Novel Fluorescent Dyes and Polymers from Dihydrophenanthrene Derivatives

Abstract

The present disclosure provides novel dihydrophenanthrene (DHP)-cyanine and DHP-squaraine fluorescent compounds and water-soluble polymers thereof. The DHP-cyanine and DHP-squaraine fluorescent compounds and polymers may be excited using UV, violet, blue, yellow, green, red, or NIR wavelengths. The fluorescent dyes may be conjugated to antibodies for detecting target analytes in biological samples and are suitable for use in flow cytometry analysis.

Description

Technical Field

[0001] This application was filed as a PCT international patent application on June 30, 2023, claiming the benefits and priority of U.S. Provisional Patent Application No. 63 / 357,980, filed on July 1, 2022, and U.S. Provisional Patent Application No. 63 / 490,921, filed on March 17, 2023, the entire disclosures of which are hereby incorporated by reference in their entirety.

Background Art

[0002] Background of the Invention There is a growing demand for various fluorescent dyes for use in current flow cytometers and spectral flow devices. Water-soluble fluorescent compounds and their conjugates can be used in various biological applications by generating signals that can be monitored in real time, providing a simple and rapid method for detecting biological targets and events in, for example, diagnostic kits, microscopy, cytometry, or drug screening.

[0003] Molecular recognition involves the specific binding of two molecules. Molecules having binding specificity for target biomolecules are used in various investigative and diagnostic applications, such as labeling and separation of analytes, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation, and chromatography. The target biomolecule may be detected by labeling with a fluorescent dye.

[0004] Current dihydrophenanthrene-based polymer dyes are strongly excited in the ultraviolet and violet regions of 350 - 450 nm. For example, U.S. Patent No. 11,208,527 describes water-soluble dihydrophenanthrene (DHP)-based fluorescent polymer dyes that exhibit an excitation maximum between 395 - 415 nm and emit light between approximately 415 - 475 nm. U.S. Patent No. 11,584,825 describes water-soluble DHP-based violet-excitable polymers and tandem dyes.

[0005] In both conventional flow cytometry and spectral flow cytometry, there is an increasing demand for multi-color panels that require additional fluorescent dyes that are excitable with other laser lights (e.g., 488, 563, 638, and 808 nm). Parameters that may be considered by users in the selection of fluorescent dyes include the excitation wavelength maximum, the emission wavelength maximum, the brightness of the dye, and the fluorescence lifetime. The brightness of the dye is the overall contribution from the extinction coefficient (ε, a measure of the amount of light absorbed at a specific wavelength) and the fluorescence quantum yield (Φ, a measure of the light emitted in the form of radiation from its singlet excited state).

[0006] By attempting to further shift the excitation range into the red region by incorporating modified unit monomers into the existing DHP violet-excitable polymer backbone, the original absorption of the polymer and the absorption from the acceptor dye were retained. By directly exciting the resulting DHP-modified unit polymer dye, strong UV and violet emissions from the core were obtained. Attempts to design a core polymer shifted to red using other aromatic cyclic molecules as monomers faced synthetic challenges. Therefore, more careful design was required to shift the excitation maximum into the blue, green, and red regions of the spectrum and beyond. Organic fluorescent dyes and water-soluble polymer dyes that can be excited using UV, violet, blue, yellow, green, red, and near-infrared (NIR) wavelengths are desirable.

Prior Art Documents

Patent Documents

[0007] [Patent Document 1] U.S. Patent No. 11,208,527 [Patent Document 2] U.S. Patent No. 11,584,825 [Summary of the Invention] [Means for Solving the Problems]

[0008] Summary of the Invention The present disclosure generally provides dihydrophenanthrene (DHP)-cyanine (Cy) and DHP-squaraine compounds and polymeric dyes conjugated to specific binding partners, water-soluble DHP-cyanine and DHP-squaraine compounds and water-soluble fluorescent polymers, complexes thereof, and complexes comprising water-soluble DHP-Cy and DHP-squaraine fluorescent compounds or polymers conjugated to binding partners for detecting an analyte in a sample. DHP-Cy and DHP-squaraine fluorescent compounds, polymers, or tandem dyes comprising labeled specific binding partners provided by the present disclosure are also provided. DHP-Cy and DHP-squaraine compounds, polymers, labeled specific binding partners, and tandem dyes provided by the present disclosure are useful in biological applications and diagnostic kits, including those for detecting target analytes. The kits may include DHP-Cy compounds, DHP-squaraine compounds, polymers, labeled specific binding partners, and / or tandem dyes provided by the present disclosure and may optionally have conjugation tags.

[0009] The present disclosure provides a fluorescent compound comprising a structure according to formula (I): [Chemical Formula] (wherein, D is an aryl group, a heteroaryl group, [Chemical Formula] and; J is an aryl group, a heteroaryl group, or [Chemical formula] and; at least one of D or J is [Chemical formula] independently selected from the group consisting of Ar as required and as required [Chemical formula] is independently selected from the group consisting of substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl groups, polycyclic aryl groups, monocyclic heteroaryl groups, and polycyclic heteroaryl groups, and at least one of D or J [Chemical formula] exists, [Chemical formula] or any available [Chemical formula] and derivatives thereof containing additional aryl or heteroaryl groups condensed with each T is independently selected from the group consisting of C, C(R 1 ), N, P, O, S, and Si(R 1 ); each U is independently selected from the group consisting of NR 10 , O, P, and S; each V is independently selected from the group consisting of NR 11 , CR 11 , C(R 11 )2, S, SO2, O, and Si(R 11 )2; each V’ is independently SO2, SO, S, NR 11 , CR 11 , C(R11 ) 2, O, Si(R 11 ) 2, >C=O, >Se=O, -CH=CH-, or -N=CH-; Each X is independently CR 1 R 2 or SiR 1 R 2 ; Each Y is independently CR 8 R 9 or SiR 8 R 9 ; Each R 1 , R 2 , R 8 , and R 9 are independently selected from the group consisting of a water-solubilizing moiety, a linker moiety, linked E, hydrogen, hydroxy, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, sulfonamide-PEG, phosphoramidate-PEG, alkylammonium salt, alkyloxyammonium salt, carbonyl, acyl, oligoetherammonium salt, alkylsulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, sulfonamide, sulfinamido, phosphonamidate, phosphinamido, alkoxysulfonamide PEG, alkoxysulfonate, alkylsulfonate, alkylsulfonate,

Chemical Structure

Chemical Structure

[0010] The present disclosure provides a fluorescent compound comprising a structure according to formula (II):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0011] Any available

Chemical formula

Chemical formula

Chemical formula

[0012] The present disclosure provides DHP-cyanine and DHP-squarylium compounds selected from the group consisting of formula (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIi), (IIj), (Ik), (IIl), (IIm), (IIn), (IIo), (IIp), (IIq), (IIr), (IIs), (IIt), (IIu), (IIv), (IIw), (IIx), (IIy), and (IIz).

Chemical formula

Chemical formula

Chemical formula

[0013] The present disclosure provides DHP-cyanine and DHP-squarylium compounds selected from the group consisting of formula (IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIi), (IIIj), (IIIk), (IIIl), (IIIm), (IIIn), (IIIo), (IIIp), (IIIq), (IIIr), (IIIs), (IIIt), (IIIu), (IIIv), (IIIw), (IIIx), (IIIy), (IIIz), (IIIaa), (IIIbb), (IIIcc), (IIIdd), and (IIIee).

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chem.

[0014] In some embodiments, the

Chem.

Chem.

[0015] In some embodiments, the

Chem.

Chem.

[0016] In some embodiments, the

Chem.

Chem.

[0017] In some embodiments, D is

Chem.

Chem.

Chem.

[0018] In some embodiments, J is [Chemical formula] and the [Chemical formula] of J is an unsubstituted or substituted benzene or unsubstituted or substituted naphthalene; U is N; V is N, O, or S.

[0019] In some embodiments, J is unsubstituted or substituted quinoline, benzoxazole, benzothiazole, benzimidazole, indole, benzindole, pyridinium, benzopyrylium, thiopyrylium, fluorene, 9H-fluorene, phenanthrene, dihydrophenanthrene, 9,10-dihydrophenanthrene, 6,8-dihydro-5H-naphtho[2,1-f]indole, 4,5-dihydro-3H-naphtho[2,1-e]indole, 6,7-dihydro-3H-naphtho[2,1-g]indole, 5,6-dihydrophenanthro[3,2-d]thiazole, 4,5-dihydrophenanthro[2,1-d]thiazole, 6,7-dihydrophenanthro[4,3-d]thiazole, 5,6-dihydrophenanthro[3,2-d]oxazole, 4,5-dihydrophenanthro[2,1-d]oxazole, 6,7-dihydrophenanthro[4,3-d]oxazole, 5,6-dihydronaphtho[2,1-g]quinoline, 7,8-dihydronaphtho[2,1-h]quinoline, 5,6-dihydronaphtho[2,1-f]quinoline, 5,6-dihydro-8λ2-phenanthro[3,2-d]imidazole, 4,5-dihydro-3λ2-phenanthro[2,1-d]imidazole, 6,7-dihydro-3λ2-phenanthro[4,3-d]imidazole, 5,6-dihydronaphtho[1,2-g]quinoxaline, 5,6-dihydronaphtho[2,1-f]quinoxaline, 7,8-dihydronaphtho[1,2-f]quinoxaline, phenyl, 1H-benzo[e]indol-3-ium, 1H-benzo[e]indole, and 9,10-dihydro-8H-thieno[2’,3’,4’,5’:4,5]phenanthro[2,1-b]pyrrole 4,4-dioxide.

[0020] In some embodiments, D is aryl, heteroaryl or [Chemical formula] and the [Chemical formula] is a substituted or unsubstituted benzene, benzene derivative, monocyclic aryl group, polycyclic aryl group, monocyclic heteroaryl group, and polycyclic heteroaryl group; J is

Chem.

Chem.

Chem.

[0021] In some embodiments, D is

Chem.

Chem.

[0022] In some embodiments, D is selected from the group consisting of unsubstituted or substituted quinoline, benzoxazole, benzothiazole, benzimidazole, indole, benzindole, pyridinium, benzopyrylium, thiopyrylium, fluorene, 9H-fluorene, phenanthrene, dihydrophenanthrene, 9,10-dihydrophenanthrene, 6,8-dihydro-5H-naphtho[2,1-f]indole, 4,5-dihydro-3H-naphtho[2,1-e]indole, 6,7-dihydro-3H-naphtho[2,1-g]indole, 5,6-dihydrophenanthro[3,2-d]thiazole, 4,5-dihydrophenanthro[2,1-d]thiazole, 6,7-dihydrophenanthro[4,3-d]thiazole, 5,6-dihydrophenanthro[3,2-d]oxazole, 4,5-dihydrophenanthro[2,1-d]oxazole, 6,7-dihydrophenanthro[4,3-d]oxazole, 5,6-dihydronaphtho[2,1-g]quinoline, 7,8-dihydronaphtho[2,1-h]quinoline, 5,6-dihydronaphtho[2,1-f]quinoline, 5,6-dihydro-8λ2-phenanthro[3,2-d]imidazole, 4,5-dihydro-3λ2-phenanthro[2,1-d]imidazole, 6,7-dihydro-3λ2-phenanthro[4,3-d]imidazole, 5,6-dihydronaphtho[1,2-g]quinoxaline, 5,6-dihydronaphtho[2,1-f]quinoxaline, 7,8-dihydronaphtho[1,2-f]quinoxaline, phenyl, 3,5,6,8-tetrahydrophenanthro[2,3-e:7,6-e’]diindole, 5,6-dihydropentapheno[3,4-d:10,9-d’]bis(oxazole), 5,6-dihydropentapheno[3,4-d:10,9-d’]bis(thiazole), 6,7-dihydrophenanthro[2,3-f:7,6-f’]diquinoline, 5,6-dihydropentapheno[3,2,1-cd:10,11,12-c’d’]diindole, 2,6,7,11-tetrahydrobenzo[1,2-g:4,3-g’]dichromene, 2,6,7,11-tetrahydrobenzo[1,2-g:4,3-g’]bis(thiochromene), and 9,10-dihydro-8H-thieno[2’,3’,4’,5’:4,5]phenanthro[2,1-b]pyrrole 4,4-dioxide.

[0023] In some embodiments, D is

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0024] In some embodiments, D is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0025] In some embodiments, D is

Chemical formula

[0026] In some embodiments, D is

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0027] In some embodiments, J is [Chem.] and [Chem.] the group is [Chem.] any available [Chem.] at the position of [Chem.] condensed with [Chem.] a group or a derivative thereof. [Chem.] The derivative of [Chem.] is condensed with an additional aryl or heteroaryl group, optionally [Chem.] may contain, and V' is SO2, SO, S, NR 11 , CR 11 , C(R 11 )2, O, Si(R11 ) 2, >C=O, >Se=O, -CH=CH-, or -N=CH-, and T, V, X, Y, and m are as defined above in this specification.

[0028] In some embodiments, J is

Chemical formula

[0029] In some embodiments, at least one

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0030] In some embodiments, D is

Chemical formula

Chemical formula

Chemical formula

[0031] In some embodiments, J is

Chemical formula

Chemical formula

Chemical formula

[0032] In some embodiments, R 10 is a solubilizing moiety, a linker moiety, a linked solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, aryl, heteroaryl, (hetero)arylamino, sulfonamide-PEG, phosphoramidate-PEG, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkylsulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamido, alkoxysulfonamide PEG, alkoxysulfonate, alkylsulfonate, alkylsulfonate salt,

Chemical formula

Chemical formula

[0033] In some cases, [Chemical formula] [Chemical formula] A fluorescent dye is provided that includes a structure by any one of.

[0034] In some cases, an acceptor dye is provided that includes a DHP-cyanine compound having a structure according to formula (II).

[0035] The present disclosure provides a polymeric dye that includes a monomer having a structure according to formula (IV) or (V) [Chemical formula] (wherein each in (IV) or (V) [Chemical formula] is a bonding point to the polymeric dye skeleton; each D 1 and D 2 are independently selected from the group consisting of an aryl group, a heteroaryl group, and [Chemical formula] is independently selected from the group consisting of, and each [Chemical formula] is independently selected from the group consisting of a substituted or unsubstituted benzene, a benzene derivative, a monocyclic aryl group, a polycyclic aryl group, a monocyclic heteroaryl group, and a polycyclic heteroaryl group, and at least one of D 1 or D 2 is [Chemical formula] and

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0036]

Chem.

Chem.

Chem.

[0037] In some embodiments, the monomer of formula (IV) or (V) is of formula (A1), (A2), (A3), (A4), (A5), (A6), (A7), (A8), (A9), (A10), (A11), (A12), (A13), (A14), (A15), (A16), (A17), (A18), and (A19):

Chem.

Chem.

Chem.

Chem.

Chem.

[0038] The present disclosure provides a polymeric dye comprising a structure according to formula (VI) or (VII)

Chem.

Chem.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0039] This disclosure provides a polymeric dye having a structure of formula (VI’) [Chemical formula] (wherein, A is of formula (IV) or (V) [Chemical formula] is a monomer containing a structure according to each in formula (IV) or (V) [Chemical formula] is a bonding point to the polymer dye skeleton; each D 1 and D 2 are an aryl group, a heteroaryl group, and [Chemical formula] is independently selected from the group consisting of [Chemical formula] is independently selected from the group consisting of substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl groups, polycyclic aryl groups, monocyclic heteroaryl groups, and polycyclic heteroaryl groups, and at least one of D 1 or D 2 is [Chemical formula] where [Chemical formula] is an additional aryl or heteroaryl group condensed with any available [Chemical formula] including [Chemical formula] or a derivative thereof, [Chemical formula] the group or its derivative is [Chemical formula] Any available on a base or its derivative

Chem.

Chem.

[0040] In some embodiments, G 1 and G 2is independently selected from the group consisting of aryl or heteroaryl substituted with one or more pendant chains terminated with a functional moiety selected from hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronic acid-substituted aryl, boronic ester-substituted aryl, boronic ester, boronic acid, optionally substituted tetrahydropyran (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protecting groups thereof, conjugated to a substrate or binding partner as needed.

[0041] In some embodiments, D 1 and D 2 are the same. In some embodiments, D 1 and D 2 are different.

[0042] In some embodiments, at least one or at least two of R 1 , R 2 , R 3 , R 4 , R 8 , R 9 , R 10 , and R 11 contain a solubilizing moiety or a linked solubilizing moiety.

[0043] The present disclosure provides a labeled specific binding partner comprising a fluorescent compound or polymer according to the present disclosure; and a specific binding partner covalently attached to the fluorescent compound or polymer. The specific binding partner may be selected from the group consisting of proteins, peptides, affinity ligands, antibodies, antibody fragments, carbohydrates, lipids, nucleic acids, and aptamers. The specific binding partner may be an antibody. The specific binding partner may be an antibody specific for a target analyte.

[0044] The present disclosure provides a tandem dye comprising a fluorescent compound, polymer, or labeled specific binding partner according to the present disclosure; and an acceptor chromophore covalently attached to the fluorescent compound, polymer, or labeled specific binding partner. In some cases, the fluorescent compound of the present invention is an acceptor dye.

[0045] The fluorescent compound, polymer, labeled specific binding partner, or tandem dye may be water-soluble.

[0046] A method for detecting a target analyte in a sample, comprising the steps of preparing a sample suspected of containing the analyte; and contacting the sample with a specific binding partner conjugated to a water-soluble fluorescent compound, polymer, or tandem dye according to the present disclosure, wherein the binding partner is capable of interacting with the target analyte.

[0047] The method may include one or more of the following: the method is configured for flow cytometry; the water-soluble fluorescent polymer is bound to a substrate; the analyte is a protein expressed on the cell surface; the method is configured as an immunoassay; and / or the method further includes the step of preparing an additional binding partner for simultaneously detecting an additional analyte.

[0048] The present disclosure provides a kit comprising at least one fluorescent compound, polymer, labeled specific binding partner, or tandem dye according to the present disclosure. The compounds, polymers or tandem dyes according to the present disclosure may comprise a conjugation tag.

Brief Description of the Drawings

[0049]

Figure 1

[0050]

Figure 2

[0051]

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[0052]

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[0055]

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[0056]

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Figure 10

DETAILED DESCRIPTION OF THE INVENTION

[0059] DETAILED DESCRIPTION OF THE INVENTION I. General Matters The present disclosure provides novel DHP-cyanine and DHP-squaraine fluorescent compounds and polymers thereof. In some embodiments, the DHP-cyanine and DHP-squaraine fluorescent compounds and polymer dyes are designed to be water-soluble. The present disclosure also provides labeled specific binding partners that include DHP-cyanine and DHP-squaraine fluorescent compounds and polymers thereof. The present disclosure also provides tandem dyes that include DHP-cyanine and DHP-squaraine fluorescent compounds and polymers thereof. The DHP-cyanine and DHP-squaraine fluorescent compounds may be acceptor dyes.

[0060] A method is provided for detecting a target analyte in a sample using a fluorescent DHP-cyanine and DHP-squaraine compound or polymer conjugated to a binding partner. The various DHP-cyanine and DHP-squaraine fluorescent compounds and polymers of the present disclosure have demonstrated water solubility and can be excited using UV, violet, blue, yellow, green, red, or NIR wavelengths. There is a growing need for various fluorescent dyes for use in current flow cytometers and spectral flow devices.

[0061] In the present disclosure, the dihydrophenanthrene monomer was modified to include a condensed heterocyclic ring. For example, the DHP monomer was modified to an electron-rich indole containing a DHP-indole moiety using the Fischer indole synthesis that produces an aromatic heterocyclic indole from a substituted phenylhydrazine and an aldehyde or ketone under acidic conditions. This can be done in two steps using bromodihydrophenanthrene as the starting material, as shown, for example, in Scheme 2. Subsequently, classical cyanine dye synthesis was performed on the DHP-indole ring, enabling the formation of DHP-cyanine dyes that can be excited using UV, violet, blue, green, red, or NIR light depending on the number of carbon atoms in the linker molecule used for the cyanine synthesis.

[0062] The various chemical modifications of the core DHP-cyanine molecules have enabled the preparation of dyes that can be excited using different laser lights. Both small molecule organic dyes and polymeric dyes have been prepared using this approach. Other families of dyes, such as squaraines, have also been prepared from the electron-rich DHP ring core. Symmetric as well as asymmetric dyes have been made using this approach that allows for fine-tuning of the absorption and emission of the dyes. The DHP-cyanine and DHP-squaraine dyes according to the present disclosure exhibit excitation maxima in the range of about 400-900 nm for monomeric dyes.

[0063] There is also provided a kit comprising at least one fluorescent DHP-cyanine compound, DHP-squaraine fluorescent compound or polymers thereof, labeled specific binding partner, or tandem dye according to the present disclosure. The fluorescent DHP-cyanine, DHP-squaraine compound, or polymers thereof, or tandem dye may comprise a conjugation tag. II. Definitions

[0064] The abbreviations used herein have their conventional meanings within the scope of the fields of chemistry and biology.

[0065] Certain embodiments of the disclosed subject matter will be described in detail, examples of which are illustrated in part in the accompanying drawings. The disclosed subject matter will be described in conjunction with the recited claims, but it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0066] Throughout this document, values expressed in a range format are to be understood in a flexible manner that includes not only the numerical values explicitly recited as the limits of the range, but also all individual numerical values, or sub-ranges that are included within the range as if each numerical value and sub-range were explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" is to be understood to include not only about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and sub-ranges within the indicated range (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%). The phrase "about X to Y" has the same meaning as "about X to about Y" unless otherwise indicated. Similarly, the phrase "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z" unless otherwise indicated.

