CN117062878A

CN117062878A - Water-soluble yellow-green absorbing dye

Info

Publication number: CN117062878A
Application number: CN202280022997.3A
Authority: CN
Inventors: 格伦·巴塞洛缪; 布瑞恩·沃尔; 乔恩·霍林格; 梁永超
Original assignee: Becton Dickinson and Co
Current assignee: Becton Dickinson and Co
Priority date: 2021-04-07
Filing date: 2022-03-21
Publication date: 2023-11-14

Abstract

Provided herein are water-soluble dipyrromethene-based dyes. In some cases, the water-soluble dye is a dipyrromethene-boron dye. Embodiments of the subject water-soluble dipyrromethene-based dyes have a narrow absorption maximum in the near 561nm laser line or the yellow-green range at 561nm laser line and are suitable for tandem dye systems. Embodiments of the subject dyes are effective to transfer energy to the acceptor chromophore, while a second dipyrromethene-type dye having the same or a different chemical structure is within its proximity to the energy acceptor. Also provided herein are tandem dyes comprising the subject water-soluble dipyrromethene-type dyes, e.g., as donor chromophores. Methods of assessing the presence of a target analyte in a sample and methods of labeling a target molecule using a subject dye are also provided. Systems and kits for practicing the subject methods are also provided.

Description

Water-soluble yellow-green absorbing dye

Cross Reference to Related Applications

According to 35 U.S. C. ≡119 (e), the present application claims priority from the filing date of U.S. provisional patent application Ser. No. 63/171,704 filed on 7 th 4 th year 2021 and U.S. provisional patent application Ser. No. 63/248,041 filed on 24 th 9 th year 2021; the disclosures of these applications are incorporated herein by reference in their entirety.

Background

The fluorescent dye is the following compound: fluorescent dyes emit (typically) light of different wavelengths when illuminated by light of the wavelength they absorb. Fluorescent dyes can be used in a variety of applications in the biochemical, biological and medical fields, 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 for the desired purpose. Parameters of interest include maximum excitation wavelength, maximum emission wavelength, stokes shift, extinction coefficient, fluorescence quantum yield, and fluorescence lifetime. The dye may be selected according to the application of interest, for example to allow penetration of excitation radiation into the biological sample, to minimize background fluorescence and/or to achieve a high signal-to-noise ratio.

Molecular recognition involves specific binding of two molecules. Molecules with binding specificity for target biomolecules are useful in a variety of research and diagnostic applications, such as labeling and separation of analytes, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assays (ELISA), western blot analysis, magnetic cell separation, and chromatography. The target biomolecule may be detected by labeling with a fluorescent dye.

Disclosure of Invention

Provided herein are water-soluble dipyrromethene-based dyes. In some cases, the water-soluble dye is a dipyrromethene-boron dye. Embodiments of the subject water-soluble dipyrromethene-based dyes have a narrow absorption maximum in the near 561nm laser line or the yellow-green range at 561nm laser line and are suitable for tandem dye systems. Embodiments of the subject dyes are effective to transfer energy to the acceptor chromophore, while a second dipyrromethene-type dye having the same or a different chemical structure is within its proximity to energy reception (energy-receiving proximity). Also provided herein are tandem dyes comprising the subject water-soluble dipyrromethene-type dyes, e.g., as donor chromophores. The tandem dye includes a pendant acceptor fluorophore configured to be an energy acceptor that is received in proximity to the energy of the donor water-soluble dipyrromethene-based dye. Methods of assessing the presence of a target analyte in a sample and methods of labeling a target molecule using a subject dye are also provided. Systems and kits for practicing the subject methods are also provided.

Drawings

It is to be understood that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1 shows an exemplary yellow-green water-soluble dipyrromethene-based dye of an embodiment of the present invention.

Fig. 2 shows absorption spectra of four exemplary dipyrromethene-boron dyes according to embodiments of the present invention.

Definition of the definition

As used herein, the terms "chemoselective functional group" and "chemoselective tag" are used interchangeably and refer to a functional group that, in some cases, after optionally activating one of the functional groups, can selectively react with another compatible functional group to form a covalent bond. Chemoselective functional groups of interest include, but are not limited to, thiols and maleimides or iodoacetamides, amines and carboxylic acids or activated esters thereof, as well as groups that can react with each other via click chemistry (e.g., azide and alkyne groups (e.g., cyclooctyne groups)), tetrazine, trans-cyclooctene, dienes and dienophiles, azide, sulfur (VI) fluoride exchange chemistry (SuFEX), sulfonyl fluoride, as well as hydroxyl, hydrazide, hydrazino, aldehyde, ketone, azide, alkyne, phosphine, epoxide, and the like.

As used herein, the term "sample" refers to a material or mixture of materials containing one or more analytes of interest, in some cases in liquid form. In some embodiments, the term is used in its broadest sense to refer to any plant, animal or bacterial material that contains cells or produces cellular metabolites, such as tissues or fluids isolated from individuals (including but not limited to plasma, serum, cerebrospinal fluid, lymph, tears, saliva, and tissue sections) or tissues or fluids isolated from cell culture components in vitro, as well as samples from the environment. The term "sample" may also refer to "biological samples". As used herein, the term "biological sample" refers to a whole organism or a subset of its tissues, cells, or components (e.g., bodily fluids, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). "biological sample" may also refer to a homogenate, lysate or extract prepared from: a subset of whole organisms or tissues, cells or components thereof, or fractions or parts thereof, including but not limited to plasma, serum, cerebrospinal fluid, lymph, external sections of skin, respiratory, intestinal and genitourinary tracts, tears, saliva, milk, blood cells, tumors and organs. In certain embodiments, the sample has been removed from an animal or plant. The biological sample may comprise cells. The term "cell" is used in its conventional sense to refer to the basic building block of living organisms (eukaryotic and prokaryotic organisms) which have at least a nucleus and a cell membrane. In certain embodiments, the cells include prokaryotic cells, such as cells from bacteria. In other embodiments, the cells include eukaryotic cells, such as cells obtained from a biological sample of an animal, plant, or fungus.

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

The term "polypeptide" refers to a polymeric form of amino acids of any length, including peptides ranging from 2 to 50 amino acids in length and polypeptides greater than 50 amino acids in length. The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" includes polymers of encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, as well as polypeptides having modified peptide backbones in which the conventional backbone has been replaced with a non-naturally occurring or synthetic backbone. The polypeptide may have any convenient length, e.g., 2 or more amino acids, e.g., 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. The "peptide" may 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 peptide is 5 to 30 amino acids in length.

As used herein, the term "isolated" refers to at least 60%, at least 75%, at least 90%, at least 95%, at least 98%, even at least 99% free of the portion of interest of other components associated with the portion of interest prior to purification.

The "plurality" contains at least 2 members. In some cases, the plurality may have 5 or more, 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.

Numerical ranges include numbers defining the range.

The term "specific binding" refers to the ability of a capture agent (or first member of a specific binding pair) to preferentially bind a particular analyte (or second member of a specific binding pair) present, for example, in a homogeneous mixture of different analytes. In some cases, the specific binding interaction will distinguish between desired and undesired analytes in the sample, with a specificity for the desired analyte that is 10-fold or more, such as 100-fold or more, or 1000-fold or more, than for the undesired analytes. In some cases, when the capture agent and analyte bind specifically with the capture agent/analyte complex, the affinity between them is at least 10 ^-8 M, at least 10 ^-9 M, e.g. up to 10 ^-10 M。

"affinity" refers to the strength of binding, with increased binding affinity being associated with lower Kd. In embodiments, affinity is determined by Surface Plasmon Resonance (SPR), for example as used by the Biacore system. The affinity of one molecule for another is determined by measuring the binding kinetics of the interaction, for example at 25 ℃.

The methods described herein include a number of steps. Each step may be performed after a predetermined amount of time has elapsed between steps, as desired. Thus, the time between performing each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, and include 5 hours or more. In certain embodiments, each subsequent step is performed immediately after the previous step is completed. In other embodiments, the steps may be performed after incubation or waiting time (e.g., a few minutes to overnight waiting time) after completion of the previous step.

As used herein, the terms "evaluate," "determine," "measure," "assess," and "determine" are used interchangeably and include quantitative and qualitative determinations.

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

The term "linker" or "linker" refers to a linking moiety that connects two groups and has a backbone length of 100 atoms or less. The linker or linker may 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 may be a straight chain, branched, cyclic or a single atom. In some cases, the linker is a branched linker, meaning a linking moiety that links three or more groups. In some cases, one, two, three, four, five or more carbon atoms of the linker backbone may be optionally substituted with a heteroatom of sulfur, nitrogen or oxygen. In some cases, the linker backbone comprises a linking functional group such as an ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester, or imine. The bonds between the backbone atoms may be saturated or unsaturated, and in some cases, no more than one, two, or three unsaturated bonds are present in the linker backbone. The linker may comprise one or more substituents, such as alkyl, aryl or alkenyl groups. The linker may include, but is not limited to, polyethylene glycol; ethers, thioethers, tertiary amines, alkyl groups, which may be straight or branched, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1-dimethylethyl (t-butyl), and the like. The linker backbone may comprise a cyclic group, e.g., aryl, heterocycle, or cycloalkyl, wherein the backbone comprises 2 or more atoms of the cyclic group, e.g., 2, 3, or 4 atoms. The linker may be cleavable or non-cleavable.

As used herein, the terms "water-solubilizing group," "water-soluble group," and WSG are used interchangeably and refer to a group or substituent that is sufficiently solvated in an aqueous environment (e.g., under physiological conditions) and imparts improved water solubility to the molecule to which it is attached. WSG increases the solubility of the multichromophore in aqueous host (predominant) solutions compared to control multichromophores lacking WSG. The water-solubilizing group can be any convenient hydrophilic group that is substantially solvated in an aqueous environment.

The terms "polyethylene oxide," "PEO," "polyethylene glycol," and "PEG" are used interchangeably and are meant to encompass compositions of the formula- - (CH) ₂ ----O--) _n -a polymer group of the chain described or a derivative thereof. In some embodiments, "n" is 5000 or less, such as 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, such as 3 to 15, or 10 to 15. It is to be understood that the PEG polymer groups can have any convenient length and can contain a variety of end groups and/or other substituents including, but not limited to, alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amidyl end groups and/or substituents. PEG groups that may be suitable for use in the subject multichromophores include those described by S.Zalipsky, "Functionalized poly (ethylene glycol) for preparation of biologically relevant conjugates", bioconjugate Chemistry 1995,6 (2), 150-165; and Zhu et al, "Water-Soluble Conjugated Polymers for Imaging, diagnostics, and Therapy", chem. Rev.,2012,112 (8), pages 4687-4735.

The term "alkyl" by itself or as part of another substituent refers to a saturated branched or straight chain monovalent hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane. Alkyl groups of interest include, but are not limited to, methyl; ethyl, propyl, such as propan-1-yl or propan-2-yl; and butyl, such as but-1-yl, but-2-yl, 2-methyl-prop-1-yl or 2-methyl-prop-2-yl. In some embodiments, the alkyl group contains 1 to 20 carbon atoms. In some embodiments, the alkyl group contains 1 to 10 carbon atoms. In certain embodiments, the lower alkyl group contains 1 to 6 carbon atoms, such as 1 to 4 carbon atoms. The term includes, for example, straight and branched chain hydrocarbon groups such as methyl (CH) ₃ (-), ethyl (CH) ₃ CH ₂ (-), n-propyl (CH) ₃ CH ₂ CH ₂ (-), isopropyl ((CH) ₃ ) ₂ CH-), n-butyl (CH) ₃ CH ₂ CH ₂ CH ₂ (-), isobutyl ((CH) ₃ ) ₂ CHCH ₂ (-), sec-butyl ((CH) ₃ )(CH ₃ CH ₂ ) CH-), tert-butyl group ((CH) ₃ ) ₃ C-), n-pentyl (CH) ₃ CH ₂ CH ₂ CH ₂ CH ₂ (-) and neopentyl ((CH) ₃ ) ₃ CCH ₂ -)。

The term "substituted alkyl" refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain have been optionally replaced by, for example, -O-, -N-, -S (O) _n - (wherein n is 0 to 2), -NR- (wherein R is hydrogen or alkyl) and having 1 to 5 substituents selected from: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -aryl, -SO ₂ -heteroaryl and-NR ^a R ^b Wherein R 'and R' may be the same or different and are selected from the group consisting of hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl.

"alkoxy" refers to the group-O-alkyl, wherein alkyl is as defined herein. Alkoxy groups include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to the group: alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, wherein alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

The term "substituted alkoxy" refers to a group: 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.

"alkynyl" or "alkyne" refers to a straight or branched monovalent hydrocarbyl group (hydrocarbyl groups) having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and having at least 1, preferably 1 to 2 triple bond unsaturation sites. Examples of such alkynyl groups include ethynyl (-C≡CH) and propargyl (-CH) ₂ C≡CH)。

The term "substituted alkynyl" or "substituted alkyne" refers to an alkynyl group as defined herein having 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, 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, -SO- ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl and-SO ₂ -heteroaryl.

"amino" means a radical-NH ₂ . 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.

"aryl" by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of an aromatic ring system. Interest inAryl groups include, but are not limited to, groups derived from: acenaphthene, acenaphthene, acephenanthrene, anthracene, azulene, benzene,Coronene, fluoranthene, fluorene, acene, hexaphene, hexadiene (hexaene), asymmetric indacene (as-indacene), symmetrical indacene (s-indacene), indane, indene, naphthalene, octaacene, octaphene, cyclooctatetraene, egg benzene, penta-2, 4-diene, pentacene, pentalene, perylene, phenalene, phenanthrene, picene, heptadiene (pleiadiene), pyrene, pyranene, rubicene, benzo [9,10 ]]Phenanthrene, trinaphthalene, and the like. In certain embodiments, the aryl group comprises from 6 to 20 carbon atoms. In certain embodiments, the aryl group contains 6 to 12 carbon atoms. Examples of aryl groups are phenyl and naphthyl.

Unless otherwise limited by the definition of aryl substituent, "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, acylamino, alkylaryl, aryl, aryloxy, azido, carboxy, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyamido, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioaheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl, -SO ₂ Heteroaryl and trihalomethyl.

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

"heterocycle", "heterocyclic", "heterocycloalkyl" and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple condensed rings (including fused bridged and spiro ring systems) and having from 3 to 20 ring atoms (including 1 to 10 heteroatoms). These ring atoms are selected from nitrogen, sulfur or oxygen, wherein in the fused ring system one or more of the rings may be cycloalkyl, aryl or heteroaryl, provided that the point of attachment passes through the non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atoms of the heterocyclic group are optionally oxidized to provide an N-oxide, -S (O) -or-SO ₂ -a portion.

Examples of heterocycles and heteroaryls include, but are not limited to, azetidines, pyrroles, imidazoles, pyrazoles, pyridines, pyrazines, pyrimidines, pyridazines, indolines, indoles, indolines, indazoles, purines, quinolizines, isoquinolines, quinolines, phthalazines, naphthyridines, quinoxalines, quinazolines, cinnolines, pteridines, carbazoles, carbolines, phenanthridines, acridines, phenanthrolines, isothiazoles, phenazines, isoxazoles, phenoxazines, phenothiazines, imidazolidines, imidazolines, piperidines, piperazines, indolines, phthalimides, 1,2,3, 4-tetrahydroisoquinolines, 4,5,6, 7-tetrahydrobenzo [ b ] thiophenes, thiazoles, thiazolidines, thiophenes, benzo [ b ] thiophenes, morpholines, thiomorpholines (also known as thiomorpholines), 1-dioxothiomorpholines, piperidines, pyrrolidines, tetrahydrofurans, and the like.

Unless otherwise limited by the definition of substituent, a "substituted heteroaryl" refers to a heteroaryl 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, alkylaryl, aryl, aryloxy, azido, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyamido, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioaheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl, -SO ₂ Heteroaryl and trihalomethyl.

The term "alkylaryl" or "aralkyl" refers to a group: -alkylene-aryl and substituted alkylene-aryl, wherein alkylene, substituted alkylene and aryl are as defined herein.

"alkylene" means a divalent aliphatic hydrocarbon group preferably having 1 to 6, more preferably 1 to 3 carbon atoms, which is straight or branched and optionally interrupted by one or more groups selected from-O-, -NR ¹⁰ -、-NR ¹⁰ C(O)-、-C(O)NR ¹⁰ -groups of the like. For example, the term includes methylene (-CH) ₂ (-), ethylene (-CH) ₂ CH ₂ (-), n-propylene (-CH) ₂ CH ₂ CH ₂ (-), isopropylidene (-CH) ₂ CH(CH ₃ )-)、(-C(CH ₃ ) ₂ CH ₂ CH ₂ -)、(-C(CH ₃ ) ₂ CH ₂ C(O)-)、(-C(CH ₃ ) ₂ CH ₂ C(O)NH-)、(-CH(CH ₃ )CH ₂ (-), etc. "substituted alkylene" refers to an alkylene group having 1 to 3 hydrogens substituted with a substituent as described below for carbon in the definition of "substituted" below。

"substituted" refers to a group in which one or more hydrogen atoms are independently replaced with 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 、-CF ₃ 、-CN、-OCN、-SCN、-NO、-NO ₂ 、＝N ₂ 、-N ₃ 、-S(O) ₂ O ^- 、-S(O) ₂ OH、-S(O) ₂ R ⁶⁰ 、-OS(O) ₂ O ^- 、-OS(O) ₂ R ⁶⁰ 、-P(O)(O ^- ) ₂ 、-P(O)(OR ⁶⁰ )(O ^- )、-OP(O)(OR ⁶⁰ )(OR ⁶¹ )、-C(O)R ⁶⁰ 、-C(S)R ⁶⁰ 、-C(O)OR ⁶⁰ 、-C(O)NR ⁶⁰ R ⁶¹ 、-C(O)O ^- 、-C(S)OR ⁶⁰ 、-NR ⁶² C(O)NR ⁶⁰ R ⁶¹ 、-NR ⁶² C(S)NR ⁶⁰ R ⁶¹ 、-NR ⁶² C(NR ⁶³ )NR ⁶⁰ R ⁶¹ and-C (NR) ⁶² )NR ⁶⁰ R ⁶¹ Wherein M is halogen; r is R ⁶⁰ 、R ⁶¹ 、R ⁶² And R is ⁶³ Independently is 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 is ⁶¹ Together with the nitrogen atom to which they are bonded, form a cycloheteroalkyl or substituted cycloheteroalkyl ring; and R is ⁶⁴ And R is ⁶⁵ Independently is hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, or optionally R ⁶⁴ And R is ⁶⁵ Together with the nitrogen atom to which they are bonded form 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 、-CF ₃ 、-CN、-OCN、-SCN、-NO、-NO ₂ 、＝N ₂ 、-N ₃ 、-S(O) ₂ R ⁶⁰ 、-OS(O) ₂ O ^- 、-OS(O) ₂ R ⁶⁰ 、-P(O)(O ^- ) ₂ 、-P(O)(OR ⁶⁰ )(O ^- )、-OP(O)(OR ⁶⁰ )(OR ⁶¹ )、-C(O)R ⁶⁰ 、-C(S)R ⁶⁰ 、-C(O)OR ⁶⁰ 、-C(O)NR ⁶⁰ R ⁶¹ 、-C(O)O ^- 、-NR ⁶² C(O)NR ⁶⁰ R ⁶¹ . In certain embodiments, the substituents include-M, -R ⁶⁰ 、＝O、-OR ⁶⁰ 、-SR ⁶⁰ 、-NR ⁶⁰ R ⁶¹ 、-CF ₃ 、-CN、-NO ₂ 、-S(O) ₂ R ⁶⁰ 、-P(O)(OR ⁶⁰ )(O ^- )、-OP(O)(OR ⁶⁰ )(OR ⁶¹ )、-C(O)R ⁶⁰ 、-C(O)OR ⁶⁰ 、-C(O)NR ⁶⁰ R ⁶¹ 、-C(O)O ^- . In certain embodiments, the substituents include-M, -R ⁶⁰ 、＝O、-OR ⁶⁰ 、-SR ⁶⁰ 、-NR ⁶⁰ R ⁶¹ 、-CF ₃ 、-CN、-NO ₂ 、-S(O) ₂ R ⁶⁰ 、-OP(O)(OR ⁶⁰ )(OR ⁶¹ )、-C(O)R ⁶⁰ 、-C(O)OR ⁶⁰ 、-C(O)O ^- Wherein R is ⁶⁰ 、R ⁶¹ And R is ⁶² As defined above. For example, the substituted group may bear methylenedioxy substituents, 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".

It is to be understood that, of all the substituted groups defined hereinabove, polymers obtained by defining substituents having other substituents themselves (e.g., substituted aryl groups having a substituted aryl group as a substituent, the substituent itself being substituted with a substituted aryl group, the substituted aryl group being further substituted with a substituted aryl group, etc.) are not intended to be included herein. In this case, the maximum number of such substitutions is three. For example, the sequential substitution of substituted aryl groups specifically contemplated herein is limited to substituted aryl- (substituted aryl) -substituted aryl groups.

Unless otherwise indicated, the naming of substituents not explicitly defined herein results from the terminal portion of a functional group being preceded and subsequently named towards the adjacent functional group at the point of attachment. For example, the substituent "arylalkoxycarbonyl" refers to the group (aryl) - (alkyl) -O-C (O) -.

With respect to any of the groups disclosed herein that contain one or more substituents, it is of course understood that these groups do not contain any sterically impractical and/or synthetically infeasible substitution or substitution patterns. Furthermore, the subject compounds include all stereochemical isomers resulting from the substitution of these compounds.

It is to be understood that the definitions provided herein are not intended to be mutually exclusive. Thus, some chemical moieties may fall within the definition of more than one term.

Other definitions of terms may appear throughout the specification.

Detailed Description

As outlined above, provided herein are water-soluble dipyrromethene-based dyes. In some cases, the water-soluble dye is a dipyrromethene-boron dye. Embodiments of the subject water-soluble dipyrromethene-based dyes have a narrow absorption maximum in the near 561nm laser line or the yellow-green range at 561nm laser line and are suitable for tandem dye systems. Embodiments of the subject dyes are effective to transfer energy to the acceptor chromophore, while a second dipyrromethene-type dye having the same or a different chemical structure is within its proximity to the energy acceptor. Also provided herein are tandem dyes comprising the subject water-soluble dipyrromethene-type dyes, e.g., as donor chromophores. The tandem dye includes a pendant acceptor fluorophore configured to be an energy acceptor that is received in proximity to the energy of the donor water-soluble dipyrromethene-based dye. Methods of assessing the presence of a target analyte in a sample and methods of labeling a target molecule using a subject tandem dye are also provided. Systems and kits for practicing the subject methods are also provided.

Before the present invention is described in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

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

Certain ranges of values provided herein are preceded by the term "about". The term "about" is used herein to provide literal support for the exact number following it, as well as numbers near or approximating the end of the term. In determining whether a number is close or approximate to a specifically recited number, the close or approximate non-recited number may be a number that provides a substantial equivalent of the specifically recited number in the context in which it is presented.

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

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

Note 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 the claims may be drafted to exclude any optional element. Accordingly, this description is intended to serve as a prerequisite basis for the use of exclusive terminology (e.g., "solely," "only," etc.) or the use of "negative" limitations in connection with the recitation of claim elements.

As will be apparent to those of skill in the art upon reading this disclosure, each of the various embodiments described and illustrated herein has discrete components and features that may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any of the enumerated methods may be performed in the order of enumerated events or in any other order that is logically possible.

Although the apparatus and methods have been and will be described for the purpose of grammatical fluidity and functional explanation, it is to be expressly understood that the claims, unless explicitly stated by 35u.s.c. -) 112, are not to be construed as necessarily limited in any way by the interpretation of the terms of "means" or "steps," but are to be given the full scope of the meaning and equivalents of the definitions provided by the claims under judicial doctrine of equivalents, and that the claims, if explicitly stated by 35u.s.c. -) 112, are to be given full legal equivalents by 35u.s.c. -) 112.

In further describing aspects of the invention, embodiments of the subject dyes are first reviewed in greater detail, followed by review of tandem dyes comprising the subject dyes, and methods of using the dyes.

Dipyrromethene-based dyes

As summarized above, the present disclosure provides water-soluble dipyrromethene-based dyes. The term "dipyrromethene-based dye" refers to a dye comprising the following core structure:

in some cases, the subject dipyrromethene-type dye is a yellow-green absorbing dye. In some cases, the subject water-soluble dipyrromethene-type dye absorbs in the yellow-green range at 561 nm. In some cases, the water-soluble dipyrromethene-based dye has an absorbance maximum of 540nm to 590nm, 555 to 585nm, e.g., 555 to 575nm, e.g., 555 to 574nm, 555 to 573nm, 555 to 572nm, 555 to 571nm, 555 to 570nm, 555 to 569nm, 555 to 568nm, 555 to 567nm, 555 to 566nm, 555 to 565nm, 555 to 564nm, 555 to 563nm, or 555 to 562nm. In some cases, the water-soluble dipyrromethene-based dye has an absorbance maximum of 558 to 568nm, such as 558 to 567nm, 558 to 566nm, 558 to 565nm, 558 to 564nm, 558 to 563nm, or 558 to 562nm. In some cases, the absorption maximum of the subject water-soluble dipyrromethene-based dye is, for example, about 561nm or 562nm or thereabout.

In certain embodiments, the subject water-soluble dipyrromethene-based dyes exhibit narrow absorption spectra. The narrow absorption spectrum is an absorption spectrum having an absorption maximum peak with a full width at half maximum (FWHM) of 35nm or less, e.g., 25nm or less, 20nm or less, 15nm or less, 12nm or less, or 10nm or less, e.g., as measured in ethanol or Phosphate Buffered Saline (PBS).

In some cases, the extinction coefficient of the dipyrromethene-based dye is 5×10 ⁵ cm ^-1 M ^-1 Or greater, e.g. 6X 10 ⁵ cm ^-1 M ^-1 Or greater, 7X 10 ⁵ cm ^-1 M ^-1 Or greater, 8X 10 ⁵ cm ^-1 M ^-1 Or larger, 9X 10 ⁵ cm ^-1 M ^-1 Or greater, e.g. 1X 10 ⁶ cm ^-1 M ^-1 Or greater, 1.5X10 ⁶ cm ^-1 M ^-1 Or greater, 2X 10 ⁶ cm ^-1 M ^-1 Or greater, 2.5X10 ⁶ cm ^-1 M ^-1 Or greater, 3X 10 ⁶ cm ^-1 M ^-1 Or greater, 4X 10 ⁶ cm ^-1 M ^-1 Or greater, 5X 10 ⁶ cm ^-1 M ^-1 Or greater, 6X 10 ⁶ cm ^-1 M ^-1 Or greater, 7X 10 ⁶ cm ^-1 M ^-1 Or greater, or 8X 10 ⁶ cm ^-1 M ^-1 Or larger. In some embodiments, the molar extinction coefficient of the polymeric tandem dye is 5X 105M-1cm-1 or greater. In certain embodiments, the tandem dye has a molar extinction coefficient of 1×10 ⁶ M ^- ¹ cm ^-1 Or larger.

The subject water-soluble dipyrromethene dyes are substituted with one or more water-solubilizing groups (WSG). The water-soluble dyes of the present disclosure are soluble under aqueous conditions, which makes them particularly useful in a variety of bioassays. The subject water-soluble dyes, tandem dyes, and conjugates thereof are resistant to undesired aggregation, which provides advantageous fluorescence and spectral characteristics in various bioassays. Aggregation of the dye is undesirable because it can lead to a decrease in fluorescence signal, e.g., quenching of dye fluorescence via aggregation. The subject water-soluble dyes and tandem dyes containing them are useful as fluorescent reporter molecules for a variety of biosensors and provide excellent brightness signals with excitation and emission wavelength selective ranges for applications such as flow cytometry and imaging.

