Polymer dye modification and application

Abstract

To provide water-soluble photoactive polymers, included in polymer tandem dyes, and methods for their preparation and use.SOLUTION: The photoactive polymers can be prepared by direct modification of core polymers (e.g., violet excitable polymers) with dyes or other functional groups. Methods of detecting analytes using the polymers are also described. Provided herein are new polymer dyes which can be made via direct modification of core polymers (e.g., violet excitable polymers) with dyes or other functional groups. Dyes can be introduced by attaching them to functional groups already present in the polymer backbone, eliminating the need for an extra category of monomers to effect dye attachment.SELECTED DRAWING: None

Description

[Background technology]

[0001] Background of the Invention Water-soluble fluorescent polymers can be used in a variety of biological applications by generating signals that can be monitored in real time, providing a simple and rapid method for detecting biological targets and events. Water-soluble fluorescent polymers are generally prepared by polymerizing monomers containing polyethylene glycol units (e.g., purple-excitable dihydrophenanthrene monomers) to solubilize the polymer. A common approach for preparing polymers of various colors / emission wavelengths involves covalently attaching acceptor dye molecules to a common polymer backbone to enable efficient fluorescence resonance energy transfer (FRET). These polymers are often referred to as "tandem polymer dyes." The tandem polymer dye approach requires the introduction of an additional reactive monomer with one or more chemically modifiable functional groups for receptor dye attachment. However, the introduction of a new monomer has several drawbacks. For example, the preparation of the new monomer requires an additional synthetic step, which can be costly and time-consuming, and the polymerization conditions must be adjusted or completely redesigned due to changes in the monomer structure. Furthermore, replacing a monomer with a water-solubilizing group for dye attachment can affect the solubility of the desired product. See, for example, U.S. Patent Nos. 8,362,193 and 9,896,538. Identifying the appropriate combination and amount of various monomers to prepare a polymer with acceptable FRET properties and solubility levels requires a considerable amount of trial and error. [Prior art documents] [Patent documents]

[0002] [Patent Document 1] U.S. Patent No. 8,362,193 [Patent Document 2] U.S. Patent No. 9,896,538 Summary of the Invention [Means for solving the problem]

[0003] Described herein are water-soluble photoactive polymers contained in polymer tandem dyes, as well as methods for their preparation and use. The photoactive polymers can be prepared by direct modification of the core polymer (e.g., a purple excitable polymer) with dyes or other functional groups. Methods for detecting analytes using the polymers are also described. Provided herein are compounds of Formula I: [ka] [In the formula, each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; L 1 , L 2 and L 3 is a linker moiety, W is a water-solubilizing moiety; each E is an independently selected chromophore, functional moiety, or linking agent; each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene and ethynylene; G 1 and G 2 are independently selected from unmodified polymer ends and modified polymer ends, and the subscripts n and m are independently integers ranging from 1 to 10,000; the subscript p is an integer ranging from 0 to 10,000; the sum of the subscripts n, m, and p is in the range of 2 to 10,000; the subscript q is 1, 2, 3 or 4; The subscript r is 1, 2, 3, or 4, the subscript s is 0, 1, 2, or 3; The subscript t is 1 or 2, the sum of the subscripts r and s ranges from 1 to 4; A and B are randomly or regularly distributed in the conjugated polymer. The water-soluble photoactive polymer comprises a conjugated polymer according to

[0004] Some embodiments of the present disclosure provide compounds of formula II: [ka] The method provides a method for making a conjugated polymer according to formula IIa: [ka] converting a conjugated polymer according to formula II into a polymer according to formula II, A, B, G 1 , G 2 , L 2 , W, E, and the subscripts n, m, p, q, r, and s are as defined above; L 1a is a linker moiety, R 1 is selected from the group consisting of H and an amine protecting group.

[0005] Also provided is a method for detecting an analyte in a sample. The method includes providing a sample suspected of containing the analyte and combining the sample with a conjugated polymer complex comprising a binding agent conjugated to a water-soluble conjugated polymer described herein. Assay techniques such as flow cytometry can be used to detect fluorescence associated with the polymer bound to the analyte of interest. For example, the present disclosure provides: (Item 1) Formula I: [ka] [In the formula, each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; L 1 , L 2 and L 3is a linker moiety, W is a water-solubilizing moiety; each E is an independently selected chromophore, functional moiety, or linking agent; each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene, and optionally substituted ethynylene; G 1 and G 2 are independently selected from unmodified polymer ends and modified polymer ends, and the subscripts n and m are independently integers ranging from 1 to 10,000; the subscript p is an integer ranging from 0 to 10,000; the sum of the subscripts n, m, and p is in the range of 2 to 10,000; the subscript q is 1, 2, 3 or 4; The subscript r is 1, 2, 3, or 4, the subscript s is 0, 1, 2, or 3; The subscript t is 1 or 2, the sum of the subscripts r and s ranges from 1 to 4; A and B are randomly or regularly distributed in the conjugated polymer. Conjugated polymers by. (Item 2) L 1 comprises a sulfonamide, sulfinamide, disulfonamide, disulfinamide, sultam, amide, secondary amine, phosphonamide, phosphinamide, phosphonamidate, selenoneamide or seleninamide. (Item 3) L 3 3. The conjugated polymer according to claim 1 or 2, wherein (Item 4) The subscript q is equal to the sum of the subscripts r and s, the subscript r is 1 or 2, If the subscript r is 1, then the subscript s is 0 or 1; If the subscript r is 2, then the subscript s is 0. 4. The conjugated polymer according to any one of items 1 to 3. (Item 5) Formula II: [ka] [In the formula, L 1a is a linker moiety, R 1 is selected from the group consisting of H and an amine protecting group. 5. The conjugated polymer according to any one of items 1 to 4, having a structure according to (Item 6) L 1a is a covalent bond, C 1~8 Alkylene, 2-8 membered heteroalkylene, -NHC(O)L a -, -C(O)NHL a - and -C(O)L a - selected from the group consisting of L 2 is a covalent bond, C 1~8 Alkylene, 2-8 membered heteroalkylene, -L b NHC(O)-, -L b C(O)NH-, -L b C(O)-, -C(O)NHL b - and -C(O)L b - selected from the group consisting of L a and L b But C 1~8 independently selected from the group consisting of alkylene and 2- to 8-membered heteroalkylene; Item 6. The conjugated polymer according to item 5. (Item 7) 7. The conjugated polymer of any one of items 1 to 6, wherein W comprises one or more ethylene glycol monomers. (Item 8) 8. The conjugated polymer of any one of items 1 to 7, wherein W comprises poly(ethylene glycol). (Item 9) 9. The conjugated polymer of any one of items 1 to 8, wherein each A is the same comonomer. (Item 10) Item 11. The conjugated polymer of any one of items 1 to 9, wherein A is a purple fluorescent monomer. 11. The conjugated polymer of any one of items 1 to 10, wherein A is a 9,10-phenanthrene dione-based monomer, a dihydrophenanthrene oxepin-based monomer, a fluorene-based monomer, or a fluorenoxepin-based monomer. (Item 12) Formula III: [ka] wherein each subscript t is an integer ranging from 1 to 20. Item 1. The conjugated polymer according to item 1, having a structure according to (Item 13) G 1 and G 2 13. The conjugated polymer of any one of items 1 to 12, wherein one or both of (Item 14) G 1 and G 2 one of which is modified with a capping moiety, and G 1 and G 2 14. The conjugated polymer according to any one of items 1 to 13, wherein one of the (Item 15) 15. The conjugated polymer of any one of items 1 to 14, wherein each E is an independently selected chromophore. (Item 16) Formula II: [ka] 1. A method of making a conjugated polymer according to The method comprises reacting a compound of formula IIa: [ka] converting a conjugated polymer according to formula II into a polymer according to formula II, During the ceremony, A is a fluorescent monomer, L 1a and L 2 is a linker moiety, W is a water-solubilizing moiety; each E is an independently selected chromophore; each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene, and optionally substituted ethynylene; G 1 and G 2 are independently selected from unmodified polymer ends and modified polymer ends, and R 1 is selected from the group consisting of H and an amine protecting group; the subscripts n and m are independently integers ranging from 1 to 10,000; the subscript p is an integer ranging from 0 to 10,000; the sum of the subscripts n, m, and p is in the range of 2 to 10,000; the subscript q is 1, 2, 3 or 4; The subscript r is 1, 2, 3, or 4, the subscript s is 0, 1, 2, or 3; The subscript t is 1 or 2, the sum of the subscripts r and s ranges from 1 to 4; A and B are randomly or regularly distributed in the fluorescent polymer; method. [Brief explanation of the drawings]

[0006] [Figure 1] Figure 1 shows an overlay of flow cytometry histograms generated using CD4-conjugated tandem polymer dyes (5), including BV786.

[0007] [Figure 2] FIG. 2 shows the UV-vis absorbance spectrum of a polymer-dye tandem containing the Dy752 chromophore, a sulfonamide (PEG) DHP polymer.

[0008] [Figure 3] Figure 3 shows the fluorescence emission spectrum of a CD4 mAb conjugated to a sulfonamide (PEG) DHP polymer, a polymer-dye tandem containing the Dy752 chromophore. The tandem polymer exhibited a significant quenching effect at 450 nm due to the presence of the acceptor chromophore. DETAILED DESCRIPTION OF THE INVENTION

[0009] Detailed Description of the Invention Provided herein are novel polymeric dyes that can be made through direct modification of core polymers (e.g., purple excitable polymers) with dyes or other functional groups. Dyes can be introduced by attaching them to functional groups already present in the polymer backbone, eliminating the need for an additional category of monomers to effect dye attachment. The starting polymer can be, for example, a purple polymeric dye having a 9,10-dihydrophenanthrene dione (DHP) backbone with solubilizing polyethylene glycol (PEG) groups attached via sulfonamide linkages. Modification of the sulfonamide groups with dye molecules or other functional groups leads to novel tandem polymeric dyes.

[0010] The compositions and methods described herein offer several significant advantages. For example, only one polymer batch needs to be produced for the purple polymer and purple tandem polymer. A common polymer platform can then be used to create tandem polymers with any desired dye, avoiding the need for new monomers. The overall composition of the polymer backbone remains unaffected, and the polymer remains soluble and easy to handle throughout the synthesis and experimental use of the final product. Furthermore, the chemical reactions used for direct modification of the polymer backbone are themselves rapid and efficient.

[0011] I. Definition As used herein, the term "alkyl," by itself or as part of another substituent, refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. 1~2 , C 1~3 , C 1~4 , C 1~5 , C 1~6 , C 1~7 , C 1~8 , C 1~9 , C 1~10 , C 2~3 , C 2~4 , C 2~5 , C 2~6 , C 3~4 , C 3~5 , C 3~6 , C 4~5 , C 4~6 and C 5~6 For example, C 1~6Alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, and the like. Alkyl can also refer to alkyl groups having up to 20 carbon atoms, such as, but not limited to, heptyl, octyl, nonyl, decyl, and the like. Alkyl groups can be substituted or unsubstituted. Unless otherwise specified, "substituted alkyl" groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0012] As used herein, the term "alkoxy," by itself or as part of another substituent, refers to a group having the formula --OR, where R is alkyl.

[0013] 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 can be attached to the same carbon atom or different carbon atoms of the alkylene group.

