / or T 6 is a C1-C12-alkyl linker, for example, a C1-C6-alkyl linker, wherein one or more backbone atoms are optionally substituted with a heteroatom (e.g., —O—). In some embodiments of the WSG, each R 11 are H. In some embodiments of the WSG, each R 11 All are methyl.

[0435] It should be understood that in any of the above WSG groups, hydroxyl-terminated PEG chains can be used instead of methoxy-terminated PEG chains. In certain cases of any of the formulas described herein, one or more repeat units or side groups are replaced by WSG, which is a dendron selected from any of the following structures:

[0436]

[0437] Where R=H, CH3

[0438] In certain instances of any of the formulae described herein, one or more repeating units or side groups are substituted with WSG, which is a polyol selected from any of the following structures:

[0439]

[0440] In certain instances of any of the formulae described herein, one or more repeat units or side groups are substituted with WSG, which is an oxazoline as shown in the following structure:

[0441] In certain instances of any of the formulae described herein, one or more repeating units or side groups are substituted with WSG, which is a peptoid selected from any of the following structures:

[0442]

[0443] The water-soluble group (WSG) can make the solubility of the multi-chromophore or polymer tandem dye in water exceed 10 mg / mL, for example, exceed 20 mg / mL, exceed 30 mg / mL, exceed 40 mg / mL, exceed 50 mg / mL, exceed 60 mg / mL, exceed 70 mg / mL, exceed 80 mg / mL, exceed 90 mg / mL or exceed 100 mg / mL. In some cases, the branched nonionic water-soluble group (WSG) can make the solubility of the multi-chromophore or polymer tandem dye in water (for example, solubility in aqueous buffer) reach 20 mg / mL or more, for example, 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 higher. It should be understood that under certain conditions, the water-solvated multi-chromophore can form discrete water-soluble nanoparticles in an aqueous system. In certain cases, the water-solvated nanoparticles are resistant to aggregation and can be used in various biological assays.

[0444] The polymer backbone of the multichromophore can have any suitable length. In some cases, the specific number of monomer repeat units of the multichromophore can range from 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000 or 2 to 1,000 units or segments, or such as 5 to 100,000, 10 to 100,000, 100 to 100,000, 200 to 100,000 or 500 to 50,000 units or segments. In some cases, the specific number of monomer repeating units or segments of the polymer backbone of the multichromophore can range from 2 to 1,000, e.g., 2 to 500, 2 to 500, 3 to 500, 4 to 500, 5 to 500, 6 to 500, 7 to 500, 8 to 500, 9 to 500, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, or 20 to 100 units or segments.

[0445] The multichromophore can have any suitable molecular weight (MW). In some cases, the MW of the multichromophore can be expressed as an average molecular weight. In some cases, the average molecular weight range of the polydye is 500 to 500,000, for example, 1,000 to 100,000, 2,000 to 100,000 (for example, 2,000 to 10,000 or 10,000 to 100,000), or even an average molecular weight range of 50,000 to 100,000. In some cases, a polymer backbone having a multichromophore of a specific discrete monomer sequence is prepared so that the MW of the multichromophore can be expressed as an accurate molecular weight. In some cases, the polymeric dye has an exact molecular weight range of 500 to 500,000, for example, 1,000 to 100,000, 1,000 to 50,000, 2,000 to 50,000 (eg, 2,000 to 10,000 or 10,000 to 50,000), or even an average molecular weight range of 50,000 to 100,000.

[0446] Polymer tandem dyes

[0447] As described above, the target light-harvesting multichromophore may include a polymer backbone comprising non-conjugated repeating units, and may include a plurality of pendant donor chromophores, each of which may be attached to a repeating unit. As described above, the target water-soluble light-harvesting multichromophore is capable of homogeneous energy transfer between pendant donor chromophores, which may result in continuous reversible energy transfer between the same chromophores, rather than emission from a single chromophore. Therefore, although the multichromophore system itself may emit fluorescence, the emission of Fig. 6A Due to the self-quenching process shown, the quantum yield of the multichromophore system can be significantly lower than the quantum yield observed for a single isolated chromophore.

[0448] The water-soluble light-harvesting multichromophore is capable of transferring energy to an attached acceptor fluorophore. For details, see, e.g., Figure 6B Thus, the subject polymer tandem dye further comprises a covalently linked acceptor signaling fluorophore that is proximal to the donor water-solvated light-harvesting multichromophore system upon energy reception (i.e., proximal to at least one pendant donor chromophore upon energy reception). The terms "acceptor fluorophore" and "acceptor chromophore" are used interchangeably herein.

[0449] The receptor signaling fluorophore can be attached as a side group to a non-conjugated repeating unit of the polymer backbone. Excitation of the multi-chromophore donor can transfer energy to the covalently attached receptor signaling fluorophore or cause the fluorophore to emit energy. The number of repeating units of the donor water-solvated light-harvesting multi-chromophore with attached receptor signaling fluorophores may vary, wherein, 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%.

[0450] Energy transfer mechanisms between the light-harvesting chromophores of the multichromophore and from the donor chromophore to the attached acceptor signaling fluorophore include, for example, resonance energy transfer (e.g., (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. The transfer distance of the energy transfer mechanism may be relatively short; that is, the chromophores of the light-harvesting multi-chromophore system must be very close to each other and / or the acceptor fluorophore to achieve effective energy transfer.

[0451] Under efficient energy transfer conditions, amplified acceptor fluorophore emission may occur, wherein the emission intensity of the luminescent acceptor fluorophore is higher when the wavelength of the incident light ("pump light") is absorbed and transferred by the chromophore of the light-harvesting multichromophore, compared to when the luminescent acceptor fluorophore is directly excited by the pump light.

[0452] "Efficient" energy transfer refers to 10% or more of the energy captured by the donor chromophore and transferred to the acceptor, for example, 20% or more, 30% or more, 40% or more, 50% or more. "Amplification" refers to the signal emitted by the acceptor fluorophore excited by energy transfer from the donor light-harvesting multi-chromophore system being 1.5 times higher or more than when the acceptor fluorophore is directly excited with incident light of equivalent intensity. The signal can be measured by any suitable method. In some cases, the signal of 1.5 times or more refers to the intensity of the emitted light. In some cases, the signal of 1.5 times or more refers to an increased signal-to-noise ratio. In certain embodiments of the polymer tandem dye, the emission of the acceptor fluorophore excited by the multichromophore is 1.5 times or more higher, e.g., 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 8 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, or even higher (compared to the case where the acceptor fluorophore is excited directly with incident light), as compared to the case where the acceptor fluorophore is excited directly with incident light.

[0453] Any suitable fluorescent dye can be used as an acceptor fluorophore in the polymer tandem dye. The terms "fluorescent dye" and "fluorophore" are used interchangeably herein. The acceptor fluorophore (e.g., each A 1 ) can be a small molecule fluorophore. The acceptor fluorophore (e.g., each A 1 ) can be a dye molecule selected from the group consisting of rhodamine, coumarin, xanthene, cyanine, polymethine, pyrene, thiazine, acridine, dipyrromethene boron difluoride, naphthylimide, phycobiliprotein, peridinium-chlorophyll-protein, conjugates thereof, and combinations thereof. In certain embodiments, the acceptor fluorophore (A 1 ) is a cyanine dye, a xanthene dye, a coumarin dye, a thiazine dye, or an acridine dye. In some cases, the acceptor fluorophore (A 1)Selected from DY431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy3.5 and diethylaminocoumarin. Fluorescent dyes of interest include, but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyparamethylamino acid, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridin-chlorophyll-a protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa 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, their conjugates and combinations thereof. Lanthanide chelates of interest 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 the group consisting of Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa 647, and Alexa 700.In certain embodiments, the polymer tandem dye comprises a multichromophore linked to an acceptor fluorophore selected from the group consisting of a Dyomics 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, Cy3.5, and diethylaminocoumarin.

[0454] In some cases, the acceptor fluorophore is a BODIPY group, for example, a BODIPY group as shown in any of Formulas (XI)-(XIXa) or any of the Examples described herein. It should be understood that any suitable BODIPY group described herein with a suitable absorption and emission spectrum can be configured as an acceptor fluorophore in proximity to a donor water-solvated light-harvesting multichromophore system (i.e., in proximity to at least one compatible side-group donor chromophore in proximity to energy reception) upon energy reception.

[0455] In some embodiments, the maximum emission wavelength of the selected acceptor fluorophore is in the range of 300 to 900 nm, for example, 350 to 850 nm, 350 to 600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm, 390 to 500 nm, or 400 to 500 nm, wherein specific examples of emission maxima of the target signal transduction chromophore include, but are not limited to, 395 nm ± 5 nm, 420 nm ± 5 nm, 430 nm ± 5 nm, 440 nm ± 5 nm, 450 nm ± 5 nm, 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.

[0456] The quantum yield of the emission of the linked luminescent acceptor fluorophore of the polymer tandem dye is 0.03 or more, for example, the quantum yield is 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.3 or more, or even higher. In some cases, the extinction coefficient of the polymer tandem dye is 5×10 5 cm -1 M -1 or more, for example, 6×10 5 cm -1 M -1 or above, 7×10 5 cm -1 M -1 or above, 8×10 5 cm -1 M -1 or above, 9×10 5 cm -1 M -1 or more, for example 1×10 6 cm -1 M -1 or above, 1.5×10 6 cm -1 M -1 or above, 2×10 6 cm -1 M -1 or above, 2.5×10 6 cm -1 M -1 or above, 3×10 6 cm -1 M -1 or above, 4×10 6 cm -1 M -1 or above, 5×10 6 cm -1 M -1 or above, 6×10 6 cm -1 M -1 or above, 7×10 6 cm -1 M -1 or above or 8×10 6 cm -1 M -1 In some embodiments, the molar extinction coefficient of the polymer tandem dye is 5×10 5 M -1 cm -1In some embodiments, the molar extinction coefficient of the polymer tandem dye is 1×10 6 M -1 cm -1 or above.

[0457] The target polymer tandem dye can provide fluorescence emission from the luminescent signaling chromophore dye, the brightness of which is higher than the emission that may come from the separated luminescent dye. The brightness of the emission of the linked luminescent signaling chromophore of the polymer tandem dye can be 50 mM -1 cm -1 or above, for example, 60 mM -1 cm -1 or above, 70mM -1 cm -1 or above, 80mM -1 cm -1 or above, 90mM -1 cm -1 or above, 100mM -1 cm -1 or above, 150mM -1 cm -1 or above, 200mM -1 cm -1 or above, 250mM -1 cm -1 or above, 300mM -1 cm -1 In some cases, the brightness of the emission of the linked signaling chromophore of the polymer tandem dye is at least 5 times higher, for example, at least 10 times higher, at least 20 times higher, at least 30 times higher, at least 50 times higher, at least 100 times higher, at least 300 times higher, or even higher (compared to the brightness of the directly excited luminescent dye).

[0458] The polymer tandem dye of interest can provide a fluorescence emission with a Stokes shift of 100 nm or more, for example, 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, for example, 200 nm or less.

[0459] In some embodiments, the polymer tandem dye comprises a water-soluble light-harvesting multichromophore represented by any one of formulas (I)-(IX), wherein Z 1 The chemically selective label is stained with an acceptor fluorophore (A 1) is substituted. It should be understood that for the polymer tandem dyes described in the present invention, any embodiment of the target multi-chromophore shown in formula (I)-(IX) can also be implemented. In certain embodiments of formula (I)-(IX), one or more Z 1 The group can be conjugated with an acceptor fluorophore precursor to obtain an acceptor fluorophore (A 1 ). Therefore, the polymer tandem dye may include a segment as shown in formula (Ia):

[0460]

[0461] in:

[0462] Each M 1 and M 2 are each independently an unsaturated comonomer (e.g., an aromatic or heteroaromatic comonomer);

[0463] Each S 1 and S 2 are all independently non-conjugated spacer units;

[0464] Each D 1 are independently 1 an attached pendant donor chromophore (e.g., as described herein);

[0465] Each A 1 are independently 2 the attached acceptor fluorophore;

[0466] x is 75 mol% or more; and

[0467] y is 25 mol% or less.

[0468] First (M 1 -S 1 ) and the second repeating unit (M 2 -S 2 ) can be arranged in a random configuration or a co-block configuration. In certain cases of formula (Ia), D of the first repeating unit 1 The side groups include two or more (eg, two or three) different types of side group light absorbing chromophores, which together constitute a light-harvesting multi-chromophore system. In certain cases of formula (Ia), D of the first repeating unit 1 The side groups are all identical.

[0469] In some cases of Formula (Ia), x is 80 mol% or more, e.g., 85 mol% or more, 90 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more, or 99 mol% or more. In some cases of Formula (Ia), y is 20 mol% or less, e.g., 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, or 1 mol% or less.

[0470] In some cases, the polymer tandem dye comprises a segment represented by formula (IIa):

[0471]

[0472] in:

[0473] The polymer backbone containing non-conjugated repeating units comprises SM 1 , SM 2 and SM 3 comonomers, each of which is independently a non-conjugated comonomer;

[0474] Each D 1 Both are independently 1 an attached side-group donor chromophore;

[0475] Each A 1 Both are independently 2 the attached acceptor fluorophore;

[0476] Each Z 2 Both are related to SM 3 attached optional side chain groups;

[0477] x is 50 mol% or more; and

[0478] y+z is 50 mol% or less.

[0479] Z 2 It may be absent or may be any suitable side chain group, for example, a light absorbing chromophore, a chemical selective tag, a linker, a linked biomolecule, an acceptor fluorophore, WSG, etc. In certain cases of Formula (IIa), SM 3 is a spacer comonomer, wherein Z 2 In certain cases of Formula (IIa), SM 3 Yes, including Z 2 A comonomer of a group (i.e., a second side-group light-absorbing chromophore), wherein each D 1 and each Z 2Together they form a light-harvesting multichromophore system. In some cases, SM 3 is a second chemoselective tag (Z 2 ) comonomers, for example, protected functional groups or with Z 1 Orthogonal tags that can be selectively placed into the intended part.

[0480] In some cases of formula (IIa), x is 60mol% or more, for example, 65mol% or more, 70mol% or more, 75mol% or more, 80mol% or more, 85mol% or more, 90mol% or more, 95mol% or more, or even higher. In some cases of formula (IIa), y+z is 40mol% or less, for example, 30mol% or less, 25mol% or less, 20mol% or less, 15mol% or less, 10mol% or less, 5mol% or less, or even lower. In some cases of formula (IIa), y is at least 1mol% and no more than 25mol%, for example, 20mol% or less, 15mol% or less, 10mol% or less, 5mol% or less, or even lower. In some cases of formula (IIa), z is at least 1mol% and no more than 10mol%, for example, 5mol% or less, or even lower.

