HaloTag ligand capable of being clicked as well as preparation method and application of HaloTag ligand

Abstract

The invention relates to the technical field of fluorescence labeling, in particular to a click HaloTag ligand and a preparation method and application thereof, the click HaloTag ligand is chloralkane-R, and R is a click chemical group capable of reacting with TCO dye or DBCO dye. The HaloTag ligand capable of being clicked, provided by the invention, can penetrate through a cell membrane and carry out click chemical marking in a living cell, so that living cell microtubule imaging is realized. In the aspect of biocompatibility, the ligand is low in molecular weight, fast in cell membrane penetration, free of obvious cytotoxicity and free of intracellular retention; the ligands all have chloralkane parts, can efficiently target the structure of fusion expression HaloTag protein in living cells and are stably combined through covalent bonds, so that non-specific adsorption is avoided; in the marking strategy level, the ligand base has a click chemical functional group and can react with any cell membrane permeable fluorescent probe with TCO or DBCO to mark the subcellular structure in the living cell, and the cell membrane impermeable dye can also be realized by modifying the cell penetrating peptide.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent labeling technology, and in particular to a clickable HaloTag ligand and a preparation method and application thereof. Background Art

[0002] With the continuous advancement of life sciences, in-depth understanding of the internal structure and function of cells has become increasingly important. Fluorescent labeling techniques for live cells have become indispensable tools in these studies. Common labeling techniques include fluorescent protein labeling, fluorescent dye labeling, bioorthogonal labeling, fluorescent-conjugated antibody labeling, bioluminescent labeling, and nanoparticle labeling. Fluorescent protein labeling has become a popular tool for studying the structure and function of living cells due to its high specificity, ease of use, and ability to dynamically observe the localization and movement of specific proteins or structures within cells in real time. However, traditional fluorescent proteins pose challenges in many studies due to their poor photostability, limited spectral range, and difficulty in modification. Nanoparticles (particularly quantum dots) offer excellent photostability, a wide spectral range, and ease of functionalization, and are currently overcoming these challenges. However, potential cytotoxicity and biocompatibility still limit their application in live cell labeling. To address these challenges, self-labeling protein tagging technologies such as HaloTag have gained widespread attention and application in recent years, providing more reliable solutions for cell labeling and imaging.

[0003] HaloTag is a specific protein tag derived from the genetically engineered dehalogenase (DhaA) of Rhodococcus rhodopsin. It consists of 297 amino acids and a molecular weight of 33.6 kDa. Within its surface, a 15-Å-deep pocket houses the catalytic amino acid active site, aspartic acid (Asp106). Its carboxyl group can undergo dehalogenation reactions with halogenated alkanes, catalyzed by asparagine (Asn41) and tryptophan (Trp107), to form an ester. Through this ester bond, it forms a stable covalent label with the dehalogenated substrate. However, the number of chloroalkyl substrates that can covalently bind to the HaloTag protein is limited, and the direct synthesis of fluorescent probes bearing chloroalkyl moieties is time-consuming and has limited applications.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a clickable HaloTag ligand and its preparation method and application, aiming to solve the problems that the number of existing chloroalkyl substrates that can covalently bind to HaloTag proteins is limited and the direct synthesis of fluorescent probes with chloroalkyl moieties is not only time-consuming but also has a single purpose.

[0006] The technical solutions of the present invention are as follows: A clickable HaloTag ligand, wherein the clickable HaloTag ligand is a chloroalkane-R, wherein R is a click chemistry group that can react with a TCO dye or a DBCO dye.

[0007] The clickable HaloTag ligand, wherein the clickable HaloTag ligand is chloroalkane-tetrazine or chloroalkane-azide.

[0008] A method for preparing a clickable HaloTag ligand comprises the steps of: 2-(2-aminoethoxy)ethanol and di-tert-butyl carbonate are reacted in an organic solvent to obtain a first product ; The first product is dissolved in an organic solvent, potassium tert-butoxide is added under an inert atmosphere, and 1-chloro-6-iodohexane is added after stirring to react to obtain the second product. ; The second product is dissolved in an organic solvent, and trifluoroacetic acid is added to react to obtain CA-NH2; The CA-NH2 and Tz-COOH are dissolved in an organic solvent, and EDC·HCl and DMAP are added under an inert atmosphere and stirred to obtain chloroalkane-tetrazine.

[0009] The preparation method of the clickable HaloTag ligand, wherein the 2-(2-aminoethoxy)ethanol and di-tert-butyl carbonate are reacted in an organic solvent to obtain a first product The steps include: Dissolving 2-(2-aminoethoxy)ethanol in an organic solvent, adding di-tert-butyl carbonate, stirring at 0° C. for 10-30 minutes, and then stirring at room temperature for 12-24 hours to obtain a reaction solution; The reaction solution is washed, dried, and purified by column chromatography to obtain the first product .