[0014] As used herein, the term "heteroalkyl," by itself or as part of another substituent, refers to an alkyl group of any suitable length having 1 to 3 heteroatoms, such as N, O, and S. For example, heteroalkyls can include ethers, thioethers, and alkyl-amines. Additional heteroatoms, including but not limited to, B, Al, Si, and P, may also be useful. Heteroatoms can be oxidized to form moieties such as, but not limited to, -S(O)- and -S(O)2-. The heteroatom portion of a heteroalkyl can replace a hydrogen atom of an alkyl group to form a hydroxy, thio, or amino group. Alternatively, the heteroatom portion can be the connecting atom or can be inserted between two carbon atoms.

[0015] As used herein, the term "heteroalkylene" refers to a heteroalkyl group (i.e., a divalent heteroalkyl radical), as defined above, that links at least two other groups. The two moieties linked to the heteroalkylene group can be linked to the same atom or different atoms of the heteroalkylene group.

[0016] As used herein, the term "cycloalkyl," by itself or as part of another substituent, refers to a saturated or partially unsaturated, monocyclic, fused bicyclic, or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated. Cycloalkyl refers to a 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 3~12 The cycloalkyl group can contain any number of carbons, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated monocyclic cycloalkyl rings include, for example, norbornane, [2.2.2]bicyclooctane, decahydronaphthalene, and adamantane. The cycloalkyl group can also be partially unsaturated with one or more double 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, cyclopeftadiene, cyclooctene, cyclooctadiene (1,3-, 1,4-, and 1,5-isomers), norbornene, and norbornadiene. When the cycloalkyl is a saturated monocyclic C 3~8 When cycloalkyl, exemplary groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. 3~6For cycloalkyl, exemplary groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can be substituted or unsubstituted. Unless otherwise specified, "substituted cycloalkyl" groups can be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy. The term "lower cycloalkyl" refers to cycloalkyl radicals having 3 to 7 carbons, including, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0017] As used herein, the term "cycloalkylene" refers to a cycloalkyl group (i.e., a divalent cycloalkyl radical), as defined above, that links at least two other groups. The two moieties linked to the cycloalkylene group can be linked to the same atom or different atoms of the cycloalkylene group.

[0018] The terms "halo" and "halogen," as used herein, by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0019] As used herein, the term "haloalkyl," by itself or as part of another substituent, refers to an alkyl group in which some or all of its hydrogen atoms have been replaced by halogen atoms. 1~6 The alkyl group may have any suitable number of carbon atoms, such as 1,1,1-trifluoromethyl, ...

[0020] As used herein, the term "haloalkoxy," by itself or as part of another substituent, refers to an alkoxy group in which some or all of its hydrogen atoms have been replaced by halogen atoms.

[0021] As used herein, the term "aryl," by itself or as part of another substituent, refers to an aromatic ring system having any suitable number of carbon ring atoms and any suitable number of rings. Aryl groups include C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C219, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C53, C54, C55, C56, C57, C58, C59, C60, C61, C62, C63, C64, C65, C66, C67, C68, C69, C70, C71, C72, C73, C74, C75, C76, C77, C78, ​​C79, C80, C81, C82, C83, C84, C85, C86, C87, C88, C89, C90, C91, C92, C93, C94, C95, C96, C97, C98, C9 ...100, C111, C112, C113, C114, C115, C116, C117, C1 10 , C 11 , C 12 , C 13 , C 14 , C 15 or C 16 , and C 6~10 , C 6~12 or C 6~14 Aryl groups can contain any suitable number of carbon ring atoms, such as aryl, aryl ...

[0022] As used herein, the term "arylene" refers to an aryl group (ie, a divalent aryl radical), as defined above, that links at least two other groups.

[0023] As used herein, the term "heteroaryl," by itself or as part of another substituent, refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, in which 1 to 5 of the ring atoms are heteroatoms such as N, O, or S. Additional heteroatoms may also be useful, including, but not limited to, B, Al, Si, and P. The heteroatoms may be oxidized to form moieties such as, but not limited to, -S(O)- and -S(O)2-. A heteroaryl group includes C 5~6 , C 3~8 , C 4~8 , C 5~8 , C 6~8 , C 3~9 , C 3~10 , C 3~11 or C 3~12 and any number of ring atoms, such as 1, 2, 3, 4, or 5, or any suitable number of heteroatoms, such as 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, or 3-5, can be included in a heteroaryl group. For example, a heteroaryl group can be a C, in which 1-4 carbon ring atoms are replaced by heteroatoms. 5~8 Heteroaryl or C in which 1-3 carbon ring atoms are replaced by heteroatoms 5~8 Heteroaryl or C in which 1-4 carbon ring atoms are replaced by heteroatoms 5~6 Heteroaryl or C in which 1-3 carbon ring atoms are replaced by heteroatoms 5~6It can be heteroaryl. Heteroaryl groups can include groups such as pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, carbazole, pyridazine, triazine (1,2,3-, 1,2,4-, and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. Heteroaryl groups can also be fused to aromatic ring systems, such as phenyl rings, to form members including, but not limited to, benzopyrroles such as indole and isoindole, benzopyridines such as quinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine (quinazoline), benzopyridazines such as phthalazine and cinnoline, benzothiophene, and benzofuran. Other heteroaryl groups include heteroaryl rings connected by bonds, such as bipyridine. Heteroaryl groups can be substituted or unsubstituted. Unless otherwise specified, "substituted heteroaryl" groups may be substituted with one or more groups selected from halo, hydroxy, amino, alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0024] The heteroaryl group can be linked through any position on the ring. For example, pyrrole includes 1-, 2-, and 3-pyrrole, pyridine includes 2-, 3-, and 4-pyridine, imidazole includes 1-, 2-, 4-, and 5-imidazole, pyrazole includes 1-, 3-, 4-, and 5-pyrazole, triazole includes 1-, 4-, and 5-triazole, tetrazole includes 1- and 5-tetrazole, pyrimidine includes 2-, 4-, 5-, and 6-pyrimidine, pyridazine includes 3- and 4-pyridazine, 1,2,3-triazine includes 4- and 5-triazine, 1,2,4-triazine includes 3-, 5-, and 6-triazine, 1,3,5-triazine includes 2-triazine, thiophene includes 2- and 3-thiophene, and furan includes 2- and 3-furan. thiazoles include 2-, 4-, and 5-thiazoles, isothiazoles include 3-, 4-, and 5-isothiazoles, oxazoles include 2-, 4-, and 5-oxazoles, isoxazoles include 3-, 4-, and 5-isoxazoles, indole includes 1-, 2-, and 3-indole, isoindole includes 1- and 2-isoindole, quinoline includes 2-, 3-, and 4-quinoline, isoquinoline includes 1-, 3-, and 4-isoquinoline, quinazoline includes 2- and 4-quinazoline, cinnoline includes 3- and 4-cinnoline, benzothiophene includes 2- and 3-benzothiophene, and benzofuran includes 2- and 3-benzofuran.

[0025] Some heteroaryl groups include those having 5 to 10 ring members and 1 to 3 ring atoms containing N, O, or S, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4-, and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, isoxazole, indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, cinnoline, benzothiophene, and benzofuran. Other heteroaryl groups include those with 5 to 8 ring members and 1 to 3 heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4-, and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. Some other heteroaryl groups include those with 9 to 12 ring members and 1 to 3 heteroatoms, such as indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, cinnoline, benzothiophene, benzofuran, and bipyridine. Still other heteroaryl groups include those having 5 to 6 ring members and 1 to 2 ring atoms containing N, O, or S, such as pyrrole, pyridine, imidazole, pyrazole, pyrazine, pyrimidine, pyridazine, thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole.

[0026] Some heteroaryl groups contain 5 to 10 ring members and only nitrogen heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4-, and 1,3,5-isomers), indole, isoindole, quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine, and cinnoline. Other heteroaryl groups contain 5 to 10 ring members and only oxygen heteroatoms, such as furan and benzofuran. Some other heteroaryl groups contain 5 to 10 ring members and only sulfur heteroatoms, such as thiophene and benzothiophene. Still other heteroaryl groups contain 5 to 10 ring members and at least two heteroatoms, such as imidazole, pyrazole, triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiazole, isothiazole, oxazole, isoxazole, quinoxaline, quinazoline, phthalazine, and cinnoline.

[0027] As used herein, the term "heteroarylene" refers to a heteroaryl group (ie, a divalent heteroaryl radical), as defined above, that links at least two other groups.

[0028] As used herein, the term "heterocyclyl," by itself or as part of another substituent, refers to a saturated ring system having 3 to 12 ring members and 1 to 4 heteroatoms of N, O, and S. Additional heteroatoms may be useful, including, but not limited to, B, Al, Si, and P. The heteroatoms may be oxidized to form moieties such as, but not limited to, -S(O)- and -S(O)2-. A heterocyclyl group is a 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 or C 3~12and wherein at least one of the carbon atoms is replaced by a heteroatom. Any suitable number of carbon ring atoms, such as 1, 2, 3, or 4, or 1-2, 1-3, 1-4, 2-3, 2-4, or 3-4, can be replaced by a heteroatom in a heterocyclyl group. Heterocyclyl groups may include groups such as aziridine, azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3-, and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine, dioxane, or dithiane. Heterocyclyl groups may also be fused to aromatic or non-aromatic ring systems to form members including, but not limited to, indoline. Heterocyclyl groups may be substituted or unsubstituted. Unless otherwise specified, "substituted heterocyclyl" groups may be substituted with one or more groups selected from halo, hydroxy, amino, oxo (=O), alkylamino, amido, acyl, nitro, cyano, and alkoxy.

[0029] The heterocyclyl group may be linked through any position on the ring. For example, aziridine may be 1- or 2-aziridine, azetidine may be 1- or 2-azetidine, pyrrolidine may be 1-, 2-, or 3-pyrrolidine, piperidine may be 1-, 2-, 3-, or 4-piperidine, pyrazolidine may be 1-, 2-, 3-, or 4-pyrazolidine, imidazolidine may be 1-, 2-, 3-, or 4-imidazolidine, and piperazine may be 1-, 2-, 3-, or 4-piperazine. the tetrahydrofuran may be 1- or 2-tetrahydrofuran, the oxazolidine may be 2-, 3-, 4- or 5-oxazolidine, the isoxazolidine may be 2-, 3-, 4- or 5-isoxazolidine, the thiazolidine may be 2-, 3-, 4- or 5-thiazolidine, the isothiazolidine may be 2-, 3-, 4- or 5-isothiazolidine, and the morpholine may be 2-, 3- or 4-morpholine.

[0030] When a heterocyclyl contains 3 to 8 ring members and 1 to 3 heteroatoms, representative members include, but are not limited to, pyrrolidine, piperidine, tetrahydrofuran, oxane, tetrahydrothiophene, thiane, pyrazolidine, imidazolidine, piperazine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, morpholine, thiomorpholine, dioxane, and dithiane. Heterocyclyls can also form rings having 5 to 6 ring members and 1 to 2 heteroatoms, representative members include, but are not limited to, pyrrolidine, piperidine, tetrahydrofuran, tetrahydrothiophene, pyrazolidine, imidazolidine, piperazine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, and morpholine.

[0031] As used herein, the term "heterocyclylene" refers to a heterocyclyl group (i.e., a divalent heterocyclyl radical), as defined above, that links at least two other groups. The two moieties linked to the heterocyclylene group can be linked to the same atom or different atoms of the heterocyclylene group.

[0032] As used herein, the term "amine protecting group" refers to a chemical moiety that renders an amino group unreactive but is also removable to restore the amino group. Examples of amine protecting groups include, but are not limited to, benzyloxycarbonyl; 9-fluorenylmethyloxycarbonyl (Fmoc); tert-butyloxycarbonyl (Boc); and allyloxycarbonyl (Alloc).