[0481] In some cases, the polymer tandem dye comprises a segment represented by formula (IIIa):

[0482]

[0483] in:

[0484] The polymer backbone containing non-conjugated repeating units comprises SM 1 and SM 2 comonomers, each of which is independently a non-conjugated comonomer;

[0485] Each D 1 Both are independently 1 an attached side-group donor chromophore;

[0486] Each A 1 Both are independently 2 the attached acceptor fluorophore;

[0487] x is 75 mol% or more; and

[0488] y is 25 mol% or less.

[0489] In certain embodiments of Formula (IIIa), SM 1 and SM 2Each 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 is independently a partially saturated non-conjugated comonomer, for example, a comonomer that provides isolated double C=C covalent bonds in a saturated covalent bond-containing backbone. The first and second repeating units (SM 1 and SM 2 ) can be arranged in a random configuration, a block configuration, a co-block configuration, or in a specific sequence. In certain cases of formula (IIIa), SM 1 D 1 The side groups include two or more (eg, two or three) different types of side group light absorbing chromophores, which together constitute a light-harvesting multichromophore system. In certain cases of formula (IIIa), D of the first repeating unit 1 The side groups are all identical.

[0490] In some cases of formula (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 formula (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, or 1 mol% or less.

[0491] In some cases, the polymer tandem dye has a structural formula as shown in formula (IVa):

[0492]

[0493] in:

[0494] Each D 1 are all independently side-donating chromophores;

[0495] Each A 1 All are independently acceptor fluorophores;

[0496] Each L 1 and L 2 are independently linkers;

[0497] p1 and q1 are independently 0 or 1, where p1+q1≤1;

[0498] p2 and q2 are independently 0 or 1, where p1+q1≤1;

[0499] x is 75 mol% or more;

[0500] y is 25 mol% or less; and

[0501] G 1 and G 2 Each is independently selected from a terminal group, a polymer segment, a light absorbing (eg, donor) chromophore, an acceptor fluorophore, a linker, and a linked specific binding member.

[0502] 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 has a structural formula as shown in formula (Va):

[0503]

[0504] in:

[0505] Each D 1 are all independently side-donating chromophores;

[0506] Each A 1 All are independently acceptor fluorophores;

[0507] L 1 and L 2 each independently a linker;

[0508] x is 75 mol% or more;

[0509] y is 25 mol% or less; and

[0510] G 1 and G 2 Each is independently selected from an end group, a polymer segment, a light absorbing (e.g., donor) chromophore, a linker, and a linked specific binding member. It should be understood that the multichromophore represented by formula (Va) includes any suitable comonomer arranged in a defined linear sequence, which has x and y totaling to a defined mol% ratio. In some cases, A 1 The comonomers of are spaced throughout the polymer backbone sequence and are therefore always flanked on both sides by one or more comonomers containing D1.

[0511] In certain instances of Formula (Va), the polymeric tandem dye comprises a segment as shown in Formula (VIa):

[0512]

[0513] in:

[0514] Each D 1 are all independently side-donating chromophores;

[0515] Each A 1 Each is independently an acceptor chromophore;

[0516] Each L 1 and L 2 are independently linkers;

[0517] n and p are each independently an integer between 1 and 20, wherein n+p≥2; and

[0518] m is 1 or 2.

[0519] In some instances of Formula (VIa), n and p are each independently 1 to 10, e.g., 2 to 20, 3 to 10, or 3 to 6. In some instances of Formula (VIa), n+p is an integer between 2 to 20, e.g., 3 to 20, 4 to 20, 5 to 20, 5 to 15, or 5 to 12. In certain embodiments of Formula (VIa), m is 1.

[0520] The target polymer tandem dye may include multiple segments as shown in formula (VIa), wherein each segment includes a separate A 1 The comonomer is flanked by D 1 In some cases, the multichromophore comprises two or more segments of formula (VIa) oriented adjacent to each other to provide two separate segments containing A 1 The comonomer contains 2-20 D 1 Blocks of comonomers (e.g., containing 3 to 20, 4 to 20, 5 to 20, 5 to 15, or 5 to 12 D 1 Therefore, in certain embodiments, the polymer tandem dye comprises a q segment of a block copolymer, and its structural formula is shown in Formula (VIIa):

[0521]

[0522] Where: Each (n) q and each (p) q are independently integers between 1 and 20, wherein for each q segment, (n) q +(p) q ≥3; and q is an integer between 1 and 100.

[0523] In certain embodiments, the polymer tandem dye has a structural formula as shown in Formula (VIIIa):

[0524]

[0525] in

[0526] Each D 1 are all independently side-donating chromophores;

[0527] Each A 1 Each is independently an acceptor chromophore;

[0528] Each L 1 and L 2 are all independently linkers;

[0529] x is 75 mol% or more;

[0530] y is 25 mol% or less; and

[0531] G 1 and G 2 Each is independently selected from the group consisting of an end group, a polymer segment, a donor chromophore, a linker, and a linked specific binding member.

[0532] In certain embodiments, the polymer tandem dye has a structural formula as shown in Formula (IXa):

[0533]

[0534] in:

[0535] Each D 1 Each is independently a pendant BODIPY donor chromophore;

[0536] Each A 1 All are independently acceptor fluorophores;

[0537] Each L 1 and L 2 are all independently linkers;

[0538] x is 75 mol% or more;

[0539] y is 25 mol% or less; and

[0540] G 1 and G 2 Each is independently selected from the group consisting of: an end group, a polymer segment, a donor chromophore, an acceptor fluorophore, a linker, and an attached specific binding member.

[0541] In some cases of Formula (IVa), (Va), (XIIIa) and (IXa), x is 80 mol% or more, e.g., 85 mol% or more, 90 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more or 99 mol% or more. In some cases of Formula (IV), (V), (XIII) and (IX), y is 20 mol% or less, e.g., 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, 1 mol% or less.

[0542] In certain embodiments, the polymer tandem dye has a structural formula as shown in Formula (Xa):

[0543]

[0544] in:

[0545] Each D 1 Each is independently a side-group donor chromophore;

[0546] Each A 1 All are independently acceptor fluorophores;

[0547] Each L 1 , L 2 and L 3 are all independently linkers;

[0548] a, b and c are the mol% values ​​of each comonomer;

[0549] d represents the total polymerization or average length of the polymer (e.g., d is 2-1000, such as 2-500, 2-200, 2-100 or 2-50);

[0550] WSG is a water solubilizing group (e.g., as described herein); and

[0551] G 1 and G 2 Each is independently selected from the group consisting of: an end group, a polymer segment, a donor chromophore, an acceptor fluorophore, a linker, and an attached specific binding member.

[0552] In some cases of formula (Xa), c = 0. In some cases of formula (Xa), a>0 and b>0. In some cases of formula (Xa), a is 80mol% or more, for example, 85mol% or more, 90mol% or more, 95mol% or more, 96mol% or more, 97mol% or more, 98mol% or more or 99mol% or more. In some cases of formula (Xa), b is 20mol% or less, for example, 15mol% or less, 10mol% or less, 5mol% or less, 4mol% or less, 3mol% or less, 2mol% or less, 1mol% or less. In some cases of formula (Xa), a is 65-95mol%, b is 5-35mol%, c is 0-30mol%, wherein a+b+c=100%.

[0553] In certain cases of Formula (Xa), each D 1 are all BODIPY groups that have been attached (e.g., as described herein). In some cases, each acceptor dye A 1 Each is selected from DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY800, 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 cases of Formula (Xa), L 1 -D 1 Select from the following:

[0554]

[0555] Where R 4 is H, lower alkyl, substituted lower alkyl and WSG.

[0556] Furthermore, in certain instances of formula (Xa), the donor and acceptor dye pairs D 1 and A 1Can be selected from the following: Dyomics dyes (DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY701, 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 water-solubilizing group as described in any of the Examples, the structure of which is as described herein.

[0557] In some cases, the polymer tandem dye is derived from a multichromophore of formula (XXI), wherein SM 2 Chemoselective labeling of Z 1 Conjugated with an acceptor fluorophore. Therefore, the exemplary polymer tandem dye structure is detailed in Example 3 of the experimental section, and its structure is as follows:

[0558]

[0559] in:

[0560] G 1 is a terminal group (e.g., as described herein);

[0561] L 1 and L 2 independently a linker;

[0562] D 1 is a pendant chromophore (e.g., as described herein);

[0563] A 1 is the acceptor fluorophore;

[0564] Each of d, e, f, g and h is independently 1-6;

[0565] Each n and m is independently 1-1000 (e.g., 2-1000, 2-500, 2-100 or 2-50);

[0566] Each R 41 are selected from alkyl, substituted alkyl and WSG; and

[0567] A "linker" is a linker that includes an optional chemoselective functional group (e.g., a functional group for conjugation to a comonomer or biomolecule). 1 are BODIPY chromophores (e.g., as described herein). In some cases, the structure of a polymeric tandem dye derived from formula (XXI) is as shown in the following general structure:

[0568]

[0569] wherein each n and m is independently 1-1000 (eg, 2-1000, 2-500, 2-100 or 2-50). In some cases, the donor chromophore is a BODIPY chromophore (e.g., as described herein) and the acceptor dye is selected from DY 431, DY485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY731, 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.

[0570] Labeled SBM

[0571] Aspects of the present invention include labeled specific binding members. Labeled specific binding members are conjugates composed of target polydye (e.g., as described herein) and specific binding members. Any polydye or polymer tandem dye described herein can be conjugated with specific binding members. The specific binding members and the polydye can be conjugated to each other (e.g., covalently linked) at any suitable position of the two molecules through an optional linker. In certain embodiments, the labeled specific binding members have anti-polymerization. "Anti-polymerization" used herein refers to a labeled specific binding member with a concentration of 1 mg / ml or more in an aqueous buffer of interest, which can form a homogenous aqueous composition, and no polymeric precipitate, for example, 2 mg / ml or more, 3 mg / ml or more, 4 mg / ml or more, 5 mg / ml or more, 6 mg / ml or more, 7 mg / ml or more, 8 mg / ml or more, 9 mg / ml or more, 10 mg / ml or more, or even higher concentrations of labeled specific binding members.

[0572] In certain embodiments, the labeled specific binding member comprises: a water-solvable polymeric dye having a deep ultraviolet excitation spectrum and comprising a segment of a π-conjugated comonomer and a conjugated modified repeating unit; and a specific binding member covalently linked to the multichromophore.

[0573] As used herein, the term "specific binding member" refers to one member of a pair of molecules that have binding specificity for each other. One member of the pair of molecules may have an area on its surface or in a cavity that specifically binds to a corresponding area on the surface or in a cavity of the other member of the pair of molecules. Thus, the members of the pair of molecules have the property of specifically binding to each other to produce a binding complex. In some embodiments, the affinity between the specific binding members in the binding complex is characterized by a K d (dissociation constant) is 10 -6 M or less, e.g. 10 -7 M or below, including 10 -8 M or less, e.g. 10 -9 M or below, 10 -10 M or below, 10 -11 M or below, 10 -12 M or below, 10 -13 M or below, 10 -14 M or below, including 10 - 15 M or less. In some embodiments, the specific binding member specifically binds with high affinity. High affinity refers to a binding member that specifically binds with an apparent affinity characterized by an apparent K d is 10×10 -9 M or less, e.g. 1×10 -9 M or below, 3×10 -10 M or less, 1×10 -10 M or below, 3×10 -11 M or less, 1×10 -11 M or below, 3×10 -12 M or less or 1×10 -12 M or below.

[0574] The specific binding member can be a protein portion. The term "protein" as used herein refers to a portion consisting of amino acid residues. The protein portion can be a polypeptide. In some cases, the protein-specific binding member is an antibody. In certain embodiments, the protein-specific binding member is an antibody fragment, for example, a binding fragment of an antibody that specifically binds to a polymeric dye. The terms "antibody" and "antibody molecule" used herein are interchangeable and refer to a protein consisting of one or more polypeptides that are substantially encoded by all or part of a recognized immunoglobulin gene. The recognized immunoglobulin genes (e.g., the corresponding genes in the human body) include κ (k), λ (I) and heavy chain genetic loci, which together constitute countless variable region genes, as well as constant region genes μ (u), δ (d), γ (g), σ (e) and α (a) encoding IgM, IgD, IgG, IgE and IgA isotypes, respectively. The immunoglobulin light chain or heavy chain variable region is composed of a "framework" region (FR) interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). The extent of the framework region and CDR has been precisely defined (for details, see "Protein Sequences of Immunological Significance", E. Kabat et al., U.S. Department of Health and Human Services, (1991)). The numbering of all antibody amino acid sequences discussed herein conforms to the Kabat system. The sequences of different light or heavy chain framework regions are relatively conserved within the same species. The framework region of an antibody (i.e., the combined framework region of the constituent light and heavy chains) is used to position and adjust the CDR. The CDR is primarily responsible for binding to the antigenic epitope. The term "antibody" is intended to include full-length antibodies, and may refer to natural antibodies, engineered antibodies, or recombinantly produced antibodies from any organism used for experiments, treatments, or other purposes further defined below.

[0575] The antibody fragment of interest includes, but is not limited to, Fab, Fab', F(ab')2, Fv, scFv or other antigen-binding subsequences of an antibody, which are produced by modifying a whole antibody or synthesizing de novo using recombinant DNA technology. The antibody may be a monoclonal antibody or a polyclonal antibody, and may have other specific activities against cells (e.g., antagonists, agonists, neutralizing antibodies, inhibitory antibodies or stimulating antibodies). It should be understood that antibodies may undergo other conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions.

[0576] In certain embodiments, the specific binding member is a Fab fragment, a F(ab')2 fragment, a 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 a binding fragment thereof. In some cases, the specific binding member is a recombinant antibody or a binding fragment thereof.

[0577] In some embodiments, the labeled specific binding member comprises: a water-solvated light-harvesting multichromophore (e.g., as described herein); a signaling chromophore covalently linked to the multichromophore in proximity upon energy reception (e.g., as described herein); and a specific binding member covalently linked to the multichromophore. In some cases of the labeled specific binding member, the multichromophore of any formula described herein, wherein: G 1 and G 2 Each is independently selected from the group consisting of a terminal group (eg, a terminal group), a linker, and a linked specific binding member, wherein G 1 and G 2 At least one of the two is a linked specific binding member.

