Arylation coupling agents, fluorescent dye conjugates and related applications

Abstract

The present invention provides an aryl coupling agent, a fluorescent dye conjugate, and related applications. The aryl coupling agent has the following structural formula: #imgabs0# R1 is H or a protecting group for an amino group; R2 is selected from an alkyl group; R3 is selected from a substituted or unsubstituted arylene group, alkylene group, heteroalkylene group, or heterocycloalkenylene group; when R3 contains a substituent, the substituent is located at any substitutable position of the arylene group, alkylene group, or heterocycloalkenylene group; the substituent is selected from any one or more of a C1-C6 alkyl group containing or not containing a heteroatom, a halogen, an amino group, and a nitro group; the heteroatoms in the heterocycloalkenylene group or the C1-C6 alkyl group containing a heteroatom are each independently selected from O, S, or N, and the heteroatoms are located at any carbon atom position; X is F or Cl. Under the same conditions, this type of aryl coupling agent reacts rapidly with the dye, and the resulting product is more stable than the reaction product of maleimide.

Description

Technical Field

[0001] The present invention relates to the field of protein labeling, and in particular to an arylated coupling agent, a fluorescent dye conjugate and related applications. Background Art

[0002] The development of fluorescence imaging technology relies on the joint progress of fluorescence microscopes and fluorescent dyes, and at the same time, the two promote and restrict each other at the technical level. For example, the emergence of fluorescent dye technology capable of photoswitching has made STORM (stochastic optical reconstruction microscopy) super-resolution microscopy possible. The more powerful lasers used in confocal microscopes and STED (stimulated emission depletion microscopy) super-resolution microscopes compared to conventional fluorescence microscopes also place higher demands on the photostability of dyes. Currently, the consensus in the imaging field is to achieve large-scale, in vivo (from cells to tissues at all levels), real-time, long-term imaging with high spatiotemporal resolution (super-resolution at the cellular level). Therefore, the development of highly photostable, low-phototoxic, high-brightness, and biocompatible fluorescent dyes has become a common pursuit in the field.

[0003] Bioconjugation reactions enable the covalent attachment of small dye molecules to biomacromolecules. Temporal and spatial monitoring of fluorescent dyes attached to biomacromolecules in vivo or in vitro via fluorescence imaging or detection has become a crucial component of fluorescence imaging. Covalent modification of proteins is crucial for fluorescence microscopy, whether at the single-molecule level in smFRET (single molecule fluorescence resonance energy transfer) or at the cellular or tissue level in immunofluorescence imaging. Generally, lysine and cysteine ​​are the two most commonly used strategies for labeling specific proteins due to the favorable affinity of side chain groups. A key difference lies in the higher abundance of lysine (5.9%) compared to the lower abundance of cysteine ​​(1.9%). Due to its hydrophilic and hydrophobic nature, lysine is highly prevalent on nearly all protein surfaces, often providing up to 20 sites for attachment to dyes, biotin, polymers, nanoparticles, or glass surfaces. Therefore, it is widely used for nonspecific protein labeling. As for cysteine, due to its low abundance, site-directed mutagenesis is often allowed to achieve specific site labeling of proteins for research that requires higher spatial precision.

[0004] Specifically, the basic principles of the above two protein modification technologies are as follows:

[0005] (1) Succinimidyl ester and lysine modification

[0006] The oldest and most versatile protein modification techniques are based on groups that react with primary amines. This is largely due to the fact that deprotonated primary amines are the most nucleophilic of all functional groups in proteins. Among all amino group modification methods, the most classic is the NHS ester (N-Hydroxysuccinimide)-based method. Generally speaking, due to the poor leaving ability of alkoxy groups, alkyl esters of carboxylic acids are difficult to react with amines in aqueous solutions. Therefore, it is crucial to use groups with stronger leaving abilities and relatively stable aqueous systems. NHS esters were first developed in 1963 as an alternative to p-phenyl esters in amide bond-forming reactions. Compared to other amine-selective reagents, they offer several significant advantages: First, they react at a pH of 7-8, close to physiological conditions and significantly lower than other reagents, such as isothiocyanates. Second, any molecule containing a carboxyl functional group can be readily converted to a corresponding NHS ester, making it very convenient to use. Third, NHS esters are highly stable in water, with a half-life of 4-5 hours at pH 7, approximately 1 hour at pH 8, and 10 minutes at pH 8.6—times sufficient for completion of the reaction. Fourth, the formed amide bond is extremely stable, with a half-life of 7 years in water, allowing for the long-term storage of the conjugated product. Because of these advantages, NHS esters are currently considered one of the most powerful protein modification reagents and represent the gold standard for bioconjugation reactions.

[0007] (2) Cysteine ​​modification

[0008] The biggest difference between cysteine ​​and lysine in protein modification stems from their differing abundance. Cysteine, the second-lowest-abundant amino acid (1.9%), can be easily introduced into specific positions through site-directed mutagenesis, making it the preferred method for specific labeling. Furthermore, in terms of reactivity, sulfhydryl groups, particularly in their deprotonated thiolate form, significantly outperform any other nucleophilic functions found in proteins. Furthermore, considering the dissociation capacity of sulfhydryl groups, the pKa value of cysteine ​​sulfhydryl groups in proteins is approximately 8-9, similar to that of protonated amino groups, and exhibits excellent reactivity at physiological pH.

[0009] Since the reaction between sulfhydryl and maleimide (MAL) was developed as a specific reaction for cysteine ​​in 1949, it has been widely used in various bioconjugation strategies due to its rapid kinetics and good selectivity. It may even be the most commonly used bioconjugation method.

[0010] Because of the advantages of the above two reactions, reagents based on these two reactions have been widely commercialized. For fluorescence technology, dyes containing maleimide and succinimidyl ester as linkers can be easily obtained commercially and used in biological experiments.

[0011] However, the applicants have discovered for the first time that the specific labeling conditions of the MAL labeling strategy have a significant disadvantage of reducing the photostability of the dye. Therefore, under the existing labeling strategy, how to improve the luminescence stability of the fluorescent dye has become a problem that needs to be solved. Summary of the Invention

[0012] The main purpose of the present invention is to provide an arylated coupling agent, a fluorescent dye conjugate and related applications to solve the problem in the prior art that when the Mal coupling agent is coupled with a fluorescent dye, the dye luminescence stability is poor.

[0013] In order to achieve the above object, according to one aspect of the present invention, an aryl coupling agent is provided. The aryl coupling agent has a structural formula shown in Formula I, Formula II or Formula III:

[0014]

[0015] wherein R1 is H or any amino protecting group, R2 is selected from an alkyl group, R3 is selected from a substituted or unsubstituted arylene group, a substituted or unsubstituted alkylene group, a substituted or unsubstituted heteroalkylene group, or a substituted or unsubstituted heterocycloalkenylene group, and when R3 is a substituted arylene group, a substituted alkylene group, or a substituted heterocycloalkenylene group, the substituent is located at any substitutable position of the arylene group, the alkylene group, or the heterocycloalkenylene group, and the substituent is selected from any one or more of a C1-C6 alkyl group not containing heteroatoms, a C1-C6 alkyl group containing heteroatoms, a halogen, an amino group, and a nitro group, and the heteroatoms in the heteroalkylene group, the heterocycloalkenylene group, or the C1-C6 alkyl group containing heteroatoms are each independently selected from O, S, or N, and the heteroatoms are located at any carbon atom position; and X is F or Cl.

[0016] Furthermore, R1 is any one of Boc, Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB or Bn; and R2 is any one of methyl, ethyl, propyl or butyl.

