Method for preparing macromers and macromers prepared thereby

Abstract

The invention relates to a method for preparing macromonomers and macromonomers prepared by the method. The method includes combining a plurality of reactive monomers to form a reaction mixture wherein a first portion of the reactive monomers carries a first reactive group and a second portion of the monomers carries a second reactive group wherein the first and second reactive groups are different and capable of reacting with each other to form a macromonomer.

Description

[0001] This application is a divisional application of Chinese patent application No. 201880049895.4, filed on July 26, 2018, with the invention name “Conjugated polymers and methods of use thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Patent Application No. 62 / 538,583, filed on July 28, 2017, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0004] The invention relates to a method for preparing a macromonomer and the macromonomer prepared by the method. Background of the Invention

[0006] Such as Pacific Blue TM , Common dyes such as GFP and Cy5 have high quantum yields but limited extinction coefficients. Alternative reporter molecules such as phycobiliproteins offer larger absorption cross-sections, producing brighter signals, but are limited by rapid photobleaching and sensitivity to fixation.

[0007] Fluorophores are important in various forms of bioassays. In fact, fluorescence is a common method for bioanalysis and biodetection, and fluorescent labels are important in these applications. Some assays rely on the fluorescence resonance energy transfer (FRET) mechanism using two fluorophores. In these assays, energy is transferred between a donor fluorophore and an acceptor fluorophore if the two fluorophores are in close proximity to each other. If the two fluorophores are within a given proximity range, excitation of the "donor" by an energy source (e.g., UV light) results in energy transfer to the "acceptor." In turn, the acceptor emits light of its characteristic wavelength. In order for FRET to occur, the fluorescence emission spectrum of the donor molecule must overlap with the absorption or excitation spectrum of the acceptor chromophore.

[0008] Polyindene polymers or macromonomers have well-defined structures, unique optical properties, and are stable. Since the extinction coefficient of the macromonomer is proportional to the degree of polymerization (or number of repeating units), macromonomers are designed to improve brightness. In addition, since these materials are derived from common synthetic organic and polymer chemistry techniques, more defined and reproducible reagents can be made in terms of size, binding sites, physical properties, and optical properties.

[0009] Unlike quantum dots, conjugated macromonomers have discrete excitation spectra, similar to those of organic dyes, which minimizes potential problems with cross-beam compensation.

[0010] In view of the above, there is a need in the art for new polyindenylfluorene macromonomers that are water soluble and brighter than currently available technology. The present disclosure satisfies these and other needs. Summary of the invention

[0011] In one embodiment, the present disclosure provides a macromer of Formula I or II:

[0012]

[0013] Where R 1 , R 2 , R 3 and R 4 are each independently selected from hydrogen, optionally substituted C1-C 10 Alkyl, optionally substituted C1-C 18 Alkoxy, optionally substituted C6-C 18 Aryl, optionally substituted C4-C 18 Acyl, optionally substituted C4-C 18 Acyloxy, NR a R b (where R a and R b are each independently hydrogen or lower alkyl), a water-soluble group, an ethylene oxide oligomer, and an ethylene oxide oligomer methyl ether;

[0014] Or, R 1 and R 2 and / or R 3 and R 4 Together with the carbon atoms to which they are attached, they form an optionally substituted 4-, 5-, or 6-membered ring;

[0015] Each R 5 independently selected from halogen, alkyl, alkoxy, amino, ethylene oxide oligomer and ethylene oxide oligomer methyl ether;

[0016] Each p is a value from 0 to 3;

[0017] m is a value selected from 1-10,000;

[0018] A and B each may be present or absent and each may be the same or different, and each is selected from an aromatic group or a heteroaromatic group, which completes the π-conjugated backbone;

[0019] 1, 2 and 3 may each be present or absent and may each be the same or different, and are each selected from an aromatic group or a heteroaromatic group, which completes the π-conjugated backbone;

[0020] n and o are each independently a value selected from 0 to 10,000, wherein the monomers within m, n and o may be the same or different;

[0021] E 1 and E 2 each independently selected from hydrogen, halogen, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, halogen, substituted aryl, boronic acid substituted aryl, borate substituted aryl, borate, boronic acid, optionally substituted fluorene, and aryl or heteroaryl substituted with one or more side chains terminated with: i) a functional group selected from the group consisting of amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protecting groups thereof, for conjugation with a molecule or biomolecule; or ii) conjugation with an organic dye or biomolecule; and

[0022] The macromonomer of formula I or II may be E 1 and E 2 An alternating copolymer, a block copolymer, a random copolymer or a graph copolymer.

[0023] In another embodiment, the present disclosure provides a method for detecting an analyte in a sample, the method comprising:

[0024] (a) combining a sample with a macromer of formula I or formula II;

[0025] (b) exciting the macromonomer with light; and

[0026] (c) Detecting the fluorescence from the macromer to detect the analyte.

[0027] Macromers conjugated to biomolecules (eg, antibodies) can be used, for example, as direct reporter molecules in biological assays (eg, immunoassays). Excitation of the macromer with light can result in emission from the macromer, indicating the presence of the antibody in the assay or assay solution.

[0028] In yet another embodiment, the present invention provides a method for detecting a target biomolecule in a sample, the method comprising:

[0029] Providing a sample suspected of containing the target analyte;

[0030] providing a macromer of formula I or II conjugated to a capture molecule, wherein the capture molecule is capable of interacting with the target analyte;

[0031] contacting the sample with the capture molecule and the conjugated macromer under conditions where the capture molecule can bind to the target analyte (if present); applying a light source to the sample that excites the conjugated macromer; and

[0032] Whether light is emitted from the conjugated macromer is detected.

[0033] In some aspects, the method is carried out in vivo or alternatively in vitro. In some aspects, the sample contains living cells. In some aspects, the analyte is a nucleic acid that comprises a genetic point mutation, a deletion or an insertion relative to a control nucleic acid. The control nucleic acid may contain a genetic mutation. In some aspects, the detection of the nucleic acid indicates that cancer is present in the sample.

[0034] These and other aspects, objects and advantages will become more apparent when the following detailed description and accompanying drawings are read. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 An embodiment of the present disclosure is shown in which an antibody labeled with a dye is reacted with a macromer of the present disclosure.

[0036] Figure 2 A schematic diagram showing a bioconjugated macromer according to one embodiment of the present disclosure.

[0037] Figure 3 AD shows the antibody ( Figure 3 A), avidin ( Figure 3 B) Nucleic Acids ( Figure 3 C), and dyes ( Figure 3 D) Exemplary macromers conjugated.

[0038] Figure 4 AD shows an embodiment of the present disclosure; Figure 4 A shows an antibody labeled with a dye and a macromer according to the present disclosure; Figure 4 B shows streptavidin labeled with a dye and with a macromer according to the present disclosure; Figure 4 C shows a nucleic acid probe sequence labeled with a quencher molecule conjugated to a macromer of the present disclosure; and Figure 4 D shows a nucleic acid probe sequence labeled with a quencher molecule and a macromer according to the present disclosure.

[0039] Figure 5 AD show schematic diagrams depicting various methods for analyzing target biomolecules or analytes; Figure 5 A shows the macromer linked to the primary antibody and conjugated to the secondary antibody with a dye; Figure 5 B shows that the macromer and dye-labeled antibodies recognize a common target; Figure 5 C shows an antibody with attached dye and biotin and a second bioconjugate of streptavidin with a macromer attached thereto; Figure 5D shows a nucleic acid with a dye and biotin bound thereto and a macromer with streptavidin conjugated thereto.

[0040] Figure 6 A schematic diagram according to the present disclosure is shown.

[0041] Figure 7 AC shows various schematic diagrams of the present disclosure; Figure 7 A describes a macromer with attached dye and biomolecule, which produces energy transfer; Figure 7 B shows the macromer with conjugated streptavidin and attached dye; Figure 7 C shows the macromer conjugated to nucleic acid and dye.

[0042] Figure 8 AF show schematics depicting indirect association with a macromer attached to a biomolecule; Figure 8 A shows biotinylated antibody interacting with the covalent conjugate of the macromer; Figure 8 B shows a biotinylated antibody bound to a macromer-linked moiety with a dye attached; Figure 8 C shows biotinylated nucleic acid interacting with the covalent part of the macromer; Figure 8 D shows a biotinylated nucleic acid bound to a linker moiety of a macromer with an attached dye; Figure 8 E shows a nucleic acid having a digoxigenin moiety that interacts with the covalently attached antibody of macromer 801; and Figure 8 F shows a nucleic acid with a digoxigenin moiety and a covalent antibody attached to the macromer dye tandem complex.

[0043] Fig. 9 AB shows experimental embodiments of the present disclosure; Fig. 9 A shows the primary antibody 905 bound to the analyte, to which the secondary antibody with attached macromers was added; Fig. 9 B shows the target analyte bound to the primary antibody using the linked macromer.

[0044] Fig.10 AB shows a sandwich type composite embodiment; Fig.10 A shows the primary antibody binding to the analyte, followed by the addition of a secondary antibody with biotin, followed by the addition of a macromer with streptavidin to create a sandwich-type complex; Fig.10 B shows the biotinylated primary antibody bound to the analyte, followed by the addition of streptavidin linked to the macromer.

[0045] Fig.11A-C shows CD3 staining of lysed washed blood (LWB) gated on lymphocytes: 0.5 μg; Fig.11A UCHT1 Pacific Blue (clone: ​​UCHT1; conjugate: Pacific BlueTM; signal-to-noise ratio: 60; staining index: 10; antibody concentration: μg) is shown; Fig. 11B P43 is shown (clone: ​​UCHT1; conjugate: P43; signal-to-noise ratio: 507; staining index: 71; antibody concentration: 0.5 μg); Fig. 11C P100 is shown (clone: ​​UCHT1; conjugate: P100; signal-to-noise ratio: 517; staining index: 76; antibody concentration: 0.5 μg).

[0047] Fig. 12A -C shows perforin staining of peripheral blood mononuclear cells (PBMCs) gated on lymphocytes: Fig. 12A Pacific Blue was displayed (clone: ​​dG9; conjugate: Pacific BlueTM; signal-to-noise ratio: 33; staining index: 9; antibody concentration: 0.25 μg); Fig. 12B P43 (clone: ​​dG9; conjugate: P43; signal-to-noise ratio: 46; staining index: 17; antibody concentration: 0.25 μg; Fig. 12C P100 is shown (clone: ​​dG9; conjugate: P100; signal-to-noise ratio: 41; staining index: 15; antibody concentration: 0.25 μg).

[0048] Fig.13 The absorption curve of the macromonomer (L18) of the present disclosure is shown.

[0049] Fig.14 The absorption curve of the macromer (P100) of the present disclosure is shown. DETAILED DESCRIPTION

[0050] I. Definitions

[0051] As used herein, the terms "a", "an" or "the" include not only aspects having one member but also aspects having more than one member.

[0052] The term "about" used to modify a numerical value herein represents a limited range around the value. If "X" is the value, then "about X" represents a value of 0.9X-1.1X, more preferably a value of 0.95X-1.05X. Any reference to "about X" specifically represents a value of at least X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Therefore, "about X" is intended to teach and provide written description support for a claim limitation of, for example, "0.98X".

[0053] When the modifier "about" is used to describe the beginning of a numerical range, it applies to both ends of the range. Thus, "about 500 to 850 nm" is equivalent to "about 500 nm to about 850 nm". When "about" is used to describe the first value in a group of values, it applies to all values ​​in the group. Thus, "about 580, 700, or 850 nm" is equivalent to "about 580 nm, about 700 nm, or about 850 nm". However, when the modifier "about" is used to describe only the end of the range or only a later value in the group of values, it only applies to that value or the end of the range. Thus, the range of "about 2 to about 10" is the same as "about 2 to about 10", but not the same as the range of "2 to about 10".

[0054] As used herein, "activated acyl" includes -C(O)-LG groups. A "leaving group" or "LG" is a group that is easily displaced by nucleophilic acyl substitution (i.e., nucleophilic addition to the carbonyl of -C(O)-LG followed by elimination of the leaving group). Representative leaving groups include halogens, cyano, azido, carboxylic acid derivatives, such as tert-butylcarboxyl, and carbonate derivatives, such as i-BuOC(O)O-. Activated acyl groups can also be activated esters as defined herein or activated by carbodiimide to form anhydrides (preferably cyclic) or mixed anhydrides -OC(O)R a or -OC(NR a )NHR b (preferably cyclic) carboxylic acid, wherein R a and R b Independently selected from C1-C6 alkyl, C1-C6 perfluoroalkyl, C1-C6 alkoxy, cyclohexyl, 3-dimethylaminopropyl or N-morpholinoethyl. Preferred activated acyl groups include activated esters.

