Fluorescence enhancement anchoring agent based on BODIPY-tetrazine dye and used for expansion microscopic imaging technology

Abstract

The invention discloses a fluorescence enhancement anchoring agent based on a BODIPY-tetrazine dye for an expansion microimaging technology, and belongs to the technical field of biochemical analysis. A fluorophore of the fluorescence enhanced anchoring agent is shown as a formula (I), a formula (II) or a formula (III): # imgabs 0 # imgabs 1 #; a tetrazine group carried by the fluorescence enhanced anchoring agent and a drug probe are subjected to a biological orthogonal reaction to remove fluorescence quenching; the drug probe is shown as a formula (IV): # imgabs2. The fluorescence enhancement anchoring agent can anchor covalent and non-covalent small molecules in a hydrogel network based on tetrazine biological orthogonal reaction, and meanwhile, expansion microscopic fluorescence imaging of a dual fluorescence enhancement mechanism is realized. Besides, according to the drug probe, a non-covalent drug gefitinib is selected as a probe core, a BCN click handle is modified, in-situ fluorescence expansion microscopic imaging of EGFR drug-target is successfully achieved, and a new tool is provided for non-covalent drug target co-localization information research and clinical research of exosomes.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical analysis, and in particular to the design of a functional boron dipyrrol-tetrazine (BODIPY-TZ) compound and its application in expansion microscopy imaging technology. Background Art

[0002] Due to the limitations of optical diffraction, conventional fluorescence microscopy cannot accurately observe the distribution and interactions of biomolecules in subcellular structures. Super-resolution fluorescence imaging techniques can reveal biomolecules and structural details within 200 nanometers, but these techniques often require expensive equipment and complex algorithms. Expansion microscopy overcomes the limitations of optical diffraction by improving sample preparation methods and utilizing the biocompatibility and swelling properties of hydrogels, enabling conventional fluorescence microscopy to achieve super-resolution imaging. Despite this, existing expansion microscopy techniques have not yet developed a universal method to anchor non-covalently interacting biomolecules in hydrogels.

[0003] Swelling microscopy achieves uniform physical amplification of biological samples by uniformly filling hydrogel monomers into biological samples and polymerizing them to form a gel. When the gel absorbs water and swells, the relative positions of the biological molecules remain unchanged, but the distance between them is pulled farther, so that when the swollen sample is imaged using a conventional fluorescence microscope, a super-resolution effect can be achieved. Currently, a series of expansion microscopy techniques for cell and tissue biological imaging have been developed, including Click-ExM, Magnify, Iterative-ExM, TREx, and proExM. However, these techniques face challenges in studying the mechanism of action of non-covalent small molecule drugs in cells and exosomes because they lack the ability to in situ label drug-target interactions, making it difficult to achieve high-resolution ultra-sensitive analysis. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a fluorescence enhancement anchoring agent based on fluoroboron dipyrrole-tetrazine dye for expansion microscopy imaging technology.

[0005] According to one aspect of the present invention, a fluorescence enhancing anchoring agent based on fluoroboron dipyrrole-tetrazine dye is provided, wherein the fluorophore of the fluorescence enhancing anchoring agent is represented by formula (I), formula (II) or formula (III):

[0006] The tetrazine group carried by the fluorescence enhancement anchoring agent and the drug probe relieve fluorescence quenching through a bioorthogonal reaction. The drug probe is represented by formula (IV):

[0007]

[0008] According to the second aspect of the present invention, a method for preparing the fluorescence enhancing anchoring agent is provided. The method for preparing the fluorophores of formula (I) and formula (II) is as follows:

[0009] A1, the fluorophore of formula (I): dissolving compound 1 and 4-aminobenzaldehyde with toluene and N,N-dimethylformamide DMF, then adding piperidine and acetic acid dropwise under nitrogen protection for reaction, then extracting with dichloromethane, and separating by silica gel column to obtain black solid 1, i.e., the fluorophore of formula (I);

[0010] A2, the fluorophore of formula (II): dissolving compound 1 and 4-aminobenzaldehyde in N,N-dimethylformamide DMF, then adding piperidine and acetic acid dropwise under nitrogen protection for reaction, then extracting with dichloromethane, and separating by silica gel column to obtain black solid 2, i.e. the fluorophore of formula (II);

[0011] The reaction scheme of the preparation method is as follows:

[0012]

[0013] Furthermore, the volume ratio of toluene to N,N-dimethylformamide DMF in A1 is (4.5-5):(0.3-0.8), and the mass ratio of compound 1 to 4-aminobenzaldehyde, piperidine and acetic acid is (9-11):(11-12):(22-23):(16-18).