[0010] The method for preparing the clickable HaloTag ligand, wherein the CA-NH2 and Tz-COOH are dissolved in an organic solvent, and EDC·HCl and DMAP are added under an inert atmosphere and stirred to obtain the chloroalkane-tetrazine step, specifically comprises: The CA-NH2 and Tz-COOH are dissolved in an organic solvent, and EDC·HCl and DMAP are added under a nitrogen atmosphere to obtain a mixed solution; The mixture is stirred at room temperature for 12-24 hours, washed, dried, and purified by column chromatography using a mixture of methanol and dichloromethane as an eluent to obtain chloroalkane-tetrazine.

[0011] The method for preparing the clickable HaloTag ligand further comprises the steps of: The CA-NH2 and Dissolve in an organic solvent, add EDC·HCl and DMAP under an inert atmosphere and stir to obtain chloroalkane-azide.

[0012] The preparation method of the clickable HaloTag ligand, wherein the The preparation method comprises the steps of: Ethyl-4-hydroxybenzoic acid and N3-OTs were dissolved in acetonitrile, potassium carbonate was added under an inert atmosphere, and the third product was obtained after stirring and reacting. ; The third product is mixed with potassium hydroxide and ethanol, and stirred at 80-100° C. to obtain the fourth product. .

[0013] Application of a clickable HaloTag ligand in the preparation of drugs for treating tumors.

[0014] Application of a clickable HaloTag ligand in super-resolution optical wave imaging.

[0015] Application of a clickable HaloTag ligand in magnetic resonance imaging.

[0016] Beneficial Effects: The present invention provides a clickable HaloTag ligand, its preparation method, and application. The clickable HaloTag ligand is a chloroalkane-R, where R is a click chemistry group that reacts with a TCO dye or a DBCO dye. The clickable HaloTag ligand provided by the present invention can penetrate cell membranes and perform click chemistry labeling in living cells, enabling live cell microtubule imaging. In terms of biocompatibility, the above-mentioned ligands have low molecular weight, penetrate the cell membrane quickly, have no obvious cytotoxicity and no intracellular retention; in terms of labeling specificity and stability, the above-mentioned ligands all carry chloroalkane (CA) moieties, which can efficiently target the structure of HaloTag protein fused and expressed in living cells and stably bind through covalent bonds, avoiding nonspecific adsorption; in terms of labeling strategy, the above-mentioned ligands carry click chemistry functional groups, which can react with any cell membrane-permeable fluorescent probe with TCO or DBCO to label subcellular structures inside living cells. For cell membrane-impermeable dyes, this can also be achieved by modifying cell-penetrating peptides; in terms of imaging applications, fourth-order super-resolution optical wave imaging (SOFI) can be achieved by using clickable HaloTag ligands and dye-labeled cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The synthetic route for chloroalkane-tetrazine (CA-Tz) is shown below; Figure 2 The synthetic route for chloroalkane-azide (CA-N3) is shown below; Figure 3 is the H NMR spectrum of the first product; Figure 4 is the H NMR spectrum of the second product; Figure 5 is the H NMR spectrum of CA-NH2; Figure 6 is the H NMR spectrum of CA-Tz; Figure 7 is the H NMR spectrum of the fourth product; Figure 8 is the H NMR spectrum of CA-N3; Figure 9 This is the result of confocal imaging of CA-Tz labeled living cells; Figure 10 This is the result of confocal imaging of CA-N3 labeled living cells; Figure 11 The confocal imaging results of living cells labeled with different concentrations of CA-Tz; Figure 12 This is the confocal imaging result of live cells labeled with 10 μM CA-Tz for 0.5 h; Figure 13 SOFI images of microtubules in living cells labeled with green fluorescent protein and CA-Tz; Figure 14 A magnified image showing the difference in wide-field and fourth-order image retention between green fluorescent protein and CA-Tz labeled microtubules in living cells; Figure 15 Quantitative analysis of image retention rate and resolution data of green fluorescent protein and CA-Tz labeled microtubules in living cells. DETAILED DESCRIPTION

[0018] The present invention provides a clickable HaloTag ligand, its preparation method, and application. To clarify the objectives, technical solutions, and effects of the present invention, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention.

[0019] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art in the art to which the present invention belongs. It should also be understood that terms such as those defined in common dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0020] Currently, there are only a limited number of chloroalkyl substrates that can covalently bind to HaloTag proteins, and the direct synthesis of fluorescent probes bearing chloroalkyl moieties is time-consuming and limited in application. The development of the concept of bioorthogonal reactions offers a novel approach to address this problem. Bioorthogonal reactions do not interfere with native biological functions and are characterized by high biocompatibility, specificity, and versatility. These reactions, including the ring-strain-promoted azide-alkyne cycloaddition (SPAAC) in click chemistry and the inverse electron demand Diels-Alder reaction (iEDDA) between tetrazines (Tz) and trans-cycloolefins (TCOs), are gaining increasing attention in live-cell imaging and molecular labeling. Therefore, the development of bifunctional ligands with clickable functional groups that can bind to HaloTag proteins is crucial.

