Fluorescent dyes for detecting presence of analyte in sample and compositions comprising dendrimers

Abstract

The present disclosure relates to compounds comprising A and B; wherein the one or more A groups are linked to the one or more B groups by electron-conducting conjugate bonds configured to delocalize the one or more electrons between A and B; a method of detecting the presence or absence of an analyte in a sample or organism, the method comprising: contacting the sample with a composition comprising a dendrimer structure and a compound; exposing the sample to light of an optical wavelength capable of exciting the compound in the composition; and detecting the presence or absence of light emitted by the composition; and a method of forming a dendrimer composition, the method comprising: contacting a dendrimer with a compound comprising a compound as provided herein, thereby forming a dendrimer composition; and contacting the dendrimer composition with the probe under the condition that the dendrimer composition is combined with the probe.

Description

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to U.S. Provisional Application No. 63 / 543,191, filed October 9, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This specification relates to compounds having the desired fluorescent properties, their preparation methods, and their usage methods. Background Technology

[0004] Imaging technologies for detecting, tracking, and identifying biological materials are becoming increasingly important in medical diagnostics, treatment, and research. Among mechanisms for specific target detection, fluorescence techniques have demonstrated powerful detection capabilities and are safer than other existing technologies. Many probe molecules can be labeled, including antibodies, peptides, and nucleic acids, which can be readily labeled to specifically bind to targets, thereby identifying the presence, location, and quantity of that particular target in cells, tissues, or subjects.

[0005] Labeling these probe molecules requires robust fluorophores that not only possess excellent specificity but also the ability to be easily detected against complex backgrounds. Conventional UV dyes may have low extinction coefficients and low quantum yields, leading to reduced brightness and limiting their use as functional dyes in certain applications. The effective fluorescence / photon ratio of these conventional dyes may also be low, partly due to quenching upon close contact. Conventional compositions including such dye molecules directly conjugated with the probe may exhibit low brightness, thus limiting their use in detection methods.

[0006] Therefore, there is still a need to continue developing improved compositions with tunable excitation and emission profiles and / or increased brightness, as well as methods for using such compositions. Summary of the Invention

[0007] The following overview of this disclosure is provided to aid in understanding some of its unique innovations and is not intended to be a complete description. A full understanding of the various aspects of this disclosure can be obtained by considering the entire specification, claims, drawings, and abstract as a whole.

[0008] Improved compositions and methods for detecting the presence or absence of analytes using these compositions are disclosed. These compositions can provide improved solubility in aqueous solvent systems, reduced quenching, increased extinction coefficients, and / or tunable excitation and emission profiles, thereby improving methods for using these compositions.

[0009] This document provides compounds comprising A and B; wherein one or more A groups are connected to one or more B groups via an electron-conjugated bond, the electron-conjugated bond being configured to delocalize one or more electrons between A and B; wherein A is an electron-accepting group, an electron-donating group, or a fluorophore; wherein B is an electron-accepting group, an electron-donating group, or a fluorophore; and wherein the compound does not contain repeating units of A, B, or combinations thereof, and optionally wherein A and B are structurally different. Optionally, each conjugated bond is independently selected from n conjugated bond units selected from single bonds, triple bonds, C=N groups, amide bonds, click-reactive bonds, benzene ring bonds, heterocyclic bonds, and combinations thereof; and wherein n ≥ 1. Optionally or additionally, the compound further comprises one or more water-soluble groups connected to A, B, or both. Optionally or additionally, each conjugated bond is independently composed of n conjugated bond units selected from single bonds, triple bonds, C=N groups, amide bonds, click-reactive bonds, benzene ring bonds, heterocyclic bonds, and combinations thereof; where n ≥ 1, and optionally n ≥ 2. Optionally or additionally, the electronically conducted conjugated bond is a conjugated single bond. Optionally or additionally, A is a conjugated ring dye. Optionally or additionally, B is a conjugated ring dye.

[0010] In any aspect of the foregoing, the compound optionally has maximum absorption in the wavelength ranges of 300 nm and 450 nm, and optionally 365 nm and 830 nm. In any aspect of the foregoing, the compound optionally has maximum emission in the wavelength ranges of 365 nm and 830 nm. Optionally, when not conjugated with B, A has maximum absorption in the wavelength ranges of 300 nm and 450 nm, and optionally 300 nm and 600 nm. Optionally, when not conjugated with A, B has maximum absorption in the wavelength ranges of 150 nm and 450 nm, and optionally, when not conjugated with A, B has maximum emission in the wavelength ranges of 150 nm and 830 nm.

[0011] In any of the foregoing aspects, the compound optionally has a structure comprising or consisting of A groups linked to 1 to 10 B groups, optionally having the following formula:

[0012] The compound said compound comprises or consists of B groups linked to 1 to 10 A groups, and optionally has the following structure:

[0013] Optionally, A is connected to two or more Bs. Optionally, B is connected to two or more A's.

[0014] Optionally, in any of the foregoing, one or more water-soluble groups are connected to A, and one or more water-soluble groups are connected to B.

[0015] In any of the foregoing embodiments, if measured at the maximum emission of the compound, the molar extinction coefficient of the compound may be greater than 5000 cm⁻¹. -1 M -1 .

[0016] In any of the foregoing, A, B, or both are optionally linked to one or more dendrimer structures. The dendrimer structure may be a branched structure. In some aspects, both A and B are linked to one or more dendrimer structures. Optionally, the average molecular weight of the dendrimer is from 5 kilodaltons (kDa) to 100 kDa, optionally from 5 kDa to 30 kDa, or optionally from 10 kDa to 20 kDa. In some aspects, the dendrimer comprises a polyethylene glycol chain. Optionally, the dendrimer and the compound are linked by a polyethylene glycol chain. Optionally, the polyethylene glycol chain comprises at least three ethylene glycol repeating units. In some aspects, the composition comprising the dendrimer structure further comprises a probe linked to one or more of the dendrimer structures. Optionally, the probe molecule is a protein or fragment thereof, including but not limited to antibodies or fragments thereof, nucleic acids, phospholipids, polysaccharides, triglycerides, aptamers, avidin, streptavidin, neutral avidin, avidin DN, avidin D, or cells. Optionally, the F / P ratio of the composition is from 5 to 100, optionally from 10 to 30, wherein the F / P ratio is defined as the total number of molecules of the compound present in the composition divided by the total number of molecules of the probe present in the composition. In some aspects, the dendritic polymer also contains functional groups.

[0017] In any of the foregoing aspects, the dendritic polymer optionally comprises a compound of the following formula.

[0018] Wherein: each n is an independent integer from 10 to 300; R63, R64, R65, R66, R67, R68, R69, and R70 are independently selected from amines, azides, linkages to A or B, linkages to a probe, and one or more other dendritic polymers, optionally 1 to 10 other dendritic polymer structures. Optionally, at least one of R63, R64, R65, R66, R67, R68, R69, and R70 is a linkage to A or B, and at least one of R63, R64, R65, R66, R67, R68, R69, and R70 is a linkage to a probe. Optionally, the probe is a protein or a fragment thereof, optionally including but not limited to antibodies or fragments thereof, nucleic acids, phospholipids, polysaccharides, triglycerides, aptamers, avidin, streptavidin, neutral avidin, avidin DN, avidin D, or cells. Optionally, the linkage is selected from amides, thiols, succinimides, maleimides, azides, carboxyl esters, carboxyl / EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sulfonyl-SMCC, BMPH, sulfonyl-SBED, trans-cyclooctene / tetraazine, and amine / epoxide. In some respects, A or B is selected from fluorescein, 6-FAM, rhodamine, Texas red, tetramethylrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade blue, Cascade yellow, coumarin, 2-[3-[3-[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]-2(3H)-benzoxazolyl]-1-propen-1-yl]-3-ethyl-benzoxazolium, monoiodide (Cy2), thioanthocyanin 3 (Cy3), anthocyanin 3.5 (Cy3.5), thioanthocyanin 5 (Cy5), anthocyanin 5.5 (Cy5.5), Cy-chrome yellow, phycoerythrin, PerCP (chlorophyll protein-a protein), and PerCP-Cy5.5. JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Fluorescein Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, 7-amino-3-[2-(2,5-dioxopyrrolidine-1-yl)oxy-2-oxoethyl]-4-methyl-2-oxochromene-6-sulfonic acid (Alexa Fluor® 350), N,N-diethylethylamine-[9-{6-[(2,5-dioxo-1-pyrrolidine)oxy]-6-oxohexyl}-8,8-dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H-pyrano[3,2-g]quinoline-6-yl]methanesulfonate (Alexa Fluor®) 430) Dilithium; 3-(3-amino-6-imino-5-sulfon-4-sulfonyloxanthracene-9-yl)-4-carboxybenzoate (Alexa Fluor® 488), 5-(4-{[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl}phenyl)-2,3,3,7,7,8-hexamethyl-2,3,7,8-tetrahydro-1H-pyrrolo[3′,2′:6,7]chromeno[3,2-f]indole-10,12-disulfonic acid dihydrogen salt (Alexa Fluor®) 532), Sodium 6-(2-carboxy-3,4,6-trichloro-5-{[2-({6-[(2,5-dioxopyrrolidone-1-yl)oxy]-6-oxohexyl}amino)-2-oxoethyl]thio}phenyl)-2,2,4,8,10,10-hexamethyl-3,4,5a,8,9,10,11,12a-octahydro-2H-pyrano[3,2-g:5,6-g']diquinoline-1-onthium-12,14-disulfonic acid (Alexa Fluor®) 546), 4-(2,5-dioxopyrrolidone-1-yl)oxycarbonyl-2-[7,7,19,19-tetramethyl-9,17-bis(sulfonyl)-2-oxa-6,20-diazapentacyclo[12.8.0.03,12.05,10.016,21]docosa-1(14),3,5,8,10,12,15,17,21-nonen-13-yl]benzoic acid (Alexa Fluor®) 568), [13-[2-carboxy-4-(2,5-dioxopyrrolidone-1-yl)oxycarbonylphenyl]-6,7,7,19,19,20-hexamethyl-17-(sulfonylmethyl)-2-oxa-20-aza-6-azaonium pentacyclic [12.8.0.03,12.05,10.][016,21] docosa-1(14),3,5,8,10,12,15,17,21-nonen-9-yl]methanesulfonate (Alexa Fluor® 594), Alexa Fluor® 633 (CHEBI:137393), 3-[(2Z)-2-[(2E,4E)-5-[3,3-dimethyl-5-sulfon-1-(3-sulfopropyl)indol-1-onthium-2-yl]penta-2,4-dienidel]-3-[5-(2,5-dioxopyrrolidone-1-yl)oxy-5-oxopentyl]-3-methyl-5-sulfonoindol-1-yl]propane-1-sulfonate (Alexa Fluor® 647), Alexa Fluor 660, Alexa Fluor 680, 7-amino-4-methylcoumarin-3-acetic acid, (3-{2-[(3,5-dimethyl-1H-pyrrolo-2-yl-κN)methylene]-2H-pyrrolo-5-yl-κN}propionate)(difluoro)boron (BODIPY® FL), BODIPY FL-Br2, CAS No. 216961-93-2 (BODIPY® 530 / 550), 12-(2,2-difluoro-12-thiophene-2-yl-3-aza-1-aza-2-boron anion tricyclic [7.3.0.03,7]dodecane-1(12),4,6,8,10-pentene-4-yl)dodecanoate; hydron (BODIPY® 558 / 568), CAS#:150173-90-3 (BODIPY® 564 / 570), CAS#:150173-78-7 (BODIPY® 576 / 589), CAS No.: 217075-36-0 (BODIPY® 581 / 591), CAS#:2183512-02-7 (BODIPY® 630 / 650), CAS No.: 1818267-45-6 (BODIPY® 650 / 665), difluoro(3-{2-[(5-phenyl-1H-pyrrolo-2-yl-κN)methylene]-2H-pyrrolo-5-yl-kappaN}propionate)boron (BODIPY® R6G), CAS#:485397-12-4 (BODIPY® TMR), CAS#:2183473-18-7 (BODIPY® TR), SPK dyes, cf514, DY405, DY396XL, cf570, cf405, their conjugates, and combinations thereof.

