Water-soluble π-conjugated fluorescent 1,1'-binaphthyl-based tandem polymers

Abstract

To provide dyes which can be tuned to a desired excitation energy by choosing a modifying unit (MU).SOLUTION: The invention is directed to a conjugate having the general formula (I) in the figure. In the formula, AR, MU and MU* are repeating units of a polymer, where MU and MU* are polymer modifying units or band gap modifying units which are evenly or randomly distributed along the polymer main chain; G1 and G2 represent hydrogen, halogen or an antigen recognizing moiety, with the proviso that at least one of G1 and G2 is an antigen recognizing moiety.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to water-soluble fluorescent polymer dyes, as well as their use in conjugates with antibodies for cell detection and analysis.

[0002] Background of the Invention Immunofluorescence labeling is crucial in both research and clinical applications for detailed analysis and specific isolation of target cells from biological samples. These method workflows typically utilize fluorescent tags bound to target molecules, which are then used to identify the desired analytes in the biological sample. Both sensitivity to analyte detection and the ability to multiplex multiple analytes simultaneously are highly desirable features in cell analysis workflows. While analytical sensitivity largely depends on the brightness of the fluorescent tags, the ability to multiplex multiple analytes simultaneously depends on the optical differentiation between different fluorescent tags. Water-soluble π-conjugated polymers offer the potential to provide solutions for both sensitivity and multiplexed analysis.

[0003] π-conjugated polymers as fluorescent probes have become a cornerstone of the toolbox for the fluorescence detection of biologically relevant analytes. Due to their strong interactions and light-absorbing ability, fluorescent polymers, coupled with their bright fluorescence intensity, offer enhanced sensitivity to analytes compared to previous techniques. Current trends in the detection of biological analytes are increasing the demand for fluorescent systems with color-tunable properties, enabling the detection of multiplexed analytes using individual fluorescence colors. Therefore, to be useful in applications detecting biological analytes, fluorescent systems must have a controllable design, allowing both absorption and emission to be separately tuned to desired excitation and emission levels. No fluorescent system currently exists that combines highly tunable excitation energy and separately highly tunable emission energy within the same system, enabling highly sensitive multiplexed detection of biomolecular analytes.

[0004] An ideal π-conjugated polymer suitable for this challenge inherently possesses a high-energy bandgap that allows excitation by ultraviolet (UV) light. Simultaneously, by using bandgap-changing units, it should be easily possible to shift the excitation energy to red across the entire visible spectrum. Furthermore, the fluorescence emission energy of such a polymer should be independently tunable by incorporating fluorescence energy acceptor dyes. These acceptor dyes can be used to channel energy from the polymer backbone and generate fluorescence at specific wavelengths for detection in a color-tunable format. In this configuration, the π-conjugated polymer would have the ability to independently control the excitation wavelength to overlap with common laser excitations (e.g., lasers in the 355 nm, 405 nm, 488 nm, 561 nm, or 640 nm lines) while simultaneously independently tuning the fluorescence emission wavelength. This color-tunable π-conjugated polymer system would be extremely useful for the detection of multiplexed biomolecular analytes.

[0005] Such water-soluble π-conjugated polymers are described, for example, in “Synthesis of Binaphthyl-Oligothiophene Copolymers with Emissions of Different Colors: Systematically Tuning the Photoluminescence of Conjugated Polymers” by KY Musick, Q.-S. Hu, L. Pu, Macromolecules 1998, 31, 2933-2942.

[0006] Furthermore, U.S. Patent Publication No. 20180009990 and U.S. Patent No. 10604657 describe 4,4'-polymerized binaphthyl substituted with PEG at the 2,2' position. This is sp 3This invention refers to binaphthyl-based polymers in which 4,4' bonds are attached to adjacent monomers via polyparaethynilline (PPE) bonds utilizing hybrid orbitals. The binaphthyl-based polymers disclosed in this invention are directly bonded to adjacent comonomers without ethynilline bonds, and as a result sp 2 Hybridized and fully aromatic conjugated systems are obtained. Water-solvable polymer dyes and polymer tandem dyes are provided. Polymer dyes include water-solvable light-harvesting multiple chromophores having conjugated segments of aryl or heteroaryl comonomers linked via covalent bonds, vinylene groups, or ethynylene groups. Polymer tandem dyes further include signal transduction chromophores covalently bonded to a nearby multiple chromophore that accepts its energy.

[0007] U.S. Patent No. 1,0481,161 discloses 4,4'-polymerized binaphthyl substituted with substituents at the 2,2' position. Water-solvable polymer dyes and polymer tandem dyes are provided. Polymer dyes include water-solvable light-harvesting multiple chromophores having a conjugated segment of aryl or heteroaryl comonomer linked via covalent bonds, vinylene groups, or ethynylene groups. Polymer tandem dyes further include a signaling chromophore covalently bonded to a nearby multiple chromophore that accepts its energy.