[0578] In certain embodiments of the formulae described herein, G 1 and / or G 2 is a linker, for example, a linker comprising a functional group suitable for conjugation with a specific binding moiety. It should be understood that the polychromophore G may be selected to be located 1 and / or G 2 The linker is positioned so that it can be combined with any other linker (which may be present on a multi-chromophore side chain, for example, at Z) including a chemoselective tag (e.g., as described herein) 2 In some embodiments, at G 1 and / or G 2 Introduce an amino functional group or its derivative at Z 2 In certain embodiments, a carboxylic acid functional group or a derivative thereof is introduced at G 1 and / or G 2 A carboxylic acid functional group or its derivative is introduced at Z 2 An amino functional group or its derivative is introduced.

[0579] In some embodiments of the formulae described herein, G 1 and G 2 At least one of the following is -L 3 -Z 4 , where L 3 is a linker (eg, as described herein), Z 4 is a specific binding member (e.g., as described herein). In some embodiments of the formulae described herein, G 1 and G 2 At least one of the following is -L 3 -Z 3 , where L 3 is a linker (eg, as described herein), Z 3is a chemoselective tag (e.g., as described herein). Any suitable chemoselective tag and conjugation chemistry can be used in the target multichromophore. Chemoselective tags of interest include, but are not limited to, amines, activated esters, maleimides, thiols, hexavalent sulfur-fluoride exchange chemistry (SuFEX), fluorinated sulfonyls, Diers Alder cycloaddition click reagents and click chemistry, tetrazines, trans-cyclooctenes, aldehydes, alkoxyamines, alkynes, cyclooctynes, azides, and the like. In some cases, Z 3 Selected from the group consisting of carboxylic acid, activated ester (eg, N-hydroxysuccinimide ester (NHS) or sulfo-NHS), amino, maleimide, iodoacetyl, and thiol.

[0580] The biomolecules of interest include, but are not limited to, polypeptides, polynucleotides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs thereof, and combinations thereof. 4 is an antibody. In some cases, Z 4 is an antibody fragment or a binding derivative thereof. In some cases, the antibody fragment or a binding derivative thereof is selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a scFv, a diabody, and a triabody.

[0581] How to use

[0582] As described above, aspects of the invention include methods for assessing the presence of a target analyte in a sample. Aspects of the method include contacting the sample with a polymeric dye conjugate that specifically binds to the target analyte to produce a labeled composition contacting the sample. The terms "polymeric dye conjugate" and "labeled specific binding member" as used herein are used interchangeably. Thus, the polymeric dye conjugate may include: (i) a water-solvated polymeric dye (e.g., as described herein); and (ii) a specific binding member (e.g., as described herein). In some cases, the polymeric dye conjugate further comprises a signaling chromophore covalently linked to a multichromophore of the polymeric dye that is in proximity to energy reception.

[0583] Any suitable method can be used to contact the sample with a polymeric dye conjugate that specifically binds to the target analyte to produce a labeled composition contact sample. In some cases, under the condition that the specific binding member is specifically bound to the target analyte (if any), the sample is contacted with the polymeric dye conjugate. In order to make the specific binding member of the conjugate specifically bind to the target analyte, an appropriate solution can be used to maintain the biological activity of the sample components and the specific binding member. The solution can be a balanced salt solution, for example, normal saline, PBS, Hank's balanced salt solution, etc., wherein fetal bovine serum, human platelet lysate or other factors and low concentrations (for example, 5-25mM) of acceptable buffer are appropriately supplemented. Suitable buffers include HEPES, phosphate buffer, lactate buffer, etc. Various culture media can be purchased on the market, and can be used according to the properties of the target analyte, including dMEM, HBSS, dPBS, RPMI, Iscove culture media, etc., and in some cases, it is necessary to supplement fetal bovine serum or human platelet lysate. The final component of the solution can be selected according to the sample components included.

[0584] The temperature at which the specific binding member of the conjugate specifically binds to the target analyte may vary, and in some cases, the temperature range may be 5°C to 50°C, such as 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, such as 20°C, 25°C, 30°C, 35°C or 37°C (e.g., as described above). In some 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 some cases, the temperature is 25°C, 30°C, 35°C or 37°C. In some cases, the specific binding member is an antibody or fragment thereof, and the temperature at which specific binding occurs is room temperature (e.g., 25°C), 30°C, 35°C or 37°C. Any suitable incubation time suitable for specific binding may be selected so as to obtain the desired amount of binding complex, and in some cases, the time may be 1 minute (min) or more, such as 2 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, or even 6 hours or more.

[0585] Any suitable specific binding member can be used in conjugate.Purpose specific binding members include but are not limited to reagents specifically bound to cell surface proteins of various cell types, and the cell types include but are not limited to stem cells (e.g., pluripotent stem cells, hematopoietic stem cells), T cells, regulatory T 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 erythroid cells.Purpose target cells include cells with suitable cell surface markers or antigens, which can be captured by suitable specific binding member conjugates. In certain embodiments, the target cell is selected from cells containing HIV, Treg cells, antigen-specific T cell colonies, tumor cells or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow or umbilical cord blood.In the target method, any suitable cell surface protein or cell marker can be targeted to specifically bind to a polymeric dye conjugate. In certain embodiments, the target cell includes a cell surface marker selected from a cell receptor and a cell surface antigen. 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-terminal), SSEA-3, TRA-1-60 antigen, disialylganglioside GD2, and CD71.

[0586] Any suitable target can be selected for evaluation using the target method. The target of interest includes, but is not limited to, nucleic acids, such as RNA, DNA, PNA, CNA, HNA, LNA or ANA molecules; proteins, such as fusion proteins, modified proteins (e.g., phosphorylated proteins, glycosylated proteins, ubiquitinated proteins, SUMOylated proteins or acetylated proteins); or antibodies, peptides, polymeric biomolecules, cells, small molecules, vitamins and drug molecules. The term "target protein" as used herein refers to all members of the target family and fragments thereof. The target protein can be any target protein, such as a therapeutic or diagnostic target, including but not limited to: hormones, growth factors, receptors, enzymes, cytokines, osteogenic induction factors, colony stimulating factors and immunoglobulins. The term "target protein" is intended to include recombinant and synthetic molecules, which can be prepared or commercially available using any suitable recombinant expression method or any suitable synthesis method. In some embodiments, the polymeric dye conjugate includes an antibody or an antibody fragment. In the target method, any suitable target analyte that specifically binds to the target antibody or antibody fragment can be targeted.

[0587] In some embodiments, the target analyte is associated with a cell. In some cases, the target analyte is a cell surface marker of a cell. In some cases, the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen. In some cases, the target analyte is an intracellular target, and the method further comprises lysing the cell.

[0588] In some embodiments, the sample may include a heterogeneous cell population from which target cells are separated. In some cases, the sample includes peripheral whole blood, peripheral whole blood, cord blood, bone marrow, density gradient purified peripheral blood mononuclear cells or homogenized tissues in which red blood cells have been cracked before cell separation. In some cases, the sample includes hematopoietic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow or cord blood. In certain embodiments, the sample includes tumor cells in peripheral blood. In some cases, the sample is a sample including (or suspected to include) viral cells (e.g., HIV).

[0589] The labeled specific binding members can be used in the method of the label, for example, with polymeric dyes or polymer tandem dyes to label target cells, particles, targets or analytes. For example, the labeled specific binding members can be used to label cells to be processed (for example, detected, analyzed and / or sorted) in a flow cytometer. The labeled specific binding members can include antibodies specifically bound to cell surface proteins such as various cell types (for example, as described herein). The labeled specific binding members can be used to study various biological (for example, cell) characteristics or processes, for example, cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression and / or presentation, etc. The labeled specific binding members can be used in any application including (or possibly including) antibody-mediated labeling of cells, particles or analytes.

[0590] In some cases of the methods, the labeled specific binding member comprises a multichromophore as described herein (e.g., a multichromophore according to any one of Formulas (I)-(IX)). 1 and G 2 Each is independently selected from the group consisting of an end group, a polymer segment, a linker, a chemoselective tag, and a linked specific binding member, wherein G 1 and G 2 At least one of the two is a linked specific binding member.

[0591] Aspects of the method include determining whether there is a polymeric dye conjugate-target analyte binding complex in the sample contacted with the labeling composition to assess whether there is a target analyte in the sample. Once the sample is contacted with the polymeric dye conjugate, any suitable method can be used to determine whether there is a polymeric dye conjugate-target analyte binding complex in the sample contacted with the labeling composition. The polymeric dye conjugate-target analyte binding complex is a binding complex produced when the specific binding member of the conjugate specifically binds to the target analyte (if any). Determining whether the labeling composition contacts the sample may include detecting a fluorescent signal (if any) from the binding complex. In some cases, the determination includes a separation step in which the target analyte (if any) is separated from the sample. Various methods can be used to separate the target analyte from the sample, for example, by fixing it on a support. The purpose of the determination method includes, but is not limited to, any suitable method and determination format, which involves a specific binding member pair, for example, avidin-biotin or hapten-anti-hapten antibody can be used. Methods and assay formats of interest suitable for use in conjunction with the subject compositions include, but are not limited to, flow cytometry methods, in situ hybridization methods, enzyme-linked immunosorbent assays (ELISA), Western blot analysis, magnetic cell sorting assays, and fluorescent dye purification chromatography analysis.

[0592] In certain embodiments, the method further includes contacting the sample with a second specific binding member that specifically binds to the target analyte. In some cases, the second specific binding member has been bound to a support. Any suitable support can be used to fix the component (e.g., the second specific binding member) described in the method of the target. In some cases, the support is a particle, e.g., magnetic powder. In some cases, the second specific binding member and the poly-dye conjugate produce a sandwich complex, and the complex can be separated and detected (if any) by any suitable method. In certain embodiments, the method further includes analyzing the poly-dye conjugate-target analyte binding complex by flow cytometry, i.e., a fluorescently labeled target analyte. The determination of the presence of the poly-dye conjugate-target analyte binding complex can provide a corresponding determination result (e.g., qualitative or quantitative determination data), which can be used to assess whether there is a target analyte in the sample.

[0593] Any suitable support can be used in the target method to fix any suitable component shown in the method, for example, a labeled specific binding member, a target, a second specific binding member, etc. The target support includes, but is not limited to: a solid matrix, wherein the matrix can have a variety of configurations, for example, a sheet, a microsphere or other structure, such as a plate with holes; microspheres, polymers, particles, fiber grids, hydrogels, porous matrices, needles, microarray surfaces, chromatographic support, etc. In some cases, the support is selected from the group consisting of particles, planar solid matrices, fiber grids, hydrogels, porous matrices, needles, microarray surfaces, and chromatographic support. The support can be introduced into a system, and the system can be separated by cells with the assistance of any suitable method, for example, a manually operated syringe, a centrifuge, or an automated liquid handling system. In some cases, the support can be used in an automated liquid handling system (e.g., a flow cytometer) to achieve high-throughput separation of cells.

[0594] In some embodiments of the method, the separation step includes applying an external magnetic field to fix the magnetic powder. Any suitable magnet can be used as the external magnetic field source (e.g., a magnetic field gradient). In some cases, the external magnetic field is generated by a magnetic source, for example, by a permanent magnet or an electromagnet. In some cases, fixing the magnetic powder means that the magnetic powder is accumulated near the surface closest to the magnetic field gradient source (i.e., the magnet).

[0595] The separation may further include one or more optional washing steps to remove unbound material from the support. Any suitable washing method may be used, for example, washing the fixed support with a biocompatible buffer that can retain the specific binding interaction between the polymeric dye and the specific binding member. The unbound material of the sample is separated and optionally washed from the support to provide an enriched target cell population, wherein undesirable cells and materials may be removed.

[0596] In certain embodiments, the method further comprises detecting a labeled target. Detecting a labeled target may include exciting a multichromophore with one or more lasers, followed by detecting fluorescence emission from a polymer tandem dye with one or more optical detectors. The labeled target may be detected using any suitable instrument and method, including but not limited to flow cytometry, a FACS system, fluorescence microscopy; fluorescence, cold light, ultraviolet light and / or visible light detection using a microplate reader; high performance liquid chromatography (HPLC); and mass spectrometry. When fluorescently labeled components are used in the methods and compositions described herein, it is generally believed that different types of fluorescence detection systems can be used to practice the target method. In some cases, high throughput screening may be performed, for example, using a system of microtiter plates with 96 or more wells. Fluorescent substances can be measured using a variety of methods, for example, those described in the following publications: 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, eds., Taylor, DL and Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, NJ, Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Col, Inc. (1978), pp. 296-361.

[0597] 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 an excitation optical device. The excitation optical device causes the excitation radiation to excite the sample. In response, the wavelength of radiation emitted by the fluorescently labeled target in the sample is different from the excitation wavelength. Then, the collection optical device collects the emission from the sample. Related equipment may include a temperature controller to keep the sample at a specific temperature while scanning the sample. In some cases, a multi-axis translation stage moves a microtiter plate containing multiple samples in order to locate different wells to be exposed. The multi-axis translation stage, temperature controller, autofocus function, and electronic equipment associated with imaging and data acquisition can all be managed by a properly programmed digital computer. The computer can also convert the data collected during the determination process into another format for display.

[0598] In some embodiments, the method of assessing the presence of a target analyte in a sample further comprises detecting fluorescence in a flow cytometer. In some embodiments, the method of assessing the presence of a target analyte in a sample further comprises imaging the label composition contact sample by fluorescence microscopy. Fluorescence microscopy imaging can be used to identify the poly-dye conjugate-target analyte binding complex in the contact sample to assess the presence of the target analyte. The target microscopy method used in the target method comprises laser scanning confocal microscopy.

[0599] The present invention also provides a method for labeling a target molecule. The target polydyes can be used in various labeling, separation, detection and / or analysis methods. In some embodiments, the method comprises: contacting the target molecule with a polydye (e.g., as described herein) to produce a labeled target molecule, wherein the polydye comprises a conjugated tag covalently linked to the target molecule. In some cases, the polydye further comprises a signal transduction chromophore covalently linked to a multichromophore of the polydye that is close to the energy reception. In some cases of the method, the polydye member comprises a multichromophore according to any one of formulas (I)-(IX) (e.g., as described herein), wherein G 1 and G 2 One of them is a terminal group, and G 1 and G 2 The other term in is the conjugate label.

[0600] The term "conjugated tag" as used herein refers to a group comprising a chemoselective functional group (e.g., as described herein) that can be covalently linked to a compatible functional group of a target molecule after optional activation and / or deprotection. Any suitable conjugated tag can be used in the labeled polydye to conjugate the dye to the target molecule of interest. In some embodiments, the conjugated tag includes a terminal functional group selected from the following: amino, carboxylic acid or derivatives thereof, thiol, hydroxyl, hydrazine, hydrazide, azide, alkyne, and protein reactive groups (e.g., amino reactive groups, thiol reactive groups, hydroxy reactive groups, imidazole reactive groups, or guanidinium reactive groups).