[0019] A method for detecting the presence or absence of an analyte in a sample or organism is also provided, the method comprising: (a) contacting the sample with a composition comprising a dendritic polymer structure and a fluorophore; (b) exposing the sample to light of a wavelength capable of exciting the fluorophore in the compound; and (c) detecting the presence or absence of light emitted by the composition. Optionally, the fluorophore is linked to the dendritic polymer structure. In some aspects, the dendritic polymer structure is a branched structure. Optionally, the average molecular weight of the dendritic polymer structure is from 5 kilodaltons (kDa) to 100 kDa, optionally from 5 kDa to 30 kDa, and optionally from 10 kDa to 20 kDa. Optionally, the dendritic polymer structure comprises a polyethylene glycol chain. The polyethylene glycol chain optionally comprises at least three ethylene glycol repeating units. Optionally, the composition further comprises a probe linked to one or more of the dendritic polymer structures. The probe is optionally a protein or a fragment thereof, including but not limited to antibodies or fragments thereof, nucleic acids, phospholipids, polysaccharides, triglycerides, aptamers, avidin, streptavidin, neutral avidin, avidin DN, avidin D, or cells. The F / P ratio of the composition is optionally from 5 to 100, optionally from 10 to 30, wherein the F / P ratio is defined as the total number of fluorophore molecules present in the composition divided by the total number of probe molecules present in the composition.

[0020] In any of the foregoing embodiments, the dendritic polymer structure optionally further comprises functional groups. Optionally, the dendritic polymer structure comprises one or more branches, wherein the one or more said branches terminate at said functional groups.

[0021] In any of the foregoing methods, the composition is any of the compounds provided herein or above.

[0022] A method for forming a dendritic polymer composition is also provided, the method comprising: (a) contacting a dendritic polymer with a compound containing a fluorophore under conditions in which the compound is bound to the dendritic polymer, thereby forming a dendritic polymer composition; and (b) contacting the dendritic polymer composition with a probe under conditions in which the dendritic polymer composition is bound to the probe, thereby forming a targeted dendritic polymer composition. Optionally, the dendritic polymer structure is any of the foregoing. Optionally, the fluorophore is any of the compounds provided in any of the foregoing or otherwise provided herein. Attached Figure Description

[0023] Figure 1 The excitation and emission spectra of compounds based on one aspect are described; Figure 2 Flow cytometry plots of compounds and comparative examples according to one aspect are described, with the detector as the x-axis and the mean fluorescence intensity (MFI) as the y-axis; Figure 3AFlow cytometry diagrams of comparative embodiments are described; Figure 3B Flow cytometry diagrams of comparative embodiments are described; Figure 3C A flow cytometry plot is described according to an embodiment of one aspect; Figure 4A Flow cytometry diagrams of comparative embodiments are described; Figure 4B Flow cytometry diagrams of comparative embodiments are described; Figure 4C A flow cytometry plot is described according to an embodiment of one aspect; Figure 5A Flow cytometry diagrams of comparative embodiments are described; Figure 5B Flow cytometry diagrams of comparative embodiments are described; Figure 5C A flow cytometry plot is described according to an embodiment of one aspect; Figure 6A Flow cytometry diagrams of comparative embodiments are described; Figure 6B A flow cytometry plot according to an embodiment of one aspect is described. Detailed Implementation

[0024] A persistent challenge in fluorescence detection techniques is the ability to absorb light at specific wavelengths that can penetrate complex backgrounds to emit at easily detectable and valuable wavelengths, providing a robust fluorescence signal. A unique structure is disclosed that not only specifically binds to target molecules but also more completely saturates the target with fluorophores to improve detectability, which is particularly useful for low-concentration analytes. A unique fluorescent dendritic polymer structure is also disclosed that can or is capable of being linked to antibodies, peptides, nucleic acids, or other probes, providing multiple fluorescent molecules to the probe and thus improving the localization of fluorescent molecules to the target in its presence. This dendritic polymer structure not only presents multiple probes but also displays a large number of fluorophores capable of providing the desired signal.

[0025] Unique fluorescent molecules are also disclosed, capable of absorption at a desired wavelength and tunable emission at one or more desired alternative wavelengths simultaneously. As a non-limiting example, very few existing dye structures can absorb in the 350 nm wavelength range and emit in the 430 nm to 600 nm wavelength range. Those previously identified dye structures possessing the desired properties of these examples exhibit low quantum yields and low extinction coefficients, rendering them ineffective for sensitive detection techniques. The disclosed dyes address these issues by being robust absorbers at the desired wavelength while also possessing sufficient quantum yield for detection at the desired wavelength. Therefore, the disclosed compounds and methods improve the detectability of target molecules both in vitro and in vivo.

[0026] The term "independently chosen" as used in this article means that the R groups can be the same or different (e.g., R1, R2, R7, and R8 can all be linkages to B, or R2 and R7 can both be linkages to B, and R1 can be (C1-C2)). 20 ) hydrocarbon group, and R8 can be (C1-C 20 (such as heterohydrocarbon groups).

[0027] When used to describe certain carbon-containing chemical groups, the "(C" symbol is used. x -C y The bracketed representation in the form of ")" indicates that the unsubstituted form of the chemical group has x to y carbon atoms, inclusive. For example, (C1-C 20 A hydrocarbon group is a hydrocarbon group having 1 to 20 carbon atoms in its unsubstituted form.

[0028] The term "(C1-C)" 20 "Hydrocarbon group" refers to a hydrocarbon group with 1 to 20 carbon atoms, wherein each hydrocarbon group is aromatic or non-aromatic, saturated or unsaturated, straight or branched, cyclic (including monocyclic and polycyclic, fused and non-fused polycyclic, including bicyclic; 3 or more carbon atoms) or acyclic, and is unsubstituted or substituted by one or more substituents.

[0029] The term "substitution" refers to the substitution of at least one hydrogen atom bonded to a carbon atom or heteroatom of the corresponding unsubstituted compound or functional group by a substituent.

[0030] dye

[0031] Conventional UV dyes emitting at 430 nm to 600 nm may have low extinction coefficients and quantum yields, which can lead to reduced brightness and limit their use as functional dyes in certain applications. Conventional compositions comprising dye molecules directly conjugated with the target analyte may exhibit lower brightness, increased quenching, and / or a lower effective F / P ratio, partly due to quenching of the dye in close contact, thus limiting their use in detection methods.

[0032] The compounds disclosed herein have utility, for example, as fluorescent imaging molecules and / or as fluorescent dyes, exhibiting increased extinction coefficients and tunable excitation and emission. Furthermore, fluorophores such as those in the compositions disclosed herein can be coupled with dendritic polymers to form compositions with improved solubility and reduced quenching in aqueous solvent systems, both of which can increase utility for improved analytical detection methods. Dendritic polymer compositions can also be formed by conjugating a targeting molecule to the disclosed compositions. The compositions described and disclosed herein can be used as ultraviolet (UV) dyes with tunable emission and / or increased brightness.

[0033] The compounds disclosed herein may include A groups and B groups, wherein one or more A groups are connected to one or more B groups via an electron-conductive conjugated bond. The A and B groups may have different structures or other properties. The electron-conductive conjugated bond may be configured to delocalize one or more electrons between A and B. Each A group may be an electron-accepting group, an electron-donating group, or a fluorophore. Each B group may be an electron-accepting group, an electron-donating group, or a fluorophore. The compound may not contain one or more repeating units of A, B, AB, BA, or combinations thereof. Therefore, in some aspects, the fluorophores disclosed herein do not include polymeric fluorophores.

[0034] According to several aspects, the compounds disclosed herein comprise one or more A groups linked to one or more B groups. Throughout the molecule, each A group can independently function as an electron acceptor, electron donor, or fluorophore. Each B group can independently function as an electron acceptor, electron donor, or fluorophore. Where A, B, or both are fluorophores, the fluorophore can contribute a high extinction coefficient to the compound. The choice of A and B can also be used to tune the emission of the compound.

[0035] Groups A and B can be independently connected by an electronically conductive conjugated bond (connecting bond). When used to connect A and B, the conjugated bond is provided in units of N units in length and can be a single bond, triple bond, C=N group, amide bond, click-reactive bond, benzene ring bond, heterocyclic bond, or a combination thereof, where n ≥ 1. Optionally, n ≥ 2. Optionally, n is not greater than 2. Optionally, n is 1.

[0036] In some respects, the electronically conducted conjugated bond connecting A and B is a single bond, triple bond, C=N group, amide bond, or click reaction bond, and n = 1. In other respects, n can be greater than or equal to 2. The electronically conducted conjugated bond can be a conjugated single bond.

[0037] As disclosed herein, A, B, or both can be conjugated ring structure dyes. Conjugated ring structure dyes are optionally formed of one, two, three, or more ring structures, wherein they are conjugated within one or more such ring structures and optionally between the ring structures. Optionally, the conjugated ring structure dye includes at least one 6-membered ring. Optionally, the conjugated ring structure dye includes at least one 5-membered ring. Optionally, the conjugated ring structure dye includes at least one 6-membered ring and at least one 5-membered ring. In some aspects, the conjugated ring structure dye contains one, two, three, four, five, six, seven, or more ring structures within the conjugated ring structure dye.

[0038] Compounds including A and B can be characterized as having a maximum absorption at one wavelength and a maximum emission at one wavelength. Tuning of the absorption and emission properties of a compound can be provided in part by structural changes in A and B. For example, a compound may have a maximum absorption at a wavelength greater than or equal to 300 nm. Optionally, a compound may have a maximum absorption wavelength in the range of 300 nm to 830 nm. Optionally, a compound may have a maximum absorption wavelength from 300 nm to 400 nm, and optionally 300 nm and 450 nm. Optionally, a compound may have a maximum absorption wavelength from 365 nm to 400 nm. Optionally, a compound may have a maximum absorption at wavelengths greater than or equal to 300 nm, 325 nm, 350 nm, 365 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 700 nm, or 800 nm. The disclosed compounds may have maximum absorption at wavelengths less than or equal to 450 nm, 600 nm, 700 nm, 800 nm or 830 nm.