A variety of water-soluble polymer groups may be suitable for WSG of the subject dye. Any convenient Water Solubilising Group (WSG) may be included in the dyes described herein to provide increased water solubility. Although the increase in solubility may vary, in some cases the increase (as compared to the compound without WSG) is 2-fold or more, e.g., 5-fold, 10-fold, 25-fold, 50-fold, 100-fold or more. In some cases, the hydrophilic water solubilizing group is charged, e.g., positively or negatively charged. In some cases, the hydrophilic water solubilizing group is a neutral hydrophilic group. In some embodiments, the WSG is branched (e.g., asAs described herein). In some cases, the WSG is linear. In some embodiments, the WSG is a hydrophilic polymer, e.g., polyethylene glycol, modified PEG, peptide sequences, peptoids, carbohydrates, oxazolines, polyols, dendritic units, dendritic polyglycerols, cellulose, chitosan, or derivatives thereof. Water-solubilizing groups of interest include, but are not limited to, carboxylate, phosphonate, phosphate, sulfonate, sulfate, sulfinate, sulfonium, esters, polyethylene glycol (PEG) and modified PEG, hydroxyl, amine, amino acid, ammonium, guanidinium, pyridinium, polyamine and sulfonium, polyols, linear or cyclic sugars, primary, secondary, tertiary or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols, including polyethers, -COOM', -SO ₃ M'、-PO ₃ M'、-NR ₃ ⁺ 、Y'、(CH ₂ CH ₂ O) _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 alkyl (e.g., methyl), and M' can be a cationic counterion or hydrogen, - - (CH) ₂ CH ₂ O) _yy CH ₂ CH ₂ XR ^yy 、--(CH ₂ CH ₂ O) _yy CH ₂ CH ₂ X--、--X(CH ₂ CH ₂ O) _yy CH ₂ CH ₂ -, diols and polyethylene glycols, wherein yy is selected from 1 to 1000, X is selected from O, S and NR ^ZZ And R is ^ZZ And R is ^YY Independently selected from H and C _1-3 An alkyl group. In some cases, WSG is (CH ₂ ) _x (OCH ₂ CH ₂ ) _y OCH ₃ Wherein each x is independently an integer from 0-20 and each y is independently an integer from 0 to 50. In some cases, the water-solubilizing group comprises a nonionic polymer (e.g., a PEG polymer) substituted at the terminus with an ionic group (e.g., sulfonate).

In some embodiments of this formula, the pendent group according to interest comprises a moiety selected from (CH ₂ ) _x (OCH ₂ CH ₂ ) _y OCH ₃ Wherein each x is independently an integer from 0 to 20 and each y is independently an integer from 0 to 50A number; and optionally by one or more halogens, hydroxy, C ₁ -C ₁₂ Alkoxy Or (OCH) ₂ CH ₂ ) _z OCH ₃ A substituted benzyl group, wherein each z is independently an integer from 0 to 50. In some cases, the substituent is (CH ₂ ) ₃ (OCH ₂ CH ₂ ) ₁₁ OCH ₃ . In some embodiments, one or more of the substituents are at least one member selected from (CH ₂ ) _x (OCH ₂ CH ₂ ) _y OCH ₃ A benzyl substituted with a WSG group (e.g., one or two WSG groups), wherein each x is independently an integer from 0-20, and each y is independently an integer from 0 to 50.

Multiple WSGs may be included at a single location in the subject dye via branched linkers. In certain embodiments, the branched linker is an aralkyl substituent further disubstituted with a water solubilizing group. Thus, in some cases, a branched linker group is a substituent of a dye that links the dye to two or more water-solubilizing groups. In certain embodiments, the branched linker is an amino acid, e.g., a lysine amino acid linked to three groups via an amino group and a carboxylic acid group. In some cases, incorporation of multiple WSGs via branched linkers imparts desirable solubility to the dye. In some cases, WSG is a nonionic side chain group that is capable of imparting solubility in water in excess of 50 mg/mL. In some cases, WSG is a nonionic side chain group that is capable of imparting solubility in water in excess of 100 mg/mL. In some embodiments, the dye comprises substituents selected from alkyl, aralkyl, and heterocyclic groups, each of which is further substituted with a hydrophilic polymer group comprising a water-solubilizing group, such as polyethylene glycol (PEG) (e.g., a 6-24 unit PEG group).

Water-soluble polymers of interest that can be used in WSG include polyethylene glycol (PEG) groups or modified PEG groups. Water-soluble polymers of interest include, but are not limited to, polymers based on polyalkylene oxides, such as polyethylene glycol "PEG" (see, e.g., "Poly (ethylene glycol) Chemistry: biotechnical and Biomedical Applications", J.M.Harris, plenum Press, new York, N.Y. (1992); and "Poly (ethylene glycol) Chemistry and Biological Applications", J.M.Harris and S.Zalipsky, ACS (1997); and 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. Pat. Nos. 4,179,337; 5,075,046; 5,089,261; 5,100,992; 5,134,192; 5,166,309; 5,171,264; 5,213,891; 5,219,564; 5,275,838; 5,281,698; 5,298,643; 5,312,808; 5,321,095; 5,324,844; no. 5,349,001; 5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805; no. 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662; 5,637,749; no. 5,643,575; 5,650,388; 5,681,567; 5,686,110; no. 5,730,990; 5,739,208; no. 5,756,593; no. 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; no. 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; 6,214,966).

Examples of water-soluble polymers of interest include, but are not limited to, those containing polyalkylene oxides, polyamide alkylene oxides, or derivatives thereof, including those comprising the formula- (CH) ₂ -CH ₂ -O) -alkylene oxides and polyamide alkylene oxides of ethylene oxide repeat units. Further examples of polymers of interest include those having the formula- [ C (O) -X-C (O) -NH-Y-NH]n-or- [ NH-Y-NH-C (O) -X-C (O)] _n Polyamides having a molecular weight of greater than 1,000 daltons, wherein X and Y are identical or different and may be branchedN is a non-continuous integer from 2 to 100 (e.g., from 2 to 50), and wherein either or both of X and Y comprise biocompatible, substantially non-antigenic, water-soluble repeat units that may be linear or branched. Further examples of water-soluble repeating units include the formula- (CH) ₂ -CH ₂ -O) -or- (O-CH) ₂ -CH ₂ ) -ethylene oxide. The number of such water-soluble repeat units can vary significantly, with the number of such units ranging from 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 6-100, e.g., 2 to 50 or 6 to 50. Examples of embodiments are those wherein one or both of X and Y are selected from: - ((CH) ₂ ) _n1 -(CH ₂ -CH ₂ -O) _n2 -(CH ₂ ) -or- ((CH) ₂ ) _n1 -(O-CH ₂ -CH ₂ ) _n2 -(CH ₂ ) _n-1 (-), wherein n1 is 1 to 6, 1 to 5, 1 to 4, or 1 to 3, and wherein n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2 to 8, or 2 to 5. Further examples of embodiments are those wherein X is- (CH) ₂ -CH ₂ ) -, and wherein Y is- (CH) ₂ -(CH ₂ -CH ₂ -O) ₃ -CH ₂ -CH ₂ -CH ₂ ) -or- (CH) ₂ -CH ₂ -CH ₂ -(O-CH ₂ -CH ₂ ) ₃ -CH ₂ )-。

The term modified polymer (e.g., modified PEG) refers to a water-soluble polymer that has been modified or derivatized at either or both termini, e.g., to include terminal substituents (e.g., terminal alkyl, substituted alkyl, alkoxy, or substituted alkoxy, etc.) and/or terminal linking functionalities (e.g., suitable for forming an attached amino or carboxylic acid group via an amide bond) suitable for linking the polymer to a molecule of interest (e.g., to a light harvesting chromophore via a branching group). The subject water-soluble polymers may be adapted to contain any convenient linking group. It will be appreciated that in some cases, the water-soluble polymer may include some dispersibility relative to the polymer length, depending on the method of preparing and/or purifying the polymer starting material. In some cases, the water-soluble polymer is monodisperse.

The water-soluble polymer may comprise one or more spacers or linkers. Examples of spacers or linkers include linear or branched moieties comprising one or more repeating units for use in a water-soluble polymer, diamino and/or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, and the like, which may contain, for example, up to 18 carbon atoms or even contain additional polymer chains.

The water-soluble polymer moiety or one or more spacers or linkers of the polymer moiety (when present) may comprise a biostable or biodegradable polymer chain or unit. For example, polymers with repeating linkers have varying degrees of stability under physiological conditions, depending on the bond instability. Polymers having such linkages can be classified according to their relative hydrolysis rate under physiological conditions, based on the known hydrolysis rate of low molecular weight analogues, e.g., from less stable to more stable, e.g., polyurethane (-NH-C (O) -O-) > polyorthoester (-O-C ((OR) (R')) -O-) > polyamide (-C (O) -NH-). Similarly, the linking system linking the water-soluble polymer to the target molecule may be biostable or biodegradable, e.g., from less stable to more stable: carbonate (-O-C (O) -O-) > ester (-C (O) -O-) > urethane (-NH-C (O) -O-) > orthoester (-O-C ((OR) (R')) -O-) > amide (-C (O) -NH-). In general, it may be desirable to avoid the use of sulfated polysaccharides, depending on the instability of the sulfuric acid groups. Furthermore, the use of polycarbonates and polyesters may be less desirable. These bonds are provided by way of example and are not intended to limit the types of bonds that may be employed in the polymer chains or linking systems of the water-soluble polymers useful in the WSGs disclosed herein.

The water-soluble group (WSG) is capable of imparting solubility to the subject dye or polymeric tandem dye in water in excess of 10mg/mL, such as in excess of 20mg/mL, in excess of 30mg/mL, in excess of 40mg/mL, in excess of 50mg/mL, in excess of 60mg/mL, in excess of 70mg/mL, in excess of 80mg/mL, in excess of 90mg/mL, or in excess of 100 mg/mL. In certain instances, branched nonionic water-soluble groups (WSGs) are capable of imparting solubility to the subject dye or polymeric tandem dye of 20mg/mL or greater, such as 30mg/mL or greater, 40mg/mL or greater, 50mg/mL or greater, 60mg/mL or greater, 70mg/mL or greater, 80mg/mL or greater, 90mg/mL or greater, 100mg/mL or greater, or even greater, in water (e.g., aqueous buffer). It is understood that water-soluble dipyrromethene-based dyes may form discrete water solvated nanoparticles in aqueous systems under certain conditions. In some cases, the water-solvated nanoparticles are anti-aggregated and can be used in a variety of bioassays.

In some cases, the dipyrromethene-based dye is a dipyrromethene-boron-based dye. In certain instances, the water-soluble dipyrromethene-based dye has formula (I):

wherein:

R ¹ -R ⁷ each independently selected from H, alkyl, halogen, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, amido, substituted amido, thiol, substituted thiol, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water Solubilizing Group (WSG), and-T ¹ -J ¹ Or (b)

Optionally selected from R ⁶ And R is ⁷ 、R ² And R is ³ 、R ⁵ And R is ⁶ 、R ³ And R is ⁴ 、R ² And R is ¹ R is as follows ⁷ And R is ¹ Any one or more pairs of substituents together form a divalent group and are cyclic and taken together with the carbon atom to which they are bound provide a 5 or 6 membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5 or 6 membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N) which may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substitutedAlkenyl, alkynyl, substituted alkynyl, water Solubilising Groups (WSG) and-T ¹ -J ¹ Is substituted by a substituent of (a);

T ¹ is an optional linker;

J ¹ is a repeating unit of a compound of formula (I); 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 Water Solubilizing Group (WSG), or

Y ¹ And Y ² Together with the boron atom to which they are bound, form a 5-or 6-membered ring (e.g., a 5-or 6-membered ring containing a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-solubilizing group (WSG), and-T ¹ -Z ¹ Is substituted by a substituent of (a); and

wherein is selected from Y ¹ 、Y ² And R is ¹ -R ⁷ At least one moiety of (2) is WSG, or is selected from Y ¹ 、Y ² And R is ¹ -R ⁷ Is substituted with WSG, wherein WSG has formula (Ia):

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ¹ ) _m (Ia)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ¹ is a water-soluble polymer comprising 6 to 24 monomer units; and

m is an integer from 1 to 3.

In some cases of WSG of formula (Ia), L ¹ 、L ² And X ¹ Is absent (i.e., n1-n3 are 0), and WSG is Z ¹ I.e. a water-soluble polymer comprising 6-24 monomer units. In some cases, is selected from Y ¹ 、Y ² And R is ¹ -R ⁷ Is at least partially Z ¹ Substituted with radicals and Y ¹ 、Y ² And R is ¹ -R ⁷ As defined above. In some cases, Y ¹ And Y ² Are all Z ¹ Group substitution (e.g., as described herein). In some cases, Z ¹ Is a PEG or modified PEG polymer of 6-24 monomer units, such as 10-30, 10-24, 10-20, 12-24, 12-20, 12-16 or 16-20 monomer units.

In certain cases of WSG of formula (Ia), L ² And X ¹ Absent, n1 is 1, and WSG is-L ¹ -Z ¹ Wherein L is ¹ Is a linker having a backbone of 20 atoms or less in length (e.g., as described herein), and Z ¹ Is WSG (e.g., as described herein). In some cases, L ¹ Selected from the group consisting of an alkyl or substituted alkyl linker, an alkenyl or substituted alkenyl linker, an alkynyl or substituted alkynyl linker, an alkoxy or substituted alkoxy linker, a PEG linker, a sulfonylamino-alkyl or substituted sulfonylamino-alkyl linker, an acylamino-alkyl or substituted acylamino-alkyl linker, and an alkyl-acylamino-alkyl or substituted alkyl-acylamino-alkyl linker. In some cases, L ¹ Selected from C ₁ -C ₁₂ Alkyl or substituted alkyl linker, C ₁ -C ₁₂ Alkenyl or substituted alkenyl linker, C ₁ -C ₁₂ Alkynyl or substituted alkynyl linkers, C ₁ -C ₁₂ Alkoxy or substituted alkoxy linker, C ₁ -C ₁₂ Amido-alkyl or substituted amido-alkyl linker, and C ₁ -C ₁₂ Alkyl-amido-alkyl or substituted alkyl-amido-alkyl linkers. Z is Z ¹ Is a water-soluble polymer (e.g., as described herein). In some cases, the water-soluble polymer is a PEG group. In some cases, Z ¹ Is a monomer of 6 to 24 monomersA PEG or modified PEG polymer of 10-30, 10-24, 10-20, 12-24, 12-20, 12-16 or 16-20 monomer units.

In some other embodiments of the WSG of formula (Ia), n1 and n2 are each 1, n3 is 1 or 0, and m is 2, such that the WSG has formula (Ic) or (Id):

Wherein L is ¹ 、X ¹ 、L ² And Z ¹ As defined above. In some cases, L ¹ Is a linker having a main chain of 20 atoms or less in length, and Z ¹ Is WSG (e.g., as described herein). In some cases, L ¹ Selected from the group consisting of an alkyl or substituted alkyl linker, an alkenyl or substituted alkenyl linker, an alkynyl or substituted alkynyl linker, an acyl or substituted acyl, an alkoxy or substituted alkoxy linker, a PEG linker, a sulfonylamino-alkyl or substituted sulfonylamino-alkyl linker, an acylamino-alkyl or substituted acylamino-alkyl linker, and an alkyl-acylamino-alkyl or substituted alkyl-acylamino-alkyl linker. In some cases, L ¹ And L ² Each independently selected from C ₁ -C ₁₂ Alkyl or substituted alkyl linker, C ₁ -C ₁₂ Alkenyl or substituted alkenyl linker, C ₁ -C ₁₂ Alkynyl or substituted alkynyl linkers, C ₁ -C ₁₂ Acyl or substituted acyl linker, C ₁ -C ₁₂ Alkoxy or substituted alkoxy linker, C ₁ -C ₁₂ Amido-alkyl or substituted amido-alkyl linker, and C ₁ -C ₁₂ Alkyl-amido-alkyl or substituted alkyl-amido-alkyl linkers. In some cases, the linker comprises a carbonyl group. In some cases, L ¹ Containing carbonyl groups, and L ² Is C ₁ -C ₁₂ Alkyl or substituted alkyl. In some cases, the branching point X ¹ Selected from N, CR', C (=O) N, SO ₂ N, trisubstituted aryl groups (e.g., 1,3, 5-phenyl), tetrasubstituted aryl groups, and trisubstituted aryl groupsSubstituted heteroaryl groups. In some cases, the branching point X ¹ Is a nitrogen atom. In other cases, the branching point X ¹ Is CR 'wherein R' is selected from hydrogen, alkyl, substituted alkyl or-L ² -Z ¹ (e.g., as described herein). Z is Z ¹ Is a water-soluble polymer (e.g., as described herein). In some cases, the water-soluble polymer is a PEG group. In some cases, each Z ¹ PEG or modified PEG polymers independently of 6 to 24 monomer units, for example 10 to 30, 10 to 24, 10 to 20, 12 to 24, 12 to 20, 12 to 16 or 16 to 20 monomer units. In some cases, R ⁵ And R is ⁴ Comprises WSG of formula (Ic) or (Id).

In some other embodiments, WSG is a branched group of formula (Ie):

wherein L is ¹ 、X ¹ 、L ² N3 and Z ¹ Each as defined above; l (L) ^2' N3' and Z ^1' Respectively as for L herein ² N3 and Z ¹ Defined as follows.

In some other embodiments, WSG is a branched group of formula (If):

wherein L is ¹ 、X ¹ 、L ² N3 and Z ¹ Each as defined above; and X is ¹ Is a branching group selected from carbon atoms and tetra-substituted aryl groups (e.g., 1,3,4, 5-phenyl).

In some embodiments, the water-soluble dipyrromethene-type dye of formula (I) further comprises WSG of formula (Ib):

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ² ) _m (Ib)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ² is a water-soluble polymer comprising 1 to 50 monomer units; and

m is an integer from 1 to 3.

In some cases of WSG of formula (Ib), L ¹ 、L ² And X ¹ Is absent (i.e., n1-n3 are 0), and WSG is Z ² I.e. a water-soluble polymer comprising 1 to 50 monomer units. In some cases, Z ² Is a PEG or modified PEG polymer of 1-50 monomer units, such as 1-40, 1-30, 1-20, 2-24, 2-20, 2-10 or 2-6 monomer units.

In some cases of WSG of formula (Ib), L ² And X ¹ Absent, n1 is 1, and WSG is-L ¹ -Z ² Wherein L is ¹ Is a linker having a backbone of 20 atoms or less in length (e.g., as described herein), and Z ² Is WSG (e.g., as described herein). In some cases, L ¹ Selected from the group consisting of an alkyl or substituted alkyl linker, an alkenyl or substituted alkenyl linker, an alkynyl or substituted alkynyl linker, an alkoxy or substituted alkoxy linker, a PEG linker, a sulfonylamino-alkyl or substituted sulfonylamino-alkyl linker, an acylamino-alkyl or substituted acylamino-alkyl linker, and an alkyl-acylamino-alkyl or substituted alkyl-acylamino-alkyl linker. In some cases, L ¹ Selected from C ₁ -C ₁₂ Alkyl or substituted alkyl linker, C ₁ -C ₁₂ Alkenyl or substituted alkenyl linker, C ₁ -C ₁₂ Alkynyl or substituted alkynyl linkers, C ₁ -C ₁₂ Alkoxy or substituted alkoxy linker, C ₁ -C ₁₂ Amido-alkyl or substituted amido-alkyl linker, and C ₁ -C ₁₂ Alkyl-amido-alkyl or substituted alkyl-amido-alkyl linkers. Z is Z ¹ Is a water-soluble polymerAn object (e.g., as described herein). In some cases, the water-soluble polymer is a PEG group. In some cases, Z ² Is a PEG or modified PEG polymer of 1-50 monomer units, such as 1-40, 1-30, 1-20, 2-24, 2-20, 2-10 or 2-6 monomer units.

In some other embodiments of the WSG of formula (Ib), n1 and n2 are each 1, n3 is 1 or 0, and m is 2, such that the WSG has formula (Ig) or (Ih):

wherein L is ¹ 、X ¹ 、L ² And Z ² As defined above. In some cases, L ¹ Is a linker having a main chain of 20 atoms or less in length, and Z ² Is WSG (e.g., as described herein). In some cases, L ¹ Selected from the group consisting of an alkyl or substituted alkyl linker, an alkenyl or substituted alkenyl linker, an alkynyl or substituted alkynyl linker, an acyl or substituted acyl, an alkoxy or substituted alkoxy linker, a PEG linker, a sulfonylamino-alkyl or substituted sulfonylamino-alkyl linker, an acylamino-alkyl or substituted acylamino-alkyl linker, and an alkyl-acylamino-alkyl or substituted alkyl-acylamino-alkyl linker. In some cases, L ¹ And L ² Each independently selected from C ₁ -C ₁₂ Alkyl or substituted alkyl linker, C ₁ -C ₁₂ Alkenyl or substituted alkenyl linker, C ₁ -C ₁₂ Alkynyl or substituted alkynyl linkers, C ₁ -C ₁₂ Acyl or substituted acyl linker, C ₁ -C ₁₂ Alkoxy or substituted alkoxy linker, C ₁ -C ₁₂ Amido-alkyl or substituted amido-alkyl linker, and C ₁ -C ₁₂ Alkyl-amido-alkyl or substituted alkyl-amido-alkyl linkers. In some cases, the linker comprises a carbonyl group. In some cases, L ¹ Containing carbonyl groups, and L ² Is C ₁ -C ₁₂ Alkyl or substituted alkyl. In some cases, the branching point X ¹ Selected from N, CR', C (=O) N, SO ₂ N, trisubstituted aryl groups (e.g., 1,3, 5-phenyl), tetrasubstituted aryl groups, and trisubstituted heteroaryl groups. In some cases, the branching point X ¹ Is a nitrogen atom. In other cases, the branching point X ¹ Is CR 'wherein R' is selected from hydrogen, alkyl, substituted alkyl or-L ² -Z ² (e.g., as described herein). Z is Z ² Is a water-soluble polymer (e.g., as described herein). In some cases, the water-soluble polymer is a PEG group. In some cases, Z ² Is a PEG or modified PEG polymer of 1-50 monomer units, such as 1-40, 1-30, 1-20, 2-24, 2-20, 2-10 or 2-6 monomer units. In some cases, R ⁵ And R is ⁴ Comprises WSG of formula (Ig) or (Ih).

In some other embodiments, WSG is a branched group of formula (Ii):

wherein L is ¹ 、X ¹ 、L ² N3 and Z ² Each as defined above; l (L) ^2' N3' and Z ^2' Respectively as for L herein ² N3 and Z ² Defined as follows.

In some other embodiments, WSG is a branched group of formula (Ij):

wherein L is ¹ 、X ¹ 、L ² N3 and Z ² Each as defined above; and X is ¹ Is a branching group selected from carbon atoms and tetra-substituted aryl groups (e.g., 1,3,4, 5-phenyl).

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (I), the water-solubilizing group (WSG) comprises a PEG moiety (e.g., as described herein). In some cases, the subject water-soluble dipyrromethene-type dye is substituted with one or more water-solubilizing groups (WSG) independently selected from the following formulas:

/>

wherein:

L ¹ and L ² Each independently is an optional linker;

R ^a and R are each independently H, alkyl or substituted alkyl; and

each p is an integer from 6 to 24.

In some cases, each p is independently 6 to 24, e.g., 6 to 20, 11 to 20, 12 to 18, or 12 to 16. In some cases, each p is independently 6 to 24, e.g., 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 p is independently 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some cases, each p 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, even greater, and in some cases, up to 24 monomer units, e.g., up to 20, up to 15, or up to 12 monomer units. In some cases, each p is the same. In some embodiments of WSG, L ¹ And/or L ² Is a C1-C12 alkyl linker, e.g., a C1-C6 alkyl linker, wherein one or more backbone atoms are optionally substituted with heteroatoms (e.g., -O-). In some embodiments of WSG, L ¹ And/or L ² The linker comprises a carbonyl group. In some embodiments of WSG, each R ^a Is H. In some embodiments of WSG, each R ^a Is methyl.

It is understood that hydroxyl-terminated PEG chains can be used in place of methoxy-terminated PEG chains in any of the WSG groups described above. In certain instances, any of the formulae described herein may be substituted with WSG, which is a dendritic unit selected from one of the following structures:

in certain instances, any of the formulae described herein can be substituted with WSG, which is a polyol selected from one of the following structures:

in certain instances, any of the formulae described herein can be substituted with WSG, which is an oxazoline of the structure:

in certain instances, any of the formulae described herein may be substituted with WSG, which is a peptoid selected from one of the following structures:

in certain embodiments of the subject water-soluble dipyrromethene-based dyes, Y ¹ And Y ² Each comprising a Water Solubilising Group (WSG). In certain other cases, R ¹ Comprising a Water Solubilising Group (WSG). In some cases, all Y ¹ 、Y ² And R is ¹ Comprising WSG. In some cases, R ⁴ Or R is ⁵ Comprises WSG. In some cases, is selected from R ⁶ And R is ⁷ 、R ² And R is ³ 、R ⁵ And R is ⁶ 、R ³ And R is ⁴ 、R ² And R is ¹ R is as follows ⁷ And R is ¹ Any one or more pairs of substituents of (a) together with the atoms to which they are attachedA cyclic group is formed (e.g., as described herein), and the cyclic group is substituted with WSG (e.g., as described herein).

In some cases, Y of the subject water-soluble dipyrromethene-based dye ¹ Is alkynyl substituted with WSG. Y is Y ² Can be combined with Y ¹ The same or different. In some cases of the subject water-soluble dipyrromethene-based dyes, Y ² 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 Selected from F, CN, phenyl and substituted phenyl.

In some cases of the subject water-soluble dipyrromethene-based dyes, Y ¹ And Y ² Each comprising a Water Solubilising Group (WSG). In some cases, Y ¹ And/or Y ² Is alkynyl substituted (e.g., as described herein) with WSG. In some cases, Y ¹ And/or Y ² Is alkynyl substituted with polyethylene glycol (PEG) or modified PEG. In some cases of any of the subject water-soluble dipyrromethene-based dyes, Y ¹ And/or Y ² Has the formula:

wherein:

s1 is 1 to 12;

q1 is 0 to 50; and

R ³¹ is H, alkyl or substituted alkyl. In some cases s1 is 1 to 6, for example 1, 2 or 3. In some cases, q1 is 1 to 50, 1 to 30, 2 to 30, 4 to 30, 6 to 30, 8 to 30, 10 to 20, or 11 to 16. In some cases, q1 is 10 to 50, e.g., 10 to 30, 10 to 20, or 11 to 16.

In certain embodiments, the water-soluble dipyrromethene-based dye has formula (II):

wherein R is ¹ -R ⁷ As defined herein, and p is an integer from 6 to 24.

In some cases, each p is independently 6 to 20, e.g., 11 to 20, 12 to 18, or 12 to 16. In some cases, each p is independently 8 to 24, e.g., 10 to 24, 12 to 24, 13 to 24, 14 to 24, 15 to 22, or 16 to 20. In some cases, each p is independently 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, each p 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, even greater, and in some cases, up to 24 monomer units, e.g., up to 20, up to 15, or up to 12 monomer units. In some cases, each p is the same. In some cases, each p is 11. In some cases, each p is 12. In some cases, each p is 13. In some cases, each p is 14. In some cases, each p is 15. In some cases, each p is 16. In some cases, each p is 17. In some cases, each p is 18. In some cases, each p is 19. In some cases, each p is 20.

In certain embodiments, a water-soluble dipyrromethene-type dye of formula (I) or (II), R ⁵ Selected from the group consisting of halogen, substituted alkyl, substituted alkenyl, alkenylaryl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, spiroheterocyclic, substituted spiroheterocyclic, heteroaryl, substituted amino, substituted amido, and substituted thiol. In some cases, R ⁵ Is halogen. In some cases, R ⁵ Is a chloride. In some cases, R ⁵ Is a substituted alkyl or substituted alkenyl. In some cases, R ⁵ Is an alkenyl aryl group such as alkenyl-phenyl (e.g., styrene derivatives). In some cases, R ⁵ Is aryl or substituted aryl. In some cases, R ⁵ Is a substituted aryl group, wherein the aryl group is substituted with one or more groups selected from C _1-6 Alkyl, halogen, hydroxy, cyano, amino, carboxamide, and PEG groups (e.g., as described herein). In some cases, R ⁵ Is a heterocycle or a substituted heterocycle. In some cases, R ⁵ Is a spiroheterocycle or a substituted spiroheterocycle. In some cases, the spiroheterocycle is spiro [3,3]Heptane core. In some cases, R ⁵ Is heteroaryl or substituted heteroaryl. In some cases, R ⁵ Is a substituted amino group. In some cases, R ⁵ Is a substituted amide group. In some cases, R ⁵ Is a substituted thiol. In certain other cases, R ⁵ And R is R ⁶ Together with the atoms to which they are attached form a cyclic group, for example, a 5 or 6 membered carbocyclic or heterocyclic group.