[0055] As used herein, "activated esters" include carboxyl derivatives that are more easily displaced by nucleophilic addition and elimination than ethyl esters (e.g., NHS esters, sulfo-NHS esters, PAM esters, or halophenyl esters). Representative carbonyl substituents of activated esters include succinimidyloxy (-OC4H4NO2), sulfosuccinimidyloxy (-OC4H3NO2SO3H), -1-oxybenzotriazolyl (-OC6H4N3); 4-sulfo-2,3,5,6-tetrafluorophenyl; or aryloxy (e.g., pentafluorophenyloxy, 2,3,5,6-tetrafluorophenyloxy) optionally substituted one or more times with electron-withdrawing substituents (e.g., nitro, fluorine, chlorine, cyano, trifluoromethyl, or a combination thereof). Preferred activated esters include succinimidyloxy, sulfosuccinimidyloxy, and 2,3,5,6-tetrafluorophenyloxy esters.

[0056] As used herein, "acyl" includes alkanoyl, aroyl, heterocyclic acyl or heteroaroyl as defined herein. Representative acyl groups include acetyl, benzoyl, nicotinoyl, and the like.

[0057] As used herein, "alkanoyl" includes alkyl-C(O)- groups, wherein the alkyl group is as defined herein. Representative alkanoyl groups include acetyl, ethoxy, and the like.

[0058] As used herein, "alkenyl" includes straight or branched aliphatic hydrocarbon groups of 2 to about 15 carbon atoms containing at least one carbon-carbon double bond or triple bond. Preferred alkenyl groups have 2 to about 12 carbon atoms. More preferred alkenyl groups contain 2 to about 6 carbon atoms. In one aspect, hydrocarbon groups containing carbon-carbon double bonds are preferred. In a second aspect, hydrocarbon groups (i.e., alkynyl) containing carbon-carbon triple bonds are preferred. "Lower alkenyl" as used herein includes alkenyl groups of 2 to about 6 carbon atoms. Representative alkenyl groups include vinyl, allyl, n-butenyl, 2-butenyl, 3-methylbutenyl, n-pentenyl, heptenyl, octenyl, decenyl, propynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl, heptynyl, etc.

[0059] Alkenyl can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms (e.g., 1 to 4, 1 to 2, or 1) of alkenyl can be independently substituted by a moiety selected from the group consisting of fluorine, hydroxyl, alkoxy, amino, alkylamino, acylamino, sulfenyl, and alkylthio.

[0060] As used herein, "alkenylene" includes a straight or branched divalent hydrocarbon chain containing at least one carbon-carbon double bond or triple bond. Preferred alkenylene groups contain 2 to about 12 carbons in the chain, and more preferred alkenylene groups contain 2 to 6 carbons in the chain. In one aspect, hydrocarbon groups containing carbon-carbon double bonds are preferred. In a second aspect, hydrocarbon groups containing carbon-carbon triple bonds are preferred. Representative alkenylene groups include -CH=CH-, -CH2-CH=CH-, -C(CH3)=CH-, -CH2CH=CHCH2-, ethynylene, propynylene, n-butynylene, and the like.

[0061] As used herein, "alkoxy" includes alkyl-O- groups, wherein the alkyl group is as defined herein. Representative alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, heptoxy, and the like.

[0062] Alkoxyl can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms (e.g. 1-4, 1-2, or 1) of alkoxyl can be independently substituted by a moiety selected from fluorine, hydroxyl, alkoxyl, amino, alkylamino, acylamino, sulfenyl, and alkylthio.

[0063] As used herein, "alkoxyalkyl" includes alkyl-O-alkylene- groups wherein the alkyl and alkylene are as defined herein. Representative alkoxyalkyl groups include methoxyethyl, ethoxymethyl, n-butoxymethyl and cyclopentylmethoxyethyl.

[0064] As used herein, "alkoxycarbonyl" includes ester groups, i.e., alkyl-O-CO- groups, wherein the alkyl group is as defined herein. Representative alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, and the like.

[0065] As used herein, "alkoxycarbonylalkyl" includes alkyl-O-CO-alkylene-groups, wherein the alkyl and alkylene are as defined herein. Representative alkoxycarbonylalkyl groups include methoxycarbonylmethyl, ethoxycarbonylmethyl, methoxycarbonylethyl, and the like.

[0066] As used herein, "alkyl" includes aliphatic hydrocarbon groups, which may be straight or branched, having from about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups have from 1 to about 12 carbon atoms in the chain. More preferred alkyl groups have from 1 to 6 carbon atoms in the chain. As used herein, "branched" includes one or more lower alkyl groups, such as methyl, ethyl or propyl groups attached to a straight alkyl chain. As used herein, "lower alkyl" includes from 1 to about 6 carbon atoms in the chain, preferably 5 or 6 carbon atoms, which may be straight or branched. Representative alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl and 3-pentyl.

[0067] Alkyl can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms (e.g., 1 to 4, 1 to 2, or 1) of the alkyl can be independently substituted by a moiety selected from the group consisting of fluorine, hydroxyl, alkoxy, amino, alkylamino, acylamino, sulfenyl, and alkylthio.

[0068] As used herein, "alkylene" includes straight or branched divalent hydrocarbon chains of 1 to about 6 carbon atoms. Preferred alkylene groups are lower alkylene groups of 1 to about 4 carbon atoms. Representative alkylene groups include methylene, ethylene, and the like.

[0069] As used herein, "alkylthio" includes alkyl-S-groups, wherein the alkyl group is as defined herein. Preferred alkylthio groups are those in which the alkyl group is a lower alkyl group. Representative alkylthio groups include methylthio, ethylthio, isopropylthio, heptylthio, and the like.

[0070] As used herein, "alkylthioalkyl" includes alkylthio-alkylene- groups, wherein the alkylthio and alkylene are as defined herein. Representative alkylthioalkyl groups include methylthiomethyl, ethylthiopropyl, isopropylthioethyl, and the like.

[0071] As used herein, "amide" includes groups of the formula Y1Y2N-C(O)-, wherein Y1 and Y2 are independently hydrogen, alkyl or alkenyl; or Y1 and Y2 are combined with the nitrogen connecting Y1 and Y2 to form a 4-membered nitrogen heterocyclic group to a 7-membered nitrogen heterocyclic group (e.g., piperidinyl). Representative amides include primary amides (H2N-C(O)-), methylamides, dimethylamides, diethylamides, and the like. Preferably, "amide" is a -C(O)NRR' group, wherein R and R' are independently selected from H and alkyl. More preferably, at least one of R and R' is H.

[0074] As used herein, "amidoalkyl" includes amido-alkylene- groups wherein the amido and alkylene are as defined herein. Representative amidoalkyl groups include amidomethyl, amidoethylene, dimethylamidomethyl, and the like.

[0075] As used herein, "amino" includes groups of the formula Y1Y2N-, wherein Y1 and Y2 are independently hydrogen, acyl or alkyl; or Y1 and Y2 are combined with the nitrogen connecting Y1 and Y2 to form a 4-membered nitrogen heterocyclic group to a 7-membered nitrogen heterocyclic group (e.g., piperidinyl). Optionally, when Y1 and Y2 are independently hydrogen or alkyl, additional substituents may be added to nitrogen to produce a quaternary ammonium ion. Representative amino groups include primary amino (H2N-), methylamino, dimethylamino, diethylamino, and the like. Preferably, "amino" is a -NRR' group, wherein R and R' are members independently selected from H and alkyl. Preferably, at least one of R and R' is H.

[0076] As used herein, "aminoalkyl" includes amino-alkylene-groups wherein the amino and alkylene groups are as defined herein. Representative aminoalkyl groups include aminomethyl, aminoethyl, dimethylaminomethyl, and the like.

[0077] As used herein, "aroyl" includes an aryl-CO- group wherein the aryl group is as defined herein. Representative aroyl groups include benzoyl, naphth-1-yl and naphth-2-yl.

[0078] As used herein, "aryl" includes aromatic monocyclic or multicyclic ring systems of 6 to about 14 carbon atoms, preferably 6 to about 10 carbon atoms. Representative aryl groups include phenyl and naphthyl.

[0079] As used herein, "aromatic ring" includes 5-12 membered aromatic monocyclic or fused polycyclic moieties, which may include 0-4 heteroatoms selected from oxygen, sulfur, selenium and nitrogen. Exemplary aromatic rings include benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, naphthalene, benzothiazoline, benzothiophene, benzofuran, indole, benzindole, quinoline, etc. The aromatic ring group may be substituted at one or more positions by halogen, alkyl, alkoxy, alkoxycarbonyl, haloalkyl, cyano, sulfonic acid, aminosulfonyl, aryl, sulfonyl, aminocarbonyl, carboxyl, acylamino, alkylsulfonyl, amino and substituted or unsubstituted substituents.

[0080] As used herein, "biomolecules" include natural or synthetic molecules used in biological systems. Preferred biomolecules include antibodies, antigens, proteins, peptides, enzyme substrates, hormones, haptens, avidin, streptavidin, carbohydrates, carbohydrate derivatives, oligosaccharides, polysaccharides, and nucleic acids. More preferred biomolecules include antibodies, proteins, peptides, avidin, streptavidin, or biotin. In certain aspects, biomolecules include, but are not limited to, proteins, peptides, affinity ligands, antibodies, antibody fragments, sugars, lipids, enzymes, and nucleic acids (as hybridization probes and / or aptamers).

[0081] As used herein, "carboxy" and "carboxyl" include a HOC(O)- group (ie, a carboxylic acid) or a salt thereof.

[0082] As used herein, "carboxyalkyl" includes HOC(O)-alkylene-groups, wherein the alkylene is as defined herein. Representative carboxyalkyl groups include carboxymethyl (ie, HOC(O)CH2-) and carboxyethyl (ie, HOC(O)CH2CH2-).

[0083] As used herein, "conjugated macromonomers" include macromonomers containing a series of extended unsaturated bonds. The main chain of the conjugated macromonomer or polymer may contain alternating double bonds and single bonds. The conjugated polymer may be conjugated along the entire length of its backbone or may contain conjugated segments and non-conjugated segments.

[0084] As used herein, "conjugated" includes unsaturated organic systems of adjacent atoms with pi electrons where there is overlap of a p orbital with another orbital across an intervening sigma bond. In larger atoms, d orbitals may be involved. Atoms may be sp 2 Or sp hybridized carbon atoms or other atoms with unshared electron pairs that can hybridize into p orbitals.

[0085] As used herein, "cycloalkyl" includes non-aromatic monocyclic or polycyclic ring systems of about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms. More preferred cycloalkyl rings contain 5 or 6 ring atoms. Cycloalkyl optionally contains at least one sp 2 -hybridized carbon (e.g., a ring introducing an endocyclic or exocyclic olefin). Representative monocyclic cycloalkyls include cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, and the like. Representative multicyclic cycloalkyls include 1-decalin, norbornyl, adamantyl, and the like.

[0088] As used herein, "cycloalkylene" includes divalent cycloalkyl groups having about 4 to about 8 carbon atoms. Preferred cycloalkylene groups include 1,2-, 1,3- or 1,4-cis or trans-cyclohexylene.

[0089] As used herein, "halo" or "halogen" includes fluorine, chlorine, bromine or iodine.

[0090] "Heteroatom" as used herein includes atoms other than carbon or hydrogen. Representative heteroatoms include O, S and N. The nitrogen or sulfur atom of the heteroatom is optionally oxidized to the corresponding N-oxide, S-oxide (sulfoxide) or S, S-dioxide (sulfone). In preferred aspects, the heteroatom has at least two bonds with the alkylene carbon atom (e.g., -C1-C9 alkylene-O-C1-C9 alkylene-). In some embodiments, the heteroatom is further substituted by acyl, alkyl, aryl, cycloalkyl, heterocyclic or heteroaryl (e.g., -N(Me)-, -N(Ac)-).

[0091] As used herein, "heteroaryl" refers to an aryl group in which at least one carbon atom in at least one aromatic ring is replaced by a heteroatom (e.g., nitrogen, oxygen, sulfur, and phosphorus) such that the aromaticity of the compound is retained and optionally substituted at one or more substitutable positions. Exemplary heteroaryl groups include furanyl, thienyl, pyridyl, pyridazinyl, pyrrolyl, N-lower alkyl-pyrrolyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, triazolyl, tetrazolyl, imidazolyl, bipyridyl, tripyridyl, tetrapyridyl, phenazinyl, phenanthroline, purinyl, perylene, perylene diimide, pyrrolopyrrolyl, benzothiadiazole, benzoxadiazole, thienopyrazine, and the like. Other examples of heteroaryl groups include fused ring systems such as benzofuranyl, benzothienyl, benzopyrrolyl, dibenzofuranyl, dibenzothienyl, phenanthrolinyl, carbazolyl, and azacarbazolyl.

[0092] As used herein, "heteroaroyl" includes heteroaryl-C(O)- groups, wherein the heteroaryl group is as defined herein. Representative heteroaroyl groups include thiophenoyl, nicotinoyl, pyrrol-2-ylcarbonyl, pyridinoyl, and the like.

[0093] As used herein, "heterocyclic acyl" includes heterocyclic-C(O)- groups, wherein the heterocyclic group is as defined herein. Representative heterocyclic acyl groups include N-methylprolyl, tetrahydrofuranoyl, and the like.

[0094] As used herein, "hydroxyalkyl" includes alkyl groups as defined herein substituted with one or more hydroxy groups. Preferred hydroxyalkyl groups contain a lower alkyl group. Representative hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.