[0014] Furthermore, the mass ratio of compound 1 in A2 to 4-aminobenzaldehyde, piperidine and acetic acid is (9-11):(16-18):(23-25):(16-18).

[0015] Furthermore, the preparation method of the fluorophore of formula (III) is as follows:

[0016] B1, the fluorophore of formula (III): compound 2 and 4-aminobenzaldehyde were dissolved in toluene and N,N-dimethylformamide DMF, and then piperidine and acetic acid were added dropwise under nitrogen protection to react, and then extracted with dichloromethane, and separated and purified by reverse phase preparative high performance liquid chromatography RP-HPLC to obtain a black solid 3, i.e., the fluorophore of formula (III);

[0017] The reaction scheme of the preparation method is as follows:

[0018]

[0019] Furthermore, the volume ratio of toluene to N,N-dimethylformamide DMF in B1 is (4.5-5):(0.3-0.8), and the mass ratio of compound 2 to 4-aminobenzaldehyde, piperidine and acetic acid is (10-11):(11-12):(15-17):(11-13).

[0020] Furthermore, the preparation method of the drug probe of formula (IV) is as follows:

[0021] C1, gefitinib EGFR react with melted pyridine hydrochloride, then dissolve with sodium hydroxide solution, then extract with ethyl acetate, and separate by silica gel column chromatography to obtain a yellow-green solid, namely formula (IV-1);

[0022] C2, dissolving the formula (IV-1), N-Boc-3-aminopropyl bromide and K2CO3 obtained from C1 with acetonitrile ACN, then extracting with ethyl acetate, and separating through a silica gel column to obtain a transparent solid 1, namely, formula (IV-2);

[0023] C3, dissolving the formula (IV-2) obtained by C2 in anhydrous dichloromethane, adding trifluoroacetic acid TFA dropwise for reaction, treating under reduced pressure to obtain a transparent solid 2, dissolving the transparent solid 2 in N,N-dimethylformamide DMF, and then sequentially adding N,N-diisopropylethylamine DIEA and (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate BCN-NHs dissolved in N,N-dimethylformamide DMF dropwise for reaction, and separating by silica gel chromatography to obtain a transparent solid 3, i.e., the drug probe of formula (IV);

[0024] The reaction scheme of the preparation method is as follows:

[0025]

[0026] Furthermore, the mass ratio of pyridine hydrochloride and gefitinib EGFR in C1 is (8-10):1, and the mass ratio of formula (IV-1), N-Boc-3-aminopropyl bromide and K2CO3 in C2 is (1-1.5):(0.8-1.2):(1-1.3).

[0027] According to the third aspect of the present invention, a method for applying the fluorescence enhancing anchoring agent in expansion microscopy is proposed, characterized in that the application method includes removing fluorescence quenching by a bioorthogonal reaction between the tetrazine group carried by the fluorescence enhancing anchoring agent and the drug probe.

[0028] Furthermore, the application method also includes reacting the amino group carried by the fluorescence enhancement anchoring agent with glutaraldehyde to release the fluorescence quenching again, and the fluorescence enhancement anchoring agent is covalently linked to the hydrogel grid.

[0029] Beneficial effects of the present invention:

[0030] The present invention realizes super-resolution in situ imaging analysis of the action and distribution of non-covalent small molecule probes in cells by designing fluorescence-enhanced anchoring agents and expansion microscopy techniques. Among them, the technical key lies in the double quenching mechanism of tetrazine and amino groups, which not only realizes the covalent connection of anchoring agents, probes and hydrogel networks, but also significantly enhances the fluorescence signal. The present invention utilizes expansion microscopy technology to expand cells isotropically in space by 7-8 times, breaking through the optical diffraction limit (280nm) of the fluorescence microscope to achieve an improvement in resolution, and realizing super-resolution imaging of non-covalent drug action. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The H-NMR spectrum of the fluorophore of formula (I) provided by the present invention;

[0032] Figure 2 The nuclear magnetic hydrogen spectrum of the fluorophore of formula (II) provided by the present invention;

[0033] Figure 3 The nuclear magnetic hydrogen spectrum of the fluorophore of formula (III) provided by the present invention;

[0034] Figure 4 The nuclear magnetic hydrogen spectrum of the drug probe of formula (IV) provided by the present invention;