[0039] Independently or in addition to the maximum absorption wavelengths described above, the compounds according to this disclosure may have a maximum emission at a wavelength greater than or equal to 300 nm. For example, the compounds may have a maximum emission at wavelengths from 300 nm to 830 nm. Optionally, the compounds may have a maximum emission at wavelengths from 300 nm to 600 nm, and optionally from 365 nm to 600 nm. Optionally, the compounds may have a maximum emission at wavelengths from 300 nm to 450 nm, and optionally from 365 nm to 450 nm. Optionally, such compounds may have a maximum emission wavelength greater than or equal to 300 nm, 365 nm, 400 nm, 500 nm, or 600 nm. The compounds may have a maximum emission at wavelengths less than or equal to 450 nm, 600 nm, 700 nm, 800 nm, or 830 nm.

[0040] Without wishing to be bound by any particular theory, compounds with maximum absorption and emission as described herein are believed to be suitable for applications using UV-excited fluorescent dyes, such as detection methods employing fluorescence excitation.

[0041] Unit A of the compounds disclosed herein may have a maximum absorption at a wavelength greater than or equal to 300 nm, or less than or equal to 450 nm, or both, wherein the maximum absorption is measured when not conjugated with B. Optionally, when not conjugated with B, A may have a maximum absorption at a wavelength greater than or equal to 325 nm, greater than or equal to 350 nm, or greater than or equal to 375 nm. Optionally, when not conjugated with B, A may have a maximum absorption at a wavelength less than or equal to 425 nm, less than or equal to 400 nm, or less than or equal to 475 nm.

[0042] Unit A of the compounds disclosed herein may have maximum emission at a wavelength greater than or equal to 300 nm, or less than or equal to 600 nm, or both, wherein the maximum emission is measured when not conjugated with B. Optionally, when not conjugated with B, A may have maximum emission at a wavelength greater than or equal to 350 nm, greater than or equal to 400 nm, or greater than or equal to 450 nm. Optionally, when not conjugated with B, A may have maximum emission at a wavelength less than or equal to 550 nm, less than or equal to 500 nm, or less than or equal to 450 nm.

[0043] Unit B of the compounds disclosed herein may have a maximum absorption at a wavelength greater than or equal to 150 nm, or less than or equal to 450 nm, or both, wherein the maximum absorption is measured when not conjugated with A. Optionally, when not conjugated with A, B may have a maximum absorption at a wavelength greater than or equal to 200 nm, 250 nm, 300 nm, 350 nm, or 400 nm. Optionally, when not conjugated with A, B may have a maximum absorption at a wavelength less than or equal to 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, or 200 nm.

[0044] Unit B of the compounds disclosed herein may have maximum emission at a wavelength greater than or equal to 150 nm, or less than or equal to 830 nm, or both, wherein the maximum emission is measured when not conjugated with A. Optionally, when not conjugated with A, B may have maximum emission at a wavelength greater than or equal to 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, or 500 nm.

[0045] Optionally, when not conjugate with A, B may have maximum emission at wavelengths less than or equal to 830 nm, 800 nm, 750 nm, 700 nm, 650 nm, 600 nm, 550 nm, or 500 nm.

[0046] In some respects, the disclosed compounds may have an emission rate greater than or equal to 5000 cm⁻¹ measured at the maximum emission of the compound. -1 M -1 The molar extinction coefficient, or optionally, greater than or equal to 5500 cm⁻¹ -1 M -1 The molar extinction coefficient, or optionally, greater than or equal to 6000 cm⁻¹ -1 M -1 The molar extinction coefficient.

[0047] One or more water-soluble groups may be attached to A, or B, or both. Without wishing to be bound by any particular theory, it is believed that including water-soluble groups attached to A, B, or both can increase the water solubility of the compound, which can increase the concentration of the compound in certain applications or impart improved targeting ability to the target site. The water-soluble group optionally comprises a straight-chain or branched group of 0-5 consecutive carbons, optionally 2-5 consecutive carbons, optionally separated by one or more heteroatoms. Optionally, the water-soluble group comprises one or more alcohols, amides, amines, carboxylic acids, esters, ethers, aldehydes, ketones, and / or nitriles, optionally present alone or attached as a side group to or separating two or more carbons on the carbon backbone. In some aspects, the water-soluble group comprises one or more 1-3 carbon groups linked by oxygen, nitrogen, or sulfur. In some aspects, the water-soluble group comprises one or more ethylene units linked by oxygen. Optionally, the water-soluble group includes...

[0048] Where n is 1-10, and optionally the water-soluble group is connected to group A or B via a carbon linker of 1-5 carbon atoms. Optionally, the water-soluble group is straight-chain or branched (C1-C2). 20 ) hydrocarbon group, (C1-C 20 ( ) heterocyclic group or combination thereof, wherein the heteroatom optionally comprises O, N or S, and wherein the heterocycle optionally comprises -SO3, -NH2, -OH or others.

[0049] According to some aspects, the compounds disclosed herein comprise at least one A unit linked to at least one B unit. Optionally, A may be linked to two or more B units, or B may be linked to two or more A units, or both. Optionally, A may be linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more B units, or B may be linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more A units, or both.

[0050] Optionally, the compound may have the following structure.

[0051] In other respects, the compound can have the following structure:

[0052] The compounds disclosed herein comprise one or more A-unit structures. An A-unit can be illustratively defined as:

[0053] Among them, R1, R2, R7, R8, R9, R13, R14, R15, R19, R20, R23, R24, R29, R30, R52, R53, R72, R73, R74, and R75 can be independently selected from the linkage with B, the water-soluble group, the linkage with the probe, the linkage with the dendritic polymer, and (C1-C) 20 ) hydrocarbon group, (C1-C 20 ( ) Heteroalkyl groups, forming ring structures and combinations thereof; at least one of R1 and R2 can be a bond with B; at least one of R7, R8 and R9 can be a bond with B; at least one of R13, R14 and R15 can be a bond with B; at least one of R19 and R20 can be a bond with B; at least one of R23 and R24 can be a bond with B; at least one of R29 and R30 can be a bond with B; at least one of R52 and R53 can be a bond with B; at least one of R72, R73, R74 and R75 can be a bond with B; R3, R4, R5, R6 R10, R11, R12, R16, R17, R18, R21, R22, R25, R26, R27, R28, R31, R33, R35, R37, R39, R40, R42, R44, R46, R47, R48, R50, R51, R54, and R55 can independently be H, water-soluble groups, (C1-C) 50 ( ) Heteroalkyl groups, or may be fused to form a ring optionally including R54, R55 or both; R32, R34, R36, R38, R41, R43, R45 and R49 may be linkages with B; X may be C, Si, O, S, P, N, Se or Te.

[0054] In some respects, R1, R2, R7, R8, R9, R13, R14, R15, R19, R20, R23, R24, R29, R30, R52 and R53 can be independently selected from the linkage bond with B and the (C1-C5) hydrocarbon group.

[0055] The linking bond with B allows fluorophore A to be conjugated with B. The linking bond between A and B can be designed to provide an electron conduction conjugation bond between A and B, as disclosed herein.

[0056] Optionally, R3, R4, R5, R6, R10, R11, R12, R16, R17, R18, R21, R22, R25, R26, R27, R28, R31, R33, R35, R37, R39, R40, R42, R44, R46, R47, R48, R50, R51, R54, and R55 can be selected independently to increase the solubility of the compound in water.

[0057] Optionally, R3, R4, R5, R6, R10, R11, R12, R16, R17, R18, R21, R22, R25, R26, R27, R28, R31, R33, R35, R37, R39, R40, R42, R44, R46, R47, R48, R50, R51, R54, and R55 can be independently water-soluble groups. Water-soluble groups can have the following structures:

[0058] Where n is an integer from 1 to 15, m is an integer from 1 to 5, and R71 is independently selected from –H and the connection bond with the second structure, which is optionally a probe or a dendritic polymer structure. In some aspects, each n can be 10 or 11.

[0059] In some respects, the compound is conjugated with a second structure such as a probe or a dendritic polymer, for example, R1, R3, R4, R5, R6, R10, R11, R12, R16, R17, R18, R21, R22, R25, R26, R27, R28, R31, R33, R35, R37, R39, R40, R42, R44, R46, R47, R48, R50, R51, R54, and R55 can be independently selected from (C1-C50). 50 (Heterohydrocarbon group and the linking bond with the second structure)

[0060] The compounds disclosed herein include an A unit linked to a B unit. The B unit is optionally an electron-accepting group, an electron-donating group, or a fluorophore. Optionally, the B unit is a rhodamine dye, a fluorescein dye, a coumarin dye, an indole dye, any other dye, or any combination thereof. Optionally, the B unit is any fluorescent dye listed in U.S. Patent No. 8,354,239. In some aspects, B is selected from the group consisting of:

[0061] R56, R58, and R60 are linkages to A; and R57, R59, R61, and R62 are independently water-soluble groups, optionally (C1-C2). 50 ) heterohydrocarbon group, (C1-C 40 (C5-C) heterohydrocarbon group, (C5-C) 40 ) heterohydrocarbon group, (C 10 -C 40 ) heterohydrocarbon group, (C1-C 30 (C5-C) heterohydrocarbon group, (C5-C) 30 ) heterohydrocarbon group or (C 10 -C 30 ) heterohydrocarbon group.

[0062] The linking bond with B allows fluorophore A to be conjugated with B. The linking bond between A and B can be designed to provide an electron conduction bond between A and B, as disclosed herein.

[0063] In some respects, R57, R59, R61 and R62 can be selected independently to increase the solubility of the compound in water.

[0064] In some respects, R57, R59, R61 and R62 can be independently selected from the following structures:

[0065] Where n is an integer from 1 to 15, m is an integer from 1 to 5, and R71 is independently selected from –H and the connection bond with the second structure, which is optionally a dendritic polymer or a probe. In some aspects, each n can be 10 or 11.

[0066] When the disclosed compound is conjugated with a second structure such as a dendritic polymer, R57, R59, R61, and R62 can be independently selected from (C1-C2). 50 (Heterohydrocarbon group and the linking bond with the second structure)

[0067] In specific respects, the disclosed compounds may be or include structures according to the following formula:

[0068] Each n is independently selected from an integer from 4 to 100, optionally from 5 to 15. Optionally, each n is independently selected from 10 or 11. This structure can be connected to a second structure such as a probe or a dendritic polymer.

[0069] In some respects, the compound has the structure of groups shown in Table 1 below, optionally each having these groups.

[0070] Table 1: Exemplary compound structures.

