Water-soluble π-conjugated fluorescent 1,1'-binaphthyl-based polymers with tunable absorption

Abstract

To provide a conjugate and a detection method for detecting a target moiety in a biological specimen.SOLUTION: A conjugate having the general formula (I) in the figure is applied to analysis. In the formula, AR and MU are repeating units of a polymer; MU is a polymer modifying unit or band gap modifying unit that is evenly or randomly distributed along the polymer main chain; G1 and G2 stand for hydrogen, halogen or an antigen recognizing moiety, with the proviso that at least one of G1 or G2 is an antigen recognizing moiety; a is 10 to 100 mol% and b is 0 to 90 mol%, with the proviso that a+b=100 mol %; and c is 1 to 10000.SELECTED DRAWING: None

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 separation of target cells from biological samples. The sensitivity of the analysis depends primarily on the brightness of the fluorophore. Water-soluble π-conjugated polymers are used as high-brightness fluorophores and are also utilized in biological applications.

[0003] 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.

[0004] 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 3 This 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 2Hybridized 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.

[0005] The polymer dyes disclosed in this manner are expensive to manufacture, and the synthesis of 4,4'-functionalized binaphthyl monomers in particular is cumbersome and costly. Furthermore, special comonomers are required to use these polymer dyes with ultraviolet excitation light, and the resulting polymer dyes are very complex. Therefore, the object of the present invention is to provide a simpler system that offers greater flexibility in adjusting absorption and emission properties toward the UV region of the spectrum, and is also easier and less expensive to manufacture.

[0006] Summary of the Invention π-conjugated polymers based on binaphthyl monomers allow for tuning of absorption wavelengths over a very wide range. The energy of this system is very high due to the unique structure of the binaphthyl monomer, which consists of two twisted naphthyl units. In contrast to prior art polymers based on polyfluorene and polyphenanthrene, this structural feature allows for the design of polymers with absorption in the deep ultraviolet region.

[0007] The energy of the system can be precisely controlled by copolymerization of binaphthyl monomers with appropriate comonomers. Strategically selecting comonomers allows for the ability to tune the polymer's absorption bands from the deep ultraviolet to the visible spectrum.

[0008] By using unique monomers and combining them with specific comonomers, the position of the absorption bands in the spectrum can be controlled to precisely match the desired excitation laser, thereby providing a means to solve technical challenges.

[0009] The use of π-conjugated polymers as fluorescent probes has become a cornerstone of the fluorescence detection toolbox. The absorption band of a useful polymer needs to match the wavelength of the light source, for example, lasers at wavelengths of 355 nm, 405 nm, 488 nm, 561 nm, or 640 nm. However, current polymers have limited light absorption bands, confined to a narrow violet region, thus limiting their usefulness. Novel π-conjugated polymers with tunable absorption bands would expand the repository of water-soluble polymer dyes for biological applications, including the detection of analytes.

[0010] Brief description of the invention Therefore, the object of the present invention is the general formula (I): [ka] [In the formula, AR and MU are repeating units of the polymer, MUs are bandgap-changing 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%, Assuming that a + b = 100 mol%, c is between 1 and 10000. In a conjugate having, AR is given by general formula (II): [ka] [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, CORa, CO2Ra, COCl, CONRaRb, 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 C6H4NRaRb, and is substituted with the same or different residues selected from the group consisting of Ra-c are, independently, hydrogen, alkyl-, alkenyl-, alkynyl-, heteroalkyl-, aryl-, heteroaryl-, cycloalkyl-, alkylcycloalkyl-, heteroalkylcycloalkyl-, heterocycloalkyl-, aralkyl-, or heteroaralkyl residues, or (CH2) x (OCH2CH2) y O(CH2)[[ID=​​​​​​​​​​​​​​In a variant of the present invention, at least two positions 2,2'; 3,3'; 4,4'; 5,5'; 6,6'; 7,7' and 8,8' are of the general formula (IV) [Chemical formula] [where n = 5 to 15] and are substituted with residues according to

[0014] The conjugate of the present invention is preferably water-soluble.