[0035] Figure 5 This is an emission spectrum diagram of the fluorescence enhancement anchoring agent provided by the present invention before and after the reaction with BCN;

[0036] Figure 6 This is the relationship diagram of the expansion multiple after the content of each monomer in the hydrogel is changed;

[0037] Figure 7 Confocal microscopy imaging of compounds 1 and 2 with the fluorophores of formula (I) and formula (III) in A549 cells incubated with Gefitinib-BCN;

[0038] Figure 8 This is the expansion microscopy imaging of the fluorophore of formula (III) in HeLa cells incubated with Gefitinib-BCN. DETAILED DESCRIPTION

[0039] The present application will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It should also be noted that, for ease of description, only the parts related to the relevant invention are shown in the accompanying drawings.

[0040] 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 application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0041] In view of the challenges often faced in studying the mechanism of action of non-covalent small molecule drugs in cells and exosomes, the existing technology lacks the ability to in situ label drug-target interactions and it is difficult to achieve high-resolution ultra-sensitive analysis. Therefore, the first object of the present invention is to propose a fluorescence enhancement anchoring agent based on fluoroboron dipyrrole-tetrazine dye to achieve dual fluorescence enhancement.

[0042] The second purpose of the present invention is to propose a drug probe targeting EGFR target, using the non-covalent drug gefitinib as the drug probe core, modified with a BCN click handle, while maintaining the drug activity to achieve in situ reaction and fluorescence imaging of the drug probe and tetrazine fluorescent reporter group.

[0043] The third object of the present invention is to provide a preparation method based on the fluorescence enhancer and the drug probe.

[0044] The fourth object of the present invention is to propose an application of the fluorescence enhanced anchoring agent in expansion microscopy imaging, which can isotropically expand the cells by 7-8 times in space, break through the optical diffraction limit (280nm) of the fluorescence microscope to achieve improved resolution and realize super-resolution imaging of non-covalent drug effects.

[0045] Compound 1 described in the example was prepared by optimizing the synthesis method described in the reference (Carlson, JC, et al., BODIPY-tetrazine derivatives as superbright bioorthogonal turn-on probes. Angew Chem Int Ed Engl, 2013. 52(27): p. 6917-20); Compound 2 described in the example was prepared by optimizing the synthesis method described in the reference (Kim, D., H. Son and SB Park, Ultrafluorogenic Monochromophore-Type BODIPY-Tetrazine Series for Dual-Color Bioorthogonal Imaging with a Single Probe. Angew Chem Int Ed Engl, 2023. 62(52): p. e202310665).

[0046] Example 1: Preparation of fluorescence enhancing anchoring agent

[0047] 1. Preparation of the Fluorophores of Formula (I) and Formula (II) of the Present Invention

[0048] The preparation of the fluorophores of formula (I) and formula (II) of the present invention is based on compound 1. Since the yield of the synthesis method of compound 1 in the prior art is too low, the synthesis method is optimized. The specific steps are as follows:

[0049] (1) Add 3-cyanobenzaldehyde (328 mg) to a flask, dissolve it in 25 mL of dry dichloromethane, and slowly drop 0.55 mL of 2,4-dimethylpyrrole (516.6 mg). Under nitrogen protection, add 3 drops of trifluoroacetic acid at -40°C, react for 2 hours, and monitor the reaction by TCL. Add 20 mL of water, extract twice with dichloromethane, combine the organic phases, wash with saturated sodium chloride water, dry with anhydrous sodium sulfate, filter, and concentrate. Separate on a silica gel column (ethyl acetate: petroleum ether = 7%, iodine staining) to obtain 687 mg of red solid compound (1-1), with a yield of 90.8%. 1HNMR (500MHz, CDCl3) δ7.72 (s, 2H), 7.61–7.50 (m, 3H), 7.47 (t, J = 7.8Hz, 1H), 5.87 (s, 2H), 5.65 (s, 1H), 2.31 (s, 6H), 2.01 (s, 6H).