[0021] Based on this, the present invention provides a clickable HaloTag ligand, wherein the clickable HaloTag ligand is a chloroalkane-R, wherein R is a click chemistry group that can react with a TCO dye or a DBCO dye.

[0022] In this embodiment, the provided clickable HaloTag ligand can penetrate the cell membrane and perform click chemistry labeling in living cells, enabling live-cell microtubule imaging. In terms of biocompatibility, the ligand has a low molecular weight, rapidly penetrates the cell membrane, has no significant cytotoxicity, and exhibits no intracellular retention. In terms of labeling specificity and stability, the ligands all carry a chloroalkane (CA) moiety, which can efficiently target structures fused to and expressed in living cells with the HaloTag protein and stably bind via covalent bonds, avoiding nonspecific adsorption. In terms of labeling strategy, the ligands carry click chemistry functional groups that can react with any cell-permeable fluorescent probe bearing TCO or DBCO to label subcellular structures within living cells. Cell-impermeable dyes can also be achieved by modifying cell-penetrating peptides. In terms of imaging applications, fourth-order super-resolution optical wave imaging (SOFI) can be achieved using cells labeled with clickable HaloTag ligands and dyes.

[0023] Specifically, the present invention selects HaloTag protein as the target for synthetic small molecules for labeling living cells. Plasmids can be synthesized and cells can be transfected by genetic engineering means. It has strong specificity and is easy to operate. The clickable HaloTag ligand based on self-labeling protein tag technology and click chemistry reaction is synthesized for live cell labeling. Compared with the direct use of fluorescent proteins, nanoparticles or probes with CA groups for labeling, chloroalkane-R has better biocompatibility, exhibits stronger targeting specificity, and achieves stable binding through covalent bonds. In addition, these compounds can be combined with various dyes, drugs or chelating agents with TCO or DBCO groups, which can break the limitation of HaloTag substrates and thus be widely used in super-resolution, nuclear magnetic resonance imaging, and tumor diagnosis and treatment of living cells and living animals. At the same time, by directly incubating chloroalkane-R with cells expressing HaloTag, a handle for click chemistry reaction can be introduced into the cells without affecting the physiological functions of living cells. This method is more suitable for studying physiological activities inside living cells.

[0024] In some embodiments, the clickable HaloTag ligand is a chloroalkane-tetrazine (CA-Tz) or a chloroalkane-azide (CA-N3). In terms of biocompatibility, chloroalkane-tetrazine and chloroalkane-azide have low molecular weight, rapidly penetrate cell membranes, lack significant cytotoxicity, and exhibit no intracellular retention. In terms of labeling specificity and stability, both ligands carry a chloroalkane moiety (CA), which can efficiently target structures fused to and expressed in living cells with HaloTag proteins and stably bind via covalent bonds, avoiding nonspecific adsorption. In terms of labeling strategy, the two ligands, respectively bearing click chemistry functional groups tetrazine (Tz) and azide (N3), can react with any cell-permeable fluorescent probe bearing TCO or DBCO to label subcellular structures within living cells. Cell-impermeable dyes can also be achieved by modifying cell-penetrating peptides. In terms of imaging applications, cells labeled with clickable HaloTag ligands and dyes can achieve fourth-order super-resolution optical wave imaging (SOFI).

[0025] Specifically, the chemical structural formula of the chloroalkane-tetrazine (CA-Tz) is The chemical structural formula of the chloroalkane-azide (CA-N3) is .

[0026] In some embodiments, live cell microtubule imaging can be achieved by synthesizing CA-Tz or CA-N3 with different structural formulas for click chemistry labeling. For example, the structural formulas may contain different numbers of polyethylene glycol molecules or the Tz or N3 may be replaced with other click chemistry groups that react with TCO or DBCO dyes.

[0027] In addition, the present invention also provides a method for preparing a clickable HaloTag ligand, comprising the steps of: Step S10: reacting 2-(2-aminoethoxy)ethanol with di-tert-butyl carbonate in an organic solvent to obtain a first product ; Step S20: dissolving the first product in an organic solvent, adding potassium tert-butoxide under an inert atmosphere, stirring, and then adding 1-chloro-6-iodohexane to react to obtain a second product. ; Step S30: dissolving the second product in an organic solvent, adding trifluoroacetic acid to react, and obtaining CA-NH2; Step S40: dissolving the CA-NH2 and Tz-COOH in an organic solvent, adding EDC·HCl and DMAP under an inert atmosphere and stirring to obtain chloroalkane-tetrazine.

[0028] In this embodiment, the chloroalkane-tetrazine prepared using this method can react with any cell-permeable fluorescent probe bearing TCO or DBCO to label subcellular structures within living cells. Cell-impermeable dyes can also be modified with cell-penetrating peptides. This overcomes the substrate limitations of HaloTag, enabling widespread application in super-resolution imaging of living cells and animals, magnetic resonance imaging, and tumor diagnosis and treatment.