[0067] In this document, the terms "a", "an", or "the" are used to include one or more than one, unless the context clearly indicates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. The phrases "at least one of A and B" or "at least one of A or B" have the same meaning as "A, B, or A and B". Further, terms or technical terms used herein and not specifically defined are for illustrative purposes only and are not to be construed as limiting. Any use of section headings is intended to assist in the interpretation of the document and is not to be construed as limiting, and appropriate information for a section heading may be found within and outside that particular section. All publications, patents, and patent documents referred to in this document are hereby incorporated by reference in their entirety as if individually incorporated by reference. If there is a conflict in use between this document and the documents incorporated by reference, the use in the incorporated reference should be considered supplementary to that of this document; if the conflict cannot be resolved, the use in this document prevails. In the methods described herein, acts can be performed in any order without departing from the principles of the disclosure, except where the temporal or operational order is explicitly recited. Further, the specified acts may be performed simultaneously, unless they are not individually recited in the exact claim language. For example, the claimed act of performing X and the claimed act of performing Y may be performed simultaneously within a single operation, and the resulting method will fall within the literal scope of the claimed method. The term "about", as used herein, can tolerate a degree of variation within a value or range, for example, within 10%, 5%, or 1% of the defined limits of the defined value or range, and includes the precisely defined value or range.As used herein, the term "substantially" refers to mostly or mainly, e.g., at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999%, or more than this, or 100%. As used herein, the term "substantially free of" means having none at all or having such a small amount that the amount of the material present does not affect the material properties of the composition containing the material, e.g., from about 0 wt% to about 5 wt% of the composition is the material, or from about 0 wt% to about 1 wt%, or about 5 wt% or less, or about 4.5 wt% or less, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt% is the material.

[0068] In some cases, the term "reactive group" refers to a functional group that can selectively react with another compatible functional group to form a covalent bond after activation as required for one of the functional groups. The chemical-selective functional groups of interest include, but are not limited to, thiol, maleimide, halogenated maleimide, iodoacetamide, amine, alkyl carboxylate, alkyl sulfonate, carboxylic acid amine, carbamate, carboxylic acid ester, N-hydroxysuccinimidyl (NHS), imido ester, halogen, boronic acid ester, boronic acid, hydrazonyl, carboxylic acid or its active ester, and groups that can react with each other via another click chemistry, e.g., azide and alkyne groups (e.g., cyclooctyne group), tetrazine and alkene groups (e.g., cyclooctene group), diene and dienophile, sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluoride, and hydroxyl, hydrazide, hydrazino, aldehyde, ketone, azide, alkyne, phosphine, epoxide, etc., or their protecting groups. The reactive group may be a conjugation tag. The chemical-selective functional groups may or may not be protected.

[0069] The term "amine-reactive group" refers to any group that forms a chemical bond with a primary amine. Examples of amine-reactive groups include, but are not limited to, isothiocyanate, isocyanate, acyl azide, NHS ester, imido ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imido ester, carbodiimide, anhydride, and fluorophenyl ester. The amine-reactive group may be an NHS ester or an imido ester.

[0070] In some cases, the non-covalent bond may be accompanied by specific binding between two moieties of interest (e.g., between two affinity moieties such as between a hapten and an antibody or between a biotin moiety and streptavidin). In certain cases, the non-covalent bond may be accompanied by absorption to a substrate.

[0071] The term "symmetric" in reference to the compounds of the present disclosure means that each terminal heterocyclic ring system is the same and the substituents may be the same or different. In some symmetric compounds, n = 2.

[0072] The term "asymmetric" in reference to the compounds of the present disclosure means that each terminal heterocyclic ring system is different. In some asymmetric compounds, n = 3.

[0073] The term "counterion" refers to an ion that provides charge balance to the fluorescent compounds according to the present disclosure. The counterion may be a cation. The counterion may be an anion. In some cases, the counterion may be a halogen ion, perchlorate ion, PF 6- , phosphate ion, sulfate ion, etc. The counterion may be F - , Cl - , Br - , I - , ClO4 - , CF3CO2 - , CH3CO2 - , PO43- , SO4 2- , BF4 - and the like may also be used. In some cases, the counter ion is Na + , K + , Mg ++ , Ca ++ and the like may also be used.

[0074] For example, the DHP-cyanine dyes or polymer-labeled antibodies according to the present disclosure are used in flow cytometry as reagents that exhibit a fluorescence signal. Furthermore, it may contain an orthogonal "functional group" that can be used for bioconjugation of binding partners or for the binding of acceptor signal transduction chromophores in donor-acceptor tandem dyes.

[0075] The term "organic group", as used herein, refers to any carbon-containing functional moiety. Examples include oxygen-containing groups such as alkoxy groups; aryloxy groups; aralkyloxy groups; oxo (carbonyl) groups; amine groups including alkylamines and amine esters, and sulfonamide groups; carboxyl groups including carboxylic acids, carboxylates, and carboxylic acid esters; sulfur-containing groups such as alkyl and aryl sulfide groups, thiols, thiol-reactive groups, and sulfone groups; maleimide; iodoacetamide; azide groups; alkyne groups; and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, OC(O)N(R)2, CN, CF3, OCF3, R, C(O)R, methylenedioxy, ethylenedioxy, N(R)2, N3, S(H)R, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2) 0~2 N(R)C(O)R, (CH2) 0~2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, C(=NOR)R, and substituted or unsubstituted (C1-C 100 ) hydrocarbyl, where R may be hydrogen (e.g., including other carbon atoms) or a carbon-based moiety, and the carbon-based moiety may or may not be substituted.

[0076] As used herein, the term "heteroatom" refers to any suitable atom other than carbon that is inserted between adjacent carbon atoms in an organic group, such as N, O, S, Se, P, B, Al, Si, and Ge. The organic group may be cyclic, aryl, or a straight-chain or branched-chain group (e.g., alkyl or alkene). More than one heteroatom (e.g., 1, 2, 3, 4, or 5 heteroatoms) may be inserted between adjacent carbon atoms. The heteroatom can be oxidized to form, for example, but not limited to, -S(O)- and S(O)2-, sulfinate, and sulfonamide.

[0077] As used herein in combination with a molecule or organic group as defined herein, the term "substituted" refers to a situation where one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms, such as, for example, alkyl, aryl, or a functional group. A "substituted" group may include one or more groups selected from halogen, hydroxy, amino, alkylamino, amide, acyl, nitro, cyano, and alkoxy.

[0078] The terms "functional group", "functional moiety", or "substituent", as used herein, refer to a group that may be substituted onto or is a substituent of a molecule or organic group. Examples of substituents or functional groups include, but are not limited to, halogen (e.g., F, Cl, Br, and I); alkene; cycloalkene; alkyne; cycloalkyne; atoms in groups such as oxygen atoms in a hydroxyl group, an alkoxy group, an aryloxy group, an aralkyloxy group, an oxo (carbonyl) group, a carboxylic acid, a carboxylate, and a carboxyl group including a carboxylic acid ester; sulfur atoms in groups such as a thiol group, an alkyl and aryl sulfide group, a sulfoxide group, a sulfone group, a sulfonyl group, and a sulfonamide group; nitrogen atoms in groups such as an amine group, a hydroxyamine group, a nitrile group, a nitro group, an N-oxide group, a hydrazide group, an azide group, an imide group, and an enamine group; and other heteroatoms in various other groups. Non-limiting examples of substituents that may be attached to a substituted carbon atom (or other atom) include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azide, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2) 0~2 N(R)C(O)R, (CH2) 0~2 N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R, where R may be hydrogen or a carbon-based moiety; for example, R is hydrogen, (C1-C 100)which may be hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or two R groups attached to a nitrogen atom or adjacent nitrogen atoms may together with one or more nitrogen atoms form a heterocyclyl. The fluorescent compound of interest may contain one or more "functional groups" (also referred to as "conjugation tags") that provide for bioconjugation dyes. In some cases, such functionality may be used to covalently attach a biomolecule or binding partner, such as a protein, peptide, affinity ligand, antibody, antibody fragment, polynucleotide, or aptamer. In some cases, the functional group or conjugation tag may be selected from the group consisting of amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, isothiocyanato, azide, alkyne, cycloalkyne (e.g., alkene, cycloalkene (e.g., cyclooctene), tetrazine, aldehyde, thiol, and protecting groups thereof for conjugating to a substrate, acceptor dye, functional moiety, or binding partner. The functional group may or may not be protected. The functional group may be a reactive or chemoselective functional group that can react with another group via copper-free click chemistry, including strain-promoted azide-alkyne cycloaddition (SPAAC) and inverse electron demand Diels-Alder (iEDDA) reactions, which enable rapid and specific chemical conjugates. See Kim et al., Chem. Sci., 2019, 10, 7835; and Davis et al., J. Org. Chem. 2016, 81, 6816-6819, both of which are hereby incorporated by reference in their entireties.The functional group or conjugation tag may be, for example, a cycloalkene (e.g., cyclooctene); an alkyne; a cycloalkyne (e.g., a cyclooctyne group, e.g., bicyclo[6.1.0]nonyne (BCN)), dibenzocyclooctyne (DBCO)); a cycloalkene (e.g., a cyclooctene group, e.g., trans-cyclooctene (TCO)); an azide group; or a tetrazine group.

[0079] As used herein, the term "activated ester" or "active ester" refers to a carboxyl activating group used in peptide chemistry to facilitate the facile condensation of a carboxyl group with the free amino group of an amino acid derivative, either by itself or as part of another substituent. Descriptions of these carboxyl activating groups can generally be found in textbooks of peptide chemistry, such as K. D. Kopple, "Peptides and Amino Acids", W. A. Benjamin, Inc., New York, 1966, pp. 50-51 and E. Schroder and K. Lubke, "The Peptides"; Vol. 1, Academic Press, New York, 1965, pp. 77-128.

[0080] As used herein, the term "ammonium" refers to a cation having the formula NHR3 + wherein each R group is independently hydrogen, or a substituted or unsubstituted alkyl group, aryl group, aralkyl group, or alkoxy group. Preferably, each R group is hydrogen.

[0081] The term "hydrocarbon" or "hydrocarbyl", as used herein, refers to a molecule or functional group containing carbon and hydrogen atoms. The term may also refer to a molecule or functional group that normally contains both carbon and hydrogen atoms, but in which some or all of the hydrogen atoms are replaced by other functional groups. The term "hydrocarbyl" refers to a functional group derived from a straight-chain, branched, or cyclic hydrocarbon, and may be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. A hydrocarbyl group may be represented as (C a ~C b ), where a and b are integers and mean having any number of carbon atoms from a to b. For example, (C1-C4) hydrocarbyl means that the hydrocarbyl group may be methyl (C1), ethyl (C2), propyl (C3), or butyl (C4), and (C0-C b ) hydrocarbyl means, in certain embodiments, that no hydrocarbyl group is present. A hydrocarbylene group is a diradical hydrocarbon, for example a hydrocarbon that is bonded at two locations.

[0082] As used herein, the term "alkyl" refers to a straight or branched saturated aliphatic radical having the indicated number of carbon atoms, either by itself or as part of another substituent. The alkyl group may be an optionally substituted alkyl group. For example, C1-C6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, decyl, etc. Alkyl may contain any number, for example, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6, and 5-6 carbons. Alkyl may also refer to an alkyl group having up to 20 carbon atoms, for example, but not limited to, heptyl, octyl, nonyl, decyl, etc. The alkyl group may or may not be substituted. Unless otherwise specified, the "substituted alkyl" group may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amide, acyl, nitro, cyano, and alkoxy. The alkyl group is typically monovalent, but may be divalent, for example, when the alkyl group links two moieties together.

[0083] As used herein, the term "alkylene" refers to an alkyl group (i.e., a divalent alkyl radical) as defined above that links at least two other groups. The two moieties linked to the alkylene group may be linked to the same or different carbon atoms of the alkylene group.

[0084] As used herein, the term "alkoxy" refers to an alkyl group as defined above having an oxygen atom connecting the alkyl group to the point of attachment, either by itself or as part of another substituent. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexyloxy, and the like. The alkoxy group may be further substituted with various substituents described within this specification. For example, the alkoxy group can be substituted with a halogen to form a "halo-alkoxy" group.

[0085] As used herein, the term "alkene" or "alkenyl" refers to a straight-chain, branched-chain, or cyclic hydrocarbon having at least one double bond between two carbon atoms, either by itself or as part of another substituent. Examples of alkene groups include, but are not limited to, vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. The alkene group is typically monovalent, but may be divalent, for example, when the alkenyl group connects two moieties together.

[0086] As used herein, the term "alkyne" or "alkynyl" refers to a straight-chain or branched hydrocarbon having at least one triple bond between two carbon atoms, either by itself or as part of another substituent. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatriynyl. An alkynyl group is typically monovalent, but may be divalent, for example, when two alkynyl groups link two moieties together.

[0087] As used herein, the term "acyl" refers to a group containing a carbonyl moiety and attached through the carbonyl carbon atom. The carbonyl carbon atom is bonded to hydrogen to form a "formyl" group, or to another carbon atom which may be part of, for example, an alkyl group, an aryl group, an aralkylcycloalkyl group, a cycloalkylalkyl group, a heterocyclyl group, a heterocyclylalkyl group, a heteroaryl group, a heteroarylalkyl group, etc. The acyl group may contain from 0 to about 12, from 0 to about 20, or from 0 to about 40 additional carbon atoms bonded to the carbonyl group. The acyl group may contain a double bond or a triple bond within the meaning herein. The acyl group may also contain heteroatoms as needed within the meaning herein. Examples of acyl groups include, but are not limited to, nicotinoyl group (pyridyl-3-carbonyl), acetyl group, benzoyl group, phenylacetyl group, pyridylacetyl group, cinnamoyl group, and acryloyl group. When the group containing a carbon atom bonded to the carbonyl carbon atom contains a halogen, the group is called a "haloacyl" group. An example is the trifluoroacetyl group.

[0088] As used herein, the term "aldehyde" refers to a chemical compound having a -CHO group, either by itself or as part of another substituent.

[0089] As used herein, the term "aryl" refers to a cyclic aromatic hydrocarbon group that does not contain heteroatoms in the aromatic ring assembly, either by itself or as part of another substituent. An "aryl" group may be a monocyclic or fused bicyclic, tricyclic, tetracyclic, pentacyclic or higher cyclic aromatic ring assembly containing 6 to 22, 14 to 22, 17 to 22, or 6 to 16 ring carbon atoms. For example, aryl may be, but is not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylene, anthracenyl, benzyl, or naphthyl, etc. An aryl group may include a substituted aryl group. Examples of substituted aryl groups include, but are not limited to, alkoxy, phenyl, halogen, alkyl or trifluoromethyl, hydroxyl, C1-C 12 alkyl, C2-C 12 alkene, C2-C 12 alkyne, C3-C 12 cycloalkyl, C1-C 12 haloalkyl, C1-C 12 alkoxy, C2-C 18 (hetero)aryloxy, C2-C 18 (hetero)arylamino, carboxylate, carboxylic acid, C2-C 12 alkylcarboxylic acid, C2-C 12 alkylcarboxylate, C2-C 12 alkylcarboxylic acid ester, C1-C 12 alkoxy, water-soluble group (WSG), functional group, sulfonic acid, sulfonate, C1-C 12There is naphthyl or phenyl optionally mono- or di-substituted by an alkyl sulfonate. In some cases, the substituted aryl group, such as naphthyl or phenyl, may be mono- or di-substituted by a functional group, a WSG, a WSG optionally containing a functional group, an alkoxy, a halogen or a trifluoromethyl. The WSG may be a branched WSG optionally containing a functional group, such as PEG and a WSG containing a functional group.

[0090] Preferred as aryl is naphthyl, phenyl or phenyl mono- or di-substituted by alkoxy, phenyl, halogen, alkyl or trifluoromethyl, especially phenyl or phenyl mono- or di-substituted by alkoxy, halogen or trifluoromethyl, especially phenyl.

[0091] The term "monocyclic heteroaryl" refers to an unsubstituted or substituted heteroaryl ring system containing one aryl ring and a monocyclic ring system containing one or more, two or more, three or more, or four or more heteroatoms. Non-limiting examples of monocyclic heteroaryl groups are substituted or unsubstituted pyridinyl, pyranyl, thiophenyl, furanyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl.

[0092] The term "polycyclic aryl" refers to an unsubstituted or substituted polycyclic ring system containing from 2 to 9, from 2 to 8, or from 2 to 6 aryl rings, with or without condensed cycloalkyl or cycloalkenyl rings. Examples of polycyclic aryl groups can be fluorene, 9H-fluorene, phenanthrene, dihydrophenanthrene, 9,10-dihydrophenanthrene, naphthalene, anthracene, tetracene, pentacene, etc.

[0093] The term "polycyclic heteroaryl" refers to an unsubstituted or substituted polycyclic ring system containing 2 to 9, 2 to 8, or 2 to 6 aryl rings, with or without a fused cycloalkyl or cycloalkenyl ring, and containing one or more, two or more, three or more, or four or more heteroatoms. Non-limiting examples of polycyclic heteroaryl systems include quinoline, benzoxazole, benzothiazole, benzimidazole, indole, benzindole, pyridinium, benzopyrylium, thiopyrylium, 6,8-dihydro-5H-naphtho[2,1-f]indole, 4,5-dihydro-3H-naphtho[2,1-e]indole, 6,7-dihydro-3H-naphtho[2,1-g]indole, 5,6-dihydrophenanthro[3,2-d]thiazole, 4,5-dihydrophenanthro[2,1-d]thiazole, 6,7-dihydrophenanthro[4,3-d]thiazole, 5,6-dihydrophenanthro[3,2-d]oxazole, 4,5-dihydrophenanthro[2,1-d]oxazole, 6,7-dihydrophenanthro[4,3-d]oxazole, 5,6-dihydronaphtho[2,1-g]quinoline, 7,8-dihydronaphtho[2,1-h]quinoline, 5,6-dihydronaphtho[2,1-f]quinoline, 5,6-dihydro-8λ2-phenanthro[3,2-d]imidazole, 4,5-dihydro-3λ2-phenanthro[2,1-d]imidazole, 6,7-dihydro-3λ2-phenanthro[4,3-d]imidazole, 5,6-dihydronaphtho[1,2-g]quinoxaline, 5,6-dihydronaphtho[2,1-f]quinoxaline, 7,8-dihydronaphtho[1,2-f]quinoxaline, 5,6-dihydropentapheno[3,2,1-cd:10,11,12-c’d’]diindole, 3,8-dihydrophenanthro[2,3-e:7,6-e’]diindole, 3,5,6,8-tetrahydrophenanthro[2,3-e:7,6-e’]diindole, 1,2,3,5,6,8-hexahydrophenanthro[2,3-e:7,6-e’]diindole-1,10-dium salt, 3,5,6,8-tetrahydrophenanthro[2,3-e:7,6-e’]diindole--ethane (1 / 1), 5,6-Dihydropentapheno[3,4-d:10,9-d']bis(oxazole), 1,2,5,6,9,10-hexahydropentapheno[3,4-d:10,9-d']bis(thiazole)-1,10-dium, pentapheno[3,4-d:10,9-d']bis(thiazole), 1,2,9,10-tetrahydropentapheno[3,4-d:10,9-d']bis(thiazole)-1,10-dium, 6,7-dihydrophenanthro[2,3-f:7,6-f']diquinoline-1,12-dium, 6,7-dihydrophenanthro[2,3-f:7,6-f']diquinoline, 2,6,7,11-tetrahydrobenzo[1,2-g:4,3-g']dichromene, 2,11-dihydrobenzo[1,2-g:4,3-g']dichromene, 5,10-dihydro-6H-naphtho[2,1-g]chromene, 10H-naphtho[2,1-g]chromene, 2,6,7,11-tetrahydrobenzo[1,2-g:4,3-g']bis(thiochromene), 2,11-dihydrobenzo[1,2-g:4,3-g']bis(thiochromene) and the like may be present.,

[0094] The term "arylene" refers to a divalent radical derived from an aryl group. The aryl group may be mono-substituted, di-substituted or tri-substituted by one, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy and oxy-C2-C3-alkylene; all of these may in turn be further substituted as necessary, for example as defined above in the present specification; or may be 1- or 2-naphthyl; or 1- or 2-phenanthrenyl. Alkylenedioxy is a divalent substitution bonded to two adjacent carbon atoms of phenyl, for example, methylenedioxy or ethylenedioxy. Oxy-C2-C3-alkylene is also a divalent substituent bonded to two adjacent carbon atoms of phenyl, for example, oxyethylene or oxypropylene. An example of oxy-C2-C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl. The polycyclic heteroaryl may or may not be substituted.,

[0095] As used herein, the term "aryloxy" refers to an O-aryl group where the aryl, by itself or as part of another substituent, is as defined above. The aryloxy group may or may not be substituted with one or two suitable substituents. The term "phenoxy" refers to an aryloxy group where the aryl moiety is a phenyl ring. The term "(hetero)aryloxy", as used herein, means an -O-heteroaryl group where the heteroaryl is as defined below. The term "(hetero)aryloxy" is used to indicate that the moiety is either an aryloxy or a (hetero)aryloxy group.

[0096] As used herein, the term "aralkyl" refers to an alkyl group as defined herein where a hydrogen or carbon bond of the alkyl group is replaced by a bond to an aryl group as defined herein. Representative aralkyl groups include the benzyl group, the phenylethyl group, and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. An aralkenyl group is an alkenyl group as defined herein where a hydrogen or carbon bond of the alkyl group is replaced by a bond to an aryl group as defined herein.

[0097] As used herein, the term "amine", when used by itself or as part of another substituent, refers to an alkyl group as defined herein that has one or more amino groups. The amino group may be primary, secondary or tertiary. The alkylamine may be further substituted with a hydroxy group. Amines useful in the present disclosure include, but are not limited to, ethylamine, propylamine, isopropylamine, ethylenediamine, and ethanolamine. The amino group may be linked to the point of attachment of the alkylamine to the remainder of the compound, present at the omega position of the alkyl group, or link together at least two carbon atoms of the alkyl group. One of ordinary skill in the art will understand that other alkylamines are useful in the present disclosure.

[0098] As used herein, the term "amino group" refers to -NH2, -NHR, -NR2, -NR3, in each protonated form except where each R is independently selected and cannot be protonated -NR3 + substituents of the form. Thus, any compound substituted with an amino group can be considered an amine. The "amino group" may be a primary, secondary, tertiary, or quaternary amino group, as defined herein. The "alkylamino" group may include a monoalkylamino group, a dialkylamino group, or a trialkylamino group. +

[0099] The term "amide" refers to a functional group having a carbonyl group bonded to an amine group and having the general formula RC(=O)NR’R’’, where R, R’, and R’’ represent an organic group or a hydrogen atom. The term "amide" refers to a substituent containing an amide group.