[0095] The term "optionally substituted" means that the substituent may be substituted or unsubstituted. For example, the hydrogen atoms of an alkyl group (e.g., 1-4, 1-2, or 1) may be independently substituted by a portion selected from fluorine, hydroxyl, alkoxyl, amino, alkylamino, acylamino, sulfenyl, and alkylthio. The aromatic ring group may be substituted by halogen, alkyl, alkoxyl, alkoxycarbonyl, haloalkyl, cyano, sulfonic acid, aminosulfonyl, aryl, sulfonyl, aminocarbonyl, carboxyl, acylamino, alkylsulfonyl, amino, and substituted or unsubstituted substituents at one or more positions. In a similar manner, each substituent mentioned herein may be substituted by amino, halogen, hydroxyl, alkoxyl, cyano, nitro, alkylamino, acylamino, sulfenyl, alkylthio, alkyl, alkoxycarbonyl, haloalkyl, sulfonic acid, aminosulfonyl, aryl, sulfonyl, aminocarbonyl, carboxyl, acylamino, alkylsulfonyl, amino, and substituted or unsubstituted substituents.

[0096] The "number average molecular weight" comprises the arithmetic mean or average of the molecular weights of the individual macromolecules. It is determined by measuring the molecular weight of n polymer molecules, adding the masses, and then dividing by n. The weight average molecular weight (Mw), on the other hand, takes into account the molecular weight of the chains when determining the contribution to the molecular weight average. The larger the chain, the greater the contribution of the chain to the Mw. Mw is determined by methods that are sensitive to the size of the molecules and not just their number, such as light scattering techniques.

[0097] As used herein, "water-soluble" includes materials that are soluble in water-based solutions such as water, water-based solutions or buffered solutions, including those used in biological or molecular detection systems. A "water-soluble" solution refers to a homogeneous solution containing a completely dissolved substance. The "water-soluble" macromers of the present disclosure are soluble in water-based solutions at concentrations >0.10 mg / mL. Introducing at least one "water-solubilizing group" into a material can increase the hydrophilicity of the material and can improve the solubility or dispersibility of the material in an aqueous environment.

[0098] A. Compounds

[0099] The present disclosure provides polyindenofluorene macromonomers, methods for preparing macromonomers, and methods for using macromonomers. In some aspects, the macromonomers of formula I or II include one or more water solubilizing groups. The water-soluble conjugated macromonomers including one or more water-soluble groups make the polyindenofluorene macromonomers soluble in aqueous media (e.g., water or buffer). The macromonomers are suitable for various types of biological applications. The macromonomers emit bright visible light (e.g., generated by violet laser irradiation) when UV excited, and can exhibit high extinction coefficients and quantum efficiencies (e.g., quantum yields>50%).

[0100] Thus, in one embodiment, the present disclosure provides a macromer of Formula I or Formula II:

[0101]

[0102] In certain aspects, the macromonomers of Formula I and Formula II can be 1 and E 2 Between in alternating, block, random or graphic copolymer configuration combination. Advantageously, various connection modes and the characteristics and contents of the second and third monomers lead to different conjugated structures with structural polymer differences and spectral properties such as absorption, emission and quantum yield differences. Various copolymer configurations are schematically shown in the following Scheme I. In this illustrative scheme, the fragment A in the following Scheme I can represent the fragment "m" in Formula I or II, and the fragment B in Scheme I can represent the fragment "n" in Formula I or II, as shown below:

[0103]

[0104] Although a segment representing segment "o" is not shown, one skilled in the art will immediately recognize that a segment o from Formula I or II can be incorporated into the copolymer configuration as shown in Scheme I.

[0105] The term "copolymer" is a term known in the art. It includes polymers comprising two or more different monomeric units, such as segments m, n and o (depicted as, for example, A, B, C), which are polymerized in a process known as copolymerization. Since copolymers contain at least two different monomeric units, copolymers can be classified based on how the monomers are arranged to form a polymer chain. These classifications include "alternating copolymers" (wherein the monomeric units are repeated in a regular alternating pattern, such as ABABAB or ABCABC), "block copolymers" (wherein two or more homopolymer subunits are connected, such as AAABBB or AAABBBCCC), "random copolymers" (wherein the monomeric units are connected in a random order, such as ABAAABBAB or ABAAABBAB-CCC-AB), and "patterned copolymers" (wherein a homopolymer is grafted to a second polymer, such as BBB grafted to AAAA or CCC grafted to AAAABBB). The copolymers of the present disclosure may be E 1 and E 2 In some cases, the copolymer is an alternating copolymer, a block copolymer, a random copolymer or a graft copolymer. In some cases, the copolymer is an alternating copolymer. In some cases, the copolymer is a random copolymer.

[0106] In Formula I or Formula II, R 1 , R 2 , R 3 and R 4 are each independently selected from hydrogen, optionally substituted C1-C 10 Alkyl, optionally substituted C1-C 18 Alkoxy, optionally substituted C6-C 18 Aryl, optionally substituted C4-C 18 Acyl, optionally substituted C4-C 18 Acyloxy, NR a R b , where R a and R b Each is independently hydrogen or a lower alkyl group, a water-soluble group, an ethylene oxide oligomer or an ethylene oxide oligomer methyl ether. The dotted circle means that the ring is an optional embodiment.

[0107] In another embodiment, R 1 and R 2 and / or R 3 and R 4 Together with the carbon atoms to which they are attached, they are joined to form an optionally substituted 4-membered ring, 5-membered ring, or 6-membered ring. 1 and R 2 and R 3 and R4 are connected to form an optionally substituted 4-membered ring, 5-membered ring or 6-membered ring. 1 and R 2 are connected to form an optionally substituted 4-membered ring, 5-membered ring or 6-membered ring. 3 and R 4 The rings are linked to form an optionally substituted 4-, 5- or 6-membered ring.

[0108] In certain aspects, the optionally substituted 4-membered ring, 5-membered ring or 6-membered ring is selected from optionally substituted C4-C6 cycloalkyl and optionally substituted C4-C6 heterocyclyl. 1 and R 2 and / or R 3 and R 4 In one aspect, R 1 and R 2 and / or R 3 and R 4 An optionally substituted 4-, 5- or 6-membered ring is formed which results in a bicyclic ring system (within a larger ring system) in which the two rings are linked by a single common atom. This common atom linking the bicyclic rings is often referred to as a "spiro" atom.

[0109] In Formula I or Formula II, each R 5 Independently selected from halogen, alkyl, alkoxy, amino, water solubilizing group, ethylene oxide oligomer and ethylene oxide oligomer methyl ether.

[0110] In some cases, R 5 It is an ethylene oxide oligomer, such as -(CH2) y -(OCH2CH2) x OCH3 groups wherein y is a value of 1 to 20 and x is a value of 1 to 50. For example, y can have a value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. x can have a value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. The ethylene oxide oligomers enhance the water solubility of the macromonomers.

[0111] In some aspects, C4-C6 heterocyclic group is a member selected from the group consisting of azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, oxolanyl, thiolanyl, piperidinyl, oxanyl and thianyl or optionally substituted tetrahydropyranyl, such as xanthenyl. In some preferred aspects, C4-C6 heterocyclic group is optionally substituted pyrrolidinyl or piperidinyl.

[0112] In certain aspects, the C4-C6 heterocyclic group is replaced by R 5 Replace. 5 It may be at least one ethylene oxide oligomer, such as -(CH2) y -(OCH2CH2) x OCH3 group, wherein the value of y is 1-20 and the value of x is 1-50.

[0113] In one aspect, the optionally substituted 4-, 5- or 6-membered rings are C4-C6 heterocyclic radicals selected from: azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, oxolanyl, thiolanyl, piperidinyl, oxane and thiolanyl or optionally substituted tetrahydropyranyl, such as xanthanyl. In some preferred aspects, the C4-C6 heterocyclic radical is optionally substituted pyrrolidinyl or piperidinyl.

[0114] In Formula I or Formula II, each p is a value of 0-3, such as 0, 1, 2 or 3.

[0115] In Formula I or Formula II, m is a value selected from 1 to 10,000. In certain aspects, the value of m is 1-10,000, such as 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 70, 1 to 80, 1 to 90, 1 to 100, 1 to 200, 1 to 300, 1 to 400, 1 to 500, 1 to 600, 1 to 700, 1 to 800, 1 to 900, 1 to 1000, 1 to 1 to 2000, 1 to 3000, 1 to 4000, 1 to 5000, 1 to 6000, 1 to 7000, 1 to 8000, 1 to 9000, or 1 to 10000.

[0116] In Formula I or Formula II, n and o are each independently a value selected from 0-10,000, wherein the monomers within m, n and o can be the same or different. In certain aspects, n and o are each independently a value selected from 0-10,000, such as 0, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000. When n is 0, fragment A-2 is absent. When o is 0, fragment B-3 is absent. Thus, A-2 and B-3 may each be present or absent and may each be the same or different, and are each selected from aromatic or heteroaromatic groups, which complete the π-conjugated backbone.

[0117] In certain aspects, in Formula I or Formula II, m is 1-50, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and / or 50.

[0118] 41 , 42, 43, 44, 45, 46, 47, 48, 49, and / or 50.

[0119] 41 , 42, 43, 44, 45, 46, 47, 48, 49, and or 50. In some aspects, in Formula I or Formula II, o is 0-50, such as about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, and or 50.

[0120] In some cases, the polyindenofluorene macromonomer of formula I or formula II comprises at least one monomer residue substituted with one or more water solubilizing groups. In some cases, the main chain of the polyindenofluorene macromonomer of formula I or II comprises a plurality of monomer residues. The macromonomer main chain may comprise a conjugated segment. The polymer backbone may further comprise at least one monomer residue comprising an optionally substituted aromatic or heteroaromatic group attached to the indenofluorene monomer, the indenofluorene monomer being A and / or B.

[0121] In some aspects, A and B in Formula I or Formula II may or may not exist. If present, A and B may be the same or different. A and B represent a divalent substituent, which is a divalent group selected from benzene, naphthalene, anthracene, benzodithiazolyl, fluorene, indenofluorene, thiophene, thienothiophene, dithienothiophene, 3,4-ethylenedioxythiophene, furan, pyridine, pyrrole, fused pyrrole, tetrahydropyrene, quinoxaline, benzothiadiazole, thieno[3,4-b]-pyrazine and oxadiazole. Each of the aforementioned groups may be optionally substituted.

[0122] In the case of copolymers, these polymers can be alternating, block and random copolymers or combinations thereof. Fluorescence properties such as molar extinction coefficient, fluorescence quantum yield, absorption and emission maxima and shape are expected to be different.

[0123] In certain aspects, segments m, n and o may also have moieties 1, 2 and / or 3 present, which are divalent aromatic or heteroaromatic groups. 1, 2 or 3 may each be present or absent. Moieties 1, 2 and 3 are each independently selected from benzene, naphthalene, anthracene, benzodithiazolyl, fluorene, indenofluorene, thiophene, thienothiophene, dithienothiophene, 3,4-ethylenedioxythiophene, furan, pyridine, pyrrole, fused pyrrole, tetrahydropyrene and oxadiazole. Each of the foregoing groups may be optionally substituted.

[0124] In Formula I and Formula II, E 1 and E 2 Each is independently selected from hydrogen, halogen, alkynyl, optionally substituted aryl, optionally substituted heteroaryl, halogen, substituted aryl, boronic acid substituted aryl, borate substituted aryl, borate, boronic acid, optionally substituted fluorene and aryl or heteroaryl substituted by one or more side chains, which are terminated by the following groups: i) a functional group selected from the following: amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimide, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol and its protecting group, for conjugation with molecules or biomolecules; or ii) conjugation with organic dyes or biomolecules.

[0125] In one aspect, E 1 and E 2Each is independently selected from hydrogen and a functional moiety selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, alkyl halide and protecting groups thereof for conjugation to a molecule or biomolecule.

[0126] Biomolecules include, but are not limited to, antibodies, antigens, proteins, peptides, enzyme substrates, hormones, haptens, avidin, streptavidin, carbohydrates, carbohydrate derivatives, oligosaccharides, polysaccharides, and nucleic acids. More preferred biomolecules include antibodies, proteins, peptides, avidin, streptavidin, or biotin. In certain aspects, biomolecules include, but are not limited to, proteins, peptides, affinity ligands, antibodies, antibody fragments, sugars, lipids, enzymes, and nucleic acids (as hybridization probes and / or aptamers).

[0127] Compounds of Formula I and Formula II can react with biomolecules using conjugation chemistry well known in the art. For example, activated esters (NHS esters) can react with primary amines to form stable amide bonds. Maleimide and thiol can react together and prepare thioethers. Alkyl halides react with amines and thiols to prepare alkylamines and thioethers, respectively. Any derivative that provides a reactive portion that can be conjugated to a protein or biomolecule can be used herein.

