A clickable halo tag ligand and its preparation method and application

Abstract

The present application relates to the field of fluorescent labeling technology, and particularly relates to a clickable HaloTag ligand, a preparation method and application thereof, the clickable HaloTag ligand is chloroalkane-R, wherein R is a click chemistry group capable of reacting with TCO dye or DBCO dye. The clickable HaloTag ligand provided by the present application can penetrate cell membranes and perform click chemistry labeling in living cells, and realizes live cell microtubule imaging. In terms of biocompatibility, the above ligand has low molecular weight, fast penetration of cell membranes, no obvious cytotoxicity and no intracellular retention; the above ligand all has a chloroalkane part, can efficiently target the structure of HaloTag protein fusion expression in living cells and stably combine through a covalent bond, and avoids non-specific adsorption; in terms of labeling strategy, the above ligand has a click chemistry functional group, can react with any cell membrane permeable fluorescent probe with TCO or DBCO to label subcellular structures in living cells, and for cell membrane impermeable dyes, cell penetrating peptides can also be modified to achieve the purpose.

Description

Technical Field

[0001] This invention relates to the field of fluorescent labeling technology, and in particular to a clickable HaloTag ligand, its preparation method, and its application. Background Technology

[0002] With the continuous development of life sciences, the importance of in-depth research into the internal structure and function of cells is becoming increasingly prominent. In these studies, live-cell fluorescent labeling technology has become an indispensable tool. Commonly used labeling techniques include fluorescent protein labeling, fluorescent dye labeling, bioorthogonal labeling, fluorescent conjugated antibody labeling, bioluminescent labeling, and nanoparticle labeling. Among these, fluorescent protein labeling, due to its high specificity, ease of operation, and ability to observe the localization and movement of specific proteins or structures within cells in real time and dynamically, has become a commonly used tool for studying the structure and function of live cells. However, traditional fluorescent proteins have presented challenges in many studies due to their poor photostability, limited spectral range, and difficulty in modification. Nanoparticles (especially quantum dots) possess excellent photostability, a wide spectral range, and ease of functionalization, and are overcoming these problems. However, their potential cytotoxicity and biocompatibility issues still limit their application in live-cell labeling. Based on these challenges, self-labeled protein tagging technologies such as HaloTag have received widespread attention and application in recent years, providing a more reliable solution for cell labeling and imaging.

[0003] HaloTag is a specific protein tag derived from the dehalogenase (DhaA) of *Rhodococcus purpureus* through genetic engineering. It contains 297 amino acids and a molecular weight of 33.6 kDa. Within a 15 Å deep pocket on its surface lies the catalytic amino acid active site aspartic acid (Asp106). Its carboxyl group can undergo a dehalogenation reaction with haloalkanes under the catalysis of asparagine (Asn41) and tryptophan (Trp107), forming an ester. This ester bond then forms a stable covalent tag with the dehalogenated substrate. However, the number of chloroalkyl substrates that can covalently bind to the HaloTag protein is limited, and directly synthesizing fluorescent probes with chloroalkyl moieties is not only time-consuming but also has limited applications.

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

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a clickable HaloTag ligand, its preparation method and application, in order to solve the problems that the number of existing chloroalkyl substrates that can covalently bind to HaloTag proteins is limited and that directly synthesizing fluorescent probes with chloroalkyl moieties is not only time-consuming but also has limited applications.

[0006] The technical solution of the present invention is as follows:

[0007] A clickable HaloTag ligand, wherein the clickable HaloTag ligand is a chloroalkane-R, wherein R is a clickable chemical group that can react with TCO dyes or DBCO dyes.

[0008] The clickable HaloTag ligand is, in this case, a chloroalkane-tetraazine or a chloroalkane-azide.

[0009] A method for preparing a clickable HaloTag ligand includes the following steps:

[0010] 2-(2-aminoethoxy)ethanol was reacted with di-tert-butyl carbonate in an organic solvent to give the first product. ;

[0011] The first product was dissolved in an organic solvent, and potassium tert-butoxide was added under an inert atmosphere. After stirring, 1-chloro-6-iodohexane was added to react and give the second product. ;

[0012] The second product was dissolved in an organic solvent, and trifluoroacetic acid was added to react with it to obtain CA-NH2;

[0013] The CA-NH2 and Tz-COOH were dissolved in an organic solvent, and EDC·HCl and DMAP were added under an inert atmosphere. After stirring, chloroalkyl-tetraazine was obtained.