[0100] ​As used herein, the term "carbamate" refers to a functional group having the structure -NR’’CO2R’ by itself or as part of another substituent, where R’ and R’’ are independently selected from hydrogen, (C1-C8) alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4) alkyl, and (unsubstituted aryl)oxy-(C1-C4) alkyl. Examples of carbamates include t-Boc, Fmoc, benzyloxy-carbonyl, alloc, methyl carbamate, ethyl carbamate, 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, Tbfmoc, Climoc, Bimoc, DBD-Tmoc, Bsmoc, Troc, Teoc, 2-phenylethyl carbamate, Adpoc, 2-chloroethyl carbamate, 1,1-dimethyl-2-haloethyl carbamate, DB-t-BOC, TCBOC, Bpoc, t-Bumeoc, Pyoc, Bnpeoc, V-(2-pivaloylamino)-1,1-dimethylethyl carbamate, NpSSPeoc.

[0101] As used herein, the term "carboxylic acid" refers to a structure R-COOH by itself or as part of another substituent, where R is a group of carbon-containing atoms.

[0102] As used herein, the term "carboxylate" refers to the general formula RCOO by itself or as part of another substituent -Refers to the conjugate base of a carboxylic acid that can be represented by. For example, the term "magnesium carboxylate" refers to the magnesium salt of a carboxylic acid. The term "carboxylic acid ester", as used herein, refers to a compound derived from a carboxylic acid that can generally be represented by the formula RCOOR', either by itself or as part of another substituent, where R' can be alkyl, alkene, alkyne, haloalkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, (unsubstituted aryl)alkyl, and (unsubstituted aryl)oxy-alkyl or other carbon-containing atomic groups. R' may optionally contain a functional group.

[0103] As used herein, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic, fused bicyclic or bridged polycyclic ring assembly that contains from 3 to 12 ring atoms or the number of atoms indicated, either by itself or as part of another substituent. Cycloalkyl can have any number of carbons, e.g., C 3~6 、C 4~6 、C 5~6 、C 3~8 、C 4~8 、C 5~8 、C 6~8 、C 3~9 、C 3~10 、C 3~11 、and C 3~12It may contain. Examples of the saturated monocyclic cycloalkyl ring include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Examples of the saturated bicyclic and polycyclic cycloalkyl rings include norbornane, [2.2.2]bicyclooctane, decahydronaphthalene, and adamantane. The cycloalkyl group may also be partially unsaturated having one or more double bonds or triple bonds in the ring. Representative partially unsaturated cycloalkyl groups include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene, and norbornadiene. When cycloalkyl is saturated monocyclic C 3~8 When it is cycloalkyl, exemplary groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. When cycloalkyl is saturated monocyclic C 3~6 When it is cycloalkyl, exemplary groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The cycloalkyl group may or may not be substituted. Unless otherwise specified, the "substituted cycloalkyl" group may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amide, acyl, nitro, cyano, and alkoxy. The term "lower cycloalkyl" refers to a cycloalkyl radical having 3 to 7 carbons and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Examples of the monocyclic ring include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Examples of the bicyclic and polycyclic rings include norbornane, decahydronaphthalene, and adamantane. For example, C 3~8Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane. The polycyclic ring system may or may not be substituted.

[0104] As used herein, the term "cycloalkylene" refers to a cycloalkyl group as defined above that links at least two other groups (i.e., a divalent cycloalkyl radical). The two moieties linked to the cycloalkylene group may be linked to the same atom or different atoms of the cycloalkylene group.

[0105] As used herein, the term "haloalkyl" refers to an alkyl as defined above, wherein some or all of the hydrogen atoms are replaced by halogen atoms, either by itself or as part of another substituent. Halogen (halo) preferably represents chloro or fluoro, but may also be bromo or iodo. For example, examples of haloalkyl include trifluoromethyl, fluoromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term "perfluoro" defines a compound or radical having at least two available hydrogens replaced by fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethane refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy.

[0106] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, and iodine, either by itself or as part of another substituent.

[0107] As used herein, the term "heteroaryl", by itself or as part of another substituent, refers to a monocyclic or fused polycyclic, e.g., bicyclic, tricyclic, tetracyclic, or pentacyclic aromatic ring assembly containing, e.g., from 5 to 22, 14 to 22, 17 to 22, 6 to 16, or 5 to 16 ring atoms, wherein from 1 to 4 of the ring atoms may be heteroatoms, e.g., N, O, or S. Additional heteroatoms including, but not limited to, B, Al, Si, or P may also be useful. Heteroaryl may or may not be substituted. Substituted heteroaryl has one or more K-R 13 , halogen, O-C 1~6 alkyl, S-C 1~6 alkyl, O-aryl, S-aryl, NHC 1~6 alkyl, Ph-NCS, Ph-CO2H, Ph-(CH2) 1~4It may contain a CO2H substituent. The heteroatom can be oxidized to form, in part, for example, but not limited to, -S(O)- and -S(O)2-. For example, heteroaryl may include pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radical, especially monosubstituted or disubstituted, substituted by, for example, alkyl, nitro or halogen. Pyridyl may represent 2-, 3- or 4-pyridyl, preferably 2- or 3-pyridyl. Thienyl may represent 2- or 3-thienyl. Quinolinyl may preferably represent 2-, 3- or 4-quinolinyl. Isoquinolinyl may preferably represent 1-, 3- or 4-isoquinolinyl. Benzopyranyl and benzothiopyranyl each preferably represent 3-benzopyranyl or 3-benzothiopyranyl. Thiazolyl may preferably represent 2- or 4-thiazolyl, most preferably 4-thiazolyl. Triazolyl may preferably represent 1-, 2- or 5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl. Heteroaryl may contain an aryloxy or arylamino group. In some embodiments, heteroaryl is any of pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or a substituted, especially monosubstituted or disubstituted radical.

[0108] As used herein, the terms "heteroalkyl" or "heteroalkoxy", by themselves or as part of another substituent, refer to an alkyl or alkoxy group, preferably a C1-C 12 alkyl group or a C1-C 12“alkoxy group” refers to, and C is substituted by a heteroatom, for example, N, O or S. For example, heteroalkyl or heteroalkoxy may include ethers, thioethers and alkyl-amines. Without being limited thereto, additional heteroatoms including B, Al, Si, or P may also be useful. The heteroatom can be oxidized to form, in part, for example, but not limited to, -S(O)-, -S(O)2-, sulfinates, sulfonamides. The heteroatom moiety of heteroalkyl can replace the hydrogen atom of the alkyl group to form a hydroxy, thio, or amino group. Alternatively, the heteroatom moiety may be a connecting atom or inserted between two carbon atoms.

[0109] As used herein, the term “heteroalkylene” refers to a heteroalkyl group as defined above (i.e., a divalent heteroalkyl radical) that links at least two other groups. The two moieties linked to the heteroalkylene group may be linked to the same atom or different atoms of the heteroalkylene group.

[0110] As used herein, the term “(hetero)aryl amino” refers to an amine radical (e.g., -NH-aryl) substituted by an aryl group, either by itself or as part of another substituent. Aryl amino may also be an aryl radical substituted by an amine group (e.g., -aryl-NH2). Aryl amino may or may not be substituted.

[0111] In some embodiments, the substituents on the aryl, heteroaryl, and heteroalkylene groups are diverse and are selected from 0 to the total number of open valences on the aromatic ring system from - halogen, - OR’, - OC(O)R’, - C(O)R’, - NR’R’’, - SR’, - R’, - CN, - NO2, - CO2R’, - CONR’R’’, - C(O)R’, - OC(O)NR’R’’, - NR’’C(O)R’, - NR’’C(O)2R’, - NR’ - C(O)NR’’R’’’, - NH - C(NH2)=NH, - NR’C(NH2)=NH, - NH - C(NH2)=NR’, - S(O)R’, - S(O)2R’, - S(O)2NR’R’’, - N3, - CH(Ph)2, perfluoro(C1 - C4)alkoxy, and perfluoro(C1 - C4)alkyl; R’, R’’ and R’’’ are independently selected from hydrogen, (C1 - C5)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1 - C4)alkyl, and (unsubstituted aryl)oxy-(C1 - C4)alkyl.

[0112] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by substituents of the formula -T - C(O)-(CH2) q -U-, where T and U are independently -NH-, -O-, -CH2- or a single bond, and q is an integer from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by substituents of the formula -A-(CH2) r -B-, where A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)2-, -S(O)2NR’- or a single bond, and r is an integer from 1 to 3. One of the single bonds of the newly formed ring may optionally be replaced by a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be of the formula -(CH2) s -X’-(CH2) t- may be optionally replaced by a substituent, s and t are independently integers from 0 to 3, and X’ is -O-, -NR’-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR’-. The substituent R’ in -NR’- and -S(O)2NR’- is selected from hydrogen or unsubstituted (C1-C6) alkyl.

[0113] As used herein, the term "oligoether" is understood to mean an oligomer containing a structural repeating unit having an ether functionality. As used herein, "oligomer" is understood to mean a molecule containing one or more identifiable structural repeating units of the same or different formulas.

[0114] As used herein, the term "polyethylene glycol", "PEG", "polyethylene oxide" or "PEO" refers to the formula -(CH2-CH2-O-) n-refers to a family of ethylene glycol monomer unit-based biocompatible water-soluble linear polymers or derivatives 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. The PEG polymer group can be of any convenient length and includes, but is not limited to, various end groups and / or additional substituents including alkyl, alkoxy, aryl, hydroxyl, amino, acyl, carboxylic acid, carboxylic acid ester, acyloxy, and amide termini and / or substituents. As used herein, PEG groups include, but are not limited to, PEG, modified PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramidate-PEG, alkylsulfonamide-PEG, and alkoxysulfonamide-PEG. The PEG polymer moiety can be of any convenient length and includes, but is not limited to, various end groups and / or additional substituents including alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amide termini and / or substituents. PEG groups that may be adapted for use with the subject compounds include S. Zalipsky, "Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates", Bioconjugate Chemistry 1995, 6 (2), 150-165; Zhu et al, "Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy", Chem. Rev., 2012, 112 (8), pp 4687-4735;There are PEGs described by J.M. Harris, "Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications", Plenum Press, New York, N.Y. (1992); and J.M. Harris and S Zalipsky, "Poly(ethylene glycol) Chemistry and Biological Applications", ACS (1997). In some cases, PEG and modified PEG moieties are described, for example, in International Patent Applications: WO90 / 13540, WO92 / 00748, WO92 / 16555, WO94 / 04193, WO94 / 14758, WO94 / 17039, WO94 / 18247, WO94 / 28937, WO95 / 11924, WO96 / 00080, WO96 / 23794, WO98 / 07713, WO98 / 41562, WO98 / 48837, WO99 / 30727, WO99 / 32134, WO99 / 33483, WO99 / 53951, WO01 / 26692, WO95 / 13312, WO96 / 21469, WO97 / 03106, WO99 / 45964, U.S. Patent 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; 5,349,001; 5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662; 5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110; 5,730,990; 5,739,208;It may also be what is taught in the following: U.S. Patent Nos. 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,442; 5,919,455; 5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011,042; 6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; 6,214,966, and each of these applications is hereby incorporated by reference in its entirety).;

[0115] As used herein, the terms "sulfonate functionality" or "sulfonate" refer to both the free sulfonate anion (-S(=O)2O-) and its salts, either by themselves or as part of another substituent. Thus, the term "sulfonate" includes sulfonates such as sodium sulfonate, lithium sulfonate, potassium sulfonate, and ammonium sulfonate.

[0116] As used herein, the term "sulfonamide" refers to a group of the formula -SO2NR2, either by itself or as part of another substituent, where each R is independently, for example, a solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or a functional group, and may contain a carboxylic acid group. R may be, but is not limited to, a polymer containing 6 or more monomer units, a nonionic water-soluble polymer, such as PEG, or a water-soluble polymer containing a modified PEG terminated with a carboxylic acid, carboxylic acid ester, or alkoxy group (e.g., O-methyl or O-ethyl). The "sulfonamide" is attached to another molecule by a linker or bond. The "sulfonamide" may be, for example, sulfonamide-PEG, alkylsulfonamide, alkoxysulfonamide, alkylsulfonamide PEG, alkoxysulfonamide PEG, alkylsulfonamide PEG carboxylate, alkoxysulfonamide PEG carboxylate.

[0117] As used herein, the term "sulfonamide" refers to a group of the formula -SO2NR-, either by itself or as part of another substituent, where R may be, for example, a solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or a functional group, and may contain a carboxylic acid group. R may be, but is not limited to, a polymer containing 6 or more monomer units, a nonionic water-soluble polymer, such as PEG, or a water-soluble polymer containing a modified PEG terminated with a carboxylic acid or carboxylic acid ester. The "sulfonamide" may be attached to another molecule by a linker or bond. The "sulfonamide" may be, for example, sulfonamide-PEG, alkylsulfonamide, alkoxysulfonamide, alkylsulfonamide PEG, alkoxysulfonamide PEG, alkylsulfonamide PEG carboxylate, alkoxysulfonamide PEG carboxylate.

[0118] As used herein, the term "sulfinamide" refers to a group of the formula -SONR2, either by itself or as part of another substituent, where each R is independently, for example, a solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or a functional group, and may contain a carboxylic acid group. R may be, but is not limited to, a polymer containing 6 or more monomer units, a nonionic water-soluble polymer such as PEG, or a water-soluble polymer containing a modified PEG terminated with a carboxylic acid or carboxylic acid ester. "Sulfinamide" may be attached to another molecule by a linker or bond. "Sulfinamide" may be, for example, sulfinamide-PEG, alkylsulfinamide, alkoxysulfinamide, alkylsulfinamide PEG, alkoxysulfinamide PEG, alkylsulfinamide PEG carboxylate, alkoxysulfinamide PEG carboxylate.

[0119] As used herein, the terms "hydrazine" and "hydrazide" refer to a compound containing a singly-bonded nitrogen, either by itself or as part of another substituent, one of which is a primary amine functional group. For example, the term "hydrazine" refers to a moiety having the structure -NHNH2.

[0120] As used herein, the term "thiol" refers to a compound containing a functional group composed of a sulfur-hydrogen bond, either by itself or as part of another substituent. The general chemical structure of a thiol functional group is R-SH, where R represents alkyl, alkene, aryl, or other carbon-containing atomic groups.

[0121] As used herein, the term "silyl" refers to Si(R z )3, either by itself or as part of another substituent, where each R z is independently alkyl, aryl, or other carbon-containing atomic groups.

[0122] As used herein, the term "diazonium salt" refers to a group of an organic compound having the structure R-N2 + X’ - by itself or as part of another substituent, where R may be any organic group (e.g., alkyl or aryl), and X’ is an inorganic or organic anion (e.g., halogen).

[0123] As used herein, the term "triflate" refers to a group having the formula CF3SO3, also known as trifluoromethanesulfonate, by itself or as part of another substituent.

[0124] As used herein, the term "boronic acid" refers to the structure -B(OH)2 by itself or as part of another substituent. It is recognized by those skilled in the art that boronic acids can exist as boronate esters at various stages in the synthesis of the quencher. Boronic acid is meant to include such esters. The terms "boronic acid ester" or "boronate ester" as used herein refer to a chemical compound containing the -B(Z 1 )(Z 2 ) moiety, where Z 1 and Z 2 together form a moiety where the atoms bonded to boron are oxygen atoms in each case. In some embodiments, the boronic acid ester moiety is a 5-membered ring. In some other embodiments, the boronic acid ester moiety is a 6-membered ring. In some other embodiments, the boronic acid ester moiety is a mixture of 5-membered and 6-membered rings.

[0125] As used herein, the term "maleimide" refers to the structure

Chemical Formula

[0126] As used herein, the term "hydrazone" refers to the structure

Chemical Structure

[0127] As used herein, the term "azide" refers to the structure -N3, either by itself or as part of another substituent.

[0128] As used herein, the term "tetrazine" refers to a compound having the molecular formula C2H2N4 consisting of a 6-membered heteroaromatic ring structure with 4 nitrogen atoms, either by itself or as part of another substituent. The term "tetrazine" includes all of its isomers, namely, 1,2,3,4-tetrazine, 1,2,3,5-tetrazine, and 1,2,4,5-tetrazine.

[0129] As used herein, the term "N-hydroxysuccinimidyl" refers to the structure

Chemical Structure

[0130] As used herein, the term "phosphoramidate" refers, by itself or as part of another substituent, to the structure

Chemical formula

[0131] As used herein, the term "phosphonamidate" refers, by itself or as part of another substituent, to the structure

Chemical formula

[0132] As used herein, the term "phosphinamido" refers to the structure [Chemical Formula] refers to, and R may be, for example, a water-solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other group, and may contain a carboxylic acid group. R may be, but is not limited to, a polymer containing 6 or more monomer units, a nonionic water-soluble polymer, PEG, or a water-soluble polymer terminated with a carboxylic acid or carboxylic acid ester-modified PEG. "Phosphinamido" may be attached to another molecule by a linker or bond. "Phosphinamido" may be, for example, phosphinamido-PEG, alkylphosphinamido, alkoxyphosphinamido, alkylphosphinamido PEG, alkoxyphosphinamido PEG, alkylphosphinamido PEG carboxylate, alkoxyphosphinamido PEG carboxylate.

[0133] As used herein, the term "fluorescence" refers to a compound that emits light (typically) at a different wavelength when irradiated with light of a wavelength absorbed by the compound. Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. In most cases, the emitted light has a longer wavelength than the absorbed light.

[0134] The term "absorbance maximum" or "Abs λ max" or "max λ abs" refers to the wavelength of the maximum absorbance measured by UV Vis spectroscopy.

[0135] The term "excitation wavelength" or "λ ex" refers to a wavelength that can excite a compound to induce fluorescence emission, which is not necessarily the Abs λ max.

[0136] The term "chromophore" refers to a molecular entity or a part thereof that consists of an atom or group of atoms in which the electronic transition responsible for a given spectral band is substantially localized. In some cases, a "chromophore" may itself fluoresce. As used herein, the terms "fluorescent chromophore" and "fluorescent dye" are used interchangeably and refer to a compound having a structure that can collect light having a specific absorption maximum wavelength and convert it into light emitted at a longer emission maximum wavelength. A chromophore may have reactive groups (e.g., carboxylate moieties, amino moieties, haloalkyl moieties, etc.) that can form covalent bonds. Examples of suitable chromophores include, but are not limited to, those described in U.S. Patent Nos. 7,687,282; 7,671,214; 7,446,202; 6,972,326; 6,716,979; 6,579,718; 6,562,632; 6,399,392; 6,316,267; 6,162,931; 6,130,101; 6,005,113; 6,004,536; 5,863,753; 5,846,737; 5,798,276; 5,723,218; 5,696,157; 5,658,751; 5,656,449; 5,582,977; 5,576,424; 5,573,909; and 5,187,288, the entire disclosures of which are incorporated herein by reference.

[0137] The term "moiety" refers to a group as part of a molecule that can be a functional group or a part of a molecule that includes a plurality of groups sharing common structural and / or functional aspects. Examples of groups or moieties include, but are not limited to, linker moieties, functional groups, solubilizing moieties, PEG moieties according to the present disclosure.

[0138] The terms "linker", "linked" or "linking" refer to a connecting moiety that connects two groups and has a backbone of 100 atoms or less in length. The linker or linkage may be a covalent bond that connects two groups or a chain of 1 to 100 atoms in length, e.g., a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms, and the linker may be linear, branched, cyclic or a single atom. In some embodiments, the linker is a branched linker that refers to a connecting moiety that connects three or more groups. In some embodiments, the linker backbone includes a linking functional group, e.g., ether, thioether, amino, amide, carbonyl, acyl, sulfonamide, sulfinamide, disulfonamide, disulfinamide, sultam, amide, secondary amine, phosphonamide, phosphinamid, phosphonamidate, selenonamide, seleninamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester or imine. In some embodiments, the linker backbone includes a linking functional group, e.g., amino, amide, carbonyl, sulfonamide, sulfinamide, disulfonamide, disulfinamide, sultam, amide, secondary amine, phosphonamide, phosphinamid, phosphonamidate, selenonamide or seleninamide. In certain cases, one, two, three, four or five or more carbon atoms of the linker backbone may optionally be substituted with sulfur, nitrogen or oxygen heteroatoms. In some embodiments, the linker backbone includes a linking functional group, e.g., ether, thioether, amino, amide, carbonyl, acyl, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester or imine. The bonds between the backbone atoms may be saturated or unsaturated, and in some cases one, two, or three or fewer unsaturated bonds are present in the linker backbone. The linker may optionally include one or more substituents having, e.g., an alkyl group, an aryl group or an alkenyl group.Examples of linkers include, but are not limited to, polyethylene glycol, ether, thioether, tertiary amine, and alkyl, which may be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), etc. The linker backbone may contain a cyclic group of two or more atoms, for example, a cyclic group containing two, three, or four atoms in the backbone, such as an aryl group, a heterocyclic group, or a cycloalkyl group. The linker may or may not be cleavable.

[0139] The linker moiety may be attached to "L" or "A" as taught in U.S. Patent No. 11,584,825B2, which is hereby incorporated by reference in its entirety. The linker moiety may contain a covalent bond, alkoxy, sulfonamide, disulfonamide, selenonamide, sulfinamide, sultam, disulfinamide, amide, carbonyl, seleninamide, phosphonamide, phosphinamide, phosphonamidate, or secondary amine.

[0140] As described therein, when each is related to the linker moiety, the term "sulfonamide" refers to the -S(O)2NR- moiety; the term "disulfonamide" refers to the -S(O)2NRS(O)2- moiety; the term "selenonamide" refers to the -Se(O)2NR- moiety; the term "sulfinamide" refers to the -S(O)NR2 moiety; the term "disulfinamide" refers to the -S(O)NRS(O)- moiety; the term "seleninamide" refers to the -Se(O)NR- moiety; the term "phosphonamide" refers to the -NR-PR(O)NR- moiety; the term "phosphinamide" refers to the -PR(O)NR- moiety; the term "phosphonamidate" refers to the -O-PR(O)NR- moiety; the term "sultam" refers to a cyclic sulfonamide (e.g., the R group is attached to the sulfur atom via an alkylene moiety); for each term, the R group is independently H, alkyl, haloalkyl, or aryl.

[0141] The water-soluble fluorescent polymers of interest feature terminals on the conjugated polymer chain that can include functional groups that provide for bioconjugation. In some cases, such functionalities are referred to as terminal linkers or terminal groups. These terminal linkers can be used to form covalent bonds for binding biomolecules, such as proteins, peptides, affinity ligands, antibodies, antibody fragments, polynucleotides, or aptamers. For example, polymer dye-labeled antibodies are used in flow cytometry as reagents that exhibit high brightness. Furthermore, orthogonal functional groups can be introduced along the conjugated polymer chain and can be used for either bioconjugation or binding of acceptor signaling chromophores in donor-acceptor polymer tandem dyes.