[0071] In Table 1, R1, R2, R7, R8, R9, R13, R14, R15, R19, R20, R23, R24, R29, R30, R52, R53, R72, R73, R74, and R75 can be independently selected from bonds with B, water-soluble groups, bonds with probes, bonds with dendritic polymers, and (C1-C2) bonds. 20 ) hydrocarbon group, (C1-C 20( ) Heteroalkyl groups, forming ring structures and combinations thereof; at least one of R1 and R2 can be a bond with B; at least one of R7, R8 and R9 can be a bond with B; at least one of R13, R14 and R15 can be a bond with B; at least one of R19 and R20 can be a bond with B; at least one of R23 and R24 can be a bond with B; at least one of R29 and R30 can be a bond with B; at least one of R52 and R53 can be a bond with B; at least one of R72, R73, R74 and R75 can be a bond with B; R3, R4, R5, R6 R10, R11, R12, R16, R17, R18, R21, R22, R25, R26, R27, R28, R31, R33, R35, R37, R39, R40, R42, R44, R46, R47, R48, R50, R51, R54, and R55 can independently be H, water-soluble groups, (C1-C) 50 The heteroalkyl group, or may be fused to form a ring optionally including R54, R55 or both; R32, R34, R36, R38, R41, R43, R45 and R49 may be a linking bond to B; X may be C, Si, O, S, P, N, Se or Te; and wherein R56, R58 and R60 are linking bonds to A; and R57, R59, R61 and R62 are independently water-soluble groups, optionally (C1-C1) 50 ) heterohydrocarbon group, (C1-C 40 (C5-C) heterohydrocarbon group, (C5-C) 40 ) heterohydrocarbon group, (C 10 -C 40 ) heterohydrocarbon group, (C1-C 30 (C5-C) heterohydrocarbon group, (C5-C) 30 ) heterohydrocarbon group or (C 10 -C 30 The heteroalkyl group, optionally wherein R3, R4, R5, R6, R10, R11, R12, R16, R17, R18, R21, R22, R25, R26, R27, R28, R31, R33, R35, R37, R39, R40, R42, R44, R46, R47, R48, R50, R51, R54, R55, R57, R59, R61 and / or R62 may be independently selected to increase the solubility of the compound in water, optionally wherein R3, R4, R5, R6 R10, R11, R12, R16, R17, R18, R21, R22, R25, R26, R27, R28, R31, R33, R35, R37, R39, R40, R42, R44, R46, R47, R48, R50, R51, R54, R55, R57, R59, R61 and / or R62 can be

[0072] Where n is an integer from 1 to 15, m is an integer from 1 to 5, and R71 is independently selected from –H and the connection bond with the second structure, which is optionally a dendritic polymer or a probe. In some aspects, each n can be 10 or 11.

[0073] The disclosed compounds containing A and B may also be conjugated with one or more second structures. The second structure is optionally a probe. A probe is any molecule that can be used to bind a target. A target is any structure that needs to be labeled with the compounds disclosed herein, optionally a tissue, cell, protein, nucleic acid, lipid, or any other desired structure. The probe optionally binds to a group A, a group B, or both groups A and B.

[0074] In some respects, the A group is (26) Wherein R13, R14, and R15 are bonds attached to B groups, and each B group is independently selected from one or more of the following: (XXVI) (27) R57 is a water-soluble group, and R56 is a linking bond with A. (28) Where R59 is a water-soluble group, R58 is a bond to A, and (29) Wherein R61 and R62 are each independently water-soluble groups, and R60 is a linking bond to A, wherein optionally the water-soluble groups have the following structure:

[0075] Where n is an integer from 1 to 15, m is an integer from 1 to 5, and R71 is independently selected from –H and the linking bond with the second structure, which is optionally a probe or dendritic polymer structure, wherein n can optionally be 10 or 11. Optionally, R14 is XXVI, and R13 and R15 are each B groups 29.

[0076] In some respects, the A group is (30) Wherein R52 and R53 are bonds attached to the B group, and wherein the B group is selected from one or more of the following: (XXVI) (27) R57 is a water-soluble group, and R56 is a linking bond with A. (28) Where R59 is a water-soluble group, R58 is a bond to A, and (29) Wherein R61 and R62 are each independently water-soluble groups, and R60 is a linking bond to A, wherein optionally the water-soluble groups have the following structure:

[0077] Where n is an integer from 1 to 15, m is an integer from 1 to 5, and R71 is independently selected from –H and the connection bond with the second structure, which is optionally a probe or a dendritic polymer structure, wherein n can optionally be 10 or 11.

[0078] In some respects, group A, group B, or both are compounds as described in U.S. Patent No. 10,053,484 or U.S. Patent Application Publication No. 2021 / 0102873.

[0079] The compound may optionally be linked to one or more probes, optionally linked to a group A, a group B, or both. A probe is any molecule capable of binding to a target, and optionally selectively binding to a target. Illustrative examples of probes include, but are not limited to, antibodies, such as any suitable antibody known in the art, including other immunologically active fragments of antibodies or single-chain antibodies. A probe may be a single moiety, such as a polypeptide or protein, or it may comprise two or more moietyes, such as a pair of polypeptides, or a pair of single-chain antibody domains. The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody or a fragment thereof may be derived from an organism, such as a sheep, goat, rabbit, or rat polyclonal antibody.

[0080] Probes can be antibody fragments, such as single-chain antibodies (scFv), Fab and scFv antibodies, single-domain antibodies (VHH), or chimeric antibodies. Probes can be derived from naturally occurring proteins or peptides; they can be redesigned (…). de novo Alternatively, the probe can be derived from an antibody, single-chain antibody (scFv), single-domain antibody (VHH), lipid transport protein, single-chain MHC molecule, or anticalin. TM (Pieris), Affibody TM Nanobodies (Ablynx) or Trinectin TM (Phylos).

[0081] In some respects, a probe is a fragment of an antibody, optionally an antigen-binding fragment or a variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments. Other probe molecules include biantibodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0082] Optionally, the compounds provided herein may be combined with one or more second structures. The second structure is optionally one or more dendritic polymers. Conjugation of the disclosed compounds with dendritic polymers may improve the solubility of the compounds in water, increase the brightness of the compounds, and / or reduce fluorescence quenching of the compounds, among other benefits.

[0083] Not wanting to be bound by any particular theory, it is believed that the conjugation of this compound containing A and B with the dendritic polymer can reduce dye-dye interactions by keeping each compound containing A and B at a certain distance from multiple compounds containing A and B.

[0084] In some aspects, the composition may include a dendritic polymer and one or more compounds containing A and B as disclosed herein.

[0085] Dendrimer

[0086] This document also discloses dendritic polymers or dendritic polymer structures that can be labeled with one or more fluorophores, optionally wherein said one or more fluorophores are compounds as provided herein. The dendritic polymer structures are sufficiently soluble in water for in vivo or in vitro biological imaging and, due to the number of potential binding sites on the dendritic polymer structures, can optionally bind to one or more fluorophores, probes, or other molecules. Therefore, the dendritic polymer structures disclosed herein optionally bind to one or more fluorophores. Optionally, the dendritic polymer structures bind to one or more fluorophores disclosed herein, said fluorophores comprising one or more A units as defined herein and one or more B units as defined herein.

[0087] The average molecular weight of dendritic polymers can be greater than or equal to 5 kilodaltons (kDa) and less than or equal to 100 kDa, for example greater than or equal to 10 kDa and less than or equal to 30 kDa, or greater than or equal to 10 kDa and less than or equal to 20 kDa.

[0088] In some aspects, the dendritic polymer can be straight-chain or branched and can include one or more polyethylene glycol chains, which not only provide sufficient spacing to allow the binding of one or more probe molecules and / or one or more fluorescent molecules, but also provide sufficient water solubility of the composition for probing biological samples. The one or more polyethylene glycol chains may include at least three repeating ethylene glycol units, optionally at least five ethylene glycol units, and / or optionally at least ten ethylene glycol units. Optionally, the dendritic polymer and the fluorophore or probe can be linked by one or more polyethylene glycol chains of the dendritic polymer.

[0089] Dendritic polymers may include probes as disclosed herein. Probes may be conjugated with the dendritic polymer. Optionally, probes may be proteins or fragments thereof, including but not limited to antibodies or fragments thereof, nucleic acids, phospholipids, polysaccharides, triglycerides, aptamers, avidin, streptavidin, neutral avidin, avidin DN, avidin D, or cells.

[0090] The F / P ratio of the dendritic polymer composition can be from 5 to 100, for example from 10 to 30, wherein the F / P ratio is defined as the total number of molecules of the compound present in the composition divided by the total number of molecules of the fluorescent molecule or probe present in the composition.

[0091] The dendritic polymer may also include one or more functional groups. The dendritic polymer may include one or more branches, wherein one or more of said branches terminate at a functional group. A functional group is any group that can be used to attach the dendritic polymer to another molecule, optionally to a second dendritic polymer, a probe, a fluorophore, or a combination thereof. The functional group may be or include amides, thiols, succinimides, maleimides, azides, carboxyl esters, carboxyl / EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sulfonyl-SMCC, BMPH, sulfonyl-SBED, trans-cyclooctene / tetraazine, amines / epoxides, or combinations thereof.

[0092] The dendritic polymers disclosed herein optionally include the following compounds:

[0093] Each n is independently an integer from 1 to 300 (optionally 10-300), z is 1-50, optionally 1-10, and R63, R64, and R65 are independently selected from the functional groups disclosed herein, optionally amines, azides, linkages to A or B, linkages to probes, and one or more additional dendritic polymers and / or optionally 1 to 10 additional dendritic polymer structures. The probe may be an antibody or a fragment thereof, nucleic acid, phospholipid, polysaccharide, triglyceride, aptamer, avidin, streptavidin, neutral avidin, avidin DN, avidin D, or a cell. Optionally, at least one of R63, R64, or R65 may be a linkage to A or B, and at least one of R63, R64, or R65 may be a linkage to a probe. Optionally, the linking bond may be selected from amides, thiols, succinimides, maleimides, azides, carboxyl esters, carboxyl / EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sulfonyl-SMCC, BMPH, sulfonyl-SBED, trans-cyclooctene / tetraazine, amine / epoxide, or any combination thereof.

[0094] In other respects, the dendritic polymers disclosed herein may include compounds of the following formula:

[0095] Each n is independently an integer from 1 to 300 (optionally 10-300), and R63, R64, R65, R66, R67, R68, R69, and R70 are independently selected from the functional groups disclosed herein, optionally amines or azides, linker A or B, linker to the probe, and one or more additional dendritic polymers and / or optionally 1 to 10 additional dendritic polymer structures. The probe can be a protein or a fragment thereof, optionally including but not limited to antibodies or fragments thereof, nucleic acids, phospholipids, polysaccharides, triglycerides, aptamers, avidin, streptavidin, neutral avidin, avidin DN, avidin D, or cells. Optionally, at least one of R63, R64, R65, R66, R67, R68, R69, and R70 may be a linkage to A or B, and at least one of R63, R64, R65, R66, R67, R68, R69, and R70 may be a linkage to the probe. Optionally, the linkage may be selected from amides, thiols, succinimides, maleimides, azides, carboxylic acid esters, carboxyl / EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sulfonyl-SMCC, BMPH, sulfonyl-SBED, trans-cyclooctene / tetraazine, amine / epoxide, or any combination thereof.