[0029] In some embodiments, in step S10, 2-(2-aminoethoxy)ethanol and di-tert-butyl carbonate are reacted in an organic solvent to obtain a first product. The steps include: Step S11: 2-(2-aminoethoxy)ethanol ( ) is dissolved in an organic solvent, and di-tert-butyl carbonate is added, stirred at 0° C. for 10-30 minutes, and then stirred at room temperature for 12-24 hours to obtain a reaction solution; Step S12: washing, drying, and column chromatography purification of the reaction solution to obtain the first product .

[0030] Specifically, 2-(2-aminoethoxy)ethanol is dissolved in an anhydrous organic solvent, di-tert-butyl carbonate is added to the above solution, and the mixture is stirred at 0°C for 10 to 30 minutes, and then stirred at room temperature for 12 to 24 hours; after the reaction is completed, the solution is washed with a saturated sodium bicarbonate solution, brine and water, and dried over anhydrous sodium sulfate; after filtering and evaporating the solvent, the crude product is purified by column chromatography using an ethyl acetate / petroleum ether mixture as an eluent to finally obtain a colorless oily liquid, which is the first product. .

[0031] In some embodiments, the step S20 specifically includes: dissolving the first product in an anhydrous organic solvent, then cooling at 0°C, adding potassium tert-butoxide in a nitrogen atmosphere, then stirring at 0°C for 1 to 4 hours, then adding 1-chloro-6-iodohexane dropwise, and stirring at this temperature for 3 to 5 hours. Then, stirring is continued at room temperature for 12 to 24 hours. Subsequently, the solvent is evaporated and the residue is added to water; the residue is extracted with ethyl acetate (EtOAc) 3 to 4 times, and the organic layers are combined and dried over anhydrous sodium sulfate. After filtering and evaporating the solvent, the crude product is purified by column chromatography using an ethyl acetate / petroleum ether mixture as an eluent to finally obtain a colorless oily liquid, i.e., the second product. .

[0032] In some embodiments, step S30 specifically includes: dissolving the second product in an anhydrous organic solvent, adding trifluoroacetic acid (TFA) at 0°C; then, stirring the mixture at room temperature for 6 to 12 hours; then, removing the solvent and extracting with ethyl acetate (EtOAc) 3 to 4 times; after combining the organic layers, washing with saturated sodium carbonate (Na2CO3) and water, and drying over anhydrous sodium sulfate. After filtering and evaporating the solvent, the crude product is purified by column chromatography using a methanol / dichloromethane mixture as an eluent. The resulting CA-NH2 is a colorless oily liquid with the chemical formula of CA-NH2. .

[0033] In some embodiments, in step S40, the step of dissolving the CA-NH2 and Tz-COOH in an organic solvent, adding EDC·HCl and DMAP under an inert atmosphere and stirring to obtain the chloroalkane-tetrazine specifically includes: Step S41: dissolving the CA-NH2 and Tz-COOH in an organic solvent, adding EDC·HCl and DMAP under a nitrogen atmosphere to obtain a mixed solution; wherein the structural formula of Tz-COOH is ; Step S42: stirring the mixture at room temperature for 12-24 hours, washing, drying, and purifying by column chromatography using a mixture of methanol and dichloromethane as an eluent to obtain chloroalkane-tetrazine.

[0034] Specifically, CA-NH2 and Tz-COOH are dissolved in an appropriate amount of an organic solvent. Subsequently, EDC·HCl and DMAP are added under a nitrogen atmosphere to obtain a mixed solution. The mixed solution is stirred at room temperature for 12 to 24 hours. Thereafter, the solution is washed with saturated brine and water and dried over anhydrous sodium sulfate. After filtering and evaporating the solvent, the crude product is purified by column chromatography using a methanol / dichloromethane mixture as an eluent. The obtained CA-Tz is a pink oily liquid. The structural formula of CA-Tz is .

[0035] In some embodiments, the method for preparing the clickable HaloTag ligand further comprises the steps of: Step S50: The CA-NH2 is mixed with Dissolve in an organic solvent, add EDC·HCl and DMAP under an inert atmosphere and stir to obtain chloroalkane-azide.

[0036] In some embodiments, the step S50 specifically comprises: Dissolve in an organic solvent. Subsequently, EDC·HCl and DMAP were added under a nitrogen atmosphere. The mixture was stirred at room temperature for 12 to 24 hours. The solution was washed with saturated brine and water and dried over anhydrous sodium sulfate (Na2SO4). After filtration and evaporation of the solvent, the crude product was purified by column chromatography using a methanol / dichloromethane (DCM) mixture (volume ratio 1:80) as an eluent to obtain a colorless oily liquid CA-N3. The chemical formula of CA-N3 is .

[0037] In some embodiments, in step S50, the The preparation method comprises the steps of: Step S51: dissolving ethyl-4-hydroxybenzoic acid and N3-OTs in acetonitrile, adding potassium carbonate under an inert atmosphere, and stirring to react to obtain the third product ; Step S52: Mix the third product with potassium hydroxide and ethanol, and stir at 80-100° C. to obtain the fourth product. .