[0142] The phrase "conjugated water-soluble fluorescent polymer" refers to a water-soluble fluorescent polymer having a binding partner conjugated to it.

[0143] In the chemical structure, " [ka] " represents either a single bond or a double bond.

[0144] In the chemical structure, " [ka] " represents an optional aryl group.

[0145] The term "binding partner" or "binding member" refers to any molecule or molecular complex that can specifically bind to a target analyte. Binding partners of the present disclosure include, for example, proteins (such as antibodies or antibody fragments), carbohydrates (such as polysaccharides), oligonucleotides, polynucleotides, lipids, affinity ligands, aptamers, and the like. In some embodiments, the binding partner is an antibody or a fragment thereof. Specific binding in the context of the present disclosure refers to a binding reaction that determines the presence of a target analyte in the presence of a heterogeneous population. Thus, under certain assay conditions, a defined binding partner preferentially binds to a specific protein or an isoform of a specific protein and does not bind in significant amounts to other proteins or other isoforms present in the sample.

[0146] In some cases, antibodies include intravenous immunoglobulin (IVIG) and / or antibodies from IVIG (such as enriched from IVIG, purified from IVIG, for example, affinity purified from IVIG). IVIG is a blood product containing IgG (immunoglobulin G) pooled from plasma (in some cases, containing no other proteins at all) from many (for example, sometimes more than 1,000 to 60,000) normal and healthy blood donors. IVIG is commercially available. Aspects of IVIG are described, for example, in U.S. Patent Application Publication Nos. 2010 / 0150942; 2004 / 0101909; 2013 / 0177574; 2013 / 0108619; and 2013 / 0011388.

[0147] In some cases, the antibody is a monoclonal antibody of a defined subclass (such as IgG1, IgG2, IgG3, or IgG4). When a combination of antibodies is used, the antibodies may be from the same subclass or from different subclasses. For example, the antibody may be an IgG1 antibody. In some embodiments, the monoclonal antibody is humanized.

[0148] The phrase "water-soluble fluorescent polymer conjugate" refers to the water-soluble fluorescent polymer of the present disclosure conjugated with a binding partner.

[0149] The phrase "protected group" (also called "protecting group") refers to a reversibly formed derivative of a functional group present in a molecule in which the reactivity is decreased and as a result the protected functional group is attached so that it does not react under the synthetic conditions to which the molecule is subjected. Examples of amine protecting groups include, but are not limited to, benzyloxycarbonyl; 9-fluorenylmethyloxycarbonyl (Fmoc); tert-butyloxycarbonyl (Boc); allyloxycarbonyl (Alloc); p-toluenesulfonyl (Tos); 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc); 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf); mesityl-2-sulfonyl (Mts); 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr); acetamide; phthalimide, and the like. These and other protecting groups for amines, carboxylic acids, alcohols, and further functional groups can be added to and removed from the polymers of the present disclosure using known techniques such as those described in, for example, Green and Wuts (Protective Groups in Organic Synthesis, 4 th Ed. 2007, Wiley-Interscience, New York).

[0150] The term "sample" refers to a material or mixture of materials, in some cases in liquid form, containing one or more analytes of interest. In some embodiments, the term, when used in its broad sense, refers to any plant, animal or bacterial material that contains cells or produces cell metabolites, such as tissue or body fluid (including but not limited to plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue fragments) isolated from an individual or from in vitro cell culture components, as well as samples from the environment. The term "sample" may also refer to a "biological sample". As used herein, the term "biological sample" refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids including but not limited to blood, mucus, lymph fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal discharge and semen). A "biological sample" may also refer to a whole organism or a subset of its tissues, cells or component parts, including but not limited to plasma, serum, spinal fluid, lymph fluid, outer fragments of skin, respiratory, intestinal, and urogenital tracts, tears, saliva, milk, blood cells, tumors and organs, or a homogenate, lysate or extract prepared from a fraction or part thereof. In certain embodiments, the sample is taken from an animal or a plant. A biological sample may contain cells. The term "cell" is used in its conventional sense to refer to the basic structural unit of both eukaryotic and prokaryotic organisms having at least a nucleus and a cell membrane. In certain embodiments, the cells include those from prokaryotic cells, such as bacteria. In other embodiments, the cells include eukaryotic cells, such as cells obtained from biological samples from animals, plants or fungi.

[0151] The term "substrate" refers to solid materials having various spatial arrangements. The substrate may be, for example, a sheet, beads, or other structures, such as a plate with wells, a polymer, a particle, a semiconductor surface, a nanotube, a fiber mesh, a hydrogel, a porous matrix, a pin, a microarray surface, a chromatography support, and the like. In some cases, the substrate is selected from the group consisting of a particle, a planar solid substrate, a fiber mesh, a hydrogel, a porous matrix, a pin, a microarray surface, and a chromatography support.

[0152] The term "water", as used herein, mainly refers to water and refers to any aqueous solution that conforms to physiological conditions. In some cases, the aqueous solution contains more than 50% water, for example, more than 60% water, more than 70% water, more than 80% water, more than 90% water, or more than 95% water. The term "water" includes, for example, biological buffers and other aqueous solutions that may contain additives, such as salts, detergents, stabilizers, and other water-soluble components, such as sugars, proteins, amino acids, and nucleotides. In some cases, "water" may be an aqueous solution containing up to 10% miscible organic solvent (for example, up to 10% DMSO in water). The term "water" does not include a pure solvent or a combination of solvents different from water, such as pure alcohol, such as pure methanol or ethanol, pure ether, such as pure diethyl ether or tetrahydrofuran, or any other pure solvent that may or may not be miscible with water.

[0153] The term "water solubilizing moiety", as used herein, either by itself or as part of another group, refers to any hydrophilic group that can be sufficiently solvated and bound in an aqueous environment, such as under physiological conditions, to enhance the water solubility of the molecule to which it is attached. The increase in the water solubility of the molecule may vary depending on the moiety attached. In some cases, the increase in water solubility (compared to the solubility of the molecule without the attached moiety) may be 2-fold or more, 5-fold or more, 10-fold or more, 25-fold or more, 50-fold or more, or 100-fold or more. Examples of "water solubilizing moieties" include, but are not limited to, carboxylic acids and carboxylates, polyvinyl alcohol, glycols, peptides, polyphosphates, polyalcohols, sulfonates, phosphonates, boronate, amines, ammonium, sulfonium, phosphonium, alcohols, polyols, oxazolines, zwitterionic derivatives, carbohydrates, nucleotides, polynucleotides, PEG groups, carboxy groups, substituted carboxylic acids and carboxylates, substituted glycols, substituted peptides, substituted polyphosphates, substituted polyalcohols, substituted sulfonates, substituted phosphonates, substituted boronate, substituted amines, substituted ammonium, substituted sulfonium, substituted phosphonium, alcohols, substituted zwitterionic derivatives, substituted carbohydrates, substituted nucleotides, substituted polynucleotides, substituted PEG groups, substituted carboxy groups, and combinations thereof.

[0154] The term "water solubilizable moiety" or "water soluble group" (WSG or W), when used herein either by itself or as part of another group, refers to any hydrophilic group that can be sufficiently solvated and bound in an aqueous environment, such as under physiological conditions, to enhance the water solubility of the molecule to which it is attached. To enhance water solubility, any convenient WSG may be included in the dyes described herein. The water solubilizing moiety can primarily enhance the solubility of the compound in an aqueous solution when compared to a control compound lacking the water solubilizing moiety. The water solubilizing moiety may be any convenient hydrophilic moiety that is sufficiently solvated in an aqueous environment. In some cases, the water solubilizing moiety may be able to provide solubility in water (e.g., an aqueous buffer) of >1 mg / mL, >2 mg / mL, >3 mg / mL, >4 mg / mL, >5 mg / mL, >6 mg / mL, >7 mg / mL, >8 mg / mL, >9 mg / mL, or >10 mg / mL. In some cases, the water solubilizing moiety may be able to provide solubility in water of >10 mg / mL, >20 mg / mL, >30 mg / mL, >40 mg / mL, >50 mg / mL, >60 mg / mL, >70 mg / mL, >80 mg / mL, >90 mg / mL or >100 mg / mL.

[0155] The increase in the water solubility of the molecule can be varied depending on the moiety attached. In some cases, the increase in water solubility (when compared to the solubility of a molecule without the attached moiety) may be 2-fold or greater, 5-fold or greater, 10-fold or greater, 25-fold or greater, 50-fold or greater, or 100-fold or greater. In some cases, the water solubilizing moiety is charged, e.g., a positively or negatively charged hydrophilic moiety. In some cases, the water solubilizing moiety is a neutral hydrophilic moiety. In some cases, the water solubilizing moiety is branched (e.g., such as those described herein). In some cases, the water solubilizing moiety is linear. Examples of water solubilizing moieties include, but are not limited to, those taught in U.S. Patent Publication No. 2022 / 0348770, which is hereby incorporated by reference in its entirety.

[0156] A "water-soluble compound" may exhibit solubility in water (e.g., an aqueous buffer) at ambient room temperature of >1 mg / mL, >2 mg / mL, >3 mg / mL, >4 mg / mL, >5 mg / mL, >6 mg / mL, >7 mg / mL, >8 mg / mL, >9 mg / mL, or >10 mg / mL. In some cases, a water-soluble compound can exhibit solubility in water at ambient room temperature of >10 mg / mL, >20 mg / mL, >30 mg / mL, >40 mg / mL, >50 mg / mL, >60 mg / mL, >70 mg / mL, >80 mg / mL, >90 mg / mL, and / or >100 mg / mL.

[0157] To enhance water solubility, any convenient WSG may be included in the dyes described herein. WSGs include, but are not limited to, carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfonium, esters, polyethylene glycol (PEG) and modified PEG, linear PEG groups, branched PEG groups, hydroxyls, amines, amino acids, ammoniums, guanidiniums, pyridiniums, polyamines and sulfonium, polyalcohols, linear or cyclic saccharides, primary, secondary, tertiary, or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols including polyether, zwitterionic derivatives, peptide sequences, nucleotides (DNA and RNA), peptoids, carbohydrates, oxazolines, polyols, dendrons, dendritic polyglycerols, celluloses, chitosans, -COOM’, -SO3M’, -PO3M’, -NR 3 + , Y’, (CH2CH2O) pR and these mixtures may also be used. Y’ may be any halogen, sulfate, sulfonate, or oxygen-containing anion. p may be from 1 to 500. Each R may independently be H or alkyl (e.g., methyl). M’ may be a cationic counterion or hydrogen, -(CH2CH2O) yy CH2CH2XR yy 、-(CH2CH2O) yy CH2CH2X-, -X(CH2CH2O) yy CH2CH2-, glycol, and polyethylene glycol may also be used. yy is selected from 1 to 1000. X is selected from O, S, and NR ZZ and R ZZ and R YY are independently selected from H and C 1~3 alkyl, and combinations or derivatives thereof. In some cases, the WSGs include, but are not limited to, PEG, modified PEG, peptide sequences, peptoids, carbohydrates, oxazolines, polyols, dendrons, dendritic polyglycerols, cellulose, chitosan, or derivatives thereof. The WSG may or may not be substituted.

[0158] In some cases, the WSG may be a hydrophilic polymer. For example, hydrophilic polymers that may be used in the WSG include, but are not limited to, polyalkylene oxide-based polymers containing ethylene oxide repeat units of the formula -(CH2-CH2-O) n - or -(O-CH2-CH2) n -, such as PEG, polyamidoalkylene oxide, or derivatives thereof. Further examples of the polymer of interest include the formula -[C(O)-X-C(O)-NH-Y-NH] n - or -[NH-Y-NH-C(O)-X-C(O)] nThere is a polyamide having a molecular weight of more than 1,000 Daltons, where X and Y may be the same or different and may be branched or linear divalent radicals, n is an individual integer from 2 to 100, for example from 2 to 50, and either or both of X and Y contain a biocompatible and substantially non-antigenic water-soluble repeating unit that may be linear or branched. The number of such water-soluble repeating units can be varied significantly, and the number of such units is from 2 to 500, from 2 to 400, from 2 to 300, from 2 to 200, from 2 to 100, from 6 to 100, for example, from 2 to 50 or from 6 to 50. Examples of embodiments are where one or both of X and Y are -((CH2) n1 -(CH2-CH2-O) n2 -(CH2)- or -((CH2) n1 -(O-CH2-CH2) n2 -(CH2) n1 -), n1 is from 1 to 6, from 1 to 5, from 1 to 4, or from 1 to 3, and n2 is from 2 to 50, from 2 to 25, from 2 to 15, from 2 to 10, from 2 to 8, or from 2 to 5. In some cases, the water-soluble polymer is a group of 1 to 50 monomer units, for example, 1 to 40, 1 to 30, 1 to 20, 2 to 24, 2 to 20, 2 to 10, or 2 to 6 monomer units. Further examples of embodiments are where X is -(CH2-CH2)- and Y is -(CH2-(CH2-CH2-O)3-CH2-CH2-CH2)- or -(CH2-CH2-CH2-(O-CH2-CH2)3-CH2)-. In certain cases, any one of the formulas described herein may be substituted with a water-solubilizing moiety that is a dendron known in the art.

[0159] In some cases, the hydrophilic polymer may be, for example, PEG, a peptide sequence, a peptoid, a carbohydrate, an oxazoline, a polyol, a dendron, a dendritic polyglycerol, cellulose, chitosan, or a derivative thereof.

[0160] In some cases, the WSG is (CH2) x(OCH2CH2) y is OCH3, each x is independently an integer from 0 to 20, and each y is independently an integer from 0 to 50. In some cases, the water-soluble polymer is a PEG group or a modified PEG polymer having 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.

[0161] In some cases, the WSG comprises a non-ionic polymer (e.g., a PEG polymer) substituted at a terminal having an ionic group (e.g., sulfonate). In some embodiments of the formula, the WSG is (CH2) x (OCH2CH2) y comprises a substituent selected from OCH3, each x is independently an integer from 0 to 20, and each y is independently an integer from 0 to 50; benzyl is optionally substituted with one or more halogens, hydroxyls, C1-C 12 alkoxy, or (OCH2CH2) z OCH3, and each z is independently an integer from 0 to 50. In some cases, the WSG is (CH2)3(OCH2CH2) 11 OCH3. In some embodiments, one or more of the substituents are benzyl substituted with at least one WSG group (e.g., one or two WSG groups) selected from (CH2) x (OCH2CH2) y OCH3, each x is independently an integer from 0 to 20, and each y is independently an integer from 0 to 50. It is understood that a hydroxy-terminated polymer chain (e.g., a PEG chain) may be utilized in any of the solubilizing moieties instead of a methoxy-terminated polymer chain (e.g., a PEG chain).

[0162] The term "modified polymer", such as modified PEG, refers to a water-soluble polymer that is modified or derivatized at one or both ends to include terminal substituents (e.g., terminal alkyl, substituted alkyl, alkoxy or substituted alkoxy, etc.) and / or terminal linking functional groups (e.g., amino or carboxylic acid groups suitable for bonding via amide bond formation) that are suitable for bonding the polymer to a target molecule (e.g., to a light-harvesting chromophore via a branching group). The water-soluble polymer of interest may be adapted to include any convenient linking group. In some cases, it is understood that the water-soluble polymer may include a degree of polydispersity in relation to the polymer length, depending on the method of preparation and / or purification of the polymer starting material. In some cases, the water-soluble polymer is monodisperse.

[0163] The water-soluble polymer may include one or more spacers or linkers. Examples of spacers or linkers include linear or branched moieties containing one or more repeating units used in the water-soluble polymer, diamino units and / or diacid units, natural or non-natural amino acids or their derivatives, and aliphatic moieties containing alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, etc., for example, containing up to 18 carbon atoms and optionally additional polymer chains.

[0164] The water-soluble polymer moiety, or, if present, one or more of the spacers or linkers of the polymer moiety, may include biostable or biodegradable polymer chains or units. For example, a polymer having repeating linkages may vary in degree of stability under physiological conditions depending on linkage lability. Such polymers having such linkages may be classified, based on the known hydrolysis rates of low molecular weight analogs, for example, from least stable to most stable, for example, polyurethane (-NH-C(O)-O-) > polyorthoester (-O-C((OR)(R’))-O-) > polyamide (-C(O)-NH-), by their relative rates of hydrolysis under physiological conditions. Similarly, a linking system that attaches a water-soluble polymer to a target molecule may be, for example, from least stable to most 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-), and may be biostable or biodegradable. Generally, depending on the lability of sulfate groups, it may be desirable to avoid the use of sulfated polysaccharides. Further, the use of polycarbonates and polyesters may be undesirable. These linkages are provided by way of example and are not intended to limit the types of linkages that can be used in the polymer chains or linking systems of water-soluble polymers useful in the WSGs disclosed herein.

[0165] In some cases, the water-solubilizing moieties include, but are not limited to, hydroxy, alkoxy, (hetero)aryloxy, (hetero)arylamino, PEG, linked PEG, amide-PEG, sulfonamide-PEG, phosphoramidate-PEG, alkylammonium salts, alkyloxyammonium salts, oligoetherammonium salts, carbonyl, acyl, sulfonate, alkylsulfonate, alkylcarboxylate, alkylsulfonate salts, alkoxysulfonate salts, oligoether sulfonate salts, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamid, alkoxysulfonamide PEG, alkylcarboxylate, alkylamide, alkoxysulfonate, alkylsulfonate, alkylsulfonate salts,

Chemical formula

Chemical formula

[0166] In some cases, the target compound may contain a plurality of water-solubilizing moieties attached at a single position in the target compound via, for example, a branched linker, such as an aralkyl substituent further disubstituted with a solubilizing group. Thus, in some cases, the branched linker group is a substituent of the dye that connects the dye to two or more solubilizing groups. In some cases, the plurality of water-solubilizing moieties may be attached to the target compound via a group having, for example, the following formula

Chemical formula

[0167] In some cases, one or more solubilizing moieties may be attached to the subject compound via a group comprising a linker according to the present disclosure, such as those taught in U.S. Published Application No. 2020 / 0190253A1, which is hereby incorporated by reference in its entirety. The linker moiety may be attached to the cyanine bridge or heterocycloaryl group of the fluorescent compounds of the present disclosure. The linker may be cleavable or non-cleavable.

[0168] One or more solubilizing moieties may also be a linker, such as, but not limited to, the following linker formula (VIe): -(L 3 ) m -(X 1 ) m’ -((L 1 ) m” -(W 1 ) s ) t -R 3 (VIe) and may be attached to the subject compound via a group containing (wherein each optional L 1 and L 3 are independently selected linker moieties; X 1 is optionally present and is a branch point; W 1 is a water-soluble moiety comprising a water-soluble polymer containing from 2 to 50, from 4 to 30, or from 6 to 24 monomer units, including but not limited to; each m is independently 0 or 1; each m' is independently 0 or 1; each m'' is independently 0 or 1; each s is independently 1 or 2; each t is independently 0, 1, 2, or 3; R 3 is as defined herein).

[0169] In some cases, L 1 , L 3, and X is absent, and W 1 is a water-solubilizing moiety, e.g., a water-soluble polymer containing 2 to 50, 4 to 30, or 6 to 24 monomer units, e.g., 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, the water-solubilizing moiety may be a linear water-solubilizing moiety. For example, L 1 and X may be absent, and L 3 is a linker (e.g., as disclosed herein), and W 1 is a water-solubilizing moiety.

[0170] In some cases, L 1 , L 2 and / or L 3 at least one, at least two, or all three of may each independently be selected from alkyl or substituted alkyl linkers, alkenyl or substituted alkenyl linkers, alkynyl or substituted alkynyl linkers, alkoxy or substituted alkoxy linkers, PEG linkers, sulfonamide-alkyl or substituted sulfonamide-alkyl linkers, amide-alkyl or substituted amide-alkyl linkers, and alkyl-amide-alkyl or substituted alkyl-amide-alkyl linkers. In certain cases, the linker contains a carbonyl group. The linker moiety may be a covalent bond, alkoxy, sulfonamide, disulfonamide, selenamide, sulfinamide, sultam, disulfinamide, amide, carbonyl, seleninamide, phosphonamide, phosphinamide, phosphonamidate, or secondary amine.

[0171] In some cases, L 2 and L 3 may each independently be a linker moiety selected from the group consisting of a covalent bond, C 1~8 alkylene, 2- to 8-membered heteroalkylene, and chains having a backbone atom length between 2 and 200, and the chains may include linear chains, branched chains, and / or cyclic moieties.

[0172] In some cases, L 1 may be a sulfonamide, sulfinamide, disulfonamide, disulfinamide, sultam, amide, secondary amine, phosphonamide, phosphinamid, phosphonamidate, selenonamide, or seleninamide.

[0173] In some cases, L 3 may be a linker having a backbone of 20 atoms or less in length, and W 1 is a water-solubilizing moiety (e.g., as described herein). In some cases, L 3 may be selected from 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 sulfonamide-alkyl or substituted sulfonamide-alkyl linker, an amide-alkyl or substituted amide-alkyl linker, and an alkyl-amide-alkyl or substituted alkyl-amide-alkyl linker. In some cases, L 3 may be a bond. In some cases, L 3 may be an alkyl or substituted alkyl linker, an alkenyl or substituted alkenyl linker, an alkynyl or substituted alkynyl linker, an alkoxy or substituted alkoxy linker, and X may be an aryl group.

[0174] In some cases, L 1 and L 3 are each independently a C1-C 12 alkyl or substituted alkyl linker, a C1-C 12 alkenyl or substituted alkenyl linker, a C1-C 12 alkynyl or substituted alkynyl linker, a C1-C 12 acyl or substituted acyl linker, a C1-C 12 alkoxy or substituted alkoxy linker, a C1-C 12 amide-alkyl or substituted amide-alkyl linker, a C1-C 12It is selected from alkyl-amido-alkyl or substituted alkyl-amido-alkyl linkers, sulfonamides, sulfinamides, disulfonamides, disulfinamides, sultams, amides, secondary amines, phosphonamides, phosphinamides, phosphonamidates, selenonamides, and seleninamides. In certain cases, L 3 contains a carbonyl group or an alkoxy group, and L 1 is C1-C 12 alkyl or substituted alkyl, sulfonamide, sulfinamide, disulfonamide, disulfinamide, sultam, amide, secondary amine, phosphonamide, phosphinamide, phosphonamidate, selenonamide, and seleninamide. In some cases, L 3 may be an alkoxy or substituted alkoxy linker, X may be absent, and L 1 may be sulfonamide, sulfinamide, disulfonamide, disulfinamide, sultam, amide, secondary amine, phosphonamide, phosphinamide, phosphonamidate, selenonamide, or seleninamide.