[0050] (2) Add compound (1-1) (1.15 g) to a flask and dissolve it with 3.97 mL of acetonitrile (3.12 g) and 3 mL of ethanol. Slowly drop 331 μL of 3-mercaptopropionic acid (403.3 mg) under ice bath. After 5 minutes, slowly drop 2.96 mL of hydrazine hydrate (3.04 g). Under nitrogen protection, react overnight at 45°C for 18 hours. TCL monitoring shows that the reaction is complete. After post-treatment, add 5 mL of saturated ammonium chloride solution, then add 20 mL of water and 20 mL of ethyl acetate, extract twice, combine the organic phases, wash with saturated sodium chloride water, and then dry with anhydrous sodium sulfate, filter, and concentrate to obtain 1.28 g of reddish brown solid compound (1-2). Compound (1-2) was transferred to a 100 mL flask, dissolved in 20 mL tetrahydrofuran, and a solution of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (1.29 g) prepared with 5 mL tetrahydrofuran was added dropwise under an ice bath, and reacted at room temperature for 2 hours. After the intermediate product was completely oxidized, 5.28 mL triethylamine (3.845 g) was slowly added dropwise under an ice bath, and 10 minutes later, 7.19 mL boron trifluoride ether (8.09 g) was slowly added dropwise. The mixture was then reacted for 2 hours under a 40°C water bath, monitored by TCL, and the reaction was complete. 30 mL water and 30 mL ethyl acetate were added, extracted twice, the organic phases were combined, washed with saturated ammonium chloride water, dried with anhydrous sodium sulfate, filtered, and concentrated. Silica gel column separation (ethyl acetate: petroleum ether = 7%, iodine staining) gave 450 mg of red solid compound 1, with a yield of 28.3%.

[0051] The optimized synthesis route of compound 1 is as follows:

[0052]

[0053] The specific steps of the preparation method of the fluorophore of formula (I) and formula (II) are as follows:

[0054] The fluorophore of formula (Ⅰ): Add compound 1 (100 mg) and 4-aminobenzaldehyde (115.8 mg) into a flask and dissolve with 4.9 mL toluene and 0.4 mL DMF (to promote the dissolution of 4-aminobenzaldehyde). Subsequently, add 283 μL piperidine (244.2 mg) and 164 μL acetic acid (172.1 mg) dropwise. Under nitrogen protection, react at 120°C for 5 minutes. After cooling to room temperature, add 10 mL and 15 mL dichloromethane, extract twice, combine the organic phases, wash with saturated ammonium chloride water, and then dry with anhydrous sodium sulfate, filter, and concentrate. Separate on a silica gel column (ethyl acetate: petroleum ether = 1:2, UV) to obtain 25 mg of a black solid, i.e., the fluorophore of formula (Ⅰ), with a yield of 20.1%. The H NMR spectrum shows Figure 1. 1HNMR (500MHz, DMSO) δ8.63(d,J=8.0Hz,1H),8.39(s,1H),7.86(t,J=7.7Hz,1H),7.76(d,J=7.5Hz,1H),7.51–7.42(m,1H),7.33(d,J=8.3Hz,2 H),7.22(d,J=16.1Hz,1H),6.94(s,1H),6.62(d,J=8.2Hz,2H),6.13(s ,1H),5.88(s,2H),3.00(s,3H),2.48(s,3H),1.45(s,3H),1.39(s,3H).

[0055] The fluorophore of formula (II): Add compound 1 (100 mg) and 4-aminobenzaldehyde (173.7 mg) into a flask and dissolve with 5 mL of DMF. Subsequently, add 283 μL of piperidine (244.2 mg) and 164 μL of acetic acid (172.1 mg) dropwise. Under nitrogen protection, react at 120°C for 10 minutes. After cooling to room temperature, add 10 mL and 15 mL of dichloromethane, extract twice, combine the organic phases, wash with saturated ammonium chloride water, and then dry with anhydrous sodium sulfate, filter, and concentrate. Separate by silica gel column chromatography (methanol: dichloromethane = 5%, UV) to obtain 23 mg of black solid, i.e., the fluorophore of formula (II); the yield is 15.5%, and the nuclear magnetic hydrogen spectrum is shown in Figure 2 . 1H NMR (500MHz, DMSO) δ8.63(d,J=7.8Hz,1H),8.40(s,1H),7.86(t,J=7.7Hz,1H),7.78(d,J=7.4Hz,1H),7.36(dd,J=22 .3,12.1Hz,6H),7.25(d,J=16.1Hz,2H),6.88(s,2H),6.64(d,J=8.1Hz,4H),5.81(s,4H),3.01(s,3H),1.45(s,6H).