[0128] In one embodiment, 1, 2, and 3 are absent in Formula I and Formula II, which provide Formula Ia and Formula II-a:

[0129]

[0130] In one aspect, A and B are absent (n=0 and o=0), and the compound has the following structure:

[0131]

[0132] The dotted line refers to R 1 and R 2 and R 3 and R 4 The two may be optionally linked to form an optionally substituted 4-membered ring, 5-membered ring or 6-membered ring, or only R 1 and R 2 are connected to form an optionally substituted 4-membered ring, 5-membered ring or 6-membered ring, or only R 3 and R 4 Connect to form an optionally substituted 4-, 5- or 6-membered ring. The optionally substituted 4-, 5- or 6-membered ring is selected from an optionally substituted C4-C6 cycloalkyl and an optionally substituted C4-C6 heterocyclic group. The optionally substituted 4-, 5- or 6-membered ring may not exist.

[0133] In formula I or II, the water solubilizing group is a group that gives the macromonomer more hydrophilicity. The water solubilizing group can be an ethylene oxide oligomer or an ethylene oxide polymer or an ethylene oxide oligomer methyl ether. In some aspects, the water solubilizing group includes one or more alkylene oxide repeating units. For example, the water solubilizing group can contain one or more ethylene glycol units, -(OCH2CH2)-. Ethylene glycol oligomers or polymers are referred to as "polyethylene glycol" (PEG) groups in this article. The PEG group can be any length, however, typically 1-5000 ethylene glycol repeating units. In certain embodiments, a PEG group with more than 20 ethylene glycol repeating units is used. In some aspects, the ethylene oxide unit can be 11 to about 550, including 11 and 550. The PEG chain can be attached to the monomer, grafted or coupled to the polymer to increase water solubility.

[0134] In certain aspects, PEG 550 -OCH3 has CH3-(O-CH2-CH2) x -, with a number average molecular weight of 550 and a terminal methoxy group. Polyethylene glycol monomethyl ether, "mPEG" has the general formula CH3(OCH2CH2) n OH, with an average Mn of 550. Other averages include, but are not limited to, 350, 750, 2000, and 5000, or the number of units may be between 11 and 550, inclusive.

[0135] In certain aspects, the compound of Formula Ia has a formula selected from:

[0136]

[0137] In certain aspects, the compound of Formula Ia has the formula:

[0138]

[0139] In some respects, R 6 is a water-soluble group, such as ethylene oxide oligomer methyl ether. In certain aspects, ethylene oxide oligomer methyl ether is in -O-PEG 11 -OCH3 and -O-PEG 550 -OCH3, and including -O-PEG 11 -OCH3 and -O-PEG 550 -OCH3, e.g. -O-PEG 11 -OCH3 and -O-PEG 550 -OCH3. In one aspect, R 6 Yes-O-PEG 11 -OCH3. On the other hand, R 6 Yes-O-PEG 550-OCH3.

[0140] In certain aspects, the compound of formula II-a has the formula:

[0141]

[0142] The dotted line represents R 1 and R 2 and R 3 and R 4 The two may be optionally linked to form an optionally substituted 4-membered ring, 5-membered ring or 6-membered ring, or only R 1 and R 2 to form an optionally substituted 4-membered ring, 5-membered ring or 6-membered ring, or only R 3 and R 4 Connect to form an optionally substituted 4-, 5- or 6-membered ring. The optionally substituted 4-, 5- or 6-membered ring is selected from an optionally substituted C4-C6 cycloalkyl and an optionally substituted C4-C6 heterocyclic group. The optionally substituted 4-, 5- or 6-membered ring may not exist.

[0143] In certain aspects, the compound of Formula II-a has a formula selected from:

[0144]

[0145] In certain aspects, the compound of formula II-a has the formula:

[0146]

[0147] In certain aspects, the compound of Formula II has the formula:

[0148]

[0149] Wherein "1" in the above formula has the following structure:

[0150]

[0151] In certain aspects, the compound of formula II-a has the formula:

[0152]

[0153] In some respects, each R 6 Independently a water-soluble group, such as an ethylene oxide oligomer methyl ether, such as mPEG 550 In certain aspects, the ethylene oxide oligomer methyl ether in -O-PEG 11 -OCH3 and -O-PEG 550 -OCH3, and including -O-PEG 11-OCH3 and -O-PEG 550 -OCH3, e.g. -O-PEG 11 -OCH3 and -O-PEG 550 -OCH3. In one aspect, R 6 Yes-O-PEG 11 -OCH3. On the other hand, R 6 Yes-O-PEG 550 -OCH3.

[0154] In certain aspects, B represents a divalent substituent selected from benzene, naphthalene, anthracene, benzodithiazolyl, fluorene, indenofluorene, thiophene, thienothiophene, dithienothiophene, 3,4-ethylenedioxythiophene, furan, pyridine, pyrrole, fused pyrrole, tetrahydropyrene and oxadiazole. Each of the foregoing groups may be optionally substituted.

[0155] In some aspects, m is 1-50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and / or 50; n is 1-50, and o is 0-50.

[0156] In certain aspects, B has the formula:

[0157]

[0158] Where R a is selected from hydrogen, optionally substituted C1-C 18 Alkyl, optionally substituted C1-C 18 Alkoxy, optionally substituted C6-C 18 Aryl, optionally substituted C4-C 18 Acyl, optionally substituted C4-C 18 acyloxy groups, water solubilizing groups, ethylene oxide oligomers, ethylene oxide polymers, and functional groups for conjugation with molecules or biomolecules;

[0159] Where R b is selected from hydrogen, optionally substituted C1-C 18 Alkyl, optionally substituted C1-C 18 Alkoxy, optionally substituted C6-C 18 Aryl, optionally substituted C4-C 18 Acyl, optionally substituted C4-C 18 acyloxy groups, water solubilizing groups, ethylene oxide oligomers, ethylene oxide polymers, or conjugation sites; and

[0160] z is a value from 0 to 4, for example, 0, 1, 2, 3 or 4.

[0161] In certain aspects, B has the formula:

[0162]

[0163] Among them, PEG 550 With CH3(-O-CH2-CH2) x The formula has a number average molecular weight of 550 and o is 1-50.

[0164] In certain aspects, B has the formula:

[0165]

[0166] wherein o is a value of 1-15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or 1-30, 1-40 or 1-50. In one aspect, E 2 is an N-hydroxysuccinimide group. The Boc group and water solubilizing groups, ethylene oxide oligomers and ethylene oxide polymers can be removed, or it can be a conjugation site. Examples of water-soluble groups are ethylene oxide oligomers, such as -(CH2) y -(OCH2CH2) x OCH3 groups wherein y is a value from 1 to 20 and x is a value from 1 to 50. For example, y can have a value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. x can have a value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. The site for conjugation can be i) a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimide, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol and its protecting group for conjugating a molecule or biomolecule; or ii) a functional group for conjugating an organic dye or biomolecule. a It is a substituted acyl -C(O)-CH(NH2)-CH2CH2CH2CH2NH2, in which the amine is protected by Boc.

[0167] In certain aspects, B has the formula:

[0168]

[0169] In certain aspects, o is a value of 1-50 or o is a value of 1-15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, and x is between 11 and 550. In one aspect, E 2 It is N-hydroxysuccinimide.

[0170] In certain aspects, the compound of Formula II has the formula:

[0171]

[0172] Where "1" has the following structure:

[0173]

[0174] In certain aspects, the compound of Formula II-a has the formula:

[0175]

[0176] 43, 44, 45, 46, 47, 48, 49, and / or 50.

[0177] 43, 44, 45, 46, 47, 48, 49, and / or 50.

[0178] 43, 44, 45, 46, 47, 48, 49, and / or 50. In some aspects, o is 0-50, such as about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and / or 50.

[0179] In some ways, each R 6In some aspects, the ethylene oxide oligomer methyl ether is a water-soluble group. 11 -OCH3 and -O-PEG 550 -OCH3, and including -O-PEG 11 -OCH3 and -O-PEG 550 -OCH3, e.g. -O-PEG 11 -OCH3 and -O-PEG 550 -OCH3. In one aspect, R 6 Yes-O-PEG 11 -OCH3. In another aspect, R6 is -O-PEG 550 -OCH3.

[0180] In certain aspects, NHBoc is tert-butyloxycarbonyl which protects NH2 and can be removed to generate NH2.

[0181] In certain aspects, the compound of Formula II has the formula:

[0182]

[0183] Where "1" has the following structure:

[0184]

[0185] The NHBoc group can be converted to an amine for optional conjugation.

[0186] In certain aspects, the compound of formula II-a has the formula:

[0187]

[0188] The NHBoc group can be converted to an amine for optional conjugation.

[0189] In certain aspects, the compound of formula II-a has the formula:

[0190]

[0191] Where R 6 is a water-soluble group, such as ethylene oxide oligomer methyl ether. In certain aspects, ethylene oxide oligomer methyl ether is in -O-PEG 11 -OCH3 and -O-PEG 550 -OCH3, and including -O-PEG 11 -OCH3 and -O-PEG 550 -OCH3, e.g. -O-PEG 11 -OCH3 and -O-PEG 550-OCH3. In one aspect, R 6 Yes-O-PEG 11 -OCH3. In one aspect, R 6 Yes-O-PEG 550 -OCH3.

[0192] In certain aspects, the compound of formula II-a has the formula:

[0193]

[0194] Where R 6 is a water-soluble group, such as ethylene oxide oligomer methyl ether. In certain aspects, ethylene oxide oligomer methyl ether is in -O-PEG 11 -OCH3 and -O-PEG 550 -OCH3, and including -O-PEG 11 -OCH3 and -O-PEG 550 -OCH3, e.g. -O-PEG 11 -OCH3 and -O-PEG 550 -OCH3. In one aspect, R 6 Yes-O-PEG 11 -OCH3.

[0195] In certain aspects, the compound of Formula Ia has the formula:

[0196]

[0197] In some aspects, the size of the macromonomer (i.e., polymer) of formula I or II can vary according to the type and amount of polymerization conditions, catalysts, and monomers used in the polymerization reaction. For example, the macromonomer (i.e., polymer) may have a number average molecular weight (Mn) of about 5,000 to about 100,000. Macromonomers with Mn of about 30,000 to about 70,000 are water-soluble and not easy to aggregate in aqueous media. Generally, the macromonomers of the present disclosure have a narrow range of molecular weights. For example, the polydispersity of the macromonomer of formula I or II can be represented by a polydispersity index (PDI). PDI can be calculated using the following equation: PDI=Mw / Mn, where Mw is the weight average molar mass and Mn is the number average molar mass.

[0198] In certain aspects, the macromonomers described herein may have a PDI of about 1.2 to 2.2, such as 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, or 2.2, wherein a polymer having a PDI of about 1.0 to about 1.5 may be considered monodisperse. In certain embodiments, the macromonomers of Formula I or Formula II have an Mn of about 42,000-70,000 Daltons, with a narrow distribution PDI <1.5.

[0199] In certain aspects, the macromers of Formula I or Formula II are soluble in common organic solvents, such as THF, dichloromethane, methanol, toluene, etc. Certain macromers described herein may also be soluble in aqueous media, such as water, saline, aqueous buffers, such as borate, carbonate or phosphate buffers, etc. Typically, the macromers of Formula I or Formula II exhibit water solubility up to 1.0 mg / mL in water or buffers, such as PBS.

[0200] In some aspects, the macromonomers of formula I or formula II include conjugated segments. The extended conjugation within the polymer backbone allows the macromonomer to display fluorescent emission under light excitation of the appropriate wavelength. In some cases, aromatic or heteroaromatic groups, such as A and / or B, complete the π-conjugated backbone when present. Fluorescent macromonomers including conjugated segments can have tunable photophysical properties in the range of near ultraviolet (UV) to far infrared (IR). Because the disclosed macromonomers exhibit many favorable optical properties, such macromonomers can be used in bioassays requiring high levels of sensitivity.

[0201] In some aspects, the macromers of Formula I or Formula II emit bright visible light upon UV excitation, and generally absorb light having a wavelength of about 500nm or less (e.g., about 300nm to about 500nm). In some embodiments, the disclosed polymers absorb light having a wavelength of about 350nm to about 450nm; or light having a wavelength of about 380nm to about 410nm. A violet laser can be used to effectively irradiate a macromer that absorbs light having a wavelength of about 405nm. When irradiated at an appropriate wavelength, the macromer can emit light having a wavelength greater than about 400nm; for example, about 400 to about 800nm; or light having a wavelength of about 400-780nm. The introduction of additional aromatic monomer residues (A and / or B, such as benzodithiazole, phenyl, or thienyl) into the macromer backbone can change the electronic properties of the macromer and change the excitation and emission wavelengths of the copolymer.

[0202] In certain aspects, the macromers of Formula I or Formula II can exhibit one or more of the following properties or characteristics: fluorescence emission when excited at an appropriate wavelength of light (e.g., less than about 500 nm); emission of light at a wavelength greater than about 400 nm; an extinction coefficient in water greater than about 2×10 6 cm-1 M-1; and a number average molecular weight (Mn) of from about 5,000 to about 70,000, e.g., from 5,000 to about 10,000, or to 15,000, or to 20,000, or to 25000, or to 30,000, or to 35,000, or to 40,000, or to 45,000, or to 50,000, or to 55,000, or to 60,000, or to 65,000, or to 70,000.