In certain embodiments, a water-soluble dipyrromethene-type dye of formula (I) or (II), R ⁴ Selected from the group consisting of halogen, substituted alkyl, substituted alkenyl, alkenylaryl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, spiroheterocyclic, substituted spiroheterocyclic, heteroaryl, substituted amino, substituted amido, and substituted thiol. In some cases, R ⁴ Is halogen. In some cases, R ⁴ Is a chloride. In some cases, R ⁴ Is a substituted alkyl or substituted alkenyl. In some cases, R ⁴ Is an alkenyl aryl group such as alkenyl-phenyl (e.g., styrene derivatives). In some cases, R ⁴ Is aryl or substituted aryl. In some cases, R ⁴ Is a substituted aryl group, wherein the aryl group is substituted with one or more groups selected from C _1-6 Alkyl, halogen, hydroxy, cyano, amino, carboxamide, and PEG groups (e.g., as described herein). In some cases, R ⁴ Is a heterocycle or a substituted heterocycle. In some cases, R ⁴ Is a spiroheterocycle or a substituted spiroheterocycle. In some cases, the spiroheterocycle is spiro [3,3]Heptane core. In some cases, R ⁴ Is heteroaryl or substituted heteroaryl. In some cases, R ⁴ Is a substituted amino group. In some cases, R ⁴ Is a substituted amide group. In some cases, R ⁴ Is substituted byIs a thiol of (a). In certain other cases, R ⁴ And R is R ³ Together with the atoms to which they are attached form a cyclic group, for example, a 5 or 6 membered carbocyclic or heterocyclic group.

In certain embodiments of the subject water-soluble dipyrromethene-based dyes, R ⁵ And R is ⁴ The same applies.

In certain embodiments, a water-soluble dipyrromethene-type dye of formula (I) or (II), R ⁵ Is an alkenyl aryl group such as alkenyl-phenyl (e.g., styrene derivatives). In certain instances, the water-soluble dipyrromethene-type dye has formula (IIa):

wherein:

R ¹ -R ⁴ 、R ⁶ -R ⁷ and p is as previously defined herein;

R ⁸ selected from H, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water Solubilizing Group (WSG), or

R ⁸ And R is ⁶ Together with the atoms to which they are bonded, form a 5-or 6-membered heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5-or 6-membered ring comprising a carbon atom and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG);

q is an integer from 0 to 5; and

each R ⁹ Independently selected from alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, thiol, substituted thiol, cyano, aryl, substituted aryl, heteroaryl, substitutedHeteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG), or

Optionally two adjacent R ⁹ The groups together form a divalent group and are cyclic linked and together with the carbon atoms to which they are bound provide a 5 or 6 membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5 or 6 membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Groups (WSG).

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (IIa), R ⁸ Is H. In certain other cases, R ⁸ And R is ⁶ Together with the atoms to which they are bonded, form a 5 membered carbocycle, which carbocycle is optionally further substituted with substituents independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG). In certain instances, the 5-membered carbocyclic ring is substituted with one or more alkyl substituents. In some cases, the alkyl substituent is C _1-6 An alkyl group. In some cases, the alkyl substituent is methyl. In some cases, the alkyl substituents are bonded to the same carbon atom of the carbocyclic ring.

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (IIa), q is 0, such that R is absent ⁹ A substituent. In some cases, q is 1, 2 or 3, and each R ⁹ Independently selected from alkyl, substituted alkyl, hydroxy, alkoxy, amino, thiol, cyano, aryl, heteroaryl, alkenyl, alkynyl, and Water Solubilizing Groups (WSG). In some cases, q is 1, 2 or 3, and each R ⁹ Independently selected from alkyl, substituted alkyl, and WSG. In certain instances, each WSG is independently a PEG group (e.g., as described herein). In some cases, each alkyl group is independentlyIs C _1-6 An alkyl group. In some cases, each alkyl is independently selected from methyl, ethyl, propyl, isopropyl, butyl, and tert-butyl. In some cases, q is 1, and R ⁹ Selected from C _1-6 Alkyl, C _1-6 Substituted alkyl groups and PEG. In some cases, C _1-6 The substituted alkyl groups are substituted with PEG groups.

In certain embodiments of formula (IIa), R ² 、R ³ 、R ⁴ 、R ⁶ And R is ⁷ Independently selected from H and C _1-6 An alkyl group. In some cases, R ² Is C _1-6 An alkyl group. In some cases, R ³ Is H. In some cases, R ⁴ Is C _1-6 An alkyl group. In some cases, R ⁶ Is H. In some cases, R ⁷ Is C _1-6 An alkyl group. In some cases, C _1-6 The alkyl group is selected from methyl, ethyl, propyl, isopropyl, butyl and tert-butyl. In some cases, C _1-6 Alkyl is methyl.

In certain embodiments of formula (IIa), the compound has any one of the following structures:

/>

in certain other embodiments of the compounds of formula (IIa), R ⁴ Is an alkenyl aryl group such as alkenyl-phenyl. In some cases, R ⁴ Is a substituted styrene derivative. In certain instances, the compound of formula (IIa) has the following structure:

In certain embodiments of the subject water-soluble dipyrromethene-based dyes, R ⁵ And R is ⁴ Identical to. In some cases, R ⁵ And R is ⁴ Are all aryl or substituted aryl. In certain instances, the water-soluble dipyrromethene-based dye has formula (IIb):

wherein:

R ¹ -R ³ 、R ⁶ -R ⁷ and p is as previously defined herein;

each q is independently an integer from 0 to 5; and

R ¹⁰ and R is ^10' Each independently selected from halogen, hydroxy, cyano, carboxamide, substituted carboxamide, amino, substituted amino, thiol, substituted thiol, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and water-soluble group (WSG), if present, or

At least one R ¹⁰ The radicals being in the ortho position and R ¹⁰ And R is ⁶ Together with the atoms to which they are bonded, form a 5 or 6 membered ring (e.g., a 5 or 6 membered ring comprising a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilizing Group (WSG); and

At least one R ^10' Is ortho-position, and R ¹⁰ And R is ³ Together with the atoms to which they are bonded, form a 5-or 6-membered ring (e.g., a 5-or 6-membered ring containing a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and water-solubilizing groups(WSG) substituent substitution.

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (IIb), each q is 0 such that R is absent ¹⁰ Or R is ^10' A substituent. In some cases, each q is independently 1, 2 or 3, and R ¹⁰ And R is ^10' Each independently selected from cyano, carboxamide, substituted carboxamide, alkyl, substituted alkyl, hydroxy, alkoxy, amino, thiol, aryl, heteroaryl, alkenyl, alkynyl, and water-solubilizing groups (WSG). In some cases, each q is 1, 2 or 3, and R ¹⁰ And R is ^10' Each independently selected from cyano, carboxamide, substituted carboxamide and WSG. In certain instances, each WSG is independently a PEG group (e.g., as described herein). In some cases, each q is 1, and R ¹⁰ And R is ^10' Each selected from cyano, carboxamide, substituted carboxamide and PEG. In some cases, R ¹⁰ And R is ^10' The same applies. In some cases, the carboxamide is substituted with a PEG group.

In some cases of formula (IIb), each q is 1, and R ¹⁰ And R is ^10' All present in their respective para-positions. In some cases, R ¹⁰ And R is ^10' Are present in their respective ortho-positions. In some cases, R ¹⁰ And R is ^10' All present in their respective meta positions.

In certain embodiments of formula (IIb), R ¹⁰ And R is ^10' Comprising a PEG moiety. In some cases, the PEG moiety is selected from one of the following formulas:

wherein:

L ¹ is an optional linker (e.g., as described herein);

R ^a and R is H, alkyl or substituted alkyl; and

each p1 is an integer of 1 to 50.

In certain instances, each p1 is independently 1 to 40, such as 1 to 30, 1 to 20, 1 to 10, or 1 to 6. In some cases, each p1 is independently 1-10, e.g., 1-9, 1-8, 1-7, 1-6, or 1-5. In some cases, each p is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, each p is 3. In some cases, each p is 4. In some cases, each p is 5. In some cases, each p is 6. In some cases, each p is 7. In some cases, each p is 8. In some cases, each p is 9. In some cases, each p is 10. In some cases, each p is 11. In some cases, each p is 12.

In certain embodiments of formula (IIb), R ² 、R ³ 、R ⁶ And R is ⁷ Independently selected from H and C _1-6 An alkyl group. In some cases, R ² Is C _1-6 An alkyl group. In some cases, R ³ Is H. In some cases, R ⁶ Is H. In some cases, R ⁷ Is C _1-6 An alkyl group. In some cases, C _1-6 The alkyl group is selected from methyl, ethyl, propyl, isopropyl, butyl and tert-butyl. In some cases, C _1-6 Alkyl is methyl. In certain other cases, all R ² 、R ³ 、R ⁶ And R is ⁷ Is H.

In certain embodiments of formula (IIb), the compound has any one of the following structures:

/>

in certain embodiments of the subject water-soluble dipyrromethene-based dyes, R ⁵ Is halogen. In some cases, R ⁶ And R is ⁷ Together with the atoms to which they are attached form a 5-or 6-membered ring group. In certain instances, the water-soluble dipyrromethene-based dye has formula (IIc):

wherein:

R ¹ -R ⁴ and p is as previously defined herein;

x is halogen; and

ring a is a 5 or 6 membered fused heterocycle, carbocycle, aryl ring or heteroaryl ring (e.g., a 5 or 6 membered ring comprising a carbon atom and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG).

In certain embodiments of formula (IIc), ring a is a 6 membered heterocyclic, carbocyclic, aryl or heteroaryl ring. In some cases, ring a is a heterocycle. In some cases, ring a is a carbocycle. In certain instances, ring a is heteroaryl. In some cases, ring a is an aryl ring.

In certain embodiments of formula (IIc), X is Cl, F, br, or I. In some cases, X is F. In some cases, X is Br. In some cases, X is I. In some cases, X is Cl.

In certain embodiments of formula (IIc), R ² 、R ³ And R is ⁴ Independently selected from H and C _1-6 An alkyl group. In some cases, R ² Is C _1-6 An alkyl group. In some cases, R ³ Is H. In some cases, R ⁴ Is C _1-6 An alkyl group. In some cases, C _1-6 The alkyl group is selected from methyl, ethyl, propyl, isopropyl, butyl and tert-butyl. In some cases, C _1-6 Alkyl is methyl.

In certain embodiments of formula (IIc), the compound has any one of the following structures:

in some cases, R ⁵ And R is ⁴ Are each selected from the group consisting of substituted amino groups, substituted amide groups, substituted thiols, and spiro heterocycles. In certain instances, the water-soluble dipyrromethene-based dye has formula (IId):

wherein:

R ¹ -R ^3' 、R ⁶ -R ⁷ and p is as previously defined herein;

X ² And X ³ Each independently selected from NR ¹³ S and O;

R ¹³ selected from H, alkyl, and substituted alkyl;

R ¹¹ and R is ¹² Each independently selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, and Water Soluble Group (WSG), or

When X is ² Is NR ¹³ When optionally R ¹³ And R is ¹¹ Together with the nitrogen atom to which they are attached, form a heterocyclic or spiro-heterocyclic ring, which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG); and

when X is ³ Is NR ¹³ When optionally R ¹³ And R is ¹² Forms, together with the nitrogen atom to which they are attached, a heterocyclic or spiro-heterocyclic ring which may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenylSubstituted alkenyl, alkynyl, substituted alkynyl, and substituents of the Water Solubilising Group (WSG).

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (Id), X ² And X ³ Each is NR ¹³ Wherein R is ¹³ Is H. In certain other embodiments, X ² And X ³ Each is O. In certain other embodiments, X ² And X ³ Each is S.

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (Id), R ¹¹ Selected from aryl, substituted aryl, and water-soluble groups (WSG). In some cases, R ¹¹ Is a phenyl group. In some cases, R ¹¹ Is a PEG group (e.g., as described herein). In certain instances, the PEG group has 2 or more monomer units, e.g., 3 or more, 4 or more, or 5 or more monomer units. In some cases, the PEG group has 1-5 monomer units, for example 2, 3 or 4 monomer units.

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (Id), R ¹² Selected from aryl, substituted aryl, and water-soluble groups (WSG). In some cases, R ¹² Is a phenyl group. In some cases, R ¹² Is a PEG group (e.g., as described herein). In certain instances, the PEG group has 2 or more monomer units, e.g., 3 or more, 4 or more, or 5 or more monomer units. In some cases, the PEG group has 1-5 monomer units, for example 2, 3 or 4 monomer units.

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (Id), R ¹¹ And R is ¹² The same applies. In some cases, R ¹¹ And R is ¹² Are all phenyl groups. In some cases, R ¹¹ And R is ¹² Are PEG groups (e.g., as described herein).

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (Id), X ² Is NR ¹³ And R is ¹³ And R is ¹¹ With nitrogen to which they are attachedAtoms together form a heterocycle or spiroheterocycle; x is as follows ³ Is NR ¹³ And R is ¹³ And R is ¹² Together with the nitrogen atom to which they are attached form a heterocyclic or spiro-heterocyclic ring. In some cases, the spiroheterocycle has a ring selected from the group consisting of spiro [3,3 ]]Heptane, spiro [3,4 ]]Octane, spiro [3,5 ]]Nonane, spiro [4,4 ]]Nonane, spiro [4,5 ]]Decane and spiro [5,5 ]]Core structure of undecane. In some cases, the spiroheterocycle is spiro [3,3]Heptane core structure.

In certain embodiments of formula (IId), R ² 、R ³ 、R ⁶ And R is ⁷ Independently selected from H and C _1-6 An alkyl group. In some cases, R ² Is C _1-6 An alkyl group. In some cases, R ³ Is H. In some cases, R ⁶ Is H. In some cases, R ⁷ Is C _1-6 An alkyl group. In some cases, C _1-6 The alkyl group is selected from methyl, ethyl, propyl, isopropyl, butyl and tert-butyl. In some cases, C _1-6 Alkyl is methyl. In certain other cases, all R ² 、R ³ 、R ⁶ And R is ⁷ Is H.

In certain embodiments of formula (IId), the compound has any one of the following structures:

/>

in some embodiments of the water-soluble dipyrromethene-type dye of formula (I), Y ¹ And Y ² Together with the boron atom to which they are bonded, form a 5 or 6 membered ring (e.g., a 5 or 6 membered ring comprising a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further substituted (e.g., as described herein). In some cases, the water-soluble dipyrromethene-based dye has formula (III):

wherein:

R ¹ -R ⁷ as previously defined herein; and

ring B is a 5 or 6 membered cyclic group (e.g., a 5 or 6 membered ring comprising a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S and N), which cyclic group may be unsubstituted or further substituted with one or more substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilizing Group (WSG).

In some cases, ring B is a 6 membered cyclic group. In certain instances, the 6-membered cyclic group is further substituted with one or more substituents selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilizing Group (WSG). In certain instances, the 6-membered cyclic group is further substituted with one or more substituents selected from aryl and substituted aryl. In some cases, the 6-membered cyclic group is substituted with two phenyl groups.

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (III), the compound has formula (IIIa):

wherein:

X ⁴ selected from O, S, NR and CR ₂ ；

Each R is independently selected from H, alkyl, and substituted alkyl;

each r is independently an integer from 0 to 4; and

each R ¹⁴ And R is ^14' Independently selected from halogen, hydroxy, cyano, amino, and substituted if presentSubstituted thiol, amide, substituted amide, acyl, substituted acyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilizing Group (WSG).

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (IIIa), R ⁵ And R is ⁶ R is as follows ⁴ And R is ³ Together form a divalent group and are cyclic, and together with the carbon atoms to which they are bonded provide a 5 or 6 membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5 or 6 membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG). In some cases, R ⁵ And R is ⁶ Or R is ⁴ And R is ³ Only one pair together form a divalent group and are cyclic and together with the carbon atoms to which they are bound provide a 5 or 6 membered ring. In certain other cases, R ⁵ And R is ⁶ R is as follows ⁴ And R is ³ All together form a divalent group and are cyclic linked and together with the carbon atom to which they are bound provide a 5 or 6 membered ring. In some cases, R ⁵ And R is ⁶ R is as follows ⁴ And R is ³ Together with the carbon atoms to which they are bonded form a 6 membered ring (e.g., as described herein). In some cases, R ⁵ And R is ⁶ R is as follows ⁴ And R is ³ Together with the carbon atoms to which they are bonded form a 5-membered ring (e.g., as described herein). In certain embodiments, R ⁵ And R is ⁶ Or R is ⁴ And R is ³ Together with the carbon atoms to which they are bonded, provide a 5-membered ring represented by one of the following structures, wherein the wavy line represents a ring with R ⁵ And R is ⁶ Or R is ⁴ And R is ³ Dipyrromethene of (2)Connection point of core:

wherein:

x is O or S;

y is O, S or NR wherein R is H, alkyl, substituted alkyl or R as in formula (I) ¹ -R ⁷ A substituent defined; and

R ²¹ -R ²³ independently selected from H and R as in formula (I) ¹ -R ⁷ A substituent as defined. In certain instances, the 5-membered ring is independently selected from the following rings:

As defined above.

In some cases of formulae (III) - (IIIa), R ⁵ And R is ⁶ Or R is ⁴ And R is ³ The same ring is formed. In some cases, R ⁵ And R is ⁶ Or R is ⁴ And R is ³ Different rings are formed.

In certain embodiments, the water-soluble dipyrromethene-type dye of the claims has formula (IIIb) or (IIIc):

wherein:

X ⁵ and X ⁶ Each independently selected from O, S, NR and CR ₂ ；

Each R is independently selected from H, alkyl, and substituted alkyl; and

R ¹⁵ and R is ¹⁶ Each independently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, amino, substituted amino, thiol, substituted thiol, amido, substituted amido, acyl, substituted acyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxySubstituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilizing Groups (WSG).

In some cases of formula (IIIb), X ⁵ Is O. In some cases, X ⁵ Is S. In some cases, X ⁵ Is NR. In certain other cases, X ⁵ Is CR (CR) ₂ 。

In some cases of formula (IIIc), X ⁵ And X ⁶ Each is O. In some cases, X ⁵ And X ⁶ Each is S. In some cases, X ⁵ And X ⁶ Each is NR. In certain other cases, X ⁵ And X ⁶ Each CR is ₂ 。

In certain cases of formulae (IIIb) and (IIIc), R ¹⁵ Selected from the group consisting of alkyl, substituted alkyl, aryl, and substituted aryl. In certain instances, the substituted alkyl or substituted aryl is substituted with a Water Solubilizing Group (WSG) (e.g., as described herein). In some cases, the WSG comprises a PEG moiety. In some cases, the WSG comprises a carboxamide group. In some cases, the substituted alkyl or substituted aryl groups are substituted with an alkoxy group or substituted alkoxy group. In some cases, the substituted aryl is a disubstituted aryl. In some cases, the disubstituted aryl is a 2, 4-disubstituted aryl moiety.

In certain cases of formulae (IIIb) and (IIIc), R ¹⁶ Selected from the group consisting of alkyl, substituted alkyl, aryl, and substituted aryl. In some cases, R ¹⁶ Is C _1-6 Alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyl. In some cases, R ¹⁶ Is methyl. In certain instances, the substituted alkyl or substituted aryl groups are substituted with a Water Solubilizing Group (WSG) (e.g., as described herein). In some cases, the WSG comprises a PEG moiety. In some cases, the WSG comprises a carboxamide group. In some cases, the substituted alkyl or substituted aryl groups are substituted with an alkoxy group or substituted alkoxy group. In some cases, the substituted aryl is a disubstituted aryl. In some cases, the disubstituted aryl is a 2, 4-disubstituted aryl Part(s).

In certain embodiments, the water-soluble dipyrromethene-based dye of formulas (III) - (IIIc) is one of the following structures:

in some embodiments of the water-soluble dipyrromethene-type dye of formula (I), R ¹ -R ⁷ At least one of which is T ¹ -J ¹ Such that the compound of formula (I) comprises at least one repeating unit of the compound of formula (I). In some cases, the compound of formula (I) is a dimer. In some cases, R ¹ Is T ¹ -J ¹ . In some cases, R ² Is T ¹ -J ¹ . In some cases, R ³ Is T ¹ -J ¹ . In some cases, R ⁴ Is T ¹ -J ¹ . In some cases, R ⁵ Is T ¹ -J ¹ . In some cases, R ⁶ Is T ¹ -J ¹ . In some cases, R ⁷ Is T ¹ -J ¹ . In some cases T ¹ Is absent and R ¹ -R ⁷ At least one of is J ¹ (e.g., repeating units of a compound of formula (I)).

In some embodiments of the subject water-soluble dipyrromethene-based dyes, R ³ Is T ¹ -J ¹ And T is ¹ Is absent, so that R ³ Only J ¹ . In some cases, the water-soluble dipyrromethene-type dye has one of the following structures.

In certain instances, the dipyrromethene-based dye has formula (IV):

wherein:

X ^- is a counter ion;

R ^1' -R ^7' each independently selected from H, alkyl, halogen, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, amido, substituted amido, thiol, substituted thiol, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water Solubilizing Group (WSG), and-T ¹ -Z ¹ Or (b)

Optionally selected from R ^6' And R is ^7' 、R ^2' And R is ^3' 、R ^5' And R is ^6' 、R ^3' And R is ^4' 、R ^2' And R is ^1' R is as follows ^7' And R is ^1' Forms a divalent group together with any one or more pairs of substituents and is cyclic and, together with the carbon atom to which they are bonded, provides a 5 or 6 membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5 or 6 membered ring comprising a carbon atom and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water Solubilising Group (WSG) and-T ¹ -Z ¹ Is substituted by a substituent of (a);

T ¹ is an optional linker;

Z ¹ is a repeating unit of a compound of formula (I); 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, or

Y ^1' And Y ^2' Together with the boron atom to which they are bonded form a 5-or 6-membered ring (e.g. containing boron atoms, carbon atoms and 0-3 members selected from O, S anda 5 or 6 membered ring of heteroatoms of N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water Solubilizing Group (WSG) and-T ¹ -Z ¹ Is substituted by a substituent of (a); and

wherein is selected from Y ^1' 、Y ^2' And R is ^1' -R ^7' At least one moiety of (2) is WSG, or is selected from Y ^1' 、Y ^2' And R is ^1' -R ^7' Is substituted with WSG, wherein WSG has formula (Ia):

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ¹ ) _m (Ia)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ¹ is a water-soluble polymer comprising 6 to 24 monomer units; and

m is an integer from 1 to 3.

In some embodiments of the water-soluble dipyrromethene-type dye of formula (IV), the dye further comprises WSG of formula (Ib):

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ² ) _m (Ib)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ² is a water-soluble polymer comprising 1 to 50 monomer units; and

m is an integer from 1 to 3.

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (IV), the WSG comprises a PEG moiety (e.g., as described herein). In certain instances, the one or more Water Solubilizing Groups (WSG) are independently selected from any of the WSG formulas disclosed herein.

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (IV), R ^1' -R ^7' As for R ¹ -R ⁷ As defined (e.g., as described herein). In some cases, Y ^1' And Y ^2' As for Y ¹ And Y ² As defined (e.g., as described herein). In some cases, Y ^1' And Y ^2' Are all hydrogen.

In certain embodiments of the water-soluble dipyrromethene-type dye of formula (IV), R ^5' And R is ^4' All contain Water Solubilising Groups (WSG). In some cases, R ^5' And R is ^4' The same applies. In some cases, R ^5' And R is ^4' Are substituted aryl groups and the aryl groups are substituted with at least one WSG (e.g., as described herein).

In some cases, the water-soluble dipyrromethene-type dye of formula (IV) has one of the following structures:

counter ion (X) in the water-soluble dipyrromethene-based dye of formula (IV) ^- ) May be any convenient counterion. In some cases, the counter ion (X ^- ) Selected from the group consisting of bromide, chloride, formate, tartrate, benzenesulfonate, methanesulfonate, acetate (e.g., trifluoroacetate), maleate, and oxalate.

In certain embodiments of the water-soluble dipyrromethene-based dye of any one of formulas (I) - (IV), R ¹ Or R is ^1' Selected from the group consisting of substituted alkyl groups and substituted aryl groups, wherein the substituents comprise a chemoselective tag. As herein describedIn any of the embodiments of formulas (I) - (IV), R ¹ Or R is ^1' May include a particular-L as described in one of the following embodiments ^a -Z ^a A group.

L ^a May be a linker having a backbone of 20 atoms or less in length (e.g., as described herein). In some cases, L ^a Selected from the group consisting of alkyl or substituted alkyl linkers, alkoxy or substituted alkoxy linkers, aryl or substituted aryl linkers, PEG linkers, sulfonylamino-alkyl or substituted sulfonylamino-alkyl linkers, amido-alkyl or substituted amido-alkyl linkers, and alkyl-amido-alkyl or substituted alkyl-amido-alkyl linkers. The linker may be substituted with WSG (e.g., a PEG group). In some cases, L ^a Selected from C ₁ -C ₁₂ Alkyl or substituted alkyl linker, C ₁ -C ₁₂ Alkoxy or substituted alkoxy linker, C ₁ -C ₁₂ Amido-alkyl or substituted amido-alkyl linkers, and C ₁ -C ₁₂ Alkyl-amido-alkyl or substituted alkyl-amido-alkyl linkers. Joint L ^a May include several linked components such as one or independently selected from lower alkylene, substituted lower alkylene, alkenylene (-ch=ch-), substituted alkenylene, alkynylene (-CC-), substituted or unsubstituted amide groups (e.g., -NRCO-or-CONR-, where R is H, alkyl or substituted alkyl), substituted or unsubstituted sulfonamide groups (e.g., -NRSO) ₂ -or-SO ₂ NR-, wherein R is H, alkyl or substituted alkyl), oxo (-O-), thio (-S-), ethylene glycol (-OCH- ₂ CH ₂ O-), polyethylene glycol (e.g., - (CH) ₂ CH ₂ O) _n -, wherein n is 2 to 20, such as 2 to 10 or 2 to 6, or 2, 3, 4, 5 or 6), arylene, substituted arylene, heteroarylene, substituted heteroarylene, alpha-amino acid residue, beta-amino acid residue, or the like.

Z ^a May be a chemically selective functional group suitable for conjugation (for conjugation to) with a molecule of interest having compatible functional groups. Chemoselective functional groups of interest that can be used in the subject dyes include, but are not limited to, amine groups(e.g., -NH) ₂ ) Carboxylic acid (-CO) ₂ H) Activated esters (e.g., NHS or thio-NHS esters), thiols, maleimides, iodoacetamides, hydroxyl, hydrazide, hydrazine, aldehyde, ketone, azide, alkyne, tetrazine, alkene, phosphine, and epoxide. It will be appreciated that in some cases, the chemoselective functional group of the subject dye is a synthetic precursor or protected form of the functional group of interest, which can be converted to a reactive functional group capable of conjugation with the molecule of interest. For example, carboxylic acids are chemically selective functional groups that can be coupled to amine groups on the molecule of interest. The carboxylic acid may be converted to an activated ester coupled in situ with the amine group or prior to coupling. In some cases, Z ^a Are protected carboxylic acids, such as ester-protected carboxylic acids. In some cases, Z ^a Is a synthetic precursor to a chemically selective functional group, such as an azido group that can be reduced to an amine, or a cyano group that can be hydrolyzed to a carboxylic acid using any convenient method.

In certain embodiments of any of the water-soluble dipyrromethene-type dyes of formulas (I) - (IV), R ¹ Or R is ^1' is-L ^a -Z ^a Wherein L is ^a Is alkylene or substituted alkylene, and Z ^a Is a carboxylic acid or an activated ester of a carboxylic acid. In some cases, -L ^a -Z ^a Is- (C) ₁ -C ₁₂ ) alkylene-CO ₂ H, e.g. (C) ₁ -C ₆ ) alkylene-CO ₂ H. In certain embodiments, L ^a Is arylene or substituted arylene, and Z ^a Is a carboxylic acid or an activated ester of a carboxylic acid. In certain embodiments, L ^a Is arylene or substituted arylene, and Z ^a Is a carboxylic acid or an activated ester of a carboxylic acid. In some cases, -L ^a -Z ^a Selected from-phenyl- (C) ₁ -C ₁₂ ) alkylene-CO ₂ H and-substituted phenyl- (C) ₁ -C ₁₂ ) alkylene-CO ₂ H。

In some embodiments of any of the water-soluble dipyrromethene-type dyes of formulas (I) - (IV), R ¹ Or R is ^1’ is-L ^a -Z ^a Wherein L is ^a Comprising functional groups (Z) chemically selective to ^a ) An attached optionally substituted carbocyclic or heterocyclic group. In some cases, L ^a Is an optionally substituted aryl or heteroaryl group. 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), and the like. Divalent aryl or heteroaryl groups of interest include, but are not limited to, 1, 4-phenyl, 1, 3-phenyl, 2, 5-pyridyl, 2, 6-pyridyl, 3, 5-pyridyl, and the like. L (L) ^a Divalent carbocyclic or heterocyclic groups or divalent aryl or heteroaryl groups of (C) may be substituted with-L ^b -Z ^a A connection, wherein L ^b Is a linking group, e.g., as described in any of the embodiments herein. In some embodiments of any of the water-soluble dipyrromethene-type dyes of formulas (I) - (IV), R ¹ Or R is ^1' Described by one of the following structures:

wherein:

R ¹⁷ is L ^b -Z ^a ；

L ^b Is a linker, and Z ^a Is a chemoselective group or molecule of interest;

t is 0-4; and each R ¹⁸ Independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, sulfonic acid, and Water Solubilizing Groups (WSG). In certain embodiments, R ¹ Or R is ^1' Described by one of the following structures:

wherein:

L ^b is a linker, and Z ^a Is a chemoselective group or molecule of interest;

t is 0-4; and each R ¹⁸ Independently selected from alkyl, substitutedAlkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, sulfonic acid, and Water Solubilizing Groups (WSG). In certain embodiments, R ¹ Or R is ^1' Described by one of the following structures:

L ^b is a linker, and Z ^a Is a chemoselective group or molecule of interest;

R ³² is H, alkyl, substituted alkyl, and Water Solubilizing Group (WSG);

L ^C is selected from alkylene (e.g., C ₁ -C ₆ -alkylene), -O-alkylene, and a substituted version of the linker;

t is 0-4; and each R ¹⁸ Independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, sulfonic acid, and Water Solubilizing Groups (WSG).