[0014] The method for preparing the clickable HaloTag ligand, wherein 2-(2-aminoethoxy)ethanol and di-tert-butyl carbonate are reacted in an organic solvent to obtain the first product. The steps specifically include:

[0015] Dissolve 2-(2-aminoethoxy)ethanol in an organic solvent, add di-tert-butyl carbonate and stir at 0°C for 10-30 minutes, then stir at room temperature for 12-24 hours to obtain a reaction solution;

[0016] The reaction solution was washed, dried, and purified by column chromatography to obtain the first product. .

[0017] The method for preparing the clickable HaloTag ligand, wherein the steps of dissolving CA-NH2 and Tz-COOH in an organic solvent, adding EDC·HCl and DMAP under an inert atmosphere and stirring to obtain a chloroalkane-tetraazine, specifically include:

[0018] The CA-NH2 and Tz-COOH were dissolved in an organic solvent, and EDC·HCl and DMAP were added under a nitrogen atmosphere to obtain a mixed solution.

[0019] The mixture was stirred at room temperature for 12-24 hours, washed, dried, and purified by column chromatography using a mixture of methanol and dichloromethane as the eluent to obtain chloroalkane-tetraazine.

[0020] The method for preparing the clickable HaloTag ligand further includes the following steps:

[0021] The CA-NH2 and Dissolved in an organic solvent, EDC·HCl and DMAP were added under an inert atmosphere and stirred to obtain chloroalkane-azide.

[0022] The method for preparing the clickable HaloTag ligand, wherein the... The preparation method includes the following steps:

[0023] Ethyl-4-hydroxybenzoic acid and N3-OTs were dissolved in acetonitrile, and potassium carbonate was added under an inert atmosphere. After stirring, the reaction yielded a third product. ;

[0024] The third product was mixed with potassium hydroxide and ethanol, and stirred at 80-100°C to obtain the fourth product. .

[0025] Application of a clickable HaloTag ligand in the preparation of drugs for treating tumors.

[0026] Application of a clickable HaloTag ligand in super-resolution optical wave imaging.

[0027] An application of a clickable HaloTag ligand in magnetic resonance imaging.

[0028] Beneficial Effects: This invention provides a clickable HaloTag ligand, its preparation method, and its application. The clickable HaloTag ligand is a chloroalkane-R, wherein R is a clickable chemical group that can react with TCO dyes or DBCO dyes. The clickable HaloTag ligand provided by this invention can penetrate cell membranes and perform clickable chemical labeling in living cells, enabling live-cell microtubule imaging. In terms of biocompatibility, the aforementioned ligands have low molecular weights, penetrate cell membranes quickly, exhibit no significant cytotoxicity, and do not remain within cells. Regarding labeling specificity and stability, all ligands contain a chloroalkyl (CA) moiety, enabling efficient targeting of HaloTag protein-expressing structures in living cells and stable covalent binding, avoiding non-specific adsorption. In terms of labeling strategy, the ligands contain clickable chemical functional groups, allowing them to react with any cell membrane-permeable fluorescent probe containing TCO or DBCO to label subcellular structures within living cells. For cell membrane-impermeable dyes, cell-permeable peptides can also be modified. 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. Attached Figure Description

[0029] Figure 1 This is a synthetic route diagram for chloroalkane-tetraazine (CA-Tz);

[0030] Figure 2 This is a synthetic route diagram for chloroalkanes-azides (CA-N3);

[0031] Figure 3 The first product is shown in the 1H NMR spectrum.

[0032] Figure 4 The first image shows the hydrogen NMR spectrum of the second product.

[0033] Figure 5 The hydrogen NMR spectrum of CA-NH2;

[0034] Figure 6 The hydrogen NMR spectrum of CA-Tz;

[0035] Figure 7 The first image is the hydrogen NMR spectrum of the fourth product.