[0017] Furthermore, in the arylated coupling agent shown in Formula I, R3 is selected from a substituted arylene group or a substituted heterocycloalkenylene group, wherein the substituent is any one or more of a C1-C6 alkyl group not containing heteroatoms, a C1-C6 alkyl group containing heteroatoms, a halogen, an amino group and a nitro group, and the heteroatoms in the heteroalkylene group, the heterocycloalkenylene group and the C1-C6 alkyl group containing heteroatoms are each independently selected from O, S or N, more preferably O.

[0018] Furthermore, in the arylated coupling agent represented by formula I, R3 is selected from unsubstituted C1-C6 alkyl or heteroalkyl, wherein the heteroatom is O.

[0019] Furthermore, R3 is selected from any one of the following groups:

[0020]

[0021]

[0022] In order to achieve the above object, according to a second aspect of the present invention, a fluorescent dye conjugate is provided. The fluorescent dye conjugate is formed by coupling a fluorescent dye and a coupling agent, wherein the coupling agent is the aforementioned arylated coupling agent.

[0023] Furthermore, the fluorescent dye is selected from any one of the following: cyanine dyes, rhodamine dyes, coumarin dyes, fluorescein dyes, squaric acid dyes, porphyrin dyes and BODIPY dyes; preferably, the cyanine dyes and rhodamine dyes are selected from any one of the following: Cy3, Cy3.5, Cy5, Cy7, Cy3B, Cy5B, tetramethylrhodamine, Alexa Fluor 488 or Alexa Fluor 568.

[0024] In order to achieve the above-mentioned purpose, according to the third aspect of the present invention, a method for labeling a biological molecule is provided, which comprises: incubating a biological molecule containing a thiol group with the aforementioned fluorescent dye conjugate, and labeling the biological molecule with the fluorescent dye through the formation of a thiol bond between the thiol group and the fluorescent dye conjugate, thereby obtaining a biological molecule labeled with the fluorescent dye.

[0025] Furthermore, the biomolecule is selected from nucleic acids, proteins or polypeptides.

[0026] In order to achieve the above object, according to a fourth aspect of the present invention, a fluorescently labeled biomolecule is provided. The fluorescently labeled biomolecule is obtained by labeling using the aforementioned method.

[0027] Furthermore, under single-molecule fluorescence test conditions, the total photon number of the fluorescently labeled biomolecule is 1.5 to 2 times the total photon number of the biomolecule labeled with the maleimide conjugate of the same fluorescent dye.

[0028] Furthermore, the electron density of sulfur atoms in thioether bonds in biomolecules ranges from -0.0032 to +0.02942.

[0029] According to a fifth aspect of the present invention, a kit is provided, which includes the aforementioned arylation coupling agent, fluorescent dye conjugate, or fluorescently labeled biological molecules.

[0030] According to a sixth aspect of the present invention, there is provided a use of the aforementioned arylated coupling agent in a kit for preparing fluorescent dye conjugates or labeling biomolecules.

[0031] To achieve the above-mentioned object, according to seven aspects of the present invention, a method for preparing an aryl coupling agent is provided, which comprises: placing compound 1 and carbon disulfide in a first solvent containing an alkaline solution for a first reaction to obtain compound 2; placing compound 2 and methyl iodide in a second solvent for a second reaction to obtain an intermediate product; placing the intermediate product and an amine bromide compound with an amino protecting group in a third solvent for a third reaction to obtain compound 3; placing compound 3 and m-chloroperbenzoic acid in a fourth solvent for a fourth reaction to obtain the aryl coupling agent represented by the aforementioned formula I; wherein, in the aryl coupling agent, R1 is H or any amino protecting group; R2 is selected from an alkyl group; and R3 is The structural formulas of compound 1, compound 2 and compound 3 are as follows:

[0032]

[0033] Furthermore, compound 1, carbon disulfide and a first base are dissolved in a first solvent for a first reaction, wherein the molar ratio of compound 1 to carbon disulfide is (2-20):1; the molar ratio of compound 1 to the first base is (2-20):1; preferably, the temperature of the first reaction is 85-95°C, and the time of the first reaction is 18-24h; preferably, the first base is selected from any one of the following: KOH or NaOH, preferably, the first solvent is selected from any one of the following: anhydrous ethanol or anhydrous methanol.

[0034] Further, compound 2 and N, N-diisopropylethylamine are dissolved in a second solvent to obtain a solution of compound 2; iodomethane is dissolved in a second solvent to obtain an iodomethane solution; the iodomethane solution is added dropwise to the solution of compound 2 to carry out a second reaction; preferably, the molar ratio of compound 2 to iodomethane is (0.8-1.2):1; the molar ratio of compound 2 to N, N-diisopropylethylamine is (2-20):1; preferably, the second reaction is carried out under ice bath conditions for 30-60 min, preferably 35-45 min; preferably, the second solvent is selected from any one of the following: tetrahydrofuran, acetone, 2-methyltetrahydrofuran or diethyl ether.

[0035] Further, the intermediate product is dissolved in a third solvent, and then the second base and the brominated amine compound with an amino protecting group are added to the third solvent in sequence to carry out a third reaction; preferably, the molar ratio of compound 2 to the second base is (2-10):1; preferably, the molar ratio of compound 2 to the brominated amine compound with an amino protecting group is (2-20):1; preferably, the temperature of the third reaction is 15-30° C., and the time of the third reaction is 2-4 hours; preferably, the brominated amine compound with an amino protecting group is selected from C2-C4 alkyl brominated amines with an amino protecting group, more preferably C 2 or C3 alkylamine bromide; preferably, the amino protecting group is any one of Boc, Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB or Bn; more preferably, the amine bromide compound with an amino protecting group is Boc-HN-CH2-CH2-Br or Boc-HN-CH2-CH2-CH2-Br; preferably, the second base is selected from any one of the following: anhydrous sodium carbonate, anhydrous potassium carbonate, anhydrous lithium carbonate or anhydrous cesium carbonate, and the third solvent is selected from any one of the following: dimethylformamide, dimethyl sulfoxide or sulfolane.

[0036] Furthermore, the molar ratio of compound 3 to m-chloroperbenzoic acid is (2-10):1; preferably, the fourth solvent is selected from any one of the following: dichloromethane, chloroform, dioxane or tetrahydrofuran.

[0037] To achieve the above object, according to an eighth aspect of the present invention, a method for preparing an arylated coupling agent is provided, the method comprising: placing compound 4 and ethylenediamine with an amino protecting group in a fifth solvent for a fifth reaction, and under the action of an acid-amine condensing agent and an organic base, obtaining an arylated coupling compound represented by the aforementioned formula II;

[0038]

[0039] Wherein, the fifth solvent is acetonitrile, dichloromethane or dimethylformamide; the acid amine condensation agent is selected from carbodiimides or onium salts, and the organic base is selected from triethylamine and N,N-diisopropylethylamine; when the acid amine condensation agent is selected from carbodiimides, the reaction system also includes an auxiliary agent, and the auxiliary agent is HOBt; preferably, the acid amine condensation agent of the carbodiimide class is selected from DCC, EDCI or DIC; the acid amine condensation agent of the onium salt class is selected from HATU, HBTU or BOP; preferably, the molar ratio of compound 4 to ethylenediamine with an amino protecting group is (2 to 20):1; preferably, the amino protecting group is any one of Boc, Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB or Bn.