[0056] The reaction routes of the preparation methods of the fluorophores of formula (I) and formula (II) are as follows:

[0057]

[0058] 2. Preparation of the Fluorophore of Formula (III) of the Present Invention

[0059] The specific steps of the preparation method of the fluorophore of formula (III) of the present invention are as follows:

[0060] Compound 2 (54 mg) and 4-aminobenzaldehyde (58.7 mg) were added to a flask and dissolved with 4.9 mL toluene and 0.4 mL DMF (to promote the dissolution of 4-aminobenzaldehyde). Subsequently, 94 μL piperidine (81.2 mg) and 55 μL acetic acid (57.3 mg) were added dropwise. Under nitrogen protection, the mixture was reacted at 120°C for 5 minutes. After cooling to room temperature, 10 mL and 15 mL dichloromethane were added, and the mixture was extracted twice. The organic phases were combined and washed with saturated ammonium chloride water, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was separated and purified by RP-HPLC (reverse phase preparative high performance liquid chromatography) to obtain a black solid, i.e., the fluorophore of formula (III), with a yield of 24.8%. The H NMR spectrum showed Figure 3 . 1HNMR (500MHz, DMSO) δ8.47(d,J=16.1Hz,1H),7.78(d,J=15.9Hz,1H),7.63(d,J=16.2Hz,1H),7.47(d,J=10. 6Hz, 3H), 7.37 (s, 1H), 7.22 (d, J = 12.9Hz, 2H), 7.01 (s, 1H), 6.67 (d, J = 7.9Hz, 2H), 2.95 (s, 3H), 2.32 (s, 3H).

[0061] The reaction route of the method for preparing the fluorophore of formula (III) is as follows:

[0062]

[0063] Example 2: Preparation of drug probes

[0064] The drug probe of the present invention targets the EGFR target, the probe core is the non-covalent drug gefitinib, and is modified with a BCN click handle. The preparation method of the drug probe of formula (IV) is as follows:

[0065] (1) Add pyridine hydrochloride (5.2 g) to a flask and heat to 150°C until completely melted. Then add Gefitinib (530 mg) solid and react for 3 hours. The reaction is complete after TCL monitoring. After cooling to room temperature, dissolve the substrate with 25 ml of 5M NaOH solution. Then, extract twice with 60 ml of ethyl acetate. Combine the organic phases and wash with saturated brine, then dry with anhydrous sodium sulfate, filter, and concentrate. Separate with silica gel column (methanol: dichloromethane = 8%, PMA) to obtain 410 mg of yellow-green solid formula (IV-1), with a yield of 79.8%. 1HNMR(500MHz,CD3OD_SPE)δ8.31(s,1H),7.95(dd,J=6.6,2.0Hz,1H),7.58(dd,J=8.5,3.1Hz,1H),7.53(s,1H),7.11(t,J =8.9Hz,1H),6.99(s,1H),4.05(t,J=5.8Hz,2H),3.73–3.58(m,4H),2.51(t,J=7.2Hz,2H),2.45(s,4H),2.03–1.89(m,2H).

[0066] (2) Add formula (IV-1) (285 mg), N-Boc-3-aminopropyl bromide (238 mg) and K2CO3 (276 mg) to a flask, dissolve with 7 mL of acetonitrile, react at 75°C overnight, monitor with TCL, and the reaction is complete. Add 30 mL of water, extract twice with 50 mL of ethyl acetate, combine the organic phases, wash with saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate. Separate with a silica gel column (methanol: dichloromethane = 5%, PMA) to obtain 304 mg of a transparent solid formula (IV-2), with a yield of 77.2%. 1H NMR (500MHz, CD3OD_SPE) δ8.34 (s, 1H), 7.98 (dd, J = 6.6, 2.1Hz, 1H), 7.64–7. 57(m,1H),7.44(s,1H),7.15(t,J=8.9Hz,1H),6.94(s,1H),4.11(t,J=5.9Hz ,2H),4.06(t,J=5.8Hz,2H),3.75–3.64(m,4H),3.28(t,J=6.3Hz,2H),2.66– 2.57(m,2H),2.52(s,4H),2.13–2.02(m,2H),2.02–1.92(m,2H),1.43(s,9H).