[0203] B. Methods of Using the Compounds

[0204] In one aspect, the macromers disclosed herein are used in FRET assays 100, such as Figure 1 As shown. For example, a biomolecule such as an antibody 110 labeled with a dye 125 is reacted with a macromer 131 of the present disclosure. The macromer of the present disclosure reacts covalently with the antibody 110 to produce an antibody with an attached dye and macromer. In this assay, energy is transferred between the donor fluorophore and the acceptor fluorophore if they are in close proximity to each other. If the two fluorophores are within a given proximity range, energy transfer occurs from the "donor", i.e., macromer 131, to the "acceptor", i.e., dye 125, by an energy source (e.g., UV light). The acceptor then emits light characterized by a star-shaped depiction of 125 at its characteristic wavelength.

[0205] Advantageously, the interaction or binding between the macromers of the present disclosure and dye-labeled antibodies increases the detection sensitivity of, for example, a biomolecular target or analyte. On the other hand, covalently attaching the macromers of the present disclosure to a dye (e.g., an antibody complex) attached to a biomolecule has several advantages, including reduced background and / or improved energy transfer. In the case of direct attachment to a biomolecule, the presence of the macromer is controlled by the biorecognition event rather than the nonspecific macromer interaction or binding event. In this way, nonspecific binding of the macromer to the biomolecule can be eliminated, thereby reducing the background emission generated by unbound or uncomplexed conjugated macromer molecules in the reaction mixture.

[0206] Now turn to Figure 2 , the schematic 200 illustrates a conjugated macromer 210 of one embodiment of the present disclosure. In certain aspects, the macromer 210 of the present disclosure is conjugated to an affinity ligand, ie, a biomolecule that has an affinity for another biomolecule. Figure 2A class of materials according to the present disclosure is illustrated, wherein a macromer 210 is attached to, for example, a dye, a biomolecule, or a biomolecule / dye complex, i.e., "X" 241. The macromer can be attached to the macromer via a first functional group 231 (A) on the macromer, which acts as a biobinding site capable of covalently attaching to a second functional group linker 236 (A') attached to a biomolecule and / or dye "X" 241. This arrangement can fix the distance between the macromer 210 and X241, thereby ensuring only a specific interaction between the macromer 210 and the portion X241 of the present disclosure. It is contemplated that the biomolecule component 241 in these embodiments can be any of the various biomolecules described herein, including but not limited to antibodies, proteins, affinity ligands, enzymes, nucleic acids, and the like.

[0207] Advantageously, linker "A" 231 can be attached anywhere on the macromer, including at a terminal position on the macromer, within a repeating subunit, between repeating subunits, or any combination thereof. Similarly, linker "A'" 236 can be connected to any suitable group on the biomolecule and / or dye X. The connection chemistry of A and A' (231, 236) to their respective macromers or biomolecules (or dyes or dye-labeled biomolecules) can include, but are not limited to, complementary reactive groups, such as maleimide / thiol; thiol / thiol; pyridyl dithiol / thiol; succinimidyl iodoacetate / thiol; N-succinimidyl ester (NHS ester), sulfodichlorophenol ester (SDP ester) or pentafluorophenyl ester (PFP ​​ester) / amine; disuccinimidyl ester / amine; imidate / amine; hydrazine or amine / aldehyde, dialdehyde or benzaldehyde; isocyanate / hydroxyl or amine; carbohydrate-periodate / hydrazine or amine; diazines / aryl azide chemistry; pyridyl dithiol / aryl azide chemistry; alkynes / azide; carboxyl-carbodiimide / amine; amine / sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) / thiol; and amine / BMPH (N-[β-maleimidopropionic acid] hydrazide·TFA) / thiol. Those skilled in the art will be aware of other complementary coupling chemistries that can be used in the present disclosure.

[0208] In some aspects, in this article, Figure 2The "X" portion 241 in can be, but is not limited to, a dye, a fluorescent protein, a nanomaterial (such as quantum dots), a chemiluminescent molecule, a conjugate between a dye and a chemiluminescent molecule, a conjugate between a fluorescent protein and a chemiluminescent molecule, a conjugate between a nanomaterial (such as quantum dots) and a chemiluminescent molecule, streptavidin, avidin, an enzyme, a substrate of an enzyme, a substrate analog of an enzyme, a receptor, a ligand of a receptor, a ligand analog of a receptor, DNA, RNA, a modified nucleic acid, a DNA aptamer, an RNA aptamer, a modified nucleic acid aptamer, a peptide aptamer, an antibody, an antigen, a bacteriophage, a bacterium, or a conjugate of any two of the above.

[0209] In certain aspects, the present disclosure provides a method for detecting an analyte in a sample, the method comprising:

[0210] (a) combining a sample of the present disclosure and a macromer;

[0211] (b) exciting the macromonomer with light; and

[0212] (c) detecting fluorescence from the macromer, thereby detecting the analyte.

[0213] Figure 3 300 is a schematic diagram depicting exemplary macromers conjugated to, for example, antibodies (A); avidin (B); nucleic acids (C); and dyes (D). In certain aspects, the present disclosure includes the use of these conjugated macromers as direct labels. In certain aspects, such as Figure 3 As shown in Figure A, macromer 307 is conjugated to antibody 301, which can be, for example, a first or second antibody. Macromers conjugated to antibodies can be used as direct reporter molecules in, for example, biological assays (e.g., immunoassays). Excitation of the macromer with light can result in emission from the macromer, indicating that the antibody is present in the assay or assay solution.

[0214] Figure 3 B and 3C further illustrate the use of the conjugated macromers of the present disclosure as biomolecular labels capable of detecting specific targets, analytes, and target-related biomolecules. Figure 3 B depicts a macromer 307 conjugated to an avidin (eg, streptavidin, neutravidin, etc.) 310, which is capable of binding a biotin-modified molecule, biomolecule, or substrate. Figure 3C describes a nucleic acid (DNA, RNA, PNA, etc.) 315 conjugate, which includes a macromer 307 of the present disclosure covalently linked to a nucleic acid 315, which is capable of hybridizing with a target nucleic acid containing a complementary nucleic acid sequence. The connection or conjugation of a fluorescent macromer to a molecule capable of recognizing a target biomolecule, an analyte, or a target-related molecule provides a direct means of detection. In other aspects, the signal generated by the excitation of the macromer is not modulated by other assay components other than those directly conjugated to the macromer. In such an embodiment, the macromer is directly used as a fluorescent marker.

[0215] On the other hand, Figure 3 As shown in FIG. 3D , the macromer 307 of the present disclosure is labeled with a dye 317, such as a chromophore. In this embodiment, the conjugated macromer 307 can act as a donor, and the dye 317 can act as an acceptor in the energy transfer process. Here, the conjugated macromer can act as a light collector, and the excitation of the conjugated macromer is followed by an energy transfer process, such as fluorescence resonance energy transfer (FRET) 333, to direct the excitation to the dye. This results in amplified dye emission (compared to direct excitation of the dye). In one embodiment, the fluorescence of the donor-conjugated macromer can be quenched (e.g., >90% quenching).

[0216] A variety of dyes can be used in the present disclosure. In some aspects, functional groups are used to label dyes to form fluorescence resonance energy transfer (FRET) pairs. The emission wavelength is preferably around about 200-900 nm, such as about 200 nm, 220 nm, 240 nm, 260 nm, 280 nm, 300 nm, 320 nm, 340 nm, 360 nm, 380 nm, 400 nm, 420 nm, 440 nm, 460 nm, 480 nm, 500 nm, 520 nm, 540 nm, 560 nm, 580 nm, 600 nm, 620 nm, 640 nm, 660 nm, 680 nm, 700 nm, 720 nm, 740 nm, 760 nm, 780 nm, 800 nm, 820 nm, 840 nm, 860 nm, 880 nm, and / or 900 nm. 80nm, 800nm, 820nm, 840nm, 860nm, 880nm and / or about 900nm.

[0217] Exemplary fluorescent dyes include, but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodamine, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy-Chrome, Phycoerythrin, PerCP (Peridin chlorophyll a protein), PerCP-Cy5.5, JOE (6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, OregonGreen 514, Alexa 350. Alexa 430. Alexa 488. Alexa 532. Alexa 546. Alexa 568. Alexa 594. Alexa 633. Alexa 647、Alexa 660、Alexa 680, 7-amino-4-methylcoumarin-3-acetic acid, FL, FL-Br2, 530 / 550, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650, 650 / 665, R6G, TMR, TR, its conjugates, and combinations thereof. Exemplary lanthanide chelates include europium chelates, terbium chelates, and samarium chelates. Those skilled in the art will be aware of other dyes that can be used to practice the present disclosure.

[0218] In some cases, the dye can be a quencher dye. Quencher dyes include, but are not limited to (maximum absorption), Dabcyl (453), QSY35 (475), BHQ-0 (495), Eclipse (530), BHQ-1 (534), QSY 7 (560), QSY 9 (562), BHQ-2 (579), Elle-Quencher (630), Iowa Black (651), QSY 21 (661) and BHQ-3 (672). In some cases, when the macromonomer of the present disclosure is substituted by one or more substituents such as nitro, cyano or optionally substituted amino, the macromonomer will be a quencher dye.

[0219] Figure 4 (A) illustrates an antibody 405 labeled with a dye 410 and a macromer 407, which can be used in a FRET assay using UV light 413, according to the present disclosure; Figure 4 (B) illustrates streptavidin (SA) 425 labeled with dye 420 and labeled with a macromer 407 according to the present disclosure, as seen in a FRET assay using UV light 413; Figure 4 (C) illustrates a nucleic acid probe sequence 427 labeled with a quencher molecule 431 conjugated to a macromer 407 of the present disclosure, with a nucleic acid probe sequence 436 hybridized to sequence 427; Figure 4(D) illustrates a tandem complex of nucleic acid probe sequence 427 labeled with quencher molecule 431 and macromer 407, and attached dye 441. Nucleic acid probe sequence 427 hybridizes to sequence 436.

[0220] In the present invention, the macromer and the dye ( Figure 3 D) or biomolecule / dye complexes (e.g. Figure 4 In the case of direct connection (as shown in the example in ), the donor-acceptor distance can be fixed, rather than depending on the intensity of the interaction or binding, and the energy transfer efficiency can be significantly increased. This has significant results in improving dye signal conduction (or quenching) and reducing the background fluorescence associated with donor-acceptor crosstalk. In this case, crosstalk refers to the overlap between the macromonomer (donor) and the dye (acceptor) emission peaks. It should be understood that non-specifically bound macromonomers at distances that are too large for energy transfer can contribute to background fluorescence (or crosstalk). The shorter (fixed) distance between the donor and the acceptor can therefore not only promote direct dye amplification, but also greatly quench the donor emission.

[0221] Figure 5 A schematic diagram illustrating various methods for measuring a target biomolecule, analyte, or target-related biomolecule. For example, Figure 5 (A) shows a macromer 510 attached to a first antibody 501 bound to a second antibody 515 using a dye 512; Figure 5 (B) illustrates that the macromer 520 and the dye 512 labeled antibody recognize a common target 525; Figure 5 (C) illustrates an antibody 515 having attached dye 512 and biotin 531 and a second bioconjugate of streptavidin 541 with macromer 510 attached thereto; and Figure 5 (D) illustrates nucleic acid 562 having dye 512 and biotin 555 bound thereto and streptavidin 541 having macromer 510 conjugated thereto.

[0222] Go to Figure 6 , which shows a schematic diagram depicting a first attachment site 605 (A) and a second attachment site 615 (B) within a macromer 601 for attaching an "X" portion 612 (e.g., a dye) and a "Y" portion 631 (e.g., an antibody) via complementary linkers 609 (A') and linkers 619 (B'), respectively.

[0223] Figure 7 (A) Schematic diagram illustrating a macromer 701 with attached dye 709 and biomolecule 710 and the resulting energy transfer; Figure 7 (B) shows the macromer 701 with conjugated streptavidin 715 and linked dye 709; and Figure 7 (C) shows macromer 701 conjugated to nucleic acid 721 and dye 709.

[0225] Figure 8 A schematic diagram is shown depicting an indirect association with a macromer attached to a biomolecule. For example, Figure 8 (A) shows the interaction of biotinylated antibody 815 with covalent conjugate 804 of macromer 801; Figure 8 (B) shows biotinylated antibody 815 interacting with moiety 809 attached to macromer 801 having attached dye 812; Figure 8 (C) shows the biotinylated nucleic acid 822 interacting with the covalent portion 809 of the macromer; Figure 8 (D) shows biotinylated nucleic acid 822 bound to linker portion 809 of macromer 801 having attached dye 812; Figure 8 (E) Nucleic acid 822 showing a digoxigenin moiety 831 that interacts with a covalently attached antibody of macromer 801; and Figure 8 (F) Nucleic acid 822 with a digoxigenin moiety 831 and a covalent antibody against the macromer dye 812 tandem complex are shown.