[0008] The object of the present invention was to provide a dye that can be adjusted to a desired excitation energy by selecting denaturing units (MUs).

[0009] Summary of the Invention Figure 1 shows a scheme illustrating a method for separately adjusting the excitation energy by the selection of denaturing units (MUs) and the emission energy by the selection of receptor dyes using a polybinaphthyl comonomer tandem system. In this method, the present invention can be used to create a color-adjustable state of a substance in which excitation and emission can be controlled separately. Black boxes represent impossible combinations. b) A schematic representation of a copolymer binaphthyl tandem system bound to a biological site (G2), in which the excitation energy of the polymer can be adjusted by the selection of comonomers (x), the emission energy of the system can be adjusted by the selection of tandem dyes (y), and the efficiency of energy transfer from the polymer backbone to the receptor dye can be adjusted by the selection of binaphthyl monomers (z) with different bonds between monomers.

[0010] Therefore, the object of the present invention is the general formula (I) [ka] [In the formula, AR(Ar), MU, and MU * is a repeating unit of polymers, MU and MU * These are polymer modification units or band gap alteration units that are uniformly or randomly distributed along the polymer backbone. G1 and G2 represent hydrogen, halogen, or antigen recognition sites, provided that at least one of G1 or G2 is an antigen recognition site. a is 10-100 mol%, b is 0-90 mol%, c is 0.1 to 90 mol%, preferably 1 to 80 mol%, more preferably 5 to 70 mol%, and most preferably 10 to 60 mol%, however, Assuming that a+b+c=100mol%, d is between 1 and 10000. In a conjugate having, The conjugate is characterized in that AR is bonded to the polymer chain via the 2, 2' or 3, 3' or 5, 5' or 6, 6' or 7, 7' or 8, 8' position according to the general formula (II). [Chemical formula] [In the formula, the remaining positions 2, 2'; 3, 3'; 4, 4'; 5, 5'; 6, 6'; 7, 7' and 8, 8' are H, SO2CF3, SO2R a , CF3, CCl3, CN, SO3H, NO2, NR a R b R c + , CHO, COR a , CO2R a , COCl, CONR a R b , F, Cl, Br, I, R a , OR a , SR a , OCOR a , NR a R b , NHCOR a , CCR a , aryl-, heteroaryl-, C6H4OR a or C6H4NR a R b substituted with the same or different residues selected from the group consisting of, R a~c is independently hydrogen, alkyl-, alkenyl-, alkynyl-, heteroalkyl-, aryl-, heteroaryl-, cycloalkyl-, alkylcycloalkyl-, heteroalkylcycloalkyl-, heterocycloalkyl-, aralkyl-, or heteroaralkyl residue, or (CH2) x (OCH2CH2) y O(CH2) z CH3, x is an integer from 0 to 20, y is an integer from 0 to 50, and z is an integer from 0 to 20.]

[0011] As can be seen from Equation II, two positions from 2,2'; 3,3'; 4,4'; 5,5'; 6,6'; 7,7' and 8,8' are required to bind the AR to the polymer chain. Therefore, the term “remaining positions” refers to the 12 positions of the AR in the polymer chain that are not used for AR binding and are available for substitution with the disclosed residues. Possible substitution patterns are shown in Figure 5. The residues used for substitution may be the same or different at each position (for example, positions 2 and 2' may have the same or different residues).

[0012] Optionally, the remaining two residues can form a common cycloalkyl or heterocycloalkyl ring system.

[0013] The phrase "ARs are bonded to the polymer chain via the 2,2' or 3,3' or 5,5' or 6,6' or 7,7' or 8,8' positions" refers to CC bonds between each carbon atom at an AR position and carbon atoms in other AR units, G1 units, or L units.

[0014] A further object of the present invention is a method for detecting a target site in a sample of a biological specimen using at least one conjugate disclosed herein, a) A step of contacting a biological sample with at least one conjugate, thereby labeling the target site recognized by the antigen recognition site with the conjugate. b) A step of exciting the labeled target site with light having a wavelength in the absorbance spectrum of the conjugate, c) A step of detecting a labeled target site by detecting the fluorescence emission emitted by the conjugate, The method is characterized by including [a certain element].