[0040] According to a ninth aspect of the present invention, a method for preparing an arylated coupling agent is provided, the method comprising: dissolving compound 5 and 4-N-tert-butyloxycarbonylaminopiperidine (tert-butyl piperidin-4-ylcarbamate) in a sixth solvent, respectively, to obtain a solution of compound 5 and a solution of 4-N-tert-butyloxycarbonylaminopiperidine; adding a base to the 4-N-tert-butyloxycarbonylaminopiperidine solution to obtain an alkaline solution; dropwise adding the solution of compound 5 to the alkaline solution at 0-4°C to obtain a mixed solution; and reacting the mixed solution at room temperature for 1.5-2.5 hours to obtain the arylated coupling agent represented by formula III in claim 1; wherein the sixth solvent is dichloromethane, dimethylformamide, acetonitrile, dioxane or tetrahydrofuran; preferably, the molar ratio of compound 5 to 4-N-tert-butyloxycarbonylaminopiperidine is: (1-20):1.

[0041] In order to achieve the above object, according to the tenth aspect of the present invention, a method for preparing a fluorescent dye conjugate is provided, the method comprising: reacting a fluorescent dye with the aforementioned arylated coupling agent to obtain the fluorescent dye conjugate.

[0042] Furthermore, the fluorescent dye is selected from any one of cyanine dyes, rhodamine dyes, coumarin dyes and BODIPY dyes; preferably, the dye is selected from any one of the following: Cy3, Cy5, Cy3B, Cy5B, AF488, AF568 and TMR.

[0043] Furthermore, the preparation method includes: deprotecting the optional amino protecting group in the aryl coupling agent to obtain a deaminated product; reacting the deaminated product with a fluorescent dye and N,N-diisopropylethylamine in a DMF solvent to obtain a fluorescent dye conjugate; when the fluorescent dye is Cy3, Cy5, Cy7, AF488, AF568 or TMR, reacting with the deaminated product in the form of Cy3-NHS, Cy5-NHS, Cy7-NHS, AF488-NHS, AF568-NHS or TMR-NHS; when the fluorescent dye is Cy3B or Cy5B, reacting with the deaminated product in the form of Cy3B-COOH or Cy5B-COOH; preferably, deprotecting the amino protecting group by placing the aryl coupling agent in dichloromethane and hydrochloric acid / dioxane; preferably, the molar ratio of the aryl coupling agent to N,N-diisopropylethylamine is (2-20):1.

[0044] By applying the technical solution of the present invention, by adjusting the electronic properties of the thioether in the biomacromolecule conjugate that requires specific fluorescent dye labeling, the compound represented by Formula I, Formula II or Formula III is used as a linker (or coupling agent) to replace the traditional maleimide coupling agent, thereby achieving different ratios of 1.2-4.5 times improvement in the photostability of the dye in the dye-biomacromolecule conjugate on different biomacromolecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0046] Figure 1 A graph showing experimental results showing that thioether groups directly affect the photostability of dyes;

[0047] Figure 2 Schematic diagram of the labeling reaction of three arylation coupling agents with traditional NHS and MAL coupling agents;

[0048] Figure 3 The results show that the photostability of Cy3 / Cy5 dyes using three different arylated coupling agents is improved compared with the MAL strategy in nucleic acid labeling systems.

[0049] Figure 4 The results show that the photostability of Cy3 / Cy5 dyes using POD coupling agents is improved compared with the MAL strategy in protein labeling systems.

[0050] Figure 5 The results show that the photostability of three commercial rhodamine dyes using POD coupling agents in nucleic acid labeling systems is improved compared to the MAL strategy.

[0051] Figure 6 The results show that the photostability of Cy3B / Cy5B dyes using POD coupling agents is improved compared to the MAL strategy in protein labeling systems. DETAILED DESCRIPTION

[0052] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0053] As mentioned in the background, existing techniques for linking dyes with maleimides and succinimidyl esters are widely used, but there have been no reports directly elucidating whether different labeling strategies affect the properties of the same dye. In this application, we experimentally discovered that the currently common thiol-based MAL coupling agent for specific biomarkers can significantly impair the properties of the dye, resulting in a several-fold loss of photostability compared to the nonspecifically labeled NHS coupling agent. Specifically, the specific labeling conditions using the MAL coupling agent have the significant disadvantage of deteriorating the dye's photostability. Therefore, we sought to identify alternatives to MAL to improve the photostability of specifically labeled dyes.

[0054] It should be noted that, at present, we believe that single-molecule fluorescence is the most direct method for detecting indicators such as dye brightness and photostability. Using single-molecule fluorescence technology, we can directly measure the brightness of a single dye molecule after excitation, the total number of photons emitted before photobleaching (we believe this indicator is the most direct reflection of the dye's photostability), the time required for photobleaching, and the signal-to-noise ratio. This characterization method can largely eliminate interference caused by factors such as labeling density and interactions between dyes in a macroscopic system. Therefore, detection in a single-molecule system is the most direct way to characterize dye properties.

[0055] In the field of single-molecule fluorescence, dye labeling is primarily applied to protein systems. While there has long been a consensus that specific and nonspecific dye labeling on proteins actually exhibit significant differences in photostability, this issue has never been systematically analyzed, nor has the underlying cause been discussed. Furthermore, due to the complexity of the protein environment, significant differences in photostability can be observed for dyes labeling at different sites, even when using the same labeling method.

[0056] To further investigate the reasons for this significant difference, the following experiments and studies were conducted in this application:

[0057] We selected the most classic cyanine dyes as representatives of dyes and labeled the ends of DNA chains where their chemical environment is simpler. We then used commercially available DNA molecules with the same sequence of sulfhydryl and amino groups at the ends to bind to the cyanine dyes Cy3 and Cy5 (structures shown in Table 1) containing two of the most classic biological conjugation groups, maleimide and succinimide ester. Figure 2) were separately labeled, and based on the single-molecule fluorescence method, the dye was labeled on the surface of the glass slide and characterized. In the same buffer containing a triplet quencher (specifically nitrobenzene, cyclooctatetraene and water-soluble vitamin E, etc.) and an oxygen scavenger (such as glucose oxidase glucose and catalase) (unless otherwise specified, the single-molecule fluorescence experiments in this application were all carried out in this buffer). The results showed that for the two dye molecules Cy3 and Cy5, the total number of photons (the total number of photons is the total number of photons that can be released for a single dye molecule before photobleaching occurs, and all single-molecule fluorescence data comparisons in this application use this indicator as the brightness of light stability) of the former was 3.2 times and 2.8 times that of the latter, respectively.

[0058] Considering that the difference between the two different labeling strategies is the introduction of thioether groups and thiol groups (both DTT and Cys introduce thiol groups into the solution), we tried to add dithiothreitol (DTT), methionine (Met) and cysteine ​​(Cys) into the buffer solution, which can also significantly reduce the total number of photons of the two cyanine dyes (the purpose of this step is to introduce thioether groups and thiol groups into the solution, proving that the presence of these two groups in the system will affect the photostability of the dye, so as to further consider that the thioether groups in the reaction products of the coupling reaction are the key factors of this phenomenon). Further analysis shows that there are no exposed thiol groups on the surface of the dye after dye labeling, so it is believed that the thioether group is the main reason for the decrease in photostability (see Figure 1 A).

[0059] Next, we conducted similar experiments on common commercially available dye molecules. The results showed that all dyes tested, including the two largest types of fluorescent dyes, rhodamine and cyanine dye molecules, all experienced a similar decrease in the total photon number, but the magnitude of the decrease was different. In addition, this phenomenon also existed for the LD550 and LD650 dye molecules (trade names) that use a self-repairing dye strategy, and was even more obvious (see Figure 1 B and C in ).