[0226] Fig. 9 A shows a primary antibody 905 bound to an analyte 951, to which a secondary antibody 912 to which a macromer 915 is attached is added. Fig. 9 In B, target analyte 951 is bound to primary antibody 912 with attached macromer 915.

[0227] Fig.10 A shows a sandwich type complex in which a primary antibody 1005 binds to an analyte 1051. A secondary antibody 1010 with biotin 1012 is then added. Next, a macromer 1015 with streptavidin 1018 is added to produce a sandwich type complex.

[0228] Fig.10 B shows a primary antibody 1005 labeled with biotin 1012 bound to an analyte 1051. Streptavidin 1118 linked to a macromer 1015 is added.

[0229] In certain aspects, the present disclosure provides a method for detecting a target biomolecule in a sample, the method comprising:

[0230] Providing a sample suspected of containing the target analyte;

[0231] providing a macromer as described herein conjugated to a capture molecule, wherein the capture molecule is capable of interacting with a target analyte; and

[0232] The sample is contacted with the capture molecule and the conjugated macromer under conditions where the capture molecule can bind to the target analyte (if present); a light source is applied to the sample to excite the conjugated macromer; and whether the conjugated macromer emits light is detected.

[0233] In some aspects, the method is performed in vivo. In some aspects, the sample contains living cells. In some aspects, the analyte is a nucleic acid that contains a genetic point mutation, a deletion, or an insertion relative to a control nucleic acid. In some aspects, the detection of nucleic acid indicates that cancer is present in the sample.

[0234] C. Preparation of Compounds

[0235] In one embodiment, the present disclosure provides a method for synthesizing a compound as described herein. In some cases, the monomer unit can be derived as a diboronate, which can then be used for polymerization, for example, by Suzuki coupling. See J.Am.Chem.Soc., 2014, 136, 14027-14030. Polyfluorenes can also be obtained by using other reaction schemes involving organometallic catalysis. For example, the Yamamoto reaction uses a nickel (0)-based catalyst for the catalytic coupling of aromatic halide monomers. In addition, Stille, Heck and Sonogashira coupling reactions can also be used to synthesize conjugated polymers. See, for example, Yamamoto et al., Macromolecules 25: 1214-1223, 1992 for the Yamamoto reaction scheme; Kreyenschmidt et al., Macromolecules 28: 4577-4582, 1995; and Pei et al., J.Am.Chem.Soc.118: 7416-7417, 1996. See also Leclerc, Polym. Sci. Part A: Polym. Chem. 39:2867-2873, 2001, for Stille reaction schemes; Mikroyannidis et al., J. Polym. Sci. Part A: Polym. Chem. 45:4661-4670, 2007, for Heck reaction schemes; and Sonogashira et al., Tetrahedron Lett. 16:4467-4470, 1975, and Lee et al., Org. Lett. 3:2005-2007, 2001, for Sonogashira reaction schemes.

[0236] In some aspects, the method for preparing the macromonomer of formula I or formula II includes combining multiple reactive monomers to form a reaction mixture, wherein the first part of the reactive monomer carries the first reactive group, and the second part of the monomer carries the second reactive group, wherein the first and second reactive groups are different and can react with each other to form a polymer. In some aspects, at least one reactive monomer includes a monomer optionally substituted with one or more water-soluble groups. Then the reaction mixture is subjected to the conditions in which the first and second reactive groups on the monomer react to form a polymer. In some methods, multiple monomers with suitable polymerizable groups are condensed to produce a macromonomer including a polymer backbone formed by connected monomer residues.

[0237] In certain aspects, various types of polymerization strategies can be used to couple the polymerizable monomers described herein. As described above, a representative method for preparing the conjugated macromonomers described herein includes Yamamoto polymerization of monomers with halide functional groups in the presence of a metal catalyst (e.g., nickel). In some cases, the Yamamoto reaction will produce a random copolymer or a graft copolymer product.

[0238] In certain aspects, the method comprises heating Ni(COD)2, Di-Py, COD in a solvent mixture of toluene and DMF at 70°C for 30 minutes, then adding the monomers to the catalyst mixture and heating the mixture under argon or an inert atmosphere for 2-3 days.

[0239] In certain aspects, the polymerization method comprises polymerizing functionalized monomers in a certain size range to produce "polymer intermediates" with a larger size range that are soluble in standard, widely used organic solvents. These polymer intermediate compounds can then be further modified by any suitable chemical method now known or later developed. These methods produce polymers that are soluble in aqueous solutions and, due to the presence of certain high levels of poly(ethylene oxide), can be used directly in, for example, antibody conjugation applications.

[0240] In certain aspects, the monomer subunits can be PEGylated or non-PEGylated monomers, with the number of monomers being from about 50 to about 30,000.

[0241] In certain aspects, PEG can be up to 5,000 kDa in size; and monomer subunits of intermediate size (MW about 300 to about 2000 kD).

[0242] In certain aspects, the polymerization process may include the same monomers to produce a homopolymer or different monomers to produce a copolymer.

[0243] In certain aspects, the reaction is carried out in an aprotic solvent such as THF, DMF, toluene, acetonitrile and other suitable organic solvents.

[0244] In some aspects, the polymerization strategy involves Suzuki polycondensation. The Suzuki reaction is a Pd-catalyzed coupling reaction between an aromatic boronic acid derivative and an aromatic halide to produce a corresponding biphenyl. Typically, Suzuki polymerization involves coupling aromatic monomers each having two reactive functional groups. Suitable functional groups for Suzuki polymerization include halides (e.g., Br or I) and boron-containing functional groups, such as boric acid, boric esters (e.g., C1-C6 boric esters), borane groups (e.g., C1-C6 boranes) and BF3 groups. In an exemplary method, a first reactive dihalide monomer is polymerized with a second monomer having two boron derivative functional groups. In this arrangement, the first and second monomers can be the same to produce homopolymers or different to produce alternating copolymers. In a second exemplary method, monomers having boron derivative functional groups and reactive halide functional groups are polymerized to form homopolymers. Copolymers can be prepared using this arrangement by polymerizing two or more different types of monomers together, each monomer containing two functional groups. In some cases, the Suzuki reaction produces an alternating copolymer product or a main homopolymer having a certain content of alternating segments by adjusting the ratio of the monomers.

[0245] In certain aspects, a representative Suzuki polymerization reaction involves forming a reaction mixture comprising (a) an aromatic monomer having at least two reactive boron-containing groups and an aromatic monomer having at least two reactive halide functional groups; or (b) an aromatic monomer having one reactive halide functional group and one reactive boron-containing group. The reaction is carried out in a solvent in which the conjugated macromonomer is soluble. Suitable solvents include water-miscible polar solvents such as THF, DMF, and toluene. A catalytic amount of a catalyst is included in the reaction mixture to catalyze the polymerization of the aromatic monomers. A soluble Pd source can be used to catalyze the reaction. Pd(II) salts (e.g., Pd acetate) or Pd(0) complexes, such as Pd(Ph3P)4 are embodiments of suitable Pd sources that can be used in the methods disclosed herein. The reaction also includes a base in an amount sufficient to convert the reactive boron-containing functional group into an anion -BX3 - A group wherein X is independently F or OH. Suitable bases include inorganic bases such as alkali metal carbonates or bicarbonates, for example potassium bicarbonate or sodium bicarbonate.

[0246] In some aspects, the monomers described herein are polymerized via Suzuki polycondensation to produce 2,7-connected macromonomers. For example, dibromo and bis(boronate) 2,7-disubstituted monomers can be polymerized as described herein to produce poly(2,7-monomers). The polymerization reaction can utilize monomers as described herein, which can optionally carry one or more water-solubilizing groups. Adding an aromatic monomer (e.g., an aromatic hydrocarbon or heteroaromatic hydrocarbon) with a suitable polymerizable group to the reaction mixture can provide a copolymer having a main chain including connected monomers and aromatic monomer residues. In some methods, additional monomers can include optionally substituted aromatic hydrocarbons (e.g., fluorene) or heteroaromatic hydrocarbon groups, thereby forming a copolymer having a polymer main chain containing residues of aromatic hydrocarbon or heteroaromatic hydrocarbon monomers. In any polymerization method described herein, a polymerizable monomer with one or more water-soluble groups can be used in a polymerization reaction to provide a water-soluble polymer and a copolymer.

[0247] The figure below lists two representative reaction schemes for producing desired representative PEGylated conjugates of the invention; the upper scheme uses Yamamoto coupling chemistry, while the lower scheme uses palladium-catalyzed Suzuki reaction chemistry. Polyindenofluorene copolymers are also included.

[0248]

[0249] In some aspects, solubility is a characteristic of macromolecular monomers of the present disclosure. There are several methods by which water-soluble groups such as polyethylene glycol (PEG) are introduced into polymers to increase their solubility in water. In some aspects, PEGylated monomers are directly polymerized onto polymers or copolymers. For other aspects, polymers or copolymers are first prepared, and then post-modification methods introduce PEG into the functional groups present in the polymer to increase water solubility. Some representative process strategies for modified polymers are provided below.

[0250] 1. Strategy I - Williamson Ether Method

[0251] In certain aspects, the polymer intermediate is formed by polymerizing hydroxyl (-OH) functionalized monomers, bromoalkyl monomers, and NH-Boc protected monomers. The resulting polymer is then modified with reactive mPEG and functional groups such as -OH, NH2, Br in the polymer backbone. It should be understood that appropriate post-modification steps are performed based on the functional group moieties and mPEG monomers attached to the functionalized backbone in the polymer chain.

[0252]

[0253] 2. Strategy II - Anionic Polymerization

[0254] In certain aspects, mPEG monomers and hydroxyl monomers are copolymerized, followed by anionic polymerization of ethylene oxide grafted onto the polymer -OH groups, as shown below.

[0255]

[0256] 3. Strategy III--Coupling reaction

[0257] In certain aspects, mPEG monomers and amine-containing monomers are copolymerized, and then the mPEG-COOH group is coupled to the amine group via EDC chemistry to form an amide bond, as shown below.

[0258]

[0259] 4. Strategy IV---Click Chemistry

[0260] In certain aspects, the mPEG monomer and the hydroxyl-containing monomer are copolymerized, and then the polymer is coupled with an alkyl bromide species to form an alkyl-containing polymer. This reaction is followed by click chemistry to form a triazine ring with the azide-functionalized mPEG molecule, as shown below:

[0261]

[0262] 5. Strategy V-Schiff Base Reduction

[0263] In certain aspects, mPEG monomers and amine-containing monomers are copolymerized. The resulting polymer is coupled to an mPEG aldehyde-functionalized moiety, after which the C=N bond can be reduced using a suitable reducing agent as shown below.

[0264]

[0265] Example

[0266] The following examples are provided to illustrate but not to limit the claimed invention.

[0267] Example 1 illustrates the synthesis of 6,12-dihydroindeno[1,2-b]fluorene.

[0268]

[0269] 4.00 g (14.17 mmol) of indeno[1,2-b]fluorene-6,12-dione was suspended in 330 mL of triethylene glycol and mixed with 19.7 g (24.80 eq., 351.41 mmol) of KOH. 26.5 mL (29.89 eq., 423.49 mmol) of hydrazine monohydrate solution (80%) was carefully added to the mixture and heated to 170°C for 24 hours. The reaction mixture was cooled to room temperature and poured into a mixture of 1 L of ice water and 60 mL of hydrochloric acid (concentrated). The precipitate was separated, dried and recrystallized from toluene (700 mL) to give 2.17 g (60% yield) of the desired compound in the form of colorless needles. 1 H-NMR (THF-d 8 ,300MHz,)(ppm),4.259(s,4H),7.574δ(t,2H),7.662(t,2H),7.862(d,2H),8.199(d,2H),8.366(s,2H).

[0270] Example 2 illustrates the synthesis of 6,6,12,12-tetrakis(3-phenoxypropyl)-6,12-dihydroindeno[1,2-b]fluorene.

[0271]

[0272] To a suspension of indenofluorene (1.5 g, 5.94 mmol) in THF (60 mL) in Example 1 was added n-BuLi (2.5 M in hexane, 7.1 mL, 17.8 mmol) via syringe at -25°C. The solution was stirred at -25°C for 15 minutes, then warmed to room temperature for 1 hour, and 3-phenoxypropyl bromide (3.828 g, 17.8 mmol) was added via syringe at -25°C. After stirring for 4 hours, a second portion of n-BuLi (2.5 M in hexane, 7.1 mL, 17.8 mmol) was added via syringe at -25°C. After 1 hour, a second portion of 3-phenoxypropyl bromide (3.828 g, 17.8 mmol) was added via syringe at -25°C. The mixture was then kept stirring for 12 h, then quenched with ammonium chloride solution and extracted with chloroform (3×50 mL). The organic layer was dried over anhydrous MgSO4, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography using silica gel and chloroform / petroleum ether as eluents. The desired product was obtained as a yellow solid (3.2 g, yield 61%). 1H NMR(DMSO-d6,400MHz,ppm):7.95(s,2H),7.87(d,2H),7.50(d,2H),7.38(t,2H),7.33(t,2H ),7.11-7.13(t,8H),6.77(t,4H),6.77(d,8H),3.64(t,8H),2.11(t,8H),1.05-1.09(m,8H).