[0038] In some embodiments, the step S51 specifically comprises: adding ethyl-4-hydroxybenzoic acid ( ) and N3-OTs ( ) is dissolved in acetonitrile. Potassium carbonate (K2CO3) is added under a nitrogen atmosphere. The mixture is stirred at 80-100°C for 12-24 hours. After filtering out the solid, the residue is purified by column chromatography using a methanol / dichloromethane mixture as the eluent to obtain a crude product, i.e., the third product. The crude product is directly used for the reaction in step S52.

[0039] In some embodiments, step S52 specifically comprises: mixing the third product, potassium hydroxide, and ethanol, and stirring at 80-100°C overnight. After cooling to room temperature, the solvent is evaporated. The residue is added to an appropriate amount of hydrochloric acid (HCl) and extracted three times with ethyl acetate (EtOAc). The combined organic layers are dried over anhydrous sodium sulfate (Na2SO4). After filtration, the solvent is evaporated to obtain a crude product. The crude product is purified by column chromatography using a methanol / dichloromethane (DCM) mixture as an eluent to obtain the fourth product as a pale yellow oily liquid.

[0040] In addition, the present invention also provides an application of a clickable HaloTag ligand in the preparation of a drug for treating tumors, an application of a clickable HaloTag ligand in super-resolution optical wave imaging, and an application of a clickable HaloTag ligand in nuclear magnetic resonance imaging.

[0041] In some embodiments, clickable BG / BC-Tz or BG / BC-N3 can be synthesized by replacing the HaloTag protein with other self-labeling protein tags such as SNAP-tag or CLIP-tag.

[0042] The present invention will be described in detail with reference to the following examples. It should also be understood that the following examples are only intended to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above disclosure of the present invention fall within the scope of protection of the present invention. Example

[0043] This embodiment provides a clickable HaloTag ligand, including chloroalkane-tetrazine (CA-Tz) or chloroalkane-azide (CA-N3), the synthesis routes of which are as follows: Figure 1 and Figure 2 As shown, the specific steps include: 1. Synthesis of chloroalkane-tetrazine (CA-Tz), the synthesis route is as follows Figure 1 shown.

[0044] 1) Synthesis of the first product 2-(2-Aminoethoxy)ethanol (2.83 mL, 1 eq) was dissolved in 100 mL of anhydrous dichloromethane (DCM). Di-tert-butyl carbonate (6.46 g, 1.1 eq) was added to the above solution and stirred at 0°C for 10 minutes, and then stirred at room temperature for 12 hours. After the reaction was completed, the solution was washed with saturated sodium bicarbonate solution, brine and water in sequence, and dried over anhydrous sodium sulfate. After filtering and evaporating the solvent, the crude product was purified by column chromatography using an ethyl acetate / petroleum ether mixture (volume ratio 1:1) as the eluent. Finally, 5.15 g of the first product was obtained as a colorless oily liquid with a yield of 93%. The hydrogen nuclear magnetic resonance spectrum of the first product is shown in FIG. Figure 3 As shown, 1 H NMR (400 MHz, CDCl3) δ 3.74 (dd, J = 5.2, 3.9 Hz, 2H), 3.60 -3.53 (m, 4H), 3.33 (J, J = 5.1 Hz, 2H), 1.45 (S, 9H).

[0045] 2) Synthesis of the second product The first product (2.42 g, 1 eq) was dissolved in anhydrous tetrahydrofuran (THF, 100 mL). After cooling at 0°C, potassium tert-butoxide (1.32 g, 1 eq) was added under a nitrogen atmosphere. Stir at 0°C for 1 hour, and then 1-chloro-6-iodohexane (3.49 g, 1.2 eq) was added dropwise and stirred at this temperature for 3 hours. Then, stirring was continued at room temperature for 12 hours. Subsequently, the solvent was evaporated and the residue was added to 100 mL of water. The residue was extracted three times with ethyl acetate (EtOAc), and the organic layers were combined and dried over anhydrous sodium sulfate. After filtering and evaporating the solvent, the crude product was purified by column chromatography using an ethyl acetate / petroleum ether mixture (volume ratio 1:6) as the eluent. The final obtained 1.76 g of the second product was a colorless oily liquid with a yield of 46%. The nuclear magnetic resonance hydrogen spectrum of the second product is shown as follows Figure 4 As shown, 1 H NMR (400 MHz, CDCl3) δ 3.62-3.52 (m, 8H), 3.47 (t, J = 6.7 Hz, 2H), 3.32 (t, J = 5.2 Hz, 2H), 1.82-1.73 (m, 2H), 1.66-1.56 (m, 2H), 1.45 (s, 13H).