[0060] Since we previously believed that the difference in photostability of cyanine dyes Cy3 and Cy5 in different labeling methods came from the influence of the thioether group, we tried to change the electronic structure of the thioether group, that is, directly adjusting the reactivity of the lone pair of electrons may directly affect the final result. Taking into account the various advantages of the maleimide labeling strategy in terms of reactivity and reaction conditions, combined with our understanding of the cysteine ​​arylation strategy, we finally chose POD (MethylsulfonylPhenyloxadiazole), PCB (p-nitrochlorobenzene) and FBP (pentafluorobenzene) molecules as coupling agents that can directly replace maleimide without changing the reaction conditions during labeling. Using quantum mechanics to analyze the key parts of the reaction products, we used the Hirshfeld charge to predict and estimate the electron density of the sulfur atom on the thioether group (see Figure 2 ), the results showed that the POD linker had the highest electropositivity, that is, the lowest electron cloud density value, which indicates that the POD linker can significantly improve the photostability of the labeled dye. At the same time, the electropositivity of PCB and FBP linkers was also significantly improved compared with the MAL linker.

[0061] On this basis, we further improved the structures of POD, PCB and FBP. Among them, various substitutions were made to the aromatic group of POD and the alkyl group containing O atoms connected thereto, namely, the NH2 or the imino group (-NH-) with amino protecting group and the group connected to the dye were replaced. The distance between the two groups is at least one carbon atom, resulting in a series of coupling agents with improved POD structures. Similarly, Hirshfeld charges were used to predict and estimate the electron density of the sulfur atoms on the thioether groups. The results (shown in the table below) show that coupling agents based on improved POD structures also have significantly higher electropositivity than MAL. This suggests that these improved structures also significantly improve the photostability of the labeled dye.

[0062] Table 1:

[0063]

[0064] It should be noted that the differences between the charge values ​​shown in Table 1 are all within the error range and therefore are not statistically significant. A comparison of structures 2 and 3 with structure 1 shows that the electron density test results are not affected by the length of the alkyl chain or whether or not the structure carries an amino protecting group.

[0065] Accordingly, the structure of PCB was improved, and it was found that the Cl atom at the para-nitro position was replaced by other halogen atoms, which also had similar electropositivity.

[0066] Improvements to the structure of FBP revealed that the structure formed by the combination of a phenyl group substituted with five fluorine atoms and a sulfonyl group has the effect of making the label more stable. If three fluorine atoms or other atoms are used to replace the fluorine atoms, it will be difficult to carry out the corresponding reaction by adjusting the conditions such as organic solvents (such as DMF or DMSO) or organic bases (such as DIPEA). If the sulfonyl group is replaced with a stronger nitro group, it will easily react with multiple sulfhydryl groups, making the reaction non-specific (for example, it may react with multiple sulfhydryl groups on the same protein or different proteins, and even after replacing it with a very strong electron-withdrawing group, it may not react with a single sulfhydryl group, and the amino group in lysine may also participate. This makes it difficult to achieve the expected results from the reaction level).

[0067] Furthermore, we verified the effect of three coupling agents (POD, PCB and FBP) on improving the photostability of simple dye Cy3 / 5 compared with MAL coupling agent on nucleic acid (see Figure 3 ), and further verified on proteins that the POD strategy can improve Cy3 / Cy5 at multiple sites in various proteins by about two times compared to the traditional maleimide strategy (see Figure 4 To further demonstrate the universality of this strategy, we synthesized POD coupling agent versions of rhodamine molecules AF488, AF568 and TMR (structures see Figure 2 ) and a cyanine iterative version of the Cy3B / Cy5B-POD molecule (structure see Figure 2 ) and its corresponding Cy3B / Cy5B-MAL (structure see Figure 2 ) and tested the photostability of the two types of dye molecules on nucleic acid or protein systems and compared them with the results of MAL commercial coupling agents. Significant improvement in photostability was also observed (see Figure 5 and Figure 6 ).

[0068] Based on the above research results, the applicant has proposed a series of protection schemes for this application. In a typical embodiment of this application, an arylation coupling agent is proposed, which has the structural formula shown in Formula I, Formula II, and Formula III:

[0069]

[0070]

[0071] wherein R1 is H or any amino protecting group; R2 is selected from an alkyl group; R3 is selected from a substituted or unsubstituted arylene group, a substituted or unsubstituted alkylene group, a substituted or unsubstituted heteroalkylene group, or a substituted or unsubstituted heterocycloalkenylene group; and when R3 is a substituted arylene group, a substituted alkylene group, or a substituted heterocycloalkenylene group, the substituent is located at any substitutable position of the arylene group, the alkylene group, or the heterocycloalkenylene group; the substituent is selected from any one or more of a C1-C6 alkyl group not containing heteroatoms, a C1-C6 alkyl group containing heteroatoms, a halogen, an amino group, and a nitro group; the heteroatoms in the heteroalkylene group, the heterocycloalkenylene group, or the C1-C6 alkyl group containing heteroatoms are each independently selected from O, S, or N, and the heteroatoms are located at any carbon atom position; and X is F or Cl.

[0072] The strategy for arylating thiol groups on biomacromolecules has developed rapidly in the past decade. In this application, three coupling agents represented by Formula I (abbreviated as POD), Formula II (abbreviated as PCB), and Formula III (abbreviated as FBP) are designed using nucleophilic substitution reactions as alternatives to the maleimide reaction. Under the same reaction conditions, all of them have the characteristics of fast reaction speed and the products formed are more stable than the reaction products of maleimide.

[0073] When R1 is H, the compounds shown in the three general formulas all contain a -NH2, which is used for coupling with the dye, that is, the coupling with the dye occurs on this group. When R1 is an amino protecting group, the compounds shown in the three general formulas are all precursor molecules. When coupling with the dye, the amino protecting group needs to be removed to carry out the coupling reaction. The specific amino protecting group can be any one of Boc, Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB or Bn. R2 is selected from any one of methyl, ethyl, propyl, or butyl.

[0074] In some embodiments, in the arylated coupling agent shown in Formula I, R3 is selected from a substituted arylene group or a substituted heterocycloalkenylene group, wherein the substituent is any one or more of a C1-C6 alkyl group not containing heteroatoms, a C1-C6 alkyl group containing heteroatoms, a halogen, an amino group, and a nitro group, and the heteroatoms in the heteroalkylene group, the heterocycloalkenylene group, and the C1-C6 alkyl group containing heteroatoms are each independently selected from O, S, or N, more preferably O.

[0075] In some embodiments, in the arylated coupling agent represented by Formula I, R3 is selected from unsubstituted C1-C6 alkyl or heteroalkyl, wherein the heteroatom is O.

[0076] In some specific examples, R3 is selected from any of the following groups:

[0077]

[0078]

[0079] -CH2-,

[0080] The above R1 is any one of H, Boc, Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB or Bn; R2 is any one of methyl, ethyl, propyl or butyl.

[0081] In a second typical embodiment of the present application, a fluorescent dye conjugate is provided. The fluorescent dye conjugate is formed by coupling a fluorescent dye and a coupling agent, wherein the coupling agent is the above-mentioned coupling agent.

[0082] Among them, the fluorescent dye includes but is not limited to any one of the following: cyanine dyes, rhodamine dyes, coumarin dyes, fluorescein dyes, squaric acid dyes, porphyrin dyes and BODIPY dyes; preferably, the cyanine dyes and rhodamine dyes are selected from any one of the following: Cy3, Cy3.5, Cy5, Cy7, Cy3B, Cy5B, tetramethylrhodamine (abbreviated as TMR), Alexa Fluor 488 (abbreviated as AF488), or Alexa Fluor 568 (abbreviated as AF568).