[0067] (3) Add formula (IV-2) (140 mg) to a flask, dissolve with 2 mL of anhydrous dichloromethane, add 300 μL of trifluoroacetic acid under nitrogen protection and ice bath, react at room temperature for 1 hour, and monitor the reaction by TCL. Post-treatment: first remove dichloromethane under reduced pressure, then add 5 mL of chloroform to fully dissolve, and then dry under reduced pressure, repeat three times to obtain a transparent solid intermediate. Add 2 mL of DMF to dissolve, add 388 μL of N,N-diisopropylethylamine (288.2 mg) under nitrogen protection and ice bath until the pH is around 9, and after 10 minutes, add (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (58 mg) dissolved with 1 mL of DMF, and after 15 minutes after the addition, transfer to room temperature for overnight reaction, and monitor the reaction by TCL. 15 mL of water and 20 mL of ethyl acetate were added, washed twice, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Silica gel column separation (methanol: dichloromethane = 4%, PMA) gave 113 mg of a transparent solid, i.e., the drug probe of formula (IV), with a yield of 85.4%. The H NMR spectrum showed Figure 4 . 1H NMR (500MHz, DMSO) δ9.57 (s), 8.49 (s), 8.13 (d, J = 5.0Hz), 7.83 (s), 7.43 (t, J = 8.9Hz), 7.19 (d, J = 14.2Hz), 4.27–4.10 (m), 4.03 (d, J = 7. 6Hz), 3.58(s), 3.21(d,J=5.3Hz), 2.41(s), 2.18(dd,J=32.9,20.5Hz), 1.96(d,J=30.2Hz), 1.51(d,J=9.7Hz), 1.31–1.22(m), 0.84(s).

[0068] The reaction route of the method for preparing the drug probe of formula (IV) is as follows:

[0069]

[0070] Example 3: Reaction of fluorescence-enhanced anchoring agent with BCN

[0071] The fluorophores of formula (I), formula (II) and formula (III) were added with 5 times the equivalent of BCN-NHs, reacted in acetonitrile for 5 hours, and then diluted to 10 μM. The emission spectra were tested using Hitachi fluorescence spectrophotometer F-7100 to obtain the excitation and emission wavelengths of the three fluorophores, as well as the emission intensity ratio (fluorescence turn-on multiple) corresponding to the emission wavelength point. The results are shown in Table 1:

[0072] Table 1 Reaction of fluorescence enhancing anchoring agent with BCN

[0073] BODIPY-TZ Excitation wavelength (nm) Emission wavelength (nm) Open multiple Formula (I) 585 642 30× Formula (II) 670 716 16× Formula (III) 650 714 126×

[0074] The results show that the fluorescence properties of BODIPY dye can be effectively quenched by resonance energy transfer through tetrazine functionalization, and then the quenching effect can be effectively removed by reaction with bicyclo[6,1,0]nonyne (BCN) or trans-cyclooctene (TCO), thereby turning on the fluorescence.

[0075] Example 4: Preparation of hydrogel

[0076] 1. Hydrogel production process

[0077] (1) All monomer mother liquors were prepared with deionized water (Note: during preparation, g / 100mL should be the total volume after adding water to 100mL, and the solute volume effect cannot be ignored), as shown in Table 2:

[0078] Table 2 Preparation of hydrogel monomer mother solution

[0079] ST(g / 100mL) DMAA(N,N-dimethylacrylamide) ～ SA(Sodium Acrylate) 45 AA (Acrylamide) 65 Bis(N,N'-methylenebisacrylamide) 2 NaCl (Sodium Chloride) 30 PBS 10× APS (Ammonium Persulfate) 10 TEMED(Tetramethylethylenediamine) 10 4-HT(Nitrogen oxide free radical piperidinol) 1

[0080] (2) Mix the hydrogel monomer mother liquors in a certain proportion to ensure that the final PBS concentration is 1× (Note: reserve the volume of TEMED, APS, and 4-HT). The concentration of sodium chloride is to fill the weight of the hydrogel, so there is no strict requirement (the prepared gel can be stored at 4°C for 1 month and at -20°C for 6 months).

[0081] (3) Transfer the desired hydrogel solution to an EP tube, add 4-HT, TEMED and APS in sequence on ice, shake to mix evenly, and place on ice for later use.

[0082] (4) Preparation of hydrogel chamber: Three drops of 4 μL water were placed on a glass slide (25.4 × 76.2 mm), three cover slips (20 × 20 mm) were placed side by side on the slide to form the first layer, and then two more cover slips were placed as the second layer to form a 10 × 20 mm rectangular cavity.

[0083] (5) Take 50 μL of hydrogel (adjust according to the size of the cavity) and drop it into the cavity. Use a cover glass to slowly push along the edge until the hydrogel fills the gel chamber and no obvious bubbles are observed (if there are bubbles, place the cover glass upright to expel them).

[0084] (6) Place the prepared glass slide on top of the partition of the tip box filled with water, and place the tip box in a 37°C oven away from light to gel for 1 hour.