[0036] Figure 8 The hydrogen NMR spectrum of CA-N3;

[0037] Figure 9 Image showing CA-Tz labeled live-cell confocal imaging results;

[0038] Figure 10 Image showing confocal imaging results of CA-N3-labeled live cells;

[0039] Figure 11 Images showing confocal imaging results of live cells labeled with different concentrations of CA-Tz;

[0040] Figure 12 Confocal imaging results of live cells labeled with 10 μM CA-Tz for 0.5 h;

[0041] Figure 13 SOFI images of live-cell microtubules labeled with green fluorescent protein and CA-Tz;

[0042] Figure 14 Magnified images showing the difference in wide-field and fourth-order image retention rates between green fluorescent protein and CA-Tz labeled microtubules in live cells;

[0043] Figure 15 Quantitative analysis of image retention rate and resolution data of microtubules in live cells labeled with green fluorescent protein and CA-Tz. Detailed Implementation

[0044] This invention provides a clickable HaloTag ligand, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0045] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0046] The number of existing chloroalkyl substrates capable of covalently binding to HaloTag proteins is limited, and the direct synthesis of fluorescent probes with chloroalkyl moieties is not only time-consuming but also limited in application. The development of the concept of bioorthogonal reactions offers a new approach to addressing this problem. Bioorthogonal reactions do not interfere with natural biological functions and are characterized by high biocompatibility, specificity, and versatility. These reactions are gaining increasing attention in live-cell imaging and molecular labeling, including ring-strain-promoted azide-alkyne cycloaddition reactions (SPAAC) and the inverse electron-demanding Diels-Alder reaction (iEDDA) between tetrazine (Tz) and trans-cycloene (TCO). Therefore, the development of bifunctional ligands with clickable functional groups that can bind to HaloTag proteins is particularly necessary.

[0047] Based on this, the present invention provides a clickable HaloTag ligand, wherein the clickable HaloTag ligand is a chloroalkane-R, wherein R is a clickable chemical group that can react with TCO dyes or DBCO dyes.

[0048] In this embodiment, the provided clickable HaloTag ligand can penetrate the cell membrane and perform click chemical labeling in living cells, enabling live-cell microtubule imaging. Regarding biocompatibility, the ligand has a low molecular weight, penetrates the cell membrane quickly, exhibits no significant cytotoxicity, and does not remain within the cell. Regarding labeling specificity and stability, all ligands contain a chloroalkyl (CA) moiety, allowing for efficient targeting of structures in living cells that fusion-express HaloTag proteins and stable covalent bonding, avoiding non-specific adsorption. Regarding labeling strategy, the ligand contains clickable chemical functional groups, enabling it to react with any cell membrane-permeable fluorescent probe containing TCO or DBCO to label subcellular structures within living cells. For cell membrane-impermeable dyes, cell-permeable peptides can also be modified. Regarding imaging applications, cells labeled with the clickable HaloTag ligand and dyes can achieve fourth-order super-resolution optical wave imaging (SOFI).

[0049] Specifically, this invention selects HaloTag protein as the target for synthesizing small molecules for live cell labeling. Plasmids can be synthesized and transfected into cells using genetic engineering techniques, offering high specificity and ease of operation. Clickable HaloTag ligands based on self-labeled protein tagging technology and click chemistry are synthesized for live cell labeling. Compared to directly using fluorescent proteins, nanoparticles, or probes with CA groups, chloroalkyl-R exhibits better biocompatibility, stronger targeting specificity, and stable binding via covalent bonds. Furthermore, these compounds can bind to various dyes, drugs, or chelating agents with TCO or DBCO groups, overcoming the limitations of HaloTag substrates and thus enabling wide application in super-resolution and magnetic resonance imaging of live cells and animals, as well as tumor diagnosis and treatment. Simultaneously, by directly co-incubating chloroalkyl-R with HaloTag-expressing cells, a handle for click chemistry can be introduced into the cells without affecting their physiological functions. This method is more suitable for studying the physiological activities within live cells.