[0096] The dendritic polymer structure may be linked to one or more fluorophores. Optionally, the fluorophores linked to the dendritic polymer structure may be compounds disclosed herein, including A and / or B, or may be rhodamine dyes, fluorescein dyes, coumarin dyes, indole dyes, or any combination thereof. Optionally, the fluorophore bound to the dendritic polymer may be or include fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy-chromium, phycoerythrin, PerCP (cyclophycocyanin chlorophyll a protein), allophycocyanin, PerCP-Cy5.5, JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, fluorescein yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY® FL, BODIPY® FL-Br2, BODIPY® 530 / 550, BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 576 / 589, BODIPY® 581 / 591, BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, SPK dyes, cf514, DY405, DY396XL, cf570, cf405, their conjugates or combinations thereof.

[0097] Illustrative examples of dyes that can be used in any aspect provided herein include, but are not limited to, Spark UV™ 387 and Spark Violet. TM 423. Spark Violet TM 500, Spark Violet TM538, Spark Blue TM 515, SparkBlue TM 550, Spark Blue TM 574, Spark YG TM 570, Spark YG TM 581, Spark YG TM 593, SparkNIR TM 685, Spark Red TM 718.

[0098] Target detection

[0099] This document also discloses methods for detecting the presence or absence of an analyte / target in a sample (optionally a biological sample) or organism. The methods may include contacting the sample with a composition comprising the compounds disclosed herein, the dendritic polymer structures disclosed herein, or combinations thereof; exposing the sample to light at a wavelength capable of exciting fluorophores in the composition; and detecting the presence or absence of light emitted by the composition. This detection can utilize various analytical methods, such as, but not limited to, flow cytometry, FISH, immunohistochemistry, sandwich assays, Southern blotting, Western blotting, microarrays, or substrate binding assays.

[0100] Methods for detecting the presence or absence of an analyte in a sample or organism may include the use of a dendritic polymer linked to a fluorophore as disclosed herein. The dendritic polymer used in methods for detecting the presence or absence of an analyte in a sample or organism may include any dendritic polymer and any compound described herein.

[0101] Without being bound by any particular theory, it is believed that including dendritic polymers in the composition can reduce fluorophore quenching, likely due to the increased distance between individual fluorophores provided by the dendritic polymer. Therefore, the compositions and methods described herein can provide increased brightness from the compounds compared to conventional compositions and methods.

[0102] Methods for detecting the presence of an analyte in a sample or organism may eliminate the need for adding a buffer solution to the sample, likely due at least in part to reduced fluorophore-fluorophore interactions between components. These factors can reduce the complexity and / or operating cost of the method.

[0103] A method for forming a targeted dendritic polymer composition is also disclosed. The method may include contacting the dendritic polymer with a compound containing a fluorophore. The contact may include contacting the dendritic polymer and the compound under conditions in which the compound is bound to the dendritic polymer, thereby forming a dendritic polymer composition. Optionally, the dendritic polymer includes at least one or more functional groups, optionally amines or azides, adapted to bind to one or more fluorophores optionally as provided herein. A targeted dendritic polymer composition can be formed by optionally reacting the dendritic polymer with one or more functional groups having fluorophores using conditions known in the art.

[0104] The method of forming a targeted dendritic polymer composition may further include contacting the dendritic polymer composition with the probe under conditions where the dendritic polymer composition is bound to the probe, thereby forming the targeted dendritic polymer composition. The contact may include contacting the dendritic polymer composition with the probe under conditions where the dendritic polymer composition is bound to the probe, thereby forming the targeted dendritic polymer composition.

[0105] Any dendritic polymer disclosed herein may be formed by methods for forming targeted dendritic polymer compositions described herein, and optionally include one or more fluorescent compounds disclosed herein or another fluorophore.

[0106] The probes used in the methods for forming targeted dendritic polymer compositions may include any aspect of the target molecule as described herein as a method for detecting the presence or absence of an analyte in a sample or organism.

[0107] Various aspects of this disclosure are illustrated by the following non-limiting examples. These examples are for illustrative purposes only and are not intended to limit any practice of this disclosure. However, it should be understood that variations and modifications may be made without departing from the spirit and scope of this disclosure. The reagents described herein are commercially available or readily synthesized from readily available precursors using well-known methods, and those skilled in the art will readily understand where such reagents are available.

[0108] Example

[0109] Example 1: Synthesis of compounds 1E and 1F: Basically, compounds 1E and 1F were synthesized as described in Scheme I:

[0110] Option 1

[0111] In short, step 1. Add 100-300 mL of acetic acid, 10-100 mL of toluene, and 10-100 mL of 48% water-soluble HBr to a 500 mL round-bottom pressure vessel. Immediately seal the vessel with the appropriate pressure vessel plastic cap. Then add 1-20 g (g) of 6,6',12,12'-tetratetra(3-phenoxypropyl)indeno[1,2-b]fluorine to the 500 mL round-bottom pressure vessel. Stir the reaction mixture under reflux overnight and collect the resulting crystals of Fr-4Br by filtration.

[0112] Step 2. In a 250 mL round-bottom flask, add 1-20 g of Fr-4Br. Pour 1-200 mL of DCM into the flask and stir continuously for 10 min. Add 1-20 mL of bromine to the flask and maintain the reaction mixture at room temperature for 2-36 hours (h). After the reaction, wash the reaction mixture with 200 mL of sodium carbonate solution and remove the solvent using a rotary evaporator. Wash the yellow crude product with DCM to obtain pure Fr-6Br.

[0113] Step 3. Add 5-50 g mPEG to a 500 mL three-necked round-bottom flask. 11 10-300 mL THF and 0.5-10 g NaH were added. The reaction mixture was stirred at 10-100 °C for 0.5-10 h. Then, 1-20 g of FR-6Br was added to the container, and the reaction was maintained at 10-100 °C for another 2-72 h. The reaction mixture was then cooled to room temperature, and the crude product was purified by column chromatography (filled with C18 silica gel), eluting with a mixture of water and MeOH to give Fr-4PEG as a yellow oil. 10 -2Br.

[0114] Step 4 was performed by adding 3.6 g of FR-2Br-4PEG10 to 100 mL of 24 / 40 solution and 1.152 g of 4-(2-carboxyethyl)phenylboronic acid pinacol ester to a two-necked round-bottom flask, and dissolving in 18 mL of anhydrous dimethylformamide (DMF). The mixture was frozen with liquid N2. 2M K2CO3 (1.73 g in 6 mL) and 58 mg of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloride, fused with dichloromethane, were added to the container. The mixture was frozen and thawed three times, then heated to 100 °C for 6 hours. 160 mL of DCM was added to the container, and the mixture was sonicated for 2 min, followed by the addition of 2 mL of acetic acid. The DCM was removed by a rotary evaporator in a water bath to give a black oily product. The crude product was redissolved in 15–20 mL of DI water. The filtrate was transferred to 50 mL plastic centrifuge tubes and separated by column chromatography. The identity of the final compound 1E was examined by HPLC and NMR.

[0115] Optionally, the synthetic protocol was continued to produce compound 1F. For step 5, 100 mg of Fr-4PEG10-2COOH and 5 mL of acetonitrile were added to a 20 mL glass vial, followed by 0.4 mL of anhydrous DMF. TSTU and DIPEA were then added to the vial, and the mixture was shaken at room temperature for 15 min. Acetic acid was then added to the vial, and the mixture was shaken to mix, followed by solvent removal by lyophilization. The product was separated using a C18 HPLC column, dried, and analyzed by HPLC and NMR. The compound obtained from step 5 was identified herein as compound 1F.

[0116] The absorption and emission spectra of compound 1F were analyzed by fluorescence spectroscopy. The results were... Figure 1 As shown in the figure. The maximum absorption of compound 1F observed is approximately 355 nm. The maximum emission of compound 1F observed is approximately 385 nm. Figure 2 The spectra of compound 1F and comparative example A (i.e., Brilliant Ultra Violet™ 395 dye (BUV395)) are shown under different detectors normalized to mean fluorescence intensity (MFI) of 1.

[0117] Example 2: Synthesis of compound 2F.

[0118] Repeat the reaction of Example 1 starting with compound 2A as follows: (2A).

[0119] As in steps 1 and 2 of Example 1, compound 2A is brominated at the desired location to form compound 2B.

[0120] (2B)

[0121] As in step 3 of Example 1, compound 2B was subjected to mPEG. 11 The addition of [a] is used to generate water-soluble groups at the desired positions, as observed in compound 2C.

[0122] (2C).

[0123] The system then reacts with the desired precursor of compound 29 to form compound 2D.

[0124] (2D).

[0125] Compound 2D was characterized by HPLC and NMR as in Example 1. Optionally, as in step 5 of Example 1, one or more terminal carboxyl groups on compound 2C were further functionalized to bind the probe.

[0126] Example 3: Synthesis of other compounds:

[0127] The steps of Example 1 or 2 will be further performed to produce various compounds having various A and B groups using similar precursor compounds A and B with appropriate modifications (as in Examples 1 and 2). The following A and B groups are used in the synthetic scheme.

[0128] Table 2: Synthesis schemes for A and B groups in Example 3.

[0129]

[0130] In Table 2, regarding group A: R1, R2, R7, R8, R9, R13, R14, R15, R19, R20, R23, R24, R29, R30, R32, R34, R36, R38, R41, R43, R45, R49, R52 or R53 or both, R72, R73, R74, and R75 are each independently bonded to group B; R3, R4, R5, and R6 are bonds that can be bonded to group B. R10, R11, R12, R16, R17, R18, R21, R22, R25, R26, R27, R28, R31, R33, R35, R37, R39, R40, R42, R44, R46, R47, R48, R50, R51, R54, and R55 are each independently connected to

[0131] Where n is 11-12, m is an integer from 1 to 2, and R71 is H. The compound was characterized by HPLC and NMR.

[0132] Example 4: Preparation of dendritic polymer composition 2: The dendritic polymer composition 2 was prepared by coupling compound 1F with a dendritic polymer to form dendritic polymer composition 2. The dendritic polymer used has the following structure: Where n is 10. In short, the above dendritic polymers are modified by reacting the dyes of Examples 1-3 to form amides in appropriate ratios (dye to dendritic polymer ratio of 2-100, depending on the target DOL). The reaction mixture is placed under neutral or alkaline conditions and reacted overnight with any of the compounds from Examples 1-3 to complete the reaction. The labeled dendritic polymer product is then purified by a size separation column before further use.