[0601] Any suitable methods and reagents are suitable for use in the target labeling method to covalently link the conjugated tag to the target molecule. The purpose method for labeling the target includes, but is not limited to, the methods and reagents described in the following publications: Hermanson, Bioconjugation Technology, Third Edition, Academic Press, 2013. The contacting step can be performed in an aqueous solution. In some cases, the conjugated tag includes an amino functional group and the target molecule includes an activated ester functional group, for example, an NHS ester or a sulfo-NHS ester, or vice versa. In some cases, the conjugated tag includes a maleimide functional group and the target molecule includes a thiol functional group, or vice versa. In some cases, the conjugated tag includes an alkyne (e.g., cyclooctynyl) functional group and the target molecule includes an azide functional group, or vice versa, which can be conjugated by a click chemistry reaction.

[0602] Any suitable target molecule can be selected for labeling using a labeling method. Target molecules include, but are not limited to, nucleic acids, for example, RNA, DNA, PNA, CNA, HNA, LNA or ANA molecules; proteins, for example, fusion proteins, modified proteins (e.g., phosphorylated proteins, glycosylated proteins, ubiquitinated proteins, SUMOylated proteins or acetylated proteins); or antibodies, peptides, polymeric biomolecules, cells, small molecules, vitamins and drug molecules. The term "target protein" used herein refers to all members of the target family and their fragments. The target protein can be any target protein, for example, a therapeutic or diagnostic target, including but not limited to: hormones, growth factors, receptors, enzymes, cytokines, osteogenic induction factors, colony stimulating factors and immunoglobulins. The term "target protein" is intended to include recombinant and synthetic molecules, which can be prepared or purchased using any suitable recombinant expression method or any suitable synthesis method. In some embodiments, the target molecule is a specific binding member (e.g., as described herein). In some cases, the specific binding member is an antibody. In some cases, the specific binding member is an antibody fragment or its binding derivative. In some cases, the antibody fragment or binding derivative thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a diabody, and a triabody.

[0603] In some cases, the method includes a separation step in which the labeled target molecule is separated from the reaction mixture, e.g., excess reagents or unlabeled target. Various methods can be used to separate the target in the sample, e.g., by immobilization on a support, precipitation, chromatography, etc.

[0604] In some cases, the method further includes detecting and / or analyzing the labeled target molecule. In some cases, the method further includes detecting the labeled target molecule by fluorescence detection. Any suitable method and target method and composition can be used to detect and / or analyze the labeled target molecule. The method of analyzing the target of interest used in the target method includes but is not limited to flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell sorting assay and fluorescent dye purification chromatography analysis. The target detection method includes but is not limited to fluorescence spectroscopy, fluorescence microscopy, nucleic acid sequencing, fluorescence in situ hybridization (FISH), protein mass spectrometry, flow cytometry, etc.

[0605] Detection can be performed directly by polymeric dyes or polymer tandem dyes, or indirectly by secondary detection systems. The latter can be performed based on any one or a combination of several different principles, including but not limited to antibody-labeled anti-species antibodies and other forms of immune or non-immune bridging and signal amplification systems (e.g., biotin-streptavidin technology, protein-A and protein-G mediated technology or nucleic acid probes / anti-nucleic acid probes, etc.). Suitable reporter molecules can be known reporter molecules in the field of immunocytochemistry, molecular biology, light, fluorescence and electron microscopy, cell immunophenotyping, cell sorting, flow cytometry, cell visualization, detection, enumeration and / or signal output quantification. More than one specific and / or non-specific antibody can be labeled, and the antibody can be used simultaneously or sequentially to enhance the detection, identification and / or analysis of the target.

[0606] Preparation method

[0607] Aspects of the invention include methods for preparing the subject multichromophores and polymer tandem dyes. An advantage of the subject multichromophores is the modularity of the underlying scaffold. In some cases, the modular scaffold is a linear polymer of repeating units with a defined sequence. The side chromophores used can be selected from a variety of dyes, which can be covalently attached to the polymer backbone before or after polymer synthesis.

[0608] In some embodiments, the polymer backbone of the target multichromophore (see, for example, Formula (IX) described herein) can be prepared using cyclic carbonate or protected carbonate monomers. The target methods and comonomers include, but are not limited to, the methods and monomers described in the following publications: Barnes et al., WO2013036532; Cooley et al., Journal of the American Chemical Society, 131, 45, 1640-3, 2009; and Rothbard et al., U.S. Patent No. 7,169,814. In the case of an initiator and a suitable cyclic carbonate monomer feed ratio, the monomer can be added to the polymerization reaction to provide a polymer backbone. In addition, protected carbonate monomers can be assembled in a stepwise synthesis process to provide a defined sequence.

[0609] The target comonomer can be connected using compatible chemoselective functional groups and chemical methods. In some cases, the comonomer is connected by a click chemistry reaction, for example, copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, olefin-azide [3+2] cycloaddition, reverse demand Diers-Alder of olefin and tetrazine, Staudinger connection, etc. (see, for example, Kolb et al., Applied Chemistry International Edition, 40: 2004-2021, 2001). Therefore, the connection of the comonomer can be achieved by conjugation of compatible chemoselective functional group pairs, for example, alkyne / azide, tetrazine / olefin, azide / olefin, phosphine / azide, etc. Non-limiting examples of azide-alkyne cycloaddition reactions include copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions and ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) reactions. CuAAC is suitable for a wide temperature range, is insensitive to aqueous environments, has a pH range of 4 to 12, and tolerates 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 using sodium ascorbate as a reducing agent, such as Cu(I) salts or Cu(II) salts. The reaction can produce 1,4-substituted products. RuAAC utilizes pentamethylcyclopentadienylruthenium chloride [Cp*RuCl] complexes, which are able to catalyze the cycloaddition of azides with terminal alkynes to regioselectively produce 1,5-disubstituted 1,2,3-triazoles (see Rasmussen et al., Comm. Org. Chem., 9:5337-5339, 2007). In addition, in contrast to CuAAC, RuAAC can also be used in combination with internal alkynes to provide fully substituted 1,2,3-triazoles.

[0610] In some embodiments, the polymer backbone of the target multichromophore is a polypeptide. Any suitable peptide synthesis method can be used to prepare the polymer backbone of the target multichromophore. In some cases, the solid phase peptide synthesis (SPPS) method is used to prepare the polymer backbone of the target multichromophore by a defined stepwise synthesis. Conventional protecting group strategies deprotect and couple the target amino acid residues according to the defined sequence, while orthogonally protecting the target side chain functional groups. In some cases, the Fmoc / tert-butyl method is used. In some cases, the Boc / benzyl method is used. The protecting group strategy can also be applied to the insertion of target side groups on the side chains of specific amino acid residues and / or at the ends of the polypeptide. In some cases, one or more side groups can be inserted into the protected amino acid monomer starting material. In some cases, after completing the SPPS of the polymer backbone, the side groups are inserted into the multichromophore. Using orthogonal protecting groups, different target groups can be independently attached to the polypeptide backbone, for example, attached to the N- and C-termini and / or different types of amino acid residues. The polypeptide may include β2-amino acid residues, β3-amino acid residues, α-amino acid residues, or a mixture thereof.

[0611] In some embodiments, a method for preparing a light-harvesting multichromophore (e.g., as described herein) comprises synthesizing a protected polypeptide having a defined amino acid sequence consisting of blocks of first amino acid residues separated by a single occurrence of a second amino acid residue. As described herein, the preparation of a defined sequence of amino acid residues can provide the desired side group donor chromophore and acceptor fluorophore configuration along the polypeptide. When the side groups are inserted after polypeptide synthesis, the chemoselective functional groups of the amino acid side chains along the polypeptide backbone are selectively conjugated to the side groups. In some cases, the light-harvesting multichromophore is a polypeptide having a defined sequence, wherein: each block of first amino acid residues comprises at least two residues; each of the first amino acid residues comprises a protected first chemoselective side chain group; and each of the second amino acid residues comprises a protected second chemoselective side chain group.

[0612] In addition, the pendant donor chromophore can be introduced using protected amino acid building blocks that already include the chromophore as part of the side chain. It should be understood that at least the pendant chromophore can be first attached to the side chain of a defined polypeptide sequence to produce a light-harvesting multi-chromophore. Various other pendant groups, including acceptor fluorophores, can then be introduced in turn by selective conjugation with chemically selective tags attached to specific residues of the sequence.

[0613] In some cases, the method further comprises coupling the reactive acceptor fluorophore moiety to the deprotected second chemoselective side chain group of the second amino acid residue to generate a side group acceptor fluorophore.

[0614] In certain instances of the methods, the defined amino acid sequence comprises one or more amino acid sequence segments selected from:

[0615]

[0616] wherein each X is a first amino acid residue having a first chemoselective functional group or a protected form thereof, and each Y is a second amino acid residue having a second chemoselective functional group or a protected form thereof.

[0617] In certain embodiments of the above sequences, each X is a lysine or ornithine residue or a protected form thereof, which has a side chain amino group that can be selectively covalently N-linked to a pendant donor chromophore. Each Y is a cysteine ​​residue or a protected form thereof, which has a side chain thiol group that can be selectively covalently linked to a pendant acceptor fluorophore.

[0618] A polypeptide sequence comprising two or more of the above sequence segments can be prepared by optionally being separated by other third amino acid residues (e.g., (segment 1)-Z-(segment 2), wherein Z is a third residue). The 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 selectively placing other moieties of interest, such as a second side group absorbing chromophore, a chemoselective tag (e.g., a bioorthogonal click chemistry tag), a linker, a connected biomolecule, a receptor fluorophore, WSG, etc. In some cases, the other moieties of interest are introduced into the third amino acid residue prior to polypeptide synthesis.

[0619] Similarly, various moieties may be placed at the N- and / or C-terminus of a polypeptide during or after peptide synthesis. In some 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-terminus of the deprotected polypeptide. G1 may be any suitable terminal group (e.g., a capping group (e.g., an alkanoyl group), e.g., an acetyl group), a donor chromophore, a linker with a specific chemically selective tag, or a target biomolecule.

[0620] In some cases, the method further comprises inserting a G2 group at the C-terminus of the polypeptide. This can be achieved in a variety of ways: for example, during SPPS, the C-terminal group G2 is placed between the solid support and the first amino acid residue of the sequence; the polypeptide is cleaved from the solid support; or after synthesis, the target moiety (e.g., a specific binding member) or a specific chemical selective tag directed thereto can be coupled to the C-terminus of the polypeptide. In some cases, native chemical ligation methods can be used to prepare C-terminal polypeptide thioesters suitable for coupling to a target polypeptide fragment or portion.

[0621] Any polypeptide multichromophore described herein can be prepared according to the subject method, for example, the polypeptides represented by formula (IV)-(VII) described herein and their synthetic precursors.

[0622] Some of the various methods that can be used to synthesize the target polypeptide multichromophore are summarized in the following publications: Steward et al., Solid Phase Peptide Synthesis, WH Freeman Co., San Francisco, 1969; Bodanszky et al., Peptide Synthesis, John Wiley & Sons, Second Edition, 1976; Meienhofer, Hormone Proteins and Peptides, Vol. 2, p. 46, Academic Press (New York), 1983; Kent, Annual Review of Biochemistry, 57, 957, 1988, for solid phase peptide synthesis; and Schroder et al., In: Peptides, Vol. 1, Academic Press (New York), 1965, for liquid phase synthesis. Any suitable protecting group strategy can be used, such as, but not limited to, Fmoc solid phase peptide synthesis and Boc solid phase peptide synthesis strategies. In Boc solid phase peptide synthesis, a Boc-amino protecting group is used at the amino terminus, and a benzyl or benzyl-based protecting group or other suitable protecting groups can be used to protect side chain functional groups. In Fmoc solid phase peptide synthesis, an Fmoc-amino protecting group is used at the amino terminus, and tert-butyl or benzyl-based protecting groups or other suitable protecting groups may be used to protect side chain functional groups. Suitable protecting groups used in the synthetic methods are described in the above references and in the following publications: McOmic, Protecting Groups Used in Organic Chemistry, Plenum Press, New York, 1973; and Greene and Wuts, Protecting Groups Used in Organic Synthesis, John Wiley & Sons, 4th edition, 2006.

[0623] system

[0624] Aspects of the present invention further include systems for implementing target methods and composition-related processes. The sample analysis system may include a sample field of view or flow path loaded with a sample and a labeled specific binding member. In some embodiments, the system is a flow cytometry system, comprising: a flow cytometer including a flow path; a composition in the flow path, wherein the composition comprises: 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 microscopy system, comprising: a fluorescence microscope including a sample field of view; a composition disposed in the sample field of view, wherein the composition comprises a sample; and a labeled specific binding member (e.g., as described herein).

[0625] In some cases of the system, the labeled specific binding member comprises: a water-solvated light-harvesting multichromophore (e.g., as described herein) and a specific binding member (which specifically binds to a target analyte covalently linked to the multichromophore). In some cases, the labeled specific binding member further comprises a signaling chromophore covalently linked to the multichromophore of the polymeric dye in proximity upon energy reception. In some cases of the system, the structural formula of the labeled specific binding member and the multichromophore is as shown in any one of Formulas (I)-(IX) (e.g., as described herein), wherein: G 1 and G 2 Each is independently selected from the group consisting of an end group, a polymer conjugated segment, a linker, and a linked specific binding member, wherein G 1 and G 2 At least one of the two is a linked specific binding member.

[0626] In certain embodiments of the system, the composition further comprises a second specific binding member that has been bound to a support and specifically binds to the target analyte. In some cases, the support comprises magnetic particles. Thus, in some cases, the system may further comprise a controllable external paramagnetic field that is configured to be applicable to the assay region of the flow channel.

[0627] The sample may include cells. In some cases, the sample is a biological sample containing cells. In some cases, the sample includes a labeled specific binding member specifically bound to a target cell. In some cases, the target analyte specifically bound to a specific binding member is a cell surface marker of the cell. In some cases, the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0628] In certain aspects, the system may further include a light source configured to direct light to the assay region of the flow channel or the sample field of view. The system may include a detector configured to receive a signal from the assay region of the flow channel or the sample field of view, wherein the signal is provided by the fluorescent composition. Optionally, the sample analysis system may further include one or more additional detectors and / or light sources to detect one or more additional signals.