[0015] A further object of the present invention is the use of methods in fluorescence microscopy, flow cytometry, fluorescence spectroscopy, cell isolation, pathology, or histology. [Brief explanation of the drawing]

[0016] [Figure 1] This figure shows a scheme demonstrating a method in which the excitation energy can be separately adjusted by selecting the denaturing units (MUs) using a polyvinaphthyl comonomer tandem system. [Figure 2] This figure shows an example of a tandem binaphthyl comonomer polymer system bound to a target site. Depending on the selected bond between the binaphthyl monomers, the polymer can transition from a predominantly linear structure to a predominantly helical structure, thereby allowing for adjustment of the energy transfer efficiency from the polymer system donor to the receptor dye. [Figure 3] This figure shows examples of the use of multiple binaphthyl tandem dye systems of different colors, conjugated to different target sites, in combination with biological samples for the detection of multiplexed analytes. [Figure 4a] This figure shows the fluorescence resonance energy transfer from these compounds according to the embodiments of the present invention. [Figure 4b] This figure shows the fluorescence resonance energy transfer from these compounds according to the embodiments of the present invention. [Figure 4c] This figure shows the fluorescence resonance energy transfer from these compounds according to the embodiments of the present invention. [Figure 4d] This figure shows the fluorescence resonance energy transfer from these compounds according to the embodiments of the present invention. [Figure 4e] This figure shows the fluorescence resonance energy transfer from these compounds according to the embodiments of the present invention. [Figure 4f] This figure shows the fluorescence resonance energy transfer from these compounds according to the embodiments of the present invention. [Figure 5] This figure shows the performance of the polymer-based dye monoclonal antibody (anti-CD19) conjugate according to the present invention in flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) excited at 355 nm.

[0017] Detailed explanation General structure of a binaphthyl (BNP)-based water-soluble fluorescent π-conjugated polymer dye represented by formula (I), having both a tunable absorption band and a tunable emission wavelength. MU is a modification unit for tuning the absorption band. Ar is an aryl unit based on 1,1'-binaphthyl. L is an optional linker, and G1 and G2 are both terminal sites. FL is the fluorescent dye for color-tunable emission. MU * This is a modified unit having a chemoselective functional group that can be used to selectively add fluorescent dyes for color-adjustable emission.

[0018] In a modified version of the present invention, at least two positions 2,2';3,3';4,4';5,5';6,6';7,7' and 8,8' are given by General Formula (IV) [ka] [In the formula, n = 5 to 15] It is substituted with a residue according to [the specified formula].

[0019] The conjugate of the present invention is preferably water-soluble.

[0020] MU and MU as bandgap changing units * MU and MU * This is a bandgap-shifting unit that shifts the absorption band of a conjugate.

[0021] MU * MU is a bandgap-changing unit similar to or identical to MU, but can be bonded to a chemoselective site via any modifiable atom not used in polymerization. Chemoselective sites include, but are not limited to, amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazine azides, alkynes, aldehydes, thiols, thiocyanates, and their protected groups for conjugate to fluorescent dyes and for variability of fluorescence emission.

[0022] To shift the absorption band of the conjugate, MU and MU * Each comprises the same or different aromatic systems having at least one benzene ring or thiophene ring that provides a conjugated π system, which is optionally substituted with electron-withdrawing residues, electron-donating residues, sterically hindering groups, and / or residues that enhance water solubility.

[0023] More preferably, MU and MU * Each comprises the same or different aromatic systems having at least two condensed, cyclized, or conjugated benzene or thiophene rings, which provide a conjugated π system that is optionally substituted with electron-withdrawing residues, electron-donating residues, sterically hindering groups, and / or residues that enhance water solubility.

[0024] One or more residues, such as alkyl or ether (glycol) residues, or halogen atoms or alkyl halides, can be used as electron-withdrawing residues, electron-donating residues, sterically hindered groups, and / or residues that enhance water solubility.

[0025] MU and MU * The repeating units may be uniformly or randomly distributed in the conjugate of the present invention. To that extent, MU and MU * It functions not only as a bandgap-changing unit, but also as a polymer-modifying unit.

[0026] MU and MU * The preferred residues (optionally substituted as disclosed) are as follows: [ka] [ka] [ka]

[0027] Terminal groups G1 and G2 G1 and G2 are both independently selected from the group of hydrogen, halogen, or antigen recognition sites. Antigen recognition sites can be selected from antibodies, fragmented antibodies, fragmented antibody derivatives, peptide / MHC complexes, cell adhesion or costimulatory molecule receptors, receptor ligands, antigens, hapten conjugates, avidin, streptavidin, aptamers, primers and ligase substrates, peptide / MHC complexes targeting TCR molecules, cell adhesion receptor molecules, costimulatory molecule receptors, or artificially designed binding molecules.

[0028] Fluorescent site FL Suitable fluorescent sites (FLs) are those known in the field of immunofluorescence techniques, such as flow cytometry or fluorescence microscopy. In the method of the present invention, the conjugate-labeled target site is detected by exciting the polymer backbone, the fluorescent site (FL), or both, and detecting the light emission (photoluminescence) obtained from the FL or polymer.