[0033] As used herein, the term "carbonyl," alone or as part of another substituent, refers to -C(O)-, i.e., a carbon atom double-bonded to oxygen and bonded to two other groups in the carbonyl-bearing moiety.

[0034] As used herein, the term "amino" refers to the moiety -NR2, where each R group is H or alkyl. The amino moiety can be ionized to form the corresponding ammonium cation.

[0035] As used herein, the term "sulfonyl" refers to a -SO2R moiety, where the R group is alkyl, haloalkyl, or aryl.

[0036] The term "sulfonamide," when it belongs to a linker moiety as depicted herein, refers to the moiety -S(O)NR-, where the R group is H, alkyl, haloalkyl, or aryl. The term "sultam" refers to a cyclic sulfonamide (e.g., the R group is attached to the sulfur atom via an alkylene moiety).

[0037] The term "disulfonamide" as it pertains to the linker moieties depicted herein refers to the moiety -S(O)2NRS(O)2-, where the R groups are H, alkyl, haloalkyl, or aryl.

[0038] The term "selenone amide," as it pertains to the linker moieties depicted herein, refers to the moiety -Se(O)2NR-, where the R group is H, alkyl, haloalkyl, or aryl.

[0039] The term "sulfinamide," as it pertains to the linker moieties depicted herein, refers to the moiety --S(O)NR--, where the R group is H, alkyl, haloalkyl, or aryl.

[0040] The term "disulfinamide," as it pertains to a linker moiety as depicted herein, refers to a -S(O)NRS(O)- moiety, where the R groups are H, alkyl, haloalkyl, or aryl.

[0041] The term "seleninamide," as it pertains to the linker moieties depicted herein, refers to the moiety --Se(O)NR--, where the R group is H, alkyl, haloalkyl, or aryl.

[0042] The term "phosphonamide," as it pertains to a linker moiety as depicted herein, refers to the moiety --NR--PR(O)NR--, where each R group is independently H, alkyl, haloalkyl, or aryl.

[0043] The term "phosphinamide," as it pertains to a linker moiety as depicted herein, refers to the moiety --PR(O)NR--, where each R group is independently H, alkyl, haloalkyl, or aryl.

[0044] The term "phosphonamidate," as it pertains to the linker moieties depicted herein, refers to the moiety --O--PR(O)NR--, where each R group is independently H, alkyl, haloalkyl, or aryl.

[0045] As used herein, the term "hydroxy" refers to an --OH moiety.

[0046] As used herein, the term "cyano" refers to a carbon atom triple bonded to a nitrogen atom (ie, a -C≡N moiety).

[0047] As used herein, the term "carboxy" refers to the moiety -C(O)OH. The carboxy moiety can be ionized to form the corresponding carboxylate anion. As used herein, the term "carboxylate" refers to the conjugate base of a carboxylic acid, which generally has the formula -C(O)O - For example, the term "magnesium carboxylate" refers to the magnesium salt of a carboxylic acid.

[0048] As used herein, the term "amide" refers to an -NRC(O)R or -C(O)NR2 moiety, where each R group is H or alkyl.

[0049] As used herein, the term "nitro" refers to a -NO2 moiety.

[0050] As used herein, the term "oxo" refers to an oxygen atom that is double bonded to a compound (ie, O=).

[0051] As used herein, the term "ammonium" refers to a compound of the formula NHR3 + wherein each R group is independently hydrogen, or a substituted or unsubstituted alkyl, aryl, aralkyl, or alkoxy group. Preferably, each R group is hydrogen.

[0052] As used herein, "oligoether" is understood to mean an oligomer containing structural repeat units with ether functionality. As used herein, "oligomer" is understood to mean a molecule containing one or more identifiable structural repeat units of the same or different formula.

[0053] As used herein, the term "sulfonate functional group" or "sulfonate" refers to both the free sulfonate anion (-S(=)O2O-) and its salts. Thus, the term sulfonate encompasses sulfonate salts such as sodium sulfonate, lithium sulfonate, potassium sulfonate, and ammonium sulfonate.

[0054] As used herein, the terms "polyethylene glycol" and "PEG" refer to a family of biocompatible, water-solubilizing linear polymers based on ethylene glycol monomer units.

[0055] As used herein, the term "carbamate" refers to a functional group having the structure -NR"COR', 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 Boc, Fmoc, benzyloxy-carbonyl, alloc, methyl carbamate, ethyl carbamate, 9-(2-sulfb)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, and dimethylethyl carbamate.

[0056] As used herein, the term "activated ester" refers to a carboxyl-activating group used in peptide chemistry to facilitate the condensation of the carboxyl group with the free amino group of an amino acid derivative. Descriptions of these carboxyl-activating groups can be found in general textbooks on peptide chemistry, such as KD 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.

[0057] The term "hydrazine" refers to a moiety having the structure -NHNH2.

[0058] As used herein, the term "aldehyde" refers to a compound having a -CHO group.

[0059] As used herein, the term "thiol" refers to a compound containing a functional group composed of a sulfur-hydrogen bond. The general chemical structure of a thiol functional group is R-SH, where R represents an alkyl, alkylene, aryl, or other carbon-containing group of atoms.

[0060] As used herein, the term "silyl" refers to Si(R z )3, R z are each independently an alkyl aryl or other carbon-containing group of atoms.

[0061] As used herein, the term "diazonium salt" refers to a compound having the structure R-N2 + X - where R can be any organic residue (e.g., alkyl or aryl) and X is an inorganic or organic anion (e.g., a halogen).

[0062] The term "triflate," also called trifluoromethanesulfonate, is a group having the formula CF3SO3.

[0063] As used herein, the term "boronic acid" refers to the structure -B(OH). It will be recognized by those skilled in the art that boronic acids may be present as boronate esters at various stages of the synthetic steps disclosed herein, and boronic acid is intended to include such esters. As used herein, the term "boronic ester" or "boronate ester" refers to a group consisting of -B(Z 1 )(Z 2 ) moiety, Z 1 and Z 2 together form a moiety in which the atom bonded to boron in each instance is an oxygen atom. In some embodiments, the boronic ester moiety is a 5-membered ring. In some other embodiments, the boronic ester moiety is a 6-membered ring. In some other embodiments, the boronic ester moiety is a mixture of 5- and 6-membered rings. II. Polymers

[0064] Provided herein are water-soluble conjugated polymers, including fluorescent polymers having monomer subunits such as dihydrophenanthrene (DHP), fluorene, and combinations thereof. Some embodiments of the present disclosure include a polymer having a structure represented by Formula I: [ka] [In the formula, each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; L 1 , L 2 and L 3 is a linker moiety, W is a water-solubilizing moiety; each E is an independently selected chromophore, functional moiety, or linking agent; each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene and ethynylene; G 1 and G 2 are independently selected from unmodified polymer ends and modified polymer ends, and the subscripts n and m are independently integers ranging from 1 to 10,000; the subscript p is an integer ranging from 0 to 10,000; the sum of the subscripts n, m, and p is in the range of 2 to 10,000; the subscript q is 1, 2, 3 or 4; The subscript r is 1, 2, 3, or 4, the subscript s is 0, 1, 2, or 3; The subscript t is 1 or 2, the sum of the subscripts r and s ranges from 1 to 4; A and B are randomly or regularly distributed in the conjugated polymer. The present invention provides a conjugated polymer according to the present invention.

[0065] In some embodiments, L 1 In some embodiments, L comprises a sulfonamide, sulfonamide, sultam, disulfinamide, amide, phosphonamide, phosphonamidate, phosphinamide, or secondary amine. 1 comprises a sulfonamide, an amide, a phosphonamide or a secondary amine.

[0066] In some embodiments, The subscript q is equal to the sum of the subscripts r and s, The subscript r is 1 or 2, If the subscript r is 1, then the subscript s is 0 or 1; If the subscript r is 2, then the subscript s is 0.

[0067] In some embodiments, L 3 are each covalent bonds.

[0068] In some embodiments, the conjugated polymer has Formula II: [ka] [In the formula, L 1a is a linker moiety, R 1 is selected from the group consisting of H and an amine protecting group. It has a structure as follows.

[0069] The various linkers L described herein 1a and L 2 can be used to synthesize polymers according to Formula I and Formula II. In some embodiments, L 1a is a covalent bond, C 1~8 Alkylene, 2-8 membered heteroalkylene (e.g., divalent alkoxy linkers), C 3~8 Cycloalkylene, C 6~10 Arylene, 5- to 12-membered heteroarylene, 5- to 12-membered heterocyclylene, -NHC(O)L a -, -C(O)NHL a -, -C(O)L a - and combinations thereof; L 2 is a covalent bond, C 1~8 Alkylene, 2-8 membered heteroalkylene (e.g., divalent alkoxy linkers), C 3~8 Cycloalkylene, C 6~10 Arylene, 5- to 12-membered heteroarylene, 5- to 12-membered heterocyclylene, -L b NHC(O)-, -L b C(O)NH-, -L b C(O)-, -C(O)NHL b -, -C(O)L b - and combinations thereof; L a and L b is C 1~8independently selected from the group consisting of alkylene and 2- to 8-membered heteroalkylene; R 1 is selected from the group consisting of H and an amine protecting group.

[0070] In some embodiments, a polymer according to Formula II is provided: L 1a is a covalent bond, C 1~8 Alkylene, 2-8 membered heteroalkylene, -NHC(O)L a -, -C(O)NHL a - and -C(O)L a - selected from the group consisting of L 2 is a covalent bond, C 1~8 Alkylene, 2-8 membered heteroalkylene, -L b NHC(O)-, -L b C(O)NH-, -L b C(O)-, -C(O)NHL b - and -C(O)L b - selected from the group consisting of L a and L b is C 1~8 independently selected from the group consisting of alkylene and 2- to 8-membered heteroalkylene; R 1 is selected from the group consisting of H and an amine protecting group.

[0071] In some embodiments, W comprises one or more ethylene glycol monomers, hi some embodiments, W comprises poly(ethylene glycol).

[0072] In some embodiments, L 3 The first L 1 Part (or first L 1a a first point of attachment to the L moiety; a second point of attachment to the L moiety; 1 Part (or second L 1a For example, some embodiments of the present disclosure include a trivalent arylalkyl moiety having a second point of attachment to the A monomer (a moiety) and a third point of attachment to the A monomer. [ka] [In the formula, L 3a is a covalent bond, C 1~8 Alkylene, 2-8 membered heteroalkylene, -NHC(O)L a -, -C(O)NHL a - and -C(O)L a - selected from the group consisting of L a and L b is C 1~8 independently selected from the group consisting of alkylene and 2- to 8-membered heteroalkylene; The wavy lines are the points of attachment to the monomers.]

[0023] A conjugated polymer is provided having two or more chromophores attached as shown in

[0073] In some embodiments, each A is the same comonomer. In some embodiments, A is a fluorescent monomer. In some embodiments, A is a 9,10-phenanthrene dione-based monomer (e.g., a dihydrophenanthrene (DHP)-based monomer), a fluorene-based monomer, or a fluorenoxepin-based monomer. In some embodiments, the "A" monomer in the polymer according to Formula I is a DHP-based monomer, such as [ka] [In the formula, Each X is independently C or Si; Y is independently CR 1 R 2 or SiR 1 R 2 and R 1 are each independently an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, or a moiety: [ka] and R 2 are each independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a PEG group, an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, or a moiety: [ka] and R 3 are each independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and a PEG group; each Z is independently selected from the group consisting of C, O, and N; Q is a bond, NH, or NR 4 and CH2, each subscript n is independently an integer from 0 to 20. is.