[0015] The term "AR is attached to the polymer chain via the 2,2' or 3,3' or 5,5' or 6,6' or 7,7' or 8,8' position" refers to a C-C bond between each C atom of the position of AR and a C atom of another AR unit, G1 unit, or L unit.

[0016] A further object of the present invention is a method for detecting a target site in a sample of a biological sample using at least one conjugate disclosed herein, comprising[[ID=​​​​​​​​​​​​​​​​​​​ [Figure 2] This figure shows the absorption (solid line) and emission (dashed line) spectra of phenylene copolymer 2 based on water-soluble binaphthyl. [Figure 3] This figure shows the absorption (solid line) and emission (dashed line) spectra of bithiofen copolymer 3 based on water-soluble binaphthyl. [Figure 4] This figure shows flow cytometry analysis of human peripheral blood mononuclear cells (PBMCs) stained with copolymer 2 anti-CD4 and anti-CD19 antibody conjugates using a commercially available flow cytometer. [Figure 5] This figure shows the substitution patterns of AR. [Figure 6] This figure shows the conjugate of the present invention, which includes a polymer portion and an antigen recognition site.

[0019] MU as a unit of bandgap change MU is a bandgap-changing unit that causes a shift in the absorption band of the conjugate. For this purpose, MU comprises an aromatic system having at least one benzene or thiophene ring that provides a conjugated π system, optionally substituted with electron-withdrawing residues, electron-donating residues, sterically hindering groups, and / or residues that enhance water solubility.

[0020] More preferably, MU comprises an aromatic system 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.

[0021] 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.

[0022] The MU repeating units can be uniformly or randomly distributed in the conjugate of the present invention. To that extent, MU functions not only as a bandgap changing unit but also as a polymer modification unit.

[0023] The preferred residues for MU are as follows: [ka] [ka] That is the case.

[0024] 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.

[0025] Linker base L In the first embodiment of the present invention, the MU unit located at the end of the conjugate is given by the following formula (III) [ka] [In the formula, G1, AR, MU, G2, a, b, and c have the same meaning as in formula (I), and L represents a molecule containing an alkyl, aryl, or heteroaryl group that links the polymer to G2.] As shown, they are bonded via a linker group L.

[0026] L is an aryl or heteroaryl group located at the terminal end of the polymer backbone and may be 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 fluorescent dye conjugated as an energy acceptor in an energy transfer system, or iii) a biomolecule.

[0027] Preferably, L is one of the following residues: [ka] [ka] One or more of these will be selected.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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)).

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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 individually or sequentially in small groups (2-10).

[0040] 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.

[0041] 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.

[0042] 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.

[0043] Examples For example, the following water-soluble binaphthyl-based polymers 1-3 [ka] [ka] [ka] The compound was synthesized and given an antigen recognition site at position G1.

[0044] Synthesis of building block M1 [ka]

[0045] 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.

[0046] Synthesis of Building Block M2 [ka]

[0047] A stirred solution of 4-(4-bromophenyl)butanoic acid (2.00 g, 8.22 mmol) in dichloromethane (20 mL) was cooled to 0°C, and N,N-diisopropylethylamine (4.40 mL, 24.7 mmol) was added, followed by the addition of 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.36 g, 12.3 mmol). The mixture was stirred for 10 minutes, and N-hydroxysuccinimide (1.42 g, 12.3 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure, and saturated sodium bicarbonate solution (50 mL) was added. The suspension was filtered, and the residue was dried under reduced pressure to obtain 2.4 g of the crude product as an off-white solid. The solid was washed with ethanol and dried under reduced pressure to obtain the NHS ester as a white solid (1.24 g). NHS ester (2.00 g, 5.89 mmol) was dissolved together with N-Boc-ethylenediamine hydrochloride (2.06 g, 6.48 mmol) in a mixture of tetrahydrofuran (40 mL) and water (20 mL) at 0°C, and sodium bicarbonate (2.47 g, 29.5 mmol) was added. The reaction temperature was slowly raised to room temperature, and the mixture was stirred for 7 hours. The reaction mixture was concentrated under reduced pressure to remove the tetrahydrofuran, then water (100 mL) was added, and the suspension was stirred for 15 minutes. The resulting solid was filtered and dried under reduced pressure to obtain the product (2.0 g) as an off-white solid. Compound M2 was obtained as a white solid (1.15 g, 38%) by recrystallization of the solid with isopropyl alcohol (24 mL).