[0029] Useful fluorescent sites (FL) may be protein systems, such as phycobiliproteins; small organic dyes, such as xanthenes like fluorescein; or rhodamine, cyanine, oxazine, coumarin, acridine, oxadiazole, pyrene, pyromethene, pyridyloxazole; or organometallic complexes, such as Ru, Eu, and Pt complexes. In addition to single molecules, clusters of fluorescent proteins or small organic dyes, as well as nanoparticles such as quantum dots, upconverting nanoparticles, gold nanoparticles, and dye polymer nanoparticles, can also be used as fluorescent sites.

[0030] In a modified version of the present invention, the fluorophore FL is substituted with one or more water-soluble substituents selected from the group consisting of sulfonates, phosphonates, phosphates, polyethers, sulfonamides, and carbonates. It is particularly advantageous to use fluorescent moieties having sulfonate substituents, such as dyes from the Alexa Fluor family supplied by Thermo Fisher Scientific Inc. The degree of sulfonate substitution per fluorophore may be 2 or more, i.e., in the case of rhodamine-based or cyanine-based dyes.

[0031] Suitable commercially available fluorescent sites can be purchased from Miltenyi Biotec BV & Co. KG's "Vio" product line, or Thermofisher's FITC, Promofluor, or Alexa dyes and / or Bodipy dyes, or Lumiprobe's cyanines, or Dyomics GmbH's DY® fluorophores, or Aberior GmbH's Star dyes.

[0032] Linker base L L is a linker located at the end of the polymer backbone, consisting of an aryl or heteroaryl group, and is substituted with one or more pendant chains terminated with i) a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazine, azide, alkyne, aldehyde, thiol, and their protected groups for conjugate to molecules or biomolecules, or ii) an organic dye conjugated as an acceptor dye, or iii) a biomolecule.

[0033] Preferably, L is one of the following residues: [ka] [ka] One or more of the following will be selected.

[0034] antigen recognition site The term “antigen recognition site” refers to any type of antibody, fragmented antibody, or fragmented antibody derivative directed toward a target site expressed in a biological sample, such as an antigen expressed intracellularly or extracellularly. This term relates to fully intact antibodies, fragmented antibodies, or fragmented antibody derivatives, e.g., Fab, Fab', F(ab')2, sdAb, scFv, di-scFv, and nanobodies. Such fragmented antibody derivatives can be synthesized by recombinant procedures involving covalent and non-covalent conjugates containing these types of molecules. Further examples of antigen recognition sites include peptide / MHC complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for costimulatory molecules, and artificially engineered binding molecules, e.g., peptides or aptamers targeting cell surface molecules.

[0035] The conjugate used in the method of the present invention may contain up to 100, preferably 1 to 20, antigen recognition sites Y. The interaction between the antigen recognition site and the target antigen may be high affinity or low affinity. The binding interaction of a single low affinity antigen recognition site is too small to obtain stable binding to the antigen. High binding activity can be obtained by polymerizing the low affinity antigen recognition site through conjugation to an enzymatically degradable spacer. When the spacer is enzymatically degraded, the low affinity antigen recognition site monomerizes, and the fluorescent marker is completely removed.

[0036] Preferably, the term “antigen recognition site” refers to antibodies against antigens expressed intracellularly, such as IL2, FoxP3, and CD154, or extracellularly, such as CD19, CD3, CD14, CD4, CD25, CD34, CD56, and CD133, in biological samples (target cells). Antigen recognition sites G1 and G2, particularly antibodies, can bind to CP via side-chain amino or sulfhydryl groups. In some cases, the glycoside side chain of the antibody may be oxidized by periodic acid to produce an aldehyde functional group.

[0037] The antigen recognition site can be conjugated covalently or non-covalently. Methods of covalent or non-covalent conjugation are known to those skilled in the art and are the same as those described for the conjugation of fluorescent markers.

[0038] The method of the present invention is particularly useful for detecting and / or isolating specific cell types from complex mixtures and may comprise multiple sequential steps a) to c). This method can utilize various combinations of conjugates. For example, a conjugate may comprise antibodies specific to two different epitopes, such as two different anti-CD34 antibodies. Different antigens can be addressed with different conjugates containing different antibodies, for example, anti-CD4 and anti-CD8 for differentiation between two different T cell populations, or anti-CD4 and anti-CD25 for determining different cell subpopulations such as regulatory T cells.