[0175] In some cases, the branching point X 1 is selected from N, CR’, C(=O)N, SO2N, trisubstituted aryl moieties (e.g., 1,3,5-phenyl), tetrasubstituted aryl moieties (e.g., 1,3,4,5-phenyl), and trisubstituted heteroaryl groups. In certain cases, the branching point X 1 is a nitrogen atom. In other cases, the branching point X 1 is CR’ and R’ is selected from hydrogen, alkyl, substituted alkyl, or -L 3 -W 1 (such as those described herein).

[0176] The term "water-soluble", when referring to a polymer as used herein, refers to a polymer having solubility in "water" as used herein of 1 mg / mL or more, for example, 3 mg / mL or more, 10 mg / mL or more, 20 mg / mL or more, 30 mg / mL or more, 40 mg / mL or more, 50 mg / mL or more, 60 mg / mL or more, 70 mg / mL or more, 80 mg / mL or more, 90 mg / mL or more, 100 mg / mL or more, or more than these. It is understood that water-soluble polymers may, under certain conditions, form individual water-solvated nanoparticles in an aqueous solution system and may be resistant to aggregation. The fluorescent compounds of the present disclosure may be water-soluble. The fluorescent polymers of the present disclosure may be water-soluble.

[0177] The term "cyanine", as used herein, refers to a substituted or unsubstituted bridging unit that enables delocalization across the molecules or monomers of the present invention. In some embodiments, "cyanine" is a substituted or unsubstituted methine or polymethine unit, such as a tri-, penta- or heptamethine unit. For example, in some embodiments, "cyanine" is a substituted and unsubstituted group, such as the following

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0178] The present disclosure provides DHP-cyanine and DHP-squaraine organic dye monomer compounds, polymers, and methods for making them. According to the methods provided in the present disclosure, both small molecule organic dyes and polymer dyes can be prepared. The obtained DHP-cyanine and DHP-squaraine dyes exhibit an excitation maximum in the range of about 420 - 900 nm for the monomer dyes. In addition to the specific structures disclosed herein, structural isomers of the disclosed structures are also included.

[0179] The DHP-cyanine and DHP-squaraine compounds of the present disclosure each contain at least one 9,10-dihydrophenanthrene (DHP) moiety or a derivative thereof. A DHP-cyanine or DHP-squaraine compound according to the present disclosure may include a structure according to formula (I)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0180] [Chemistry] is at any available

Chem.

Chem.

[0181] Each T may be independently selected from the group consisting of C(R 1 ), N, P, O, S, and Si(R 1 ). Each T may independently be C(R 1 ) or N.

[0182] Each U may be independently selected from the group consisting of NR 10 , O, P, and S. Each U may independently be NR 10 .

[0183] Each V may be independently selected from the group consisting of NR 11 , CR 11 , C(R 11 )2, S, O, and Si(R 11 )2. Each V may independently be CR 11 , C(R 11 )2, S, or O. Each V may independently be CR 11 or C(R 11 )2.

[0184] Each V' may be independently selected from the group consisting of SO2, SO, S, NR 11 , CR 11 , C(R 11 )2, O, Si(R 11 )2, >C=O, >Se=O, -CH=CH-, and -N=CH-. Each V' may be independently selected from the group consisting of SO2, SO, and S.

[0185] Each X is independently CR 1 R 2 or SiR 1 R 2 and may be. In some cases, each X is independently CR 1 R 2 and may be.

[0186] Each Y is independently CR 8 R 9 or SiR 8 R 9 and may be. In some cases, each Y is independently CR 8 R 9 and may be.

[0187] Each R 1 R 2 R 8 and R 9 are a water-solubilizing moiety, a linker moiety, a linked E, hydrogen, hydroxy, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, sulfonamide-PEG, phosphoramidate-PEG, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, carbonyl, acyl, alkylsulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamido, alkoxysulfonamide PEG, alkoxysulfonate, alkylsulfonate, alkylsulfonate,

Chemical formula

[0188] Each R 3 may be independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a water solubilizing moiety, a chromophore, a functional moiety, a binding partner, and a PEG group.

[0189] Each Q is independently a bond, NH, NR 4 , C1-C 12 alkylene, CHR 4 , and CH2CH2, CHR 4 , O, NR 4 , or NH; each Z is independently CH2, CHR 4 , O, NR 4 , or NH.

[0190] Each W 1 may independently be a water solubilizing moiety.

[0191] Each L 1 , L 2 , and L 3 may each independently be a selected linker moiety; each E is independently selected from the group consisting of a chromophore, a functional moiety, a substrate, and a binding partner.

[0192] Each R 4 may be independently selected from the group consisting of H, alkyl, PEG, a water solubilizing moiety, a linker moiety, a chromophore, a carboxylic acid amine, an amine, a carbamate, a carboxylic acid, a carboxylic acid ester, maleimide, an activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, and thiol, or a protecting group thereof.

[0193] Each R 7 is H, hydroxyl, C1-C 12 alkyl, C2-C 12 alkene, C2-C 12 alkyne, C3-C 12Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (Hetero)arylamino, C2-C 12 Carboxylic acid, C2-C 12 Carboxylic acid ester, and C1-C 12 Alkoxy, water-solubilizing moiety, PEG moiety, functional group, chemoselective functional group, conjugation tag, linked conjugation tag, linker, sulfonic acid, sulfonate, C1-C 12 It may be independently selected from the group consisting of alkyl sulfonate and sulfonamide. In some cases, each R7 is H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkene, C2-C 12 Alkyne, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (Hetero)arylamino, C2-C 12 Carboxylic acid, C2-C 12 Carboxylic acid ester, and C1-C 12 It may be independently selected from the group consisting of alkoxy. In some cases, each R7 is a functional group, chemoselective functional group, conjugation tag, linked conjugation tag, linker, sulfonic acid, sulfonate, C1-C 12 It may be independently selected from the group consisting of alkyl sulfonate and sulfonamide.

[0194] Each R 10 may be independently selected from the group consisting of hydrogen, linker moiety, linked reactive group, linked ionic group, linked chromophore, linked water-solubilizing moiety, water-solubilizing moiety, chromophore, binding partner, linked binding partner.

[0195] Each R 11may be independently selected from the group consisting of hydrogen, a linker moiety, a linked reactive group, a linked ionic group, a linked chromophore, a linked solubilizing moiety, a solubilizing moiety, a chromophore, a binding partner, and a linked binding partner.

[0196] Each R 12 and R 13 may be independently selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6 alkyl, C3-C 10 cycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; or R 12 and R 13 together form an unsubstituted or substituted unsaturated cycloalkyl group having 3 to 8 ring members or a substituted or unsubstituted heterocycloalkyl having 3 to 8 ring members, optionally substituted with O. Each K may independently be a covalent bond, O, S, P, or CH2.

[0197] Each f may independently be an integer from 0 to 50, 1 to 40, 2 to 30, 3 to 25, or 4 to 20.

[0198] Each m may independently be 0 or 1.

[0199] Each n may independently be 0, 1, 2, 3, or 4.

[0200] Each s may independently be 1 or 2.

[0201] Each t may independently be 0, 1, 2, or 3.

[0202] In some examples, R 1 , R 2 , R 3 , R 4 , R 8 , R 9 , R 10 , and R 11At least one of which comprises a water-solubilizing moiety or a linked water-solubilizing moiety.

[0203] Any available [Chemical formula] Containing an additional aryl or heteroaryl group condensed with [Chemical formula] Derivatives of [Chemical formula] May optionally contain, and each V' is SO2, SO, S, NR 11 , CR 11 , C(R 11 )2, O, Si(R 11 )2, >C=O, >Se=O, -CH=CH-, or -N=CH-, and T, V, X, Y, and m are as defined above in this specification.

[0204] The DHP-cyanine or DHP-squaraine compounds according to the present disclosure may contain a structure according to formula (II) [Chemical formula] (wherein Each [Chemical formula] Is independently selected from the group consisting of substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl groups, polycyclic aryl groups, monocyclic heteroaryl groups, and polycyclic heteroaryl groups, and at least one [Chemical formula] Is [Chemical formula] , or a derivative thereof, and the derivative is any available [Chemistry] optionally including an additional aryl or heteroaryl group condensed with

[0205] In some cases, [Chemistry] derivatives of [Chemistry] are (wherein, each V' is independently SO2, SO, S, NR 11 , CR 11 , C(R 11 )2, O, Si(R 11 )2, >C=O, >Se=O, -CH=CH-, or -N=CH-; each T is independently selected from the group consisting of C, C(R 1 ), N, P, O, S, and Si(R 1 ); each U is independently selected from the group consisting of NR 10 , O, P, Se, Te, and S; each V is independently selected from the group consisting of NR 11 , CR 11 , C(R 11 )2, S, SO2, O, Se, Te, and Si(R 11 ); each X is independently CR 1 R 2 or SiR 1 R 2 ; each Y is independently CR 8 R 9 or SiR 8 R 9 ; each m is independently 0 or 1).

[0206] In some cases, each [Chemistry] is, independently,

Chem.

[0207] In some cases, each

Chem.

Chem.

[0208] In some cases, one

Chem.

Chem.

Chem.

Chem.

Chem.

[0209] In some cases, V is CR 11 or C(R 11 )2. Each R1 , R 2 , R 8 , and R 9 are independently selected from the group consisting of a water-solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, a binding partner, a linked binding partner, a functional group, hydrogen, hydroxy, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, carbonyl, acyl, alkylsulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamid, alkoxysulfonamide PEG, alkoxysulfonate, alkylsulfonate, alkylsulfonate, [Chemical formula] [Chemical formula] may be independently selected from the group consisting of. In some cases, each R 1 , R 2 , R 8 , and R 9 are independently selected from the group consisting of a water-solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, a binding partner, a linked binding partner, a functional group, hydrogen, hydroxy, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, and haloalkyl. In some cases, each R 1 , R 2 , R 8 , and R 9is independently selected from the group consisting of watr sulfonamide-PEG, phosphoramidate-PEG, alkylammonium salts, alkyloxyammonium salts, oligoetherammonium salts, carbonyl, acyl, alkylsulfonates, alkoxysulfonates, oligoethersulfonates, sulfonamido oligoethers, sulfonamides, sulfinamides, phosphonamidates, phosphinamides, alkoxysulfonamide PEG, alkoxysulfonates, alkylsulfonates, alkylsulfonates,

Chemical formula

[0210] In some cases, R 1 and R 8 together form an unsubstituted or substituted cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, cycloalkoxy, aryl, or heteroaryl having 3 to 9 ring members.

Chemical formula

[0211] In some cases, each R 3is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a solubilizing moiety, a chromophore, a functional moiety, a linking partner, and a PEG group. In some cases, each R 3 is independently selected from the group consisting of a solubilizing moiety, a chromophore, a functional moiety, a linking partner, and a PEG group (e.g., (PEG) f -OMe and (PEG) f -OEt).

[0212] In some cases, each Q is independently a bond, NH, NR 4 , C1-C 12 alkylene, CHR 4 , or CH2. In some cases, each Q is independently a bond, NH, or NR 4 .

[0213] In some cases, each Z is independently CH2, CHR 4 , O, NR 4 , or NH. In some cases, Z is O.

[0214] In some cases, each W 1 is independently a solubilizing moiety.

[0215] In some cases, L 1 , L 2 , and L 3 are each independently selected linker moieties.

[0216] In some cases, each E is independently selected from the group consisting of a chromophore, a functional moiety, a conjugation tag, a substrate, and a linking partner.

[0217] In some cases, each R 4is independently selected from the group consisting of H, alkyl, PEG, solubilizing moiety, linker moiety, chromophore, conjugated chromophore, functional group, conjugation tag, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, cycloalkyne, alkene, cycloalkene, tetrazine, aldehyde, and thiol, or protecting groups thereof.

[0218] In some cases, each R 7 is independently selected from the group consisting of H, hydroxyl, C1-C 12 alkyl, C2-C 12 alkene, C2-C 12 alkyne, C3-C 12 cycloalkyl, C1-C 12 haloalkyl, C1-C 12 alkoxy, C2-C 18 (hetero)aryloxy, C2-C 18 (hetero)arylamino, C2-C 12 carboxylic acid, C2-C 12 carboxylic acid ester, and C1-C 12 alkoxy.

[0219] In some cases, each R 10 is independently selected from the group consisting of hydrogen, linker moiety, linked reactive group, linked ionic group, linked chromophore, linked solubilizing moiety, solubilizing moiety, chromophore, binding partner, and linked binding partner.

[0220] In some cases, each R 11 is independently selected from the group consisting of hydrogen, linker moiety, linked reactive group, linked ionic group, linked chromophore, linked solubilizing moiety, chromophore, binding partner, and linked binding partner.

[0221] In some cases, each R 12 , R 13 , and R 14is hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C1-C6 alkene, substituted or unsubstituted C3-C 10 cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, CO2R 1 , CONR 1 R 2 , -O-CH2CH2-PEG-R 7 , -S-CH2CH2-PEG-R 7 , -N-CH2CH2-PEG-R 7 , O-aryl, S-aryl, N-aryl, -O-alkyl, S-alkyl, N-alkyl, independently selected from the group consisting of, each alkyl or aryl is one or more R 7 , PEG, PEG-R 7 , or may be optionally substituted with a linking group, optionally, each R 12 , R 13 and R 14 is, R 7 groups are independently substituted; or are not limited to but R 10 , R 11 , R 12 , R 13 , and R 14 of two, three, or four of, R 10 , R 11 , R 12 , R 13 , and R 14 of at least two of are taken together to form an unsubstituted or substituted unsaturated or partially unsaturated C3-C 10 cycloalkyl group, unsubstituted or substituted unsaturated or partially unsaturated C3-C 10 heterocycloalkyl which is C3-C 10 heterocycloalkyl optionally substituted with O, unsubstituted or substituted unsaturated or partially unsaturated cycloalkyl group having 3 to 8 ring members, or substituted or unsubstituted heterocycloalkyl having 3 to 8 ring members which is heterocycloalkyl optionally substituted with O.

[0222] In some cases, each K is independently a covalent bond, O, S, P, NR 1 , Se, Te, CR 1 R 2 , or CH2.

[0223] In some cases, each f is independently an integer from 0 to 50.

[0224] In some cases, each m is independently 0 or 1.

[0225] In some cases, each n is independently 0, 1, 2, 3, or 4.

[0226] In some cases, each s is independently 1 or 2.

[0227] In some cases, each t is independently 0, 1, 2, or 3.

[0228] In some examples, the DHP-cyanine and DHP-squarylium compounds of the present disclosure are of formula (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIi), (IIj), (Ik), (IIl), (IIm), (IIn), (IIo), (IIp), (IIq), (IIr), (IIs), (IIt), (IIu), (IIv), (IIw), (IIx), (IIy), or (IIz):

Chemical formula

Chemical formula

Chemical formula

[0229] In some examples, the DHP-cyanine and DHP-squaraine compounds of the present disclosure are of formula (IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIi), (IIIj), (IIIk), (IIIl), (IIIm), (IIIn), (IIIo), (IIIp), (IIIq), (IIIr), (IIIs), (IIIt), (IIIu), (IIIv), (IIIw), (IIIx), (IIIy), (IIIz), (IIIaa), (IIIbb), (IIIcc), (IIIdd), or (IIIee):

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0230] In some examples, D is

Chem.

Chem.

Chem.

Chem.

Chem.

[0231] In some examples, J is

Chem.

Chem.

[0232] In some examples, J is unsubstituted or substituted quinoline, benzoxazole, benzothiazole, benzimidazole, indole, benzindole, pyridinium, benzopyrylium, thiopyrylium, fluorene, 9H-fluorene, phenanthrene, dihydrophenanthrene, 9,10-dihydrophenanthrene, 6,8-dihydro-5H-naphtho[2,1-f]indole, 4,5-dihydro-3H-naphtho[2,1-e]indole, 6,7-dihydro-3H-naphtho[2,1-g]indole, 5,6-dihydrophenanthro[3,2-d]thiazole, 4,5-dihydrophenanthro[2,1-d]thiazole, 6,7-dihydrophenanthro[4,3-d]thiazole, 5,6-dihydrophenanthro[3,2-d]oxazole, 4,5-dihydrophenanthro[2,1-d]oxazole, 6,7-dihydrophenanthro[4,3-d]oxazole, 5,6-dihydronaphtho[2,1-g]quinoline, 7,8-dihydronaphtho[2,1-h]quinoline, 5,6-dihydronaphtho[2,1-f]quinoline, 5,6-dihydro-8λ2-phenanthro[3,2-d]imidazole, 4,5-dihydro-3λ2-phenanthro[2,1-d]imidazole, 6,7-dihydro-3λ2-phenanthro[4,3-d]imidazole, 5,6-dihydronaphtho[1,2-g]quinoxaline, 5,6-dihydronaphtho[2,1-f]quinoxaline, 7,8-dihydronaphtho[1,2-f]quinoxaline, phenyl, 1H-benzo[e]indol-3-ium, and 1H-benzo[e]indole.

[0233] In some examples, D is aryl, heteroaryl or

Chem.

Chem.

Chem.

Chem.

Chem.

[0234] In some examples, D is

Chem.

Chem.

[0235] In some examples, D is selected from unsubstituted or substituted quinoline, benzoxazole, benzothiazole, benzimidazole, indole, benzindole, pyridinium, benzopyrylium, thiopyrylium, fluorene, 9H-fluorene, phenanthrene, dihydrophenanthrene, 9,10-dihydrophenanthrene, 6,8-dihydro-5H-naphtho[2,1-f]indole, 4,5-dihydro-3H-naphtho[2,1-e]indole, 6,7-dihydro-3H-naphtho[2,1-g]indole, 5,6-dihydrophenanthro[3,2-d]thiazole, 4,5-dihydrophenanthro[2,1-d]thiazole, 6,7-dihydrophenanthro[4,3-d]thiazole, 5,6-dihydrophenanthro[3,2-d]oxazole, 4,5-dihydrophenanthro[2,1-d]oxazole, 6,7-dihydrophenanthro[4,3-d]oxazole, 5,6-dihydronaphtho[2,1-g]quinoline, 7,8-dihydronaphtho[2,1-h]quinoline, 5,6-dihydronaphtho[2,1-f]quinoline, 5,6-dihydro-8λ2-phenanthro[3,2-d]imidazole, 4,5-dihydro-3λ2-phenanthro[2,1-d]imidazole, 6,7-dihydro-3λ2-phenanthro[4,3-d]imidazole, 5,6-dihydronaphtho[1,2-g]quinoxaline, 5,6-dihydronaphtho[2,1-f]quinoxaline, 7,8-dihydronaphtho[1,2-f]quinoxaline, phenyl, 3,5,6,8-tetrahydrophenanthro[2,3-e:7,6-e’]diindole, 5,6-dihydropentapheno[3,4-d:10,9-d’]bis(oxazole), 5,6-dihydropentapheno[3,4-d:10,9-d’]bis(thiazole), 6,7-dihydrophenanthro[2,3-f:7,6-f’]diquinoline, 5,6-dihydropentapheno[3,2,1-cd:10,11,12-c’d’]diindole, 2,6,7,11-tetrahydrobenzo[1,2-g:4,3-g’]dichromene, or 2,6,7,11-tetrahydrobenzo[1,2-g:4,3-g’]bis(thiochromene).

[0236] In some examples, D is [Chemistry] is, or J is [Chemistry] (wherein, at least one [Chemistry] is [Chemistry] group or any available [Chemistry] at the position of [Chemistry] is condensed with [Chemistry] group or its derivative).

[0237] In some examples, D is [Chemistry] and [Chemistry] group or its derivative [Chemistry] group or any available [Chemistry] at the position of [Chemistry] is condensed with

[0238] In some examples, D is [Chem.] selected from the group consisting of.

[0239] In some examples, D is [Chem.] and of D [Chem.] is [Chem.] any available [Chem.] at the position of [Chem.] condensed to [Chem.] a group or its derivative.

[0240] In some examples, J is [Chem.] and [Chem.] the group is [Chem.] any available [Chem.] at the position of [Chemistry] condensed with [Chemistry] is a group or its derivative.

[0241] In some examples, J is [Chemistry] selected from the group consisting of J selected from the group consisting of

[0242] In some examples, at least one [Chemistry] group or its derivative is [Chemistry] [Chemistry] [Chemistry] [Chemistry] selected from the group consisting of (wherein each R 5 is halogen, hydroxyl, C1 - C 12 alkyl, C2 - C 12 alkene, C2 - C 12 alkyne, C3 - C 12 cycloalkyl, C1 - C 12 haloalkyl, C1 - C 12 alkoxy, C2 - C 18 (hetero)aryl group, C2 - C 18 (hetero)aryloxy, C2 - C 18 (hetero)arylamino, carboxylic acid, carboxylic acid ester, (CH2) x’ (OCH2 - CH2) y’OCH3 and (CH2) x’ (OCH2-CH2) y’ independently selected from the group consisting of OCF3, each x' is independently an integer from 0 to 20, and each y' is independently an integer from 0 to 50).

[0243] In some examples, D is

Chemical formula

Chemical formula

[0244] In some examples, J is

Chemical formula

Chemical formula

[0245] In some examples, each R 10 and each R 11 is a solubilizing moiety, a linker moiety, a linked solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, aryl, heteroaryl, (hetero)arylamino, sulfonamide-PEG, phosphoramidate-PEG, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkylsulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, sulfonamide, sulfinamido, phosphonamidate, phosphinamido, alkoxysulfonamide PEG, alkoxysulfonate, alkylsulfonate, alkylsulfonate salt,

Chemical formula

Chemical formula

[0246] In some cases, each R 10 and each R 11 are independently sulfonamide-PEG, phosphoramidate-PEG, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkylsulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamid, alkoxysulfonamide PEG, alkoxysulfonate, alkylsulfonate, alkylsulfonate, [Chemical formula] [Chemical formula] includes a moiety selected from the group consisting of.

[0247] In some cases, each R 10 and each R 11 are independently sulfonamide-PEG, sulfonamide oligoether, sulfonamide, sulfinamide, alkoxysulfonamide PEG, [Chemical formula] [Chemical formula] includes a moiety selected from the group consisting of.