[0048] Synthesis of Binaphthyl Homopolymer 1 [ka]

[0049] Under an argon atmosphere, bis(1,5-cyclooctadienyl)nickel(0) (105 mg, 382 μmol), 2,2'-bipyridyl (60.0 mg, 382 μmol), and 1,5-cyclooctadiene (41.0 mg, 382 μmol) were dissolved in dimethylformamide (8 mL) and stirred at 70°C for 30 minutes. Compounds M1 (250 mg, 173 μmol) and M2 (1.3 mg, 3.4 μmol) were dissolved in dimethylformamide (4 mL) and added to the reaction mixture. The solution was stirred at 70°C for 3 hours. The solvent was removed by distillation, and the polymer was dissolved in a mixture of dichloromethane (32 mL) and piperidine (8 mL). Volatile substances were removed under reduced pressure, and the residue was suspended in ethanol. After centrifugation, the supernatant was freeze-dried to obtain the product (101 mg, 45%). GPC (270nm): Mn: 7.9kDa, Mw: 9.7kDa, D=1.2.

[0050] Synthesis of Binaphthylbinaphthylphenylene Copolymer 2 [ka]

[0051] Under an argon atmosphere, tetrakis(triphenylphosphine)palladium(0) (10 mg, 8.6 μmol) was added to a mixture of compound M1 (250 mg, 173 μmol) and 1,4-benzenediboronic acid bis(pinacol) ester (57.0 mg, 173 μmol) in dimethylformamide (4 mL). A potassium carbonate aqueous solution (2 M, 750 μL) was added, and the mixture was stirred at 80°C for 3 hours. Compound M2 (52.0 mg, 100 μmol) was added, and the solvent was removed by distillation. The residue was suspended in a 20 vol% ethanol aqueous solution. After centrifugation, the supernatant was freeze-dried to obtain a yellowish-brown polymer. The polymer was purified by size exclusion and then freeze-dried. The polymer was dissolved in a mixture of dichloromethane (32 mL) and piperidine (8 mL). After removing the solvent by distillation, the polymer was dissolved in a 20 vol% aqueous ethanol solution and desalted by size exclusion purification to obtain a pure product (282 mg, 94.4%). GPC (270 nm): Mn=20.6 kDa, Mw=34.2 kDa, D=1.7.

[0052] Synthesis of Binaphthylbithiofen Copolymer 3 [ka]

[0053] A mixture of compound M1 (250 mg, 173 μmol) in dimethylformamide (4 mL) and 2,2'-bithiophene-5,5'-diboronic acid bis(pinacol) ester (67.0 mg, 174 μmol) was mixed with tetrakis(triphenylphosphine)palladium (0) (10 mg, 8.6 μmol, 0.050 equivalents). After adding aqueous potassium carbonate solution (2 M, 750 μL), the solution was stirred under argon at 80°C for 3 hours. The solvent was removed by distillation, and the polymer was dissolved in a mixture of dichloromethane (32 mL) and piperidine (8 mL). Volatile substances were removed under reduced pressure, and the residue was suspended in 20 vol% aqueous ethanol solution. After centrifugation, the supernatant was freeze-dried to obtain a yellowish-brown polymer (114 mg, 46%). GPC (270nm): Mn: 10.7kDa; Mw: 19.1kDa; D=1.8.

[0054] Bioconjugation of polymer 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] Consideration Figure 1 shows the absorption and emission spectra of polyvinaphthyl homopolymer 1. The absorption and emission maxima are at 315 nm and 408 nm, respectively (Table 1).

[0059] As shown in Figure 2, copolymerization of binaphthyl monomer and phenylene monomer (copolymer 2) resulted in a 15 nm deeper shift in the absorption maximum compared to homopolymer 1. The emission maxima of homopolymer 1 and phenylene copolymer 2 are very close (408 nm and 406 nm, respectively; see Table 1).