[0273] Example 3 illustrates the synthesis of 6,6,12,12-tetrakis(3-bromopropyl)-6,12-dihydroindeno[1,2-b]fluorene.

[0274]

[0275] 15 mL of HBr solution (48% by weight in water), 2.26 g of 6,6,12,12-tetrakis(3-phenoxypropyl)-6,12-dihydroindeno[1,2-b]fluorene (2.85 mmol), 100 mL of acetic acid and 10 mL of toluene were added to a flask and heated under reflux for 48 hours. The reaction mixture was poured into water and the resulting solution was extracted with chloroform (3×50 mL). The combined organic layers were washed with dilute Na2CO3 (1 M, 2×50 mL) solution, water, dried with MgSO4, and then the solvent was removed. The residue was purified by silica gel chromatography using petroleum ether / chloroform (3:1) as eluent to obtain the compound (1.1 g, yield 56%) as a white solid. 1 H NMR (CDCl3, 400MHz, ppm): 7.75 (d, J = 7.6Hz, 2H), 7.63 (s, 2H), 7.29–7.39 (m, 6H), 3.14 (t, J = 8.0Hz, 8H), 2.06 (t, J = 8.0Hz, 8H), 1.051.09 (m, 8H).

[0276] Example 4 illustrates the synthesis of 2,8-dibromo-6,6,12,12-tetrakis(3-bromopropyl)-6,12-dihydroindeno[1,2-b]fluorene.

[0277]

[0278] To a solution of 6,6,12,12-tetrakis(3-bromopropyl)-6,12-dihydroindeno[1,2-b]fluorene (1.1 g, 1.39 mmol) in 80 mL of CH2Cl2 was added Br2 (0.851 g, 3.89 mmol). The mixture was stirred at room temperature for 18 hours. The solution mixture was then washed with 2M Na2CO3. The organic solution was dried over MgSO4. The solvent was removed to give a crude product as a yellow solid. Recrystallization was carried out in a mixture of chloroform and methanol to give 1.12 g of the compound (91% yield). 1 H NMR(DMSO,400MHz,ppm):7.97(s,2H),7.84(d,2H),7.71(2H),7.57(dd,2H),3.11–3.20(t,8H),2.22(m,8H),1.02-1.06(m,8H).

[0279] Example 5 illustrates the m-PEGylation (poly(ethylene glycol) methyl ether (mPEG) of 2,8-dibromo-6,6,12,12-tetra(3-bromopropyl)-6,12-dihydroindeno[1,2-b]fluoro (PaPeg) 550 , HO-PEG 550 -OCH3)

[0280]

[0281] mPEG was added at 0°C under nitrogen. 550 Alcohol (HO-PEG 550 -OCH3) (3.331 g, 6.01 mmol) was dissolved in anhydrous THF (20 mL). Potassium tert-butoxide (7.74 mmol, 7.74 mL, 1 M in THF) was added to the mixture. After stirring for 10 minutes, 2,8-dibromo-6,6,12,12-tetrakis(3-bromopropyl)-6,12-dihydroindeno[1,2-b]fluorene (1.35 g, 1.50 mmol) in 20 mL of anhydrous THF was added via syringe. The mixture was warmed to room temperature and stirred overnight. After evaporation of THF, brine (50 mL) was added and the crude product was extracted with dichloromethane (3×40 mL). The combined organic layers were concentrated and purified by column chromatography (DCM-isopropanol) to give the product as a colorless oil (2.164 g).

[0282] Example 6 illustrates the synthesis of 11,12-dihydroindeno[2,1-a]fluorene

[0283]

[0284] This compound can be prepared according to US Pat. No. 7,754,841, which is incorporated herein by reference.

[0285] Example 7 illustrates the synthesis of 11,11,12,12-tetrakis(3-phenoxypropyl)-11,12-dihydroindeno[2,1-a]fluorene.

[0286]

[0287] The compound was prepared analogously to Example 2, while using 11,12-dihydroindeno[2,1-a]fluorene from Example 6 as the starting material.

[0288] Example 8 illustrates the synthesis of 11,11,12,12-tetrakis(3-bromopropyl)-11,12-dihydroindeno[2,1-a]fluorene.

[0289]

[0290] The compound was prepared similarly to Example 3, while using 11,11,12,12-tetrakis(3-phenoxypropyl)-11,12-dihydroindeno[2,1-a]fluorene from Example 7 as the starting material.

[0291] Example 9 illustrates the synthesis of 2,9-dibromo-11,11,12,12-tetrakis(3-bromopropyl)-11,12-dihydroindeno[2,1-a]fluorene.

[0292]

[0293] The compound was prepared similarly to Example 4, while using 11,11,12,12-tetrakis(3-bromopropyl)-11,12-dihydroindeno[2,1-a]fluorene from Example 8 as the starting material.

[0294] Example 10 illustrates the m-PEGylated (polyethylene glycol) methyl ether of 2,9-dibromo-11,11,12,12-tetrakis(3-bromopropyl)-11,12-dihydroindeno[2,1-a]fluorene (mPEG11).

[0295]

[0296] The compound was prepared similarly to Example 5, while using 2,9-dibromo-11,11,12,12-tetrakis(3-bromopropyl)-11,12-dihydroindeno[2,1-a]fluorene and mPEG11-OH in Example 9 as starting materials.

[0297] Example 11 illustrates the synthesis of 2,2'-(6,6,12,12-tetrakis(3-bromopropyl)-6,12-dihydroindeno[1,2-b]fluorene-2,8-diyl)bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane). (TBpeg550)

[0298]

[0299] The product in Example 5 (0.943 g, 0.34 mmol), bis(pinacolato)diboron (190 mg, 0.75 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (97 mg, 0.12 mmol) and potassium acetate (200 mg, 2.0 mmol) in anhydrous dioxane (12 mL) were heated under reflux for 18 h under an Ar atmosphere. The reaction mixture was cooled to room temperature, filtered to remove the solid, and the filtrate was concentrated in vacuo. The crude product was purified by column chromatography, eluted with 10% methanol in dichloromethane to give an oily product.

[0300] Example 12 illustrates the synthesis of 2,2'-(6,6,12,12-tetrakis(3-bromopropyl)-6,12-dihydroindeno[1,2-b]fluorene-2,8-diyl)bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane). (CBPeg11)

[0301]

[0302] This compound was prepared analogously to that described in Example 11.

[0303] Example 13 illustrates the synthesis of 2',7'-dibromo-3,3-bis(bromomethyl)spiro[cyclobutane-1,9'-fluorene].

[0304]

[0305] Under Ar protection, in a 250ml three-necked flask, NaH (0.6g, 25mmol) was added to a mixture of 2,7-dibromofluorene (3.26g, 10mmol) in 50ml anhydrous THF in 30 minutes. The mixture was stirred at room temperature for 30 minutes, and then pentaerythritol bromide (3.87g, 10mmol) in 20ml anhydrous THF was added dropwise to the flask. The mixture was heated at 70°C overnight. After the reaction was completed, the mixed solution was quenched with ice water, then extracted three times with CH2Cl2, and the CH2Cl2 solution was dried and filtered with anhydrous magnesium sulfate. The filtrate was rotary evaporated to obtain a brown solid, and the solid was separated by column chromatography to obtain a yellow solid.

[0306] Example 14 illustrates the synthesis of 2,6-diamino(Boc)-1-(2,7-dibromospiro[fluorene-9,4'-piperidinyl]-yl)hexan-1-one. (MBoc):

[0307]

[0308] The starting material 2,7-dibromo-4-methyl-spiro[fluorene-9,4'-piperidine] was prepared according to US Patent Publication No. 2014 / 0243283, which is incorporated herein by reference.

[0309] A mixture of (Boc-Lys(Boc)-OH (6.6 g, 18 mmol) and triethylamine (5.1 ml, 36 mmol) in 100 ml of CH2Cl2 was cooled to 0°C, then EDC-HCl salt (3.43 g, 18 mmol) was added, the mixture was stirred for 20 minutes, 2,7-dibromo-4-methyl-spiro[fluorene-9,4'-piperidine] (5.0 g, 12 mmol) was added and the mixture was stirred at room temperature for 16 h. 100 ml of CH2Cl2 and 100 ml of water were added to the reaction mixture. The phases were separated and the aqueous phase was extracted again with 100 ml of CH2Cl2. The collected organic phases were washed with water (200 ml×2). The organic phase was evaporated to dryness and separated by hexane / EtOAc to give 8.21 g of a white cotton solid (yield 95%).

[0310] Example 15 illustrates the synthesis of 2',7'-dibromo-3,3-bis(O-PEG550 methyl ether)spiro[cyclobutane-1,9'-fluorene] (SqPeg).

[0311]

[0312] At 0°C, under nitrogen, mPEG550 alcohol (HO-PEG 550 -OCH3) (3.331 g) was dissolved in anhydrous THF (20 mL). Potassium tert-butoxide (7.74 mmol, 7.74 mL, 1 M in THF) was added to the mixture. After stirring for 10 minutes, 2',7'-dibromo-3,3-bis(bromomethyl)spiro[cyclobutane-1,9'-fluorene] (1.45 g, 2.579 mmol) in 20 mL of anhydrous THF was added via a syringe. The mixture was warmed to room temperature and stirred overnight. After evaporation of THF, brine (50 mL) was added and the crude product was extracted with dichloromethane (3×40 mL). The combined organic layers were concentrated and purified by column chromatography (DCM-isopropanol) to give a colorless oil product (2.164 g).

[0313] Example 16 illustrates the synthesis scheme of the macromonomer P14 of the present disclosure.

[0314]

[0315] COD (246 μl, 2.0 mmol) was added to a stirred solution of Ni(COD)2 (552 mg, 2.0 mmol) and 2,2'-bipyridine (314 mg, 2.0 mmol) in anhydrous DMF (35 ml), and the resulting solution was stirred at 75 °C for 1 h under nitrogen. 2,6-Diamino(Boc)-1-(2,7-dibromospiro[fluorene-9,4'-piperidinyl]-1'-yl)hexan-1-one (72 mg, 0.01 mmol) and 2',7'-dibromo-3,3-bis(O-PEG550 methyl ether)spiro[cyclobutane-1,9'-fluorene] (0.551, 1.388 g, 0.50 mmol) were dissolved in DMF (6 ml) in a separate flask under nitrogen and then added to the mixture. The mixture was stirred at 75 °C under nitrogen. ℃ and stirred overnight for 24 hours. Bromobenzene (95μl, 0.91mmol) was added to the mixture and stirred for 12 hours under the same conditions. The mixture was slowly added dropwise to the stirred solvent 150ml6NHCl by pipette, and the resulting mixture was stirred for 2 hours. The mixture was extracted with DCM and the organic layer was washed with 50ml1NHCl. The solvent was evaporated and further dried under high vacuum to obtain a light wax polymer P14. (820mg), GPC (Mn=73,000; PDI=2.2).

[0316] The polymer P14 of the above compound was then dissolved in a mixture of 30 ml of DCM and 30 ml of TFA and then stirred overnight at room temperature. The solvent was removed under reduced pressure and then dried overnight under a high vacuum pump. This polymer required further purification before labeling. For purification, the dried polymer was resuspended in DI water and kept under stirring overnight. Most of the polymer was dissolved and the undissolved precipitate was removed by centrifugation at 10,000 rpm for 5 min. The supernatant was filtered through a 0.22 μm PES filter and filtered through Ultra-4 centrifugal filter unit (MWCO, 30K) (available from EMD Millipore Corporation, Billerica, MA) was concentrated. The solution was then loaded onto a G-25 column (Sephadex G-25 Fine, GE Healthcare Life Sciences) to remove catalyst impurities. The purified polymer was further fractionated by passing through a Superose 6 column (GE Life Sciences) to narrow the size distribution.

[0317] A series of novel copolymers composed of the monomer PEGylated fluorene (SqPEG) and different contents of PEGylated indenofluorene segments were synthesized by the Yamamoto and Suzuki methods. Their Mn ranged from 20K to 85K. As mentioned above, the Yamamoto reaction formed random copolymers, while the Suzuki reaction prepared alternating copolymers.

[0318] Example absorption data for polymers with precise content ratios of SqPeg and PEGylated indenofluorene segments to Mn are given in Fig.13 and 14 Shown in.

[0319] Fig.13 represents the absorption curve of polymer L18 prepared by Suzuki polymerization, and Fig.14 Shows the absorption curves of polymer P100 prepared by Yamamoto polymerization. In both cases, the indenofluorene monomer does cause a red shift in absorption and emission. Indenofluorene has a similar lowest energy transition, which can be attributed to the π-π* transition, with a λ maximum in the range of 363-400 nm and shows blue emission. More information is shown in Table 1.

[0320] Table 1

[0321]

[0322] Gel permeation chromatography (GPC) was performed in THF at 50°C using a 5 μm HR3 and HR4 GPC column system on an Agilent 1200 HPLC separation module with an Agilent UV detector using a flow rate of 0.5 mL / min. The system was calibrated with Agilent's Easical PS-2 standards in the range of 580-364,000 g / mol.