[0083] In a third exemplary embodiment of the present application, a method for labeling a biomolecule is provided. The method comprises incubating a biomolecule containing a thiol group with the aforementioned fluorescent dye conjugate, wherein the biomolecule is labeled with the fluorescent dye via a thiol-ether bond formed between the thiol group and the fluorescent dye conjugate, thereby obtaining a fluorescent dye-labeled biomolecule. Using the aforementioned conjugate to conjugate the fluorescent dye to the biomolecule helps improve the luminescence stability of the fluorescent dye. The aforementioned biomolecule includes, but is not limited to, nucleic acids, proteins, or polypeptides.

[0084] In a fourth typical embodiment of the present application, a fluorescently labeled biomolecule is provided. The fluorescently labeled biomolecule is obtained by labeling using the above method.

[0085] In a preferred embodiment, under single molecule fluorescence assay conditions, the total photon count of the fluorescently labeled biomolecule is 1.2 to 4.5 times the total photon count of the biomolecule labeled with a maleimide conjugate of the same fluorescent dye.

[0086] In a fifth typical embodiment of the present application, a kit is provided, which includes the aforementioned arylation coupling agent, or fluorescent dye conjugate, or fluorescently labeled biological molecules.

[0087] In a sixth typical embodiment of the present application, there is provided a use of the above-mentioned arylated coupling agent in a kit for preparing fluorescent dye conjugates or labeling biomolecules.

[0088] In a seventh typical embodiment of the present application, a method for preparing an aryl coupling agent is provided, the preparation method comprising: placing compound 1 and carbon disulfide in a first solvent containing an alkaline solution for a first reaction to obtain compound 2; placing compound 2 and methyl iodide in a second solvent for a second reaction to obtain an intermediate product; placing the intermediate product and an amine bromide compound with an amino protecting group in a third solvent for a third reaction to obtain compound 3; placing compound 3 and m-chloroperbenzoic acid in a fourth solvent for a fourth reaction to obtain a coupling agent of formula I; wherein R1 in the aryl coupling agent is H or any amino protecting group; R2 is selected from an alkyl group; R3 is The structural formulas of Compound 1, Compound 2 and Compound 3 are as follows: R1 is an amino protecting group.

[0089] The specific type of amino protecting group in compound 3 includes, but is not limited to, any one of Boc, Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB or Bn. In practical applications, it can be reasonably selected according to specific needs and convenience.

[0090] In a preferred embodiment, compound 1, carbon disulfide and a first base are dissolved in a first solvent to carry out a first reaction, wherein the molar ratio of compound 1 to carbon disulfide is (2-20):1; the molar ratio of compound 1 to the first base is (2-20):1; preferably, the temperature of the first reaction is 85-95°C, and the time of the first reaction is 18-24 hours; preferably, the first base is selected from any one of the following: KOH, NaOH, LiOH; preferably, the first solvent is selected from any one of the following: anhydrous ethanol, anhydrous methanol.

[0091] In a preferred embodiment, compound 2 and N,N-diisopropylethylamine are dissolved in a second solvent to obtain a solution of compound 2; iodomethane is dissolved in a second solvent to obtain an iodomethane solution; the iodomethane solution is added dropwise to the solution of compound 2 to carry out a second reaction; preferably, the molar ratio of compound 2 to iodomethane is (0.8-1.2):1; the molar ratio of compound 2 to N,N-diisopropylethylamine is (2-20):1; preferably, the second reaction is carried out in an ice bath for 30-60 min, preferably 35-45 min; preferably, the second solvent is selected from any one of the following: tetrahydrofuran, acetone, 2-methyltetrahydrofuran or diethyl ether.

[0092] In a preferred embodiment, the intermediate product is dissolved in a third solvent, and then the second base and the brominated amine compound with an amino protecting group are added to the third solvent in sequence to carry out a third reaction; preferably, the molar ratio of compound 2 to the second base is (2-10):1; preferably, the molar ratio of compound 2 to the brominated amine compound with an amino protecting group is (2-20):1; preferably, the temperature of the third reaction is 15-30° C., and the time of the third reaction is 2-4 hours; preferably, the brominated amine compound with an amino protecting group is selected from C2-C4 alkyl brominated amines with an amino protecting group, more preferably C2-C4 alkyl brominated amines with an amino protecting group. Preferably, the amino protecting group is any one of Boc, Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB or Bn; more preferably, the amine bromide compound with an amino protecting group is Boc-HN-CH2-CH2-Br or Boc-HN-CH2-CH2-CH2-Br; preferably, the second base is selected from any one of the following: anhydrous sodium carbonate, anhydrous potassium carbonate, anhydrous lithium carbonate or anhydrous cesium carbonate, and the third solvent is selected from any one of the following: dimethylformamide, dimethyl sulfoxide or sulfolane.

[0093] In a preferred embodiment, the molar ratio of compound 3 to m-chloroperbenzoic acid is (2-10):1; preferably, the fourth solvent is selected from any one of the following: dichloromethane, chloroform, dioxane or tetrahydrofuran.

[0094] In an eighth typical embodiment of the present application, a method for preparing a fluorescent dye conjugate is provided. The method comprises: reacting a fluorescent dye with the aforementioned coupling agent to obtain a fluorescent dye conjugate.

[0095] In a preferred embodiment, the fluorescent dye is selected from cyanine dyes, rhodamine dyes, coumarin dyes, and BODIPY dyes; preferably, the cyanine dyes and rhodamine dyes are selected from any one of the following: Cy3, Cy3.5, Cy5, Cy7, Cy3B, Cy5B, tetramethylrhodamine (abbreviated as TMR), Alexa Fluor 488 (abbreviated as AF488) or Alexa Fluor 568 (abbreviated as AF568).

[0096] In a preferred embodiment, the preparation method comprises: deprotecting the amino protecting group in the coupling agent to obtain a deaminated product; reacting the deaminated product with a fluorescent dye and N,N-diisopropylethylamine in a DMF solvent to obtain a fluorescent dye conjugate; when the fluorescent dye is Cy3, Cy5, Cy7, AF488, AF568 or TMR, reacting with the deaminated product in the form of Cy3-NHS, Cy5-NHS, Cy7-NHS, AF488, AF568 or TMR; when the fluorescent dye is Cy3B or Cy5B, reacting with the deaminated product in the form of Cy3B-COOH or Cy5B-COOH; preferably, deprotecting the amino protecting group by placing the coupling agent in dichloromethane and hydrochloric acid / dioxane; preferably, the molar ratio of the coupling agent to N,N-diisopropylethylamine is (2 to 20):1.

[0097] In a ninth typical embodiment of the present application, a method for preparing an arylated coupling agent is provided, the method comprising: placing compound 4 and ethylenediamine with an amino protecting group in a fifth solvent for a fifth reaction, and obtaining an arylated coupling compound represented by the aforementioned formula II under the action of an acid-amine condensing agent and an organic base;

[0098]

[0099] Wherein, the fifth solvent is acetonitrile, dichloromethane or dimethylformamide; the acid amine condensing agent is selected from carbodiimides or onium salts, and the organic base is selected from triethylamine and N,N-diisopropylethylamine (abbreviated as DIEA or DIPEA); when the acid amine condensing agent is selected from carbodiimides, the reaction system also includes an auxiliary agent, and the auxiliary agent is HOBt (1-hydroxybenzotriazole); preferably, the acid amine condensing agent of the carbodiimide class is selected from DCC (dicyclohexylcarbodiimide), EDCI (1-ethyl-3 (3-dimethylpropylamine) carbodiimide) or DIC (N,N-diisopropylcarbodiimide); The acid-amine condensing agent is selected from HATU (Chinese name: O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, CAS No.: 148893-10-1), HBTU or BOP (CAS No.: 56602-33-6); preferably, the molar ratio of compound 4 to ethylenediamine with an amino protecting group is (2-20):1; preferably, the amino protecting group is any one of Boc, Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB or Bn.