Various aspects of the disclosure include water-soluble dipyrromethene-type dyes (e.g., dyes of any one of formulas (I) - (IV)) that include a water-solubilizing group suitable for attachment to a molecule of interest, as well as tandem dyes that include the dyes. In some cases, the subject water-soluble dipyrromethene-type dye is substituted with a group or substituent that includes a chemically selective functional group that facilitates conjugation of the dye to a molecule of interest (e.g., a polymer donor dye). In some cases, the water-soluble dipyrromethene-type dye is any of the compounds of formulas (I) - (IV) or as depicted in fig. 1. It is to be appreciated that for any of the water-soluble dipyrromethene-based dyes described herein (e.g., dyes of formulas (I) - (IV)), the present disclosure includes novel water-soluble dipyrromethene-based dyes that are not conjugated to a polymeric dye backbone, and water-soluble dipyrromethene-based dye reagent derivatives thereof that include a chemoselective functional group. Such dyes and dye reagents can be used in a variety of situations. The water-soluble dipyrromethene-type dye reagent of interest may be conjugated to any convenient molecule of interest, for example, to a biomolecule for a second dye (e.g., a polymeric dye). Thus, also included are polymeric tandem dyes comprising a linked water-soluble dipyrromethene-based dye as described herein (e.g., a polymeric tandem dye comprising one of formulas (I) - (IV)).

Embodiments of the present invention include dipyrromethene yellow-green absorbing dyes that are effective to transfer energy to an acceptor chromophore, while a second dipyrromethene dye having the same or a different chemical structure is within its proximity to the energy acceptor. Thus, embodiments of the present invention provide dipyrromethene-type dyes that are effective to transfer energy to an acceptor chromophore, while a second dipyrromethene-type dye having the same or a different chemical structure is within its proximity to the energy acceptor. The energy transfer mechanism between the dipyrromethene-based dye and the acceptor chromophore includes, for example, resonance energy transfer (e.g.,(or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), and the like. These energy transfer mechanisms may be relatively short range; i.e. the close proximity of chromophores of the light harvesting multichromophore system to each other and/or to the acceptor fluorophore provides efficient energy transfer. By "effective" energy transfer is meant that 20% or more or 30% or more, 40% or more, 50% or more of the energy collected by the donor chromophore is transferred to the receptor.

The dipyrromethene-type dye provided by embodiments of the present invention is effective to transfer energy to the acceptor chromophore, while a second dipyrromethene-type dye having the same or a different chemical structure is within its proximity to the energy acceptor. Stated another way, such efficient energy transfer can occur even when the second dipyrromethene-type dye is located in close proximity to the energy reception of the first dipyrromethene-type dye. Efficient energy transfer may also occur when no second dipyrromethene-type dye is located in close proximity to energy reception. The provided dipyrromethene-based dyes can selectively transfer energy to the acceptor chromophore instead of to the second dipyrromethene-based dye, which can advantageously increase fluorescence quantum yield. For example, the provided dipyrromethene-based dyes and acceptor chromophores can have a fluorescence quantum yield of 0.01 or greater, such as 0.05 or greater, 0.1 or greater, 0.2 or greater, 0.3 or greater, 0.4 or greater, or 0.5 or greater. As used herein, the term "quantum yield" refers to the number of photons emitted by the acceptor chromophore divided by the number of photons absorbed by the provided dipyrromethene-type dye. In some cases, the second dipyrromethene-type dye has the same chemical structure as the first dipyrromethene-type dye.

In some cases, the dipyrromethene-based dye has formula (I):

wherein:

R ¹ -R ⁷ each independently selected from H, alkyl, substituted alkyl, alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkoxy, substituted alkoxy, amino, amido, substituted amido, thiol, halogen, hydroxy, cyano, water Solubilizing Group (WSG), -T ¹ -J ¹ Wherein T is ¹ Is an optional linker, and J ¹ Has the formula (I),

optionally one or more pairs selected from R ¹ And R is ² 、R ² And R is ³ 、R ³ And R is ⁴ 、R ⁵ And R is ⁶ 、R ⁶ And R is ⁷ And R is ⁷ And R is ¹ Together form a fused ring, and optionally a pair of additional substituents on the fused ring together form an additional fused ring;

R ⁸ and R is ⁹ Each independently is a poly (alkylene oxide) group.

In some cases, R ¹ Is aryl, substituted aryl, heteroarylA group or a substituted heteroaryl group. For example, in some cases, R ¹ Is a substituted aryl group, e.g. R ¹ Has the following formula:

wherein m is 0 to 10. In some cases, m is 0, in other cases, m is 1 to 10, such as 1 to 5.

In some embodiments, R ¹ Is alkyl or substituted alkyl, e.g. wherein R ¹ Is an alkyl group that has been substituted with a carboxyl group. For example, R ¹ May have the formula:

wherein r is 0 to 10. In some cases, r is 0, in other cases r is 1 to 10, for example 1 to 4.

In some cases, R ² -R ⁷ Comprising pi-bonds pi-conjugated to pi electrons of the pyrrole ring of the structure of formula (I). Such pi bonds may alter the optical properties of the dipyrromethene-based dye, such as absorption spectrum, emission spectrum, energy transfer efficiency (e.g., FRET), or a combination thereof. For example, R ⁴ There may be a cyano group having a triple bond between a carbon atom and a nitrogen atom (i.e. -c≡n), wherein two pi bonds of the triple bond are pi-conjugated with the pyrrole ring on the right side of the structure of formula (I). As another example, R ² And R is ³ Together, fused aromatic rings, e.g., aryl rings, may be formed. Due to R ² And R is ³ Is fused to the pyrrole ring on the right side of the structure of formula (I), and pi bonds of the aryl ring are also pi conjugated to pi electrons of the pyrrole ring.

In some embodiments, R ² And R is ³ Together forming a fused aromatic ring. The fused aromatic ring may be an aryl ring, a substituted aryl ring, a heteroaryl ring, or a substituted heteroaryl ring.

In some embodiments, R ⁴ Selected from alkyl, aryl, substituted aryl, heteroarylAryl, substituted heteroaryl, and cyano. In some cases, R ⁴ Is an alkyl group, for example, methyl, ethyl or propyl. In some cases, R ⁴ Is an aryl group or a substituted aryl group, for example, wherein the aryl group is substituted with an alkyl group, an alkoxy group, or a cyano group. R is R ⁴ The presence of pi bonds and unpaired electrons (e.g., unpaired electrons of an alkoxy group) can affect the electronic structure of the dipyrromethene core, which can alter the optical properties of the provided dye.

In some cases, R ⁵ And R is ⁶ Together, fused rings may be formed. In some cases, R ⁵ And R is ⁶ Is substituted with additional substituents that form additional fused rings. In some embodiments, the additional fused ring is aromatic, e.g., an aryl ring or a substituted aryl ring. In some cases, R ⁵ And R is ⁶ Configured such that they provide a first ring fused to the dipyrromethene core and a second ring fused to the first ring, e.g., wherein the second ring is aromatic and pi-conjugated to the dipyrromethene core. In some cases, R ⁵ And R is ⁶ Together, groups of the formula:

in some cases, R ⁸ And R is ⁹ Each independently is a poly (ethylene glycol) group. For example, one or more of the poly (ethylene glycol) groups can have the formula:

wherein n is 1 to 50, such as 3 to 35, 6 to 24, 10 to 20, or 12 to 16.R is R ⁸ And R is ⁹ The presence of poly (alkylene oxide) groups may increase the solubility of the dye in water.

Dipyrromethene-type dyes of formula (I) are provided. In some cases, R ¹ Is aryl, substituted aryl, heteroarylA group, a substituted heteroaryl group, an alkyl group, or a substituted alkyl group. In some cases, R ² And R is ³ Together forming a fused aromatic ring. In some cases, R ⁴ Is methyl, ethyl, aryl, substituted aryl or cyano. In some cases, R ⁵ And R is ⁶ Together form a fused ring, which is optionally substituted with a pair of further substituents forming a further fused ring, for example aromatic and pi-conjugated with the pyrrole ring of the structure of formula (I). In some embodiments, if R ⁴ Is a substituted aryl group, then R ⁵ Not substituted aryl; if R is ⁴ Is aryl, then R ⁵ Not alkyl; if R is ² 、R ⁴ And R is ⁵ Is methyl, then R ⁷ Not methyl; if R is ² And R is ³ Or R is ⁵ And R is ⁶ Together form a fused ring, R ³ And R is ⁶ Is not hydrogen; or a combination thereof.

In some embodiments, R ² And R is ³ Together form a fused ring, e.g. aryl ring, and R ⁴ Is aryl, substituted aryl or cyano. In some such cases, R ⁵ -R ⁷ Each hydrogen. In some cases, R ¹ Is a substituted alkyl or substituted aryl. Exemplary dyes having such R groups are shown below.

In some cases, R ⁵ And R is ⁶ Together form a ring, e.g., a non-aromatic ring, substituted with additional substituents forming additional rings. Such an embodiment may also be referred to as R ⁵ And R is ⁶ Together forming a first ring fused to the pyrrole ring of formula (I) and a second ring fused to the first ring. In some embodiments, the additional ring is an aromatic ring, e.g., an aryl ring.

In some cases, R ¹ Is a substituted alkyl or substituted aryl. Exemplary dyes having such R groups are shown below.

Tandem dyes

As outlined above, there is also provided a tandem dye comprising a water-soluble dipyrromethene-based dye (e.g. dyes of formulae (I) - (IV)) that absorbs in the yellow-green range at 561nm, as described above. The tandem dyes of the present invention comprise a water-soluble dipyrromethene-based dye and a second dye, wherein the dyes are in an energy transfer relationship, such as a Fluorescent Energy Transfer (FET) relationship, for example, as described further below. In such tandem dyes, the water-soluble dipyrromethene-type dye may be a donor or an acceptor. In such tandem dyes, the water-soluble dipyrromethene-based dye and the second dye may be linked to a backbone (e.g., a polymer backbone, e.g., a polypeptide backbone) that provides the desired energy transfer relationship.

In some cases, the water-soluble dipyrromethene-type dye is a donor of the tandem dye. The tandem dyes of these embodiments comprise a donor chromophore that is a water-soluble dipyrromethene-based dye (e.g., dyes of formulas (I) - (IV)) that absorbs in the yellow-green range at 561nm, e.g., as described above; the energy acceptor comprises a pendant acceptor fluorophore configured to receive energy in proximity to the donor chromophore, e.g., wherein both are linked to a common backbone. In some cases of the subject tandem dyes, there are a plurality of pendant donor chromophore groups, e.g., water-soluble dipyrromethene-based dyes (e.g., dyes of formulas (I) - (IV)) that absorb in the yellow-green range at 561nm, e.g., as described above, and are configured for adjacent energy transfer at the pendant acceptor fluorophores. The term "pendant" refers to a side chain group that is attached to the backbone but is not part of the backbone itself.

The present disclosure provides polymeric tandem dyes that can achieve narrow yellow-green laser absorption characteristics (i.e., at 561 nm), minimizing absorption at other common laser sources used for applications such as flow cytometry. The subject polymeric tandem dye allows absorption in the yellow-green laser range and emission at a wavelength determined by the selected linked energy acceptor. The subject tandem dyes are water soluble and soluble in water and common aqueous buffer solutions to provide good quantum yields in aqueous media.

In certain embodiments of the tandem dye, the absorption maximum of the donor chromophore is from 555nm to 585nm, such as from 555nm to 575nm, such as from 555 to 574nm, from 555 to 573nm, from 555 to 572nm, from 555 to 571nm, from 555 to 570nm, from 555 to 569nm, from 555 to 568nm, from 555 to 567nm, from 555 to 566nm, from 555 to 565nm, from 555 to 564nm, from 555 to 563nm, or from 555 to 562nm. In some cases of tandem dyes, the absorption maximum of the donor chromophore is 558 to 568nm, such as 558 to 567nm, 558 to 566nm, 558 to 565nm, 558 to 564nm, 558 to 563nm, or 558 to 562nm. In some cases of tandem dyes, the absorption maximum of the donor chromophore is about 561nm.

In certain embodiments of tandem dyes, the donor chromophore is a water-soluble dipyrromethene-type dye as described herein (e.g., a dye of any one of formulas (I) - (IV) as described herein). In certain instances, the donor chromophore is a water-soluble dipyrromethene-boron dye (e.g., a dye of any one of formulas (I) - (IIIc) as described herein). In some cases, the donor chromophore is a water-soluble dipyrromethene-based dye as depicted in fig. 1.

The donor chromophore is capable of transferring energy to the attached acceptor fluorophore. Thus, the subject tandem dye comprises a linked acceptor signal fluorophore in the vicinity of the energy reception of the donor water solvated light harvesting chromophore system (i.e., the vicinity of the at least one linked donor chromophore group). The terms "acceptor fluorophore" and "acceptor chromophore" are used interchangeably herein.

The acceptor signal fluorophore may be attached as a pendant group to a repeating unit of the backbone. Excitation of the donor may result in energy transfer to and emission from the covalently attached acceptor signal fluorophore. The number of repeat units of the donor water solvated light harvesting chromophore having an acceptor signal fluorophore group attached can vary, with in some cases the number of repeat units ranging from 1mol% to 50mol%, such as from 1mol% to 25mol%, from 2mol% to 25mol%, from 3mol% to 25mol%, from 4mol% to 25mol%, from 5mol% to 25mol%, or from 10mol% to 25mol%.

Energy transfer mechanisms between the donor chromophore and 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), and the like. These energy transfer mechanisms may be relatively short range; i.e. the close proximity of the chromophores of the light harvesting multichromophore system to each other and/or to the acceptor fluorophore provides efficient energy transfer.

Under conditions of efficient energy transfer, when incident light ("pump light") is at a wavelength that is absorbed by and transferred from the chromophore of the light harvesting chromophore, amplification of the emission from the light-emitting acceptor fluorophore may occur more strongly than if the light-emitting acceptor fluorophore is directly excited by the pump light.

By "effective" energy transfer is meant that 10% or more, such as 20% or more or 30% or more, 40% or more, 50% or more, of the energy collected by the donor chromophore is transferred to the receptor. By "amplified" is meant that the signal from the acceptor fluorophore is 1.5x or greater when excited by energy transfer from the donor light harvesting chromophore system, as compared to direct excitation of the acceptor fluorophore with an equivalent intensity of incident light. The signal may be measured using any convenient method. In some cases, a signal of 1.5x or greater refers to the intensity of the emitted light. In some cases, a signal of 1.5x or greater refers to an increased signal-to-noise ratio. In certain embodiments of the tandem dye, when excited by a chromophore, the acceptor fluorophore emission is 1.5-fold or greater than that of the acceptor fluorophore directly excited with incident light, such as 2-fold or greater, 3-fold or greater, 4-fold or greater, 5-fold or greater, 6-fold or greater, 8-fold or greater, 10-fold or greater, 20-fold or greater, 50-fold or greater, 100-fold or greater, or even greater than that of the acceptor fluorophore directly excited with incident light.

Any convenient fluorescent dye may be used as the acceptor fluorophore in the polymeric tandem dye. The terms "fluorescent dye" and "fluorophore" are used interchangeably herein. The acceptor fluorophore (e.g., each a) may be a small molecule fluorophore. The acceptor fluorophore (e.g., each a) can be a dye molecule selected from rhodamine, perylene, diimide, coumarin, xanthene, cyanine, polymethine, pyrene, thiazine, acridine, dipyrromethene boron difluoride, naphthalimide, phycobiliprotein, polymethylalgae (peridium) chlorophyll protein, conjugates thereof, and combinations thereof. In certain embodiments, the acceptor fluorophore (a) is a cyanine dye, a xanthene dye, a coumarin dye, a thiazine dye, or an acridine dye. In some cases, the acceptor fluorophore (a) is selected from DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, biotium CF 555, cy3.5, and diethylaminocoumarin. Fluorescent dyes of interest include, but are not limited to, fluorescein, 6-FAM, rhodamine, texas Red, tetramethyl rhodamine, carboxyrhodamine 6G, carboxyacetaminophen, carboxyrhodamine 110, cascade Blue, cascade Yellow, coumarin, cy2, cy3, cy3.5, cy5, cy5.5, cy-Chrome, phycoerythrin, perCP (polymannuin-chlorophyll-a-protein), perCP-Cy5.5, JOE (6-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein), NED, ROX (5- (and-6) -carboxy-X-rhodamine), HEX, fluorescein, beach 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-Br.sub.2, BODIPY 530/550, BODIPY 558/570, BODIPY 576/589, BODY 581/591, BODY/650, BODY conjugated 650, BODY 6/BODY 665, BODY 65, and combinations thereof. Lanthanide chelates of interest include, but are not limited to, europium chelates, terbium chelates, and samarium chelates. In some embodiments, the polymeric tandem dye comprises a multichromophore attached to an acceptor fluorophore selected from Cy3, cy3.5, cy5, cy5.5, cy7, alexa 488, alexa 647, and Alexa 700. In certain embodiments, the polymeric tandem dye comprises a multichromophore attached to an acceptor fluorophore selected from the group consisting of Dyomics dyes (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.

In some cases, acceptor fluorophore (A) is selected from fluorescein, 6-FAM, rhodamine, texas Red, california Red, iFluor594, tetramethyl rhodamine, carboxyrhodamine 6G, carboxyparacetamol, carboxyrhodamine 110, cascade blue, cascade yellow, coumarin,Cy-Chrome, dyLight, dylight 405, dylight 488, dylight 549, dylight 594, dylight 633, dylight 649, dylight 680, dylight 750, dylight 800, phycoerythrin, perCP (polymorphin-chlorophyll-a-protein), perCP-Cy5.5, JOE (6-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein), NED, ROX (5- (and-6) -carboxy-X-rhodamine), HEX, fluorescein, beach blue, oreg green 488, oreg green 500, oreg green 514, alexa @, NED>350、Alexa />430、Alexa />488、Alexa />532、Alexa />546、Alexa 568、Alexa />594、Alexa />633、Alexa />647、Alexa />660、Alexa />680. 7-amino-4-methylcoumarin-3-acetic acid, -/->FL、/>FL-Br2、/>530/550、/>558/568、/>564/570、/>576/589、581/591、/>630/650、/>650/665、/>R6G、/>TMR、/>TR, conjugates thereof, and combinations thereof.

In some cases, the acceptor fluorophore is a dipyrromethene-type dye, e.g., a water-soluble dipyrromethene-type dye of any one of formulas (I) - (IV) or any embodiment thereof described herein. It is to be understood that any convenient dipyrromethene-based dye described herein having suitable absorption and emission curves can be configured as an acceptor fluorophore that solvates adjacent energy reception of the light harvesting chromophore system (i.e., adjacent energy reception of at least one compatible pendant donor chromophore group) at the donor water.

In some cases, the tandem dye exhibits an effective stokes shift of 100nm or greater, e.g., 110nm or greater, 120nm or greater, 130nm or greater, 140nm or greater, 150nm or greater, 160nm or greater, 170nm or greater, 180nm or greater, 190nm or greater, 200nm or greater, 250nm or greater, when the chromophore is collected by direct excitation of light with incident light. In some cases, the effective Stokes shift of the tandem dye is up to about 300nm, for example 100-300nm, 100-250nm or 100-200nm.

The emission of the polymeric tandem dye may have a quantum yield of 0.03 or greater, such as 0.04 or greater, 0.05 or greater, 0.06 or greater, 0.07 or greater, 0.08 or greater, 0.09 or greater, 0.1 or greater, 0.15 or greater, 0.2 or greater, 0.3 or greater,or even greater quantum yields. In some cases, the extinction coefficient of the polymeric tandem dye is 5×10 ⁵ cm ^-1 M ^-1 Or greater, e.g. 6X 10 ⁵ cm ^-1 M ^-1 Or greater, 7X 10 ⁵ cm ^-1 M ^-1 Or greater, 8X 10 ⁵ cm ^-1 M ^-1 Or larger, 9X 10 ⁵ cm ^-1 M ^-1 Or greater, e.g. 1X 10 ⁶ cm ^-1 M ^-1 Or greater, 1.5X10 ⁶ cm ^-1 M ^-1 Or greater, 2X 10 ⁶ cm ^-1 M ^-1 Or greater, 2.5X10 ⁶ cm ^-1 M ^-1 Or greater, 3X 10 ⁶ cm ^-1 M ^-1 Or greater, 4X 10 ⁶ cm ^-1 M ^-1 Or greater, 5X 10 ⁶ cm ^-1 M ^-1 Or greater, 6X 10 ⁶ cm ^-1 M ^-1 Or greater, 7X 10 ⁶ cm ^-1 M ^-1 Or greater, or 8X 10 ⁶ cm ^-1 M ^-1 Or larger. In some embodiments, the molar extinction coefficient of the polymeric tandem dye is 5×10 ⁵ M ^-1 cm ^-1 Or larger. In certain embodiments, the tandem dye has a molar extinction coefficient of 1×10 ⁶ M ^-1 cm ^-1 Or larger.

In embodiments, the subject tandem dyes provide fluorescent emissions from acceptor fluorophores that are brighter than the emissions that might result from such fluorescent dyes being isolated. The brightness of the polymer tandem dye emission may be 50mM ^-1 cm ^-1 Or greater, e.g. 60mM ^-1 cm ^-1 Or greater, 70mM ^-1 cm ^-1 Or greater, 80mM ^-1 cm ^-1 Or greater, 90mM ^-1 cm ^-1 Or greater, 100mM ^-1 cm ^-1 Or greater, 150mM ^-1 cm ^-1 Or greater, 200mM ^-1 cm ^-1 Or greater, 250mM ^-1 cm ^-1 Or greater, 300mM ^-1 cm ^-1 Or larger, or even larger. In some cases, the tandem dye emits a luminance that is at least 5 times greater than the luminance of the directly excited acceptor fluorophore, e.g., more than directThe brightness of the excited acceptor fluorophore is at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 50-fold greater, at least 100-fold greater, at least 300-fold greater, or even greater.

The terms "light harvesting chromophore" and "polymeric dye" are used interchangeably and refer to a polymer of the present disclosure having a plurality of pendant chromophore groups capable of harvesting light having a specific maximum absorption wavelength and converting it to emitted light having a longer maximum emission wavelength. The polymer may have a saturated or partially unsaturated backbone.

In certain embodiments of the tandem dye, the backbone is a linear polymer. In some cases, the linear polymer is selected from the group consisting of peptides, peptoids, hydrocarbon polymers, and PEG polymers. In some cases, the linear polymer is a peptide. In some cases, the linear polymer is a peptoid. In some cases, the polymer is a hydrocarbon polymer. In certain other cases, the polymer is a PEG polymer.

The subject tandem dyes may comprise a linear polymer backbone of any number of units. As used herein, the term "unit" refers to a structural subunit of a polymer. The term "unit" is meant to include monomers, comonomers, copolymer blocks, conjugated segments, repeat units, and the like. "repeat units" are subunits of a polymer, defined by the minimum number of different structural features required for a unit to be considered a monomer, such that when the unit is repeated n times, the resulting structure describes the polymer or a block thereof. In some cases, the polymer may comprise two or more different repeating units, e.g., when the polymer is a multi-block polymer or a random arrangement of units, each block may define different repeating units, e.g., an n-block and an m-block. It will be appreciated that a variety of arrangements of n and/or m repeating units or blocks are possible, and that any convenient linear arrangement of copolymerized blocks of various lengths is included within the overall polymer structure in the formulae described for the subject chromophores described herein. It will be appreciated that the polymer may also be represented by the formula expressed in mol% value per unit in the polymer, and that such formula may represent various arrangements of repeating units, such as random or multi-block polymers. In some cases, the repeating units of the polymer comprise a single monomer group. In some cases, the repeating units of the polymer include two or more monomer groups, i.e., comonomer groups, such as two, three, four, or more comonomer groups. As used herein, the term "comonomer" or "comonomer group" refers to a structural unit of a polymer, which itself may be part of a repeating unit of a polymer. In some embodiments, the conjugated polymer comprises a block copolymer composed of blocks of polymerized monomers. In this case, the block copolymer may be described as having different repeating units each corresponding to a different copolymerized block of the polymer. In some cases, the polymer is a diblock copolymer containing two different copolymer blocks. In this case, the polymer may be described as comprising co-blocks, wherein each co-block may be composed of co-monomers, such as one, two, three or more co-monomers.

The backbone of the tandem dye may have any convenient length. In some cases, a specific number of monomeric repeat units or segments of the chromophore may fall within a range of 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000, or 2 to 1,000 units or segments, or for example 5 to 100,000, 10 to 100,000, 100 to 100,000, 200 to 100,000, or 500 to 50,000 units or segments. In some cases, the specific number of monomeric repeat units or segments of the backbone may fall within the range of 2 to 1,000, such as 2 to 500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to 100, 9 to 100, or 10 to 100 units or segments. In some cases, the specific number of monomeric repeat units or segments of the backbone may fall within the range of 2 to 500, such as 2 to 400, 2 to 300, 2 to 200, or 2 to 100 units or segments. In some cases, the specific number of monomeric repeat units or segments of the backbone may fall within the range of 2 to 100 repeat monomeric units, such as 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, or 2 to 30 units or segments.

The polymer backbone may have a random configuration of non-conjugated repeat units. The polymer backbone may comprise a block or co-block configuration of non-conjugated repeat units. Alternatively, the polymer backbone may comprise a specific defined sequence of non-conjugated repeat units, e.g., amino acid residues of a polypeptide sequence. These configurations may be characterized by polymer segments of repeating units (e.g., as described herein), which segments themselves may be repeated throughout the module support.

By "non-conjugated" is meant that at least a portion of the repeating units comprise saturated backbone groups (e.g., groups having two or more consecutive covalent single bonds) that preclude pi-conjugation or extended delocalized electron structures along the polymer backbone from one repeating unit to the next. It will be appreciated that even though one repeat unit may not be conjugated to an adjacent repeat unit, such repeat unit may comprise one or more separate unsaturated groups comprising an unsaturated bond (e.g., alkenylene or alkynylene) and/or an aryl or heteroaryl group, which groups may be part of the backbone. In some cases, each repeat unit of the polymer backbone comprises a side chain comprising a pendant group attached or a chemoselective tag for attachment to the pendant group.

In certain embodiments of the tandem dye, the polymer backbone is a linear polymer. In some cases, the linear polymer is selected from the group consisting of peptides, peptoids, hydrocarbon polymers, and PEG polymers. In some cases, the linear polymer is a peptide. In some cases, the linear polymer is a peptoid. In some cases, the polymer is a hydrocarbon polymer. In certain other cases, the polymer is a PEG polymer. Further details regarding polymer backbones that can be used in embodiments of the present invention are found in PCT application serial No. PCT/US2019/024662, published as WO2019/191482, and PCT application serial No. PCT/US2020/019510, published as WO 2020/222894; the disclosures of these applications are incorporated herein by reference.

In some cases, the tandem dye comprises a linear peptide backbone of 2 to 100 amino acids, such as 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, or 2 to 30 amino acids. In some cases, the linear peptide backbone comprises 2 or more amino acids, such as 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, up to a maximum of 100 amino acids. In some cases, the tandem dye comprises a linear peptide backbone of 5 to 30 amino acids, for example 5 to 25, 5 to 20, 5 to 15, or 5 to 10 amino acids.