[0248] The DHP-cyanine and DHP-squaraine compounds of the present disclosure may include any of the following compounds. [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula]

[0249] The DHP-cyanine and DHP-squaraine compounds of the present disclosure may be water-soluble.

[0250] A method for preparing the DHP-cyanine and DHP-squaraine compounds of the present disclosure is also provided.

[0251] Figure 1 shows an exemplary synthetic scheme 1 including selected steps in the synthesis of DHP-cyanine and DHP-squaraine from a modified dihydrophenanthrene (DHP) core. The DHP-indole (Va), DHP-thiazole (Vb), DHP-oxazole (Vc), and DHP-quinoline (Vd) intermediates can be prepared from the DHP core molecule. Treatment with either a suitable aldehyde dianiline hydrochloride or squaric acid yields the DHP-cyanine compound of formula (VI) (where n = 0, 1, 2, or 3) or the DHP-squaraine compound of formula (VII), respectively.

[0252] Figure 2 shows a representative synthetic scheme 2 used to modify dihydrophenanthrene and subsequently synthesize a DHP-Cy5 type monomer dye. Commercially available 3-bromophenanthrene-9,10-dione 6 may be reduced using sodium borohydride NaBH4 in water-ethanol to obtain the 3-bromo-9,10-dihydrophenanthrene-9,10-diol intermediate 7. The DHP-diol 7 may be treated with, for example, an alkyl iodide in the presence of sodium hydride NaH to give 3-bromo-DHP-OR 1Intermediate 8 is obtained. In the presence of sodium t-butoxide, Pd(OAc)2, and (±)-BINAP in toluene, treatment of 8 with benzophenone hydrazone gives DHP-hydrazine intermediate 9. Treatment of 9 with methyl isopropyl ketone under acidic conditions gives DHP-indole intermediate 10. Treatment of 10 with 1,3-propanesulfone gives 3-sulfopropyl-naphthindole intermediate 11. In CH3CN / CH2Cl2, in the presence of acetic anhydride, sodium acetate, and DIPEA, treatment of 11 with an appropriate aldehyde dianiline hydrochloride (e.g., glutaconic aldehyde dianilide hydrochloride) gives the DHP-cyanine dye of structure (VIII) according to the present disclosure. Using this protocol and its variations, compounds 1-5 as shown in Table 1 may be prepared.

[0253] Figure 5 shows synthetic scheme 5 for preparing an asymmetric DHP-squarine monomer dye of formula (X) from a dihydrophenanthrene type core and semisquaraine. Figure 5 also shows synthetic scheme 6 for preparing a symmetric DHP-squarine monomer dye of formula (XI) from a dihydrophenanthrene type core and squaric acid.

[0254] Table 1 shows exemplary symmetric and asymmetric DHP-cyanine compounds 1-5 of the present disclosure and their fluorescence properties including the maximum excitation wavelength (λex) and emission wavelength (λem).

[0255]

Table 1-1

Table 1-2

Table 1-3

[0256] The present disclosure provides DHP-cyanine polymers and DHP-squaraine polymers and methods of making them. DHP-cyanine and DHP-squaraine polymer dyes can exhibit an excitation λmax in the range of 420 to 900 nm. Dihydrophenanthrene having substituted bromine at the 3- and 6-positions can result in dyes that are different from dyes made using a pre-synthesized monomer A in which the bromine atoms are substituted at the 2- and 7-positions. Thus, several options for making different dyes are available from this technology.

[0257] Polymer dyes comprising monomers having a structure according to formula (IV) or (V) are provided in the present disclosure

Chemical formula

Chemical formula

[0258] Each D 1 and D 2 is independently selected from the group consisting of an aryl group, a heteroaryl group, and

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0259] any available [Chem.] and contains an additional aryl or heteroaryl group condensed with [Chem.] The derivative of

Chem.

[0260] In an example, the monomer of formula (IV) or (V) is of formula (A1), (A2), (A3), (A4), (A5), (A6), (A7), (A8), (A9), (A10), (A11), (A12), (A13), (A14), (A15), (A16), (A17), (A18), and (A19):

Chem.

Chem.

Chem.

Chem.

[0261] In some examples, the present disclosure provides DHP-cyanine or DHP-squarylium polymeric dyes according to formula (VI) or (VII)

Chem.

Chem.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0262] In some examples, the present disclosure provides a DHP-cyanine or DHP-squarylium polymer dye according to formula (VI’)

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

[0263] any available

Chem.

Chemical formula

Chemical formula

[0264] In some examples, the monomer of formula (IV) or (V) comprises a structure selected from the group consisting of formulas (A1), (A2), (A3), (A4), (A5), (A6), (A7), (A8), (A9), (A10), (A11), (A12), (A13), (A14), (A15), (A16), (A17), (A18), and (A19) shown herein.

[0265] In some examples, each G 1 and G 2is independently selected from the group consisting of aryl or heteroaryl substituted with one or more pendant chains terminated with a functional moiety selected from hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronic acid-substituted aryl, boronic ester-substituted aryl, boronic ester, boronic acid, optionally substituted tetrahydropyrrene (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protecting groups thereof, conjugated to a substrate or binding partner as needed.

[0266] In some examples, D 1 and D 2 are the same or different. In some examples, D 1 and D 2 are the same. In some examples, D 1 and D 2 are different.

[0267] The polymers according to the present disclosure may be water-soluble.

[0268] Also provided are methods for making the DHP-cyanine polymers and DHP-squaraine polymers of the present disclosure.

[0269] Figure 3 shows an exemplary synthetic scheme for manufacturing a DHP-cyanine polymer according to the present disclosure. The starting 3,6-dibromo 9,10-substituted-9,10-dihydrophenanthrene 14 may be manufactured according to U.S. Patent No. 11,208,527, which application is hereby incorporated by reference in its entirety. The production of the DHP-di-indole intermediate 16 may be carried out by any suitable protocol. For example, the DHP-di-indole 16 may be manufactured in a manner similar to Scheme 2. In the presence of DIEA and NaOAc, treatment with a suitable aldehyde dianiline hydrochloride results in the formation of the DHP-cyanine polymer according to formula (IXa) as shown in Scheme 3, where p may be an integer from 2 to 1,000; 5 to 500; or 10 to 100.

[0270] Figure 4 shows Synthetic Scheme 4 for manufacturing a DHP-cyanine polymer according to formula (IXb). Commercially available 3,6-dibromophenanthrene-9,10-dione 12 is reduced in water-ethanol using sodium borohydride NaBH4 to obtain the 3,6-di-bromo-9,10-dihydrophenanthrene-9,10-diol intermediate 13. The dibromo-DHP-diol 13 is treated, for example, with an alkyl iodide in the presence of sodium hydride NaH to obtain the 3,6-dibromo-DHP-OR 1 intermediate 14. Treatment of 14 with benzophenone hydrazone in toluene in the presence of sodium t-butoxide, Pd(OAc)2, (±)-BINAP gives the DHP-dihydrazine intermediate 15. Treatment of 15 with methyl isopropyl ketone in acidic conditions gives the DHP-diindole intermediate 16. Treatment of 16 with 1,3-propanesulfone gives the disulfopropyl-naphthylindole intermediate 17. Treatment of 17 with a suitable aldehyde dianiline hydrochloride (e.g., glutaconaldehyde dianilide hydrochloride) in CH3CN / CH2Cl2 in the presence of acetic anhydride, sodium acetate, and DIPEA gives the DHP-cyanine dye of structure (IXb), where p may be an integer from 2 to 1,000; 5 to 500; or 10 to 100. V. Capping Unit

[0271] Linkers and capping units may be conjugated to the fluorescent polymer backbone of the present disclosure via similar mechanisms as described above. For example, bromo- and boronic acid ester capping units of the capping unit may be used to attach to one or both ends of the polymer. Utilizing both capping units of bromoester and boronic acid ester will result in attachment to both ends of the polymer. Utilizing only one form of the bromoester or boronic acid ester capping unit of the capping unit will attach only to that end terminated with its individual complement, and for symmetric polymerization, it can be used to statistically modify only one end of the polymer. For asymmetric polymers, this approach is used to chemically ensure that the polymer is modified only at a single chain end. The capping unit may be added asymmetrically by first reacting a bromo-capping unit with a polymer having a Y-terminus and then reacting the polymer with a boronic acid ester capping unit.

[0272] For example, the capping agent of the present disclosure can be prepared as shown in Scheme (VII). [Chemical formula] VI. Binding Partner

[0273] A "binding partner" or "specific binding partner" of the present disclosure may be any molecule or molecular complex that can specifically bind to a target analyte. Examples of binding partners of the present disclosure include, for example, proteins, small molecule organic molecules, carbohydrates (including polysaccharides), oligonucleotides, polynucleotides, lipids, affinity ligands, antibodies, antibody fragments, aptamers, and the like. In some embodiments, the binding partner is an antibody or a fragment thereof. Specific binding in the context of the present disclosure refers to a binding reaction that determines the presence of a target analyte in the presence of a heterogeneous population. Thus, under specified assay conditions, a defined binding partner preferentially binds to a specific protein or an isoform of a specific protein and does not bind in significant amounts to other proteins or other isoforms present in the sample.

[0274] When the binding partner is an antibody, it may be a monoclonal or polyclonal antibody. The term antibody, as used herein, refers to an immunoglobulin molecule and the immunologically active portions of an immunoglobulin (Ig) molecule. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific polyclonal antibodies, antibody mimetics, chimeras, single chains, Fab, Fab’ and F(ab’)2 fragments, Fv, and Fab expression libraries.

[0275] Generally, the water-soluble fluorescent compounds and polymers of the present disclosure may be conjugated to a binding partner using techniques known to those of ordinary skill in the art or methods known to those of ordinary skill in the art in combination with the methods described herein to form a conjugated water-soluble fluorescent compound or polymer complex.

[0276] Provided is a labeled specific binding partner comprising a fluorescent compound or polymer according to the present disclosure; and a specific binding partner covalently attached to the fluorescent compound or polymer. The specific binding partner may be an antibody. The specific binding partner may be specific for a target analyte.

[0277] In some embodiments, the fluorescent compounds or water-soluble fluorescent polymers of the present disclosure may be conjugated to a binding partner using a method that directly modifies the core polymer described in US2020 / 0190253, which application is hereby incorporated by reference in its entirety. For example, DHP-cyanine dye-antibody conjugates can be prepared according to a general scheme as shown in Scheme 12 (Figure 9). As another example, polymer-antibody conjugates can be prepared according to a general scheme as shown in Scheme 13 (Figure 9). As a further example, sulfoxide-bridged DHP-cyanine dye antibody conjugates can be prepared as shown in Scheme 10 (Figure 8).

[0278] For example, the preparation of the polymer NHS ester may proceed as follows. Using a clean vial, dissolve 5 mg of the polymer in 1 mL of dry CH3CN. Add 15 mg of N,N,N’,N’-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) thereto and stir for an additional 2 minutes. Add 100 uL of N,N-diisopropylethylamine (DIPEA) thereto and continue stirring overnight while capping with parafilm. Thereafter, evaporate the organic solvent in the reaction mixture. Dissolve the crude NHS in approximately 750 uL of 1×BBS buffer (pH 8.8) by rapid vortexing and transfer it to a Zeba column 40K MWCO. Spin down the sample at 2200 RPM for 2 minutes and use this polymer NHS immediately.

[0279] The conjugate of the polymer NHS and the anti-CD4 antibody may proceed as follows. Take the polymer NHS in 1×BBS (about 800 uL), spin down, add to 0.6 mg of CD4, and mix with 100 uL of 0.5 M borate buffer (pH 9.0). Vortex rapidly for 30 seconds and mix in a thermomixer for 3-4 hours.

[0280] Purification of the polymer-antibody conjugate by a Histrap HP column may proceed as follows. Approach 1: After the crude reaction, use a Histrap HP column to purify the conjugate. Load the sample using 1×PBS buffer and collect the unbound fraction. This may be done using 20 column volumes (CV) of buffer. Then, exchange the buffer to wash the bound fraction that contains both the conjugate and the free antibody. This may be done by flowing through 10 CV using a mixture of 1×PBS and 0.25 M imidazole.

[0281] Approach 2: SP Sepharose FF column. Equilibrate the column, load the sample using 20 mM citrate buffer at pH 3.5, and collect the unbound fraction. This may be done using 20 CV of buffer. Then, exchange the buffer to elute the bound fraction that contains both the conjugate and the free antibody. This may be done by flowing through 20 CV using 20 mM Tris buffer at pH 8.5.

[0282] Approach 3: Load the crude conjugate into a tangential flow filtration system equipped with a 300K molecular weight cut-off (MWCO) membrane. Wash the conjugate using 1×PBS until the filtrate shows no absorption at 405 nm or 355 nm. Then, concentrate the compound.

[0283] Purification of the polymer-antibody conjugate by a size exclusion chromatography (SEC) column may proceed as follows. Load the crude conjugate containing the free antibody onto a size exclusion column using 1×PBS. After checking the absorption spectrum, pool the tubes and concentrate in an Amicon Ultra-15 with a 30KDa MWCO centrifugal concentrator.

[0284] Purification of the polymer-antibody conjugate by a Nuvia HR-S column (Bio-Rad Laboratories, Inc.) may proceed as follows. The crude polymer-antibody conjugate mixture is loaded onto a Nuvia HR-S column using a biological buffer having a pH between about 2 and about 14 and a conductivity of less than 3 mS / cm. Due to the charge-charge interaction between the matrix and the biomolecule, the polymer antibody conjugate will bind to the resin, while at the same time the free polymer dye will pass through without interacting with the resin. The charge-charge interaction between the matrix and the polymer antibody conjugate is disrupted by using a salt (e.g., NaCl, KCl, phosphate, etc.) at a concentration in the range of about 0.1 to 2 M. The salt concentration may be decreased by adjusting the pH of the elution buffer. For example, the conjugate may be eluted using a gradient of a biological buffer and salt (e.g., NaCl, KCl) at a concentration of about 100 to 1000 mM and a pH between about 6 and about 10.

[0285] Purification of the conjugate by a Nuvia cPrime column (Bio-Rad Laboratories, Inc.) may proceed as follows. The crude polymer-antibody conjugate mixture is loaded onto a Nuvia cPrime column using a biological buffer having a pH between about 2 and about 14 and a salt (e.g., NaCl, KCl) concentration in the range of 0 to about 1 M. The unreacted polymer will pass through the column, while at the same time the polymer-antibody conjugate will bind to the column. The polymer-antibody conjugate is eluted by increasing the pH of the elution buffer. For example, the crude polymer antibody conjugate may be loaded into a Nuvia cPrime column using a biological buffer at pH 5.0, 5 mM NaCl, and eluted with a biological buffer having a pH of 7.0 and a gradient of salt concentration from 5 to 500 mM.

[0286] Purification of the conjugate by anti-mouse anti-H+L antibody-agarose beads may proceed as follows. Mix the crude polymer-antibody conjugate mixture with anti-mouse anti-H+L antibody-agarose beads in a biological buffer having a pH between about 6 and about 8 at room temperature for about 30 minutes. The anti-mouse anti-H+L antibody-agarose beads will bind to the polymer antibody conjugate. Unreacted polymer is removed by washing with the above-described biological buffer at a speed of 300 g for 3 minutes using bench-top centrifugation. Repeat the washing process at least 3 times. To elute the polymer-antibody conjugate, apply an IgG elution buffer having a pH range from about 2 to about 4, wash the antibody-agarose beads, and incubate for about 10 to 15 minutes. Centrifugation is performed to collect the flow-through containing the polymer antibody conjugate. VII. Methods for Detecting Analytes

[0287] The present disclosure provides a method for detecting a target analyte in a sample, comprising: preparing a sample suspected of containing the target analyte; contacting the sample with a specific binding partner conjugated to a fluorescent compound or polymer of the present disclosure, wherein the binding partner is capable of interacting with the target analyte; and detecting light emitted from the conjugated fluorescent compound or polymer complex by applying a light source to the sample capable of exciting the fluorescent compound or polymer. In a typical assay, the water-soluble fluorescent compound or polymer of the present disclosure is readily excitable with light having a wavelength between about 420 nm and about 900 nm, and the emitted light is typically between about 450 nm and about 1000 nm.

[0288]

[0289] ​Alternatively, the excitation light may have a wavelength between about 500 nm and about 850 nm, and the emitted light may have a wavelength between about 550 nm and about 950 nm. The fluorescent compounds and polymers of the present disclosure may have an excitation spectrum tuned to UV, violet, blue, yellow, green, red, and NIR or another laser light, depending on the design of the compound or polymer.

[0290] In the method of the present disclosure, the fluorescent compound or polymer may be any water-soluble fluorescent compound or polymer of the present disclosure disclosed herein.

[0291] A method for detecting a target analyte in a sample, the method comprising the steps of preparing a sample suspected of containing the analyte; and contacting the sample with a specific binding partner conjugated to a fluorescent compound, polymer, or tandem dye according to the present disclosure, wherein the binding partner can interact with the target analyte. The binding partner may be a protein, peptide, affinity ligand, antibody, antibody fragment, carbohydrate, lipid, nucleic acid, or aptamer. If the binding partner is an antibody, the method may be configured for flow cytometry; the water-soluble fluorescent polymer may be bound to a substrate; the analyte may be a protein expressed on the cell surface; the method may be configured as an immunoassay; or the method may further comprise the step of preparing an additional specific binding partner for simultaneously detecting an additional analyte. (VIII) Tandem dye

[0292] The compounds, polymers, and labeled specific binding partners of the present disclosure can transfer energy to a linked acceptor chromophore or a linked donor chromophore. The compounds, polymers, and labeled specific binding partners of the present disclosure may be covalently bound to an acceptor or donor chromophore at an energy-receiving proximity such that upon excitation of the donor, energy is transferred to a covalently linked acceptor signaling chromophore and emitted therefrom. Mechanisms for energy transfer between the compounds, polymers, and labeled specific binding partners of the present disclosure and a donor or acceptor chromophore linked thereto include, for example, resonance energy transfer (e.g., Förster (or fluorescence) resonance energy transfer, FRET), quantum charge transfer (Dexter energy transfer), and the like. The terms "acceptor chromophore" and "acceptor fluorophore" are used interchangeably herein. The terms "donor chromophore" and "donor fluorophore" are used interchangeably herein.

[0293] Accordingly, the present disclosure provides tandem dyes comprising a fluorescent compound, polymer, or labeled specific binding partner according to the present disclosure, and an acceptor chromophore or donor chromophore covalently bound to the fluorescent compound, polymer, or labeled specific binding partner. In some embodiments, the fluorescent compounds, polymers, or water-soluble fluorescent compounds or polymers of the present disclosure, and conjugates thereof are donor dyes. In some cases, the fluorescent compounds, polymers, or water-soluble fluorescent compounds or polymers of the present disclosure, and conjugates thereof are acceptor dyes. In some cases, the fluorescent compounds, polymers, or water-soluble fluorescent compounds or polymers of the present disclosure, and conjugates thereof include an additional acceptor dye (e.g., a fluorophore or chromophore) bound to a donor dye having the structure of the present disclosure, or an additional donor dye (e.g., a fluorophore or chromophore) bound to an acceptor dye having the structure of the present disclosure.

[0294] When the light source excites the donor compound or the polymer backbone, the fluorophore, acceptor dye, or chromophore can absorb energy of an appropriate wavelength and emit or transfer the energy. The fluorophore (FP), chromophore, donor, or acceptor dye linked to the fluorescent dye of the present invention may have an absorption or emission profile that overlaps with the absorption or emission profile of the DHP-cyanine compound or polymer of the present disclosure. The FP, chromophore, donor, or acceptor dye linked to the fluorescent dye of the present invention has an absorption maximum longer than 405 nm or 575 nm and an emission maximum longer than 428 nm, 450 nm, or 600 nm, and optionally, a fluorescence quantum yield greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 10%. It may be a fluorescent dye that exhibits.