[0074] In some embodiments, R 1 has the structure shown below: [ka] wherein Q is NH. It has.

[0075] In some embodiments, the DHP-based monomer has the structure: [ka] [In the formula, each subscript f is independently an integer from 0 to 50; each subscript n is independently an integer from 0 to 20; R5 are each independently H, C1 to C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C3-C 20 Cycloalkyl, C1-C 20 Haloalkyl, C1-C 20 Alkoxy, C2-C 26 Aryloxy, C2-C 26 Heteroaryloxy, C2-C 26 Arylamino or C2-C 26 heteroarylamino] It has.

[0076] In some embodiments, the DHP monomer has the structure: [ka] [In the formula, each subscript f is independently an integer from 0 to 50; each subscript n is independently an integer from 0 to 20; R 5 are each independently H, C1 to C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C3-C 20 Cycloalkyl, C1-C 20 Haloalkyl, C1-C 20 Alkoxy, C2-C 26 Aryloxy, C2-C 26 Heteroaryloxy, C2-C 26 Arylamino or C2-C 26 heteroarylamino] It has.

[0077] In some embodiments, the "A" monomer in the polymer according to Formula I is a fluorene-based monomer, such as [ka] [where X, Z, R 1 , R2 , R 5 , subscript n, and subscript f are as defined above.] is.

[0078] R 1 Groups and R 2 Groups such as ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers or the structure: [ka] may confer solubility in water / buffer. In some embodiments, for example, the polymer is soluble at levels greater than 10 mg / mL, greater than 15 mg / mL, greater than 20 mg / mL, greater than 25 mg / mL, greater than 30 mg / mL, greater than 35 mg / mL, greater than 40 mg / mL, greater than 45 mg / mL, greater than 50 mg / mL, greater than 60 mg / mL, greater than 70 mg / mL, greater than 80 mg / mL, greater than 90 mg / mL, or greater than 100 mg / mL.

[0079] In some embodiments, the monomers of the present invention also include cross-linking monomers. For example, the cross-linking monomers of the present invention include: [ka] Includes:

[0080] In some embodiments, the "A" monomer in the polymer according to Formula I is an oxepin-based monomer (e.g., a fluorenoxepin-based monomer), such as [ka] [where X, R 1 and R 2 is as defined above] is.

[0081] Prior to polymerization according to methods including those described below, the monomers are independently terminated with a halogen atom, a boronic ester or acid, a silyl group, a diazonium salt, a triflate group, an acetyloxy group, a sulfonate group, or a phosphate, which are capable of undergoing palladium or nickel catalyzed polymerization.

[0082] In some embodiments, the conjugated polymer has Formula III: [ka] wherein each subscript t is an integer ranging from 1 to 20. It has a structure as follows.

[0083] In some embodiments, the "B" monomer of the conjugated polymer according to Formula I can alter the polymer bandgap. The bandgap altering monomer is: [ka] [ka] [In the formula, R 4 are each independently H, C1 to C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C3-C 20 Cycloalkyl, C1-C 20 Haloalkyl, C2-C 26 Aryl, C2-C 26 Heteroaryl, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether or (CH2) x (OCH2-CH2) y OCH3, R 5 are each independently H, halogen, hydroxyl, C1-C 20 Alkyl, C2-C20 Alkenyl, C2-C 20 Alkynyl, C3-C 20 Cycloalkyl, C1-C 20 Haloalkyl, C1-C 20 Alkoxy, C2-C 26 Aryl, C2-C 26 Heteroaryl, C2-C 26 Aryloxy, C2-C 26 Heteroaryloxy, C2-C 26 Arylamino, C2-C 26 Heteroarylamino, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether or (CH2) x (OCH2-CH2) y OCH3, Each x is independently an integer of 0 to 20; and each y is independently an integer from 0 to 50. It includes structures such as:

[0084] In some embodiments, the "B" monomer of the conjugated polymer is an optionally substituted ethylene moiety, i.e., a group of the formula -CR=CR-, where each R is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a PEG group, an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, or a moiety as defined above: [ka] In some embodiments, the "B" monomer of the conjugated polymer may include an ethynylene moiety, i.e., a carbon-carbon triple bond having the formula -C≡C-.

[0085] In some embodiments, G 1 and G 2 In some embodiments, one or both of G 1 and G 2 one of which is modified with a capping moiety, and G 1 and G 2 One of the capping units G is modified with a reactive group for conjugation. 1 and G 2 may be, for example, hydrogen, halogen, alkynyl, optionally substituted aryl (e.g., halogen-substituted aryl), optionally substituted heteroaryl, silyl, diazonium salt, triflate, acetyloxy group, azide, sulfonate, phosphate, boronic acid-substituted aryl group, boronic ester-substituted aryl group, boronic ester, or boronic acid. The capping unit may also contain one or more reactive groups for conjugation (e.g., functional groups such as amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, e.g., N-hydroxysuccinimidyl ester, hydrazine, azide, alkyne, aldehyde, or thiol), which may be covalently attached to binder and substrate materials, as described in more detail below.

[0086] In some embodiments, the polymers described herein are characterized by a minimum number average molecular weight of greater than 5,000 g / mol, greater than 10,000 g / mol, greater than 15,000 g / mol, greater than 20,000 g / mol, greater than 25,000 g / mol, greater than 30,000 g / mol, greater than 40,000 g / mol, greater than 50,000 g / mol, greater than 60,000 g / mol, greater than 70,000 g / mol, greater than 80,000 g / mol, greater than 90,000 g / mol, or greater than 100,000 g / mol.

[0087] In some embodiments, the polymers described herein are characterized by a minimum weight average molecular weight of greater than 5,000 g / mol, greater than 10,000 g / mol, greater than 15,000 g / mol, greater than 20,000 g / mol, greater than 25,000 g / mol, greater than 30,000 g / mol, greater than 40,000 g / mol, greater than 50,000 g / mol, greater than 60,000 g / mol, greater than 70,000 g / mol, greater than 80,000 g / mol, greater than 90,000 g / mol, or greater than 100,000 g / mol. Number average and weight average molecular weight values ​​can be determined by gel permeation chromatography (GPC) using polymer standards (e.g., polystyrene or similar materials). III. Methods for preparing polymers

[0088] Also provided herein are methods for preparing conjugated polymers. A. Monomer Synthesis

[0089] The DHP monomers of the present invention can be made as shown below. [ka]

[0090] For example, 2,7-dibromo-trans-9,10-dihydrophenanthrene-9,10-diol (DHP-OH) can be prepared as follows: In a conical flask (2 L), approximately 26 g of NaBH4 is added to a stirred water-ethanol mixture (1:6.5 v:v). To this solution, approximately 24 g of 2,7-dibromophenanthrene, 9,10-dione is added in small portions over a period of approximately 5 minutes. The reaction mixture is stirred for approximately 24 hours, during which time the color of the solution changes from orange-red to pale yellow and then to white at the end of the reaction. The reaction is stopped, and the reaction mixture is neutralized with dilute HCl acid. After neutralization, the white precipitate is filtered and washed with excess water. The isolated precipitate is washed with very cold (below -15 °C) ethanol (100 mL) and methanol (100 mL). [ka]

[0091] DHP-OSO3H can be prepared as follows: In a two-neck round-bottom flask, DHP-OH (3.6 g) and 18-crown-6 (500 mg) are dissolved in 120 mL of THF. The solution is purged with nitrogen (20 min), and NaH (2 g) is added while the nitrogen purge is continued. The color of the solution changes from colorless to light pink, dark pink, brown, and dark green over 10-15 min. In a separate flask, 12 g of 1,3 propane sultone is dissolved in 20 mL of THF and purged with nitrogen. This sultone solution is added to the DHP-OH solution via an addition funnel over 20-30 min. The reaction is stirred at room temperature for 4-5 h. The solvent is evaporated, and the resulting solid is dissolved in water. Acetone is added to obtain a white precipitate in the form of the disodium salt. The precipitate is filtered, redissolved in a minimum amount of water, neutralized with HCl, and reprecipitated in acetone. Repeated precipitation (2-3 times) followed by centrifugation affords the product as a white solid. [ka]

[0092] DHP-OSO2Cl can be prepared as follows: 5 g of DHP-OSO3H is weighed into a round-bottom flask and mixed with 25 mL of DMF. To this, approximately 10 mL of SOCl2 is added dropwise, and the mixture is stirred overnight. The reaction mixture is then poured into 200 mL of water, and the resulting precipitate is filtered and dried. [ka]

[0093] DHP-sulfonamide PEG can be prepared as follows: DHP-OSO2Cl is mixed with 2.2 equivalents of PEG amine in a dichloromethane / TEA mixture. After 3 hours of sonication, the crude product is extracted with dichloromethane and then subjected to column chromatography (silica gel, MeOH-CHCl3). [ka]

[0094] DHP-sulfonamide PEG, diboronic ester can be prepared as follows: Dibromo-functionalized DHP-sulfonamide is mixed with DMSO under nitrogen, to which 3 equivalents of bis-pinacolatodiboron are added. The reagent is reacted with 12 equivalents of potassium acetate and 4 equivalents of Pd(dppf)Cl catalyst at 80°C for 5 hours. The reaction mixture is cooled and extracted with CHCl / water. The organic layer is concentrated and purified by column chromatography (silica gel, MeOH-CHCl).

[0095] Similarly, fluorene (FL) monomers of the present invention can be made as described below. For example, FL-OSO3H can be prepared as follows: In a two-neck round-bottom flask, 5 g of fluorene is dissolved in 70 mL of DMSO. The solution is purged with nitrogen (20 min), and 50% NaOH (12 eq.) is added while continuing the nitrogen purge. The solution color changes from colorless to dark brown. Propane sultone (3 eq.) is weighed and dissolved in DMSO. This is added dropwise to the fluorene reaction mixture over a 5-minute period. The reaction is stirred at room temperature for 4-5 hours. The solvent is evaporated, and the precipitate is dissolved in water. Acetone is added to obtain a white precipitate of DPS in the form of a disodium salt. The precipitate is filtered, redissolved in a small amount of water, neutralized with HCl, and reprecipitated in acetone. Repeated precipitation (2-3 times) followed by centrifugation yields FL-OSO3H as a white solid.

[0096] FL-OSO2Cl can be prepared as follows: 5 g of FL-OSO3H is placed in a round-bottom flask and mixed with 25 mL of DMF. To this, approximately 10 mL of SOCl2 is added dropwise, and the mixture is stirred overnight. The reaction mixture is then poured into 200 mL of water, and the precipitate is filtered and dried.

[0097] FL-sulfonamide PEG can be prepared as follows: FL-OSO2Cl is mixed with 2.2 equivalents of PEG amine in a dichloromethane / TEA mixture. After 3 hours of sonication, the crude product is extracted with dichloromethane and then subjected to column chromatography (silica gel, MeOH-CHCl3).