[0039] Cell detection method A conjugate-labeled target is detected by exciting either the fluorescent site (FL) or the skeletal structure (CP) and analyzing the resulting fluorescence signal. The excitation wavelength is typically selected according to the absorption maximum of the fluorescent site (FL) or CP, and is provided by a laser or LED source known in the art. When multiple different detection sites (FL) are used for multiple color / parameter detection, care should be taken to select fluorescent sites whose absorption spectra do not overlap, or at least whose absorption maxima do not overlap. In the case of a fluorescent site, the target can be detected, for example, by a fluorescence microscope, flow cytometer, spectrofluorometer, or fluorescence scanner. Light emitted by chemiluminescence can be detected by similar instruments without excitation.

[0040] target site The target sites detected by the method of the present invention may be located on any biological sample, such as tissue sections, cell aggregates, suspension cells, or adherent cells. The cells may be alive or dead. Preferably, the target sites are antigens expressed intracellularly or extracellularly in biological samples such as whole animals, organs, tissue sections, cell aggregates, or single cells of invertebrates (e.g., nematodes (Caenorhabditis elegans), fruit flies (Drosophila melanogaster)), vertebrates (e.g., zebrafish (Danio rerio), African clawed frogs (Xenopus laevis)), and mammals (e.g., mice (Mus musculus), humans (Homo sapiens)).

[0041] Method of Use The method of the present invention can be used in a variety of applications in research, diagnosis, and cell therapy, such as in fluorescence microscopy, flow cytometry, fluorescence spectroscopy, cell isolation, pathology, or histology.

[0042] In a first variant of the present invention, a biological sample, such as cells, is detected for the purpose of counting, i.e., determining the amount of cells from a sample having a specific set of antigens recognized by the antigen recognition site of the conjugate. In another variant, the biological sample detected by the conjugate in step c) is separated from the sample by optical, electrostatic, pneumatic, mechanical, or acoustic means.

[0043] In another variant of the present invention, the location of a target site, such as an antigen on a biological sample, is determined by the antigen recognition site of the conjugate. Such techniques are known as "Multi Epitope Ligand Cartography," "Chip-based Cytometry," or "Multiomyx," and are described, for example, in European Patent No. 0810428, European Patent No. 1181525, European Patent No. 1136822, or European Patent No. 1224472. In this technique, cells are immobilized and contacted with an antibody bound to a fluorescent site. The antibody is recognized by each antigen on the biological sample (e.g., the cell surface), and after the unbound marker is removed and the fluorescent site is excited, the location of the antigen is detected by the fluorescence emission of the fluorescent site. In certain variants, instead of an antibody bound to a fluorescent site, an antibody bound to a site detectable by MALDI-Imaging or CyTOF can be used. Those skilled in the art are aware of methods for modifying techniques based on fluorescent sites to handle these detection sites.

[0044] The target site is positioned using a digital imaging device with sufficient resolution and sensitivity to the wavelength of fluorescence emission. This digital imaging device can be used, for example, with a fluorescence microscope, with or without optical magnification. The resulting images are saved in RAW, TIF, JPEG, or HDF5 format to a suitable storage device such as a hard drive.

[0045] To detect different antigens, different antibody conjugates having the same or different fluorescence sites or antigen recognition sites can be provided. Because parallel detection of fluorescence emission at different wavelengths is limited, antibody-fluorescent dye conjugates are used separately or sequentially in small groups (2-10).

[0046] In yet another variation of the method according to the present invention, a biological sample, particularly a sample of suspended cells, is immobilized by trapping it within a microcavity or by adhesion.

[0047] Typically, the method of the present invention can be carried out in several modified forms. For example, conjugates that are not recognized by the target site can be removed by washing with a buffer, for example, before the target site labeled with the conjugate is detected.

[0048] In a modified version of the present invention, at least two conjugates are provided simultaneously or in a subsequent staining sequence, with each antigen-recognizing site recognizing a different antigen. In another modified version, at least two conjugates may be provided to the sample simultaneously or in a subsequent staining sequence. In either case, labeled target sites can be detected simultaneously or sequentially. [Examples]

[0049] As an example, the following compounds were synthesized using different receptor dyes FL in the following general method and conjugated to the antigen recognition portion. [ka] [Table 1]

[0050] The table shows physical data for various polymer-receptor dye tandem systems. The poly(binaphthyl-co-phenylene-NH2) polymer shown above was conjugated to various receptor dyes FL, and its fluorescence emission properties were measured to determine emission wavelength (lem), fluorescence quantum yield (QY), and energy transfer efficiency (I). donor / I acceptor The following was determined. All measurements were performed by exciting the binaphthyl polymer at 325 nm.