Any convenient polymer backbone may be suitable for including the linked water-soluble dipyrromethene-type dye (e.g., as described herein). In some embodiments, the subject tandem dye is described by formula (VA) or (VB):

wherein:

each J is a repeating unit of the backbone;

L ³ -L ⁵ each independently is a linker;

each D is a donor chromophore (e.g., as described herein);

each a is an acceptor fluorophore;

G ¹ and G ² Each independently selected from the group consisting of a terminal group, a polymer segment, a donor chromophore, an acceptor fluorophore, a linker, and a linked specific binding member;

x is 75mol% or more;

X1+ X2 is 75% or more; and

y is 25mol% or less.

In certain cases of tandem dyes of formula (VA) or (VB), the donor chromophore (D) is a water-soluble dipyrromethene-type dye of any one of formulas (I) - (IV) or any compound depicted in fig. 1 (e.g., as described herein). In some cases of tandem dyes of formula (VA) or (VB), each D-side donor chromophore of each repeat unit is the same.

In certain cases of tandem dyes of formula (VA), x is 80mol% or more, such as 85% or more, 90% or more, 95% or more. In certain embodiments of formula (VA), x is 90% or more, such as 92% or more, 94% or more, 96% or more.

In certain cases where the tandem dye has formula (VB), the combination of x1+X2 is 80mol% or more, such as 85% or more, 90% or more, 95% or more. In certain embodiments of formula (VB), the combination of x1 and x2 is 90% or more, such as 92% or more, 94% or more, 96% or more.

In certain cases of tandem dyes of formula (VA), y is 20mol% or less, such as 18% or less, 15% or less, 10% or less. In certain embodiments of formula (VA), y is 10% or less, such as 8% or less, 6% or less, 5% or less.

In certain cases of tandem dyes of formula (VB), y is 20 mole% or less, e.g., 18% or less, 15% or less, 10% or less. In certain embodiments of formula (VB), y is 10% or less, e.g., 8% or less, 6% or less, 5% or less.

In certain embodiments of the tandem dye of formula (VA) or (VB), G ¹ Or G ² Is a linked specific binding member (e.g., as described herein). In certain other cases of light harvesting chromophores of formula (VA) or (VB), J, L ³ -L ⁵ Any of D or a comprises a linked specific binding member.

Any convenient comonomer may be used to provide the repeating units of backbone (J) in formula (VA) or (VB). Comonomers of interest 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, polystyrene, ROMP (ring opening metathesis polymerization) monomers, ADMET (acyclic diene metathesis) monomers, cyclic carbonates, monomers derived from polyethylene glycols, and monomers derived from polyethylenimines. The comonomer may optionally be substituted, for example, with a chemoselective tag. The comonomers may be polymerized or attached using any convenient chemical method including, but not limited to, olefin polymerization, ring-opening polymerization, free radical polymerization, and conjugation between click chemistry or compatible chemistry-selective functionalities or tags. ADMET monomers of interest include, but are not limited to, those described by Mutlu et al ("Acyclic diene metathesis: a versatile tool for the construction of defined polymer architectures", chem. Soc. Rev.,2011,40,1404-1445). In some cases, each J has the formula:

Wherein:

R ²¹ is-L ³ -D、–L ⁵ -D or-L ⁴ -a, wherein D is a pendant donor chromophore (e.g., as described herein), a is an acceptor chromophore, and L ³ -L ⁵ Is an optional linker;

n and m are independently integers from 1 to 6 (e.g., 1 or 2); and

* Is a linkage to the polymeric backbone of the chromophore.

ROMP monomers of interest include, but are not limited to, those described by Song et al ("Scope of the Ring-Opening Metathesis Polymerization (ROMP) Reaction of 1-Substituted Cyclobutenes", j.am. Chem. Soc.,2010,132 (30), pages 10513-10520). In some cases, each J independently has one of the following formulas:

wherein:

x is CH ₂ Or O; and

* Is a linkage to the polymeric backbone of the chromophore.

In some cases, each J includes one of the following formulas:

wherein:

R ²¹ is-L ³ -D、–L ⁵ -D or-L ⁴ -A；

Each D is a pendant donor chromophore;

a is an acceptor chromophore;

L ³ -L ⁵ is an optional linker;

x is O or NR';

r' is H or lower alkyl (e.g., methyl);

r' is H, lower alkyl, substituted lower alkyl and WSG; and

* Is a linkage to the polymeric backbone of the chromophore. In some cases, the polymer backbone comprises a mixture of polystyrene and acrylate or acrylamide derived comonomers.

Tandem dyes may have hydrocarbon backbones prepared using any convenient polymerization method. In some cases, the hydrocarbon backbone is derived from an acrylate, acrylamide, or styrene comonomer or derivative thereof. In some cases, the chromophore is described by formula (VC):

wherein:

each D is independently a pendant donor chromophore (e.g., as described herein);

each a is an acceptor chromophore;

each L ³ 、L ⁴ And L ⁵ Independently a linker;

a. b and c are the mol% value of each comonomer;

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

G ¹ and G ² Each independently selected from the group consisting of end groups, polymer segments, donor chromophores, acceptor fluorophoresA linker and a linked specific binding member.

In some cases of formula (VC), c=0. In some cases of formula (VC), a>0, and b>0. In some cases of formula (VC), a is 80mol% or more, such as 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 (VC), b is 20mol% or less, such as 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 (VC), a is 65-95mol%, b is 5-35mol%, and c is 0-30mol%, where a+b+c=100%. In some cases of formula (VC), L ³ -L ⁵ Comprising a linker to a polymer backbone selected from the group consisting of: -COO-, -CONR '-, -Ph-, -O-, wherein R' is H, lower alkyl, substituted lower alkyl or WSG. Such linkers can be used to attach the D and a groups to the polymer backbone.

In some cases of formula (VC), L ³ D is described by one of the following:

wherein R is ⁴ Is H, lower alkyl, substituted lower alkyl or WSG.

Tandem dyes may have backbones derived from comonomers that are linked via linkers or groups derived from click chemistry conjugation reactions. Any convenient divalent comonomer group can be derivatized with a terminal chemoselective tag and polymerized via conjugation of a compatible chemoselective tag. In some cases, the comonomer comprises one or more ethylene oxide or ethyleneamino groups that form part of the polymer backbone. Such groups may provide the desired water solubility of the resulting polymeric dye. The comonomer may also comprise trivalent units for attachment to side chain groups such as donor or acceptor dyes or WSG. In some cases, the comonomer comprises propylene oxide or a propylene amino group in the backbone, which is further substituted at the 2-position with a side chain group or substituent. The group may comprise a linked chemoselective tag, a linked donor or acceptor dye, or a linked WSG.

In some cases, the tandem dye has the formula (VD):

wherein:

the polymer backbone of the repeating unit comprises J each linked via a group T ² 、J ³ And J ⁴ A comonomer, group T is the product of a click chemistry or a chemoselective group conjugation reaction (e.g., azide-alkyne click chemistry);

J ⁴ optionally comprising a linked WSG;

each D ¹ Independently of J ² A linked pendant light absorbing chromophore;

each Z ¹ Independently from J ³ A linked chemoselective tag;

x is 50mol% or more; and

y+z is 50mol% or less, where x is the linkage to the polymeric backbone or terminal group of the multichromophore, e.g., as described herein. In some cases of the formula (VD), J ² 、J ³ And J ⁴ Comprising repeating units selected from the group consisting of ethylene oxide, ethyleneamino, 2-substituted propylene oxide and 2-substituted propyleneamino. In some cases of formula (VD), each T is a 1, 4-substituted 1,2, 3-triazole, the product of an azide-alkyne click chemistry conjugation reaction.

In some cases of formula (VD), J ² 、J ³ And J ⁴ The structure is as follows:

wherein:

each X is independently OOr NR (NR) ³¹ Wherein R is ³¹ Is H, alkyl, substituted alkyl, alkanoyl or substituted alkanoyl;

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

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

t is 0 or 1;

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

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

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

each L ¹ 、L ² And L ³ Independently a linker; and

* Is a linkage to a 1, 4-substituted 1,2, 3-triazole (T).

It will be appreciated that for deriving J as described above ² -J ⁴ Either of the structural comonomers, an azide or alkyne group can be used at the end of the comonomer for attachment during polymerization. Thus, 1, 4-substituted 1,2, 3-triazoles (T) may exist in one of two possible orientations:

the ends of the polymer backbone may comprise any convenient end groups, such as azide or alkyne groups, linkers or attached specific binding moieties.

In some cases, the tandem dye comprises one of the following structures:

wherein:

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

L ¹ and L ² Independently a linker;

D ¹ is a donor chromophore (e.g., as described herein);

each d, e and f is independently 1-6;

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

Each R ⁴¹ Selected from alkyl, substituted alkyl, and WSG; and

the "linker" comprises an optional chemically selective functional group, e.g., for conjugation with a comonomer or biomolecule. In some cases of formula (VD), L ¹ -L ³ Comprising a linker to a polymer backbone selected from-NHCO-alkyl.

Cyclic carbonates and protected carbonate monomers of interest that may be suitable for use in preparing the polymeric backbone of the subject light harvesting chromophore include, but are not limited to, those described in WO2013036532 by Barnes et al, cooley et al (j.am. Chem. Soc.,131,45,1640-3,2009) and U.S. patent 7,169,814 to Rothbard et al. Such monomers are used in polymerization reactions using initiators and suitable cyclic carbonate monomer feed ratios to provide a polymer backbone. Alternatively, the protected carbonate monomers may be combined in a stepwise synthesis to provide a defined sequence. In some cases of the subject tandem dyes, the backbone has a polycarbonate backbone. Thus, the light harvesting chromophore can have the formula (VE):

wherein:

each D is independently a pendant donor chromophore group (e.g., as described herein);

each a is independently an acceptor fluorophore;

each L ³ And L ⁴ Independently a linker;

x is 75mol% or more;

y is 25mol% or less; and

G ¹ and G ² Each independently selected from the group consisting of end groups, polymer segments, donor chromophore groups, acceptor fluorophoresA linker and a linked specific binding member.

In certain embodiments of the subject tandem dyes, the repeat units (e.g., J or J ² -J ⁴ ) Arranged in a defined linear sequence. Any convenient comonomer that can polymerize in a defined stepwise manner can be used to construct the subject light harvesting chromophores (e.g., light harvesting chromophores of formula (VA) or (VB)). The comonomer may be derived from amino acids, peptoid monomers or protected or cyclic carbonate monomers.

In some cases of the subject tandem dyes, the backbone is a polypeptide having a defined sequence of alpha-amino acid residues and/or beta-amino acid residues. Two types of β -amino acids and polypeptides can be used in the polymer backbone of the subject light harvesting chromophore: those with side chain groups immediately adjacent to the amine are referred to as β3-peptides/residues and those with side chain groups immediately adjacent to the carbonyl group are referred to as β2-peptides/residues. In certain embodiments, the light harvesting chromophore has the formula (VF):

wherein:

each D is independently a pendant light absorbing chromophore group (e.g., as described herein);

Each a is an acceptor fluorophore;

each L ³ And L ⁴ Independently a linker;

p ₁ and q ₁ Independently 0 or 1, wherein p ₁ +q ₁ ≤1；

p ₂ And q ₂ Independently 0 or 1, wherein p ₁ +q ₁ ≤1；

x is 75mol% or more;

y is 25mol% or less; and

G ¹ and G ² Each independently selected from the group consisting of a terminal group, a polymer segment, a light absorbing (e.g., donor) chromophore, an acceptor fluorophore, a linker, and a linked specific binding member.

In certain embodiments of the tandem dye of formula (VA) or (VB), each repeating unit J is an amino acid or peptide unit.

In some embodiments of formula (VA), the polymeric tandem dye has formula (VA 1):

wherein:

each a is independently an acceptor fluorophore;

L ³ and L ⁴ Each independently is a linker;

x is 75mol% or more;

y is 25mol% or less; and

G ¹ and G ² Each independently selected from the group consisting of a terminal group, a polymer segment, a donor chromophore, an acceptor fluorophore, a linker, and a linked specific binding member. It is understood that the chromophore described by formula (VA 1) includes any convenient comonomer arranged in a defined linear sequence, which together have a defined mole% ratio of x and y. In some cases, the a-containing comonomers are spaced throughout the sequence of the polymer backbone, and thus are always flanked on both sides by one or more D-containing comonomers.

In certain instances of formula (VB), the polymeric tandem dye comprises a segment of formula (VB 1):

/>

wherein:

each a is independently an acceptor chromophore;

each L ³ -L ⁵ Each independently is a linker;

x1 and x2 are each independently integers from 1 to 20, wherein x1+x2.gtoreq.2; and

y is 1 or 2.

In some cases of formula (VB 1), x1 and x2 are each independently 1 to 10, such as 2 to 10, 3 to 10, or 3 to 6. In some cases of formula (VB 1), x1+x2 is an integer from 2 to 20, such as from 3 to 20, from 4 to 20, from 5 to 15, or from 5 to 12. In certain embodiments of formula (VB 1), y is 1.

The subject polymeric tandem dye may comprise a plurality of segments of formula (VB 1), wherein each segment comprises a separate A-containing comonomer flanked by blocks containing D-comonomers. In some cases, the chromophore comprises two or more segments of formula (VB 1) immediately adjacent to each other to provide two separate a-containing comonomers separated by 2-20D-containing comonomer blocks, such as 3 to 20, 4 to 20, 5 to 15, or 5 to 12D-containing comonomer blocks. Thus, in certain embodiments, the polymeric tandem dye comprises q segments of a block copolymer and has the formula (VB 2):

(VB2)

Wherein: each (x 1) _q And each (x 2) _q Independently an integer of 1 to 20, wherein for each of the q segments, (x 1) _q +(x2) _q Not less than 3; and q is an integer from 1 to 100.

Any convenient amino acid may be suitable for providing a polymer backbone to which pendant groups may be covalently attached. The amino acids may be naturally occurring or non-naturally occurring. For example, amino acids (e.g., lysine, ornithine) have side chain amino groups suitable for conjugation to amino reactive groups (e.g., activated carboxylic acids). Cysteine contains a side chain thiol group suitable for conjugation to a thiol-reactive group (e.g., maleimide or haloacetyl). Aspartic acid and glutamic acid have pendant carboxylic acid groups that can be conjugated to an affinity group (e.g., an amino group). Described herein are methods of making subject multichromophores, including peptide synthesis methods.

In some cases, the polymeric backbone of the multichromophore has one or more of the following polypeptide sequence segments:

XYXX

XXYXX

XXXYXXX

XXXYXXXX

XXXXYXXX

XXXXYXXXX

XXXXXYXXXXX

XXXXXXYXXXXXX

XXXXXXXYXXXXXXX

XXXXXXXXYXXXXXXXX

XXXXXXXXXYXXXXXXXXX

Y(X) _n Y

XY(X) _n YX

XXY(X) _n YXX

XXXY(X) _n YXXX

XXXXY(X) _n YXXXX

XXXXXY(X) _n YXXXXX

wherein:

each X is a first amino acid residue having a side-chain attached first chemoselective tag or a side-chain attached pendant donor chromophore;

each Y is a second amino acid residue having a side-chain attached second chemoselective tag or a side-chain attached acceptor fluorophore; and

n is an integer from 2 to 20, such as from 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.

In certain embodiments, a third type of amino acid residue (Z) may be incorporated into the polymer backbone in addition to one or more polypeptide segments described herein, e.g., between two of the segments. In some cases, a single isolated third amino acid residue is incorporated between any two of the segments described herein, e.g., (segment 1) -Z- (segment 2). The third amino acid residue may be a spacer residue or a residue having a chemically selective functional group suitable for selectively mounting an additional moiety of interest, such as a second pendant light absorbing chromophore, a chemically selective tag (e.g., a bioorthogonal click chemistry tag), a linker, a linked biomolecule, an acceptor fluorophore, WSG, or the like. In some cases, the additional moiety of interest is incorporated into the third amino acid residue prior to preparation.

In certain instances, the first amino acid residue (X) comprises a side chain amino group, e.g., lysine or ornithine or protected forms thereof. During SPPS of the polymer backbone, these side chain amino groups can remain protected (e.g., with Cbz or Boc protecting groups) followed by orthogonal deprotection to provide conjugation of the first residue to the pendant donor chromophore. In some cases, the second amino acid residue comprises a side chain thiol group, e.g., cysteine or a protected form thereof. Similarly, during SPPS of the polymer backbone, these side chain thiol groups can remain protected, followed by orthogonal deprotection to provide conjugation of the second residue to the pendent group of interest (e.g., acceptor fluorophore).

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

KCKK(SEQ ID NO:1)

KKCK(SEQ ID NO:2)

KKYKK(SEQ ID NO:3)

KKKYKK(SEQ ID NO:4)

KKYKKK(SEQ ID NO:5)

KKKYKKK(SEQ ID NO:6)

KKKYKKKK(SEQ ID NO:7)

KKKKYKKK(SEQ ID NO:8)

KKKKCKKKK(SEQ ID NO:9)

KKKKKCKKKKK(SEQ ID NO:10)

KKKKKKCKKKKKK(SEQ ID NO:11)

KKKKKKKCKKKKKKK(SEQ ID NO:12)

KKKKKKKKCKKKKKKKK(SEQ ID NO:13)

KKKKKKKKKCKKKKKKKKK(SEQ ID NO:14)

KKKKCKKKKKKKKKKCKKKKK(SEQ ID NO:15)

C(K) _n C(SEQ ID NO:16)

KC(K) _n CK(SEQ ID NO:17)

KKC(K) _n CKK(SEQ ID NO:18)

KKKC(K) _n CKKK(SEQ ID NO:19)

KKKKC(K) _n CKKKK(SEQ ID NO:20)

KKKKKC(K) _n CKKKKK(SEQ ID NO:21)

wherein: each K is a lysine residue, a protected lysine residue, or a lysine residue covalently linked to a pendant donor chromophore group via a side chain amino group; n is an integer from 2 to 20 (e.g., from 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.

OCOO(SEQ ID NO:22)

OOCO(SEQ ID NO:23)

OOCOO(SEQ ID NO:24)

OOOCOO(SEQ ID NO:25)

OOCOOO(SEQ ID NO:26)

OOOCOOO(SEQ ID NO:27)

OOOCOOOO(SEQ ID NO:28)

OOOOCOOO(SEQ ID NO:29)

OOOOCOOOO(SEQ ID NO:30)

OOOOOCOOOOO(SEQ ID NO:31)

OOOOOOCOOOOOO(SEQ ID NO:32)

OOOOOOOCOOOOOOO(SEQ ID NO:33)

OOOOOOOOCOOOOOOOO(SEQ ID NO:34)

OOOOOOOOOCOOOOOOOOO(SEQ ID NO:35)

OOOOOOOOOOCOOOOOOOOOO(SEQ ID NO:36)

C(O) _n C(SEQ ID NO:37)

OC(O) _n CO(SEQ ID NO:38)

OOC(O) _n COO(SEQ ID NO:39)

OOOC(O) _n COOO(SEQ ID NO:40)

OOOOC(O) _n COOOO(SEQ ID NO:41)

OOOOOC(O) _n COOOOO(SEQ ID NO:42)

wherein:

o is an ornithine residue, a protected ornithine residue or an ornithine residue covalently linked via a side chain amino group to a pendant donor chromophore group; n is an integer from 2 to 20 (e.g., from 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.

In some cases of the subject tandem dyes, the polymer backbone is a peptoid backbone. Thus, the chromophore of formula (VA) may have formula (VA 2):

Wherein the method comprises the steps of

each a is independently an acceptor fluorophore;

each L ³ And L ⁴ Independently a linker;

x is 75mol% or more;

y is 25mol% or less; and

G ¹ and G ² Each independently selected from the group consisting of a terminal group, a polymer segment, a light absorbing (e.g., donor) chromophore group, an acceptor fluorophore, a linker, and a linked specific binding member. It is understood that the chromophore described by formula (VA 2) may comprise any convenient arrangement of comonomers in a defined linear sequence, which together have a defined mole% ratio of x and y. In some cases, the a-containing comonomers are spaced throughout the sequence of the polymer backbone, and thus are always flanked on both sides by one or more D-containing comonomers.

In some cases of formula (VA 2), x is 80mol% or more, such as 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 (VA 2), y 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.

As described above, the subject tandem dye provides a donor chromophore having an absorbance maximum of 555nm to 585nm, such as 555nm to 575nm, such as 555 to 574nm, 555 to 573nm, 555 to 572nm, 555 to 571nm, 555 to 570nm, 555 to 569nm, 555 to 568nm, 555 to 567nm, 555 to 566nm, 555 to 565nm, 555 to 564nm, 555 to 563nm, or 555 to 562nm. In some cases of tandem dyes, the absorption maximum of the donor chromophore is 558 to 568nm, such as 558 to 567nm, 558 to 566nm, 558 to 565nm, 558 to 564nm, 558 to 563nm, or 558 to 562nm. In some cases of tandem dyes, the absorption maximum of the donor chromophore is about 561nm.

The various emission curves depend on a variety of factors, such as the selected comonomers, linking groups, substituents, and attached acceptor fluorophores that make up the tandem dye. In some embodiments, the maximum emission wavelength of the chromophore is 300 to 900nm, such as 350 to 850nm, 350 to 600nm, 360 to 500nm, 370 to 500nm, 380 to 500nm, 390 to 500nm, or 400 to 500nm, with specific examples of emission maxima of interest including, but not limited to: 395 nm.+ -. 5nm, 460 nm.+ -. 5nm, 490 nm.+ -. 5nm, 550 nm.+ -. 5nm, 560 nm.+ -. 5nm, 605 nm.+ -. 5nm, 650 nm.+ -. 5nm, 680 nm.+ -. 5nm, 700 nm.+ -. 5nm, 805 nm.+ -. 5nm. In some cases, the maximum emission wavelength of the chromophore is selected from 395nm, 460nm, 490nm, 550nm, 560nm, 605nm, 650nm, 680nm, 700nm, and 805nm. In some cases, the chromophore has a maximum emission wavelength of 395 nm.+ -.5 nm. In some cases, the chromophore itself has a maximum emission wavelength of 375 to 900nm (e.g., 380nm to 900nm, 390nm to 900nm, or 400nm to 900 nm).

In certain embodiments of the subject tandem dyes, the ratio of donor chromophore to acceptor fluorophore is selected from the group consisting of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 20:2. In some cases, the ratio of donor chromophore to acceptor fluorophore is 5:1. In some cases, the ratio of donor chromophore to acceptor fluorophore is 6:1. In some cases, the ratio of donor chromophore to acceptor fluorophore is 7:1. In some cases, the ratio of donor chromophore to acceptor fluorophore is 8:1. In some cases, the ratio of donor chromophore to acceptor fluorophore is 9:1. In some cases, the ratio of donor chromophore to acceptor fluorophore is 10:1.

In certain embodiments of the tandem dye, the light harvesting chromophore further comprises a specific binding member. In some cases of tandem dyes, the specific binding member is selected from an antibody, antibody fragment, or binding derivative thereof (e.g., as described below). In some cases of tandem dyes, the specific binding member is attached to a terminal group of the light harvesting chromophore. In certain other cases of tandem dyes, the specific binding member is attached to the backbone at any point between the end groups of the light harvesting chromophore. In some cases, the specific binding member is attached to the backbone at a central point of the light harvesting chromophore.

End group

Any convenient end group (e.g., G ¹ And G ² ) Can be used at the end of the subject tandem dye. As used herein, the terms "end group" and "terminal group" are used interchangeably to refer to a group located at the end of a polymer structure of a light harvesting chromophore, e.g., as described herein. G of interest ¹ And G ² Groups include, but are not limited to, terminal end capping groups, pi conjugated segments, linkers, and linked specific binding members. In some embodiments, the terminal end capping group is a monovalent group conjugated to the backbone of the light harvesting chromophore after polymerization. In some cases, the terminal end capping group is an aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, or substituted alkyl group. In some cases, the terminal comonomer is directly linked to a chemoselective tag or linker. In some cases, the terminal end-capping end groups are derived from monomers used in the polymerization process, e.g., terminal groups, such as halogens (e.g., br), boric acid, or boronates, which are capable of further conjugation. In some cases, G ¹ And/or G ² Is pi co-A yoke segment. As used herein, pi conjugated segment refers to any convenient segment of a conjugated polymer to which a light harvesting chromophore can be conjugated, i.e., to allow pi electrons to delocalize across adjacent units. In certain embodiments, G ¹ And/or G ² Is a linker, such as a linker comprising a functional group suitable for conjugation to a specific binding moiety. It will be appreciated that G located in tandem dye may be selected ¹ And/or G ² The linker at a position so as to be orthogonal to any other linker that may be present in the side chain of the light harvesting chromophore, including the chemoselective tag (e.g., as described herein). In certain embodiments, at G ¹ And/or G ² Containing an amino function or derivative thereof. In certain embodiments, at G ¹ And/or G ² Containing carboxylic acid functional groups or derivatives thereof.

In some embodiments of the formulae described herein, G ¹ And G ² At least one of them is-L ⁶ -Z ⁴ Wherein L is ⁶ Is a linker (e.g., as described herein), and Z ⁴ Is a specific binding member (e.g., as described herein). In some embodiments of the formulae described herein, G ¹ And G ² At least one of them is-L ⁶ -Z ³ Wherein L is ⁶ Is a linker (e.g., as described herein), and Z ³ Is a chemoselective tag (e.g., as described herein). Any convenient chemoselective label and conjugated chemical composition may be suitable for the subject light harvesting chromophore. Chemoselective tags of interest include, but are not limited to, amines, activated esters, maleimides, thiols, sulfur (VI) fluoride exchange chemistry (superfex), sulfonyl fluorides, diers Alder cycloaddition click reagents and click chemistry, tetrazines, trans-cyclooctenes, aldehydes, alkoxyamines, alkynes, cyclooctyne, azides, and the like. In some cases, Z ³ Selected from carboxylic acids, activated esters (e.g., N-hydroxysuccinimide ester (NHS) or sulfo-NHS), amino groups, maleimides, iodoacetyl groups, and thiols. In certain embodiments of the formulas described herein, G ¹ And G ² Is described by the following structure:

*-Ar-L-Z

wherein Ar is a pi-conjugated aryl group, L is a linker, and Z is a chemoselective tag or specific binding member. In some cases, the L-Z group may be directly attached to the terminal comonomer. In certain embodiments of the formulas described herein, G ¹ And G ² Is described by the following structure:

wherein:

q is 0 or an integer from 1 to 12;

l is an optional linker; and

z is a chemoselective tag or a specific binding member.

In certain embodiments, Z is a biomolecule. Biomolecules of interest include, but are not limited to, polypeptides, polynucleotides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives thereof, structural analogs, and combinations thereof. In certain instances, Z is an antibody. In some cases, Z is an antibody fragment or binding derivative thereof. In some cases, the antibody fragment or binding derivative thereof is selected from the group consisting of Fab fragment, F (ab') ₂ Fragments, scFv, diabodies, and triabodies.

It is to be appreciated that for any of the structures and formulas depicted herein, in some cases of the subject light harvesting chromophores, the depicted end groups may be located at opposite ends of those shown, e.g., end group G ¹ and-Ph-L-Z may be exchanged. In some embodiments of the light harvesting chromophores described herein, at least one end group (e.g., G ¹ 、G ² ) Or a side chain group selected from one of the following structures:

wherein r is 0 or an integer from 1 to 50; k is 0 or an integer from 1 to 50 (e.g., 1 to 20); r is R ¹ Is as defined herein for any one of the fluorene comonomers; and R is ¹⁶ Selected from H, OH, NH ₂ 、-NH(CH ₂ )r-NH ₂ and-NH (CH) ₂ ) _r COOH. In some cases, r is 1 to 20, such as 3 to 20, 3 to 15, 3 to 12, or 6 to 12. In certain instances, r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some cases, r is 3. In some cases, r is 4. In some cases, r is 5. In some cases, r is 6. In some cases, r is 7. In some cases, r is 8. In some cases, r is 9. In some cases, r is 10. In some cases, r is 11.

In some embodiments, the tandem dye comprises one or more of the following groups, for example, as a terminal or side chain group for conjugation to a compatible functional group on another molecule:

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Labeled specific binding members

Aspects of the disclosure include labeled specific binding members. Labeled specific binding members are conjugates of a subject polymer dye (e.g., as described herein) and a specific binding member. Any of the polymeric dyes or polymeric tandem dyes described herein may be conjugated to a specific binding member. The specific binding member and the light harvesting chromophore may be conjugated (e.g., covalently linked) to each other at any convenient location of the two molecules via an optional linker.