[0085] (7) Take out the sample, remove the coverslip with a blade, cut the hydrogel into a rectangle, record the size before expansion, and place it in deionized water to allow it to absorb water and expand for 15 min × 3 until it stops expanding. Then record the size after expansion to obtain a physical expansion multiple.

[0086] 2. Hydrogel formula

[0087] In order to test the effect of the change of different monomer contents in the gel formula on the swelling ratio of the hydrogel, the present invention changes the ratio of SA:AA while keeping the total amount of SA and AA unchanged (total amount is 35 g / 100 mL) and other components unchanged, and observes the swelling ratio under different Bis crosslinker concentrations, such as Figure 6 As shown, the ordinate represents the physical expansion multiple before and after expansion, and the abscissa represents the overall concentration of the cross-linking agent. It can be seen that as the overall concentration of the Bis cross-linking agent increases, the physical expansion multiple of the hydrogel gradually decreases. In order to further improve the resolution in expansion microscopy imaging, the hydrogel formula is determined as shown in Table 3:

[0088] Table 3 Hydrogel formulation

[0089] Final concentration (g / 100mL) DMAA(N,N-dimethylacrylamide) 3 SA(Sodium Acrylate) 23 AA (Acrylamide) 12 Bis(N,N'-methylenebisacrylamide) 0.01 NaCl (Sodium Chloride) 3.024 PBS 1× APS (Ammonium Persulfate) 0.25 TEMED(Tetramethylethylenediamine) 0.2 4-HT(Nitrogen oxide free radical piperidinol) 0.02

[0090] Example 5: Microscopic Imaging

[0091] Swelling microscopy imaging was performed based on the hydrogel formulation determined in Example 4. Human non-small cell lung cancer A549 cells were cultured to a density of 50%-60% in a cell imaging glass dish, incubated with the Gefitinib-BCN probe for 1 hour, and then incubated with compounds 1, 2, the fluorophores of formula (I) and formula (III) for 0.5 hour, washed with PBS, and then fixed with 4% paraformaldehyde, stained with Hoechst 33342 for 15 minutes, and washed with PBST. The slides were sealed at room temperature. Figure 7 Shown are fluorescence imaging of A549 cells labeled with different BODIPY anchors after co-incubation with EGFR probes (scale bar: 20 μm), wherein all images were acquired on different detection channels of the Leica-DMi8 confocal microscope system.

[0092] A cell slide was placed in a 24-well plate, and cervical cancer HeLa cells were cultured to a density of 50%-60%, incubated with the Gefitinib-BCN probe for 1 hour, and then incubated with the fluorophore of formula (III) for 0.5 hour, washed with PBS, and then fixed with 3% paraformaldehyde + 0.1% (v / v) glutaraldehyde for 10 minutes, and then reduced with 0.1% (w / v) sodium borohydride (PBS) for 7 minutes, and then treated with 100mM glycine (PBS) for 10 minutes, and finally anchored with 0.25% (v / v) glutaraldehyde (PBS) for 10 minutes before gelling. After the above steps were completed, the cell slide was taken out and placed in the cavity containing the hydrogel. The slide was gelled at 37°C for 1 hour, and then cut into rectangles. The slide was treated in a digestion solution (50mM Tris (pH=8), 1Mm EDTA, 0.1% Triton X-100, 0.8M Guanidine HCl and 8units / mL ProK) at 37°C for 2 hours, and then incubated in 5μg / mL Hoechst 33342 (PBS) for 1 hour to facilitate the subsequent confocal imaging of the cells. Finally, the Leica-DMi8 confocal microscope was used for expansion microscopy imaging (the images were all taken at 20× magnification, and some pictures were magnified). The results are shown in the figure. Figure 8 As shown, the scale bar before expansion is 11 μm, the scale bar after expansion is 11 μm, the actual physical size is 90 μm, and the expansion multiple is 8.18 times.