[0323] Example 17 illustrates the m-PEGylation of 2,5-dibromohydroquinone (BPeg11).

[0324]

[0325] This compound was prepared using 2,5-dibromohydroquinone and mPEG11-iodine was used as the starting material.

[0326] Example 18 illustrates the m-PEGylation of 1,4-dibromo-2,5-bis(bromomethyl)benzene (Bpeg550).

[0327]

[0328] The compound was prepared similarly to Example 15, using 1,4-dibromo-2,5-bis(bromomethyl)benzene and mPEG550-OH as starting materials.

[0329] Example 19 illustrates the synthesis of a macromonomer of the present disclosure.

[0330]

[0331] Under argon, K was added to the solution of CBPeg11 (0.273 g, 1 mmol) in Example 12 and BPeg11 (0.130 g, 1.1 mmol) in Example 17 in DMF (6 mL) in a Schlenk flask. Aqueous solution (2 M, 4 mL) was then added, followed by tetrakis (triphenylphosphine) palladium (12 mg, 0.012 mmol). The mixture was degassed by three freeze-pump-thaw cycles, then heated to 80 ° C for 12 hours, 4- (N-Boc-aminomethyl) phenylboronic acid pinacol ester (50 mg) was added, and the mixture was heated at 80 ° C for another 5 hours. At room temperature, EDTA (100 mg, 0.33 mmol) was added to 20% EtOH / H2O (20 mL), and stirred at room temperature for 1 hour. The resulting mixture was then filtered through a 0.45 m cup filter. 20% EtOH / H2O was used to dilute the filtered solution to a concentration of 2 mg / mL. The resulting dilution was then dialyzed into 20% EtOH / H2O using a tangential flow filtration system with a 30,000 kD molecular weight cutoff membrane until less than 0.1 mg / mL of polymer was present in the eluent. The solution was concentrated and lyophilized to give a yellow fibrous solid polymer P15 (0.41 g). The molecular weight was determined relative to a polystyrene standard (MW=65,000, D=2.0) by SEC analysis.

[0332] Polymer 15 was treated similarly to polymer P14 in Example 17 above and then used for activation and further bioconjugation.

[0333] Example 20 illustrates the synthesis of a macromonomer of the present disclosure.

[0334] This compound was prepared analogously to Example 19, whereas TBpeg550 from Example 11 and Bpeg550 from Example 18 were used as starting materials. Absorption in PBS at 350 nm, emission at 409 nm.

[0335]

[0336] Example 21 Conjugation of polymer dye (Compound P14) to antibody

[0337] Methods for conjugating antibodies to polymeric dyes are described. Mouse monoclonal antibodies against human CD4 (UCHT1) or anti-human IFN-γ antibodies (4S.B3) are used for conjugation. Antibody solutions (5 mg / ml in PBS, pH 7.2) are reduced by DTT activation (dithiothreitol) at a molar ratio of 160:1 for 30 minutes. They are purified and the buffer is exchanged into 50 mM phosphate, 5 mM EDTA pH 7.0 using a G-25 column (Sephadex G-25 Fine, GE Healthcare Life Sciences). The polymeric dye in 50 mM phosphate, 5 mM EDTA pH 7.0 is activated for 2 hours by 25-fold molar excess of sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate, Cova Chem). The free Sulfo-SMCC reagent is then removed by a G-25 column. For conjugation, DTT-reduced antibodies were mixed with SMCC-activated polymer dyes at a molar ratio of 4:1 for 18-20 hours. After purification with protein G resin, the conjugates were separated from free antibodies by Superose 6 (GE Life Sciences) and concentrated by centrifugal filters (30KMw cutoff, Millipore). These antibody-polymer conjugates were evaluated by flow cytometry.

[0338] Example 22 Lysed Washed Blood (LWB Staining)

[0339] 100 μL of anticoagulated whole blood is added to the tube containing the fluorophore-conjugated antibody and gently mixed. The test tube is incubated at room temperature in the dark for 15 minutes, and then 2mL of room temperature 1XRBC lysis buffer (BioLegend, Inc.) is directly added to the mixture. The test tube is incubated at room temperature in the dark for 15 minutes, and then centrifuged at 1200-1500rpm for 5 minutes to precipitate cells. The supernatant is removed, the test tube is vortexed gently to loosen the cell precipitation, and 2mL of FACS washing buffer (Biolegend, Inc) is added. With 1200-1500rpm centrifugal test tube for 5 minutes, the supernatant is sucked, and the test tube is vortexed again to loosen the precipitation. The cells are resuspended from the precipitation by adding 300-500 μL 1% paraformaldehyde 1XPBS solution (NaN3, pH7.2) in each FACS tube. Then prepare to analyze cells by flow cytometry. Sample signal-to-noise ratio (S / N) is calculated from the mean fluorescence intensity (MFI) of the positive staining population divided by the MFI of the unstained population. The staining index (SI) calculation is derived from the mean fluorescence intensity (MFI) of the positive staining population minus the MFI of the negative staining population, divided by twice the robust standard deviation of the negative staining population. Flow cytometry analysis is performed using a BD LSR II instrument equipped with a 355nm ultraviolet excitation laser source, a 405nm violet excitation laser source, a 488nm blue excitation laser source, a 532nm green excitation laser source, and a 640nm red excitation laser source. Lymphocytes are gated based on the forward (FSC) and side scatter (SSC) spectra of lymphocytes. The results show that, compared with Pacific BlueTM controls, polymer fluorophores P43 or P100 can be used to successfully identify CD3+ (UCHT1+) cells present in complex heterogeneous cell mixtures such as LWB preparations.

[0340] result

[0341] Cell surface flow cytometric analysis of human lysed washed blood samples stained with P43 or P100 polymer fluorophores coupled to anti-CD3 antibodies. Human lysed washed blood (LWB) samples were stained with 0.5 μg / test of P43 or P100 polymer fluorophores conjugated to UCHT1 monoclonal antibodies. Flow cytometric analysis was performed using a BD LSRII instrument equipped with a 355nm ultraviolet excitation laser source, a 405nm violet excitation laser source, a 488nm blue excitation laser source, a 532nm green excitation laser source, and a 640nm red excitation laser source. Lymphocytes were gated based on their forward (FSC) and side scatter (SSC) spectra. Results showed that compared with Pacific BlueTM control ( Fig.11A ) compared to the polymer fluorophore P43 ( Fig. 11B ) or P100( Fig. 11C) can be used to successfully identify CD3+(UCHT1+) cells present in complex heterogeneous cell mixtures such as LWB preparations. The percentages represent the relative frequencies of the negatively stained cell population (M1) and the positively stained cell population (M2).

[0342] Example 23 illustrates PBMC isolation

[0343] According to the manufacturer's instructions, human peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque PLUS (GE Healthcare) gradient. The cells were suspended in FACS wash buffer (BioLegend) and counted. The cells were centrifuged at 1200-1500rpm for 5 minutes and the wash supernatant was removed. The cell pellet was loosened by gentle mixing, and then the cells were resuspended in 3mL of 4% formaldehyde fixation buffer while vortexing. The cells were fixed in the dark for 20 minutes at room temperature. The cells were washed twice with 1× permeabilization buffer (BioLegend) by precipitating the cells at 1200-1500rpm for 5 minutes, removing the supernatant, and vortexing to loosen the precipitate. The cell density was adjusted to 5 million-10 million cells / mL with 1x permeabilization buffer. 100 μL of cell suspension was added to a tube containing a dilution of an appropriate antibody for CD3 and for perforin (clone dG9). The cells were incubated in the dark for 20-30 minutes at room temperature and then washed twice with FACS wash buffer. Cells were resuspended in 500 μL of FACS wash buffer and analyzed by flow cytometry. Flow cytometry analysis was performed using a BD LSR II instrument equipped with a 355nm ultraviolet excitation laser source, a 405nm violet excitation laser source, a 488nm blue excitation laser source, a 532nm green excitation laser source, and a 640nm red excitation laser source. Lymphocytes were gated based on the forward (FSC) and side scatter (SSC) spectra of lymphocytes. The results show that polymer fluorophores P43 or P100 can be used to successfully identify perforin (clone dG9+) cells present in complex heterogeneous cell mixtures such as PBMCs compared to Pacific BlueTM controls.

[0344] result

[0345] Intracellular flow cytometric analysis of perforin (clone dG9) conjugated with P43 or P100 polymer fluorophores using anti-perforin antibodies. Conjugated antibodies were used to stain intracellular perforin in human peripheral blood mononuclear cell (PBMC) samples. Samples were also surface stained with anti-CD3 antibodies conjugated with allophycocyanin (APC) to simultaneously identify T cells. Conjugated anti-perforin antibodies were used at 0.25 μg / test. Flow cytometric analysis was performed using a BD LSR II instrument equipped with a 355nm ultraviolet excitation laser source, a 405nm violet excitation laser source, a 488nm blue excitation laser source, a 532nm green excitation laser source, and a 640nm red excitation laser source. Lymphocytes were gated based on their forward (FSC) and side scatter (SSC) spectra. Data were plotted as Perforin on the ordinate and CD3 on the abscissa. The results showed that compared with Pacific BlueTM control ( Fig. 12A ) compared to P43( Fig. 12B ) or P100( Fig. 12C ) polymer fluorophores can be used in intracellular staining protocols in complex heterogeneous mixtures of cells (e.g. PBMCs) together with surface staining antibodies conjugated with different fluorophores to identify perforin-producing CD3+ T cells. The percentages represent the relative frequency of each population within a quadrant.

[0346] Example 24 illustrates the synthesis of 2',8'-dibromo-3,3,3",3"-tetrakis(bromomethyl)bispiro[cyclobutane-1,6'-indeno[1,2-b]fluorene-12',1"-cyclobutane].

[0347]

[0348] 2',8'-dibromo-3,3,3",3"-tetrakis(bromomethyl)bispiro[cyclobutane-1,6'-indeno[1,2-b]fluorene-12',1"-cyclobutane]

[0349] The above compounds can be prepared in a similar manner to Examples 2, 3 and 4 above.

[0350] Example 25 illustrates the synthesis of 2',9'-dibromo-3,3,3",3"-tetrakis(bromomethyl)bispiro[cyclobutane-1,1'-indeno[2,1-a]fluorene-12',1"-cyclobutane].

[0351]

[0352] 2',9'-Dibromo-3,3,3",3"-tetrakis(bromomethyl)dispiro[cyclobutane-1,1'-indeno[2,1-a]fluorene-12',1"-cyclobutane]

[0353] The above compounds can be prepared in a similar manner to Examples 2, 3 and 4 above.

[0354] Example 26 illustrates the PEGylation of the compound in Example 25.

[0355]

[0356] This compound can be prepared in a similar manner to Example 10.

[0357] Example 27 illustrates the PEGylation of the compound in Example 24.

[0358]

[0359] This compound can be prepared in a similar manner to Example 10.

[0360] All patents, patent applications, and publications mentioned in this specification are indicative of the levels of ordinary skill in the art to which the present disclosure pertains. All patents, patent applications, and publications are hereby incorporated by reference in their entirety for all purposes, to the same extent as if each publication were specifically and individually indicated to be incorporated herein by reference in its entirety for any and all purposes.

Claims

1. A method for preparing a macromonomer, the method comprising: A plurality of reactive monomers are combined to form a reaction mixture, wherein a first portion of the reactive monomers bear a first reactive group and a second portion of the monomers bear a second reactive group, wherein the first and second reactive groups are different and capable of reacting with each other to form a macromonomer having a molecular weight (M) of n ) is 20K to 85K, and its structure has one or more of the following monomers: wherein m is 5-30, n is 0-30, and o is 0-30.

2. The method for preparing a macromonomer according to claim 1, wherein the method is carried out under Yamamoto polymerization conditions in the presence of a metal catalyst, wherein the monomer has a halide functional group.

3. The method for preparing a macromonomer according to claim 1, wherein the method is carried out under Suzuki reaction conditions, wherein a Pd-catalyzed coupling reaction between an aromatic boronic acid derivative and an aromatic halide is used. 4 . The method for preparing a macromonomer according to claim 1 , wherein the reaction mixture comprises only SqPeg monomer, wherein m and n are both 0. 5 . The method for preparing a macromonomer according to claim 1 , wherein the macromonomer comprises a SqPeg monomer, a PaPeg monomer and an MBoc, wherein both m and n are greater than 0.

6. The method for preparing a macromonomer according to claim 1, wherein the macromonomer comprises the following structure:

7. The method for preparing a macromonomer according to claim 1, wherein the macromonomer comprises the following structure:

8. The method for preparing a macromonomer according to claim 1, wherein the macromonomer is a member selected from the following group: P114, P111, P100, P102, P151, P14, P43 or L18, and each has a corresponding structure and property:

9. The method for preparing a macromonomer according to claim 1, wherein the macromonomer has the following structure:

10. A macromonomer prepared by the method of any one of claims 1 to 9.

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