[0133] Example 5: Preparation of antibody dendritic polymer composition: The preparation of the antibody dendritic polymer composition was achieved by conjugating the dendritic polymer (labeled or dendritic polymer composition 2) of Example 2 with an antibody. Briefly, the dendritic polymer was modified with azide functional groups, and the antibody was modified with DBCO functional groups. The azide-modified dendritic polymer was added to the DBCO-modified antibody solution in an appropriate ratio (the ratio of dendritic polymer to antibody is optionally 2-60, depending on the target F / P). The reaction mixture was placed under neutral or alkaline conditions and reacted overnight to achieve conjugation. The conjugate was then purified before further characterization.

[0134] Example 6: Flow cytometry assays of the antibody dendritic polymer composition and comparative examples: In short, Comparative Example A (BUV395), Compound 1F, and the dendritic polymer composition 2 bound to Compound 1F were each individually conjugated with the SK3 antibody, and the resulting conjugated products are represented as Comparative Example 4A, Comparative Example 4B, and Example 4C, respectively. Figure 3 depicts the flow cytometry plots of the samples, with the side-scattered area (SSC-A) as the x-axis and the BUV395 area (BUV395-A) as the y-axis, and BUV395-A as the x-axis and the count as the y-axis. Specifically, the flow cytometry plots of Comparative Example 4A, Comparative Example 4B, and Comparative Example 4C are described in [the figures provided]. Figure 3A , 3B In 3C, the staining index of each sample was also evaluated. The staining index of Example 4C was 129, which was brighter than that of Comparative Example 4A, which had a staining index of 92. Furthermore, the dendritic polymer contained in Example 4C increased the staining index by approximately 2.7 times compared to Comparative Example 4B, which contained the same compound F but did not contain the dendritic polymer.

[0135] Intracellular staining (cytoplasm and nucleus) of Comparative Example A (BUV395), Compound 1F, and dendritic polymer composition 2 was also evaluated. In short, the conjugated products of Comparative Example A (BUV395), Compound 1F, and dendritic polymer composition 2 conjugated with Compound 1F were represented as Comparative Example 4D, Comparative Example 4E, and Example 4F, respectively, by conjugating the 4S.B3 antibody. Figure 4 illustrates the flow cytometry plots of the samples from Example 4, with the allophycocyanin area (APC-A) as the x-axis and the BUV395 area (BUV395-A) as the y-axis. Specifically, the flow cytometry plots of Comparative Example 4D, Comparative Example 4E, and Example 4F are described in [the figures provided]. Figure 4A , 4B In 4C, the F / P ratio of Comparative Example 4D was 11.1, and the F / P ratio of Example 4E was 21.51. As shown in Figure 4, intracellular staining of the fluorophore attached to the dendritic polymer structure in Example 4F was improved compared to Comparative Example 4D, which only included the fluorophore.

[0136] Example 7: Flow cytometry assays of antibody dendritic compositions using Spark Blue™ 550 and comparative examples using RB545 or Spark Blue™ 550: In short, RB545 and Spark Blue 550 were conjugated with the probe antibody, and the resulting conjugated products are denoted as Comparative Example 5A and Comparative Example 5B. To prepare Example 5C, Spark Blue™ 550 was coupled with the dendritic polymer used in Example 6, and then the dendritic polymer dye composition was conjugated with the probe. Figure 5 depicts flow cytometry plots for the samples of Example 7, with the side-scattered area (SSC-A) as the x-axis and the fluorescein isothiocyanate area (FITC-A) as the y-axis, and FITC-A as the x-axis and count as the y-axis. Specifically, the flow cytometry plots for Comparative Example 5A, Comparative Example 5B, and Comparative Example 5C are described in [reference to Figure 5]. Figure 5A , 5B In Example 5C, the staining index of each sample was also evaluated. The staining index of Example 5C was 129, which was brighter than that of Comparative Example 5A, which had a staining index of 68. Furthermore, the dendritic polymer included in Example 5C increased the staining index by approximately 7.1 times compared to Comparative Example 5B, which contained the same dye but did not contain the dendritic polymer. The F / P ratio of both Comparative Examples 5B and 5C was approximately 14. As shown in Figure 5, the addition of the dendritic polymer reduced the dye quenching effect.

[0137] Example 8: Flow cytometry assays using an antibody dendritic polymer composition with BL351-387 and a comparative example using BUV395: In short, RB545 was conjugated with the 4S.B3 antibody, and the resulting conjugated product is denoted as Comparative Example 6A. To prepare Example 6B, BL351-387 was coupled with the dendritic polymer used in Example 4, and subsequently, the dendritic polymer dye composition was conjugated with the 4S.B3 antibody. Figure 6 depicts the flow cytometry plots of the sample from Example 6, with the allophycocyanin area (APC-A) as the x-axis and the BUV395 area (BUV395-A) as the y-axis. Specifically, the flow cytometry plots of Comparative Example 6A and Example 6B are described in [reference to Figure 6]. Figure 6A and 6B As shown in Figure 6, the intracellular staining in Example 6B is improved compared to Comparative Example 6A.

[0138] Additional exemplary aspects

[0139] 1. A compound comprising A and B; One or more A groups are connected to one or more B groups via an electronically conducted conjugated bond, wherein the electronically conducted conjugated bond is configured to delocalize one or more electrons between A and B; Where A is an electron acceptor group, an electron donor group, or a fluorophore; Wherein B is an electron acceptor group, an electron donor group, or a fluorophore; and wherein the compound does not contain repeating units of A, B, or combinations thereof.

[0140] 2. According to aspect 1, each conjugated bond is independently composed of n conjugated bond units selected from single bonds, triple bonds, C=N groups, amide bonds, click-reactive bonds, benzene ring bonds, heterocyclic bonds, and combinations thereof; and

[0141] Where n≥1.

[0142] 3. The compound according to aspect 1 or 2, wherein the compound further comprises one or more water-soluble groups connected to A, B or both.

[0143] 4. The compound according to any one of aspects 1-3, wherein each conjugated bond is independently a group of n conjugated bond units selected from single bonds, triple bonds, C=N groups, amide bonds, click-reactive bonds, benzene ring bonds, heterocyclic bonds, and combinations thereof; and

[0144] Where n≥1.

[0145] 5. According to the compound of aspect 4, where n≥2.

[0146] 6. The compound according to any one of aspects 1-5, wherein the electronically conducted conjugated bond is a conjugated single bond.

[0147] 7. The compound according to any one of aspects 1-6, wherein A is a conjugated ring dye.

[0148] 8. The compound according to any one of aspects 1-7, wherein B is a conjugated ring dye.

[0149] 9. The compound according to any one of aspects 1-8, wherein the compound has maximum absorption in the wavelength range of 300 nm and 450 nm.

[0150] 10. The compound according to any one of aspects 1-8, wherein the compound has maximum absorption in the wavelength ranges of 365 nm and 830 nm.

[0151] 11. The compound according to any one of aspects 1-8, wherein the compound has maximum emission in the wavelength range of 365 nm and 830 nm.

[0152] 12. The compound according to any one of aspects 1-11, wherein A has maximum absorption in the wavelength ranges of 300 nm and 450 nm when not conjugated with B.

[0153] 13. The compound according to any one of aspects 1-11, wherein A has maximum emission in the wavelength ranges of 300 nm and 600 nm when not conjugated with B.

[0154] 14. A compound according to any one of aspects 1-13, wherein the compound has a structure comprising or consisting of A groups linked to 1 to 10 B groups, optionally having the following formula:

[0155] 15. The compound according to any one of aspects 1-13, wherein the compound comprises or consists of B linked to 1 to 10 A groups, optionally having a structure of the following formula:

[0156] 16. The compound according to any one of aspects 1-15, wherein B is a fluorophore.

[0157] 17. The compound according to any one of aspects 1-16, wherein B has maximum absorption in the wavelength ranges of 150 nm and 450 nm when not conjugated with A.

[0158] 18. The compound according to any one of aspects 1-16, wherein B has maximum emission in the wavelength ranges of 150 nm and 830 nm when not conjugated with A.

[0159] 19. The compound according to any one of aspects 1-18, wherein A is connected to two or more Bs.

[0160] 20. The compound according to any one of aspects 1-18, wherein B is connected to two or more A's.

[0161] 21. The compound according to any one of aspects 1-20, wherein one or more water-soluble groups are connected to A, and one or more water-soluble groups are connected to B.

[0162] 22. The compound according to aspect 21, wherein the one or more water-soluble groups comprise one or more ethylene units linked by oxygen.

[0163] 23. The compound according to any one of aspects 1-22, wherein, as measured at the maximum emission of the compound, the molar extinction coefficient of the compound is greater than 5000 cm⁻¹. -1 M-1 .

[0164] 24. The compound according to any one of aspects 1-23, wherein A is a structure selected from:

[0165] in: R1, R2, R7, R8, R9, R13, R14, R15, R19, R20, R23, R24, R29, R30, R52, and R53 are independently selected from the bonds connecting to B that form the ring structure, (C1-C 20 )-hydrocarbon group, (C1-C 20 )-heterohydrocarbon groups and their combinations; At least one of R1 and R2 is a connection key to B; At least one of R7, R8 and R9 is a connection key to B; At least one of R13, R14 and R15 is a connection key to B; At least one of R19 and R20 is a connection key to B; At least one of R23 and R24 is a connection key to B; At least one of R29 and R30 is a connection key to B; At least one of R52 and R53 is a connection key to B; One or more of R3, R4, R5, R6, R10, R11, R12, R16, R17, R18, R21, R22, R25, R26, R27, R28, R31, R33, R35, R37, R39, R40, R42, R44, R46, R47, R48, R50, R51, R54, and R55 may be independently H, (C1-C 50 )- Heterohydrin groups or fused to form optional rings including R54, R55, or both; or R32, R34, R36, R38, R41, R43, R45, and R49 are connection keys to B; and Where X is C, Si, O, S, P, N, Se, or Te.

[0166] 25. The compound according to aspect 24, wherein R1, R2, R7, R8, R9, R13, R14, R15, R19, R20, R23, R24, R29, R30, R52 and R53 are independently selected from the linkage bond with B and the (C1-C5) hydrocarbon group.

[0167] 26. The compound according to aspect 24 or 25, wherein R3, R4, R5, R6, R10, R11, R12, R16, R17, R18, R21, R22, R25, R26, R27, R28, R31, R33, R35, R37, R39, R40, R42, R44, R46, R47, R48, R50, R51, R54 and R55 are independently water-soluble linking groups, optionally

[0168] Where n is an integer from 5 to 15, m is an integer from 1 to 5, and R71 is independently selected from H and the connection bond with the second structure, which is optionally a dendritic polymer or a probe.

[0169] 27. The compound according to aspect 26, wherein each n is 10 or 11.

[0170] 28. The compound according to any one of aspects 1-27, wherein B is a structure selected from:

[0171] in: R56, R58, and R60 are connection keys to A; and R57, R59, R61, and R62 are independently water-soluble groups, optionally (C1-C2). 50 ) heterohydrocarbon group.