[0098] The diboronic ester of FL-sulfonamide PEG can be prepared as follows: The corresponding dibromo-substituted compound is mixed with DMSO under nitrogen, to which 3 equivalents of bis-pinacolatodiboron are added. The reagent is reacted with 12 equivalents of potassium acetate and 4 equivalents of Pd(dppf)Cl2 at 80°C for 5 hours. The reaction mixture is cooled and extracted with CHCl3 / water. The organic layer is concentrated and purified by column chromatography (silica gel, MeOH-CHCl3). B. Polymerization

[0099] Generally, the polymerized monomer units described above can be obtained using polymerization techniques known to those skilled in the art, or by using methods known in the art in combination with the methods described herein. For example, the synthesis of diboronic ester derivatives from dihalogenated monomers can be achieved by Suzuki coupling with bis(pinacolato)diboron: [ka] This can be achieved via:

[0100] Similarly, polymerization can also be performed using Suzuki coupling: [ka] (In the formula, J 1 and J. 2 are independently H, Br, B(OH)2, or a boronic ester. This can be achieved via:

[0101] For example, polymerization can proceed as follows: In a round-bottom flask, both bromo and boronic acid monomers are dissolved in a DMF-water mixture and purged with nitrogen for 10 minutes. Approximately 20 equivalents of CsF and Pd(OAc)2 (10 mol%) are mixed under nitrogen and heated to 80°C. Polymerization is monitored using UV-Vis spectroscopy and SEC chromatography. After polymerization, a first capping agent containing an appropriate functional group is added, followed three hours later by a second capping agent. After the reaction, the solvent is removed from the crude mixture via evaporation, and the crude is passed through a gel filtration column to remove small organic molecules and low MW oligomers. C. Capping Unit

[0102] Capping units can be conjugated to the polymer backbone of the present invention through a similar mechanism as previously described. For example, bromo and boronic ester capping units can be added to one or both ends of the polymer. By using both the bromo and boronic ester capping units, both ends of the polymer are modified. By using only one form of capping unit, either the bromo or boronic ester, only the end terminating with its corresponding complementary group is modified, which can be used in symmetric polymerization to statistically modify only one end of the polymer. In asymmetric polymers, this approach is used to chemically ensure that the polymer is modified only at one chain end. Capping units can also be added asymmetrically by first reacting a bromo capping unit with the polymer at the Y end, and then reacting the polymer with a boronic ester capping unit.

[0103] For example, the capping agent of the present invention can be prepared as shown below. [ka] [ka] D. Polymer Functionalization

[0104] Tandem polymer dyes and other functionalized polymers can be prepared by post-polymerization modification of polymer intermediates as described herein, for example, those of Formula IV: [ka] The pendant solubilizing group according to formula V: [ka] where W is a water-solubilizing moiety and L 1 and L 2 is a linking moiety. In some embodiments, each E is an independently selected chromophore, functional moiety, or linking agent. In some embodiments, each E is an independently selected chromophore (e.g., an independently selected fluorophore). In some embodiments, all E moieties in the polymer have the same fluorophore structure.

[0105] The water-solubilizing moiety W in the groups according to Formula IV and Formula V can be, for example, an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, an oligo(ethylene glycol), or a poly(ethylene glycol). 1 , L 2 and L 3 is a bond including, but not limited to, a covalent bond, C 1~8The linker may be an alkylene or a 2-8 membered heteroalkylene. In some embodiments, the linker is a single atom, a straight chain, a branched chain, or a cyclic moiety. In some embodiments, the linker is a chain of between 2 and 100 backbone atoms (e.g., carbon atoms) in length, such as between 2 and 50 backbone atoms in length or between 2 and 20 backbone atoms in length. In certain cases, 1, 2, 3, 4, or 5 or more carbon atoms of the linker backbone may be optionally replaced with sulfur, nitrogen, or oxygen. The bonds between the backbone atoms may be saturated or unsaturated, and typically, no more than 1, 2, or 3 unsaturated bonds are present in the linker backbone. The linker may contain one or more substituents (e.g., alkyl or aryl groups). The linker may include, without limitation, oligo(ethylene glycol), ether, thioether, tertiary amine, and alkylene groups (i.e., divalent alkyl radicals), which may be straight chain or branched. The linker backbone can include a cyclic group, e.g., a divalent aryl radical, a divalent heterocyclic radical, or a divalent cycloalkyl radical, wherein two or more atoms of the cyclic group, e.g., two, three, or four atoms, are included in the backbone.

[0106] In some embodiments, L 1 comprises a sulfonamide, sulfinamide, disulfonamide, disulfinamide, sultam, amide, secondary amine, phosphonamide, phosphinamide, phosphonamidate, selenoneamide, or seleninamide. 1 In some such embodiments, L comprises a sulfonamide, an amide, a secondary amine, or a phosphonamide. 2 is linear or branched, saturated or unsaturated C 1~30 Contains an alkylene group, C 1~30 One or more carbon atoms in an alkylene group may be O, S, NR a are independently replaced as needed by C 1~30 The grouping of two or more adjacent carbon atoms in an alkylene is —NR a (CO)- or -(CO)NRa - is independently replaced as needed by R a are H and C, respectively. 1~6 alkyl.

[0107] In some embodiments, the polymer is prepared in a first step by dissolving L in a pendant solubilizing group according to formula IV. 1 The internal position of the linker moiety L 2 and then in a second step, a dye or other functional group E is covalently attached to the first end of the linker moiety L. 2 In some embodiments, L may be functionalized by covalently attaching it to the second end of 1 The nitrogen atom in (e.g., amide nitrogen, sulfonamide nitrogen, or phosphonamide nitrogen) is linked to a linker moiety L having a suitable leaving group at a first end of the linker moiety. 2 In some embodiments, for example, the leaving group is a halogen (e.g., chloro, bromo, or iodo). In some embodiments, the leaving group is a sulfonate (i.e., —OS(O)R, where R is alkyl, haloalkyl, aryl, or substituted aryl). Suitable sulfonates include, but are not limited to, mesylate (methanesulfonate), triflate (trifluoromethanesulfonate), besylate (benzenesulfonate), tosylate (p-toluenesulfonate), and brosylate (4-bromobenzenesulfonate).

[0108] Any suitable solvent can be used for the alkylation step during polymer functionalization. Suitable solvents include, but are not limited to, toluene, methylene chloride, ethyl acetate, acetonitrile, tetrahydrofuran, benzene, chloroform, diethyl ether, dimethylformamide, dimethyl sulfoxide, petroleum ether, and mixtures thereof. The alkylation reaction is typically carried out at a temperature ranging from approximately 25° C. to about 100° C., with the linking moiety L 2 or a linking functional group -L 2The reaction is carried out for a time sufficient for -E to be incorporated into one or more pendant groups of the polymer. The reaction can be carried out for a period of time ranging from a few minutes to several hours or longer, depending on the polymer and reagents used in the reaction. For example, the reaction can be carried out at approximately 40°C, or approximately 50°C, or approximately 60°C, or approximately 70°C, or approximately 80°C, for approximately 10 minutes, or approximately 30 minutes, or approximately 1 hour, or approximately 2 hours, or approximately 4 hours, or approximately 8 hours, or approximately 12 hours.

[0109] Connecting part L 2 The second terminus of may contain a functional group (e.g., an amine or a carboxylic acid) that is used in protected form during the first step (e.g., the alkylation step) and then deprotected prior to covalent attachment of the dye or other functional group E to the second terminus of the linking moiety. 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 are described, for example, in Green's and Wuts (Protective Groups in Organic Synthesis, 4 th The polymers may be added to or removed from the polymers of the present disclosure using known techniques as described by G. W., Ed. 2007, Wiley-Interscience, New York.

[0110] Attachment of dyes and other functional groups can be accomplished using any suitable method. In some embodiments, an amide linkage is provided between L 2The deprotected primary amine group of L is formed between the deprotected primary amine group and a carboxylate-functionalized dye. The dye can be used in an activated form, for example, using the reagent EC(O)X, where X is a leaving group. Activated carboxylate-functionalizing reagents include, but are not limited to, acid anhydrides (including symmetric, mixed, or cyclic anhydrides), activated esters (e.g., p-nitrophenyl esters, pentafluorophenyl esters, N-succinimidyl esters, etc.), acyl azoles (e.g., acylimidazoles prepared using carbonyldiimidazole), acyl azides, and acid halides (e.g., acid chlorides). Alternatively, a coupling agent can be used to convert L to a carboxylate-functionalized dye. 2An amide linkage bond can be formed between the deprotected primary amine group of the carboxylate-functionalized chromophore EC(O)OH. A coupling agent can be used to form the activated dye reagent prior to reaction with the polymer amine group. Any suitable coupling agent can be used. In some embodiments, the coupling agent is a carbodiimide, a guanidinium salt, a phosphonium salt, or a uronium salt. Examples of carbodiimides include, but are not limited to, N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and the like. Examples of phosphonium salts include, but are not limited to, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), bromotris(dimethylamino)phosphonium hexafluorophosphate (BroP), and the like. Examples of guanidinium / uronium salts include, but are not limited to, N,N,N,',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 1-[(1-cyano-2-ethoxy-2-oxoethylidene-aminooxy)dimethylaminomorpholino)]uronium hexafluorophosphate (COMU), etc. Solvents, reaction times, and other reaction conditions can vary as described above depending on factors such as the nature of the particular polymer and dye / functional group.

[0111] Some embodiments of the present disclosure provide compounds of formula II: [ka] The method provides a method for making a conjugated polymer according to formula IIa: [ka] converting a conjugated polymer according to formula II into a polymer according to formula II, A is a fluorescent monomer, L 1a is a covalent bond, C 1~8 Alkylene, 2-8 membered heteroalkylene, -NHC(O)L a -, -C(O)NHL a - and -C(O)L a - selected from the group consisting of L 2 is a covalent bond, C 1~8 Alkylene, 2-8 membered heteroalkylene, -L b NHC(O)-, -L b C(O)NH-, -L b C(O)-, -C(O)NHL b - and -C(O)L b - selected from the group consisting of L a and L b is C 1~8 independently selected from the group consisting of alkylene and 2- to 8-membered heteroalkylene; W is a water-solubilizing moiety; each E is an independently selected chromophore, functional moiety, or linking agent; each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene, and optionally substituted ethynylene; G 1 and G 2 are independently selected from unmodified polymer ends and modified polymer ends, and R 1 is selected from the group consisting of H and an amine protecting group; the subscripts n and m are independently integers ranging from 1 to 10,000; the subscript p is an integer ranging from 0 to 10,000; the sum of the subscripts n, m, and p is in the range of 2 to 10,000; the subscript q is 1, 2, 3 or 4; The subscript r is 1, 2, 3, or 4, the subscript s is 0, 1, 2, or 3; The subscript t is 1 or 2, A and B are randomly or regularly distributed in the fluorescent polymer.

[0112] In some embodiments, converting the conjugated polymer of Formula IIa to a conjugated polymer according to Formula II comprises one or more alkylation steps or one or more amide formation steps described above.

[0113] Any suitable chromophore or fluorophore can be used for polymer functionalization. Generally, suitable chromophores and fluorophores are covalently attached to a pendant solubilizing group (e.g., the linking moiety L described above). 2 and 6,004,531. Examples of suitable chromophores and fluorophores 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, and 6,004,531. Nos. 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, which are incorporated herein by reference in their entireties.

[0114] In some embodiments, the chromophore E has the structure: [ka] [In the formula, R 6a , R 6b , R6c , R 6d , R 6e , R 6f and R 6g Six of them are H, halogens, and C. 1~6 Alkyl, C 3~8 Cycloalkyl, C 6~10 Aryl, C 7~16 Aryl alkyl, C 1~6 independently selected from acyl and -SO3H; R 6a , R 6b , R 6c , R 6d , R 6e , R 6f and R 6g One of them is the linking part -L 2 -is] is a boron-dipyrromethene moiety having the formula:

[0115] In some embodiments, R 6a and R 6c is C 1~6 alkyl (e.g., methyl or ethyl), and R 6e , R 6f and R 6g One of them is the linking part -L 2 In some embodiments, R 6a and R 6c is methyl and R 6g is the connecting part -L 2 -It is.