In some embodiments, the labeled specific binding member is anti-aggregated. As used herein, "anti-aggregation" refers to a labeled specific binding member that is capable of forming a homogeneous aqueous composition free of aggregation precipitates at a concentration of 1mg/mL or more in an aqueous buffer of interest, such as 2mg/mL or more, 3mg/mL or more, 4mg/mL or more, 5mg/mL or more, 6mg/mL or more, 7mg/mL or more, 8mg/mL or more, 9mg/mL or more, 10mg/mL or more, or even more of the labeled specific binding member. In certain embodiments, the anti-aggregate labeled specific binding member comprises: a polymeric tandem dye comprising a light harvesting chromophore (e.g., as described herein) and an acceptor fluorophore attached to the chromophore in its vicinity to the energy reception; and a specific binding member covalently linked to the chromophore.

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 a region or cavity on its surface that specifically binds to a region or cavity on the surface of the other member of the pair of molecules. The members of the pair thus have the property of specifically binding to each other to create a binding complex. In some embodiments, the affinity between specific binding members in the binding complex is characterized by a Kd (dissociation constant) of 10 ^-6 M or less, e.g. 10 ^-7 M or less, including 10 ^-8 M or less, e.g. 10 ^-9 M or less, 10 ^-10 M or less, 10 ^-11 M or less, 10 ^-12 M or less, 10 ^-13 M or less, 10 ^-14 M or less, including 10 ^-15 M or less. In some embodiments, the specific binding member specifically binds with high affinity. High affinity refers to binding members that specifically bind with an apparent affinity characterized by an apparent Kd of 10X 10 ^-9 M or less, e.g. 1X 10 ^- ⁹ M or less, 3×10 ^-10 M or less, 1×10 ^-10 M or less, 3×10 ^-11 M or less, 1×10 ^-11 M or less, 3×10 ^-12 M or less, or 1X 10 ^-12 M or less.

The specific binding member may be proteinaceous. As used herein, the term "proteinaceous" refers to a moiety consisting of amino acid residues. The proteinaceous moiety may 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, e.g., a binding fragment of an antibody that specifically binds to a polymeric dye. As used herein, the terms "antibody" and "antibody molecule" are used interchangeably and refer to a protein consisting of one or more polypeptides that are encoded substantially by all or part of a recognized immunoglobulin gene. Recognized immunoglobulin genes (e.g., those recognized in humans) include kappa (k), lambda (l), and heavy chain genetic loci, which together comprise innumerable variable region genes, as well as constant region genes mu (u), delta (d), gamma (g), sigma (e), and alpha (a), which encode IgM, igD, igG, igE and IgA isoforms, respectively. Immunoglobulin light or heavy chain variable regions are composed of a "framework" region (FR) that is interrupted by three hypervariable regions (also known as "complementarity determining regions" or "CDRs"). The framework regions and the scope of the CDRs have been precisely defined (see, "Sequences of Proteins of Immunological Interest," e.kabat et al, U.S. department of health and public service (u.s.device of Health and Human Services), (1991)). All antibody amino acid sequences discussed herein are numbered in accordance with the Kabat system. The sequences of the different light or heavy chain framework regions are relatively conserved in one species. The framework regions of antibodies, i.e., the combined framework regions that make up the light and heavy chains, are used to position and align the CDRs. CDRs are primarily responsible for binding to epitopes of the antigen. The term antibody is meant to include full length antibodies, and may refer to natural antibodies, engineered antibodies from any organism, or recombinantly produced antibodies for experimental, therapeutic, or other purposes, as further defined below.

Antibody fragments of interest include, but are not limited to, fab ', F (ab') 2, fv, scFv, or other antigen-binding subsequences of antibodies, which are produced by modification of intact antibodies or synthesized de novo using recombinant DNA techniques. Antibodies may be monoclonal or polyclonal, and may have other specific activities on the cell (e.g., antagonists, agonists, neutralizing, inhibitory, or stimulating antibodies). It will be appreciated that antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions.

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 murine antibody or binding fragment thereof. In some cases, the specific binding member is a recombinant antibody or binding fragment thereof.

Method

As summarized above, aspects of the invention include methods of assessing the presence of a target analyte in a sample. Aspects of the method include contacting the sample with a polymer dye conjugate that specifically binds to the target analyte to produce a sample contacted with the labeling composition. As used herein, the terms "polymer dye conjugate" and "labeled specific binding member" are used interchangeably. Thus, the polymeric dye conjugate may comprise: (i) A water-solvated polymer tandem dye (e.g., as described herein); and (ii) a specific binding member (e.g., as described herein). In some cases, the polymer tandem dye conjugates comprise an acceptor fluorophore covalently linked to a chromophore of the polymer dye in its vicinity of the energy reception (e.g., as described herein).

The sample may be contacted with the polymer dye conjugate that specifically binds to the target analyte using any convenient method to produce a sample contacted with the labeling composition. In some cases, the sample is contacted with the polymer dye conjugate under conditions in which the specific binding member specifically binds the target analyte (if present). For specific binding of the specific binding member of the conjugate to the target analyte, an appropriate solution may be used to maintain the biological activity of the sample components and the specific binding member. The solution may be a balanced salt solution conveniently supplemented with fetal bovine serum, human platelet lysate or other factors, e.g., physiological saline, PBS, hank's balanced salt solution, etc., as well as a low concentration (e.g., 5-25 mM) of acceptable buffer. Convenient buffers include HEPES, phosphate buffer, lactate buffer, and the like. Various media are commercially available and may be used depending on the nature of the target analyte, including dMEM, HBSS, dPBS, RPMI, iscove's media and the like, in some cases supplemented with fetal bovine serum or human platelet lysate. The final composition of the solution may be selected according to the sample composition contained.

The temperature at which specific binding of the conjugate to the target analyte occurs may vary, and in some cases may be from 5 ℃ to 50 ℃, such as from 10 ℃ to 40 ℃, from 15 ℃ to 40 ℃, from 20 ℃ to 40 ℃, e.g., 20 ℃,25 ℃, 30 ℃, 35 ℃, or 37 ℃ (e.g., as described above). In some cases, the temperature at which specific binding occurs is selected to be compatible with the biological activity of the specific binding member and/or target analyte. In some cases, the temperature is 25 ℃, 30 ℃, 35 ℃, or 37 ℃. 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 ℃), 30 ℃, 35 ℃, or 37 ℃. Any convenient incubation time for specific binding may be selected to allow for the formation of the desired amount of binding complex, and in some cases, the incubation time may be 1 minute (min) or longer, such as 2min or longer, 10min or longer, 30min or longer, 1 hour or longer, 2 hours or longer, or even 6 hours or longer.

Any convenient specific binding member may be used in the conjugate. Specific binding members of interest include, but are not limited to, those agents that specifically bind cell surface proteins of a variety of cell types, including, but not limited to, stem cells (e.g., pluripotent stem cells, hematopoietic stem cells), T cells, T regulatory cells, dendritic cells, B cells (e.g., memory B cells, antigen-specific B cells), granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest include cells having a convenient cell surface marker or antigen that can be captured by a convenient specific binding member conjugate. In some embodiments, the target cells are selected from HIV-containing cells, treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (cd34+) from whole blood, bone marrow, or cord blood. In the subject methods, any convenient cell surface protein or cell marker can be targeted to specifically bind to the polymer dye conjugate. In some embodiments, the target cell comprises a cell surface marker selected from a cell receptor and a cell surface antigen. In some cases, the target cell may include a cell surface antigen, 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, bisialoganglioside GD2, and CD71.

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

In some embodiments, the target analyte is associated with a cell. In some cases, the target analyte 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. In some cases, the target analyte is an intracellular target, and the method further comprises lysing the cells.

In some embodiments, the sample may comprise a heterogeneous population of cells that separate target cells. In some cases, the sample comprises peripheral whole blood, peripheral whole blood in which red blood cells have been lysed prior to cell separation, umbilical cord blood, bone marrow, density gradient purified peripheral blood mononuclear cells, or tissue homogenates. In some cases, the sample includes hematopoietic progenitor cells (e.g., cd34+ cells) in whole blood, bone marrow, or umbilical cord blood. In certain embodiments, the sample comprises tumor cells in peripheral blood. In certain instances, the sample comprises (or is suspected of comprising) a viral cell (e.g., HIV).

Labeled specific binding members can be used in the subject methods, for example, to label target cells, particles, targets, or analytes with a polymeric dye or a polymeric tandem dye. For example, labeled specific binding members may be used to label cells to be treated (e.g., detected, analyzed, and/or sorted) in a flow cytometer. Labeled specific binding members may include antibodies that specifically bind, for example, cell surface proteins of a variety of cell types (e.g., as described herein). Labeled specific binding members can be used to study a variety of biological (e.g., cell) properties or processes, such as cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression and/or presentation, and the like. The labeled specific binding member may be used for any application, including (or may include) antibody-mediated labeling of cells, particles, or analytes.

Aspects of the method include determining the presence of a dye conjugate-target analyte binding complex in a sample contacted by the labeling composition to assess the presence or absence of the target analyte in the sample. Once the sample has been contacted with the dye conjugate, any convenient method may be used to determine the sample contacted by the labeling composition that is generated against the presence of the dye conjugate-target analyte binding complex. Dye conjugate-target analyte binding complexes are binding complexes that are generated when a specific binding member of the conjugate specifically binds to a target analyte (if present). Determining the sample contacted by the labeling composition may include detecting a fluorescent signal from the binding complex (if present). In some cases, the assay includes a separation step in which the target analyte (if present) is separated from the sample. The target analyte may be isolated from the sample using a variety of methods, for example, via immobilization on a support. The assay methods of interest include, but are not limited to, any convenient method and assay format of interest wherein specific binding member pairs, such as avidin-biotin or hapten-anti-hapten antibodies, can be used. Methods and assay formats of interest that may be suitable for use in 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 separation assays, and fluorescent dye purification chromatography.

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

Any convenient support may be used in the subject methods to immobilize any convenient component of the method, e.g., a labeled specific binding member, a target, a second specific binding member, etc. Supports of interest include, but are not limited to: a solid substrate, wherein the substrate can have a variety of configurations, e.g., sheets, beads, or other structures, such as a plate with holes; beads, polymers, particles, webs, hydrogels, porous matrices, needles, microarray surfaces, chromatographic supports, and the like. In some cases, the support is selected from the group consisting of particles, planar solid matrices, fiber networks, hydrogels, porous matrices, needles, microarray surfaces, and chromatographic supports. The support may be incorporated into a system that provides cell separation assisted by any convenient method, such as a manually operated syringe, centrifuge, or automated liquid handling system. In some cases, the support may be used in an automated liquid handling system to isolate cells at high throughput, such as a flow cytometer.

In some embodiments of the method, the separating step comprises applying an external magnetic field to immobilize the magnetic particles. Any convenient magnet may be used as a source of external magnetic fields (e.g., magnetic field gradients). In some cases, the external magnetic field is generated by a magnetic source, such as a permanent magnet or an electromagnet. In some cases, fixed magnetic particles refer to magnetic particles that accumulate near the surface closest to the magnetic field gradient source (i.e., the magnet).

The separation may also include one or more optional washing steps to remove unbound material of the sample from the support. Any convenient washing method may be used, for example washing the immobilization support with a biocompatible buffer that maintains the specific binding interactions of the polymer dye and the specific binding member. The separation and optional washing of unbound material of the sample from the support provides an enriched target cell population in which unwanted cells and material can be removed.

In certain embodiments, the method further comprises detecting the labeled target. Detecting the labeled target may include exciting a chromophore with one or more lasers, followed by detecting fluorescent emissions from the polymeric tandem dye using one or more optical detectors. The labeled targets can be detected using any convenient instrument and method, including but not limited to flow cytometry, FACS systems, fluorescence microscopy; detecting fluorescence, luminescence, ultraviolet and/or visible light using an enzyme-labeled instrument; high Performance Liquid Chromatography (HPLC); and mass spectrometry. When using fluorescently labeled components in the methods and compositions of the present disclosure, it is recognized that the subject methods can be practiced using different types of fluorescence detection systems. In some cases, high throughput screening can be performed, for example, in systems using 96-well or larger microtiter plates. Fluorescent materials can be assayed by a variety of methods, such as Lakowicz, J.R., principles of Fluorescence Spectroscopy, new York: plenum Press (1983); herman, B., resonance energy transfer microscopy, section Fluorescence Microscopy of Living Cells in Culture, section B, methods in Cell Biology, volume 30, taylor, D.L, & Wang, J-L, san Diego: academic Press (1989), pages 219-243; turro, N.J., modern Molecular Photochemistry, menlo Park: benjamin/Cummings Publishing Col, inc. (1978), pages 296-361.

Fluorescence in the sample can be measured using a fluorometer. In some cases, excitation radiation from an excitation source having a first wavelength passes through excitation optics. Excitation optics cause excitation radiation to excite the sample. In response, the fluorescently labeled target in the sample emits radiation at a wavelength different from the excitation wavelength. The collection optics then collect the emissions from the sample. The apparatus may include a temperature controller to maintain the sample at a particular temperature as it is scanned. In some cases, a multi-axis translation stage moves a microtiter plate holding multiple samples in order to position different wells to be exposed. The multi-axis translation stage, temperature controller, auto-focus features, and electronics associated with imaging and data collection can be managed by a suitably programmed digital computer. The computer may also convert data collected during the assay to another format for presentation.

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 sample contacted with the labeling composition using a fluorescence microscope. Fluorescence microscopy imaging can be used to identify polymer dye conjugate-target analyte binding complexes in contacted samples to assess the presence or absence of target analyte. Microscopy methods of interest that can be used in the subject methods include laser scanning confocal microscopy.

Methods of labeling target molecules are also provided. The subject polymeric tandem dyes are useful in a variety of labeling, separation, detection and/or analytical methods. In some embodiments, the method comprises: contacting the target molecule with a polymeric tandem dye (e.g., as described herein) to produce a labeled target molecule, whereinThe polymeric tandem dye comprises a conjugated tag covalently linked to a target molecule. In some cases of this method, the polymeric dye member includes a chromophore according to any of formulas (VA) - (VB) (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 of (2) is a conjugate tag.

The term "conjugation tag" refers to a group comprising a chemoselective functional group (e.g., as described herein) that can be covalently attached to a compatible functional group of a target molecule after optional activation and/or deprotection. Any convenient conjugation tag may be used in the subject polymer dye to conjugate the dye to the target molecule of interest. In some embodiments, the conjugate tag comprises a terminal functional group selected from amino, carboxylic acid or derivative thereof, thiol, hydroxyl, hydrazine, hydrazide, azide, alkyne, and protein reactive groups (e.g., amino reactive, thiol reactive, hydroxyl reactive, imidazolyl reactive, or guanidinyl reactive groups).

Any convenient method and reagent may be suitable for use in the subject labeling method to covalently attach the conjugate label to the target molecule. Methods of interest for labeling targets include, but are not limited to, those described by Hermanson, bioconjugate Techniques, third edition, academic Press, 2013. The contacting step may be performed in an aqueous solution. In some cases, the conjugate tag comprises an amino functionality and the target molecule comprises an activated ester functionality, such as a NHS ester or sulfo-NHS ester, or vice versa. In some cases, the conjugate tag comprises a maleimide functional group and the target molecule comprises a thiol functional group, or vice versa. In some cases, the conjugation tag comprises an alkyne (e.g., cyclooctyne group) functional group, and the target molecule comprises an azide functional group, and vice versa, which can be conjugated via click chemistry.

Any convenient target molecule may be selected for labelling with the subject method. Target molecules of interest include, but are not limited to, nucleic acids (e.g., RNA, DNA, PNA, CNA, HNA, LNA or ANA molecules), proteins (e.g., fusion proteins), modified proteins (e.g., phosphorylated, glycosylated, ubiquitinated,SUMO or acetylated proteins), or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins, and drug molecules. As used herein, the term "target protein" refers to all members of the target family and fragments thereof. The target protein may be any protein of interest, such as a therapeutic or diagnostic target, including but not limited to: hormones, growth factors, receptors, enzymes, cytokines, osteoinductive factors, colony stimulating factors and immunoglobulins. The term "target protein" is intended to include recombinant and synthetic molecules, which may be prepared using any convenient recombinant expression method or using any convenient synthetic method, or are commercially available. 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 binding derivative thereof. In some cases, the antibody fragment or binding derivative thereof is selected from the group consisting of Fab fragment, F (ab') ₂ Fragments, scFv, diabodies, and triabodies.

In some cases, the method includes a separation step in which the labeled target molecules are separated from the reaction mixture (e.g., excess reagents or unlabeled targets). A variety of methods are available for separating targets from samples, e.g., via immobilization on a support, precipitation, chromatography, etc.

In some cases, the method further comprises detecting and/or analyzing the labeled target molecule. In some cases, the method further comprises fluorescence detection of the labeled target molecule. Any convenient method may be used in combination with the subject methods and compositions for detecting and/or analyzing the labeled target molecules. Methods that can be used in the subject methods for analyzing targets of interest include, but are not limited to, flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), western blot analysis, magnetic cell separation assay, and fluorescent dye purification chromatography. Detection methods of interest include, but are not limited to, fluorescence spectroscopy, fluorescence microscopy, nucleic acid sequencing, fluorescence In Situ Hybridization (FISH), protein mass spectrometry, flow cytometry, and the like.

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

System and method for controlling a system

Aspects of the invention also include systems for practicing the subject methods and compositions. The sample analysis system may comprise a sample field of view or a flow channel carrying a specific binding member for the sample and the label. In some embodiments, the system is a flow cytometry system comprising: a flow cytometer including a flow path; a composition in a 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 microscope system comprising: a fluorescence microscope including a sample field of view; and a composition disposed in the field of view of the sample, wherein the composition comprises the sample; and a labeled specific binding member (e.g., as described herein).

In some cases of the system, the labeled specific binding member comprises: tandem dyes (e.g., as described herein) and a specific binding member that specifically binds a target analyte covalently linked to a chromophore. In some cases of the subject systems, the labeled specific binding member, tandem dye, is described by formula (VA) or (VB) (e.g., as described herein), wherein: g ¹ And G ² Each independently selected from the group consisting of a terminal group, pi conjugated segment, linker, and linked specific binding member, wherein G ¹ And G ² At least one of which is a linked specific binding member.

In certain embodiments of the system, the composition further comprises a second specific binding member that binds to the 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 configured for application to the assay area of the flow channel.

The sample may comprise cells. In some cases, the sample is a biological sample containing cells. In some cases, the sample comprises a labeled specific binding member that specifically binds to the target cell. In some cases, the target analyte that is specifically bound by the 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.

In certain aspects, the system may further comprise a light source configured to direct light to the flow channel or the measurement region of the sample field of view. The system may include a detector configured to receive a signal from the flow channel or the measurement region of the field of view of the sample, wherein the signal is provided by the fluorescent composition. Further optionally, the sample analysis system may comprise one or more additional detectors and/or light sources for detecting one or more additional signals.

In certain aspects, the system may further comprise a computer-based system configured to detect the presence of the fluorescent signal. "computer-based system" refers to hardware devices, software devices, and data storage devices for analyzing the information of the present invention. The minimal hardware 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. One skilled in the art will readily appreciate that any of the currently available computer-based systems are suitable for use with the subject system. The data storage device may comprise any product, including a record of current information as described above, or a memory access device that may access such a product.

"recording" data, programming, or other information on a computer readable medium refers to the process of storing information using any such method known in the art. Any convenient data storage structure may be selected based on the means for accessing the stored information. A variety of data processor programs and formats may be used for storage, e.g., word processing text files, database formats, etc.

"processor" refers to any combination of hardware and/or software that will perform its desired functions. For example, any processor herein may be a programmable digital microprocessor, such as that available in the form of an electronic controller, host, server, or personal computer (desktop or portable). Where the processor is programmable, the appropriate programming may be transferred from a remote location to the processor or pre-stored in a computer program product (e.g., a portable or fixed computer readable storage medium, whether magnetic, optical, or solid state device). For example, a magnetic medium or optical disk may carry a program and may be read by a suitable reader in communication with each processor at its respective station.

In addition to the sensor devices and signal processing modules, for example, as described above, the system of the present invention may include many additional components such as data output devices (e.g., monitors and/or speakers), data input devices (e.g., interface ports, keyboards, etc.), fluid processing components, power supplies, and the like.

In certain aspects, the system comprises a flow cytometer. Flow cytometry of interest includes, but is not limited to, U.S. patent No.: 4,704,891;4,727,029;4,745,285;4,867,908;5,342,790;5,620,842;5,627,037;5,701,012;5,895,922; and 6,287,791; the disclosures of which are incorporated herein by reference.

Other systems may be used to practice the subject methods. In certain aspects, the system may be a fluorometer or microscope carrying a sample having a fluorescent composition of any of the embodiments discussed herein. The fluorometer or microscope can include a light source configured to direct light to the flow channel or the measurement region of the sample field of view. The fluorometer or microscope can further include a detector configured to receive a signal from the flow channel or the measurement zone of the field of view, wherein the signal is provided by the fluorescent composition.

Kit for detecting a substance in a sample

Aspects of the invention also include kits for practicing the subject methods and compositions. The compositions of the invention may be included in a kit as reagents, or as starting materials, or provided for use in a method such as described above.

The kit may comprise a tandem dye comprising a water-soluble dipyrromethene-based dye (e.g., as described herein) and a container. In other embodiments, the kit may comprise a water-soluble dipyrromethene-based dye (e.g., as described herein) and a container. Any convenient container may be utilized, such as a tube, bottle or well in a multi-well strip or plate, a box, a pouch, an insulated container, or the like. The subject kits may further comprise one or more components selected from the group consisting of polymeric tandem dyes, fluorophores, specific binding members, specific binding member conjugates, support-bound specific binding members, cells, supports, biocompatible aqueous elution buffers, and instructions for use. In some embodiments of the kit, the light harvesting chromophore 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 binding derivative thereof. In certain instances, 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.

In certain embodiments, the kit can be used to assess the presence of a target analyte (e.g., an intracellular target) in a sample. Thus, in some cases, the kit comprises one or more components suitable for lysing cells. One or more additional components of the kit may be provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

In certain aspects, the kit further comprises reagents for performing a flow cytometric assay. Reagents of interest include, but are not limited to, buffers for reconstitution and dilution, buffers for contacting the cell sample with chromophores, wash buffers, control cells, control beads, fluorescent beads for flow cytometer calibration, and combinations thereof. The kit may further comprise one or more cell fixation reagents, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination or buffer thereof. In addition, the kit may comprise a cell permeabilizing reagent (cell permeabilizing reagent), such as methanol, acetone, or a detergent (e.g., triton, NP-40, saponin, tween 20, digitonin, leucoderm), or any combination or buffer thereof. Other protein transport inhibitors, cell fixation reagents and cell permeabilization reagents familiar to those skilled in the art are within the scope of the subject kits.

The compositions of the kit may be provided as liquid compositions, such as any suitable buffer. Alternatively, the compositions of the kit may be provided as dry compositions (e.g., may be lyophilized), and the kit may optionally comprise one or more buffers for reconstitution of the dry compositions. In certain aspects, the kit may comprise an aliquot of the composition provided in a separate container (e.g., a separate tube, bottle, or well in a multi-well strip or plate).

Furthermore, one or more of the components may be combined in a single container, e.g., a glass or plastic vial, tube or bottle. In some cases, the kit may also comprise a container (e.g., a box, a bag, an insulated container, a bottle, a tube, etc.), in which all components (and their individual containers) are present. The kit may also comprise a package separate from or attached to the kit container, with information about the kit, kit components, and/or instructions for use printed thereon.

In addition to the components described above, the subject kits may also contain instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate (e.g., one or more sheets of paper having information printed thereon), in the packaging of the kit, in the packaging instructions, etc. Another way is to record information on a computer-readable medium, such as a magnetic disk, CD, DVD, portable flash drive, etc. Another way that may exist is a website address that may be used to access information at a remote site via the internet. Any convenient means may be present in the kit.

Practicality of use

The water-soluble dipyrromethene-based dyes, tandem dyes, compositions, methods and systems as described herein may be used in a variety of applications, including diagnostic and research applications, where labeling, detection and/or analysis of a target of interest is desired. Such applications include methods such as cytometry, microscopy, immunoassays (e.g., competitive or non-competitive), assessment of free analytes, assessment of receptor binding ligands, and the like. The compositions, systems, and methods described herein can be used to analyze any of a number of samples, including but not limited to biological fluids, cell culture samples, and tissue samples. In certain aspects, the compositions, systems, and methods described herein can be used in methods for detecting an analyte in a sample (if present) using fluorescent markers, such as fluorescence-activated cell sorting or analysis, immunoassays, immunostaining, and the like. In certain instances, the compositions and methods are useful in applications in which it is of interest to assess the presence of a target analyte in a sample.

In some cases, the methods and compositions can be used in any assay format in which it is of interest to detect and/or analyze a target from a sample, including but not limited to flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), western blot analysis, magnetic cell separation assay, and fluorescent dye purification chromatography. In certain instances, the methods and compositions can be used in any application in which fluorescent-labeled target molecules are of interest. The subject compositions may be suitable for any convenient application in which specific binding member pairs, such as biotin-streptavidin and hapten-anti-hapten antibodies, may be used.

Examples

Example 1: synthesis and characterization of yellow-green water-soluble dipyrromethene dyes

Exemplary water-soluble dipyrromethene-based dyes may be prepared according to any convenient method, including those described by Loudet and Burgess ("BODIPY Dyes and their derivative: synthesis and Spectroscopic properties", chem. Rev.2007,107, 4891-4932). An exemplary yellow-green water-soluble dipyrromethene-type dye is depicted in fig. 1.

As shown in fig. 2, the absorption characteristics of four exemplary water-soluble dipyrromethene-based dyes (as depicted in fig. 1 and reproduced below) were evaluated.

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As depicted in fig. 2, the absorbance maximum of each of compounds B487, B488, B510, and B559 was close to 561nm.

The above results are examples of water-soluble dipyrromethene-based dyes (e.g., dyes of formulas (I) - (IV)) that absorb in the yellow-green range at 561nm, e.g., as described in the above specification. These dyes have strong absorption at 561nm while minimizing absorption at other common laser source wavelengths used in flow cytometry. This allows the dye and FRET pairs made from the dye to be selectively excited by a single laser rather than several lasers. In this way, a dye excited by the YG laser and emitted at a wavelength determined by the attached acceptor can be prepared. A key advantage of the subject dyes according to embodiments is that they are rendered highly water-soluble by the attachment of polyethylene glycol oligomers. This allows the dye and other moieties attached to the dye to be water soluble and increases the quantum yield of the dye in common buffer solutions relative to non-PEGylated core dye structures.

Example 2: preparation of polypeptide light harvesting chromophores

A summary of some of the various methods that can be used to synthesize the subject polypeptide light harvesting chromophores can be found in Steward et al, "Solid Phase Peptide Synthesis", w.h. freeman co., san Francisco,1969; bodanszky et al, "Peptide Synthesis", john Wiley & Sons, second edition, 1976 and Meienhofer, "Hormonal Proteins and Peptides", volume 2, page 46, academic Press (New York), 1983; and Kent, ann.Rev.biochem.,57,957,1988, and Schroder et al, "The Peptides", volume 1, academic Press (New York), 1965. Any convenient protecting group strategy may 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 benzyl or benzyl-based or other convenient protecting groups may be used to protect the side chain functionality. In Fmoc solid phase peptide synthesis, fmoc-amino protecting groups are used at the amino terminus and tertiary butyl or benzyl based or other convenient protecting groups may be used to protect the side chain functionality. Convenient protecting groups useful in this synthetic method are described in the above references, "Protective Groups in Organic Chemistry" by McOmic, plenum Press, new York,1973; and Greene and Wuts, "Protective Groups in Organic Synthesis", john Wiley & Sons, 4 th edition, 2006.

Example 3: preparation of dyes

The dipyrromethene-based dyes may be prepared according to any convenient method, including those described by Loudet and Burgess ("BODIPY Dyes and their derivative: synthesis and Spectroscopic properties", chem. Rev.2007,107, 4891-4932).

Deep learning is used to calculate the optical properties (e.g., the primary absorption band) of dyes having BODIPY core structures. Dyes with calculated absorption maxima of 562nm + -5 nm were identified and further absorption band calculations were performed for these candidate dyes at a higher degree of refinement. These candidate dyes were synthesized experimentally and their absorption characteristics were measured and compared with predicted values. It was found that the deep learning calculation was surprisingly accurate in predicting the absorption bands of the candidate dyes measured experimentally. The chemical structures of the seven candidate dyes are shown below. The candidate dye was found to have a relatively narrow absorption spectrum, which is highly consistent with 562nm light emitted by a yellow-green laser.