[0295] Any convenient fluorescent dye may be used in the tandem dye as an acceptor chromophore or a donor chromophore. The chromophore and fluorophore may be selected from coumarin, fluorescein, rhodamine, cyanine, bodipy, or other polycyclic aromatics. Many fluorophores are commercially available, including, but not limited to, any dye available from Beckman Coulter, Inc., including, but not limited to, the SuperNova polymer dye; any dye available from Becton Dickinson Biosciences, including, but not limited to, the BD Horizon Brilliant™ polymer dye; any dye available from ThermoFisher Scientific, including, but not limited to, the Super Bright polymer dye, and including, but not limited to, 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; ATTO 390, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 532, ATTO 550, ATTO 565, ATTO 590, ATTO 594, ATTO 610, ATTO 620, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein (5-FAM), 5-carboxynaphthofluorescein, 5-carboxytetramethylrhodamine (5-TAMRA), 5-FAM (5-carboxyfluorescein), 5-ROX, 6-TAMRA, 6-carboxyrhodamine 6G, 6-CR6G, 6-JOE, 6-FAM, 6-ROX, Bodipy 492 / 515, Bodipy 493 / 503, Bodipy 500 / 510, Bodipy 505 / 515, Bodipy 530 / 550, Bodipy 542 / 563, Bodipy558 / 568, Bodipy 564 / 570, Bodipy 576 / 589, Bodipy 581 / 591, Bodipy 630 / 650-X, Bodipy 650 / 665-X, Bodipy 665 / 676, Bodipy Fl, Bodipy R6G, Bodipy TMR, Bodipy TR, CF 488A, CF 555, CF 568, CF 594ST, CF 633, CF 640R, CF 647, CF 660C, CF 680, CF680R, CF 750, CF 770, CF 790, CL-NERF, CMFDA, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, DDAO, DiA, DiD, DiI, DyLight 488, DyLight 550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 755, DyLight 800, DiO, DiR, DM-NERF, DsRed, DTAF, DY-490, DY-495, DY-505, DY-530, DY-547, DY-548, DY-549, DY-549P1, DY-550, DY-554, DY-555, DY-556, DY-560, DY-590, DY-591, DY-594, DY-605, DY-610, DY-615, DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-636, DY-647, DY-648, DY-649, DY-649P1, DY-650, DY-651, DY-652, DY-654, DY-675, DY-676, DY-677, DY-678, DY-679, DY-679P1, DY-680, DY-681, DY-682, DY-700, DY-701, DY-703, DY-704, DY-730, DY-731, DY-732, DY-734, DY-749, DY-750, DY-751, DY-752, DY-754, DY-776, DY-777, DY-778, DY-780, DY-781, DY-782, DY-800, DY-831, Eosin, Erythrosin, FITC, Fluo-3, Fluo-4, Fluor-Ruby, FluorX, FM 1-43, FM 1-46, iFluor 488, iFluor 555, iFluor 594, iFluor 647, iFluorIt may be selected from Alexa Fluor dyes including 680, iFluor 700, iFluor 750, iFluor 780, Lyso Tracker Green, Lyso Tracker Yellow, Mitotracker Green, Mitotracker Orange, Mitotracker Red, NBD, Oregon Green 488, Oregon Green 514, PKH26, PKH67, Resorufin, RH 414, Rhod-2, Rhodamine, Rhodamine 110, Rhodamine 123, Rhodamine 6G, Rhodamine B, Rhodamine Green, Rhodamine Red, Rose Bengal, Spectrum Green, Spectrum Orange, Spectrum Red, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO 23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44, SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO 80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX Green, SYTOX Orange, Texas Red, Tide Fluor 2 (TF2), Tide Fluor 2WS (TF2WS), Tide Fluor 3 (TF3), Tide Fluor 3WS (TF3WS), Tide Fluor 4 (TF4), Tide Fluor 5WS (TF5WS), Tide Fluor 6WS (TF6WS), Tide Fluor 7WS (TF7WS), Tide Fluor 8WS (TF8WS), TRITC, and XTRITC. As acceptor dyes useful in the present disclosure, for example, cyanine dyes, xanthene dyes, coumarin dyes, thiazine dyes, acridine dyes, FITC, CY3B, Cy55, Alexa 488, Texas red, Cy5, Cy7, Alexa750, Cy55, Cy3B, Cy3.5, Alexa 750, 800 CW, Biotium CF 555, diethylcoumarin, DY705 (Dyomics), DY431, DY485XL, DY500XL, DY610, DY640, DY654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831 and 800CW may be present. The acceptor dye may be a pendant acceptor dye. The tandem dye may include one or more, two or more, three or more, 1 - 30, 2 - 20, or 2.5 - 10 acceptor dye moieties of a DHP - cyanine or DHP - squarylium compound or polymer according to the present disclosure. The tandem dye may include a donor dye and one or more, two or more, three or more, 1 - 30, 2 - 20, or 2.5 - 10 acceptor DHP - cyanine or DHP - squarylium acceptor dye compounds according to the present disclosure.

[0296] The fluorescent tandem dye can be prepared using techniques known to those skilled in the art or methods known in the art in combination with the methods described herein. The tandem dye may be water - soluble.

[0297] In some embodiments, instead of attaching the acceptor dye, chromophore, fluorophore, functional moiety, and binding partner to a compound or polymer backbone, a method of directly modifying the core polymer described in U.S. Patent No. 11,584,825 may be used to attach them to the compounds or polymers of the present disclosure via a linker moiety, and this application is hereby incorporated by reference in its entirety. In some embodiments, the present disclosure provides a fluorescent compound, polymer, or labeled specific binding partner according to the present disclosure; and a tandem dye comprising an acceptor chromophore covalently attached to the fluorescent compound, polymer, or labeled specific binding partner. Sample

[0298] Samples in the methods of the present disclosure may be, for example, blood, bone marrow, spleen cells, lymphocytes, bone marrow aspirates (or any cells obtained from bone marrow), urine (washing fluid), serum, saliva, cerebrospinal fluid, urine, amniotic fluid, interstitial fluid, feces, mucus, or tissue (e.g., tumor samples, disaggregated tissue, disaggregated solid tumors). In certain embodiments, the sample is a blood sample. In some embodiments, the blood sample is whole blood. Whole blood may be obtained from a subject using standard clinical procedures. In some embodiments, the sample is one or more cell subsets of whole blood (e.g., red blood cells, white blood cells, lymphocytes (e.g., T cells, B cells or NK cells), phagocytes, monocytes, macrophages, granulocytes, basophils, neutrophils, eosinophils, platelets, or any cells having one or more detectable markers). In some embodiments, the sample may be from cell culture.

[0299] The subject may be a human (e.g., a patient suffering from a disease), or a commercially important mammal including, for example, a monkey, a cow, or a horse. The sample may be obtained from a household pet including, for example, a dog or a cat. In some embodiments, the subject is an experimental animal used as an animal model of a disease or for drug screening, such as a mouse, a rat, a rabbit, or a guinea pig. Analyte

[0300] "Analyte" or "target analyte", as used herein, refers to a substance, such as a molecule, whose abundance / concentration is determined by some analytical procedure. For example, in the present disclosure, the analyte may be a protein, a peptide, a nucleic acid, a lipid, a small carbohydrate molecule, or a target-related biomolecule.

[0301] The target analyte may be, for example, a nucleic acid (DNA, RNA, mRNA, tRNA, or rRNA), a peptide, a polypeptide, a protein, a lipid, an ion, a monosaccharide, an oligosaccharide, a polysaccharide, a lipoprotein, a glycoprotein, a glycolipid, or a fragment thereof. In some embodiments, the target analyte is a protein, for example, a structural microfilament, microtubule, and intermediate diameter filament protein, an organelle-specific marker, a proteasome, a transmembrane protein, a surface receptor, a nuclear pore protein, a protein / peptide translocase, a protein folding chaperone, a signal transduction scaffold, an ion channel, etc. The protein may be, but is not limited to, an activatable protein or a protein that is differentially expressed or activated in diseased or abnormal cells, including transcription factors, DNA and / or RNA-binding and modifying proteins, nuclear import and export receptors, regulators of apoptosis or survival, etc.

[0302] The compounds, polymers, labeled specific binding partners, or tandem dyes according to the present disclosure may be water-soluble fluorescent dyes. The fluorescent compounds, labeled specific binding partners, or tandem dyes according to the present disclosure may exhibit a solubility of >1 mg / mL, >2 mg / mL, >3 mg / mL, >4 mg / mL, >5 mg / mL, >6 mg / mL, >7 mg / mL, >8 mg / mL, >9 mg / mL, >10 mg / mL, >20 mg / mL, >30 mg / mL, >40 mg / mL, >50 mg / mL, >80 mg / mL, or >100 mg / mL in water at ambient room temperature.

[0303] Compounds, polymers, labeled specific binding partners, or tandem dyes according to the present disclosure may exhibit a maximum excitation wavelength (λex) within the range of >400 nm, >500 nm, >600 nm, >700 nm, >800 nm, >850 nm, or from about 400 nm to about 1,000 nm, or from about 600 nm to about 950 nm. DHP-cyanine compounds, polymers, labeled specific binding partners comprising a DHP-cyanine compound or a DHP-cyanine tandem dye, or DHP-cyanine acceptor dyes comprising a DHP-cyanine compound according to the present disclosure may exhibit an emission maximum (λem) within the range of >550 nm, >650 nm, >750 nm, >850 nm, or >900 nm, or between about 600 and about 1200, or from about 550 nm to about 1050 nm, or from about 650 nm to about 1050 nm. assay

[0304] The DHP-cyanine dyes, tandem dyes, labeled specific binding partners, compositions, methods, and systems described herein may be used in a variety of applications, including diagnostic and investigative applications where labeling, detection, and / or analysis of a target of interest is desirable. Such applications include methodologies such as cytometry, microscopy, immunoassays (e.g., competitive or non-competitive), fluorescence in situ hybridization (FISH), cell tracing, receptor labeling, fluorescence spectroscopy, evaluation of free analytes, evaluation of receptor-binding ligands, and the like. The compositions, systems, and methods described herein may be useful in the analysis of any of several samples, including but not limited to biological fluids, cell culture samples, and tissue samples. In certain embodiments, the compositions, systems, and methods described herein are used with a fluorescent label to detect an analyte in a sample, if present, in methods such as fluorescence-activated cell sorting or analysis, immunoassays, immunostaining, and the like. In certain instances, the compositions and methods are used in applications where the evaluation of a sample for the presence of a target analyte is the goal. In some cases, the methods and compositions are used in any assay format where the detection and / or analysis of a target from a sample is the goal, including but not limited to flow cytometry, fluorescence microscopy, in-situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescence dye purification chromatography. In certain instances, the methods and compositions are used in any application where the fluorescent labeling of a target molecule is the goal. The subject compositions may be adapted for use in any convenient application where pairs of specific binding members, such as biotin-streptavidin and hapten-anti-hapten antibodies, are used.

[0305] Assay systems that utilize binding partners and fluorescent labels to quantify binding molecules are well known. Examples of such systems include flow cytometers, scanning cytometers, imaging cytometers, fluorescence microscopes, and confocal fluorescence microscopes.

[0306] In some embodiments, fluorescence is detected using flow cytometry. Several devices suitable for this use are available and are known to those of skill in the art. Examples include the BCI Navios, Gallios, Aquios, and CytoFLEX flow cytometers. In other embodiments, an assay is used. The assay may be an immunoassay. Examples of immunoassays useful in the present disclosure include, but are not limited to, fluorescence luminescence assays (FLA). The assay may also be performed on a protein array. When the binding partner is an antibody, a sandwich assay of an antibody or antibodies can also be used. A sandwich assay refers to constructing layers of various binding partners and reporter components using sequential recognition events to signal the presence of a particular analyte. Examples of sandwich assays are disclosed in U.S. Patent No. 4,486,530 and the references cited therein. (IX) Kit

[0307] The present disclosure provides a kit comprising at least one DHP-cyanine or DHP-squaraine compound or polymer, a labeled specific binding partner, or a tandem dye according to the present disclosure. Aspects of the invention further include kits for use in the practice of the methods and compositions of interest. The compositions of the invention may be included as reagents in the kit as starting materials, or may be provided for use, for example, in the methodologies described above.

[0308] The kit may include a DHP-cyanine or DHP-squaraine compound or polymer, a labeled specific binding partner, or a tandem dye as described herein, and a container. Any convenient container, such as a tube, bottle, or well in a multi-well strip or plate, box, bag, insulated container, etc. may be utilized. In some cases, the subject kit may include one or more components selected from a DHP-cyanine or DHP-squaraine compound or polymer according to the present disclosure, a labeled specific binding partner, or a tandem dye, fluorophore, chromophore, specific binding member, specific binding member conjugate, specific binding member bound to a support, cell, support, biocompatible aqueous elution buffer, and / or instructions for use. In some embodiments of the kit, the DHP-cyanine or DHP-squaraine compound or polymer, or tandem dye according to the present disclosure is covalently bound to a specific binding partner.

[0309] In some cases, the subject kit may be a "labeled kit" that includes a DHP-cyanine or DHP-squaraine compound or polymer according to the present disclosure, or a tandem dye, and a chemoselective functional group (also referred to as a "conjugation tag") to which any convenient target moiety of interest (e.g., a donor or acceptor dye, fluorophore, chromophore, specific binding partner, support) can conjugate, such as an NHS ester of a DHP-cyanine or DHP-squaraine compound or polymer. Chemoselective functional groups can include specific functional groups on biomolecules (e.g., proteins or antibodies), such as reactive groups targeting primary amines, sulfhydryls, carboxyls, or carbohydrates (e.g., biotin). The chemoselective functional group may be one used in a "click chemistry" reaction.

[0310] In certain cases, the conjugation tag has a maleimide functional group and the target moiety has a thiol functional group, or vice versa. In some cases, the conjugation tag has an alkyne (e.g., cyclooctyne group) functional group and the target moiety has an azide functional group, or vice versa, and these may be conjugated via click chemistry. In certain cases, the conjugation tag has an alkene (e.g., cyclooctene group) functional group and the target moiety has a tetrazine functional group, or vice versa, and these may be conjugated via an inverse-demand Diels–Alder cycloaddition reaction. In some cases, the conjugation tag has an amine-reactive chemical group, such as an NHS ester (N-hydroxysuccinimide ester) or an imidoester functional group, and the target moiety has an NH2 functional group, or vice versa. In some cases, the conjugation tag has a biotin-binding protein (e.g., avidin, streptavidin, or neutravidin) and the target moiety has a biotin molecule, or vice versa, and these can interact non-covalently.

Example

[0311] (Example 1) General procedure for synthesizing DHP indole-cyanine compounds 1-5 The synthetic procedure for synthesizing the DHP indole-cyanine compound is shown in FIG. 2, Scheme 2. The synthetic procedures for preparing Compounds 1-5 as shown in Table 1 are shown below.

[0312] Synthesis of DHP-diol intermediate 7; 3-bromo-9,10-dihydrophenanthrene-9,10-diol:

Chemical formula

[0313] The commercially available 3-bromo DHP-dione was reduced using NaBH₄ according to the previously published procedure, and the yield was 85% (US11208527B2).

[0314] Synthesis of DHP-OMe intermediate 8; 3-bromo-9,10-dimethoxy-9,10-dihydrophenanthrene:

Chem.

[0315] To a solution of DHP-diol (1 mmol) in THF purged with nitrogen in ice, NaH (4 mmol) was added. After 5 minutes, a solution of methyl iodide was added dropwise. The reaction mixture was stirred at room temperature overnight. The next day, the reaction was quenched using a few drops of water. After evaporating THF, the reaction mixture was extracted using chloroform. The pure compound was obtained after column chromatography, and the yield was 80%.

[0316] Synthesis of DHP-hydrazone intermediate 9; 1-(9,10-dimethoxy-9,10-dihydrophenanthren-3-yl)-2-(diphenylmethylene)hydrazine:

Chem.

[0317] Benzophenone hydrazone (1.50 mmol), Pd(OAc)₂ (0.015 mmol), and (±)-BINAP (12.7 mg, 0.020 mmol; (2,2’-bis(diphenylphosphino)-1,1’-binaphthyl)) were suspended in toluene (2 mL). The reaction vessel was briefly purged with argon and stirred for 3 minutes. To the resulting purple solution, the DHP derivative (1.36 mmol), NaOt-Bu (1.90 mmol), and toluene (0.4 mL) were added. The reaction vessel was heated at 100 °C overnight. Then, the reaction mixture was cooled to 25 °C and filtered through a short pad of celite. The clear filtrate was concentrated, and the residue was purified by flash column chromatography, and the yield was 60%.

[0318] Synthesis of DHP - indole intermediate 10; 5,6 - dimethoxy - 8,8,9 - trimethyl - 6,8 - dihydro - 5H - naphtho[2,1 - f]indole:

Chemical formula

[0319] DHP - hydrazone (1.35 mmol), isopropyl methyl ketone (2.03 mmol), and TsOH·H₂O (6.75 mmol) were heated under reflux in ethanol (20 mL) for 15 hours. The crude product was purified by flash column chromatography to obtain the DHP - indole derivative with a yield of 25%.

[0320] Synthesis of DHP - indole sulfonate intermediate 11; 3 - (5,6 - dimethoxy - 8,8,9 - trimethyl - 6,8 - dihydro - 5H - 10l4 - naphtho[2,1 - f]indol - 10 - yl)propane - 1 - sulfonate:

Chemical formula

[0321] The DHP - indole intermediate 10 (1 mmol) was heated with 1,3 - propane sultone at 120 °C for 2 hours. Then, the reaction mixture was cooled, washed with ether, and dried to obtain the sulfonated DHP - indole intermediate compound 11.

[0322] General procedure for synthesizing Compounds 1 - 3

[0323] A solution of sulfonated DHP indole (1 mmol) 11 in MeCN was added to a refluxing mixture of the corresponding aldehyde dianiline hydrochloride (1.20 mmol), Ac2O (120 mL), DIEA (600 mL), and NaOAc (78 mg) in a mixed solvent of MeCN / DCM. The resulting mixture was stirred for an additional 2 hours, after which excess sulfonated DHP indole (1.50 mmol) was added. The mixture was refluxed again for 2 hours. The reaction was quenched with water and the solvent was removed by rotary evaporator. The residue was washed successively with EtOAc, MeCN, EtOAc, and water and then purified by column chromatography to obtain Compounds 1 - 3 as shown in Table 1.

[0324] General procedure for the synthesis of Compounds 4 - 5

[0325] A solution of sulfonated DHP indole (1 mmol) 11 in MeCN was added to a refluxing mixture of the corresponding aldehyde dianiline hydrochloride (1.20 mmol), Ac2O (120 μL), DIEA (600 μL), and NaOAc (78 mg) in a mixed solvent of MeCN / DCM. The resulting mixture was stirred for an additional 2 hours, after which excess 1-(4-sulfobutyl)-2,3,3-trimethyl-4,5-benzindolium, inner salt (1.50 mmol) was added. The mixture was refluxed again for 2 hours. The reaction was quenched with water and the solvent was removed by rotary evaporator. The residue was washed successively with EtOAc, MeCN, EtOAc, and water and then purified by column chromatography to obtain Compound 4 or 5.

[0326] Compounds 1 - 5 may be derivatized with sulfonamide PEG side groups to enhance water solubility. For example, any of Compounds 1 - 5 may be treated with thionyl chloride in DMF to form a propanesulfonyl chloride intermediate, and then treated with NH2PEG 550 OMe, TEA in CH2Cl2 to form sulfonamide PEG side groups. (Example 2) Synthesis of DHP-indole propyl sulfonate intermediate 21

[0327] Figure 6 shows synthetic scheme 7 for preparing the N-propylsulfonate DHP-indole compound 21 from dihydrophenanthrene.

[0328] Synthesis of 2-nitro DHP intermediate 17; 2-nitro-9,10-dihydrophenanthrene:

Chemical formula

[0329] To 8 mL of 1 mmol of 9,10-dihydrophenanthrene, 2 mL of acetic acid and fuming nitric acid (20%) were added. The mixture was stirred while maintaining a temperature of 30 °C. After 2 hours, the red solution was poured into crushed ice. The precipitation was completed and the aqueous layer was decanted. It was washed with additional cooling water (500 mL). It was dried and the obtained 2- and 4-nitro 9,10-dihydrophenanthrene 17 was used in the next step without further purification.

[0330] Synthesis of 9,10-dihydrophenanthrene-2-amine intermediate 18:

Chemical formula

[0331] Na2S (3 mmol) was dissolved in a minimum amount of warm water and mixed with 240 mg of Na2CO3. This was added to 2.2 mmol of nitro-9,10-dihydrophenanthrene 17 in methanol. The mixture was heated overnight. The next day, it was extracted using chloroform and dried. Chromatography was performed to separate 2- and 4-amino-9,10-dihydrophenanthrene 18.

[0332] Synthesis of (9,10-dihydrophenanthrene-2-yl)hydrazine intermediate 19:

Chemical formula

[0333] To an ice-cold suspension of 2-amino-9,10-dihydrophenanthrene 18 (1 mmol) in 20% HCl (2 mL), sodium nitrite (100 mg in 250 μL) was added dropwise. Stirring was continued at 0 °C for 1 h. A small amount of urea was added to decompose the excess nitrous acid. The temperature was maintained at -5 °C. To the diazonium salt solution, SnCl₂·2H₂O (250 mg) in a minimal amount of concentrated HCl was added and stirred for 2 h. Thereafter, the precipitate was filtered and dried. Then, the precipitate was dissolved in chloroform and extracted using water containing 10% NaOH. The organic layer was dried and subsequently chromatographed to obtain 19.

[0334] Synthesis of 2,3,3-trimethyl-4,5-dihydro-3H-naphtho[2,1-e]indole intermediate 20:

Chemical formula

[0335] 2-Hydrazino-9,10-dihydrophenanthrene 19 (1 mmol) was dissolved using 5 mL of glacial acetic acid. This was mixed with isopropyl methyl ketone (2 mmol) and the reaction was carried out at 110 °C overnight. The next day, extraction was carried out using ethyl acetate and water, and subsequently chromatography was carried out to obtain 20.

[0336] Synthesis of DHP-indolepropyl sulfonate intermediate 21; 3-(2,3,3-trimethyl-4,5-dihydro-3H-naphtho[2,1-e]indol-1-ium-1-yl)propane-1-sulfonate intermediate 21:

Chemical formula

[0337] The dihydrophenanthrene indole derivative 20 (1 mmol) obtained from the previous step was heated with 1,3-propanesultone at 120 °C for 2 h. After cooling, the product was washed with ethyl acetate to remove any unreacted starting materials and 21 was obtained. (Example 3) Synthesis of Sulfoxide-Bridged DHP-Indole Cyanine Antibody 25

[0338] Figure 7 shows synthetic schemes 8 and 9 for preparing sulfoxide-bridged DHP indole cyanine dye 25 from -propylsulfonate DHP-indole intermediate compound 21.

[0339] Synthesis of Sulfoxide-Bridged DHP-Indole Propylsulfonate Intermediate 22; 2-((l1-oxidaneyl)dioxo-l6-sulfaneyl)-6-(3-((l1-oxidaneyl)dioxo-l6-sulfaneyl)propyl)-7,8,8-trimethyl-9,10-dihydro-8H-6l4-thieno[2’,3’,4’,5’:4,5]phenanthro[2,1-b]pyrrole 4,4-dioxide 22:

[0340] 2-Aminodihydrophenanthrene 18 was reacted using NaNO2 / HCl in an ice bath to convert it to the corresponding diazonium salt. Reacting this with SnCl2 / HCl at 0 - 5 °C formed 2-hydrazinodihydrophenanthrene (19). This product was used in the next step without further purification. Then, 19 was reacted with 3-methyl-2-butanone under Fischer indole synthesis conditions to produce isomers of DHP indole (20). The formation of the indole ring could occur at the 1 and 2 positions as well as the 2 and 3 positions of DHP. Both isomers were isolated using flash chromatography. The DHP with indole formed at the 1,2-position was taken and reacted with 1,3-propanesultone to obtain intermediate DHP-indole compound 21 in a 100% yield. This was further treated with 20% oleum pre-cooled to -20 °C, and the reaction was continued at room temperature overnight. The crude product was precipitated using ether, followed by C18 reverse-phase chromatography (from 100% water to 95% water 5% methanol) to obtain compound 22.