[0116] In some embodiments, the chromophore E has the structure: [ka] [In the formula, R 6h and R 6i is H, C 1~6 Alkyl, (CH2) t COOH, (CH2) t SO3H and linking moiety L 2 are independently selected from each subscript t is independently an integer from 1 to 10; R 6j and R 6k H, halogen, C 1~6 Alkyl, optionally substituted fused C 6~10 Aryl (e.g., optionally substituted benzo), -SO3H, -PO3H2, -OPO3H2, -COOH and linking moieties L 2 are independently selected from Y is O, S, C(R 6l )2, -CH=CH- and NR 6l are independently selected from R 6l are each independently H or C 1~6 is alkyl, The subscript n is an integer from 1 to 6, provided that R 6h , R 6i , R 6j and R 6k Only one of the linking moieties -L 2 -provided that is a cyanine moiety having the formula:

[0117] In some embodiments, the chromophore E has the structure: [ka] [In the formula, W is N or CR 6p and Z is O, S or NR 6q and R 6m , R 6n , R 6o , R 6p Each of these is H, halogen, C 1~6 Alkyl, -CN, -CF3, -COOR 3v , -CON(R 3v )2, -OR 3v and the linking part -L 2 - selected independently from R 6n -OR 3v and -N(R 3v )2 is selected, R 6qare H and C, respectively. 1~6 Alkyl and Linking Moiety -L 2 - selected independently from However, R 6m , R 6n , R 6o , R 6p and R 6q Only one of the linking moieties -L 2 -provided that is a coumarin moiety having the formula:

[0118] In some embodiments, the chromophore E has the structure: [ka] [In the formula, T is O, S, C(R 6u )2 and NR 6u is selected from U is O or N(R 6u )2, R 6r are H, halogen, and C, respectively. 1~6 Alkyl, -SO3H and linking moiety -L 2 - selected independently from R 6s -H, -OH, -OR 6u , -N(R 6u )2 and linking moiety-L 2 - selected from R 6t is H, C 1~6 Alkyl, R 6v and the linking part -L 2 - selected independently from R 6u are each independently H or C 1~6 is alkyl, R 6v teeth, [ka] is selected from During the ceremony, R 6w are H and the linking moiety -L, respectively. 2- selected independently from However, R 6r , R 6s , R 6t and R 6v Only one of the linking moieties -L 2 -provided that is a xanthene moiety having the formula:

[0119] In some embodiments, the xanthene moiety is a xanthene moiety in which T and U are O and R 6s is OH and R 6t but [ka] Fluorescein is

[0120] In some embodiments, the xanthene moiety is a xanthene moiety in which T and U are O and R 6s is OH and R 6r are each halogen (e.g., bromo), and R 6t but [ka] That is, eosin.

[0121] In some embodiments, the xanthene moiety is a xanthene moiety where T is O and U is N(R 6u )2 (e.g., =NH2 + ) and R 6s -N(R 6u )2 (e.g., -NH2), and R 6t but [ka] That is, rhodamine.

[0122] In some embodiments, the xanthene moiety has the structure: [ka] [In the formula, R 6v teeth, [ka] is selected from On the other hand, R 6w is H, and the other R 6w is the linking part -L 2 -is] It is a rhodamine having the formula:

[0123] The method presented herein can be used to add other functional moieties to functionalized polymers in addition to chromophores. For example, the functional moiety "E" can be a peptide tag, oligonucleotide, or polynucleotide, such as biotin, digoxigenin, or FLAG peptide. As used herein, the term "FLAG peptide" refers to an oligopeptide or polypeptide containing the amino acid sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (i.e., DYKDDDDK). FLAG peptide and its variants are described, for example, in U.S. Patent No. 4,703,004 to Hopp et al., which is incorporated herein by reference. Other peptides that can be used in place of the FLAG peptide include, but are not limited to, the HA peptide tag containing the sequence Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala (i.e., YPYDVPDYA), the His6 peptide tag containing the sequence His-His-His-His-His-His (i.e., HHHHHH), and the Myc peptide tag containing the sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (i.e., EQKLISEEDL). Peptide tags can be recognized by antibodies or other binding moieties for use with colorimetric reagents, chemiluminescent reagents, and the like, for convenient identification and / or quantification. Nucleotides (e.g., RNA, single-stranded DNA, or double-stranded DNA) can be recognized by complementary primers or other complementary nucleotides, for example, as described in WO 2016 / 019929 (Navratil et al.), the publication of which is incorporated herein by reference. As used herein, the term "digoxigenin" refers to 3-[(3S,5R,8R,9S,10S,12R,13S,14S,17R)-3,12,14-trihydroxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,15,16,17-tetradecahydrocyclopenta[a]-phenanthren-17-yl]-2H-furan-5-one (CAS Registry Number 1672-46-4) and its substituted analogs.As used herein, the term "biotin" refers to 5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoic acid (CAS Registry Number 58-85-5) and its substituted analogs. E. Binder

[0124] A "binding agent" of the present invention can be any molecule or complex of molecules capable of specifically binding to a target analyte. Binding agents of the present invention include, for example, proteins (e.g., antibodies or antibody fragments), small organic molecules, carbohydrates (e.g., polysaccharides), oligonucleotides, polynucleotides, lipids, affinity ligands, aptamers, etc. In some embodiments, a binding agent is an antibody or fragment thereof. Specific binding, in the context of the present invention, 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 specific binding agent will preferentially bind to a particular protein or isoform of a particular protein and will not bind to a significant amount of other proteins or other isoforms present in a sample.

[0125] When the binding agents are antibodies, they may be monoclonal or polyclonal antibodies. As used herein, the term antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules. Such antibodies include, but are not limited to, polyclonal, monoclonal, monospecific polyclonal antibodies, antibody mimetics, chimeric, single chain, Fab, Fab' and F(ab')2 fragments, Fv and Fab expression libraries.

[0126] In some cases, antibodies include intravenous immunoglobulin (IVIG) and / or IVIG-derived (e.g., enriched, purified, e.g., affinity-purified) antibodies. IVIG is a blood product containing IgG (immunoglobulin G) pooled from plasma (e.g., in some cases free of any other proteins) from many (e.g., 1,000 to over 60,000) normal, healthy blood donors. IVIG is commercially available. Embodiments of IVIG are described, for example, in U.S. Patent Application Publication Nos. 2010 / 0150942, 2004 / 0101909, 2013 / 0177574, 2013 / 0108619, and 2013 / 0011388.

[0127] In some cases, the antibody is a monoclonal antibody of a defined subclass (e.g., IgG1, IgG2, IgG3, or IgG4). When a combination of antibodies is used, the antibodies may be from the same or different subclasses. For example, the antibody may be an IgG1 antibody. In some embodiments, the monoclonal antibody is humanized.

[0128] In some embodiments, the antibody is selected from the group consisting of BRCA1, CTLA4, CD4, EGF, EGFR, ERBB2 (Her-2), IFN-a, IFN-gamma, IL-1, IL1R1 (CD121a), IL1R2 (CD121b), IL-IRA, IL-2, IL2RA (CD25), IL2RB (CD122), IL2RG (CD132), IL-4, IL-4R (CD123), IL-5, IL5RA (CD125 ), IL3RB(CD131), IL-6, IL6RA, (CD126), IR6RB(CD130), IL-7, IL7RA(CD127), IL-8, CXCR1(IL8RA), CXCR2, (I L8RB / CD128), IL-9, IL9R(CD129), IL-10, IL10RA(CD210), IL10RB(CDW210B), IL-11, IL11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17A, IL17B, IL17C , IL17R, JAG1, JAK1, JAK3, mTOR, MUC1 (mucin), MYC, NOTCH, NOTCH1, NOX5, PI3 kinase, PIK3CG, PTEN, PTN, TLR10, TL R2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TNF, TNF-α, TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, TOP2A (topoisomerase Iia), VEGF, VEGFB, VEGFC, versican, VHL C5, VLA-4, Wnt-1.Examples of antibodies for polymer conjugation include, but are not limited to, adalimumab (also known as HUMIRA™), adecatumumab, alemtuzumab, bertilimumab, brentuximab, cetuximab (also known as ERBITUX™), clenoliximab, dacetuzumab, dacliximab, daclizumab (also known as ZENAPAX™), detumomab, dorlixizumab, dantumumab, gemtuzumab, infliximab (also known as REMICADE™), ipilimumab (also known as YERVOY™), lumiliximab, mapatumumab These include maslimomab, nebacumab, nerelimomab, pembrolizumab (also known as KEYTRUDA™), regavirumab, reslizumab, rituximab (also known as RITUXAN™, MabTHERA™), rovelizumab, tadocizumab, and trastuzumab (also known as HERCEPTIN™).

[0129] In general, the polymers of the invention can be conjugated to a binding agent using techniques known to those skilled in the art, or using methods known in the art in combination with the methods described herein. The binding agent can be conjugated to a linker moiety L as described above. 1 , L 2 and L 3 It can also be incorporated at the "E" position of a polymer according to formula I via:

[0130] For example, the preparation of polymeric NHS ester can proceed as follows: 5 mg of polymer is dissolved in 1 ml of dry CH3CN. To this, 15 mg of TSTU is added, and the mixture is stirred for another 2 minutes. To this, 100 μL of DIPEA is added, and stirring is continued overnight. Next, the organic solvent is removed via evaporation, and the crude NHS product is dissolved in approximately 750 μL of 1×PBS buffer (pH 8.8) by quick vortexing, and then transferred to a Zeba spin column (40K MWCO). The sample is spun at 2200 RPM for 2 minutes, and the polymeric NHS ester is used immediately.

[0131] Conjugation of the polymeric NHS ester to an antibody (e.g., CD4 mAb) can proceed as follows: The polymeric NHS ester in PBS buffer is added to 0.6 mg of antibody and mixed with 100 μL of 0.5 M borate buffer (pH 9.0). The mixture is quickly vortexed for 30 seconds and then mixed in a Coulter mixer for 3-4 hours.

[0132] Purification of His-tag antibody conjugates using a Histrap HP column can proceed as follows: Procedure 1: The crude reaction mixture is purified using a Histrap HP column. The sample is loaded using 1x PBS buffer and the unbound fraction is collected. This can be done using 20 column volumes of buffer. The buffer is then changed to wash out the bound fraction, which contains both the conjugate and free antibody. This can be done by running 10 column volumes using 1x PBS with 0.25M imidazole.

[0133] Method 2: Hitrap SP Sepharose FF column. The column is equilibrated and the crude reaction mixture is loaded using 20 mM citrate buffer (pH 3.5), and the unbound fraction is collected. This can be done using 20 column volumes of buffer. The buffer is then changed to elute the bound fraction, containing both conjugate and free antibody. This can be done by running 20 column volumes using 20 mM citrate (pH 7.6) containing 0.6 M NaCl.

[0134] Purification of the conjugate by SEC column can proceed as follows: The reaction mixture is loaded onto a size exclusion column using 1x PBS. After checking the UV-visible absorption spectrum, the fractions are pooled and concentrated in an Amicon Ultra-15 tube with a 30 kDa MWCO centrifugal concentrator.