[0046] 3) Synthesis of compound CA-NH2 The second product (1.76 g, 1 eq) was dissolved in 100 mL of anhydrous dichloromethane (DCM), and trifluoroacetic acid (TFA, 6.46 mL, 16 eq) was added at 0°C. Subsequently, the mixture was stirred at room temperature for 6 hours. Then, the solvent was removed and extracted three times with ethyl acetate (EtOAc). After combining the organic layers, they were washed with saturated sodium carbonate (Na2CO3) and water and dried over anhydrous sodium sulfate. After filtering and evaporating the solvent, the crude product was purified by column chromatography using a methanol / dichloromethane mixture (volume ratio 1:16) as an eluent. The final CA-NH2 was a colorless oily liquid with a yield of 0.54 g and a yield of 44%. The hydrogen nuclear magnetic resonance spectrum of CA-NH2 is shown in FIG. Figure 5 As shown, 1 H NMR (400 MHz, CDCl3) δ 4.18 (s, 2H), 3.69-3.44 (m, 8H), 3.10 -2.89 (m, 2H), 1.88-1.70 (m, 2H), 1.70-1.54 (m, 2H), 1.51-1.30 (m, 4H).

[0047] 4) Synthesis of compound CA-Tz Tz-COOH was synthesized according to the method in the literature. CA-NH2 (0.22 g, 1 eq) and Tz-COOH (0.33 g, 1.5eq) were dissolved in 100 mL of anhydrous dichloromethane (DCM). Subsequently, EDC·HCl (0.55 g, 3 eq) and DMAP (10 mg) were added under a nitrogen atmosphere. The mixture was stirred at room temperature for 12 hours. After that, the solution was washed with saturated brine and water and dried over anhydrous sodium sulfate. After filtering and evaporating the solvent, the crude product was purified by column chromatography using a methanol / dichloromethane mixture (volume ratio 1:100) as an eluent. The obtained CA-Tz was a pink oily liquid with a yield of 0.28 g and a yield of 68%. The hydrogen nuclear magnetic resonance spectrum of CA-Tz is shown in FIG. Figure 6 As shown, 1 H NMR (400 MHz, CDCl3) δ 8.57 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 8.3 Hz, 2H), 6.06 (s, 1H), 3.66 (s, 2H), 3.59-3.40 (m, 12H), 3.10 (s, 3H), 1.80-1.71 (m, 2H), 1.61-1.54 (m, 2H), 1.47-1.40 (m, 2H), 1.39-1.32 (m, 2H). Mass spectrum (MS): theoretical value [M+H] + The measured value is 436.2037 and the actual value is 436.2104.

[0048] 2. Synthesis of chloroalkane-azide (CA-N3), the synthesis route is as follows Figure 2 shown.

[0049] 1) Synthesis of the third product N3-OTs was synthesized according to a literature method. Ethyl-4-hydroxybenzoic acid (0.61 g, 1 eq) and N3-OTs (0.81 g, 1.3 eq) were dissolved in acetonitrile. Potassium carbonate (K2CO3, 1.54 g, 3 eq) was added under a nitrogen atmosphere. The mixture was stirred at 80°C for 12 hours. After filtering off the solid, the residue was purified by column chromatography using a methanol / dichloromethane mixture (1:300 by volume) as the eluent. The crude product was used directly in the next reaction.

[0050] 2) Synthesis of the fourth product The third product (0.54 g, 1 eq), potassium hydroxide (0.19 g, 2 eq) and ethanol (10 mL) were mixed and stirred at 80 °C overnight. After cooling to room temperature, the solvent was evaporated. The residue was added to 2 mL of hydrochloric acid (HCl) and extracted three times with ethyl acetate (EtOAc). The combined organic layers were dried over anhydrous sodium sulfate (Na2SO4). After filtration, the solvent was evaporated to obtain a crude product. The crude product was purified by column chromatography using a methanol / dichloromethane (DCM) mixture (volume ratio 1:80) as an eluent. The fourth product was obtained as a light yellow oily liquid with a yield of 0.49 g (98%). The hydrogen nuclear magnetic resonance spectrum of the fourth product is shown as follows Figure 7 As shown, 1 H NMR (400 MHz, CDCl3): δ 8.08 – 8.03 (m, 2H), 6.98-6.94 (m, 2H), 4.24-4.19 (m, 2H), 3.91 (dd, J = 5.3, 4.1 Hz, 2H), 3.78-3.74 (m, 2H), 3.72-3.67 (m, 4H), 3.42-3.37 (m, 2H).

[0051] 3) Synthesis of compound CA-N3 The synthesis of CA-N3 followed the same procedure as CA-Tz. CA-NH2 (0.31 g, 1 eq) and the fourth product (0.53 g, 1.5 eq) were dissolved in 100 mL of anhydrous dichloromethane (DCM). Subsequently, EDC·HCl (0.80 g, 3 eq) and DMAP (10 mg) were added under a nitrogen atmosphere. The mixture was stirred at room temperature for 12 hours. The solution was washed with saturated brine and water, dried, and then purified with anhydrous sodium sulfate (Na2SO4). After filtration and evaporation of the solvent, the crude product was purified by column chromatography using a methanol / dichloromethane (DCM) mixture (volume ratio 1:80) as the eluent. CA-N3 was obtained as a colorless oil in a yield of 0.40 g (58%).