[0629] In some aspects, the system may further include a computer-based system configured to detect the presence or absence of a fluorescent signal. "Computer-based system" refers to a hardware device, software device, and data storage device for analyzing the information of the present invention. The minimum hardware configuration of the computer-based system of the present invention includes a central processing unit (CPU), an input device, an output device, and a data storage device. It can be easily understood by the technician that any currently available computer-based system is suitable for the target system. The data storage device may include any product, including a record of the information of the present invention as described above, or a memory access device that can access the product.

[0630] To "record" data, programming information or other information on a computer readable medium refers to the process of storing information using any of the methods known in the art. Any suitable data storage structure may be selected based on the means used to access the stored information. Various data processor programs and formats may be used to store, for example, word processing text files, database formats, etc.

[0631] "Processor" means any combination of hardware and / or software that will perform its required functions. For example, any processor described herein may be a programmable digital microprocessor, for example, provided in the form of an electronic controller, a mainframe, a server, or a personal computer (desktop or portable). If the processor is programmable, suitable programming may be transmitted to the processor from a remote location, or pre-stored in a computer program product (e.g., a portable or fixed computer readable storage medium, whether based on a magnetic, optical or solid-state device). For example, a magnetic medium or an optical disc may carry the programming, and may be read by a suitable reader in communication with each processor at its respective base station.

[0632] In addition to sensor devices and signal processing modules (e.g., as described above), the systems described in the present invention may include multiple additional components, such as data output devices (e.g., displays and / or speakers), data input devices (e.g., interfaces, keyboards, etc.), fluid processing components, power supplies, etc.

[0633] In certain aspects, the system comprises a flow cytometer. Suitable flow cytometer systems and methods for analyzing samples include, but are not limited to, those described in the following publications: Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford University Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology 91, Humana Press (1997); Practical Flow Cytometry, 3rd Edition, Wiley-Liss (1995); Virgo et al. (2012), Annals of Clinical Biochemistry, January; 49 (pt 1): 17-28; Linden et al., Seminars in Thrombosis and Hemostasis, October 2004; 30(5): 502-11; Alison et al., Journal of Pathology, December 2010; 222(4): 335-344; and Herbig et al. (2007), Reviews of Therapeutic Drug Delivery Systems, 24(3): 203-255; the contents of these publications are incorporated herein by reference. In some cases, the flow cytometer system of interest includes a BD Biosciences FACSCanto TM Flow cytometer, BD Biosciences FACSVantage TM , BD Biosciences FACSort TM BD Biosciences FACSCount TM BD Biosciences FACScan TM and BD Biosciences FACSCalibur TM System, BD Biosciences Accuri TM System, BD Biosciences FACSCanto TM System, BD Biosciences FACSCelesta TM System, BD Biosciences FACSLyric TM System, BD BiosciencesFACSVerse TM System, BD Biosciences FACSymphony TM System, BD Biosciences LSRFortessa TM System, BD Biosciences Influx TM Cell sorter, BD Biosciences FACSJazz TMCell Sorter and BD Biosciences FACSAria TM Cell sorter, BD Biosciences FACSMelody TM Cell sorter, etc.

[0634] In certain embodiments, the subject system is a flow cytometer system incorporating one or more of the flow cytometer components described in the following U.S. Patent Nos.: 3,960,449; 4,347,935; 4,667,830; 4,704,891; 4,770,992; 5,030,002; 5,040,890; 5,047,321; 5,245,318; 5,317,162; 5,464,581; 5,483,469; 5,602,039; 5,620,842 The contents of these documents are incorporated herein by reference.

[0635] Other systems can be used to implement the method for the target. In some aspects, the system can be a fluorometer or microscope loaded with a sample (with the fluorescent composition described in any embodiment discussed herein). The fluorometer or microscope can include a light source configured to direct light to the assay area or sample field of view of the flow channel. The fluorometer or microscope can also include a detector configured to receive a signal from the assay area or field of view of the flow channel, wherein the signal is provided by the fluorescent composition.

[0636] Reagent test kit

[0637] Aspects of the invention further include kits for carrying out the processes related to the subject methods and compositions. The compositions described herein can be included in a kit as reagents, which can be used as starting materials or provided for use in methods such as those described above.

[0638] The kit can include a polymeric dye (including a water-solvated light-harvesting multichromophore (e.g., as described herein)) and a container. Any suitable container can be used, for example, a tube, a bottle, a hole in a porous strip or plate, a box, a bag, an insulated container, etc. The target kit can further include one or more components selected from the following: a polymer tandem dye, a fluorophore, a specific binding member, a specific binding member conjugate, a support-bound specific binding member, a cell, a support, a biocompatible aqueous elution buffer, and instructions for use. In some embodiments of the kit, the multichromophore is covalently linked to the specific binding member. In some cases, the specific binding member is an antibody. In some cases, the specific binding member is an antibody fragment or a binding derivative thereof. In some cases, the antibody fragment or a binding derivative thereof is selected from the group consisting of the following: a Fab fragment, a F(ab')2 fragment, a scFv, a diabody, and a triabody.

[0639] In certain embodiments, the kit can be used to assess the presence of a target analyte in a sample, e.g., an intracellular target. Thus, in some cases, the kit includes one or more components suitable for lysing cells. One or more additional components of the kit can be provided in a separate container (e.g., a separate tube, bottle, or well in a porous strip or plate).

[0640] In some aspects, the kit further includes a reagent for performing flow cytometry. Purpose reagents include but are not limited to buffer for reconstruction and dilution, buffer for contacting cell samples with multiple chromophores, wash buffer, control cells, control microspheres, fluorescent microspheres for flow cytometer calibration and combinations thereof. The kit kit may also include one or more cell fixation reagents, for example, paraformaldehyde, glutaraldehyde, methanol, acetone, formalin or any combination thereof or buffer. In addition, the kit may include cell permeabilization reagents, for example, methanol, acetone or detergents (for example, Triton, NP-40, saponin, Tween 20, digitonin, leucoperm) or any combination thereof or buffer. Other protein transport inhibitors, cell fixation reagents and cell permeabilization reagents familiar to technicians are all within the scope of the target kit.

[0641] The composition of the kit can be provided in the form of a liquid composition (e.g., any suitable buffer). In addition, the composition of the kit can be provided in the form of a dry composition (e.g., can be lyophilized), and the kit can optionally include one or more buffers for reconstitution of the dry composition. In certain aspects, the kit can include aliquots of the composition provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0642] In addition, one or more components may be mixed and contained in a single container, e.g., a glass or plastic vial, tube or bottle. In some cases, the kit may further include a container (e.g., a box, bag, insulated container, bottle, tube, etc.) containing all of the respective components (and their individual containers). The kit may further include packaging that is separate from or attached to the kit container and has printed thereon information about the kit, the components of the kit, and / or instructions for use of the kit.

[0643] In addition to the above-mentioned components, the target kit may further include instructions for implementing the target method. These instructions may be present in the target kit in various forms, one or more of which may be present in the kit. One form in which these instructions may exist is printed information on a suitable medium or substrate (e.g., a sheet or sheets of paper with information printed thereon), a kit package, a package insert, etc. Another form in which it exists is a computer-readable medium with information recorded thereon, for example, a floppy disk, a CD, a portable flash drive, etc. A form in which these instructions may also exist is a website, whereby information on a remote website can be accessed via the Internet. Any suitable device may be present in the kit.

[0644] utility

[0645] The polymeric dyes, compositions, methods and systems described herein can be used in various applications, including diagnostic and research applications, where it is necessary to label, detect and / or analyze the target of interest. The applications include various methods, such as cell counting, microscopy, immunoassays (e.g., competitive or non-competitive), free analyte evaluation, receptor binding ligand evaluation, etc. The compositions, systems and methods described herein can be used to analyze any of a variety of samples, including but not limited to biological fluids, cell culture samples and tissue samples. In some aspects, the compositions, systems and methods described herein can be used for methods of detecting analytes (if any) in samples using fluorescent markers (e.g., in fluorescence-activated cell sorting or analysis, immunoassays, immunostaining, etc.). In some cases, the compositions and methods can be used in applications involving the presence or absence of target analytes in an assessment sample.

[0646] In some cases, the methods and compositions can be used in any assay format involving detection and / or analysis of a target in a sample, including but not limited to flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell sorting assays, and fluorescent dye purification chromatography. In some cases, the methods and compositions can be used in any application involving fluorescent labeling of target molecules. The target composition is suitable for use in any suitable application in which a specific binding member pair (e.g., biotin-streptavidin and hapten-anti-hapten antibody) can be used.

[0647] Example

[0648] Example 1

[0649] The synthesis of the target polydye can be achieved by Suzuki coupling method. Other methods (eg, CH bond arylation or Stille coupling method) can also be used to construct and polymerize repeating units including aromatic or heteroaromatic comonomers and saturated PEG comonomers.

[0650] The structural formula of the tandem dye based on multichromophore A (MC) is as follows Figure 1 A series of tandem dyes with the following core structures and different acceptor fluorophores ("dyes") were prepared:

[0651]

[0652] The spectral properties of the exemplary polymer tandem dyes were evaluated, and the corresponding spectra are shown in Figure 2 and Figure 3 shown.

[0653] Table 1: Spectral data of pendant multichromophores and corresponding tandem dyes

[0654] Emitter <![CDATA[λmax (em) (nm)]]> D / A Quantum yield (%) Amplification factor MC 523 not applicable 9 not applicable MC-Dye 1 649 0.09 12 5.1 MC-Dye 2 661 0.06 23 2.1 MC-Dye 3 682 0.06 33 1.8 MC-Dye 4 717 0.08 27 2.2 MC-Dye 5 740 0.12 16 2.3 MC-Dye 6 786 0.16 14 2.1

[0655] Example 2

[0656] A series of lysine and cysteine ​​amino acid residues KCKK were prepared by solid phase peptide synthesis. Figure 4 As shown. The carboxylic acid substituted BODIPY group is conjugated to the N-terminus and side chain amino groups of the lysine residue of the peptide via amide bond coupling. The maleimide substituted acceptor fluorophore (dye 7) is conjugated to the cysteine ​​residue via maleimide-thiol coupling chemistry. The peptide is prepared using a C-terminal linker suitable for conjugation to a biomolecule (e.g., protein).

[0657] Describes Figure 4 Spectral properties of BODIPY-based tandem dyes shown. Figure 5A Absorption and emission spectra of the BODIPY donor side groups are shown. Figure 5B The absorption and emission spectra of the acceptor dye (Dye 7) are shown. Figure 5C Shows Figure 4 Absorption and emission spectra of the exemplary polymer tandem dye shown. For the emission spectrum of the chromophore with support, the main chromophore is excited. For the system, the emission of the donor is almost completely quenched, and most of the emission is the emission of the acceptor fluorophore. The quantum yield of the polymer tandem dye is comparable to that of the acceptor fluorophore (dye 7), which shows that the tandem dye exhibits an efficient energy transfer process. The fluorescence of the exemplary tandem dye is amplified by about 2 times compared to the fluorescence of the acceptor fluorophore (dye 7) alone.

[0658] Polypeptides provided with donor to acceptor ratios of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 and 20:2 were prepared and characterized by the methods described above.

[0659] Example 3

[0660] Water-soluble fluorescent polymeric dyes can be prepared by click polymerization under mild conditions. The polymeric dyes can be covalently bonded to biorecognition units (biomolecules) and used as fluorescent probes of targets. When a second dye is introduced as an acceptor, a polymer tandem dye is formed. The color of the acceptor dye can be adjusted to prepare polymers with the same excitation wavelength (see Fig.9A ) but the emission wavelengths vary greatly (see Fig. 9B ) of polymer tandem dyes.

[0661] A PEGylated BODIPY dye with the desired narrow absorption is selected as the fluorophore pendant dye. The pendant fluorophore is chemically bonded to a water-soluble glycol ether monomer (terminated with a diazide, dialkyne, or azide / alkyne). PEG polymer chains with attached BODIPY dyes can be prepared based on the appropriate MW (e.g., as described herein) using the Cu(I)-catalyzed 1,3-dipolar cycloaddition of alkynes and azides. When only one fluorophore dye is attached as a pendant, the resulting polymer fluoresces as a simple fluorescent polydye (see Figure 8 ). When a second acceptor fluorophore is attached, a polymer chain with two fluorophore side groups is obtained. One fluorophore at a higher energy level can be used as an energy donor, while the second fluorophore at a lower energy level can be used as an energy acceptor. Mixing the donor / acceptor pairs in the appropriate ratio can produce polymer tandem dyes with the desired emission wavelength.

[0662] The following polymerizable dyes can be polymerized by click polymerization using Fig. 7A and 7BThe comonomer and method described in the synthesis schematic diagram shown are prepared, wherein it should be understood that n and m together can represent the average length of the polymer and the relative ratio of the comonomer, and n and m can be selected as needed by controlling the polymerization reaction parameters (using any suitable method to achieve). It should be understood that, according to the polymerization method, n and m repeating units can exist in random configuration or block configuration. In addition, the following polymer structure can be further conjugated with any suitable target molecule (e.g., biomolecule) by terminal azide or alkynyl using click chemistry reaction. In some cases, n and m are each independently selected from 1 to 1000, for example, 1 to 500, 1 to 200, 1 to 100, 2 to 100 or 5 to 100.

[0663]

[0664] Figure 8 The absorption and emission spectra of a basic polydye including a side-group BODIPY dye having the general structure described above are shown. Other exemplary polydyes (where R is a donor dye (e.g., a BODIPY dye as described above) that has been attached and the dye includes a side-chain -NH2 group suitable for conjugation (e.g., conjugation to an acceptor dye via an amide bond and a linker) are shown below:

[0665]

[0666]

[0667] An exemplary polymer tandem dye structure is shown below, where R is a linked donor dye (e.g., a BODIPY dye as described above), and the dye includes a linked acceptor dye of interest (e.g., suitable for conjugation (e.g., conjugation with an acceptor dye)), such as DY-754 or DY-704, where DY refers to a Dyomics dye, for example, having a maximum emission wavelength of 754 nm or 704 nm, respectively. By selecting an emission spectrum that is at least partially consistent with the base polymer (see Figure 8 ) overlapping receptor dyes of interest, polymer tandem dyes with various emission wavelengths can be produced. For details, see, e.g. Figures 9A-9B .

[0668]

[0669] Other embodiments

[0670] Although the claims are appended hereto, the scope of the present invention is also limited by the following clauses:

[0671] Item 1. A water-soluble light-harvesting multichromophore comprising:

[0672] a polymer backbone comprising non-conjugating repeat units; and

[0673] A plurality of pendant donor chromophore groups are independently attached to the non-conjugated repeating units of the polymer backbone.