[0341] Synthesis of sulfoxide-bridged DHP-indole cyanine dye 25; (Z)-2-((2E,4E)-5-(2-((l1-oxidanyl)dioxo-l6-sulfanyl)-6-(3-((l1-oxidanyl)dioxo-l6-sulfanyl)propyl)-8,8-dimethyl-4,4-dioxide-9,10-dihydro-8H-6l4-thieno[2’,3’,4’,5’:4,5]phenanthro[2,1-b]pyrrol-7-yl)penta-2,4-dien-1-ylidene)-1-(3-((l1-oxidanyl)dioxo-l6-sulfanyl)propyl)-3,3-dimethylindoline-5-carboxylic acid 25:

[0342] Sulfoxide-bridged DHP indole (22) was treated with carboxylic acid-functionalized sulfonated indole (23) and 24 in a mixture of acetic acid and acetic anhydride in the presence of sodium acetate. After the reaction, the crude mixture was mixed with cold ether and the precipitate was isolated. This was purified using C18 reverse-phase chromatography (20% MeOH ~ 80% water) to obtain DHP-sulfoxide Cy5 dye 25, which had an Abs maximum of 666 nm, an emission maximum of 686 nm, and a extinction coefficient of 150450 M -1 cm -1 and was found to show. (Example 4) Synthesis of sulfoxide-bridged DHP-indole cyanine antibody conjugate 27

[0343] Figure 8 shows synthetic scheme 10 for preparing sulfoxide-bridged DHP indole cyanine dye antibody conjugate 27 from sulfoxide-bridged DHP indole cyanine dye 25.

[0344] Synthesis of sulfoxide-bridged DHP-indole cyanine dye antibody conjugate 27:

[0345] The DHP-sulfoxide Cy5 dye (25) was converted to the active NHS ester 26 using a TSTU / DIPEA / DMSO / CH3CN mixture. This was purified using a precipitation technique with diethyl ether. The active ester 26 was conjugated to the available amino groups in an antibody (Ab) at pH ~7.5 - 8. Unreacted excess dye was separated using a size exclusion spin column, and the resulting pure conjugate (27) was used in flow cytometry studies. (Example 5) Synthesis of the DHP-indole intermediate 33; 1-(3-((λ1-oxidanyl)dioxo-l6-sulfanyl)propyl)-2,3,3,5,5-pentamethyl-4,5-dihydro-3H-1λ4-naphtho[2,1-e]indole 33

[0346] Figure 10 shows an exemplary synthetic scheme 14 for preparing the N-propylsulfonate DHP-indole intermediate compound 33 from chloroaminobenzoic acid 28. Figure 10 also shows an exemplary route for preparing the sulfoxide-bridged DHP indole cyanine dye 36 from the N-propylsulfonate DHP-indole intermediate compound 33.

[0347] Preparation of 2-chloro-4-hydrazinobenzoic acid 29 [Chemical formula]

[0348] Chloroaminobenzoic acid 28 was mixed with 20% HCl at 0 °C. NaNO2 (1 eq) in water was added here and stirred for 1 hour. Then, excess urea was added and stirred for 5 minutes. The reaction mixture was further cooled to about -5 °C. Stannous chloride (2.1 eq) in concentrated HCl was added here and stirred for a further 2 hours. Then, the reaction mixture was poured into ice-cold water and filtered to obtain the hydrazine compound 29.

[0349] Preparation of 5-chloro-2,3,3-trimethyl-3H-indole-4-carboxylic acid 30 [Chemical formula]

[0350] The hydrazino compound 29 was heated with 3-methyl-2-butanone at 120 °C overnight. The next day, the crude product was precipitated, washed repeatedly with EtOAc, and then subjected to silica chromatography to obtain the indole product 30 as a mixture of isomers.

[0351] Preparation of 2,3,3,5,5-pentamethyl-4,5-dihydro-3H-naphtho[2,1-e]indole 31

Chemical formula

[0352] Under Schlenk conditions, a reaction was carried out between the indole derivative 30 and 1-(tert-butyl)-2-iodobenzene in DMF in the presence of Pd(OAc)2, K2CO3, tri(o-tolyl)phosphine (P(p-tolyl)3), and tetrabutylammonium bromide at 140 °C overnight. The next day, the crude product 31 was purified using column chromatography.

[0353] Preparation of 1-(3-((l1-oxidanidyl)dioxo-l6-sulfanyl)propyl)-2,3,3,5,5-pentamethyl-4,5-dihydro-3H-1l4-naphtho[2,1-e]indole 33

Chemical formula

[0354] Compound 31 was mixed with 1 equivalent of 1,3-propanesultone 32 in THF in a sealed tube at 120 °C for 90 minutes. Thereafter, the crude product 33 was precipitated and washed using cold ether.

[0355] Synthesis of sulfoxide-bridged DHP-indolylpropyl sulfonate intermediate 34, 2-((l1-oxidanyl)dioxo-l6-sulfanyl)-6-(3-((l1-oxidanyl)dioxo-l6-sulfanyl)propyl)-7,8,8,10,10-pentamethyl-9,10-dihydro-8H-6l4-thieno[2’,3’,4’,5’:4,5]phenanthro[2,1-b]pyrrole 4,4-dioxide.

Chem.

[0356] Sulfoxide-bridged DHP-indolylpropyl sulfonate intermediate compound 34 was prepared from intermediate compound 33 in a manner similar to that described for the synthesis of compound 22 above.

[0357] Synthesis of sulfoxide-bridged DHP-indolocyanine dye 36, 2-((l1-oxidanyl)dioxo-l6-sulfanyl)-7-((1E,3E)-5-((Z)-5-((l1-oxidanyl)dioxo-l6-sulfanyl)-1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-6-(3-((l1-oxidanyl)dioxo-l6-sulfanyl)propyl)-8,8,10,10-tetramethyl-9,10-dihydro-8H-thieno[2’,3’,4’,5’:4,5]phenanthro[2,1-b]pyrrol-6-ium 4,4-dioxide.

Chem.

[0358] Sulfoxide-bridged DHP indole (34) was treated with carboxylic acid-functionalized sulfonated indole (35) in a mixture of acetic acid and acetic anhydride in the presence of potassium acetate. The crude reaction mixture was mixed with cold ether, and the precipitate was isolated. This was purified using C18 reverse-phase chromatography to obtain DHP-sulfoxide Cy5 dye 36, which was found to exhibit Abs max 663 nm and emission max 683 nm. The foregoing specification, examples, and data provide a complete description of the manufacture and use of the compositions of the invention. Since many embodiments of the invention can be made without departing from the spirit and scope of the invention, the invention resides in the claims appended hereto.

Claims

1. A fluorescent compound comprising a structure according to formula (II) 【Chemical 370】 (wherein each 【Chemical 371】 is independently selected from the group consisting of substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl groups, polycyclic aryl groups, monocyclic heteroaryl groups, and polycyclic heteroaryl groups, and at least one 【Chemical 372】 is 【Chemical 373】 or a derivative thereof, and the derivative optionally contains an additional aryl or heteroaryl group condensed with any available 【Chemical 374】 , and optionally, the derivative is 【Chemical 375】 , Each V' is independently SO ２ , SO, S, NR １１ , CR １１ , C(R １１ ), ２ , O, Si(R １１ ), ２ , >C=O, >Se=O, -CH=CH-, or -N=CH-; Each T is independently selected from the group consisting of C, C(R １ ), N, N(R １ ), P, O, S, and Si(R １ ); Each U is independently selected from the group consisting of NR １０ , O, P, Se, Te, and S; Each V is NR １１ , CR １１ , C(R １１ ), ２ , S, SO ２ , O, Se, Te, and Si(R １１ ), ２ and is independently selected from the group consisting of; Each X is independently CR １ R ２ or SiR １ R ２ and; Each Y is independently CR ８ R ９ or SiR ８ R ９ and; wherein Each R １ , R ２ , R ８ , and R ９ are a water-solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, a binding partner, a linked binding partner, a functional group, hydrogen, hydroxy, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, sulfonamide-PEG, phosphoramidate-PEG, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, carbonyl, acyl, alkylsulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamid, alkoxysulfonamide PEG, alkoxysulfonate, alkylsulfonate, alkylsulfonate, 【Chemical 376】 【Chemical 377】 【Chemical Formula 378】 Independently selected from the group consisting of, and optionally, R １ and R ８ together form an unsubstituted or substituted cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, cycloalkoxy, aryl, or heteroaryl having from 3 to 9 ring members; Each R ３ is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a water-solubilizing moiety, a chromophore, a functional moiety, a binding partner, and a PEG group; Each Q is independently a bond, NH, NR ４ , C １ ~C １２ alkylene, CHR ４ , or CH ２ ; Each Z is independently CH ２ , CHR ４ , O, NR ４ , or NH; Each W １ is independently a water-solubilizing moiety; L １ 、 L ２ 、 and L ３ are each independently selected linker moieties; each E is independently selected from the group consisting of a chromophore, a functional moiety, a substrate, and a binding partner; Each R ４ is independently selected from the group consisting of H, alkyl, PEG, solubilizing moiety, linker moiety, chromophore, functional group, conjugation tag, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, alkene, tetrazine, aldehyde, and thiol, or a protecting group thereof; Each R ７ is independently selected from the group consisting of H, hydroxyl, C １ to C １２ alkyl, C ２ to C １２ alkene, C ２ to C １２ alkyne, C ３ to C １２ cycloalkyl, C １ to C １２ haloalkyl, C １ to C １２ alkoxy, C ２ to C １８ (hetero)aryloxy, C ２ to C １８ (hetero)arylamino, C ２ to C １２ carboxylic acid, C ２ to C １２ carboxylic acid ester, and C １ to C １２ alkoxy, functional group, chemoselective functional group, conjugation tag, linked conjugation tag, linker, sulfonic acid, sulfonate, C １ to C １２ alkyl sulfonate, sulfonamide; Each R １０ is independently selected from the group consisting of hydrogen, a linker moiety, a linked reactive group, a conjugation tag, a linked conjugation tag, a linked ionic group, a linked chromophore, a linked solubilizing moiety, a solubilizing moiety, a chromophore, a binding partner, and a linked binding partner; Each R １１ is independently selected from the group consisting of hydrogen, a linker moiety, a linked reactive group, a linked ionic group, a linked chromophore, a linked solubilizing moiety, a chromophore, a binding partner, and a linked binding partner; Each R １２ 、R １３ 、and R １４ are independently selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C １ to C ６ alkyl, substituted or unsubstituted C １ to C ６ heteroalkyl, substituted or unsubstituted C １ to C ６ alkene, substituted or unsubstituted C ３ to C １０ cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, CO ２ R １ 、CONR １ R ２ 、-O-CH ２ CH ２ -PEG-R ７ 、-S-CH ２ CH ２ -PEG-R ７ 、-N-CH ２ CH ２ -PEG-R ７ 、O-aryl, S-aryl, N-aryl, -O-alkyl, S-alkyl, N-alkyl, and each alkyl or aryl may be optionally substituted with one or more R ７ 、PEG、PEG-R ７ 、or a linking group, and optionally, each R １２ 、R １３ and R １４ are independently substituted with an R ７ group; or at least two of R １０ 、R １１ 、R １２ 、R １３ 、and R １４ are unsubstituted or substituted unsaturated or partially unsaturated C ３ to C １０ cycloalkyl group, unsubstituted or substituted unsaturated or partially unsaturated C ３ to C １０ heterocycloalkyl optionally substituted with O, C ３ to C １０ a heterocycloalkyl, an unsubstituted or substituted unsaturated or partially unsaturated cycloalkyl group having 3 to 8 ring members, or a substituted or unsubstituted heterocycloalkyl having 3 to 8 ring members, which together form a heterocycloalkyl optionally substituted with O; Each K is independently a covalent bond, O, S, P, NR １ , Se, Te, CR １ R ２ , or CH ２ ; each f is independently an integer from 0 to 50; each m is independently 0 or 1; each n is independently 0, 1, 2, 3, or 4; each s is independently 1 or 2; each t is independently 0, 1, 2, or 3).

2. The compound according to claim 1, selected from the group consisting of formula (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIk), and (IIl): 【Chemical 379】

3. The group contains the same 【Chemical 380】 group or a derivative thereof, and optionally, the derivative of the 【Chemical 381】 optionally contains an additional aryl or heteroaryl group condensed with any available 【Chemical 382】 , and optionally contains 【Chemical 383】 , the compound according to claim 1 or 2. 【Chemical 384】

4. The group contains different 【Chemical 385】 groups or derivatives thereof, and optionally, the derivative of the 【Chemical 386】 optionally contains an additional aryl or heteroaryl group condensed with any available any available 【Chemical 387】 , and optionally contains 【Chemical 388】 , the compound according to claim 1 or 2. 【Chemical 389】

5. One is unsubstituted or substituted benzene or unsubstituted or substituted naphthalene; 【Chemical 390】 U is N;

6. V is CR １１ or C(R １１ ) ２ , N, O, or S, the compound according to claim 1. The at least one group or a derivative thereof is selected from the group consisting of 【Chemical Formula 391】

7. 【Chemical 392】 【Chemical 393】 【Chemical 394】 【Chemical 395】 【Chemical 396】 The compound according to any one of claims 1 to 6, selected from the group consisting of formula (IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIy), (IIIbb), (IIIcc), (IIId), and (IIIee): Each R ５ is independently selected from the group consisting of halogen, hydroxyl, C １ to C １２ alkyl, C ２ to C １２ alkene, C ２ to C １２ alkyne, C ３ to C １２ cycloalkyl, C １ to C １２ haloalkyl, C １ to C １２ alkoxy, C ２ to C １８ (hetero)aryl group, C ２ to C １８ (hetero)aryloxy, C ２ to C １８ (hetero)arylamino, carboxylic acid, carboxylic acid ester, (CH ２ ) ｘ’ (OCH ２ -CH ２ ) ｙ’ OCH ３ , and (CH ２ ) ｘ’ (OCH ２ -CH ２ ) ｙ’ OCF ３ ; each x' is independently an integer from 0 to 20; and each y' is independently an integer from 0 to 50; a compound according to any one of claims 1 to 5

8. The compound according to any one of claims 1 to 7, independently selected from the group consisting of 【Chemical 397】 【Chemical 398】 【Chemical 399】

10. An acceptor dye comprising the structure according to any one of claims 1 to 9. Each R １０ and each R １１ is a water-solubilizing moiety, a linker moiety, a linked water-solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, aryl, heteroaryl, (hetero)arylamino, sulfonamide-PEG, phosphoramidate-PEG, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkylsulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamid, alkoxysulfonamide PEG, alkoxysulfonate, alkylsulfonate, alkylsulfonate, 【Chemical 400】 ​

9. 【Chemical Formula 401】 【Chemical 402】 ​ ​ ​

11. A polymeric dye comprising a monomer having a structure according to formula (IV) or (V) 【Chemical 403】 (wherein each 【Chemical 404】 in (IV) or (V) is a point of attachment to the polymeric dye backbone; Each D １ and D ２ are an aryl group, a heteroaryl group, and 【Chemical 405】 is independently selected from the group consisting of each 【Chemical 406】 is independently selected from the group consisting of substituted or unsubstituted benzene, benzene derivatives, monocyclic aryl groups, polycyclic aryl groups, monocyclic heteroaryl groups, and polycyclic heteroaryl groups, D １ or D ２ at least one of which is 【Chemical 407】 being 【Chemical 408】 is an additional aryl or heteroaryl group condensed with any available 【Chemical 410】 or a derivative thereof, optionally, the derivative comprising 【Chemical 409】 and the 【Chemical 411】 group or a derivative thereof is condensed with at any available 【Chemical 412】 position on the 【Chemical 415】 group or a derivative thereof; 【Chemical 414】 each E is independently selected from the group consisting of a chromophore, a functional moiety, a substrate, and a binding partner; 【Chemical 413】 each f is independently an integer from 0 to 50; Each V' is independently SO ２ , SO, S, NR １１ , CR １１ , C(R １１ ), ２ , O, Si(R １１ ), ２ , >C=O, >Se=O, -CH=CH-, or -N=CH-; Each T is independently selected from the group consisting of C, C(R １ ), N, P, O, S, and Si(R １ ); Each U is independently selected from the group consisting of NR １０ , O, P, Se, Te, and S; Each V is NR １１ , CR １１ , C(R １１ ), ２ , S, SO ２ , O, Se, Te, and Si(R １１ ), ２ and is independently selected from the group consisting of; Each X is independently CR １ R ２ or SiR １ R ２ and; Each Y is independently CR ８ R ９ or SiR ８ R ９ and; Each R １ , R ２ , R ８ , and R ９ are a water-solubilizing moiety, a linked water-solubilizing moiety, a linker moiety, a linked E, a reactive group, a linked reactive group, a binding partner, a linked binding partner, a functional group, hydrogen, hydroxy, halogen, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, aryl, heteroaryl, (hetero)aryloxy, (hetero)arylamino, sulfonamide-PEG, phosphoramidate-PEG, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, carbonyl, acyl, alkylsulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, sulfonamide, sulfinamido, phosphonamidate, phosphinamido, alkoxysulfonamide PEG, alkoxysulfonate, alkylsulfonate, alkylsulfonate, 【Chemical 416】 【Chemical 417】 Independently selected from the group consisting of, and optionally, R １ and R ８ together form an unsubstituted or substituted cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, cycloalkoxy, aryl, or heteroaryl having from 3 to 9 ring members; Each R ３ is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a water solubilizing moiety, a chromophore, a functional moiety, a binding partner, and a PEG group; Each Q is independently a bond, NH, NR ４ , C １ ~C １２ alkylene, CHR ４ , or CH ２ ; Each Z is independently CH ２ , CHR ４ , O, NR ４ , or NH; Each W １ is independently a water-solubilizing moiety; L １ 、 L ２ 、 and L ３ are each independently selected linker moieties; each m is independently 0 or 1; Each R ４ is independently selected from the group consisting of H, alkyl, PEG, a solubilizing moiety, a linker moiety, a chromophore, a functional group, a conjugation tag, a carboxylic acid amine, an amine, a carbamate, a carboxylic acid, a carboxylic acid ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, alkene, tetrazine, aldehyde, and thiol, or a protecting group thereof; Each R ７ is independently selected from the group consisting of H, hydroxyl, C １ to C １２ alkyl, C ２ to C １２ alkene, C ２ to C １２ alkyne, C ３ to C １２ cycloalkyl, C １ to C １２ haloalkyl, C １ to C １２ alkoxy, C ２ to C １８ (hetero)aryloxy, C ２ to C １８ (hetero)arylamino, C ２ to C １２ carboxylic acid, C ２ to C １２ carboxylic acid ester, and C １ to C １２ alkoxy, water-solubilizing moiety, PEG moiety, functional group, chemoselective functional group, conjugation tag, linked conjugation tag, linker, sulfonic acid, sulfonate, C １ to C １２ alkylsulfonate, sulfonamide; Each R １０ is independently selected from the group consisting of hydrogen, a linker moiety, a linked reactive group, a linked ionic group, a linked chromophore, a linked solubilizing moiety, a solubilizing moiety, a chromophore, a binding partner, and a linked binding partner; Each R １１ is independently selected from the group consisting of hydrogen, a linker moiety, a linked reactive group, a linked ionic group, a linked chromophore, a linked solubilizing moiety, a chromophore, a binding partner, and a linked binding partner; Each R １２ and R １３ are independently selected from the group consisting of hydrogen, halogen, substituted or unsubstituted C １ -C ６ alkyl, substituted or unsubstituted C １ -C ６ heteroalkyl, substituted or unsubstituted C １ -C ６ alkene, substituted or unsubstituted C ３ -C １０ cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; or R １２ and R １３ together form an unsubstituted or substituted unsaturated cycloalkyl group having 3 to 8 ring members or a substituted or unsubstituted heterocycloalkyl having 3 to 8 ring members, the heterocycloalkyl optionally substituted with O; Each K is independently a covalent bond, O, S, P, NR １ , Se, Te, CR １ R ２ , or CH ２ ; each n is independently 0, 1, 2, 3, or 4; each s is independently 1 or 2; each t is independently 0, 1, 2, or 3).

12. The monomer of formula (IV) or (V) is of formula (A1), (A2), (A3), (A4), (A5), (A6), (A7), (A8), (A9), (A10), (A11), (A12), (A13), (A14), (A15), (A16), (A17), (A18), and (A19): The polymeric dye according to claim 11, comprising a structure selected from the group consisting of

13. 【Chemical 418】 【Chemical 419】 【Chemical 420】 【Chemical 421】 A fluorescent compound or polymer according to any one of claims 1 to 12; and a specific binding partner covalently attached to the fluorescent compound or polymer comprising a labeled specific binding partner.

14. The labeled specific binding partner according to claim 13, wherein the specific binding partner is selected from the group consisting of a protein, a peptide, an affinity ligand, an antibody, an antibody fragment, a carbohydrate, a lipid, a nucleic acid, and an aptamer.

15. The labeled specific binding partner according to claim 14, wherein the specific binding partner is an antibody.

16. A fluorescent compound, polymer, or labeled specific binding partner according to any one of claims 1 to 15; and an acceptor chromophore or donor chromophore covalently attached to the fluorescent compound, polymer, or labeled specific binding partner comprising a tandem dye.

17.

18. ​ R １ 、 R ２ 、 R ３ 、 R ４ 、 R ８ 、 R ９ 、 R １０ 、 and R １１ wherein at least one of R １ , R ２ , R ３ , R ４ , R ８ , R ９ , R １０ , and R １１ comprises a water-solubilizing moiety or a linked water-solubilizing moiety, the fluorescent compound, polymer, labeled specific binding partner, or tandem dye according to any one of claims 1 to 16. ​ The fluorescent compound, polymer, labeled specific binding partner, or tandem dye according to claim 17, wherein the fluorescent compound, polymer, labeled specific binding partner, or tandem dye is water-soluble.

19. A method for detecting a target analyte in a sample, comprising: providing a sample suspected of containing the analyte; contacting the sample with a specific binding partner conjugated to a fluorescent compound, polymer, or tandem dye according to any one of claims 1 to 18, wherein the specific binding partner is capable of interacting with the target analyte; A method comprising the steps of:

20. The method according to claim 19, wherein the binding partner is a protein, peptide, affinity ligand, antibody, antibody fragment, carbohydrate, lipid, nucleic acid, or aptamer.

21. The binding partner is an antibody, and optionally, a. whether the method is configured for flow cytometry; b. whether the water-soluble fluorescent polymer is bound to a substrate; c. whether the analyte is a protein expressed on the cell surface; d. whether the method is configured as an immunoassay; or e. whether the method further comprises providing an additional binding partner for simultaneously detecting an additional analyte. The method according to claim 20.

22. A kit comprising at least one fluorescent compound, polymer, labeled specific binding partner, or tandem dye according to any one of claims 1 to 21.

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