[0050] In some embodiments, the clickable HaloTag ligand is a chloroalkyl-tetraazine (CA-Tz) or a chloroalkyl-azide (CA-N3). In terms of biocompatibility, chloroalkyl-tetraazine and chloroalkyl-azide have low molecular weights, penetrate cell membranes quickly, exhibit no significant cytotoxicity, and do not remain within cells. Regarding labeling specificity and stability, both ligands contain a chloroalkyl moiety (CA), allowing for efficient targeting of structures in living cells that fusion-express HaloTag proteins and stable covalent binding, avoiding non-specific adsorption. In terms of labeling strategy, the two ligands contain the clickable chemical functional groups tetraazine (Tz) and azide (N3), respectively, enabling them to react with any cell membrane-permeable fluorescent probe containing TCO or DBCO to label subcellular structures within living cells. For cell membrane-impermeable dyes, cell-permeable peptides can also be modified. In terms of imaging applications, cells labeled with clickable HaloTag ligands and dyes can achieve fourth-order super-resolution optical wave imaging (SOFI).

[0051] Specifically, the chemical structural formula of the chloroalkane-tetraazine (CA-Tz) is as follows: The chemical structural formula of the chloroalkane-azide (CA-N3) is as follows: .

[0052] In some implementations, live-cell microtubule imaging can be achieved by synthesizing CA-Tz or CA-N3 with slightly different structural formulas for click chemical labeling. For example, the structural formula may contain different numbers of polyethylene glycol molecules or Tz or N3 may be replaced with other click chemical groups that can react with TCO or DBCO dyes.

[0053] In addition, this invention also provides a method for preparing a clickable HaloTag ligand, comprising the following steps:

[0054] Step S10: React 2-(2-aminoethoxy)ethanol with di-tert-butyl carbonate in an organic solvent to obtain the first product. ;

[0055] Step S20: Dissolve the first product in an organic solvent, add potassium tert-butoxide under an inert atmosphere, stir, and then add 1-chloro-6-iodohexane to react and obtain the second product. ;

[0056] Step S30: Dissolve the second product in an organic solvent, add trifluoroacetic acid to react, and obtain CA-NH2;

[0057] Step S40: Dissolve the CA-NH2 and Tz-COOH in an organic solvent, add EDC·HCl and DMAP under an inert atmosphere, and stir to obtain chloroalkane-tetraazine.

[0058] In this embodiment, the chloroalkyl-tetraazine prepared by this method can react with any cell membrane-permeable fluorescent probe containing TCO or DBCO to label subcellular structures inside living cells. Cell membrane-impermeable dyes can also be labeled by modifying cell-permeable peptides. This overcomes the limitations of HaloTag substrates, thus enabling its widespread application in super-resolution and magnetic resonance imaging of living cells and animals, as well as in tumor diagnosis and treatment.

[0059] In some embodiments, in step S10, the reaction of 2-(2-aminoethoxy)ethanol with di-tert-butyl carbonate in an organic solvent to obtain the first product... The steps specifically include:

[0060] Step S11: Add 2-(2-aminoethoxy)ethanol ( Dissolve it in an organic solvent, add di-tert-butyl carbonate and stir at 0°C for 10-30 minutes, then stir at room temperature for 12-24 hours to obtain a reaction solution;

[0061] Step S12: The reaction solution is washed, dried, and purified by column chromatography to obtain the first product. .

[0062] Specifically, 2-(2-aminoethoxy)ethanol was dissolved in an anhydrous organic solvent, and di-tert-butyl carbonate was added to the solution. The mixture was stirred at 0°C for 10–30 minutes, and then stirred at room temperature for 12–24 hours. After the reaction was complete, the solution was washed successively with saturated sodium bicarbonate solution, brine, and water, and dried over anhydrous sodium sulfate. After filtration and solvent evaporation, the crude product was purified by column chromatography using an ethyl acetate / petroleum ether mixture as eluent, finally yielding a colorless oily liquid, which is the first product. .