[0093] The above describes the specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A fluorescence enhancing anchoring agent based on fluoroboron dipyrrole-tetrazine dye, characterized in that: The fluorophore of the fluorescence enhancement anchoring agent is represented by formula (I), formula (II) or formula (III): The tetrazine group carried by the fluorescence enhancement anchoring agent and the drug probe relieve fluorescence quenching through a bioorthogonal reaction. The drug probe is represented by formula (IV):

2. A method for preparing the fluorescence enhancing anchoring agent according to claim 1, characterized in that: The preparation methods of the fluorophores of formula (I) and formula (II) are as follows: A1, the fluorophore of formula (I): dissolving compound 1 and 4-aminobenzaldehyde with toluene and N,N-dimethylformamide DMF, then adding piperidine and acetic acid dropwise under nitrogen protection for reaction, then extracting with dichloromethane, and separating by silica gel column to obtain black solid 1, i.e., the fluorophore of formula (I); A2, the fluorophore of formula (II): dissolving compound 1 and 4-aminobenzaldehyde in N,N-dimethylformamide DMF, then adding piperidine and acetic acid dropwise under nitrogen protection for reaction, then extracting with dichloromethane, and separating by silica gel column to obtain black solid 2, i.e. the fluorophore of formula (II); The reaction scheme of the preparation method is as follows:

3. The method for preparing the fluorescence enhancing anchoring agent according to claim 2, characterized in that: The volume ratio of toluene to N,N-dimethylformamide DMF in A1 is (4.5-5):(0.3-0.8), and the mass ratio of compound 1 to 4-aminobenzaldehyde, piperidine and acetic acid is (9-11):(11-12):(22-23):(16-18).

4. The method for preparing the fluorescence enhancing anchoring agent according to claim 2, characterized in that: The mass ratio of compound 1 to 4-aminobenzaldehyde, piperidine and acetic acid in A2 is (9-11):(16-18):(23-25):(16-18).

5. The method for preparing the fluorescence enhancing anchoring agent according to claim 2, characterized in that: The preparation method of the fluorophore of formula (III) is as follows: B1, the fluorophore of formula (III): compound 2 and 4-aminobenzaldehyde were dissolved in toluene and N,N-dimethylformamide DMF, and then piperidine and acetic acid were added dropwise under nitrogen protection to react, and then extracted with dichloromethane, and separated and purified by reverse phase preparative high performance liquid chromatography RP-HPLC to obtain a black solid 3, i.e., the fluorophore of formula (III); The reaction scheme of the preparation method is as follows:

6. The method for preparing the fluorescence enhancing anchoring agent according to claim 5, characterized in that: The volume ratio of toluene to N,N-dimethylformamide DMF in B1 is (4.5-5):(0.3-0.8), and the mass ratio of compound 2 to 4-aminobenzaldehyde, piperidine and acetic acid is (10-11):(11-12):(15-17):(11-13).

7. The method for preparing the fluorescence enhancing anchoring agent according to claim 2, characterized in that: The preparation method of the drug probe of formula (IV) is as follows: C1, gefitinib EGFR react with melted pyridine hydrochloride, then dissolve with sodium hydroxide solution, then extract with ethyl acetate, and separate by silica gel column chromatography to obtain a yellow-green solid, namely formula (IV-1); C2, dissolving the formula (IV-1), N-Boc-3-aminopropyl bromide and K2CO3 obtained from C1 with acetonitrile ACN, then extracting with ethyl acetate, and separating through a silica gel column to obtain a transparent solid 1, namely, formula (IV-2); C3, dissolving the formula (IV-2) obtained by C2 in anhydrous dichloromethane, adding trifluoroacetic acid TFA dropwise for reaction, treating under reduced pressure to obtain a transparent solid 2, dissolving the transparent solid 2 in N,N-dimethylformamide DMF, and then sequentially adding N,N-diisopropylethylamine DIEA and (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate BCN-NHs dissolved in N,N-dimethylformamide DMF dropwise for reaction, and separating by silica gel chromatography to obtain a transparent solid 3, i.e., the drug probe of formula (IV); The reaction scheme of the preparation method is as follows:

8. The method for preparing the fluorescence enhancing anchoring agent according to claim 7, characterized in that: The mass ratio of pyridine hydrochloride and gefitinib EGFR in C1 is (8-10):1, and the mass ratio of formula (IV-1), N-Boc-3-aminopropyl bromide and K2CO3 in C2 is (1-1.5):(0.8-1.2):(1-1.3).

9. A method for using the fluorescence enhancement anchoring agent as claimed in claim 1 in expansion microscopy imaging, characterized in that: The application method comprises the following steps: the tetrazine group carried by the fluorescence enhancement anchor agent and the drug probe release fluorescence quenching through a bioorthogonal reaction.

10. The method for using the fluorescence enhanced anchoring agent in expansion microscopy according to claim 9, characterized in that: The application method also includes reacting the amino group carried by the fluorescence enhancement anchoring agent with glutaraldehyde to release the fluorescence quenching again, and the fluorescence enhancement anchoring agent is covalently linked to the hydrogel grid.

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