[0051] The receptor dyes used were known sulfocyanines and Star Red dyes. Figures 4a-f show fluorescence resonance energy transfer from these compounds. Emissions from the binaphthyl residue donors are centered at approximately 400 nm in each plot. Fluorescence emission due to energy transfer via the receptor dyes is as follows: a) fluorescein (approx. 530 nm), b) Sulfo-Cy3 (approx. 576 nm), c) Sulfo-Cy3.5 (approx. 605 nm), d) Sulfo-Cy5.5 (approx. 706 nm), e) Sulfo-Cy7 (approx. 788 nm), and f) Star Red (approx. 662 nm). The sharp emission features centered around 650 nm in graphs c, d, e, and f are artifacts of the emission measurement and not actual characteristics of the tandem polymer fluorescence.

[0052] Figure 5 shows the performance tests of polymer dye monoclonal antibody (anti-CD19) conjugates in flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) excited at 355 nm. For clarity, only CD14-negative cells are shown to exclude monocytes. A) BNP-sCy3 conjugate detected with a 585 / 42 nm emission filter; B) BNP-sCy3.3 conjugate detected with a 610 / 20 nm emission filter; C) BNP-Star Red conjugate detected with a 670 / 30 nm emission filter; D) BNP-sCy5.5 conjugate detected with a 710 / 30 nm emission filter; E) BNP-sCy7 conjugate detected with a 780 / 60 nm emission filter.

[0053] [General synthesis] [Synthesis of Building Block M1] [ka]

[0054] To a stirred solution of 6,6'-dibromo-[1,1'-binaphthalene]-2,2'-diol (3.00 g, 6 mmol) in acetone (42 mL), 12.6 g, 20 mmol of 34-iodo-2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontane (12.6 g, 20 mmol), followed by K2CO3 (2.82 g, 20 mol), the resulting reaction mixture was stirred at 70°C for 16 hours. The reaction mixture was filtered, concentrated under reduced pressure, and dried. The resulting crude substance was diluted with water (20 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting crude residue was purified by silica column chromatography eluting with methanol / dichloromethane (5:95) to obtain a pale yellow liquid. This compound was further purified by reverse-phase chromatography using acetonitrile / water (50:50) elution. By freeze-drying the compound, pure compound M1 (1.7 g, 17.6%) was obtained as a pale yellow liquid.

[0055] [Synthesis of Building Block M2] [ka]

[0056] Potassium carbonate (2.74 g, 19.9 mmol) was added to a solution of 2,5-dibromophenol (1.00 g, 3.97 mmol) in dimethylformamide (40 mL). 4-(N-Boc-amino)butyl bromide (1.2 g, 4.8 mmol, 1.2 equivalents) was added, and the solution was heated at 80°C for 72 hours. The solvent was removed under reduced pressure, the residue was suspended in dichloromethane (50 mL), and washed with water (3 × 50 mL). The organic phase was dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain a white solid (1.49 g). The crude product was purified by silica flash column chromatography using a gradient of 0% to 50% by volume ethyl acetate in n-hexane, followed by reverse-phase flash column chromatography using a gradient of 0% to 100% by volume acetonitrile in water. The product was obtained as a white solid (1.46 g, 87%).

[0057] [Synthesis of tert-butyl 4-(4-bromophenyl)butanoate (M3)] [ka]

[0058] 1,1'-carbonyldiimidazole (667 mg, 4.11 mmol) was added to a solution of 4-(4-bromophenyl)butanoic acid (1.00 g, 4.11 mmol) in dimethylformamide (4.1 mL), and the mixture was stirred at 40°C for 1 hour. t-butyl alcohol (610 mg, 772 μL, 8.23 ​​mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (626 mg, 614 μL, 4.11 mol) were added, and the mixture was stirred at 40°C for 24 hours. Diethyl ether (50 mL) was added, and the solution was washed with 10% hydrochloric acid (10 mL), water (10 mL), and 10% aqueous sodium carbonate solution (10 mL). The organic phase was dried over sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica flash column chromatography using a gradient of ethyl acetate from 0% to 10% by volume in n-hexane to obtain the product as a colorless oil (234 mg, 17%).

[0059] [Synthesis of polymer skeletons] [ka] c=0.1, a=0.9, d=27

[0060] Bis(1,5-cyclooctadiene)nickel(0) (117 mg, 424 μmol), 2,2'-bipyridyl (66.0 mg, 424 μmol), and 1,5-cyclooctadiene (46.0 mg, 424 μmol) were dissolved in dimethylformamide (8 ml) and stirred at 70°C for 30 minutes under an argon atmosphere. Monomers M1 (250 mg, 173 μmol), M2 (8 mg, 19 μmol), and M3 (1.1 mg, 3.9 μmol) were dissolved in dimethylformamide (8 mL) under an argon atmosphere and added to the Ni complex-containing solution. The mixture was stirred at 70°C for 3 hours. The solvent was removed under reduced pressure, and the residue was suspended in a 20 vol% aqueous ethanol solution. After centrifugation, the supernatant was purified using size exclusion filtration and subsequently freeze-dried to obtain the polymer as a yellow foam. The polymer was dissolved in dichloromethane (50 mL), and trifluoroacetic acid (5 mL) was added. The solution was stirred at room temperature for 2 hours. Volatile substances were removed under reduced pressure, and the residue was dissolved in a 20 vol% aqueous ethanol solution (100 mL). The solution was purified by size exclusion filtration (cutoff 10 kDa) and freeze-dried to obtain the final product (122 mg, 44%).