[0135] A polymer having a Michael acceptor, such as maleimide, can be covalently attached to a thiol present in a cysteine ​​residue of an antibody, as described in more detail below. The free thiol can be generated from the reduction of an interchain disulfide bond, or the free thiol can be introduced as an engineered (i.e., non-naturally occurring) cysteine ​​residue. The engineered cysteine ​​residue can be located in the antibody heavy chain or the antibody light chain. In certain embodiments, the engineered cysteine ​​residue is located in the Fc region of the heavy chain. For example, the engineered antibodies described in U.S. Patent Nos. 7,855,275, 8,309,300, and 9,000,130 can be used. IV. Methods for Detecting Analytes A. Overview

[0136] Also provided is a method for detecting an analyte in a sample, comprising providing a sample suspected of containing the analyte and combining the sample with a conjugated polymer conjugate comprising a binding agent conjugated to a water-soluble conjugated polymer described herein. The binding agent is capable of interacting with the analyte. A light source capable of exciting the polymer is applied to the sample, and light emitted from the conjugated polymer conjugate is detected. In a typical assay, the fluorescent polymers of the present invention can be excited with light having a wavelength between about 395 nm and about 415 nm. The emitted light is typically between about 415 nm and about 475 nm. Alternatively, the excitation light can have a wavelength between about 340 nm and about 370 nm, and the emitted light is between about 390 nm and about 420 nm. B. Sample

[0137] The sample in the methods of the present invention can be, for example, blood, bone marrow, spleen cells, lymphocytes, bone marrow aspirate (or any cells obtained from bone marrow), urine (perfusion), serum, saliva, cerebrospinal fluid, urine, amniotic fluid, interstitial fluid, feces, mucus, or tissue (e.g., tumor sample, disaggregated tissue, disaggregated solid tumor). In certain embodiments, the sample is a blood sample. In some embodiments, the blood sample is whole blood. Whole blood can be obtained from a subject using standard clinical procedures. In some embodiments, the sample is a subset of one or more cells 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 with one or more detectable markers). In some embodiments, the sample can be derived from a cell culture.

[0138] The subject may be a human (e.g., a patient suffering from a disease) or a commercially important mammal, including, for example, a monkey, cow, or horse. Samples may also be obtained from domestic pets, including, for example, dogs or cats. In some embodiments, the subject is an experimental animal, such as a mouse, rat, rabbit, or guinea pig, used as an animal model of disease or for drug screening. C. Analyte

[0139] As used herein, "analyte" refers to a substance, e.g., a molecule, whose abundance / concentration is determined by some analytical procedure. For example, in the present invention, an analyte can be a protein, peptide, nucleic acid, lipid, carbohydrate, or small molecule.

[0140] The target analyte can 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, such as a structural microfilament, a microtubule, and an intermediate 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 signaling scaffold, an ion channel, or the like. The protein can be an activatable protein or a protein differentially expressed or activated in diseased or abnormal cells, including, but not limited to, a transcription factor, a DNA and / or RNA binding and modifying protein, a nuclear import and export receptor, a regulator of apoptosis or survival, or the like. D. Assay

[0141] Assay systems that utilize binding agents and fluorescent labels to quantify bound molecules are well known, and examples of such systems include flow cytometers, scanning cytometers, imaging cytometers, fluorescence microscopes, and confocal fluorescence microscopes.

[0142] In some embodiments, flow cytometry is used to detect fluorescence. Many devices suitable for this use are available and known to those of skill in the art. Examples include BCI Includes Navios, Gallios, Aquios and CytoFLEX flow cytometers.

[0143] In other embodiments, the assay is an immunoassay. Examples of immunoassays useful in the present invention include, but are not limited to, fluorescence luminescence assays (FLA). Assays can also be performed on protein arrays.

[0144] When the binding agent is an antibody, a sandwich assay of antibody or multiple antibodies can also be used.Sandwich assay refers to the use of sequential recognition events to build layers of various binding agents and reporting elements that indicate the presence of a specific analyte.Examples of sandwich assays are disclosed in U.S. Patent No. 4,486,530 and the references cited therein. [Example]

[0145] Example 1 Preparation of NHBoc polymer 50 mg of the purple-excited base polymer (1) was prepared as described in WO2017 / 180998 and weighed into a 4 mL vial. 800 μL of anhydrous DMF was added to the vial, and the mixture was vortexed and sonicated for 5 minutes to completely dissolve the polymer. Polymer (1) contains an average of 48 PEG-functionalized DHP monomers (m = approximately 24); polymers of different sizes (e.g., m = approximately 5-50, n = approximately 5-25) can be prepared in a similar manner. [ka]

[0146] Under a nitrogen atmosphere, the polymer solution was transferred to a 10 mL reaction flask containing cesium carbonate (100 equiv.). tert-Butyl-3-iodopropyl-carbamate solution was diluted from stock (10 mg / mL in anhydrous DMF) and 10 equiv. was added to the polymer mixture. The sealed reaction flask was heated to 50 °C, and the reaction was continued for 1 h under stirring at 500 rpm. The reaction mixture was cooled to room temperature, and the DMF was evaporated in a rotary evaporator under high vacuum. The crude reaction mixture was diluted with chloroform (25 mL) and washed with 15% w / v brine solution (25 mL). The organic layer was collected in a 250 mL conical flask, additional chloroform (12 mL) was added, and the mixture was washed three times with 30% w / v brine solution (10 mL). The organic fraction was dried by adding 20 g of anhydrous sodium sulfate and then filtered through Whatman Paper 2 into a 150 mL flat-bottom flask. The filtered sodium sulfate was washed twice with chloroform (15 mL) to recover the remaining polymer dye and filtered into the same flat-bottom flask. The chloroform was evaporated in a rotary evaporator at 45 °C and 150-200 rpm. Residual DMF was removed under high vacuum at 50 °C for 30-40 min. The dried polymer was washed with diethyl ether (2 × 2 mL) and sonicated for 2 min to remove unreacted tert-butyl-3-iodopropyl-carbamate. After drying the polymer under high vacuum for 5 min, the polymer yield was calculated with respect to the initial polymer amount. The dried polymer product (2) was 1 The polymer was characterized using H NMR. The proton signal at 1.4 ppm indicates the presence of NH-Boc moieties in the polymer. Modified monomers (denoted by subscript m1) and unmodified monomers (denoted by subscript m2) were randomly distributed along the polymer backbone. [ka] Example 2 Preparation of amine-functionalized polymers

[0147] 50 mg of NHBoc polymer prepared according to Example 1 was added to a 20 mL round-bottom flask and dissolved in 1 mL of methanol and 1 mL of water by vortexing for 5 minutes and sonicating for 5 minutes. To the resulting solution, 2 mL of 12 M HCl was added, and the mixture was allowed to react at room temperature for 2 hours. The reaction mixture was then transferred to a small beaker, the pH was adjusted to 9-10 using 15% w / v K2CO3 solution, and the mixture was stirred for an additional 15 minutes. The polymer was extracted with 25 mL of chloroform in a 100 mL separatory funnel, and the organic layer was collected in a conical flask. Brine solution (15% w / v) was added to the aqueous layer, and additional chloroform was used to recover the remaining polymer. The extraction process was monitored by a UV lamp. The organic layer was dried using approximately 40 g of anhydrous sodium sulfate and filtered through Whatman filter paper 2 into a 250 mL flat-bottom flask. Additional chloroform washes (2 x 20 mL) were used to recover the remaining polymer from the filtered sodium sulfate. The combined chloroform layers were evaporated in a rotary evaporator at approximately 40°C. After complete solvent evaporation, the solid was redissolved in chloroform (10 mL) in a 15 mL Falcon tube and centrifuged at 3000 rpm for 5 minutes to remove salt impurities. The supernatant was decanted into a 20 mL vial, concentrated on a rotary evaporator, and dried under high vacuum. The yield of deprotected amine-functionalized polymer (3) was calculated in terms of the amount of protected polymer. The deprotection was 1 This was confirmed by 1 H NMR and also by determining the receptor binding A / D ratio. [ka] Example 3 Tandem polymer dye-antibody conjugation and purification

[0148] Polymer-acceptor dye formation. 10 mg of polymer was weighed into a glass vial and dissolved in 200 μL of anhydrous DMSO. To ensure complete dissolution of the polymer, a combination of vortexing, sonication, and incubation in a 50°C water bath for approximately 10–15 min was applied. To this, 200 μL of acetonitrile and 20 μL of diisopropylethylamine were added. A 10 mg / mL (w / v) solution of the acceptor dye (near-IR absorbing Dy752NHS; Dyomics GmbH) was prepared in anhydrous DMSO, and 8 equivalents of the dye were added to the polymer solution. The mixture was stirred at room temperature for 2 h, protected from light, yielding a product containing an average of 2–3 dyes per polymer chain. By adjusting the amount of acceptor dye used in the reaction, products containing 1–6 dyes per polymer chain can be prepared. Tandem polymer dyes with a Cy3.5 acceptor can also be prepared using Cy3.5-NHS (Lumiprobe Corp.), as described above for Dy752.

[0149] Preparation of polymer-acceptor dye maleimide. After 2 h, TSTU (20 mg) dissolved in 50 μL of acetonitrile was added to the polymer-acceptor dye mixture, and the activation process was carried out for 30 min at room temperature by keeping it stirred under protection from light.

[0150] Two 5 mL 40 K Zeba spin columns were equilibrated with 20 mM borate buffer (pH 8.8) and proceeded as described by the manufacturer. Concurrently, a 2 mg solution of N-(2-aminoethyl)maleimide trifluoroacetate was prepared in 20 μL of anhydrous DMSO and kept in a Zeba collection tube.

[0151] The activated tandem polymer was dissolved in 1800 μL of 20 mM borate buffer (pH 8.8) and combined with the maleimide using an equilibrated Zeba spin column. The amount of polymer after the Zeba column step was estimated by measuring the UV414 of the tandem polymer. Approximately 5 mg of tandem polymer was recovered (i.e., approximately 50 mol%). The resulting tandem polymer-maleimide mixture was incubated by rotation at room temperature for 60-120 minutes. Optionally, the reaction can be followed by 90 minutes of sonication.

[0152] During the incubation period, 30% ethanol in water, 50 mM MOPS, 100 mM sodium perchlorate, and 4 mM EDTA (pH 7.0) (MOPS buffer) was prepared. After the reaction, the tandem polymer-maleimide was washed with at least 30-40 mL of 30% ethanol in water using a 30 or 50 kDa MWCO Amicon concentrator, and then buffer exchanged into MOPS buffer using at least 30-40 mL of MOPS buffer. The final volume of the buffer-exchanged polymer-maleimide was between 2 and 4 mL. This mixture containing the maleimide-functionalized tandem dye polymer (4) was stored overnight at 4 °C before further use.

[0153] Preparation of polymer-antibody conjugates. A 0.5 mL 40K Zeba column was equilibrated using 1x PBS, and 1 mg of CD4 mAb was passed through the equilibrated Zeba column. 30 μL of 10 mg / mL (w / v) DTT (approximately 300 equivalents) prepared in 1x PBS was added to the buffer-exchanged mAb, and the resulting mixture was incubated at room temperature for 30 minutes. 50 mM MES, 0.1 M sodium perchlorate, 4 mM EDTA (pH 5.8) (MES buffer) was prepared and kept in the dark. After 30 minutes, the reduced mAb was diluted to 500 μL and passed through a 2 mL 40K Zeba column pre-equilibrated with MES buffer to remove excess DTT. [ka]

[0154] The reduced CD4 mAb in MES was mixed with the polymer-maleimide (which was brought to room temperature before mixing) and incubated for 3 hours by rotation at room temperature, protected from light, to form the conjugate (5). This unpurified conjugate can be stored overnight at 4°C. [ka]

[0155] Although the foregoing has been described in some detail by way of illustration and example, for purposes of clarity and understanding, those skilled in the art will recognize that certain changes and modifications may be practiced within the scope of the appended claims. Furthermore, each reference presented herein is incorporated by reference in its entirety to the same extent as if it were individually incorporated by reference.