[0100] It should be noted that the aforementioned acid-amine condensing agents are the most common and frequently used. Both require the addition of an organic base, most commonly triethylamine and DIPEA, with DMAP sometimes also added. Carbodiimides typically incorporate HOBt as an additive, while onium salts may or may not incorporate HOBt. In practice, conventional combinations should be selected, such as HATU + DIPEA or EDCI + triethylamine + HOBt.

[0101] In a tenth typical embodiment of the present application, a method for preparing an arylated coupling agent is provided, the preparation method comprising: dissolving compound 5 and 4-N-tert-butyloxycarbonylaminopiperidine (tert-butyl piperidin-4-ylcarbamate) in a sixth solvent, respectively, to obtain a solution of compound 5 and a solution of 4-N-tert-butyloxycarbonylaminopiperidine; adding a base to the 4-N-tert-butyloxycarbonylaminopiperidine solution to obtain an alkaline solution; dropwise adding the solution of compound 5 to the alkaline solution at 0-4°C to obtain a mixed solution; and reacting the mixed solution at room temperature for 1.5-2.5 hours to obtain the arylated coupling agent represented by formula III in claim 1; wherein the sixth solvent is dichloromethane, dimethylformamide, acetonitrile, dioxane or tetrahydrofuran; preferably, the molar ratio of compound 5 to 4-N-tert-butyloxycarbonylaminopiperidine is: (1-20):1.

[0102] The beneficial effects of the present application will be further illustrated below with reference to specific embodiments.

[0103] Example 1

[0104] Using organic synthesis methods, three coupling agents, POD, PCB and FBP, can be obtained. By simply reacting them with different commercially available or self-synthesized fluorescent dye molecules, the dye-coupler conjugate (Dye-Linker-) molecules required for specific labeling of biomacromolecules can be obtained. Such molecules can react with the sulfhydryl groups on the biomacromolecules to achieve the required specific labeling and show improved photostability.

[0105]

[0106] Synthesis of POD:

[0107] Molecule 2: 152 mg of molecule 1, 241 μL (4.0 eq) of carbon disulfide, and 112 mg of KOH (2.0 eq) were added to 5 mL of anhydrous ethanol. The reaction was sealed at 90°C overnight. After acidification, filtration, and drying, 430 mg of molecule 2 (67%) was obtained. The NMR results are as follows: 1H NMR (400MHz, DMSO-d6) δ14.55(s,1H),10.39(s,1H),7.72(m,2H),6.94(m,2H).

[0108] Molecule 3: 100 mg of molecule 2 and 200 μL (2.2 eq) of DIPEA (N,N-diisopropylethylamine) were dissolved in 3 mL of anhydrous THF (tetrahydrofuran). 35 μL (1.1 eq) of iodomethane (CH3I, methylating agent) was dissolved in 2 mL of anhydrous THF and slowly added dropwise to the solution of molecule 2 under ice-bath conditions. After 40 minutes, the reaction was monitored for completion. The mixture was spin-dried and redissolved in 5 mL of anhydrous DMF (dimethylformamide). 140 mg of anhydrous sodium carbonate (2.0 eq) and 345 mg (3.0 eq) of 2-(tert-butoxycarbonylamino)ethyl bromide were added. The reaction was allowed to proceed at room temperature for 3 h before completion. 140 mg (77%) of the pure product was obtained by column chromatography. The results of hydrogen nuclear magnetic resonance spectrum were: 1H NMR (400 MHz, Chloroform-d) δ7.99-7.93 (m, 2H), 7.04-6.95 (m, 2H), 5.01 (s, 1H), 4.11 (t, J = 5.1 Hz, 2H), 3.59 (m, 2H), 2.79 (s, 3H), 1.48 (s, 9H).

[0109] POD: 41 mg of molecule 3 was dissolved in 3 mL of anhydrous DCM (full name in Chinese: dichloromethane), and 165 mg (6.0 eq) of 75% mCPBA (full name in Chinese: meta-chloroperbenzoic acid) was added thereto. After reacting at room temperature for 5 h, 26 mg (58%) of pure product was obtained by column chromatography. The results of hydrogen nuclear magnetic resonance spectrum were: 1H NMR (400 MHz, Chloroform-d) δ8.12-8.02 (m, 2H), 7.08-6.99 (m, 2H), 4.98 (s, 1H), 4.12 (t, J = 5.2 Hz, 2H), 3.58 (m, 2H), 3.51 (s, 3H), 1.46 (s, 9H).

[0110] Synthesis of PCB:

[0111]

[0112] 100 mg of molecule 4 was dissolved in 10 mL of acetonitrile, and 128 mg (1.6 eq) of N-Boc ethylenediamine, 108 mg of HOBt (1.6 eq), 330 μL of DIPEA (N,N-diisopropylethylamine) (~4 eq) and 153 mg of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) (1.6 eq) were added. After 3 h of reaction, the reaction was basically completed. After simple water washing and drying, the product was spin-dried to obtain 120 mg (70%) of NMR-pure product, characterized by: 1H NMR (400MHz, DMSO-d6) δ8.72(t,J=5.7Hz,1H),8.35(d,J=2.8Hz,1H),8.27(dd,J=8.8,2.8H z,1H),7.81(d,J=8.8Hz,1H),6.96(t,J=5.9Hz,1H),3.27(m,2H),3.13(m,2H),1.39(s,9H).

[0113] Synthesis of FBP:

[0114]

[0115] 50 mg of molecule 5 and 36 μL of tert-butyl piperidin-4-ylcarbamate were each dissolved in 1 mL of DCM (dichloromethane). 79 μL (3.0 eq) of triethylamine was added to the tert-butyl piperidin-4-ylcarbamate solution, and then the molecule 5 solution was slowly added dropwise in an ice bath. The mixture was allowed to react at room temperature for 2 h before stopping the reaction. The mixture was then spin-dried and separated by column chromatography to obtain 17.5 mg (22%) of FBP. The results of nuclear magnetic resonance are as follows: 1H NMR (400 MHz, Chloroform-d) δ 4.46 (s, 1H), 3.88 (d, J = 12.5 Hz, 2H), 3.56 (s, 1H), 2.83 (t, J = 12.1 Hz, 2H), 2.11-1.98 (m, 2H), 1.52 (dd, J = 12.5, 3.9 Hz, 2H), 1.43 (s, 9H).

[0116] Example 2

[0117] Synthesis of Dye-Linker:

[0118]

[0119] A certain amount of arylated coupling agent molecules was stirred in 1 mL of DCM (dichloromethane) and 0.5 mL of 4 M hydrochloric acid·dioxane for 2 h for deprotection and spin-dried. 5.0 mg of dye-NHS corresponding to 1.0 eq was added, and 2.5 μL of DIPEA (N,N-diisopropylethylamine) (~20 eq) and 1 mL of DMF were added. The reaction was terminated after 2 h, and the mixture was spin-dried and dissolved in water. The mixture was separated by HPLC in a water-acetonitrile system and finally spin-dried. The amount of dye-conjugate and the mass spectrometry results were identified by LCMS as shown in the following table.