[0063] In some embodiments, step S20 specifically includes: dissolving the first product in an anhydrous organic solvent, cooling it at 0°C, adding potassium tert-butoxide under a nitrogen atmosphere, stirring at 0°C for 1-4 hours, then adding 1-chloro-6-iodohexane dropwise, and stirring at this temperature for 3-5 hours. Next, stirring is continued at room temperature for 12-24 hours. Subsequently, the solvent is evaporated and the residue is added to water; the residue is extracted 3-4 times with ethyl acetate (EtOAc), and the combined organic layers are dried over anhydrous sodium sulfate. After filtration and solvent evaporation, the crude product is purified by column chromatography using an ethyl acetate / petroleum ether mixture as eluent, finally yielding a colorless oily liquid, i.e., the second product. .

[0064] In some embodiments, step S30 specifically includes: dissolving the second product in an anhydrous organic solvent, adding trifluoroacetic acid (TFA) at 0°C; subsequently, stirring the mixture at room temperature for 6-12 hours; then, removing the solvent and extracting with ethyl acetate (EtOAc) 3-4 times; combining the organic layers, washing with saturated sodium carbonate (Na2CO3) and water, and drying with anhydrous sodium sulfate. After filtration and solvent evaporation, the crude product is purified by column chromatography using a methanol / dichloromethane mixture as eluent, and the final obtained CA-NH2 is a colorless oily liquid. The chemical structural formula of CA-NH2 is as follows: .

[0065] In some embodiments, step S40, which involves dissolving CA-NH2 and Tz-COOH in an organic solvent, adding EDC·HCl and DMAP under an inert atmosphere, and stirring to obtain a chloroalkane-tetraazine, specifically includes:

[0066] Step S41: Dissolve the CA-NH2 and Tz-COOH in an organic solvent, and add EDC·HCl and DMAP under a nitrogen atmosphere to obtain a mixed solution; wherein, the structural formula of Tz-COOH is as follows: ;

[0067] Step S42: Stir the mixture at room temperature for 12-24 hours, wash and dry it, and then purify it by column chromatography using a mixture of methanol and dichloromethane as the eluent to obtain chloroalkane-tetraazine.

[0068] Specifically, CA-NH2 and Tz-COOH were dissolved in an appropriate amount of organic solvent. Then, EDC·HCl and DMAP were added under a nitrogen atmosphere to obtain a mixture. The mixture was stirred at room temperature for 12–24 hours. Afterward, the solution was washed with saturated brine and water, and dried over anhydrous sodium sulfate. After filtration and solvent evaporation, the crude product was purified by column chromatography using a methanol / dichloromethane mixture as eluent. The obtained CA-Tz was a pink oily liquid. The structural formula of CA-Tz is [insert structural formula here]. .

[0069] In some embodiments, the method for preparing the clickable HaloTag ligand further includes the step of:

[0070] Step S50: Mix the CA-NH2 with... Dissolved in an organic solvent, EDC·HCl and DMAP were added under an inert atmosphere and stirred to obtain chloroalkane-azide.

[0071] In some embodiments, step S50 specifically includes: mixing CA-NH2 and Dissolved in an organic solvent. Then, EDC·HCl and DMAP were added under a nitrogen atmosphere. The mixture was stirred at room temperature for 12–24 hours. The solution was washed with saturated brine and water, and dried over anhydrous sodium sulfate (Na₂SO₄). After filtration and solvent evaporation, the crude product was purified by column chromatography using a methanol / dichloromethane (DCM) mixture (1:80 v / v) as eluent to obtain a colorless oily liquid, CA-N₃. The chemical structural formula of CA-N₃ is [insert chemical formula here]. .

[0072] In some embodiments, in step S50, the The preparation method includes the following steps:

[0073] Step S51: Ethyl-4-hydroxybenzoic acid and N3-OTs are dissolved in acetonitrile, potassium carbonate is added under an inert atmosphere, and the reaction is stirred to obtain the third product. ;

[0074] Step S52: Mix the third product with potassium hydroxide and ethanol, and stir at 80-100°C to obtain the fourth product. .

[0075] In some embodiments, step S51 specifically includes: dissolving ethyl-4-hydroxybenzoic acid (... ) and N3-OTs ( Dissolve in acetonitrile. Add potassium carbonate (K2CO3) under a nitrogen atmosphere. Stir the mixture at 80-100°C for 12-24 hours. After filtering off the solid, purify the residue by column chromatography using a methanol / dichloromethane mixture as the eluent to obtain the crude product, i.e., the third product. Use the crude product directly in step S52 for the reaction.