[0052] The nuclear magnetic resonance hydrogen spectrum of CA-N3 is as follows Figure 8 As shown, 1 H NMR (400 MHz, CDCl3): δ 7.78-7.71 (m, 2H), 6.96-6.90 (m, 2H), 6.62 (s, 1H), 4.20-4.15 (m, 2H), 3.91- 3.87 (m, 2H), 3.77-3.73 (m, 2H), 3.72-3.63 (m, 10H), 3.62 -3.57 (m, 2H), 3.52 (t, J =6.7 Hz, 2H), 3.46 (t, J = 6.7 Hz, 2H), 3.42 - 3.36 (m, 2H), 1.79- 1.70 (m,2H), 1.62 - 1.54 (m, 2H), 1.47 - 1.31 (m, 4H). Mass spectrum (MS): m / z theoretical value [M+H] + is 501.2402; the actual value is 501.2478.

[0053] 3. Clickable HaloTag ligand labeling of microtubules in living cells CA-Tz or CA-N3 (1 mM, dissolved in DMSO) was diluted to 50 nM to 10 μM in fresh culture medium (containing FBS). These solutions were incubated with cells transfected with the mEmerald-Halo-Ensconsin plasmid at 37°C in a humidified atmosphere with 5% CO₂ for 0.5 to 24 hours. The supernatant was then discarded, and the cells were gently washed three times with PBS. The cells were then incubated with dyes bearing TCO or DBCO functional groups (50 nM to 10 μM) in fresh culture medium (containing fetal bovine serum) for 4 to 24 hours. Prior to optical imaging, the cells were gently rinsed three times with PBS and imaged in growth medium (DMEM, 10% FBS, without phenol red).

[0054] 4. Confocal Imaging of Live Cell Microtubules Labeled with Clickable HaloTag Ligands Live cell confocal laser scanning microscopy (CLSM) images were acquired on a Zeiss 980 microscope equipped with 488 nm (green fluorescent protein channel) and 639 nm (silicon rhodamine channel) lasers using a 100x oil immersion objective and an sCMOS camera with selectable exposure time. All confocal images were recorded at a resolution of 2048 × 2048 pixels. Image processing and fluorescence intensity quantification were performed using Fiji ImageJ. The results of confocal imaging of CA-Tz-labeled live cells are shown in Figure 2. Figure 9 The results of confocal imaging of CA-N3-labeled living cells are shown in Figure 10 The confocal imaging results of living cells labeled with different concentrations of CA-Tz are shown in Figure 11 The results of confocal imaging of live cells labeled with 10 μM CA-Tz for 0.5 h are shown in Figure 12 The results demonstrate that both CA-Tz and CA-N3 can label microtubules within a short timeframe and over a wide concentration range. Furthermore, because DBCO dye is easily retained in vesicles, generating a strong nonspecific signal, CA-Tz-labeled images offer higher quality under identical labeling and imaging conditions, making it more suitable for labeling and imaging microtubules in living cells.

[0055] 5. Super-resolution fluorescence fluctuation imaging of live cell microtubules labeled with clickable HaloTag ligands Fluorescence images of microtubules in COS-7 cells were acquired on a Nikon N-STORM system equipped with a CFI Apo TIRF 100× oil-immersion objective (NA: 1.4) and an Andor Life 897 EMCCD detector. To ensure the highest signal-to-noise ratio and uniform excitation intensity across the field of view, the TIRF angle was precisely adjusted according to the selected excitation wavelength. For imaging microtubules expressing mEmerald, a 488 nm laser was used for excitation, along with a ZET405 / 488 / 561 / 640xv2 excitation filter and an FF01-711 / 25-25 emission filter. For silicon-rhodamine-labeled microtubules, a 640 nm laser and a ZET405 / 488 / 561 / 640xv2 dual-band dichroic mirror were used for excitation, and a 1.6x magnification was applied during imaging. The EMCCD detector was cooled to -80°C and the frame transfer mode was used. The region of interest (ROI) was selected based on the range of cells in the field of view to determine the size of each image. The exposure time of each test set was 20 ms, and the image sequence consisted of 1500 frames. Among them, the SOFI images of green fluorescent protein and CA-Tz labeled microtubules in living cells are shown in Figure 13 , highlighting the difference in widefield and fourth-order image retention between green fluorescent protein and CA-Tz labeled microtubules in living cells. Figure 14 Quantitative analysis of retention rate and resolution calculation of images of green fluorescent protein and CA-Tz labeled microtubules in living cells are shown in Figure 15 . The results show that both green fluorescent protein and CA-Tz labeled live cell microtubules can achieve fourth-order SOFI imaging, and the structures of CA-Tz labeled microtubules in wide-field and fourth-order SOFI images are almost the same, with minimal loss, while the structure of microtubules expressing green fluorescent protein is significantly lost. This fully demonstrates that the microtubule structure labeled with CA-Tz has a higher retention rate in the fourth-order SOFI image, further emphasizing the superiority of CA-Tz labeling. In addition, the resolution of live cell microtubules labeled with CA-Tz is significantly improved compared with the wide-field image. The resolution of the fourth-order SOFI (161 nm) is 2 times higher than that of the wide-field image (332 nm), while the resolution of the fourth-order SOFI microtubule image of green fluorescent protein is only 189 nm.