Claims

1. Formula I: 【Chemistry 1】 [In the formula, each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers, wherein each A is independently selected from the group consisting of 9,10-phenanthrene dione-based monomers, dihydrophenanthrene-based monomers, oxepin-based monomers, fluorene-based monomers, and fluorenooxepin-based monomers; L １ are each a linker moiety independently selected from the group consisting of a divalent aryl group, a divalent heterocyclic group, a divalent cycloalkyl group, a trivalent arylalkyl moiety, a substituted or unsubstituted 2-8 membered heteroalkylene, an oligo(ethylene glycol), an ether, a thioether, a tertiary amine, and an alkylene group having up to 20 carbon atoms, wherein each linker moiety is independently substituted or unsubstituted, saturated or unsaturated, and optionally wherein 1, 2, 3, 4, or 5 carbon atoms of the linker backbone are replaced by one or more of sulfur, nitrogen, oxygen, or P; L ２ are each a covalent bond, a divalent aryl group, a divalent heterocyclic group, a divalent cycloalkyl group, a trivalent arylalkyl moiety, a substituted or unsubstituted 2- to 8-membered heteroalkylene, an oligo(ethylene glycol), an ether, a thioether, a tertiary amine, and a linear or branched, saturated or unsaturated C １～３０ The alkylene group is １～３０ One or more carbon atoms in an alkylene group may be O, S, NR ａ or １～３０ Grouping of two or more adjacent carbon atoms in an alkylene may be —NR ａ (CO)- or -(CO)NR ａ - optionally and independently replaced by １～３０ alkylene groups, wherein each linker moiety can independently be substituted or unsubstituted, saturated or unsaturated, and wherein optionally 1, 2, 3, 4, or 5 carbon atoms of the linker backbone can be optionally replaced by sulfur, nitrogen, or oxygen; L ３ are each a linker moiety independently selected from the group consisting of a covalent bond, a divalent aryl group, a divalent heterocyclic group, a divalent cycloalkyl group, a trivalent arylalkyl moiety, a covalent bond, a substituted or unsubstituted 2-8 membered heteroalkylene, an oligo(ethylene glycol), an ether, a thioether, a tertiary amine, and a chain of between 2 and 50 backbone carbon atoms in length, wherein each linker moiety can independently be substituted or unsubstituted, saturated or unsaturated, and wherein optionally 1, 2, 3, 4, or 5 carbon atoms of the linker backbone can be optionally replaced by sulfur, nitrogen, or oxygen; R ａ are H and substituted or unsubstituted C, respectively. １～６ independently selected from the group consisting of alkyl, W is a water-solubilizing moiety; each E is an independently selected fluorophore; each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene and ethynylene; G １ and G ２ are independently selected from unmodified and modified polymer ends optionally substituted with a linking agent or substrate, respectively; the subscripts n and m are independently integers ranging from 1 to 10,000; the subscript p is an integer ranging from 0 to 10,000; the sum of the subscripts n, m, and p ranges from 2 to 10,000; the subscript q is 1, 2, 3, or 4; the subscript r is 1, 2, 3, or 4; the subscript s is 0, 1, 2 or 3; the subscript t is 1 or 2; the sum of the subscripts r and s ranges from 1 to 4; A and B are randomly or regularly distributed in the conjugated polymer. Conjugated fluorescent polymers.

2. L １ are each substituted or unsubstituted C １～８ are linker moieties independently selected from the group consisting of alkylene, substituted or unsubstituted 2- to 8-membered heteroalkylene, oligo(ethylene glycol), ether, thioether, tertiary amine, divalent aryl group, divalent heterocyclic group, and divalent cycloalkyl group; L ２ and L ３ are each a covalent bond, a substituted or unsubstituted C １～８ are linker moieties independently selected from the group consisting of alkylene, substituted or unsubstituted 2- to 8-membered heteroalkylene, oligo(ethylene glycol), ether, thioether, tertiary amine, divalent aryl group, divalent heterocyclic group, and divalent cycloalkyl group; The conjugated polymer of claim 1 .

3. L ２ are each independently a linear or branched, saturated or unsaturated C １～３０ an alkylene group, Said C １～３０ One or more carbon atoms in an alkylene group may be O, S, NR ａ or Said C １～３０ Two or more groups of adjacent carbon atoms in an alkylene may be —NR ａ (CO)- or -(CO)NR ａ - being replaced as needed and independently by R ａ are H and substituted or unsubstituted C, respectively. １～６ independently selected from the group consisting of alkyl, The conjugated polymer of claim 1 .

4. L ３ The conjugated polymer of claim 1 , wherein is a covalent bond.

5. L ３ But the first L １ the first point of attachment to the moiety, the second L １ 10. The conjugated polymer of claim 1, wherein the A monomer is a trivalent arylalkyl moiety having a second point of attachment to the A moiety and a third point of attachment to the A monomer.

6. 6. The conjugated polymer dye of claim 1, wherein W is selected from the group consisting of ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, oligo(ethylene glycol), and poly(ethylene glycol).

7. the subscript q is equal to the sum of the subscripts r and s; the subscript r is 1 or 2; When the subscript r is 1, the subscript s is 0 or 1; If the subscript r is 2, then the subscript s is 0; A conjugated polymer according to any one of claims 1 to 6.

8. The conjugated polymer has the formula (V): 【Chemistry 2】 wherein E is a fluorophore, and L 1 , L 2 , W and E are as defined in claim 1.

8. The conjugated polymer of claim 1, wherein the conjugated polymer is a conjugated tandem polymer dye comprising at least one functionalized solubilizing group according to

9. B is each independently 【Transformation 3】 [In the formula, R ４ are each independently H, substituted or unsubstituted C １ ~C ２０ Alkyl, C ２ ~C ２０ Alkenyl, C ２ ~C ２０ Alkynyl, C ３ ~C ２０ Cycloalkyl, C １ ~C ２０ Haloalkyl, C ２ ~C ２６ Aryl, C ２ ~C ２６ Heteroaryl, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether or (CH ２ ) ｘ (OCH ２ -CH ２ ) ｙ OCH ３ and R ５ are each independently H, halogen, hydroxyl, substituted or unsubstituted C １ ~C ２０ Alkyl, C ２ ~C ２０ Alkenyl, C ２ ~C ２０ Alkynyl, C ３ ~C ２０ Cycloalkyl, C １ ~C ２０ Haloalkyl, C １ ~C ２０ Alkoxy, C ２ ~C ２６ Aryl, C ２ ~C ２６ Heteroaryl, C ２ ~C ２６ Aryloxy, C ２ ~C ２６ Heteroaryloxy, C ２ ~C ２６ Arylamino, C ２ ~C ２６ Heteroarylamino, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether or (CH ２ ) ｘ (OCH ２ -CH ２ ) ｙ OCH ３ and Each x is independently an integer from 0 to 20; Each y is independently an integer from 0 to 50.

9. The conjugated polymer of claim 1, wherein the band gap modifying monomer is selected from the group consisting of:

10. B is independent of each other, a group of the formula -CR=CR-, wherein each R is independently H, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a PEG group, an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, an ethynylene moiety having the formula -C≡C-, and 【Chemistry 4】 part, where: Z is CH ２ , O, and NH; Q is a bond, NH, NR ４ and C.H. ２ are independently selected from the group consisting of R ３ are independently selected from the group consisting of H, substituted or unsubstituted alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG groups; R ４ are each independently H, substituted or unsubstituted C １ ~C ２０ Alkyl, C ２ ~C ２０ Alkenyl, C ２ ~C ２０ Alkynyl, C ３ ~C ２０ Cycloalkyl, C １ ~C ２０ Haloalkyl, C ２ ~C ２６ Aryl, C ２ ~C ２６ Heteroaryl, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether or (CH ２ ) ｘ (OCH ２ -CH ２ ) ｙ OCH ３ and Each x is independently an integer from 0 to 20; Each y is independently an integer from 0 to 50. an optionally substituted ethylene moiety having 10. The polymer of claim 1 selected from the group consisting of:

11. G １ and G ２ is a capping moiety selected from the group consisting of hydrogen, halogen, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, silyl, diazonium salt, triflate, acetyloxy group, azide, sulfonate, phosphate, boronic acid substituted aryl group, boronic ester substituted aryl group, boronic ester, and boronic acid.

12. G １ and G ２ is modified with one or more reactive groups selected from the group consisting of amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl ester, hydrazine, azide, alkyne, aldehyde, and thiol, wherein the one or more reactive groups are optionally covalently attached to a binder or substrate.

13. 13. The conjugated polymer of claim 1, wherein each A is independently selected from the group consisting of 9,10-phenanthrene dione-based monomers, dihydrophenanthrene-based monomers, and fluorene-based monomers.

14. 14. The conjugated polymer of claim 13, wherein each A is independently selected from the group consisting of dihydrophenanthrene-based monomers and fluorene-based monomers.

15. 15. The conjugated polymer of claim 1, wherein each A is the same comonomer.

16. A is a compound of the formula 【Chemistry 5-1】 [In the formula, Each X is independently C or Si; Each Y is independently CR １ R ２ or SiR １ R ２ and R １ are each independently an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, or a partial salt. 【Chemistry 5-2】 and R ２ are each independently H, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a PEG group, an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, or a moiety. 【Chemistry 5-3】 and R ３ are each independently selected from the group consisting of H, substituted or unsubstituted alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and a PEG group; R ４ are each independently H, substituted or unsubstituted C １ ~C ２０ Alkyl, C ２ ~C ２０ Alkenyl, C ２ ~C ２０ Alkynyl, C ３ ~C ２０ Cycloalkyl, C １ ~C ２０ Haloalkyl, C ２ ~C ２６ Aryl, C ２ ~C ２６ Heteroaryl, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether or (CH ２ ) ｘ (OCH ２ -CH ２ ) ｙ OCH ３ and Z is CH ２ , O, and NH; Q is a bond, NH, or NR ４ and C.H. ２ are independently selected from the group consisting of R １ and R ２ and each subscript n is independently an integer from 0 to 20.

16. The conjugated polymer of claim 1, wherein the dihydrophenanthrene-based monomer comprises:

17. A is a compound of the formula 【Chemistry 6-1】 [In the formula, Each X is independently C or Si; R １ are each independently an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, or a partial salt. 【Chemistry 6-2】 and R ２ are each independently H, substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a PEG group, an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, or a moiety. 【Transformation 6-3】 and R ３ are each independently selected from the group consisting of H, substituted or unsubstituted alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and a PEG group; R ４ are each independently H, substituted or unsubstituted C １ ~C ２０ Alkyl, C ２ ~C ２０ Alkenyl, C ２ ~C ２０ Alkynyl, C ３ ~C ２０ Cycloalkyl, C １ ~C ２０ Haloalkyl, C ２ ~C ２６ Aryl, C ２ ~C ２６ Heteroaryl, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether or (CH ２ ) ｘ (OCH ２ -CH ２ ) ｙ OCH ３ and Z is CH ２ , O, and NH; Q is a bond, NH, or NR ４ and C.H. ２ are independently selected from the group consisting of R １ and R ２ and each subscript n is independently an integer from 0 to 20.

16. The conjugated polymer of claim 1, wherein the fluorene-based monomer comprises:

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