[0076] In some embodiments, step S52 specifically includes: mixing the third product, potassium hydroxide, and ethanol, and stirring overnight at 80-100°C. 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 the crude product. The crude product is purified by column chromatography using a methanol / dichloromethane (DCM) mixture as the eluent to obtain the fourth product, a pale yellow oily liquid.

[0077] In addition, the present invention also provides the application of a clickable HaloTag ligand in the preparation of tumor therapeutic drugs, the application of a clickable HaloTag ligand in super-resolution optical wave imaging, and the application of a clickable HaloTag ligand in magnetic resonance imaging.

[0078] In some implementations, clickable BG / BC-Tz or BG / BC-N3 can be synthesized by replacing the HaloTag protein with other self-tagged protein tags such as SNAP-tag or CLIP-tag.

[0079] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not 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 description of the present invention are within the scope of protection of the present invention. Example

[0080] This embodiment provides a clickable HaloTag ligand, including chloroalkane-tetraazine (CA-Tz) or chloroalkane-azide (CA-N3), the synthetic routes of which are as follows: Figure 1 and Figure 2 As shown, the specific steps include the following:

[0081] 1. Synthesis of chloroalkyl-tetraazine (CA-Tz), the synthetic route is as follows: Figure 1 As shown.

[0082] 1) Synthesis of the first product

[0083] 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 the mixture was stirred at 0 °C for 10 minutes, then at room temperature for 12 hours. After the reaction was complete, the solution was washed successively with saturated sodium bicarbonate solution, brine, and water, and dried over anhydrous sodium sulfate. After filtration and solvent evaporation, the crude product was purified by column chromatography using an ethyl acetate / petroleum ether mixture (1:1 v / v) as the eluent. A final yield of 5.15 g of the first product was obtained as a colorless oily liquid, with a yield of 93%. The 1H NMR spectrum of the first product is shown below. 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).

[0084] 2) Synthesis of the second product

[0085] 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. The mixture was stirred at 0 °C for 1 hour, followed by the dropwise addition of 1-chloro-6-iodohexane (3.49 g, 1.2 eq), and stirring was continued at this temperature for 3 hours. Stirring was then 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 combined organic layers were dried over anhydrous sodium sulfate. After filtration and solvent evaporation, the crude product was purified by column chromatography using an ethyl acetate / petroleum ether mixture (v / v 1:6) as the eluent. The final 1.76 g of the second product was a colorless oily liquid, with a yield of 46%. The 1H NMR spectrum of the second product is shown below. 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).

[0086] 3) Synthesis of compound CA-NH2

[0087] 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. The mixture was then stirred at room temperature for 6 hours. The solvent was then removed, and the mixture was extracted three times with ethyl acetate (EtOAc). After combining the organic layers, the mixture was washed with saturated sodium carbonate (Na₂CO₃) and water, and dried over anhydrous sodium sulfate. After filtration and solvent evaporation, the crude product was purified by column chromatography using a methanol / dichloromethane mixture (v / v 1:16) as the eluent. The final CA-NH₂ was a colorless oily liquid with a yield of 0.54 g, representing a yield of 44%. The 1H NMR spectrum of CA-NH₂ is shown below. 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).

[0088] 4) Synthesis of compound CA-Tz

[0089] Tz-COOH was synthesized according to the method described in the literature. CA-NH2 (0.22 g, 1 eq) and Tz-COOH (0.33 g, 1.5 eq) 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. The solution was then washed with saturated brine and water, and dried over anhydrous sodium sulfate. After filtration and solvent evaporation, the crude product was purified by column chromatography using a methanol / dichloromethane mixture (1:100 v / v) as the eluent. The obtained CA-Tz was a pink oily liquid with a yield of 0.28 g, representing a yield of 68%. The 1H NMR spectrum of CA-Tz is shown below. Figure 6 As shown, 1 ¹H 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 spectrometry (MS): Theoretical value [M+H] + The value was 436.2037, while the actual measured value was 436.2104.