[0674] Item 2. The multichromophore according to Item 1, wherein the pendant donor chromophore groups are all substituted with water soluble groups.

[0675] Item 3. The multichromophore according to Item 1, wherein the polymer backbone is a linear polymer.

[0676] Item 4. The multichromophore according to Item 1, wherein the pendant donor chromophores are configured to be in proximity to each other upon energy transfer.

[0677] Item 5. The multichromophore according to Item 4, wherein the configured side-donor chromophore exhibits fluorescence quenching relative to the unquenched separate donor chromophore under excitation by incident light.

[0678] Clause 6. The multichromophore according to any one of clauses 1 to 5, wherein the pendant donor chromophore group is selected from a fused tricyclic aryl or heteroaryl group and a BODIPY group.

[0679] Item 7. The multichromophore according to Item 6, wherein the pendant donor chromophore is selected from fluorene, carbazole and silole. Item 8. The multichromophore according to any one of Items 6-7, wherein the pendant donor chromophore is a fused tricyclic aromatic or heteroaromatic group having a structural formula as shown in any one of the following:

[0680]

[0681] in:

[0682] * is the bonding point of the non-conjugated repeating unit of the polymer backbone;

[0683] 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;

[0684] Each Z is independently CH, CR or N;

[0685] Each R 13are independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amine, substituted amine, aralkyl, substituted aralkyl, PEG moiety, WSG and -L 11 -Z 1 , where L 11 is the linker, Z 1 is a non-conjugating repeat unit, or any two suitable R' groups are optionally linked to form a ring structure; and

[0686] Each R is independently H or one or more substituents, and any two suitable R groups are optionally linked to form a ring structure;

[0687] Among them, R and R 13 One of the two is connected to the non-conjugated repeating unit of the polymer backbone.

[0688] Clause 9. The multichromophore according to any one of clauses 1 to 6, wherein the pendant donor chromophore group is a BODIPY group.

[0689] Item 10. The multichromophore according to Item 9, wherein the BODIPY group has the following structural formula:

[0690]

[0691] in:

[0692] R 1 -R 7 are each independently selected from H, 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 -Z 1 ,or

[0693] Optionally, selected from 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 and R 5 and R 1Any one or more pairs of substituents of which together form a divalent radical and are connected to the carbon atoms to which they are attached to form a cyclic structure, together forming a 5- or 6-membered fused heterocyclic ring, carbocyclic ring, aryl ring or heteroaryl ring (e.g., a 5- or 6-membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), wherein the ring may be unsubstituted or further substituted with substituents independently selected from the following groups: alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water solubilizing group (WSG) and -L 1 -Z 1 ;

[0694] L 1 is a linker;

[0695] Z 1 is a non-conjugated repeating unit of the polymer backbone; and

[0696] Y 1 and Y 2 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;

[0697] where Y 1 , Y 2 and R 1 -R 7 One of the two is connected to the non-conjugated repeating unit of the polymer backbone.

[0698] Item 11. The multichromophore according to Item 10, wherein R 1 is an optionally substituted aryl or heteroaryl group attached to the non-conjugated repeat unit of the polymer backbone.

[0699] Clause 12. The multichromophore according to any one of clauses 10-11, wherein Y 1 and Y 2 Each comprises a water solubilizing group (WSG).

[0700] Clause 13. The multichromophore according to any one of clauses 10 to 12, wherein the side-donor chromophore has the following structural formula:

[0701]

[0702] in:

[0703] * is the bonding point of the non-conjugated repeating unit of the polymer backbone;

[0704] Y 1and Y 2 All are alkynyl groups substituted by WSG.

[0705] Item 14. The multichromophore according to Item 13, wherein the side-donor chromophore has the following structural formula:

[0706]

[0707] in:

[0708] R 11 YesL 1 -Z 1 (linked non-conjugated repeat units of the polymer backbone);

[0709] Each R 9 are all optional substituents selected from halogen, hydroxy, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and t is 0-4. Clause 15. The multichromophore according to any one of clauses 1-14, wherein the side-donor chromophore is substituted with one or more water-solubilizing groups (WSG) independently selected from the following formula:

[0710]

[0711]

[0712]

[0713] in:

[0714] T 5 is an optional linker;

[0715] Each T 6 All are linkers;

[0716] R 11 and R are independently H, alkyl or substituted alkyl; and

[0717] Each s is an integer between 1 and 50.

[0718] Clause 16. The multichromophore according to any one of clauses 1 to 15, comprising a segment represented by the following formula:

[0719]

[0720] in:

[0721] Each M 1 and M 2 are independently aromatic or heteroaromatic comonomers;

[0722] Each S 1 and S 2 are all independently non-conjugated spacer units;

[0723] Each D 1 are independently 1 an attached side-group donor chromophore;

[0724] Each Z 1 are independently 2 attached chemoselective tags;

[0725] x is 75 mol% or more; and

[0726] y is 25 mol% or less.

[0727] Item 17. The multichromophore according to Item 16, wherein the M of the polymer backbone is 1 -S 1 and M 2 -S 2 The repeating units have a random configuration.

[0728] Clause 18. The multichromophore according to any one of clauses 16-17, wherein:

[0729] Each M 1 and M 2 independently comprising one or more groups selected from fluorene, carbazole, silole, biphenylene and phenylene; and

[0730] Each S 1 and S 2 Each is independently a saturated spacer unit selected from divalent polyethylene glycol (PEG) and divalent modified PEG groups.

[0731] Item 19. The multichromophore according to any one of Items 1 to 15, comprising a segment represented by the following formula:

[0732]

[0733] in:

[0734] The polymer backbone containing non-conjugated repeating units comprises SM 1 and SM 2 comonomers, each of which is independently a saturated non-conjugated comonomer;

[0735] Each D 1 Both are independently 1 an attached side-group donor chromophore;

[0736] Each Z 1 Both are independently 2attached chemoselective tags;

[0737] x is 75 mol% or more; and

[0738] y is 25 mol% or less.

[0739] Item 20. The multichromophore according to Item 19, wherein the SM of the polymer backbone 1 and SM 2 The repeating units have a random configuration.

[0740] Clause 21. The multichromophore according to any one of clauses 19-20, wherein SM 1 and SM 2 is a comonomer derived from acrylates, methacrylates, acrylamides, polystyrene, ROMP monomers, ADMET monomers, or cyclic carbonates.

[0741] Item 22. The multichromophore according to Item 21, wherein SM 1 and SM 2 Select from the following formula:

[0742]

[0743] in:

[0744] R 21 Yes – L 1 -D 1 or –L 2 -Z 1 ;

[0745] D 1 It is with M 1 an attached side-group donor chromophore;

[0746] Z 1 It is with M 2 attached chemoselective tags;

[0747] L 1 and L 2 is an optional linker;

[0748] X is O or NR";

[0749] R 22 is H or lower alkyl;

[0750] R" is H or lower alkyl, substituted lower alkyl and WSG; and

[0751] * is the point of attachment to the polymer backbone.

[0752] Item 23. The multichromophore according to Item 19, wherein the multichromophore has a structural formula as shown in Formula (XXI):

[0753]

[0754] in:

[0755] The polymer backbone of the non-conjugated repeating unit comprises SM 1 , SM 2 and SM 3 Comonomers, each of which is linked via a group T, which is the product of a click chemistry reaction or a chemoselective group conjugation reaction;

[0756] SM 3 Optionally including a connected WSG;

[0757] Each D 1 Both are independently 1 a light-absorbing chromophore attached to a side group;

[0758] Each Z 1 Both are independently 2 attached chemoselective tags;

[0759] x is 50 mol% or more; and

[0760] y+z is 50 mol% or less, wherein * is the point of attachment to the polymer backbone of the multichromophore or end group.

[0761] Item 24. The multichromophore according to Item 23, wherein SM 1 , SM 2 and SM 3 Contains the following structure:

[0762]

[0763] in:

[0764] Each X is independently O or NR 31 , where R 31 is H, alkyl, substituted alkyl, alkanoyl or substituted alkanoyl;

[0765] Each r and s is independently 1-6 (e.g., 1, 2 or 3);

[0766] each d and e is independently 1-12 (e.g., 1-6, such as 1, 2, 3, 4, 5 or 6);

[0767] t is 0 or 1;

[0768] D 1It is a side-donating chromophore;

[0769] Z 1 is a chemoselective tag (e.g., as described herein);

[0770] WSG is a water solubilizing group (e.g., as described herein);

[0771] Each L 1 , L 2 and L 3 are independently linkers; and

[0772] * is the connection point to a 1,4-substituted 1,2,3-triazole (T) having any of the following structures:

[0773]

[0774] or terminal G 1 or G 2 (e.g., as described herein).

[0775] Clause 25. The multichromophore according to clause 19, wherein the repeating units of the polymer backbone have a defined linear sequence.

[0776] Item 26. The multichromophore according to Item 25, wherein SM 1 and SM 2 is a comonomer derived from an amino acid, a peptoid monomer, a protected carbonate monomer, or a cyclic carbonate monomer.

[0777] Clause 27. The multichromophore according to any one of clauses 19, 25 and 26, wherein the polymer backbone is a polypeptide having a defined sequence of -amino-amino acid residues and / or -amino acid residues.

[0778] Clause 28. The multichromophore according to any one of clauses 19 and 25-27, wherein the multichromophore has the following structural formula:

[0779]

[0780] in:

[0781] Each D 1 are all independently side-donating chromophores;

[0782] Each Z 1 Each is independently a chemoselective tag;

[0783] Each L 1 and L 2 are all independently linkers;

[0784] p1 and q1 are independently 0 or 1, where p1+q1≤1;

[0785] p2 and q2 are independently 0 or 1, where p1+q1≤1;

[0786] x is 75 mol% or more;

[0787] y is 25 mol% or less; and

[0788] G 1 and G 2 Each is independently selected from the group consisting of an end group, a polymer segment, a donor chromophore, an acceptor fluorescent group, a linker, and a linked specific binding member.

[0789] Item 29. The multichromophore according to Item 28, wherein p1 and p2 are both 0, and q1 and q2 are both 1.

[0790] Item 30. The multichromophore according to Item 28, wherein p1 and p2 are both 1, and q1 and q2 are both 0. Item 31. The multichromophore according to Item 28, wherein p1, p2, q1 and q2 are all 0, and the structural formula of the multichromophore is as follows:

[0791]

[0792] in:

[0793] Each D 1 are all independently side-donating chromophores;

[0794] Each Z 1 Each is independently a chemoselective tag;

[0795] L 1 and L 2 each independently a linker;

[0796] x is 75 mol% or more;

[0797] y is 25 mol% or less; and

[0798] G 1 and G 2 Each is independently selected from the group consisting of an end group, a polymer segment, a donor chromophore, an acceptor fluorescent group, a linker, and a linked specific binding member.

[0799] Item 32. The multichromophore according to any one of Items 27, 28 and 31, comprising a segment of the formula:

[0800]

[0801] in:

[0802] Each D 1 are all independently side-donating chromophores;

[0803] Each Z 1 Each is independently a chemoselective tag;

[0804] Each L 1 and L 2 are all independently linkers;

[0805] n and p are each independently an integer between 1 and 20, wherein n+p≥2; and

[0806] m is 1 or 2.

[0807] Clause 33. The multichromophore according to clause 32, wherein the multichromophore comprises a q segment of a copolymer and has the following structural formula:

[0808]

[0809] in:

[0810] Each (n) q and each (p) q are independently integers between 1 and 20, wherein for each q segment, (n) q +(p) q ≥3; and

[0811] q is an integer between 1 and 100.

[0812] Clause 34. The multichromophore according to any one of clauses 19, 25-28 and 31-33, wherein the polymer backbone comprises one or more amino acid sequences selected from:

[0813]

[0814]

[0815] in:

[0816] Each X is a lysine or ornithine residue covalently N-linked to a pendant donor chromophore; and

[0817] Each Y is a cysteine ​​residue or a protected cysteine ​​residue.

[0818] Clause 35. The multichromophore according to any one of clauses 19, 25 and 26, wherein the multichromophore has the following structural formula:

[0819]

[0820] in

[0821] Each D 1 are all independently side-donating chromophores;

[0822] Each Z 1 Each is independently a chemoselective tag;

[0823] Each L 1 and L 2 are all independently linkers;

[0824] x is 75 mol% or more;

[0825] y is 25 mol% or less; and

[0826] G 1 and G 2 Each is independently selected from the group consisting of an end group, a polymer segment, a donor chromophore, an acceptor fluorophore, a linker, and a linked specific binding member.

[0827] Clause 36. The multichromophore according to any one of clauses 19-21 and 26, wherein the multichromophore has the following structural formula:

[0828]

[0829] in:

[0830] Each D 1 Each is independently a side-group donor chromophore;

[0831] Each Z 1 Each is independently a chemoselective tag;

[0832] Each L 1 and L 2 are all independently linkers;

[0833] x is 75 mol% or more;

[0834] y is 25 mol% or less; and

[0835] G 1 and G 2 Each is independently selected from the group consisting of: an end group, a polymer segment, a donor chromophore, an acceptor fluorophore, a linker, and an attached specific binding member.

[0836] Clause 37. The multichromophore according to any one of clauses 16 to 36, wherein the pendant donor chromophore group is a BODIPY group.

[0837] Item 38. A polymer tandem dye comprising:

[0838] A light-harvesting multichromophore comprising:

[0839] a polymer backbone comprising non-conjugating repeat units; and

[0840] A plurality of pendant donor chromophore groups independently attached to the non-conjugated repeating units of the polymer backbone; and

[0841] An acceptor fluorophore is attached to the non-conjugated repeat unit of the polymer backbone, the acceptor fluorophore being configured to be proximal to at least one pendant donor chromophore of the light-harvesting multichromophore upon energy reception.

[0842] Item 39. The polymeric tandem dye according to Item 38, wherein the pendant donor chromophore groups are all substituted with water soluble groups.

[0843] Item 40. The polymer tandem dye of Item 38, wherein the polymer backbone is a linear polymer. Item 41. The polymer tandem dye of Item 38, wherein the pendant donor chromophores are configured to be in proximity to each other upon energy transfer.

[0844] Item 42. A polymeric tandem dye according to Item 36, having a Stokes shift of 100 nm or more. Item 43. A polymeric tandem dye according to any one of Items 36 to 40, wherein the pendant donor chromophore group is selected from a fused tricyclic aryl group, a fused tricyclic heteroaryl group, and a BODIPY group.

[0845] Item 44. The polymeric tandem dye according to Item 43, wherein the pendant donor chromophore group is selected from optionally substituted fluorene, carbazole and silole groups.