[0172] 29. The compound according to aspect 1, wherein the compound comprises:

[0173] Each n is independently selected from an integer between 4 and 100, and optionally between 5 and 15.

[0174] 30. The compound according to aspect 29, wherein each n is 10 or 11.

[0175] 31. The compound according to any one of aspects 1-30, wherein A, B or both are linked to one or more dendritic polymer structures.

[0176] 32. The compound according to aspect 31, wherein the dendritic polymer structure is a branched structure.

[0177] 33. The compound according to aspect 31 or 32, wherein both A and B are connected to one or more dendritic polymer structures.

[0178] 34. The compound according to any one of aspects 31-33, wherein the average molecular weight of the dendritic polymer is from 5 kilodaltons (kDa) to 100 kDa, optionally from 5 kDa to 30 kDa.

[0179] 35. The compound according to aspect 34, wherein the average molecular weight of the dendritic polymer is from 10 kDa to 20 kDa.

[0180] 36. The compound according to any one of aspects 31-35, wherein the dendritic polymer comprises polyethylene glycol chains.

[0181] 37. The compound according to any one of aspects 31-35, wherein the dendritic polymer and the compound are linked by a polyethylene glycol chain.

[0182] 38. The compound according to aspect 37, wherein the polyethylene glycol chain comprises at least three ethylene glycol repeating units.

[0183] 39. The compound according to any one of aspects 31-38, further comprising a probe connected to one or more of the dendritic polymer structures.

[0184] 40. The compound according to aspect 39, wherein the probe molecule is a protein or a fragment thereof, including but not limited to an antibody or a fragment thereof, nucleic acid, phospholipid, polysaccharide, triglyceride, aptamer, avidin, streptavidin, neutral avidin, avidin DN, avidin D, or cell.

[0185] 41. The compound according to any one of aspects 1-40, wherein the F / P ratio of the composition is 5 to 100, optionally 10 to 30, wherein the F / P ratio is defined as the total number of molecules of the compound present in the composition divided by the total number of molecules of the probe present in the composition.

[0186] 42. The compound according to any one of aspects 31-41, wherein the dendritic polymer further comprises functional groups.

[0187] 43. The compound according to any one of aspects 31-42, wherein the dendritic polymer comprises one or more branches, wherein one or more of the branches terminate at the functional group.

[0188] 44. The compound according to any one of aspects 31-43, wherein the dendritic polymer comprises a compound of the following formula

[0189] in: Each n is an independent integer from 10 to 300; R63, R64, R65, R66, R67, R68, R69, and R70 are independently selected from amines, azides, linkages to A or B, linkages to a probe, and one or more other dendritic polymers, optionally from 1 to 10 other dendritic polymer structures.

[0190] 45. The compound according to aspect 44, wherein the probe is a protein or a fragment thereof, optionally including but not limited to an antibody or a fragment thereof, nucleic acid, phospholipid, polysaccharide, triglyceride, aptamer, avidin, streptavidin, neutral avidin, avidin DN, avidin D, or cell.

[0191] 46. ​​The compound according to aspect 44 or 45, wherein at least one of R63, R64, R65, R66, R67, R68, R69 and R70 is a linkage to A or B, and at least one of R63, R64, R65, R66, R67, R68, R69 and R70 is a linkage to a probe.

[0192] 47. The compound according to aspect 46, wherein the linkage is selected from amides, thiols, succinimides, maleimides, azides, carboxyl esters, carboxyl / EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sulfonyl-SMCC, BMPH, sulfonyl-SBED, trans-cyclooctene / tetraazine, and amine / epoxide.

[0193] 48. The compound according to aspect 46 or 47, wherein A or B is selected from fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy-chromium, phycoerythrin, PerCP (cyclophytic chlorophyll a protein), PerCP-Cy5.5, JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, fluorescein yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680. 7-amino-4-methylcoumarin-3-acetic acid, BODIPY® FL, BODIPY® FL-Br2, BODIPY® 530 / 550, BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 576 / 589, BODIPY® 581 / 591, BODIPY®630 / 650, BODIPY® 650 / 665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, SPK dyes, cf514, DY405, DY396XL, cf570, cf405, their conjugates and combinations thereof.

[0194] 49. A method for detecting the presence or absence of an analyte in a sample or organism, the method comprising: (a) Contact the sample with a composition comprising a dendritic polymer structure and a fluorophore, wherein the composition is optionally any combination of any of aspects 1-48; (b) Exposing the sample to light of a wavelength capable of exciting the fluorophore of the composition; and (c) Detect the presence or absence of light emitted by the composition.

[0195] 50. The method according to aspect 49, wherein the detection is performed by flow cytometry, FISH, immunohistochemistry, immunofluorescence microscopy, immunocytochemistry, live cell imaging, in vivo imaging (optionally using a long-wavelength emitter), direct or indirect ELISA, Southern blotting, Western blotting, microarray, or substrate binding assay.

[0196] 51. The method according to aspect 50, wherein the detection is performed by flow cytometry.

[0197] 52. The method according to any one of aspects 49-51, wherein the fluorophore is linked to the dendritic polymer structure.

[0198] 53. The method according to any one of aspects 49-52, wherein the dendritic polymer structure is a branched structure.

[0199] 54. The method according to any one of aspects 49-53, wherein the average molecular weight of the dendritic polymer structure is from 5 kilodaltons (kDa) to 100 kDa, optionally from 5 kDa to 30 kDa.

[0200] 55. The method according to aspect 54, wherein the average molecular weight of the dendritic polymer structure is from 10 kDa to 20 kDa.

[0201] 56. The method according to any one of aspects 49-55, wherein the dendritic polymer structure comprises polyethylene glycol chains.

[0202] 57. The method according to any one of aspects 49-56, wherein the dendritic polymer structure and the fluorophore are linked by polyethylene glycol chains.

[0203] 58. The method according to aspect 57, wherein the polyethylene glycol chain comprises at least three ethylene glycol repeating units.

[0204] 59. The method according to any one of aspects 49-58, wherein the composition further comprises a probe connected to one or more of the dendritic polymer structures.

[0205] 60. The method according to aspect 59, wherein the probe is a protein or a fragment thereof, including but not limited to an antibody or a fragment thereof, nucleic acid, phospholipid, polysaccharide, triglyceride, aptamer, avidin, streptavidin, neutral avidin, avidin DN, avidin D, or cell.

[0206] 61. The method according to aspects 49-60, wherein the F / P ratio of the composition is 5 to 100, optionally 10 to 30, wherein the F / P ratio is defined as the total number of fluorophore molecules present in the composition divided by the total number of probe molecules present in the composition.

[0207] 62. The method according to any one of aspects 49-61, wherein the dendritic polymer structure further comprises functional groups.

[0208] 63. The method according to any one of aspects 49-62, wherein the dendritic polymer structure comprises one or more branches, wherein one or more of the branches terminate at the functional group.

[0209] 64. The method according to any one of aspects 49-63, wherein the dendritic polymer structure comprises a compound of the following formula

[0210] in: Each n is an independent integer from 10 to 300; R63, R64, R65, R66, R67, R68, R69 and R70 are independently selected from amines, azides, fluorophores, probes, and one or more other dendritic polymers, optionally from 1 to 10 other dendritic polymer structures.

[0211] 65. The method according to aspect 64, wherein the probe is a protein or a fragment thereof, optionally including but not limited to an antibody or a fragment thereof, a nucleic acid, a phospholipid, a polysaccharide, a triglyceride, an aptamer, avidin, streptavidin, neutral avidin, avidin DN, avidin D, or a cell.

[0212] 66. The method according to aspect 64 or 65, wherein at least one of R63, R64, R65, R66, R67, R68, R69 and R70 is a linker bond to a fluorophore, and at least one of R63, R64, R65, R66, R67, R68, R69 and R70 is a linker bond to a probe.

[0213] 67. The method according to aspect 66, wherein the linkage is an amide, thiol, succinimide ester, maleimide, azide, carboxylic acid ester, carboxyl / EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sulfonyl-SMCC, BMPH, sulfonyl-SBED, trans-cyclooctene / tetraazine, and amine / epoxide.

[0214] 68. The compound according to aspect 66, wherein the fluorophore is selected from fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy-chromium, phycoerythrin, PerCP (cyclophycocyanin chlorophyll a protein), allophycocyanin, PerCP-Cy5.5, JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, fluorescein yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY® FL, BODIPY® FL-Br2, BODIPY® 530 / 550, BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 576 / 589, BODIPY® 581 / 591, BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, SPK dyes, cf514, DY405, DY396XL, cf570, cf405, their conjugates and combinations thereof.

[0215] 69. The method according to any one of aspects 49-68, wherein the composition is any one of the compounds of aspects 1-48.

[0216] 70. A method of forming a dendritic polymer composition, the method comprising: (a) Under conditions in which the compound is bound to the dendritic polymer, the dendritic polymer is contacted with a compound containing a fluorophore to form a dendritic polymer composition; and (b) Under conditions in which the dendritic composition is bound to the probe, the dendritic composition is brought into contact with the probe to form a targeted dendritic composition.

[0217] 71. The method according to aspect 70, wherein the dendritic polymer is a branched structure.

[0218] 72. The method according to any one of aspects 70 or 71, wherein the average molecular weight of the dendritic polymer is from 5 kilodaltons (kDa) to 100 kDa, optionally from 5 kDa to 30 kDa.

[0219] 73. The method according to aspect 72, wherein the average molecular weight of the dendritic polymer is from 10 kDa to 20 kDa.

[0220] 74. The method according to any one of aspects 70-73, wherein the dendritic polymer comprises polyethylene glycol chains.

[0221] 75. The method according to any one of aspects 70-74, wherein after contact in (a), the dendritic polymer and the fluorophore are linked by a polyethylene glycol chain.

[0222] 76. The method according to aspect 75, wherein the polyethylene glycol chain comprises at least three repeating glycol units.

[0223] 77. The method according to any one of aspects 70-76, wherein the probe is a protein or a fragment thereof, including but not limited to an antibody or a fragment thereof, nucleic acid, phospholipid, polysaccharide, triglyceride, aptamer, avidin, streptavidin, neutral avidin, avidin DN, avidin D, or cell.

[0224] 78. The method according to any one of aspects 70-77, wherein the F / P ratio of the targeted dendritic polymer composition is 5 to 100, optionally 10 to 30, wherein the F / P ratio is defined as the total number of fluorophore molecules present in the targeted dendritic polymer composition divided by the total number of probe molecules present in the targeted dendritic polymer composition.

[0225] 79. The method according to any one of aspects 70-78, wherein the dendritic polymer further comprises functional groups prior to contact in (a).

[0226] 80. The method according to aspect 79, wherein the dendritic polymer comprises one or more branches, wherein one or more of the branches terminate at the functional group.

[0227] 81. The method according to any one of aspects 70-80, wherein the targeted dendritic polymer composition comprises a compound of the following formula:

[0228] in: Each n is an independent integer from 10 to 300; R63, R64, R65, R66, R67, R68, R69 and R70 are independently selected from amines, azides, fluorophores, probes, and one or more other dendritic polymers, optionally from 1 to 10 other dendritic polymer structures.