[0120] Table 2:

[0121]

[0122] Example 3

[0123] (1) Synthesis of Cy3B-POD / MAL:

[0124] Two portions of 1.25 mg of Cy3B-COOH (commercially available) were each dissolved in 1 mL of DMF, and 3.0 mg of Carter condensation agent (3.0 eq) and 6.0 μL of DIPEA (~15 eq) were added to each. Then, 1.2 mg of MAL-NH2 or 3.6 mg of deprotected POD (~4.0 eq) (deprotection conditions were the same as above) were added. The reaction was terminated after stirring at room temperature for 2 h. The mixture was spin-dried and dissolved in water. After separation by HPLC in a water-acetonitrile system, 0.31 mg of Cy3B-MAL (20%) and 0.69 mg of Cy3B-POD (37%) were obtained, respectively. ESI mass spectrometry results showed that Cy3B-MAL (M+H)+, C 37 H 39 N4O7S + , calculated value 683.2, observed value 683.5; Cy3B-POD(M+H)+, C 42 H 44 N5O9S 2+ , calculated value 826.3, observed value 826.4.

[0125] (2) Synthesis of Cy5B-POD / MAL:

[0126]

[0127] Two portions of 0.11 mg of Cy5B-COOH were each dissolved in 0.5 mL of DMF, and 1.0 mg of Carter condensation agent (~15.0 eq) and 1.0 μL of DIPEA (~30 eq) were added to each. Then, 1.0 mg of MAL-NH2 or 1.0 mg of deprotected POD (~10.0 eq) was added (deprotection conditions were the same as above). The reaction was terminated after stirring at room temperature for 2 h. The mixture was spin-dried and dissolved in water. After separation by HPLC in a water-acetonitrile system, 0.029 mg of Cy5B-MAL (23%) and 0.034 mg of Cy5B-POD (23%) were obtained, respectively. ESI mass spectrometry results showed that Cy5B-MAL, C 44 H 47 N4O 10 S 2- , calculated value 855.3, observed value 855.3; Cy5B-POD, C 49 H 52 N5O 12 S 3- , calculated value 998.3, observed value 998.4.

[0128] Example 4

[0129] For three different arylated coupling agents, we conducted dye coupling and basic photophysical property tests on simple double-stranded DNA (Chinese full name: deoxyribonucleic acid). The results are as follows Figure 3 As shown in the figure, compared with the MAL coupling agent, the three newly developed coupling agents all significantly improved the photostability of the dye (i.e., the total number of photons) under basically the same test conditions (i.e., the number of photons detected per unit time is similar to the overall signal-to-noise ratio). For two simple cyanine dyes Cy3 and Cy5, the improvement ranged from 60% to 150%. Among them, the POD coupling agent was preferred to have the best photostability improvement effect.

[0130] Therefore, the coupling products of POD coupling agent and Cy3 / Cy5 dye molecules were further labeled at different sites of various proteins and single molecule fluorescence tests were performed. The results showed (see Figure 4 ), possibly due to the complexity of protein residues, the photostability of dyes may vary greatly under the same labeling method at different sites on different proteins. However, the brightness difference between the MAL strategy and the POD strategy is not obvious in most cases, and both can meet the needs of single-molecule fluorescence experiments. The POD strategy can indeed significantly improve the photostability of cyanine dyes after thiol labeling. Overall, the total photon number is increased by about 1 times on average compared to the MAL strategy.

[0131] Furthermore, to verify the universality of the arylated coupling agent, we applied the same POD coupling agent to the other of the two largest classes of dyes, the rhodamine dyes, and formed DNA-dye conjugates. We also completed photostability tests under single-molecule fluorescence conditions. The test results showed that the POD coupling agent can improve the photostability of the three different rhodamine dyes AF488, AF568, and TMR by 20%-70% (see Figure 5 ).

[0132] We attempted to test the photostability of molecules using the MAL and POD strategies as linkers for Cy3B and Cy5B dyes (see Figure 6 ) The results showed that both dyes would also have improved photostability under the POD strategy, with the improvement ranging from 20% to 350% for different biomacromolecules.

[0133] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: for cyanine dyes, the connection method of the arylated coupling agent in this application can achieve specific labeling of biomacromolecules, and compared with the connection method of the currently commercially available industry-wide MAL coupling agent, the photostability is significantly improved. In addition, the above embodiments also prove that after different cyanine dyes and rhodamine dyes are combined with POD coupling agents, the photostability is also significantly improved compared with the dyes of the currently commercialized corresponding MAL coupling agents. The arylated coupling agents in this application have been demonstrated on different biomacromolecules and the two most widely used types of fluorescent dyes (i.e., cyanine and rhodamine dyes) to be universal in terms of photostability improvement relative to the dyes of the currently commercial MAL coupling agents. Therefore, the three arylated coupling agents of this application are suitable for the connection of a series of dye molecules.

[0134] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A fluorescent dye conjugate, characterized in that The fluorescent dye conjugate is formed by coupling a fluorescent dye and a coupling agent, wherein the coupling agent has a structural formula shown in Formula I, Formula II or Formula III: in, R1 is H or any amino protecting group: Boc, Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB or Bn; R3 is selected from any one of the following groups: -CH2-、 X is F or Cl; The R2 is selected from any one of methyl, ethyl, propyl or butyl.

2. The fluorescent dye conjugate according to claim 1, characterized in that The fluorescent dye is selected from any one of the following: cyanine dyes, rhodamine dyes, coumarin dyes, fluorescein dyes, squaric acid dyes, porphyrin dyes and BODIPY dyes.

3. The fluorescent dye conjugate according to claim 2, characterized in that The cyanine dye and rhodamine dye are selected from any one of the following: Cy3, Cy3.5, Cy5, Cy7, Cy3B, Cy5B, tetramethylrhodamine, Alexa Fluor 488 or Alexa Fluor 568.

4. A method for labeling biomolecules for non-disease diagnosis and treatment purposes, characterized in that: The marking method comprises: Co-incubating a biomolecule containing a thiol group with the fluorescent dye conjugate according to any one of claims 1 to 3, The biomolecule is labeled with a fluorescent dye via the formation of a thioether bond between the sulfhydryl group and the fluorescent dye conjugate, thereby obtaining a biomolecule labeled with the fluorescent dye.

5. The marking method according to claim 4, characterized in that The biomolecule is selected from nucleic acids, proteins or polypeptides.

6. A fluorescently labeled biomolecule, characterized in that The fluorescently labeled biomolecule is labeled by the labeling method according to claim 4 or 5.

7. The biomolecule according to claim 6, characterized in that Under the conditions of single-molecule fluorescence testing, the total photon number of the fluorescently labeled biomolecule is 1.5 to 2 times the total photon number of the biomolecule labeled with a maleimide conjugate of the same fluorescent dye.

8. The biomolecule according to claim 7, characterized in that The electron density of the sulfur atom in the thioether bond in the biomolecule is -0.0032 to +0.02942.

9. A kit, characterized in that The kit comprises the fluorescent dye conjugate according to any one of claims 1 to 3, or the fluorescently labeled biomolecule according to any one of claims 6 to 8.

10. Use of an arylated coupling agent in a kit for preparing fluorescent dye conjugates or labeling biomolecules, characterized in that: The arylated coupling agent has a structural formula shown in Formula I, Formula II or Formula III: in, R1 is any one of Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB or Bn; R3 is selected from any one of the following groups: -CH2-、 X is F or Cl; Wherein, the R2 is selected from any one of methyl, ethyl, propyl, or butyl; The biomolecule is a biomolecule containing a thiol group.