[0060] As shown in Figure 3, copolymerization of binaphthyl monomer and bithiophene monomer (copolymer 3) resulted in an 85 nm deeper color shift of the absorption maximum compared to homopolymer 1. The emission maximum of bithiophene copolymer 3 was shifted to a longer wavelength side of 103 nm compared to homopolymer 1 (Table 1).

[0061] [Table 1]

[0062] In the table, Fl.QY represents the fluorescence quantum yield of the synthesized polymer, which is a unit measurement of the fluorescence conversion efficiency by the polymer. A value of 1.00 represents 100% conversion efficiency from absorbed photons to emitted fluorescent photons, and 0.00 represents 0% conversion. Mn(kDa) means number-average molecular weight, and Mw(kDa) means mass-average molecular weight.

[0063] Compounds 1, 2, and 3 demonstrate that the absorption wavelength of water-soluble binaphthyl-based polymers can be tuned by copolymerization with different comonomers. The present invention can be used to shift the absorption band to match the wavelength of commonly used excitation lasers. For example, polymer 1 (pure binaphthyl polymer) is tuned to be optimal for a 280 nm deep ultraviolet laser; polymer 2 (phenylene comonomer) is optimal for a 355 nm UV laser; and polymer 3 (bisthiophene comonomer) is optimal for a violet laser (405 nm).

[0064] To demonstrate the usefulness of the present invention, binaphthylphenylene copolymer 2 was conjugated to human anti-CD4 and human anti-CD19 monoclonal antibodies. This conjugate had the structure shown in Figure 6.

[0065] Anti-CD4 and anti-CD19 antibody conjugates of copolymer 2 were used in flow cytometry analysis of human peripheral blood mononuclear cells (PBMCs) using a UV laser (355 nm) as the excitation source. Figure 4 shows the performance of the phenylene copolymer 2 antibody conjugate in flow cytometry analysis using a UV laser. The absorption wavelength can be adjusted depending on the selection of the comonomer to match other excitation wavelengths. For example, bithiophene copolymer 3 can be used in applications using a violet laser (405 nm).

[0066] These examples demonstrate the usefulness of the present invention, which allows for the control of the position of absorption bands in the spectrum to precisely match the desired excitation laser, thereby expanding the storage capacity of water-soluble fluorescent polymer dyes for biological applications.

Claims

1. General formula (I): 【Chemistry 1】 wherein AR and MU are repeating units of the polymer; MUs are 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%, Provided that a+b=100 mol %, and c is 1 to 10,000. In a conjugate having AR is a group represented by the 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, CORa, CO ２ Ra, COCl, CONRaRb, 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 and R a - R b are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl, or heteroaralkyl residues, 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.

10. A conjugate characterized in that the conjugate is attached to the polymer chain via the 2,2' or 3,3' or 5,5' or 6,6' or 7,7' or 8,8' positions by

2. The MU unit located at the end of the conjugate has the following formula (III): 【Transformation 3】 wherein G1, AR, MU, G2, a, b, and c have the same meaning as in formula (I), and L represents a molecule containing an alkyl, aryl, or heteroaryl group.

2. The conjugate of claim 1, wherein the conjugate is linked via a linker group L according to

3. 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.

4. The conjugate of claim 1, characterized in that MU comprises an aromatic system having at least one benzene or thiophene ring providing a conjugated π system.

5. MU is the residue 【Chemistry 4-1】 【Chemistry 4-2】 The conjugate of claim 1, characterized in that it is selected from one or more of the following:

6. 2. The conjugate of claim 1, wherein 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 5-1】 【Chemistry 5-2】 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 6】 [Wherein n=5 to 15] 2. The conjugate of claim 1, characterized in that it is substituted with a residue according to

9. 2. The conjugate of claim 1, wherein G1 and G2 are independently hydrogen, halogen, or an antigen recognition site.

10. The conjugate of claim 9, 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.

11. 11. A method for detecting a target site in a sample of a biological specimen using at least one conjugate according to any one of claims 1 to 10, 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:

12. 11. Use of the conjugate of any one of claims 1 to 10 in fluorescence microscopy, flow cytometry, fluorescence spectroscopy, cell separation, pathology, or histology.

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