[0229] 82. The method according to aspect 81, wherein at least one of R63, R64, R65, R66, R67, R68, R69 and R70 is a linker bond to a fluorophore, and at least one of R63, R64, R65, R66, R67, R68, R69 and R70 is a linker bond to a probe.

[0230] 83. The method according to aspect 81 or 82, wherein the linkage is an amide, thiol, succinimide ester, maleimide, azide, carboxylic acid ester, carboxyl / EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), sulfonyl-SMCC, BMPH, sulfonyl-SBED, trans-cyclooctene / tetraazine, and amine / epoxide.

[0231] 84. The compound according to any one of aspects 70-83, wherein the fluorophore is selected from fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy-chromium, phycoerythrin, PerCP (cyclophytic chlorophyll a protein), PerCP-Cy5.5, JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, fluorescein yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY® FL, BODIPY® FL-Br2, BODIPY® 530 / 550, BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 576 / 589, BODIPY®581 / 591, BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, SPK dyes, cf514, DY405, DY396XL, cf570, cf405, their conjugates and combinations thereof.

[0232] 85. The method according to any one of aspects 70-84, wherein the fluorophore is any one of the compounds of aspects 1-30.

[0233] 86. The method according to any one of aspects 70-85, wherein the dendritic polymer composition is any one of the compounds of aspects 31-38.

[0234] 87. The method according to any one of aspects 70-86, wherein the targeted dendritic polymer composition is any one of the compounds of aspects 39-48.

[0235] The above description of specific aspects is merely exemplary in nature and is in no way intended to limit the scope of this disclosure or any related invention, its application, or use; of course, such scope can vary. Compositions or methods are described relative to the non-limiting definitions and terms included herein. These definitions and terms are not intended to limit the scope or practice of any invention disclosed herein, but are presented solely for illustrative and descriptive purposes. While the methods or compositions are described as a sequence of individual steps or the use of specific materials, it should be understood that the steps or materials can be interchangeable, such that the specification may include multiple portions or steps arranged in many ways readily understood by those skilled in the art.

[0236] The use of the singular includes the use of the plural, and vice versa (e.g., an electron-conducting conjugate bond comprises multiple electron-conducting conjugate bonds). A group named R typically has a structure that is considered in the art to correspond to an R group having that name. These definitions are intended to be supplementary and illustrative, and do not exclude definitions known to those skilled in the art.

[0237] The terminology used herein is for descriptive purposes only and is not intended to be limiting. As used herein, the singular forms “a” and “the” are also intended to include the plural forms, including “at least one”, unless the context clearly indicates otherwise. “Or” means “and / or”. As used in the text, the term “and / or” includes any and all combinations of one or more of the associated listed items. It will also be understood that, when used in this specification, the terms “comprises and / or comprising” or “includes and / or including” specify the presence of the stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof. The term “or a combination thereof” means a combination that includes at least one of the foregoing elements.

[0238] Unless otherwise defined, all terms used herein (including technical and scientific terms) shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. It should also be understood that terms such as those defined in common dictionaries shall be interpreted as having meanings consistent with their meanings in the relevant field and in the context of this disclosure, and shall not be interpreted as having idealized or overly formal meanings, unless expressly defined herein.

[0239] Any patents or publications mentioned in this specification represent the level of expertise of a person skilled in the art.

[0240] These patents and publications are incorporated herein by reference to the same extent that each individual publication is specifically and individually indicated to be fully incorporated by reference.

[0241] Those skilled in the art will readily understand that this disclosure is highly suitable for achieving the stated objectives and advantages, as well as those inherent therein. The embodiments of the invention and the methods, procedures, treatments, molecules, and specific compounds described herein represent specific aspects and are exemplary, and are not intended to be limiting of the scope of any invention, which should be defined solely by the foregoing claims. Clearly, other aspects exist, and these aspects are included within the spirit of any invention as defined by the scope of the claims.

Claims

1. A compound comprising; One or more A groups are connected to one or more B groups via electronically conducted conjugated bonds, wherein the electronically conducted conjugated bonds are configured to delocalize one or more electrons between A and B; Where A is an electron acceptor group, an electron donor group, or a fluorophore; Where B is an electron acceptor group, an electron donor group, or a fluorophore; and The compound therein does not contain repeating units of A, B or a combination thereof.

2. The compound according to claim 1, wherein each conjugated bond is independently a group of n bond units selected from single bonds, triple bonds, C=N groups, amide bonds, click-reactive bonds, benzene ring bonds, heterocyclic bonds, and combinations thereof; and Where n≥1.

3. The compound according to claim 1, further comprising one or more water-soluble groups connected to A, B, or both.

4. The compound according to any one of claims 1-3, wherein the compound has maximum absorption in a wavelength range of 300 nm to 450 nm, optionally in a wavelength range of 365 nm to 830 nm.

5. The compound according to any one of claims 1-3, wherein the compound has maximum emission in the wavelength range of 365 nm and 830 nm.

6. The compound according to any one of claims 1-3, wherein A has maximum absorption in wavelength ranges of 300 nm and 450 nm, optionally 300 nm and 600 nm, when not conjugated with B.

7. The compound according to any one of claims 1-3, wherein the compound has a structure comprising or consisting of A groups linked to 1 to 10 B groups, optionally having the following formula: 。 8. The compound according to any one of claims 1-3, wherein the compound comprises or is composed of B linked to 1 to 10 A groups, optionally having a structure of the following formula: 。 9. The compound according to any one of claims 1-3, wherein B has maximum absorption in the wavelength ranges of 150 nm and 450 nm when not conjugated with A.

10. The compound according to any one of claims 1-3, wherein B has maximum emission in the wavelength range of 150 nm and 830 nm when not conjugated with A.

11. The compound according to any one of claims 1-3, wherein one or more water-soluble groups are connected to A, and one or more water-soluble groups are connected to B.

12. The compound of claim 11, wherein the one or more water-soluble groups comprise one or more ethylene units linked by oxygen.

13. The compound according to any one of claims 1-3, wherein the molar extinction coefficient of the compound, as measured at maximum emission, is greater than 5000 cm⁻¹. -1 M -1 .

14. The compound according to any one of claims 1-3, wherein A is a structure selected from: in: R1, R2, R7, R8, R9, R13, R14, R15, R19, R20, R23, R24, R29, R30, R52, and R53 are independently selected from the bonds connecting to B that form the ring structure, (C1-C 20 )-hydrocarbon group, (C1-C 20 )-heterohydrocarbon groups and their combinations; At least one of R1 and R2 is a connection key to B; At least one of R7, R8 and R9 is a connection key to B; At least one of R13, R14 and R15 is a connection key to B; At least one of R19 and R20 is a connection key to B; At least one of R23 and R24 is a connection key to B; At least one of R29 and R30 is a connection key to B; At least one of R52 and R53 is a connection key to B; One or more of R3, R4, R5, R6, R10, R11, R12, R16, R17, R18, R21, R22, R25, R26, R27, R28, R31, R33, R35, R37, R39, R40, R42, R44, R46, R47, R48, R50, R51, R54, and R55 may be independently H, (C1-C 50 )- Heterohydrin groups or fused to form optional rings including R54, R55, or both; or R32, R34, R36, R38, R41, R43, R45, and R49 are connection keys to B; and Where X is C, Si, O, S, P, N, Se, or Te.

15. The compound according to claim 14, wherein R3, R4, R5, R6, R10, R11, R12, R16, R17, R18, R21, R22, R25, R26, R27, R28, R31, R33, R35, R37, R39, R40, R42, R44, R46, R47, R48, R50, R51, R54, and R55 are independently water-soluble linking groups, optionally Where n is an integer from 5 to 15, m is an integer from 1 to 5, and R71 is independently selected from H and the connection bond with the second structure, which is optionally a dendritic polymer or a probe.

16. The compound according to any one of claims 1-3, wherein B is a structure selected from: in: R56, R58, and R60 are connection keys to A; as well as R57, R59, R61, and R62 are independently water-soluble groups, optionally (C1-C2). 50 ) heterohydrocarbon group.

17. The compound of claim 1, wherein the compound comprises: Each n is independently selected from an integer between 5 and 15, and optionally between 10 and 11.

18. The compound according to any one of claims 1-3, further comprising a probe.

19. The compound of claim 18, wherein the probe molecule is a protein or a fragment thereof, including but not limited to an antibody or a fragment thereof, nucleic acid, phospholipid, polysaccharide, triglyceride, aptamer, avidin, streptavidin, neutral avidin, avidin DN, avidin D, or cell.

20. The compound of claim 18, wherein the F / P ratio of the composition is 5 to 100, optionally 10 to 30, wherein the F / P ratio is defined as the total number of molecules of the compound present in the composition divided by the total number of molecules of the probe present in the composition.

21. The compound according to any one of claims 1-3, wherein A, B, or both are linked to one or more dendritic polymer structures, the average molecular weight of said dendritic polymer structures optionally being from 5 kilodaltons (kDa) to 100 kDa, optionally from 5 kDa to 30 kDa.

22. The compound of claim 21, wherein the dendritic polymer comprises polyethylene glycol chains.

23. The compound of claim 21, wherein the dendritic polymer comprises one or more branches, wherein one or more of the branches terminate at the functional group.

24. The compound of claim 21, wherein the dendritic polymer comprises a compound of the following formula: in: Each n is an independent integer from 10 to 300; R63, R64, R65, R66, R67, R68, R69, and R70 are independently selected from amines, azides, linkages to A or B, linkages to a probe, and one or more other dendritic polymers, optionally from 1 to 10 other dendritic polymer structures.

25. The compound of claim 24, wherein at least one of R63, R64, R65, R66, R67, R68, R69 and R70 is a bonding bond to A or B, and at least one of R63, R64, R65, R66, R67, R68, R69 and R70 is a bonding bond to a probe.

26. A method for detecting the presence or absence of an analyte in a sample or organism, the method comprising: (a) Contact the sample with a composition comprising a dendritic polymer structure and a compound of any one of claims 1-3; (b) Expose the sample to light of a wavelength capable of exciting the compound; as well as (c) Detect the presence or absence of light emitted by the compound.

27. The method of claim 26, wherein the detection is performed by flow cytometry, FISH, immunohistochemistry, immunofluorescence microscopy, immunocytochemistry, live cell imaging, in vivo imaging (optionally using a long-wavelength emitter), direct or indirect ELISA, Southern blotting, Western blotting, microarray, or substrate binding assay.

28. A method for forming a targeted dendritic polymer composition, the method comprising: (a) Under conditions in which the compound is bonded to the dendritic polymer, the dendritic polymer is contacted with the compound of any one of claims 1-3 to form a dendritic polymer composition; and (b) Under conditions in which the dendritic composition is bound to the probe, the dendritic composition is brought into contact with the probe to form a targeted dendritic composition.

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