Candidate dyes were also tested for their ability to act as donor chromophores and transfer energy to acceptor chromophores by FRET (Forster resonance energy transfer). The energy transfer efficiency measured by quantum yield is compared to the predicted value of quantum yield. It was found that the predictions were successful in qualitatively predicting the relative quantum yields of the candidate dyes, i.e. the calculated degree of energy transfer showed a trend consistent with the experimental results. Thus, when used as a donor chromophore, the candidate dye was found to be part of a highly efficient tandem pair. Calculations indicate that high quantum yields of candidate dyes may be caused at least in part by low amounts of ground state interactions. Additional studies showed that the candidate dye had good photostability.

Although defined by the appended claims, the present disclosure is also defined by the following clauses:

1. a water-soluble dipyrromethene-based dye that absorbs in the yellow-green range at 561nm.

2. The water-soluble dipyrromethene-type dye according to clause 1, wherein the absorption maximum is 555 to 585nm.

3. The water-soluble dipyrromethene-type dye according to clause 2, wherein the absorption maximum is 558 to 568nm.

4. The water-soluble dipyrromethene-type dye according to clause 3, wherein the absorption maximum is about 561nm.

5. The water-soluble dipyrromethene-based dye according to any one of clauses 1-4, wherein the dye is a dipyrromethene-boron dye.

6. The water-soluble dipyrromethene-based dye according to any one of clauses 1 to 5, having the formula (I):

wherein:

R ¹ -R ⁷ each independently ofIn situ selected from H, alkyl, halogen, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, amido, substituted amido, thiol, substituted thiol, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water Solubilizing Group (WSG) and-T ¹ -J ¹ Or (b)

Optionally selected from R ⁶ And R is ⁷ 、R ² And R is ³ 、R ⁵ And R is ⁶ 、R ³ And R is ⁴ 、R ² And R is ¹ R is as follows ⁷ And R is ¹ Any one or more pairs of substituents together form a divalent group and are cyclic linked and, together with the carbon atom to which they are bound, provide a 5 or 6 membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5 or 6 membered ring comprising a carbon atom and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water Solubilising Group (WSG) and-T ¹ -J ¹ Is substituted by a substituent of (a);

T ¹ is an optional linker;

J ¹ is a repeating unit of a compound of formula (I); and

Y ¹ And Y ² Together with the boron atom to which they are bound, form a 5-or 6-membered ring (e.g., a 5-or 6-membered ring containing the boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl Substituted alkenyl, alkynyl, substituted alkynyl, water Solubilising Group (WSG) and-T ¹ -J ¹ Is substituted by a substituent of (a); and

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ¹ ) _m (Ia)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ¹ is a water-soluble polymer comprising 6 to 24 monomer units; and

m is an integer from 1 to 3.

7. The water-soluble dipyrromethene-based dye according to clause 6, further comprising WSG of formula (Ib):

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ² ) _m (Ib)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ² is a water-soluble polymer comprising 1 to 50 monomer units; and

m is an integer from 1 to 3.

8. The water-soluble dipyrromethene-based dye according to clause 6 or 7, wherein the water-solubilizing group (WSG) comprises a PEG moiety.

9. The water-soluble dipyrromethene-based dye according to any one of clauses 6-8, wherein the one or more water-solubilizing groups (WSG) are independently selected from the following formulas:

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wherein:

L ¹ and L ² Each independently is an optional linker;

R ¹¹ And R are each independently H, alkyl or substituted alkyl; and

each p is an integer from 6 to 24.

10. A water-soluble dipyrromethene-based dye according to any one of clauses 6 to 9, wherein R ¹ Selected from the group consisting of substituted alkyl groups and substituted aryl groups, wherein the substituents comprise a chemoselective tag.

11. A water-soluble dipyrromethene-type dye according to any one of clauses 6 to 10, wherein Y ¹ And Y ² Each comprising a Water Solubilising Group (WSG).

12. The water-soluble dipyrromethene-based dye according to any one of clauses 6 to 11 having the formula (II):

wherein p is an integer of 6 to 24.

13. A water-soluble dipyrromethene-type dye according to any one of clauses 6 to 12, wherein R ⁵ Selected from the group consisting of halogen, substituted alkyl, substituted alkenyl, alkenylaryl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, spiroheterocyclic, substituted spiroheterocyclic, heteroaryl, substituted alkoxy, substituted amino, substituted amido, and substituted thiol.

14. According to clauses and subclauses13, wherein R is a water-soluble dipyrromethene-type dye ⁵ And R is ⁴ The same applies.

15. The water-soluble dipyrromethene-type dye according to any one of clauses 6 to 14, having the formula (IIa):

Wherein:

q is an integer from 0 to 5; and

each R ⁹ Independently selected from alkyl, substituted alkyl, hydroxy, alkoxy, substituted alkoxy, amino, substituted amino, thiol, substituted thiol, cyano, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilizing Group (WSG), if present, or

Optionally two adjacent R ⁹ The groups together form a divalent group and are cyclic attached and, together with the carbon atoms to which they are bonded, provide a 5 or 6 membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5 or 6 membered ring containing a carbon atom and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkylSubstituents for groups, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilising Groups (WSG).

16. A water-soluble dipyrromethene-based dye according to clause 15, wherein R ⁸ Is H.

17. A water-soluble dipyrromethene-based dye according to clause 15, wherein R ⁸ And R is ⁶ Together with the atoms to which they are bonded, form a 5 membered carbocycle, which carbocycle is optionally further substituted with substituents independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG).

18. The water-soluble dipyrromethene-based dye according to any one of clauses 15-17 having any one of the following structures:

19. The water-soluble dipyrromethene-type dye according to any one of clauses 6 to 14, having the formula (IIb):

wherein:

each q is independently an integer from 0 to 5; and

R ¹⁰ and R is ^10' Each, if present, is independently selected from halogen, hydroxy, cyano, carboxamide, substituted carboxamide, amino, substituted amino, thiol, substituted thiol, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy,Substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Soluble Group (WSG), or

at least one R ^10' Is ortho-position, and R ¹⁰ And R is ³ Together with the atoms to which they are bonded, form a 5-or 6-membered ring (e.g., a 5-or 6-membered ring comprising a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilising Group (WSG).

20. The water-soluble dipyrromethene-type dye according to clause 19, wherein each q is 1, and R ¹⁰ And R is ^10' All present in their respective para-positions.

21. A water-soluble dipyrromethene-based dye according to clause 20, wherein R ¹⁰ And R is ^10' Each independently selected from cyano and PEG moieties.

22. The water-soluble dipyrromethene-based dye according to any one of clauses 19-21 having any one of the following structures:

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23. the water-soluble dipyrromethene-type dye according to any one of clauses 6 to 14, having the formula (IIc):

wherein:

x is halogen; and

24. The water-soluble dipyrromethene-type dye according to clause 23, wherein the a ring is an aryl ring.

25. A water-soluble dipyrromethene-based dye according to clause 23 or 24 having any one of the following compounds:

26. The water-soluble dipyrromethene-type dye according to any one of clauses 6 to 14, having the formula (IId):

wherein:

X ² and X ³ Each independently selected from NR ¹³ S and O;

R ¹³ selected from H, alkyl, and substituted alkyl;

R ¹¹ and R is ¹² Each independently selected from the group consisting of aryl, substituted aryl, heteroaryl, substituted heteroaryl, andwater-soluble group (WSG), or

When X is ² Is NR ¹³ When optionally R ¹³ And R is ¹¹ Forms, together with the nitrogen atom to which they are attached, a heterocyclic or spiro-heterocyclic ring, which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG), and

when X is ³ Is NR ¹³ When optionally R ¹³ And R is ¹² Together with the nitrogen atom to which they are attached, form a heterocyclic or spiro-heterocyclic ring, which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG).

27. A water-soluble dipyrromethene-based dye according to clause 26, wherein X ² And X ³ Each is NR ¹³ Wherein R is ¹³ Is H.

28. A water-soluble dipyrromethene-based dye according to clause 26, wherein X ² Is NR ¹³ And R is ¹³ And R is ¹¹ Together with the nitrogen atom to which they are attached, form a heterocycle or spiroheterocycle; x is as follows ³ Is NR ¹³ And R is ¹³ And R is ¹² Together with the nitrogen atom to which they are attached form a heterocyclic or spiro-heterocyclic ring.

29. A water-soluble dipyrromethene-based dye according to clause 26, wherein X ² And X ³ S.

30. The water-soluble dipyrromethene-based dye according to any one of clauses 26-29 having any one of the following structures:

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31. the water-soluble dipyrromethene-based dye according to any one of clauses 6 to 10 having the formula (III):

wherein:

32. The water-soluble dipyrromethene-based dye according to clause 31 having the formula (IIIa):

wherein:

X ⁴ selected from O, S, NR and CR ₂ ；

Each R is independently selected from H, alkyl, and substituted alkyl;

each r is independently an integer from 0 to 4; and

each R ¹⁴ And R is ^14' Independently selected from the group consisting of halogen, hydroxy, cyano, amino, substituted amino, thiol, substituted thiol, amido, substituted amido, acyl, substituted acyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedHeteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilizing Groups (WSG).

33. A water-soluble dipyrromethene-type dye according to clause 31 or 32, wherein R ⁵ And R is ⁶ R is as follows ⁴ And R is ³ Together form a divalent group and are cyclic, and together with the carbon atoms to which they are bonded provide a 5 or 6 membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5 or 6 membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG).

34. The water-soluble dipyrromethene-type dye according to clause 33 having formula (IIIb) or (IIIc):

wherein:

X ⁵ and X ⁶ Each independently selected from O, S, NR and CR ₂ ；

Each R is independently selected from H, alkyl, and substituted alkyl; and

R ¹⁵ and R is ¹⁶ Each independently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, amino, substituted amino, thiol, substituted thiol, amido, substituted amido, acyl, substituted acyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilizing Group (WSG).

35. A water-soluble dipyrromethene-type dye according to clause 34, wherein X ⁵ And X ⁶ Is O.

36. The water-soluble dipyrromethene-based dye according to any one of clauses 31-35 having any one of the following structures:

37. a water-soluble dipyrromethene-type dye according to any one of clauses 6 to 12, wherein R ³ is-L ¹ -J ¹ 。

38. The water-soluble dipyrromethene-based dye according to clause 37 having one of the following structures:

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39. the water-soluble dipyrromethene-type dye according to any one of clauses 1 to 4, having the formula (IV):

Wherein:

X ^- is a counter ion;

T ¹ is an optional linker;

Z ¹ is a repeating unit of a compound of formula (I); and

Y ^1' And Y ^2' Together with the boron atom to which they are bound, form a 5-or 6-membered ring (e.g., a 5-or 6-membered ring containing a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-solubilizing group (WSG), and-T ¹ -Z ¹ Is substituted by a substituent of (a); and

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ¹ ) _m (Ia)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ¹ is a water-soluble polymer comprising 6 to 24 monomer units; and

m is an integer from 1 to 3.

40. The water-soluble dipyrromethene-based dye according to clause 39, further comprising WSG of formula (Ib):

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ² ) _m (Ib)

Wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ² is a water-soluble polymer comprising 1 to 50 monomer units; and

m is an integer from 1 to 3.

41. The water-soluble dipyrromethene-type dye according to clause 39 or 40, wherein the WSG comprises a PEG moiety.

42. The water-soluble dipyrromethene-based dye according to any one of clauses 39-41, wherein the one or more water-solubilizing groups (WSG) are independently selected from the following formulas:

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wherein:

L ¹ and L ² Each independently is an optional linker;

R ¹¹ and R are each independently H, alkyl or substituted alkyl; and

each p is an integer from 6 to 24.

43. A water-soluble dipyrromethene-type dye according to any one of clauses 39-42, wherein R ¹ Selected from the group consisting of substituted alkyl groups and substituted aryl groups, wherein the substituents comprise a chemoselective tag.

44. A water-soluble dipyrromethene-type dye according to any one of clauses 39-43, wherein R ^5' And R is ^4' All contain Water Solubilising Groups (WSG).

45. The water-soluble dipyrromethene-based dye according to any one of clauses 39-44 having one of the following structures:

46. a yellow-green dipyrromethene-based dye of formula (II):

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Wherein:

R ¹ -R ⁷ each independently selected from H, alkyl, halogen, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, amido, substituted amido, thiol, substituted thiol, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water Solubilizing Group (WSG), and-T ¹ -Z ¹ Or (b)

Optionally selected from R ⁶ And R is ⁷ 、R ² And R is ³ 、R ⁵ And R is ⁶ 、R ³ And R is ⁴ 、R ² And R is ¹ R is as follows ⁷ And R is ¹ Forms a divalent group together with any one or more pairs of substituents and is cyclic and, together with the carbon atom to which they are bonded, provides a 5 or 6 membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5 or 6 membered ring comprising a carbon atom and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water Solubilising Group (WSG) and-T ¹ -Z ¹ Is substituted by a substituent of (a);

T ¹ is an optional linker;

Z ¹ is a repeating unit of a compound of formula (I); and

p is an integer of 6 to 24.

47. A yellow-green dipyrromethene-based dye according to clause 46, further comprising one or more water-solubilizing groups (WSG) of formula (Ib):

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ² ) _m (Ib)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ² is a water-soluble polymer comprising 1 to 50 monomer units; and

m is an integer from 1 to 3.

48. The yellow-green dipyrromethene-based dye according to clause 47, wherein the WSG comprises a PEG moiety.

49. A yellow-green dipyrromethene-based dye according to clause 48, wherein the one or more water-solubilizing groups (WSG) are independently selected from the following formulas:

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wherein:

L ¹ and L ² Each independently is an optional linker;

R ¹¹ and R are each independently H, alkyl or substituted alkyl; and

each p is an integer from 6 to 24.

50. A yellow-green dipyrromethene-based dye according to any one of clauses 46-49 wherein R ¹ Selected from the group consisting of substituted alkyl groups and substituted aryl groups, wherein the substituents comprise a chemoselective tag.

51. A yellow-green dipyrromethene-based dye according to any one of clauses 46-50 wherein R ⁵ Selected from the group consisting of halogen, substituted alkyl, substituted alkenyl, alkenylaryl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, spiroheterocyclic, substituted spiroheterocyclic, heteroaryl, substituted amino, substituted amido, and substituted thiol.

52. The yellow-green dipyrromethene-based dye according to clause 51, wherein R ⁵ And R is ⁴ The same applies.

53. The yellow-green dipyrromethene-type dye according to any one of clauses 46-52 having formula (IIa):

wherein:

R ⁸ selected from H, halogen, cyano, alkyl, substitutedAlkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG), or

q is an integer from 0 to 5; and

54. The yellow-green dipyrromethene-based dye according to clause 53, wherein R ⁸ Is H.

55. The yellow-green dipyrromethene-based dye according to clause 53, wherein R ⁸ And R is ⁶ Together with the atoms to which they are bonded form a 5 membered carbocyclic ring, which is optionally further independently selected from alkanesSubstituents for groups, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilising Groups (WSG).

56. A yellow-green dipyrromethene-based dye according to any one of clauses 46-55 having any one of the following structures:

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57. The yellow-green dipyrromethene-type dye according to any one of clauses 46-52 having formula (IIb):

wherein:

each q is independently an integer from 0 to 5; and

R ¹⁰ and R is ^10' Each, if present, is independently selected from halogen, hydroxy, cyano, amino, substituted amino, thiol, substituted thiol, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and water-soluble group (WSG), or

At least one R ¹⁰ The radicals being in the ortho position and R ¹⁰ And R is ⁶ Together with the atoms to which they are bonded, form a 5-or 6-membered ring (e.g., a 5-or 6-membered ring containing a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substitutedAlkynyl and Water Solubilising Group (WSG); and

58. The yellow-green dipyrromethene-based dye of clause 57 wherein each q is 1 and R ¹⁰ And R is ^10' All present in their respective para-positions.

59. The yellow-green dipyrromethene-based dye according to clause 58, wherein R ¹⁰ And R is ^10' Each independently selected from cyano and PEG moieties.

60. A yellow-green dipyrromethene-based dye according to clause 59, wherein the PEG moiety is selected from one of the following formulas:

wherein:

L ¹ is an optional linker;

R ^a and R are each independently H, alkyl or substituted alkyl; and

each p1 is an integer of 1 to 50.

61. A yellow-green dipyrromethene-based dye according to any one of clauses 57-60 having any one of the following structures:

/>

62. the yellow-green dipyrromethene-type dye according to any one of clauses 46-52 having formula (IIc):

wherein:

x is halogen; and

63. The yellow-green dipyrromethene-type dye according to clause 62 wherein the a ring is an aryl ring.

64. The yellow-green dipyrromethene-based dye according to clause 62 or 63 wherein X is chloride.

65. A yellow-green dipyrromethene-based dye according to any one of clauses 62-64 having any one of the following compounds:

66. the yellow-green dipyrromethene-type dye according to any one of clauses 46-52 having formula (IId):

wherein:

X ² and X ³ Each independently selected from NR ¹³ S and O;

R ¹³ selected from HAlkyl and substituted alkyl;

67. The yellow-green dipyrromethene-based dye according to clause 66, wherein X ² And X ³ Each is NR ¹³ Wherein R is ¹³ Is H.

68. The yellow-green dipyrromethene-based dye according to clause 66, wherein X ² Is NR ¹³ And R is ¹³ And R is ¹¹ Together with the nitrogen atom to which they are attached, form a heterocycle or spiroheterocycle; x is as follows ³ Is NR ¹³ And R is ¹³ And R is ¹² Together with the nitrogen atom to which they are attached form a heterocyclic or spiro-heterocyclic ring.

69. The yellow-green dipyrromethene-based dye according to clause 66, wherein X ² And X ³ S.

70. A yellow-green dipyrromethene-based dye according to any one of clauses 66-69 having any one of the following structures:

/>

71. a yellow-green dipyrromethene-based dye according to any one of clauses 46-52 wherein R ³ is-L ¹ -Z ¹ 。

72. A yellow-green dipyrromethene-based dye according to clause 71, wherein R ⁶ Selected from the group consisting of halogen, substituted alkyl, substituted alkenyl, alkenylaryl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, spiroheterocyclic, substituted spiroheterocyclic, heteroaryl, substituted amino, substituted amido, and substituted thiol.

73. A yellow-green dipyrromethene-based dye according to clause 72, wherein R ⁶ Comprising a PEG moiety.

74. A yellow-green dipyrromethene-based dye according to clause 73, wherein the PEG moiety is selected from one of the following formulas:

wherein:

L ¹ and L ² Each independently is an optional linker;

r is H, alkyl or substituted alkyl; and

each p1 is an integer of 1 to 50.

75. A yellow-green dipyrromethene-based dye according to any one of clauses 71-74 having one of the following structures:

76. a yellow-green dipyrromethene-based dye of formula (III):

wherein:

T ¹ is an optional linker;

Z ¹ is a repeating unit of a compound of formula (I); and

ring B is a 5 or 6 membered cyclic group (e.g., a 5 or 6 membered ring comprising a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S and N), which cyclic group may be unsubstituted or further substituted with one or more substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilizing Group (WSG); and

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ¹ ) _m (Ia)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ¹ is a water-soluble polymer comprising 6 to 24 monomer units; and

m is an integer from 1 to 3.

77. A yellow-green dipyrromethene-based dye according to clause 76, further comprising WSG of formula (Ib):

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ² ) _m (Ib)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ² is a water-soluble polymer comprising 1 to 50 monomer units; and

m is an integer from 1 to 3.

78. The yellow-green dipyrromethene-based dye of clause 76 or 77 wherein the WSG comprises a PEG moiety.

79. The yellow-green dipyrromethene-based dye of any one of clauses 76-78, wherein one or more water-solubilizing groups (WSG) are independently selected from the following formulas:

/>

wherein:

L ¹ and L ² Each independently is an optional linker;

R ¹¹ and R are each independently H, alkyl or substituted alkyl; and

each p is an integer from 6 to 24.

80. The yellow-green dipyrromethene-type dye of any one of clauses 76-79, wherein R ¹ Selected from the group consisting of substituted alkyl groups and substituted aryl groups, wherein the substituents comprise a chemoselective tag.

81. The yellow-green dipyrromethene-based dye of any one of clauses 76-80 having formula (IIIa):

wherein:

X ⁴ selected from O, S, NR and CR ₂ ；

Each R is independently selected from H, alkyl, and substituted alkyl;

each r is independently an integer from 0 to 4; and

each R ¹⁴ And R is ^14' Independently selected from halogen, hydroxy, if present,Cyano, amino, substituted amino, thiol, substituted thiol, amido, substituted amido, acyl, substituted acyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and water-soluble group (WSG).

82. A yellow-green dipyrromethene-based dye according to any one of clauses 76-81 wherein R ⁵ And R is ⁶ R is as follows ⁴ And R is ³ Together form a divalent group and are cyclic, and together with the carbon atoms to which they are bonded provide a 5 or 6 membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5 or 6 membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG).

83. A yellow-green dipyrromethene-based dye according to clause 82 having formula (IIIb) or (IIIc):

wherein:

X ⁵ and X ⁶ Each independently selected from O, S, NR and CR ₂ ；

Each R is independently selected from H, alkyl, and substituted alkyl; and

R ¹⁵ and R is ¹⁶ Each independently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, amino, substituted amino, thiol, substituted thiol, amido, substituted amido, acyl, substituted acyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and water-soluble group (WSG).

84. The yellow-green dipyrromethene-based dye according to clause 83, wherein X ⁵ And X ⁶ Is O.

85. A yellow-green dipyrromethene-based dye according to any one of clauses 76-84 having any one of the following structures:

86. a tandem dye comprising:

a light harvesting chromophore, the light harvesting chromophore comprising:

a main chain;

a donor chromophore attached to the backbone, wherein the donor chromophore is a water-soluble dipyrromethene-type dye that absorbs in the yellow-green range at 561 nm; and

an energy acceptor comprising a pendant acceptor fluorophore attached to a backbone and disposed adjacent to the energy acceptor of the donor chromophore.

87. The tandem dye according to clause 86, wherein the absorption maximum of the donor chromophore is 555 to 585nm.

88. A tandem dye according to clause 87 wherein the absorption maximum of the donor chromophore is 558 to 568nm.

89. The tandem dye according to clause 88, wherein the absorption maximum of the donor chromophore is about 561nm.

90. A tandem dye according to any one of clauses 86-89 wherein the donor chromophore is a water-soluble dipyrromethene-boron dye.

91. A tandem dye according to any one of clauses 86-90, wherein the donor chromophore has the formula (I):

Wherein:

R ¹ -R ⁷ each independently selected from H, alkyl, halogen, substituted alkyl, alkoxy,Substituted alkoxy, amino, substituted amino, amido, substituted amido, thiol, substituted thiol, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water Solubilizing Group (WSG) and-T ¹ -Z ¹ Or (b)

T ¹ is an optional linker;

Z ¹ is a repeating unit of a compound of formula (I); and

Y ¹ And Y ² Together with the boron atom to which they are bound, form a 5-or 6-membered ring (e.g., a 5-or 6-membered ring containing a boron atom, a carbon atom and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-solubilizing group (WSG)) and-T ¹ -Z ¹ Is substituted by a substituent of (a); and

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ¹ ) _m (Ia)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ¹ is a water-soluble polymer comprising 6 to 24 monomer units; and

m is an integer from 1 to 3.

92. A tandem dye according to clause 91, wherein the donor chromophore of formula (I) further comprises WSG of formula (Ib):

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ² ) _m (Ib)

Wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ² is a water-soluble polymer comprising 1 to 50 monomer units; and

m is an integer from 1 to 3.

93. A tandem dye according to clause 91 or 92, wherein the one or more water-solubilizing groups (WSG) comprise a PEG moiety.

94. A tandem dye according to clause 93, wherein the one or more water-solubilizing groups (WSG) are independently selected from the following formulas:

/>

wherein:

L ¹ and L ² Each independently is an optional linker;

R ¹¹ and R are each independently H, alkyl or substituted alkyl; and

each p is an integer from 6 to 24.

95. A tandem dye according to any of clauses 91-94, wherein R ¹ Selected from the group consisting of substituted alkyl groups and substituted aryl groups, wherein the substituents comprise a chemoselective tag.

96. A tandem dye according to any of clauses 91-95, wherein Y ¹ And Y ² Each comprising a Water Solubilising Group (WSG).

97. A tandem dye according to any one of clauses 91-96, wherein the donor chromophore has the formula (II):

wherein p is an integer of 6 to 24.

98. A tandem dye according to any of clauses 91-97, wherein R ⁵ Selected from the group consisting of halogen, substituted alkyl, substituted alkenyl, alkenylaryl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, spiroheterocyclic, substituted spiroheterocyclic, heteroaryl, substituted amino, substituted amido, and substituted thiol.

99. A tandem dye according to clause 98, wherein R ⁵ And R is ⁴ The same applies.

100. A tandem dye according to any one of clauses 91-99, wherein the donor chromophore has the formula (IIa):

wherein:

R ⁸ selected from H, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilizing Group (WSG), or

q is an integer from 0 to 5; and

101. A tandem dye according to clause 100, wherein R ⁸ Is H.

102. A tandem dye according to clause 100, wherein R ⁸ And R is ⁶ Together with the atoms to which they are bonded, form a 5 membered carbocycle, which carbocycle is optionally further substituted with substituents independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG).

103. A tandem dye according to any one of clauses 100-102, wherein the donor chromophore has any one of the following structures:

104. A tandem dye according to any one of clauses 91-99, wherein the donor chromophore has the formula (IIb):

wherein:

each q is independently an integer from 0 to 5; and

At least one R ¹⁰ The radicals being in the ortho position and R ¹⁰ And R is ⁶ Together with the atoms to which they are bonded form a 5-or 6-membered ring (e.g. containing boron atoms, carbon atomsA 5 or 6 membered ring of 0 to 3 heteroatoms selected from O, S and N, which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Group (WSG); and

105. The tandem dye according to clause 104, wherein each q is 1, and R ¹⁰ And R is ^10' All present in their respective para-positions.

106. A tandem dye according to clause 105, wherein R ¹⁰ And R is ^10' Each independently selected from cyano and PEG moieties.

107. A tandem dye according to any one of clauses 104-106 wherein the donor chromophore has any of the following structures:

/>

108. a tandem dye according to any one of clauses 91-99, wherein the donor chromophore has the formula (IIc):

wherein:

x is halogen; and

109. The tandem dye according to clause 108, wherein the a ring is an aryl ring.

110. A tandem dye according to clause 108 or 109, wherein the donor chromophore has any of the following compounds:

111. a tandem dye according to any one of clauses 91-99, wherein the donor chromophore has the formula (IId):

Wherein:

X ² and X ³ Each independently selected from NR ¹³ S and O;

R ¹³ selected from H, alkyl, and substituted alkyl;

When X is ² Is NR ¹³ When optionally R ¹³ And R is ¹¹ Together with the nitrogen atom to which they are attached, form a heterocyclic or spiro-heterocyclic ring, which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and waterSubstituent substitution of solubilising group (WSG), and

112. A tandem dye according to clause 111, wherein X ² And X ³ Each is NR ¹³ Wherein R is ¹³ Is H.

113. A tandem dye according to clause 111, wherein X ² Is NR ¹³ And R is ¹³ And R is ¹¹ Together with the nitrogen atom to which they are attached, form a heterocycle or spiroheterocycle; x is as follows ³ Is NR ¹³ And R is ¹³ And R is ¹² Together with the nitrogen atom to which they are attached form a heterocyclic or spiro-heterocyclic ring.

114. The polymeric tandem dye according to clause 111, wherein X ² And X ³ S.

115. A tandem dye according to any one of clauses 111-114 wherein the donor chromophore has any one of the following structures:

/>

116. a tandem dye according to any one of clauses 91-95, wherein the donor chromophore has the formula (III):

wherein:

117. The tandem dye according to clause 116, wherein the donor chromophore has the formula (IIIa):

wherein:

X ⁴ selected from O, S, NR and CR ₂ ；

Each R is independently selected from H, alkyl, and substituted alkyl;

each r is independently an integer from 0 to 4; and

each R ¹⁴ And R is ^14' Independently selected from the group consisting of halogen, hydroxy, cyano, amino, substituted amino, thiol, substituted thiol, amido, substituted amido, acyl, substituted acyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilizing Group (WSG), if present.