[0846] Clause 45. The polymer tandem dye according to any one of clauses 43-44, wherein the pendant donor chromophore group is a fused tricyclic aromatic or heteroaromatic group having a structural formula as shown in any one of the following:

[0847]

[0848] in:

[0849] * is the bonding point of the non-conjugated repeating unit of the polymer backbone;

[0850] 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;

[0851] Each Z is independently CH, CR or N;

[0852] Each R 13 are independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amine, substituted amine, aralkyl, substituted aralkyl, PEG moiety, WSG and -L 11 -Z 1 , where L 11 is the linker, Z 1 is a non-conjugated repeating unit, or any two suitable R 3 The groups are optionally linked to form a ring structure; and

[0853] Each R is independently H or one or more substituents, and any two suitable R groups are optionally linked to form a ring structure;

[0854] Among them, R and R 13 One of the two is connected to the non-conjugated repeating unit of the polymer backbone.

[0855] Item 46. The polymeric tandem dye according to Item 43, wherein the pendant donor chromophore group is a BODIPY group.

[0856] Item 47. The polymer tandem dye according to Item 46, wherein the structural formula of the BODIPY group is as follows:

[0857]

[0858] in:

[0859] R 1 -R 7 are each independently selected from H, 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 -Z 1 ,or

[0860] Optionally, selected from 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 and R 5 and R 1Any one or more pairs of substituents of which together form a divalent radical and are connected to the carbon atoms to which they are attached to form a cyclic structure, together forming a 5- or 6-membered fused heterocyclic ring, carbocyclic ring, aryl ring or heteroaryl ring (e.g., a 5- or 6-membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), wherein the ring may be unsubstituted or further substituted with substituents independently selected from the following groups: alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water solubilizing group (WSG) and -L 1 -Z 1 ;

[0861] L 1 is a linker;

[0862] Z 1 is a non-conjugated repeating unit of the polymer backbone; and

[0863] Y 1 and Y 2 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;

[0864] where Y 1 , Y 2 and R 1 -R 7 One of the two is connected to the non-conjugated repeating unit of the polymer backbone.

[0865] Item 48. The polymer tandem dye according to Item 47, wherein R 1 is an optionally substituted aryl or heteroaryl group attached to the non-conjugated repeat unit of the polymer backbone.

[0866] Item 49. A polymer tandem dye according to any one of Items 47-48, wherein Y 1 and Y 2 Each comprises a water solubilizing group (WSG).

[0867] Clause 50. The polymer tandem dye according to any one of clauses 47 to 49, wherein the pendant donor chromophore group has the following structural formula:

[0868]

[0869] in:

[0870] * is the bonding point of the non-conjugated repeating unit of the polymer backbone;

[0871] Y1 and Y 2 All are alkynyl groups substituted by WSG.

[0872] Item 51. The polymer tandem dye according to Item 50, wherein the pendant donor chromophore has the following structural formula:

[0873]

[0874] in:

[0875] R 11 YesL 1 -Z 1 (linked non-conjugated repeat units of the polymer backbone);

[0876] Each R 9 are all optional substituents selected from halogen, hydroxy, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and t is 0-4. Clause 52. A polymer tandem dye according to any one of clauses 38-51, wherein the pendant donor chromophore is substituted with one or more water solubilizing groups (WSG) independently selected from the following formula:

[0877]

[0878]

[0879]

[0880] in:

[0881] T 5 is an optional linker;

[0882] Each T 6 All are linkers;

[0883] R 11 and R are independently H, alkyl or substituted alkyl; and

[0884] Each s is an integer between 1 and 50.

[0885] Item 53. The polymer tandem dye according to any one of items 38 to 52, comprising a segment represented by the following formula:

[0886]

[0887] in:

[0888] Each M 1 and M 2 are independently aromatic or heteroaromatic comonomers;

[0889] Each S 1 and S 2 are all independently non-conjugated spacer units;

[0890] Each D 1 are independently 1 an attached side-group donor chromophore;

[0891] Each A 1 are independently 2 the attached acceptor fluorophore;

[0892] x is 75 mol% or more; and

[0893] y is 25 mol% or less.

[0894] Item 54. The polymer tandem dye according to Item 53, wherein the M of the polymer backbone is 1 -S 1 and M 2 -S 2 The repeating units have a random configuration.

[0895] Clause 55. A polymer tandem dye according to any one of clauses 53-54, wherein:

[0896] Each M 1 and M 2 independently comprising one or more groups selected from fluorene, carbazole, silole, biphenylene and phenylene; and

[0897] Each S 1 and S 2 Each is independently a saturated spacer unit selected from divalent polyethylene glycol (PEG) and divalent modified PEG groups.

[0898] Item 56. The polymer tandem dye according to any one of items 38 to 52, comprising a segment represented by the following formula:

[0899]

[0900] in:

[0901] The polymer backbone containing non-conjugated repeating units comprises SM 1 and SM 2 comonomers, each of which is independently a non-conjugated comonomer;

[0902] Each D 1 Both are independently 1 an attached side-group donor chromophore;

[0903] Each A1 Both are independently 2 the attached acceptor fluorophore;

[0904] x is 75 mol% or more; and

[0905] y is 25 mol% or less.

[0906] Item 57. The polymer tandem dye according to Item 56, wherein the repeating units of the polymer backbone have a random configuration.

[0907] Item 58. The polymer tandem dye according to any one of Items 56-57, wherein SM 1 and SM 2 is a comonomer derived from acrylates, methacrylates, acrylamides, polystyrene, ROMP monomers, ADMET monomers, or cyclic carbonates.

[0908] Item 59. The polymer tandem dye according to Item 58, wherein SM 1 and SM 2 Select from the following formula:

[0909]

[0910] in:

[0911] R 21 Yes – L 1 -D 1 or –L 2 -Z 1 ;

[0912] D 1 Yes and M 1 an attached side-group donor chromophore;

[0913] Z 1 Yes and M 2 attached chemoselective tags;

[0914] L 1 and L 2 is an optional linker;

[0915] X is O or NR";

[0916] R' is H or lower alkyl;

[0917] R" is H or lower alkyl, substituted lower alkyl and WSG; and

[0918] * is the point of attachment to the polymer backbone.

[0919] Item 60. The polymer tandem dye according to Item 56, wherein the multichromophore has a structural formula as shown in Formula (XXI):

[0920]

[0921] in:

[0922] The polymer backbone of the non-conjugated repeating unit comprises SM 1 , SM 2 and SM 3 Comonomers, each of which is linked via a group T, which is the product of a click chemistry reaction or a chemoselective group conjugation reaction;

[0923] SM 3 Optionally including a connected WSG;

[0924] Each D 1 Both are independently 1 a light-absorbing chromophore attached to a side group;

[0925] Each A 1 Both are independently 2 the attached acceptor fluorophore;

[0926] x is 50 mol% or more; and

[0927] y+z is 50 mol% or less, wherein * is the point of attachment to the polymer backbone of the multichromophore or end group.

[0928] Item 61. The polymer tandem dye according to Item 60, wherein SM 1 , SM 2 and SM 3 Contains the following structure:

[0929]

[0930] in:

[0931] Each X is independently O or NR 31 , where R 31 is H, alkyl, substituted alkyl, alkanoyl or substituted alkanoyl;

[0932] Each r and s is independently 1-6 (e.g., 1, 2 or 3);

[0933] each d and e is independently 1-12 (e.g., 1-6, such as 1, 2, 3, 4, 5 or 6);

[0934] t is 0 or 1;

[0935] Each L1 , L 2 and L 3 are independently linkers; and

[0936] * is the connection point to a 1,4-substituted 1,2,3-triazole (T) having any of the following structures:

[0937]

[0938] Item 62. The polymer tandem dye according to Item 56, wherein the repeating units of the polymer backbone have a defined linear sequence.

[0939] Item 63. The polymer tandem dye according to Item 62, wherein SM 1 and SM 2 is a comonomer derived from an amino acid, a peptoid monomer, a protected carbonate monomer, or a cyclic carbonate monomer.

[0940] Item 64. The polymer tandem dye according to any one of items 56, 62 and 63, wherein the polymer backbone is a polypeptide having a defined sequence of -amino-amino acid residues and / or -amino-amino acid residues.

[0941] Clause 65. The polymer tandem dye according to any one of clauses 56 and 62-64, wherein the multichromophore has the following structural formula:

[0942]

[0943] in:

[0944] Each D 1 are all independently side-donating chromophores;

[0945] Each A 1 All are independently acceptor fluorophores;

[0946] Each L 1 and L 2 are all independently linkers;

[0947] p1 and q1 are independently 0 or 1, where p1+q1≤1;

[0948] p2 and q2 are independently 0 or 1, where p1+q1≤1;

[0949] x is 75 mol% or more;

[0950] y is 25 mol% or less; and

[0951] G 1 and G 2Each is independently selected from the group consisting of an end group, a polymer segment, a donor chromophore, an acceptor fluorophore, a linker, and a linked specific binding member.

[0952] Item 66. The polymer tandem dye according to Item 65, wherein p1 and p2 are both 0, and q1 and q2 are both 1.

[0953] Item 67. The polymer tandem dye according to Item 65, wherein p1 and p2 are both 1, and q1 and q2 are both 0.

[0954] Item 68. The polymer tandem dye according to Item 65, wherein p1, p2, q1 and q2 are all 0, and the structural formula of the multichromophore is as follows:

[0955]

[0956] in:

[0957] Each D 1 are all independently side-donating chromophores;

[0958] Each A 1 All are independently acceptor fluorophores;

[0959] L 1 and L 2 each independently a linker;

[0960] x is 75 mol% or more;

[0961] y is 25 mol% or less; and

[0962] G 1 and G 2 Each is independently selected from the group consisting of an end group, a polymer segment, a donor chromophore, an acceptor fluorophore, a linker, and a linked specific binding member.

[0963] Item 69. The polymer tandem dye according to any one of items 64, 65 and 66, comprising a segment of the formula:

[0964]

[0965] in:

[0966] Each D 1 are all independently side-donating chromophores;

[0967] Each A 1 All are independently acceptor fluorophores;

[0968] Each L 1 and L 2 are all independently linkers;

[0969] n and p are each independently an integer between 1 and 20, wherein n+p≥2; and

[0970] m is 1 or 2.

[0971] Clause 70. The polymer tandem dye according to clause 69, wherein the multichromophore comprises a q segment of a copolymer and has the following structural formula:

[0972]

[0973] in:

[0974] Each (n) q and each (p) q are independently integers between 1 and 20, wherein for each q segment, (n) q +(p) q ≥3; and

[0975] q is an integer between 1 and 100.

[0976] Clause 71. A polymer tandem dye according to any one of clauses 56, 62-65 and 68-70, wherein the polymer backbone comprises one or more amino acid sequences selected from:

[0977]

[0978]

[0979] in:

[0980] Each X is a lysine or ornithine residue covalently N-linked to a pendant donor chromophore; and

[0981] Each Y is a cysteine ​​residue covalently linked to a pendant acceptor fluorescent group.

[0982] Item 72. The polymer tandem dye according to any one of items 56, 62 and 63, wherein the multichromophore has the following structural formula:

[0983]

[0984] in

[0985] Each D 1 are all independently side-donating chromophores;

[0986] Each A 1 Each is independently an acceptor chromophore;

[0987] Each L 1and L 2 are all independently linkers;

[0988] x is 75 mol% or more;

[0989] y is 25 mol% or less; and

[0990] G 1 and G 2 Each is independently selected from the group consisting of an end group, a polymer segment, a donor chromophore, an acceptor fluorophore, a linker, and a linked specific binding member.

[0991] Clause 73. The polymer tandem dye according to any one of clauses 56 to 58 and 63, wherein the multichromophore has the following structural formula:

[0992]

[0993] in:

[0994] Each D 1 Each is independently a side-group donor chromophore;

[0995] Each A 1 All are independently acceptor fluorophores;

[0996] Each L 1 and L 2 are all independently linkers;

[0997] x is 75 mol% or more;

[0998] y is 25 mol% or less; and

[0999] G 1 and G 2 Each is independently selected from the group consisting of: an end group, a polymer segment, a donor chromophore, an acceptor fluorophore, a linker, and an attached specific binding member.

[1000] Clause 74. The polymeric tandem dye according to any one of clauses 53 to 73, wherein the pendant donor chromophore group is a BODIPY group.

[1001] Clause 75. A polymer tandem dye according to any one of clauses 38 to 74, wherein the acceptor fluorophore (e.g., each A 1 ) is a small molecule fluorophore.

[1002] Clause 76. A polymer tandem dye according to any one of clauses 38 to 75, wherein the acceptor fluorophore (e.g., each A 1) is selected from cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethine, pyrene, dipyrromethene boron difluoride, naphthimide, thiazine dyes and acridine dyes.

[1003] Clause 77. A labeled specific binding member comprising:

[1004] A polymer tandem dye comprising:

[1005] A light-harvesting multichromophore comprising:

[1006] a polymer backbone comprising non-conjugating repeat units; and

[1007] A plurality of pendant donor chromophore groups independently attached to the non-conjugated repeating units of the polymer backbone; and

[1008] an acceptor fluorophore attached to a non-conjugated repeat unit of the polymer backbone, the acceptor fluorophore being configured to be proximal to at least one pendant donor chromophore of the light-harvesting multichromophore upon energy reception; and

[1009] Specific binding members linked to polymer tandem dyes.

[1010] Clause 78. A labelled specific binding member according to clause 77, wherein the specific binding member is an antibody.

[1011] Clause 79. A labelled specific binding member according to clause 77, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[1012] Clause 80. A labelled specific binding member according to clause 79, wherein the antibody fragment or binding derivative thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a diabody and a triabody.

[1013] Clause 81. A labelled specific binding member according to any one of clauses 77 to 80, wherein the acceptor fluorophore is selected from cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethine, pyrene, dipyrromethene boron difluoride, naphthimide, thiazine dyes and acridine dyes.

[1014] Clause 82. A labelled specific binding member according to any one of clauses 77-81, wherein the polymer tandem dye is a dye according to any one of clauses 39-76.

[1015] Clause 83. A labeled specific binding member according to any one of clauses 77 to 81, having the following structural formula:

[1016]

[1017] in:

[1018] Each D 1 are all independently side-donating chromophores;

[1019] Each A 1 All are independently acceptor fluorophores;

[1020] Each L 1 and L 2 are all independently linkers;

[1021] p1 and q1 are independently 0 or 1, where p1+q1≤1;

[1022] p2 and q2 are independently 0 or 1, where p1+q1≤1;

[1023] x is 75 mol% or more;

[1024] y is 25 mol% or less; and

[1025] G 1 is a terminal group, a polymer segment, a donor chromophore, an acceptor fluorophore, or a linker; and

[1026] G 2 is the linked specific binding member.