[0090] 2. Synthesis of chloroalkyl-azide (CA-N3), the synthetic route is as follows: Figure 2 As shown.

[0091] 1) Synthesis of the third product

[0092] N3-OTs were synthesized according to the 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 (v / v 1:300) as the eluent. The crude product was used directly in the next reaction step.

[0093] 2) Synthesis of the fourth product

[0094] The third product (0.54 g, 1 eq), potassium hydroxide (0.19 g, 2 eq), and ethanol (10 mL) were mixed and stirred overnight at 80 °C. 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 the crude product. The crude product was purified by column chromatography using a methanol / dichloromethane (DCM) mixture (v / v 1:80) as the eluent. The fourth product was a pale yellow oily liquid with a yield of 0.49 g (98%). The 1H NMR spectrum of the fourth product is shown below. 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).

[0095] 3) Synthesis of compound CA-N3

[0096] The synthesis of CA-N3 followed the same steps 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 rinsed with anhydrous sodium sulfate (Na2SO4). After filtration and solvent evaporation, the crude product was purified by column chromatography using a methanol / dichloromethane (DCM) mixture (v / v 1:80) as the eluent. A colorless oily liquid CA-N3 was given in a yield of 0.40 g (58%).

[0097] The proton NMR spectrum of CA-N3 is as follows: Figure 8 As shown, 1H 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 spectrometry (MS): m / z theoretical value [M+H] + The given value is 501.2402; the actual value is 501.2478.

[0098] 3. Clickable HaloTag ligand labeling of live cell microtubules

[0099] Dilute CA-Tz or CA-N3 (1 mM, dissolved in DMSO) to 50 nM–10 μM with fresh medium (containing FBS). Incubate these solutions with cells transfected with the mEmerald-Halo-Ensconsin plasmid at 37°C in a humidified environment of 5% CO2 for 0.5–24 hours. Then discard the supernatant and gently wash the cells three times with PBS. Co-incubate the cells with a dye containing TCO or DBCO functional groups (50 nM–10 μM) in fresh medium (containing fetal bovine serum) for 4–24 hours. Before optical imaging, gently wash the cells three times with PBS and image them in growth medium (DMEM, 10% FBS, phenol red-free).

[0100] 4. Clickable HaloTag ligand-labeled live-cell microtubule confocal imaging

[0101] Live-cell laser confocal scanning microscopy (CLSM) images were acquired using a Zeiss 980 microscope equipped with 488 nm (green fluorescent protein channel) and 639 nm (silicon rhodamine channel) lasers on an sCMOS camera with a 100x oil immersion objective and 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. Results of CA-Tz labeled live-cell confocal imaging are shown below. Figure 9 The results of CA-N3-labeled live-cell confocal imaging are shown in [the table below]. Figure 10The results of confocal imaging of live cells labeled with different concentrations of CA-Tz are shown in the figure. Figure 11 The results of confocal imaging of live cells labeled with 10 μMCA-Tz for 0.5 h are shown in the figure. Figure 12 The results show that both CA-Tz and CA-N3 can achieve microtubule labeling within a short time or a wide concentration range. Furthermore, because DBCO dye tends to remain in vesicles and generates a strong non-specific signal, CA-Tz labeling produces higher image quality under the same labeling and imaging conditions, making it more suitable for labeling and imaging microtubules in live cells.

[0102] 5. Clickable HaloTag ligand-labeled live-cell microtubule super-resolution fluorescence fluctuation imaging