[0061] [Synthesis of polymer tandem dyes] [ka] c=10%, a=90%, d=27

[0062] A solution of polymer backbone (5.0 mg) in DMSO (0.33 μL) was mixed with a solution of NHS receptor dye (1.128 μmol) in DMSO (0.1-0.7 mL). N,N-diisopropylethylamine (10.8 μL) was added, and the mixture was stirred overnight at room temperature. The reaction mixture was poured into 20% by volume EtOH (40 mL). The excess receptor dye was removed by ultrafiltration. The polymer was purified by repeating the procedure of dissolving it in 20% by volume EtOH (15 mL) and concentrating it by ultrafiltration until the filtrate was colorless.

[0063] [Amination of tandem dyes] [ka]

[0064] Polymer tandem dye (5.0 mg) was dissolved in 0.50 mL of MES buffer (50 mM, pH 6) containing 20% ​​by volume ethanol. A solution of N-hydroxysuccinimide (2.3 mg, 20 μmol) in 50 mM MES buffer (192 μL) and a solution of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (3.8 mg, 20 μmol) in 50 mM MES buffer (190 μL) were added. The mixture was stirred at room temperature for 20 minutes. Ethylenediamine (1.73 μL, 1.56 mg, 26 μmol) was added, and the mixture was stirred at room temperature for 2 hours. The mixture was diluted with 10 mL of 50 mM MES buffer and concentrated by ultrafiltration. The polymer was purified by dissolving it in 15 mL of 20% by volume EtOH, followed by ultrafiltration and lyophilization.

[0065] [Bioconjugation of polymer tandem dyes to antibodies] Antibodies were reduced at an antibody concentration of 5 mg / mL for 30 minutes using dithiothreitol (50 mM) in MES buffer (pH 6). The reduced antibodies were purified using a Sephadex G-25 column (Cytiva Europe GmbH, Germany) in a gel filtration oven. Antibody-containing fractions were identified by Coomassi staining, and the antibody concentration of the pooled fractions was determined by measuring the absorbance at 280 nm.

[0066] The amino-functionalized polymer was coupled to a reduced antibody using succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) as a heterobifunctional crosslinking agent. Cross-coupling was performed using a commercially available SMCC cross-coupling kit (Thermo Fisher Scientific Inc., USA). Coupling was carried out according to the kit's instructions. The antibody-polymer conjugate was purified by size exclusion chromatography.

[0067] [Cell staining] Using PEB buffer, 1 × 10 per 1 mL 7 The cells were washed by vortexing at a concentration of 1 x 10⁻¹⁶ cells, followed by centrifugation at 300g for 5 minutes. 7 Resuspend the individual cells in 20 μL of PEB buffer and apply 1 × 10⁶ cells per mL for cell staining. 7 Antibody-dye conjugate and PEB buffer were added to reach the desired concentration for each cell. Cells were stained by incubation at 4°C for 10 minutes. The cells were then washed again and resuspended in 1 mL of PEB buffer for flow cytometry analysis. Stained cells were stored at 4°C until analysis. To identify dead cells, propidium iodide solution (titer 1:1000) was added by instrument immediately before measurement.

[0068] [Flow cytometry analysis] Stained cells were analyzed by flow cytometry using an LSR Fortessa (Becton, Dickinson & Company Inc, USA) with five laser excitation sources (355 nm, 405 nm, 488 nm, 561 nm, 640 nm) combined with a 379 / 28 nm bandpass filter. Flow cytometry data analysis was performed using FlowJo (Becton, Dickinson & Company Inc, USA) flow cytometry analysis software.