[1027] Clause 84. The labeled specific binding member according to clause 83, wherein p1, p2, q1 and q2 are all 0, and the structural formula of the multichromophore is as follows:

[1028]

[1029] in:

[1030] Each D 1 are all independently side-donating chromophores;

[1031] Each A 1 All are independently acceptor fluorophores;

[1032] L 1 and L 2 each independently a linker;

[1033] x is 75 mol% or more; y is 25 mol% or less;

[1034] G 1 is a terminal group, a polymer segment, a donor chromophore, an acceptor fluorophore, or a linker; and

[1035] G 2 is the linked specific binding member.

[1036] Clause 85. A method of assessing the presence or absence of a target analyte in a sample, the method comprising:

[1037] (a) contacting the sample with a labeled specific binding member that specifically binds to a target analyte to produce a labeled composition contacting the sample, wherein the labeled specific binding member comprises:

[1038] (i) a polymer tandem dye according to any one of clauses 38 to 76; and

[1039] (ii) a specific binding member linked to a polymer tandem dye; and

[1040] (b) determining the presence or absence of the labeled specific binding member-target analyte binding complex in the sample contacted with the labeled composition to assess the presence or absence of the target analyte in the sample.

[1041] Clause 86. The method according to clause 85, wherein the acceptor fluorophore of the polymer tandem dye is selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethine, pyrene, dipyrromethene boron difluoride, naphthimide, thiazine dyes, and acridine dyes.

[1042] Clause 87. The method according to any one of clauses 85-86, further comprising contacting the sample with a second specific binding member which is bound to a support and which specifically binds to a target analyte.

[1043] Clause 88. The method of Clause 87, wherein the support comprises magnetic particles.

[1044] Clause 89. The method according to any one of clauses 85-88, wherein the target analyte is associated with a cell. Clause 90. The method according to clause 89, wherein the target analyte is a cell surface marker of a cell. Clause 91. The method according to clause 90, wherein the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[1045] Clause 92. The method of clause 89, wherein the target analyte is an intracellular target and the method further comprises lysing the cells.

[1046] Clause 93. The method according to any one of clauses 85-92, wherein the method further comprises analyzing the fluorescently labeled target analyte by flow cytometry.

[1047] Clause 94. A method of labeling a target molecule, the method comprising:

[1048] contacting a target molecule with a polymer tandem dye to produce a labeled target molecule, wherein:

[1049] The polymer tandem dye is a dye according to any one of clauses 38-76 and comprises a conjugated tag covalently linked to the target molecule.

[1050] Item 95. The method according to Item 94, wherein the polymer tandem dye has the following structural formula:

[1051]

[1052] in:

[1053] Each D 1 are all independently side-donating chromophores;

[1054] Each A 1 All are independently acceptor fluorophores;

[1055] Each L 1 and L 2 are independently linkers;

[1056] p1 and q1 are independently 0 or 1, where p1+q1≤1;

[1057] p2 and q2 are independently 0 or 1, where p1+q1≤1;

[1058] x is 75 mol% or more;

[1059] y is 25 mol% or less; and

[1060] G 1 is a terminal group, a polymer segment, a donor chromophore, an acceptor fluorophore, or a linker; and

[1061] G 2 is a linker containing a conjugated tag.

[1062] Clause 96. A method of preparing a light-harvesting multichromophore, the method comprising:

[1063] a) synthesizing a protected polypeptide having a defined amino acid sequence consisting of blocks of a first amino acid residue separated by a single occurrence of a second amino acid residue, wherein:

[1064] Each block of first amino acid residues comprises at least two residues;

[1065] The first amino acid residues each comprise a protected first chemoselective side chain group; and

[1066] The second amino acid residues each comprise a protected second chemoselective side chain group;

[1067] b) deprotecting the protected polypeptide to produce a deprotected polypeptide, wherein both the first and second chemoselective side chain groups are deprotected;

[1068] c) coupling a reactive donor chromophore moiety to the deprotected first chemoselective side chain group of the first amino acid residue to generate a side group donor chromophore.

[1069] Clause 97. The method according to clause 96, further comprising, continuing with step c), coupling the reactive acceptor fluorophore moiety to the deprotected second chemoselective side chain group of the second amino acid residue to produce a side group acceptor fluorophore.

[1070] Clause 98. The method according to clause 96, further comprising, after step a), deprotecting the N-terminus of the protected polypeptide, and coupling the G1 group to the N-terminus of the N-terminus of the deprotected polypeptide, wherein G1 is a terminal group (e.g., a capping group), a donor chromophore, or a linker.

[1071] Clause 99. The method according to any one of clauses 96 to 98, further comprising, after step c), coupling a specific binding member to the C-terminus of the polypeptide.

[1072] Clause 100. The method according to any one of clauses 96-99, wherein the light-harvesting multichromophore has the following structural formula:

[1073]

[1074] in:

[1075] D 1 It is a side-donating chromophore;

[1076] Z 1 is a second chemoselective side chain group;

[1077] Each L 1 and L 2 are all independently linkers;

[1078] p1 and q1 are independently 0 or 1, where p1+q1≤1;

[1079] p2 and q2 are independently 0 or 1, where p1+q1≤1;

[1080] x is 75 mol% or more;

[1081] y is 25 mol% or less; and

[1082] G 1 and G2 Each is independently selected from the group consisting of an end group, a polymer segment, a donor chromophore, an acceptor fluorophore, a linker, and a linked specific binding member.

[1083] Clause 101. The method according to any one of clauses 96 to 100, wherein the light-harvesting multichromophore has the following structural formula:

[1084]

[1085] in:

[1086] Each (n) q and each (p) q are independently integers between 1 and 20, wherein for each q segment, (n) q +(p) q ≥3; and

[1087] q is an integer between 1 and 100.

[1088] Clause 102. A method according to any one of clauses 96 to 101, wherein:

[1089] The first amino acid residue is independently selected from lysine and ornithine; and

[1090] The second amino acid residue is cysteine.

[1091] Clause 103. A method according to any one of clauses 96 to 102, wherein the defined amino acid sequence comprises an amino acid sequence selected from:

[1092]

[1093]

[1094] in:

[1095] Each X is a lysine or ornithine residue covalently N-linked to a pendant donor chromophore; and

[1096] Each Y is a cysteine ​​residue or a protected cysteine ​​residue.

[1097] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, in view of the teachings of the invention, it will be apparent to one skilled in the art that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[1098] Therefore, the foregoing only illustrates the principles of the present invention. It should be understood that those skilled in the art will be able to design various structures, although not explicitly stated or shown here, but these designs reflect the principles of the present invention and do not exceed the spirit and scope of the present invention. In addition, all examples and conditional language listed herein are mainly to help readers understand the principles of the present invention and the ideas provided by the inventors to further expand the art, and should be interpreted as not being limited by these specific examples and conditions. Moreover, all statements citing the principles, aspects and embodiments of the present invention and their specific examples herein are intended to cover their equivalents in structure and function. In addition, the equivalents are intended to include currently known equivalents and equivalents to be developed in the future, that is, any functionally identical originals developed and have nothing to do with the structure. Moreover, regardless of whether the disclosure of the present invention is clearly described in the claims, none of the contents therein will be disclosed to the public.

[1099] Therefore, the scope of the present invention is not limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are embodied by the appended claims. In the claims, only when the phrase "means for..." or "step for..." is explicitly used at the beginning of the limitation content of the claim, "35 U.S.C. Section 112(f)" or "35 U.S.C. Section 112(6)" is explicitly defined as being cited; if the phrase is not used in the limitation content of the claim, "35 U.S.C. Section 112(f)" or "35 U.S.C. Section 112(6)" is not cited.

Claims

1. A water-soluble polymer tandem dye comprising: Peptide backbone; A plurality of pendant donor fluorophores independently attached to amino acid residues of the polymer backbone; and An acceptor fluorophore is attached to an amino acid residue of the polypeptide backbone, the acceptor fluorophore being in proximity to at least one side-group donor fluorophore upon energy acceptance.

2. The water-soluble polymer tandem dye according to claim 1, wherein: Each side-donor fluorophore is substituted with a water-soluble group; The side-group donor fluorophores are arranged to be in proximity with each other during energy transfer; and / or The pendant donor fluorophore is selected from a fused tricyclic aryl group, a fused tricyclic heteroaryl group, and a BODIPY group.

3. The water-soluble polymer tandem dye according to claim 1 or 2, wherein the side-donor fluorophore is a BODIPY group represented by the following formula: in: R 1 -R 7 are each independently selected from H, 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 -Z 1 ,or Optionally, selected from 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 and R 5 and R 1 Any one or more pairs of substituents in the group of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water solubilizing group (WSG) and -L are formed together to form a divalent group and are connected to the carbon atom to form a cyclic structure, forming a 5- or 6-membered fused heterocyclic ring, carbocyclic ring, aryl ring or heteroaryl ring (e.g., a 5- or 6-membered ring containing carbon atoms and 0-3 heteroatoms selected from O, S and N), wherein the ring may be unsubstituted or further substituted by substituents independently selected from the following groups: alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water solubilizing group (WSG) and -L 1 -Z 1 ; L 1 is a linker; Z 1 is a non-conjugated repeating unit of the polymer backbone; and Y 1 and Y 2 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; where Y 1 , Y 2 and R 1 -R 7 One of the two is linked to the non-conjugated repeating unit of the polymer backbone.

4. The water-soluble polymer tandem dye according to claim 3, wherein the structure of the side-donor fluorophore is as follows: in: * is the bonding point of the non-conjugated repeating unit of the polymer backbone; Y 1 and Y 2 All are alkynyl groups substituted by WSG.

5. The water-soluble polymer tandem dye according to claim 4, wherein the structure of the side-group fluorophore is as follows: in: R 11 YesL 1 -Z 1 (linked non-conjugated repeat units of the polymer backbone); Each R 9 are all optional substituents selected from halogen, hydroxy, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and t is 0-4.

6. The water-soluble polymer tandem dye according to any one of claims 1 to 5, wherein the side-donor fluorophore is substituted with one or more than one water-solubilizing groups (WSG) independently selected from the following formula: in: T 5 is an optional linker; Each T 6 All are linkers; R 11 and R is independently H, alkyl or substituted alkyl; and Each s is an integer between 1 and 50.

7. The water-soluble polymer tandem dye according to any one of claims 1 to 6, comprising a segment represented by the following formula (IIa): in: SM 1 , SM 2 and SM 3 Each is independently a repeating unit of amino acid residues of a polypeptide backbone; Each D 1 is one of a plurality of side-donating fluorophores; Each A 1 is the acceptor fluorophore; Each Z 2 is an optional side chain group.

8. The polymer tandem dye according to any one of claims 1 to 7, wherein the polymer tandem dye comprises a segment as shown in the following formula: in: The polymer backbone of the repeating units of non-conjugated amino acid residues comprises SM 1 and SM 2 comonomers, each of which is independently a saturated non-conjugated amino acid residue comonomer; Each D 1 Both are independently 1 an attached side-group donor chromophore; Each A 1 Both are independently 2 the attached acceptor fluorophore; x is 75 mol% or higher; and y is 25 mol% or less.

9. The polymer tandem dye according to claim 8, wherein the polymer tandem dye is represented by the following formula: in: Each D 1 are all independently side-donating chromophores; Each A 1 All are independently acceptor fluorophores; Each L 1 and L 2 are all independently linkers; p1 and q1 are independently 0 or 1, where p1+q1≤1; p2 and q2 are independently 0 or 1, where p1+q1≤1; x is 75 mol% or higher; y is 25 mol% or less; and G 1 and G 2 Each is independently selected from the group consisting of an end group, a polymer segment, a donor chromophore, an acceptor fluorescent group, a linker, and a linked specific binding member.

10. The polymer tandem dye according to claim 9, wherein p1, p2, q1 and q2 are each 0, and the polymer tandem dye is represented by the following formula: in: Each D 1 are all independently side-donating chromophores; Each A 1 All are independently acceptor fluorophores; L 1 and L 2 each independently a linker; x is 75 mol% or higher; y is 25 mol% or less; and G 1 and G 2 Each is independently selected from the group consisting of an end group, a polymer segment, a donor chromophore, an acceptor fluorescent group, a linker, and a linked specific binding member.

11. The polymer tandem dye according to claim 10, comprising a segment represented by the following formula: in: Each D 1 are all independently side-donating chromophores; Each A 1 All are independently acceptor fluorophores; Each L 1 and L 2 are all independently linkers; n and p are each independently an integer between 1 and 20, wherein n+p≥2; and m is 1 or 2.

12. The polymer tandem dye according to claim 11, wherein the polymer tandem dye comprises q segments of a copolymer, and its structural formula is as follows: in: Each (n) q and each (p) q are independently integers between 1 and 20, wherein for each of the q segments, (n) q +(p) q ≥3; and q is an integer between 1 and 100.

13. The polymer tandem dye according to any one of claims 1 to 12, wherein the polypeptide backbone comprises one or more amino acid sequences selected from the group consisting of: in: Each X is a lysine residue or an ornithine residue that is covalently N-linked to a pendant donor chromophore; and Each Y is a cysteine ​​residue or a protected cysteine ​​residue.

14. A labeled specific binding member comprising: The polymer tandem dye according to any one of claims 1 to 13; and A specific binding member is attached to the polymer in tandem with a dye.

15. The labeled specific binding member of claim 14, wherein the specific binding member is an antibody or a binding fragment thereof.

16. A method for evaluating the presence of a target analyte in a sample, the method comprising: (a) contacting a sample with a labeled specific binding member according to any one of claims 14-15 that specifically binds to a target analyte to produce a labeled composition contacting the sample; and (b) determining the presence or absence of the labeled specific binding member-target analyte binding complex in the sample contacted with the labeled composition to assess the presence or absence of the target analyte in the sample.

17. A method for labeling a target molecule, the method comprising: The target molecule is contacted with a polymer tandem dye according to any one of claims 1 to 13 to produce a labeled target molecule, wherein the polymer tandem dye comprises a conjugated tag covalently attached to the target molecule.

18. The method of claim 17, wherein the target molecule comprises a specific binding member.

19. The method of claim 18, wherein the specific binding member comprises an antibody or a binding fragment thereof.

20. A kit comprising a polymer tandem dye according to any one of claims 1-13 or a labeled specific binding member according to any one of claims 14-15.

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