[0103] Fluorescence images of COS-7 cell microtubules were obtained 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. The TIRF angle was precisely adjusted according to the selected excitation wavelength to ensure the highest signal-to-noise ratio and uniformity of excitation intensity across the entire field of view. For imaging of mEmerald-expressing microtubules, a 488 nm laser was used for excitation with a ZET405 / 488 / 561 / 640xv2 excitation filter and an FF01-711 / 25-25 emission filter. For silirodamine-labeled microtubule structures, a 640 nm laser and a ZET405 / 488 / 561 / 640xv2 dual-frequency dichroic mirror were used for excitation, introducing a 1.6x magnification during imaging. The EMCCD detector was cooled to -80°C, and a frame transmission mode was used. The size of each image was determined by selecting the region of interest (ROI) based on the extent of the cells within the field of view. The exposure time for each test set was 20 ms, and the image sequence consisted of 1500 frames. SOFI images of green fluorescent protein and CA-Tz-labeled live-cell microtubules are shown below. Figure 13 Enlarged images emphasizing the differences in wide-field and fourth-order image retention between green fluorescent protein and CA-Tz-labeled live-cell microtubules are shown below. Figure 14 Quantitative analysis of image retention rate and resolution calculation of green fluorescent protein and CA-Tz labeled live cell microtubules can be found in [link to relevant documentation]. Figure 15The results showed that both green fluorescent protein (GFP) and CA-Tz-labeled live-cell microtubules could achieve fourth-order SOFI imaging. Furthermore, the CA-Tz-labeled microtubules exhibited almost identical structures in both wide-field and fourth-order SOFI images, with minimal structural loss, while GFP-expressing microtubules showed significant structural loss. This clearly demonstrates the higher retention rate of CA-Tz-labeled microtubule structures in fourth-order SOFI images, further emphasizing the superiority of CA-Tz labeling. In addition, the resolution of CA-Tz-labeled live-cell microtubules was significantly improved compared to wide-field images. The resolution of fourth-order SOFI (161 nm) was twice that of wide-field images (332 nm), while the resolution of GFP-expressing microtubules in fourth-order SOFI images was only 189 nm.

[0104] In summary, this invention provides a clickable HaloTag ligand, its preparation method, and its applications. The clickable HaloTag ligand is a chloroalkane-R, where R is a clickable chemical group that can react with TCO dyes or DBCO dyes. The clickable HaloTag ligand provided by this invention can penetrate cell membranes and perform clickable chemical labeling in living cells, enabling live-cell microtubule imaging. In terms of biocompatibility, the aforementioned ligands have low molecular weights, penetrate cell membranes quickly, exhibit no significant cytotoxicity, and do not remain within cells. Regarding labeling specificity and stability, all ligands contain a chloroalkyl (CA) moiety, enabling efficient targeting of HaloTag protein-expressing structures in living cells and stable covalent binding, avoiding non-specific adsorption. In terms of labeling strategy, the ligands contain clickable chemical functional groups, allowing them to react with any cell membrane-permeable fluorescent probe containing TCO or DBCO to label subcellular structures within living cells. For cell membrane-impermeable dyes, cell-permeable peptides can also be modified. 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.

[0105] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing a clickable HaloTag ligand, characterized in that, Including the following steps: Dissolve 2-(2-aminoethoxy)ethanol in an organic solvent, add di-tert-butyl carbonate and stir at 0°C for 10-30 minutes, then stir at room temperature for 12-24 hours to obtain a reaction solution; The reaction solution was washed, dried, and purified by column chromatography to obtain the first product. ; The first product was dissolved in an organic solvent, and potassium tert-butoxide was added under an inert atmosphere. After stirring, 1-chloro-6-iodohexane was added to react and give the second product. ; The second product was dissolved in an organic solvent, and after reacting with trifluoroacetic acid, the following structure was obtained: CA-NH2; The CA-NH2 and the structural formula are Tz-COOH was dissolved in an organic solvent, and EDC·HCl and DMAP were added under a nitrogen atmosphere to obtain a mixture. The mixture was stirred at room temperature for 12-24 hours, washed, dried, and purified by column chromatography using a mixture of methanol and dichloromethane as eluent to obtain the structure with the following formula: Chloroalkyl tetraazine; The CA-NH2 and Dissolving in an organic solvent, and then adding EDC·HCl and DMAP under an inert atmosphere followed by stirring, yields the structure with the following formula: Chloroalkanes-azides; The The preparation method includes the following steps: Ethyl-4-hydroxybenzoic acid and N3-OTs were dissolved in acetonitrile, and potassium carbonate was added under an inert atmosphere. After stirring, the reaction yielded a third product. ; The third product was mixed with potassium hydroxide and ethanol, and stirred at 80-100°C to obtain the fourth product. .

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