[0056] In summary, the present invention provides a clickable HaloTag ligand, its preparation method, and application. The clickable HaloTag ligand is a chloroalkane-R, where R is a click chemistry group that reacts with a TCO dye or a DBCO dye. The clickable HaloTag ligand provided by the present invention can penetrate cell membranes and perform click chemistry labeling in living cells, enabling live-cell microtubule imaging. In terms of biocompatibility, the above-mentioned ligands have low molecular weight, penetrate the cell membrane quickly, have no obvious cytotoxicity and no intracellular retention; in terms of labeling specificity and stability, the above-mentioned ligands all carry chloroalkane (CA) moieties, which can efficiently target the structure of HaloTag protein fused and expressed in living cells and stably bind through covalent bonds, avoiding nonspecific adsorption; in terms of labeling strategy, the above-mentioned ligands carry click chemistry functional groups, which can react with any cell membrane-permeable fluorescent probe with TCO or DBCO to label subcellular structures inside living cells. For cell membrane-impermeable dyes, this can also be achieved by modifying cell-penetrating peptides; in terms of imaging applications, fourth-order super-resolution optical wave imaging (SOFI) can be achieved by using clickable HaloTag ligands and dye-labeled cells.

[0057] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A clickable HaloTag ligand, characterized in that: The clickable HaloTag ligand is a chloroalkane-R, wherein R is a click chemistry group that can react with a TCO dye or a DBCO dye.

2. The clickable HaloTag ligand according to claim 1, characterized in that The clickable HaloTag ligand is chloroalkane-tetrazine or chloroalkane-azide.

3. A method for preparing a clickable HaloTag ligand, characterized in that: Includes steps: 2-(2-aminoethoxy)ethanol and di-tert-butyl carbonate are reacted in an organic solvent to obtain a first product ; The first product is dissolved in an organic solvent, potassium tert-butoxide is added under an inert atmosphere, and 1-chloro-6-iodohexane is added after stirring to react to obtain a second product. ; The second product is dissolved in an organic solvent, and trifluoroacetic acid is added to react to obtain CA-NH2; The CA-NH2 and Tz-COOH are dissolved in an organic solvent, and EDC·HCl and DMAP are added under an inert atmosphere and stirred to obtain chloroalkane-tetrazine.

4. The method for preparing a clickable HaloTag ligand according to claim 3, characterized in that: The method comprises reacting 2-(2-aminoethoxy)ethanol with di-tert-butyl carbonate in an organic solvent to obtain a first product. The steps include: Dissolving 2-(2-aminoethoxy)ethanol in an organic solvent, adding di-tert-butyl carbonate, stirring at 0° C. for 10-30 minutes, and then stirring at room temperature for 12-24 hours to obtain a reaction solution; The reaction solution is washed, dried, and purified by column chromatography to obtain the first product .

5. The method for preparing a clickable HaloTag ligand according to claim 3, characterized in that: The step of dissolving the CA-NH2 and Tz-COOH in an organic solvent, adding EDC·HCl and DMAP under an inert atmosphere and stirring to obtain chloroalkane-tetrazine specifically comprises: The CA-NH2 and Tz-COOH are dissolved in an organic solvent, and EDC·HCl and DMAP are added under a nitrogen atmosphere to obtain a mixed solution; The mixed solution is stirred at room temperature for 12-24 hours, washed, dried, and purified by column chromatography using a mixture of methanol and dichloromethane as an eluent to obtain chloroalkane-tetrazine.

6. The method for preparing a clickable HaloTag ligand according to claim 3, characterized in that: The method for preparing the clickable HaloTag ligand further comprises the steps of: The CA-NH2 and Dissolve in an organic solvent, add EDC·HCl and DMAP under an inert atmosphere and stir to obtain chloroalkane-azide.

7. The method for preparing a clickable HaloTag ligand according to claim 6, characterized in that: Said The preparation method comprises the steps of: Dissolve ethyl-4-hydroxybenzoic acid and N3-OTs in acetonitrile, add potassium carbonate under an inert atmosphere, and stir to obtain the third product. ; The third product is mixed with potassium hydroxide and ethanol, and stirred at 80-100° C. to obtain a fourth product. .

8. Use of the clickable HaloTag ligand according to any one of claims 1 to 2 in the preparation of a drug for treating tumors.

9. Use of the clickable HaloTag ligand according to any one of claims 1 to 2 in super-resolution optical wave imaging.

10. Use of the clickable HaloTag ligand according to any one of claims 1 to 2 in nuclear magnetic resonance imaging.

Images