118. A tandem dye according to clause 116 or 117, wherein R ⁵ And R is ⁶ R is as follows ⁴ And R is ³ Together at least one of the pairs forming a divalent group and being cyclic and together with the carbon atom to which they are bound providing a 5 or 6 membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g. a 5 or 6 membered ring comprising a carbon atom and 0 to 3 heteroatoms selected from O, S and N) which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroarylAryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and substituents of the Water Solubilising Group (WSG).

119. The tandem dye according to clause 118, wherein the donor chromophore has formula (IIIb) or (IIIc):

wherein:

X ⁵ and X ⁶ Each independently selected from O, S, NR and CR ₂ ；

Each R is independently selected from H, alkyl, and substituted alkyl; and

120. A tandem dye according to clause 119, wherein X ⁵ And X ⁶ Is O.

121. A tandem dye according to any one of clauses 116-120, wherein the donor chromophore has any one of the following structures:

122. a tandem dye according to any of clauses 91-97, wherein R ³ is-L ¹ -Z ¹ 。

123. A tandem dye according to clause 122, wherein the donor chromophore has one of the following structures:

124. a tandem dye according to any of clauses 86-89, having the formula (IV):

wherein:

X ^- is a counter ion;

T ¹ is an optional linker;

Z ¹ is a repeating unit of a compound of formula (I); and

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ¹ ) _m (Ia)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ¹ is a water-soluble polymer comprising 6 to 24 monomer units; and

m is an integer from 1 to 3.

125. A tandem dye according to clause 124, further comprising WSG of formula (Ib):

-(L ¹ ) _n1 –(X ¹ ) _n2 –((L ² ) _n3 -Z ² ) _m (Ib)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if it isPresence) is a branching point;

Z ² is a water-soluble polymer comprising 1 to 50 monomer units; and

m is an integer from 1 to 3.

126. A tandem dye according to clause 124 or 125, wherein the WSG comprises a PEG moiety.

127. A tandem dye according to any one of clauses 124-126 wherein the one or more water-solubilizing groups (WSG) are independently selected from the following formulas:

/>

wherein:

L ¹ and L ² Each independently is an optional linker;

R ¹¹ and R are each independently H, alkyl or substituted alkyl; and

each p is an integer from 6 to 24.

128. A tandem dye according to any of clauses 124-127, wherein R ¹ Selected from the group consisting of substituted alkyl groups and substituted aryl groups, wherein the substituents comprise a chemoselective tag.

129. A tandem dye according to any of clauses 124-128, wherein R ^5' And R is ^4' All contain Water Solubilising Groups (WSG).

130. A tandem dye according to any of clauses 124-129, having one of the following structures:

131. a tandem dye according to any of clauses 86-130, wherein the backbone is a linear polymer.

132. The tandem dye according to clause 131, wherein the linear polymer is selected from the group consisting of peptides, peptoids, hydrocarbon polymers, and PEG polymers.

133. The tandem dye according to clause 132, wherein the linear polymer is a peptide.

134. A tandem dye according to clause 133, wherein the peptide is 2 to 100 amino acids in length.

135. A tandem dye according to clause 134, wherein the peptide is 5 to 30 amino acids in length.

136. A tandem dye according to any of clauses 131-133, wherein the linear polymer comprises 2 to 1,000 repeating monomer units.

137. The tandem dye according to clause 136, wherein the linear polymer comprises 2 to 500 repeating monomer units.

138. The tandem dye according to clause 137, wherein the linear polymer comprises 2 to 100 repeating monomer units.

139. A tandem dye according to any one of clauses 86-138 wherein there are a plurality of flanking donor chromophores and are disposed adjacent energy transfer to flanking acceptor fluorophores.

140. A tandem dye according to any one of clauses 86-139 wherein the flanking acceptor fluorophore is a small molecule fluorophore.

141. The tandem dye according to any one of clauses 86-140, wherein the pendant acceptor fluorophore is selected from the group consisting of cyanine dyes, rhodamine dyes, perylene diimide dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene boron difluorides, naphthalimides, thiazine dyes, and acridine dyes.

142. A tandem dye according to any one of clauses 86-141 wherein the side-acceptor fluorophore is selected from fluorescein, 6-FAM, rhodamine, texas red, california red, iFluor594, tetramethyl rhodamine, carboxy rhodamine 6G, carboxy para-methylaminophenol, carboxy rhodamine 110, cascade blue, cascade yellow, coumarin, Cy-Chrome、DyLight 350、DyLight 405、DyLight 488、DyLiLight 549, dylight 594, dylight 633, dylight 649, dylight 680, dylight 750, dylight 800, phycoerythrin, perCP (fucoxanthin-chlorophyll-a-protein), perCP-Cy5.5, JOE (6-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein), NED, ROX (5- (and-6) -carboxy-X-rhodamine), HEX, fluorescence yellow, beach blue, oregon green 488, oregon green 500, oregon green 514, alexa @ @ is preferred >350、Alexa/>430、Alexa/>488、Alexa/>532、Alexa/>546、Alexa/>568、Alexa594、Alexa/>633、Alexa/>647、Alexa/>660、Alexa/>680. 7-amino-4-methylcoumarin-3-acetic acid, -/->FL、/>FL-Br2、/>530/550、558/568、/>564/570、/>576/589、/>581/591、630/650、/>650/665、/>R6G、/>TMR、/>TR, conjugates thereof, and combinations thereof.

143. A tandem dye according to any one of clauses 86-142, wherein the ratio of donor chromophore to acceptor fluorophore is selected from the group consisting of 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 20:2.

144. A tandem dye according to any one of clauses 86-143 wherein the light harvesting chromophore further comprises a specific binding member.

145. A tandem dye according to clause 144 wherein the specific binding member is attached to a terminal group of the light harvesting chromophore.

146. A tandem dye according to clause 144 wherein the specific binding member is attached to the backbone at any point between the end groups of the light harvesting chromophore.

147. A tandem dye according to clause 146 wherein the specific binding member is attached to the backbone at a central point of the light harvesting chromophore.

148. A tandem dye according to any one of clauses 144-147 wherein the specific binding member is selected from an antibody, an antibody fragment, or a binding derivative thereof.

149. A tandem dye according to any one of clauses 86-148, wherein the light collecting chromophore has the formula (VA) or (VB):

wherein:

each J is a repeating unit of the backbone;

L ³ -L ⁵ each independently is a linker;

Each D is a donor chromophore;

each a is an acceptor fluorophore;

x is 75mol% or more;

x1+ X2 is 75% or more; and

y is 25mol% or less.

150. A tandem dye according to clause 149, wherein the light harvesting chromophore has the formula (VA) and x is 80mol% or more; or (b)

The light harvesting chromophore has formula (VB) and x1+X2 is 80mol% or more.

151. A tandem dye according to clause 150 wherein the light harvesting chromophore has the formula (VA) and x is 90mol% or more; or (b)

The light harvesting chromophore has formula (VB) and x1+X2 is 90mol% or more.

152. A tandem dye according to clause 149, wherein the light collecting chromophore has the formula (VA) and y is 20mol% or less; or (b)

The light harvesting chromophore has formula (VB) and y is 20 mole% or less.

153. A tandem dye according to clause 152 wherein the light harvesting chromophore has the formula (VA) and y is 10mol% or less; or (b)

The light harvesting chromophore has formula (VB) and y is 10 mole% or less.

154. A tandem dye according to any one of clauses 149-153 wherein G1 or G2 is a linked specific binding member.

155. A tandem dye according to any of clauses 149-153, wherein J, L ³ -L ⁵ Any of D or a comprises a linked specific binding member.

156. A tandem dye according to any one of clauses 149-155, wherein each J is an amino acid or peptide unit.

157. A method of assessing the presence of a target analyte in a sample, the method comprising:

(a) Contacting the sample with an anti-aggregation dye conjugate that specifically binds to the target analyte to produce a labeled composition contacted sample, wherein the dye conjugate comprises:

(i) A tandem dye according to any one of clauses 86-156; and

(ii) A specific binding member attached to the tandem dye; and

(b) Determining the presence of the dye conjugate-target analyte binding complex in the sample contacted by the labeling composition to assess the presence or absence of the target analyte in the sample.

158. The method of clause 157, further comprising contacting the sample with a second specific binding member that binds to the support and specifically binds to the target analyte.

159. The method according to clause 158, wherein the support comprises magnetic particles.

160. The method according to any one of clauses 157 to 159, wherein the target analyte is associated with a cell.

161. The method of clause 160, wherein the target analyte is a cell surface marker of the cell.

162. The method according to clause 161, wherein the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

163. The method of clause 160, wherein the target analyte is an intracellular target, and the method further comprises lysing the cell.

164. The method of any one of clauses 157 to 163, wherein the method further comprises flow cytometry analyzing the fluorescently labeled target analyte.

165. A method of labeling a target molecule, the method comprising:

contacting the target molecule with a tandem dye to produce a labeled target molecule, wherein:

the tandem dye is a dye according to any of clauses 86-156, and comprises a conjugated tag covalently linked to a target molecule.

166. The method of clause 165, further comprising fluorescence detecting the labeled target molecule.

167. The method according to clause 165 or 166, wherein the conjugation tag comprises a terminal functional group selected from the group consisting of amino, thiol, hydroxyl, hydrazine, hydrazide, azide, alkyne, maleimide, iodoacetyl, amine, activated ester, and protein reactive groups.

168. The method according to any one of clauses 165-167, wherein the target molecule is a specific binding member.

169. The method according to clause 168, wherein the specific binding member is an antibody.

170. The method according to clause 168, wherein the specific binding member is an antibody fragment or binding derivative thereof.

171. The method according to clause 170, 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.

172. A kit comprising:

a water-soluble dipyrromethene-based dye according to any one of clauses 1 to 45 or a tandem dye according to any one of clauses 86 to 156; and

a container.

173. The kit according to clause 172, further comprising one or more components selected from the group consisting of fluorophores, specific binding members, specific binding member conjugates, cells, supports, biocompatible aqueous elution buffers, and instructions for use.

174. The kit of clause 172 or 173, wherein the tandem dye is covalently linked to the specific binding member.

175. The kit of clause 174, wherein the specific binding member is an antibody.

176. The kit of clause 174, wherein the specific binding member is an antibody fragment or binding derivative thereof.

177. The kit of clause 176, 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.

1. a dipyrromethene-based dye that is effective to transfer energy to an acceptor chromophore, while a second dipyrromethene-based dye having the same or a different chemical structure is within its proximity to the energy acceptor.

2. The dye according to clause 1, wherein the efficient energy transfer occurs after excitation of the dye with light having a wavelength of 540nm to 590 nm.

3. The dye according to any of clauses 1-2, wherein the energy transfer isResonance Energy Transfer (FRET).

4. A dye according to any of clauses 1-3, wherein the effective energy transfer to the acceptor chromophore involves a transfer of 5% or more of the energy collected by the dipyrromethene-type dye.

5. The dye according to any of clauses 1-4, wherein the dye has the formula (I):

wherein:

R ⁸ and R is ⁹ Each independently is a poly (alkylene oxide) group.

6. The dye according to clause 5, wherein R ¹ Is aryl, substituted aryl, heteroaryl or substituted heteroaryl.

7. The dye according to clause 6, wherein R ¹ Has the following formula:

wherein m is 0 to 10.

8. The dye according to clause 7, wherein m is 0.

9. The dye according to clause 5, wherein R ¹ Is alkyl or substituted alkyl.

10. The dye according to clause 9, wherein R ¹ Has the following formula:

wherein r is 1 to 10.

11. The dye according to clause 10, wherein r is 1 to 4.

12. The dye according to any of clauses 5-11, wherein R ² -R ⁷ Comprising pi-bonds pi-conjugated to pi electrons of the pyrrole ring of the structure of formula (I).

13. The dye according to clause 12, wherein R ² And R is ³ Together forming a fused aromatic ring.

14. The dye according to clause 13, wherein R ² And R is ³ The fused aromatic ring formed is an aryl ring or a substituted aryl ring.

15. A dye according to any of clauses 5-14, wherein R ⁴ Selected from the group consisting of alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and cyano.

16. The dye according to clause 15, wherein R ⁴ Is methyl, ethyl, aryl, substituted aryl or cyano.

17. A dye according to any of clauses 5-16, wherein R ⁵ And R is ⁶ Together forming a fused ring.

18. The dye according to clause 14, wherein R ⁵ And R is ⁶ The fused ring formed is substituted with a pair of additional substituents providing additional fused rings.

19. The dye according to clause 18, wherein R ⁵ And R is ⁶ Together form a group of the formula

20. A dye according to any of clauses 5-19, wherein R ⁸ And R is ⁹ Each independently is a poly (ethylene glycol) group.

21. The dye according to clause 20, wherein R ⁸ And R is ⁹ Each independently has the formula:

wherein n is 6 to 24.

22. The dye according to clause 21, wherein n is 10 to 20.

23. The dye according to clause 22, wherein n is 12 to 16.

24. A dye according to any of clauses 5-23, wherein if R ⁴ Is a substituted aryl group, then R ⁵ Not substituted aryl.

25. A dye according to any of clauses 5-24, wherein if R ⁴ Is aryl, then R ⁵ Not an alkyl group.

26. A dye according to any of clauses 5-25, wherein if R ² 、R ⁴ And R is ⁵ Is methyl, then R ⁷ Not methyl.

27. A dye according to any of clauses 5-26, wherein if R ² And R is ³ Or R is ⁵ And R is ⁶ Together form a fused ring, R ³ And R is ⁶ Not hydrogen.

28. A polymeric tandem dye comprising:

a polymer backbone comprising non-conjugated repeat units;

a dipyrromethene-based donor chromophore attached to a non-conjugated repeat unit of the polymer backbone; and

an acceptor chromophore linked to a non-conjugated repeat unit of the polymer backbone and positioned adjacent to the energy reception of the donor chromophore,

wherein the donor chromophore is effective to transfer energy to the acceptor chromophore, and the second dipyrromethene-type donor chromophore having the same or a different chemical structure is within proximity to the energy acceptor chromophore.

29. The polymeric tandem dye according to clause 28, wherein the dipyrromethene-based donor chromophore is a dipyrromethene-based dye according to any one of clauses 1-27.

30. A polymeric tandem dye according to any one of clauses 28-29, wherein the second donor chromophore is attached to a non-conjugated repeat unit of the polymeric backbone.

31. The polymeric tandem dye according to any one of clauses 28-30, wherein the acceptor chromophore is selected from cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene boron difluoride, naphthalimides, thiazine dyes, and acridine dyes.

32. A labeled specific binding member comprising:

a dye according to any of clauses 1-27 or a polymer tandem dye according to any of clauses 28-31; and

a specific binding member attached to a dye or a polymeric tandem dye.

33. A labeled specific binding member according to clause 32, wherein the labeled specific binding member comprises a dye according to any of clauses 1-27.

34. A labeled specific binding member according to clause 32, wherein the labeled specific binding member comprises a polymeric tandem dye according to any of clauses 28-31.

35. A labeled specific binding member according to any of clauses 32-34, wherein the specific binding member is an antibody.

36. A labeled specific binding member according to any of clauses 32-35, wherein the specific binding member is an antibody fragment or binding derivative thereof.

37. A labeled specific binding member according to any of clauses 32-36, 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.

38. A labeled specific binding member according to any of clauses 32-37, wherein the acceptor chromophore is selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene boron difluorides, naphthalimides, thiazine dyes, and acridine dyes.

39. A method of assessing the presence of a target analyte in a sample, the method comprising:

(a) Contacting the sample with a labeled specific binding member that specifically binds to the target analyte to produce a labeled composition contacted sample, wherein the labeled specific binding member comprises:

(i) A dye according to any of clauses 1-27 or a polymer tandem dye according to any of clauses 28-31; and

(ii) A specific binding member attached to a dye or a polymeric tandem dye; and

(b) Determining the presence of the labeled specific binding member-target analyte binding complex in the sample contacted by the labeling composition to assess the presence or absence of the target analyte in the sample.

40. The method according to clause 39, wherein the labeled specific binding member comprises a dye according to any one of clauses 1-27.

41. A method according to clause 39, wherein the labeled specific binding member comprises a polymeric tandem dye according to any of clauses 28-31.

42. The method according to any of clauses 39-41, further comprising contacting the sample with a second specific binding member that binds to the support and specifically binds to the target analyte.

43. The method according to clause 42, wherein the support comprises magnetic particles.

44. The method according to any of clauses 39-43, wherein the target analyte is associated with a cell.

45. The method according to clause 44, wherein the target analyte is a cell surface marker of the cell.

46. The method according to clause 45, wherein the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

47. The method according to any of clauses 39-44, wherein the target analyte is an intracellular target, and the method further comprises lysing the cell.

48. The method according to any one of clauses 39-47, wherein the method further comprises flow cytometry analyzing the fluorescently labeled target analyte.

49. A method of labeling a target molecule, the method comprising:

contacting the target molecule with the dye according to any of clauses 1-27 or the polymeric tandem dye according to any of clauses 28-31 to produce a labeled target molecule, wherein the dye or polymeric tandem dye further comprises a conjugated tag covalently attached to the target molecule.

50. The method according to clause 49, wherein the method comprises contacting the target molecule with a dye according to any of clauses 1-27.

51. The method according to clause 49, wherein the method comprises contacting the target molecule with a polymeric tandem dye according to any of clauses 28-31.

52. A kit comprising:

a dye according to any of clauses 1-27, a polymer tandem dye according to any of clauses 28-31, or a combination thereof; and

a container.

53. The kit according to clause 52, wherein the kit comprises a dye according to any of clauses 1-27.

54. A kit according to clause 52, wherein the kit comprises a polymeric tandem dye according to any of clauses 28-31.

55. The kit according to any of clauses 52-54, further comprising one or more components selected from the group consisting of a fluorophore, a specific binding member conjugate, a cell, a support, a biocompatible aqueous elution buffer, and instructions for use.

56. The kit of clause 55, further comprising a specific binding member.

57. The kit according to clause 56, wherein the specific binding member is covalently linked to the dye or the polymeric tandem dye.

58. The kit of clause 57, wherein the specific binding member is an antibody.

59. The kit of clause 57, wherein the specific binding member is an antibody fragment or binding derivative thereof.

60. The kit according to clause 59, 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.

61. The kit according to any of clauses 56-60, wherein the specific binding member is a support-bound specific binding member.

Various aspects (including embodiments) of the invention described herein may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the present description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the previously or subsequently individually numbered aspects. This is intended to provide support for a combination of all of these aspects, and is not limited to the combination of aspects explicitly provided below:

in at least some of the foregoing embodiments, one or more elements used in one embodiment are used interchangeably in another embodiment unless such substitution is technically not feasible. Those skilled in the art will appreciate that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the invention as claimed. All such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.

It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally considered to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "comprising" should be interpreted as "including but not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. Furthermore, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, where a convention analogous to "at least one of A, B and C, etc." is used, such a construction is generally in the sense one skilled in the art would understand conventionally (e.g., "a system having at least one of A, B and C" would include, but is not limited to, systems having a alone, B alone, C, A and B, A and C, B and C alone, and/or A, B and C, etc.). Where a convention analogous to "at least one of A, B or C, etc." is used, such a construction is generally in the sense one skilled in the art would understand conventionally (e.g., "a system having at least one of A, B or C" would include, but is not limited to, systems having a alone, B alone, C, A and B, A and C, B and C alone, and/or A, B and C, etc.). It should also be appreciated by those skilled in the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "a or B" should be understood to include the possibilities of "a" or "B" or "a and B".

Further, while features or aspects of the present disclosure are described in terms of markush groups, those skilled in the art will recognize that the present disclosure is also thereby described in terms of any individual member or subgroup of members of the markush group.

As will be understood by those of skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also include any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be readily considered as sufficiently describing and enabling the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, an upper third, and the like. It will also be understood by those skilled in the art that all language (e.g., "up to", "at least", "greater than", "less than", etc.) includes the recited numbers and refers to ranges that can be subsequently broken down into subranges as discussed above. Finally, as will be appreciated by those skilled in the art, a range includes each individual member. Thus, for example, a group of 1-3 items refers to a group of 1, 2, or 3 items. Similarly, a group of 1-5 items refers to a group of 1, 2, 3, 4, or 5 items, etc.

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

Thus, the foregoing merely illustrates the principles of the invention. It will thus be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventor to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

Thus, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied by the appended claims. In the claims, 35u.s.c. ≡112 (f) or 35u.s.c. ≡112 (6) are explicitly defined as being invoked for such limitations in the claims only when the exact phrase "means for …" or the exact phrase "step for …" is recited at the beginning of the limitation in the claims; if such exact phrase is not used to limit the claims, 35u.s.c. ≡112 (f) or 35u.s.c. ≡112 (6) is not invoked.

Claims

2. The water-soluble dipyrromethene-based dye according to claim 1, wherein absorption maximum is 555 to 585nm.

3. The water-soluble dipyrromethene-based dye according to claim 2, wherein the absorbance maximum is 558 to 568nm.

4. The water-soluble dipyrromethene-type dye according to claim 3, wherein the absorbance maximum is about 561nm.

5. The water-soluble dipyrromethene-based dye according to any one of claims 1 to 4, wherein the dye is a dipyrromethene-boron dye.

6. The water-soluble dipyrromethene-based dye according to any one of claims 1 to 5, having formula (I):

wherein:

Optionally selected from R ⁶ And R is ⁷ 、R ² And R is ³ 、R ⁵ And R is ⁶ 、R ³ And R is ⁴ 、R ² And R is ¹ R is as follows ⁷ And R is ¹ Any one or more pairs of substituents together form a divalent group and are cyclic linked and, together with the carbon atom to which they are bound, provide a 5 or 6 membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5 or 6 membered ring comprising a carbon atom and 0-3 heteroatoms selected from O, S and N) which may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water Solubilising Group (WSG) and-T ¹ -J ¹ Is substituted by a substituent of (a);

T ¹ is an optional linker;

J ¹ is a repeating unit of a compound of formula (I); and

Y ¹ And Y ² Together with the boron atom to which they are bound form a 5-or 6-membered ring (e.g. containing boron atoms, carbon atoms and 0-3 heteroatoms selected from O, S and NThe ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water Solubilizing Group (WSG) and-T ¹ -J ¹ Is substituted by a substituent of (a); and

wherein is selected from Y ¹ 、Y ² And R is ¹ -R ⁷ At least one moiety of (2) is WSG, or is selected from Y ¹ 、Y ² And R is ¹ -R ⁷ Is substituted with a WSG, wherein the WSG has the formula (Ia):

-(L ¹ ) _n1 -(X ¹ ) _n2 ((L ² ) _n3 -Z ¹ ) _m (Ia)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ¹ Is a water-soluble polymer comprising 6 to 24 monomer units; and

m is an integer from 1 to 3.

7. The water-soluble dipyrromethene-based dye of claim 6 further comprising WSG of formula (Ib):

-(L ¹ ) _n1 -(X ¹ ) _n2 -((L ² ) _n3 -Z ² ) _m (Ib)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ² is a water-soluble polymer comprising 1 to 50 monomer units; and

m is an integer from 1 to 3.

8. The water-soluble dipyrromethene-based dye according to claim 6 or 7, wherein the Water Solubilising Group (WSG) comprises a PEG moiety.

9. The water-soluble dipyrromethene-based dye according to any one of claims 6 to 8, having formula (II):

wherein p is an integer of 6 to 24.

10. The water-soluble dipyrromethene-based dye according to any one of claims 6 to 9, wherein R ⁵ Selected from the group consisting of halogen, substituted alkyl, substituted alkenyl, alkenylaryl, aryl, substituted aryl, heterocyclic, substituted heterocyclic, spiroheterocyclic, substituted spiroheterocyclic, heteroaryl, substituted alkoxy, substituted amino, substituted amido, and substituted thiol.

11. The water-soluble dipyrromethene-based dye according to any one of claims 6 to 10, having formula (IIa):

Wherein:

R ⁸ And R is ⁶ Forms, together with the atoms to which they are bonded, a 5-or 6-membered heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g. containing carbon atoms)And 0-3 5 or 6 membered rings selected from the group consisting of O, S and N, which rings may be unsubstituted or further substituted with substituents independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilizing Group (WSG);

q is an integer from 0 to 5; and

Optionally two adjacent R ⁹ The groups together form a divalent group and are cyclic linked and together with the carbon atoms to which they are bonded provide a 5 or 6 membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5 or 6 membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilising Groups (WSG).

12. The water-soluble dipyrromethene-based dye according to any one of claims 6 to 10, having formula (IIb):

wherein:

each q is independently an integer from 0 to 5; and

R ¹⁰ and R is ^10' Each, if present, is independently selected from halogen, hydroxy, cyano, carboxamide, substituted carboxamide, amino, substituted amino, thiol,Substituted thiols, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, heteroaryl groups, substituted heteroaryl groups, alkoxy groups, substituted alkoxy groups, alkenyl groups, substituted alkenyl groups, alkynyl groups, substituted alkynyl groups, and water-soluble groups (WSG), or

At least one R ¹⁰ The radicals being in the ortho position and R ¹⁰ And R is ⁶ Together with the atoms to which they are bonded, form a 5 or 6 membered ring (e.g., a 5 or 6 membered ring comprising a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilising Group (WSG); and

13. The water-soluble dipyrromethene-based dye according to any one of claims 6 to 10, having formula (IIc):

Wherein:

x is halogen; and

ring a is a 5 or 6 membered fused heterocycle, carbocycle, aryl ring, or heteroaryl ring (e.g., a 5 or 6 membered ring comprising a carbon atom and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further substituted with substituents independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and Water Solubilizing Group (WSG).

14. The water-soluble dipyrromethene-based dye according to any one of claims 6 to 10, having formula (IId):

wherein:

X ² and X ³ Each independently selected from NR ¹³ S and O;

R ¹³ selected from H, alkyl, and substituted alkyl;

When X is ² Is NR ¹³ When optionally R ¹³ And R is ¹¹ Forms a heterocyclic or spiro-heterocyclic ring together with the nitrogen atom to which they are attached, which ring may be unsubstituted or further substituted with a substituent independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and Water Solubilizing Group (WSG), and

15. The water-soluble dipyrromethene-based dye according to any one of claims 6 to 10, having formula (III):

wherein:

16. The water-soluble dipyrromethene-based dye according to any one of claims 1 to 4, having formula (IV):

wherein:

X ^- is a counter ion;

Optionally selected from R ^6' And R is ^7' 、R ^2' And R is ^3' 、R ^5' And R is ^6' 、R ^3' And R is ^4' 、R ^2' And R is ^1' R is as follows ^7' And R is ^1' Together with any one or more pairs of substituents forming a divalent group and being cyclic and linked to the carbon to which they are bondedAtoms together provide a 5 or 6 membered fused heterocycle, carbocycle, aryl ring, or heteroaryl ring (e.g., a 5 or 6 membered ring comprising a carbon atom and 0-3 heteroatoms selected from O, S and N), which rings may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water Solubilizing Group (WSG), and-T ¹ -Z ¹ Is substituted by a substituent of (a);

T ¹ is an optional linker;

Z ¹ is a repeating unit of a compound of formula (I); and

wherein is selected from Y ^1' 、Y ^2' And R is ^1' -R ^7' At least one moiety of (2) is WSG, or is selected from Y ^1' 、Y ^2' And R is ^1' -R ^7' Is substituted with a WSG, wherein the WSG has the formula (Ia):

-(L ¹ ) _n1 -(X ¹ ) _n2 -((L ² ) _n3 -Z ¹ ) _m (Ia)

wherein:

n1, n2 and n3 are each independently 0 or 1;

L ¹ (if present) is a linker;

L ² (if present) is a linker;

X ¹ (if present) are branching points;

Z ¹ is a water-soluble polymer comprising 6 to 24 monomer units; and

m is an integer from 1 to 3.

17. A tandem dye comprising:

A light harvesting chromophore, the light harvesting chromophore comprising:

a main chain;

a donor chromophore attached to the backbone, wherein the donor chromophore is a water-soluble dipyrromethene-based dye that absorbs in the yellow-green range at 561 nm; and

an energy acceptor comprising a pendant acceptor fluorophore attached to the backbone and disposed adjacent to the energy acceptor of the donor chromophore.

18. A method of assessing the presence of a target analyte in a sample, the method comprising:

(a) Contacting the sample with an anti-aggregation dye conjugate that specifically binds to the target analyte to produce a labeled composition-contacted sample, wherein the dye conjugate comprises:

(i) The tandem dye of any one of claims 86-156; and

(ii) A specific binding member attached to the tandem dye; and

(b) Determining the presence of a dye conjugate-target analyte binding complex in a sample contacted with the labeling composition to assess the presence or absence of the target analyte in the sample.