Claims

1. General formula (I) 【Chemistry 1】 [Wherein AR, MU, and MU ＊ is a repeat unit of the polymer, MU and MU ＊ are polymer modifying or band gap modifying units that are uniformly or randomly distributed along the polymer backbone; G1 and G2 represent hydrogen, halogen, or an antigen recognition site, provided that at least one of G1 or G2 is an antigen recognition site; a is 10 to 100 mol %, b is 0 to 90 mol %, c is 0.1 to 90 mol%, Provided that a+b+c=100 mol %, d is 1 to 10,000. In a conjugate having A conjugate characterized in that AR is attached to the polymer chain via 2,2' or 3,3' or 4,4' or 5,5' or 6,6' or 7,7' or 8,8' positions according to general formula (II). 【Chemistry 2】 wherein the remaining positions 2,2'; 3,3'; 4,4'; 5,5'; 6,6'; 7,7' and 8,8' are H, SO ２ CF ３ , S.O. ２ R ａ , C.F. ３ , CCl ３ , C.N., S.O. ３ H, NO ２ , N.R. ａ R ｂ R ｃ ＋ , CHO, COR ａ , CO ２ R ａ , COCl, CONR ａ R ｂ , F, Cl, Br, I, R ａ , OR ａ , S.R. ａ , O.C.O.R. ａ , N.R. ａ R ｂ , NHCOR ａ , CCR ａ , aryl-, heteroaryl-, C ６ H ４ OR ａ or C ６ H ４ NR ａ R ｂ and R is substituted with the same or different residues selected from the group consisting of ａ～ｃ are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl, or heteroaralkyl residue, or (CH ２ ) ｘ (OCH ２ CH ２ ) ｙ O (CH ２ ) ｚ CH ３ wherein x is an integer from 0 to 20, y is an integer from 0 to 50, and z is an integer from 0 to 20.

2. 2. The conjugate of claim 1, characterized in that it has a predominant chirality with AR being provided predominantly as the R- or S-stereoisomer.

3. 3. The conjugate of claim 1, wherein the antigen recognition site is selected from the group consisting of an antibody, an antibody fragment, an antibody fragment derivative, a peptide / MHC complex, a receptor for a cell adhesion or costimulatory molecule, a receptor ligand, an antigen, a hapten binder, avidin, streptavidin, an aptamer, a primer and a ligase substrate, a peptide / MHC complex targeting a TCR molecule, a cell adhesion receptor molecule, a receptor for a costimulatory molecule, or an artificially designed binding molecule.

4. MU and / or MU ＊ 3. The conjugate according to claim 1 or 2, characterized in that:

5. MU and / or MU ＊ but independently the following residues 【Chemistry 3-1】 【Chemistry 3-2】 3. The conjugate according to claim 1 or 2, characterized in that it is selected from one or more of the following:

6. 3. The conjugate according to claim 1 or 2, characterized in that L is an aryl or heteroaryl group located at the end of the polymer backbone and can be substituted with one or more pendant chains terminating in i) a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazine, azide, alkyne, aldehyde, thiol, and protected groups thereof for conjugation to molecules or biomolecules, or ii) an organic fluorescent dye attached as an energy acceptor in an energy transfer system, or iii) a biomolecule.

7. L is the residue 【Chemistry 4-1】 【Chemistry 4-2】 The conjugate according to claim 6, characterized in that it is selected from one or more of the following:

8. at least two positions 2,2'; 3,3'; 4,4'; 5,5'; 6,6'; 7,7' and 8,8' are of the general formula (III) 【Transformation 5】 [Wherein n=5 to 15] 3. The conjugate according to claim 1 or 2, characterized in that it is substituted with a residue according to

9. 3. The conjugate of claim 1, wherein FL is selected from the group consisting of fluorescein, fluorescein derivatives, rhodamine, tetramethylrhodamine, silicon-rhodamine (SiR), coumarin, resorufin, pyrene, anthracene, phenylene, phthalocyanine, cyanine, xanthene, amidopyrylium fluorophore, oxazine, Quadrain dye, carbopyronine, 7-nitrobenz-2-oxa-1,3-diazole (NBD) fluorophore, BODIPY™ fluorophore (Molecular Probes, Inc.), ALEXA™ fluorophore (Molecular Probes, Inc.), DY™ fluorophore (Dyomics GmbH), benzopyrylium fluorophore, benzopyrylium-polymethine fluorophore, lanthanide chelate, metalloporphyrin, rhodol dye, carborodol dye, naphthalimide, and porphyrin.

10. 10. A method for detecting a target site in a sample of a biological specimen using at least one conjugate according to claim 1 or 2, comprising: d) contacting a sample of said biological specimen with at least one conjugate, thereby labeling said target site recognized by the antigen recognition site with said conjugate; e) exciting the labeled target moiety with light having a wavelength within the absorbance spectrum of the conjugate; f) detecting the labeled target moiety by detecting fluorescent radiation emitted by the conjugate; A method comprising:

11. 3. Use of the conjugate according to claim 1 or 2 in fluorescence microscopy, flow cytometry, fluorescence spectroscopy, cell separation, pathology or histology.

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