11. A method for preparing an aryl coupling agent, characterized in that: The preparation method comprises: placing compound 1 and carbon disulfide in a first solvent containing an alkaline solution to carry out a first reaction to obtain compound 2; Compound 2 and methyl iodide are placed in a second solvent for a second reaction to obtain an intermediate product; The intermediate product and the brominated amine compound with an amino protecting group are placed in a third solvent for a third reaction to obtain compound 3; The compound 3 and m-chloroperbenzoic acid are placed in a fourth solvent to carry out a fourth reaction to obtain the arylated coupling agent represented by formula I in claim 1. wherein R1 in the arylation coupling agent is any one of Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB or Bn; R2 is selected from methyl; R3 is Wherein, the structural formulas of the compound 1, the compound 2 and the compound 3 are as follows:

12. The preparation method according to claim 11, characterized in that The compound 1, the carbon disulfide and the first base are dissolved in the first solvent to carry out the first reaction, wherein the molar ratio of the compound 1 to the carbon disulfide is (2-20):1; the molar ratio of the compound 1 to the first base is (2-20):

1.

13. The preparation method according to claim 12, characterized in that The temperature of the first reaction is 85-95° C., and the time of the first reaction is 18-24 hours.

14. The preparation method according to claim 12, characterized in that The first base is selected from any one of the following: KOH or NaOH.

15. The preparation method according to claim 12, characterized in that The first solvent is selected from any one of the following: anhydrous ethanol or anhydrous methanol.

16. The preparation method according to claim 12, characterized in that Dissolving the compound 2 and N,N-diisopropylethylamine in the second solvent to obtain a solution of the compound 2; dissolving the methyl iodide in the second solvent to obtain a methyl iodide solution; The iodomethane solution is added dropwise to the compound 2 solution to carry out the second reaction.

17. The preparation method according to claim 16, characterized in that The molar ratio of the compound 2 to the methyl iodide is (0.8-1.2):1; the molar ratio of the compound 2 to the N,N-diisopropylethylamine is (2-20):

1.

18. The preparation method according to claim 16, characterized in that The second reaction is carried out in an ice bath for 30-60 min.

19. The preparation method according to claim 16, characterized in that The second reaction was carried out in an ice bath for 35-45 min.

20. The preparation method according to claim 16, characterized in that The second solvent is selected from any one of the following: tetrahydrofuran, acetone, 2-methyltetrahydrofuran or diethyl ether.

21. The preparation method according to claim 12, characterized in that The intermediate product is dissolved in the third solvent, and then a second base and the brominated amine compound with an amino protecting group are added to the third solvent in sequence to carry out the third reaction.

22. The preparation method according to claim 21, characterized in that The molar ratio of the compound 2 to the second base is (2-10):

1.

23. The preparation method according to claim 21, characterized in that The molar ratio of the compound 2 to the brominated amine compound with an amino protecting group is (2-20):

1.

24. The preparation method according to claim 21, characterized in that The temperature of the third reaction is 15-30° C., and the time of the third reaction is 2-4 hours.

25. The preparation method according to claim 21, characterized in that The brominated amine compound with an amino protecting group is selected from C2-C4 alkyl brominated amines with an amino protecting group.

26. The preparation method according to claim 21, characterized in that The brominated amine compound with an amino protecting group is selected from C2 or C3 alkyl brominated amines with an amino protecting group.

27. The preparation method according to claim 26, characterized in that The amino protecting group is selected from any one of Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB or Bn.

28. The preparation method according to claim 21, characterized in that The second base is selected from any one of the following: anhydrous sodium carbonate, anhydrous potassium carbonate, anhydrous lithium carbonate or anhydrous cesium carbonate, and the third solvent is selected from any one of the following: dimethylformamide, dimethyl sulfoxide or sulfolane.

29. The preparation method according to claim 11, characterized in that The molar ratio of the compound 3 to the m-chloroperbenzoic acid is (2-10):

1.

30. The preparation method according to claim 29, characterized in that The fourth solvent is selected from any one of the following: dichloromethane, chloroform, dioxane or tetrahydrofuran.

31. A method for preparing an aryl coupling agent, characterized in that: The preparation method comprises: Compound 4 and ethylenediamine with an amino protecting group are placed in a fifth solvent for a fifth reaction, and under the action of an acid-amine condensing agent and an organic base, the arylated coupling agent of formula II in claim 1 is obtained when X is Cl; Wherein, the fifth solvent is acetonitrile, dichloromethane or dimethylformamide; the acid amine condensation agent is selected from carbodiimides or onium salts, and the organic base is selected from triethylamine and N,N-diisopropylethylamine; When the acid amine condensing agent is selected from carbodiimides, the reaction system further includes an auxiliary agent, and the auxiliary agent is HOBt; The amino protecting group is any one of Boc, Cbz, Fmoc, Alloc, Teoc, Pht, Tfa, Trt, Dmb, PMB or Bn.

32. The preparation method according to claim 31, characterized in that The carbodiimide acid amine condensing agent is selected from DCC, EDCI or DIC; the onium salt acid amine condensing agent is selected from HATU, HBTU or BOP.

33. The preparation method according to claim 31, characterized in that The molar ratio of the compound 4 to the ethylenediamine with an amino protecting group is (2-20):

1.

34. A method for preparing an arylated coupling agent, characterized in that: The preparation method comprises: Dissolving compound 5 and 4-N-tert-butoxycarbonylaminopiperidine in a sixth solvent, respectively, to obtain a solution of compound 5 and a solution of 4-N-tert-butoxycarbonylaminopiperidine; adding a base to the 4-N-tert-butyloxycarbonylaminopiperidine solution to obtain an alkaline solution; adding the compound 5 solution dropwise to the alkaline solution at 0-4° C. to obtain a mixed solution; The mixed solution is placed at room temperature to react for 1.5-2.5 hours to obtain the arylated coupling agent represented by formula III in claim 1; Wherein, the sixth solvent is dichloromethane, dimethylformamide, acetonitrile, dioxane or tetrahydrofuran; 35. The preparation method according to claim 34, characterized in that The molar ratio of the compound 5 to the 4-N-tert-butyloxycarbonylaminopiperidine is (1-20):

1.

36. A method for preparing a fluorescent dye conjugate, characterized in that: The preparation method comprises: reacting the fluorescent dye with an arylated coupling agent to obtain the fluorescent dye conjugate; Wherein, the arylated coupling agent has the structural formula shown in Formula I, Formula II or Formula III: in, R1 is any one of Cbz, Fmoc, Alloc, Teoc, Pht, Tos, Tfa, Trt, Dmb, PMB or Bn; R3 is selected from any one of the following groups: -CH2-、 X is F or Cl; Wherein, the R2 is selected from any one of methyl, ethyl, propyl, or butyl.

37. The preparation method according to claim 36, characterized in that The fluorescent dye is selected from any one of cyanine dyes, rhodamine dyes, coumarin dyes and BODIPY dyes.

38. The preparation method according to claim 37, characterized in that The dye is selected from any one of the following: Cy3, Cy5, Cy3B, Cy5B, AF488, AF568 and TMR.

39. The preparation method according to claim 36, characterized in that The preparation method comprises: Deprotecting the optional amino protecting group in the aryl coupling agent to obtain a deaminated product; The deamination product is placed in a DMF solvent to react with a fluorescent dye and N,N-diisopropylethylamine to obtain the fluorescent dye conjugate; When the fluorescent dye is Cy3, Cy5, Cy7, AF488, AF568 or TMR, the deamination product is reacted in the form of Cy3-NHS, Cy5-NHS, Cy7-NHS, AF488-NHS, AF568-NHS or TMR-NHS; When the fluorescent dye is Cy3B or Cy5B, it reacts with the deamination product in the form of Cy3B-COOH or Cy5B-COOH.

40. The preparation method according to claim 39, characterized in that The arylation coupling agent is placed in dichloromethane and hydrochloric acid·dioxane to deprotect the amino protecting group.

41. The preparation method according to claim 39, characterized in that The molar ratio of the arylation coupling agent to the N,N-diisopropylethylamine is (2-20):1.

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