Fluorescence enhancement anchor based on fluoroboron dipyrrin-tetrazine dyes for expansion microscopy imaging

Abstract

The application discloses a fluorescence enhancement anchor agent based on a fluoroboron dipyrrin-tetrazine dye for inflation microscopy technology, and belongs to the technical field of biochemical analysis. The fluorophore of the fluorescence enhancement anchor agent is shown in formula (I), formula (II) or formula (III): the tetrazine group carried by the fluorescence enhancement anchor agent is removed from fluorescence quenching through a biological orthogonal reaction with a drug probe, and the drug probe is shown in formula (IV): the fluorescence enhancement anchor agent can covalently and non-covalently anchor small molecules in a hydrogel network based on a tetrazine biological orthogonal reaction, and realizes inflation microscopic fluorescence imaging of a double fluorescence enhancement mechanism. In addition, the drug probe selects a non-covalent drug, gefitinib, as a probe core, is modified with a BCN click handle, and successfully realizes in-situ fluorescence inflation microscopic imaging of an EGFR drug-target, thereby providing a new tool for research and clinical research on non-covalent drug target co-localization information of exosomes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biochemical analysis, and in particular to a functional BODIPY-TZ compound design and its application in expansion microscopy. BACKGROUND

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

[0003] Expansion microscopy achieves uniform physical magnification of biological samples by uniformly filling hydrogel monomers into the interior of the biological sample and polymerizing them to form a gel. When the gel swells after absorbing water, the relative positions of biomolecules remain unchanged, but the distance between them is stretched, allowing the use of conventional fluorescence microscopes to achieve super-resolution when imaging the swollen sample. Currently, a series of expansion microscopy techniques for cell and tissue bioimaging have been developed, including Click-ExM, Magnify, Iterative-ExM, TREx, proExM, etc. However, these techniques face challenges when studying the mechanism of action of non-covalent small molecule drugs in cells and exosomes, as they lack the ability to label drug-target interactions in situ, making it difficult to achieve high-resolution and super-sensitive analysis. SUMMARY

[0004] The present application aims to overcome the shortcomings of the prior art and provide a fluorescence-enhancing anchor agent based on BODIPY-TZ dye for expansion microscopy.

[0005] According to one aspect of the present application, a fluorescence-enhancing anchor agent based on BODIPY-TZ dye is provided, wherein the fluorophore of the fluorescence-enhancing anchor agent is represented by formula (I), formula (II), or formula (III):

[0006] The tetrazine group carried by the fluorescence-enhancing anchor agent removes fluorescence quenching through bioorthogonal reaction with a drug probe represented by formula (IV):

[0007]

[0008] According to a second aspect of the present application, a preparation method of the fluorescence-enhanced anchor agent is provided, and the preparation method of the fluorescence group of formula (I) and formula (II) is as follows:

[0009] A1, the fluorescence group 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, and then extracting with dichloromethane, and separating black solid 1, i.e. the fluorescence group of formula (I), through a silica gel chromatographic column;

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

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

[0012]

[0013] Further, the volume ratio of toluene to N,N-dimethylformamide DMF in the 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] Further, the mass ratio of compound 1 to 4-aminobenzaldehyde, piperidine and acetic acid in the A2 is (9-11):(16-18):(23-25):(16-18).

[0015] Further, the preparation method of the fluorescence group of formula (III) is as follows:

[0016] B1, the fluorescence group of formula (III): dissolving compound 2 and 4-aminobenzaldehyde with toluene and N,N-dimethylformamide DMF, then adding piperidine and acetic acid dropwise under nitrogen protection for reaction, and then extracting with dichloromethane, and separating and purifying black solid 3, i.e. the fluorescence group of formula (III), through reverse phase preparative high performance liquid chromatography RP-HPLC;

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

[0018]

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

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

[0021] C1, the gefitinib EGFR is reacted with the pyridine hydrochloride after melting, and then dissolved with sodium hydroxide solution, followed by extraction with ethyl acetate, and then separated by a silica gel chromatographic column to obtain a yellow-green solid, that is, formula (IV-1);

[0022] C2, the formula (IV-1) obtained in C1, N-Boc-3-aminopropyl bromide and K2CO3 are dissolved in acetonitrile ACN, and then extracted with ethyl acetate, and then separated by a silica gel chromatographic column to obtain transparent solid 1, that is, formula (IV-2);

[0023] C3, the formula (IV-2) obtained in C2 is dissolved in anhydrous dichloromethane, and then trifluoroacetic acid TFA is added dropwise to react, and then transparent solid 2 is obtained by reducing pressure treatment, and then the transparent solid 2 is dissolved in N,N-dimethylformamide DMF, and then 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 are added dropwise in sequence to react, and then transparent solid 3 is obtained by separation through a silica gel chromatographic column, that is, the formula (IV) drug probe;

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

[0025]

[0026] Further, the mass ratio of pyridine hydrochloride to gefitinib EGFR in C1 is (8-10):1, and the mass ratio of the 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 a third aspect of the present application, an application method of the fluorescence-enhanced anchor agent in expansion microscopy is provided, characterized in that the application method comprises that the tetrazine group carried by the fluorescence-enhanced anchor agent is removed from fluorescence quenching by biological orthogonal reaction with the drug probe.

[0028] Further, the application method further comprises that the amino group carried by the fluorescence-enhanced anchor agent reacts with glutaraldehyde, again releasing fluorescence quenching, and the fluorescence-enhanced anchor agent is covalently connected to the hydrogel grid.

[0029] Advantages of the present application:

[0030] The present application realizes the super-resolution in-situ imaging analysis of the action and distribution of non-covalent small molecule probes in cells by designing a fluorescence-enhanced anchor agent and its swelling microscopic imaging technology. The technical key lies in the double quenching mechanism of tetrazine and amino groups, which not only realizes the covalent connection of the anchor agent, the probe and the hydrogel network, but also significantly enhances the fluorescence signal. The present application uses the swelling microscopic imaging technology to swell the cells by 7-8 times in space isotropy, breaks through the optical diffraction limit (280 nm) of the fluorescence microscope to realize the improvement of resolution, and realizes the super-resolution imaging of non-covalent drugs. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The nuclear magnetic hydrogen spectrum of the fluorescent group of formula (I) provided by the present application;

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

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

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

[0035] Figure 5 The emission spectrum diagram of the fluorescence-enhanced anchor agent provided by the present application before and after reaction with BCN;

[0036] Figure 6 The swelling ratio relationship diagram of the hydrogel after changing the content of each monomer;

[0037] Figure 7 The confocal microscope imaging of compound 1 and 2 with the fluorescent group of formula (I) and formula (III) and Gefitinib-BCN incubated A549 cells;

[0038] Figure 8 The swelling microscopic imaging of the fluorescent group of formula (III) in Gefitinib-BCN incubated HeLa cells. DETAILED DESCRIPTION

[0039] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0040] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0041] Given the challenges often encountered in studying the mechanisms of action of non-covalent small molecule drugs in cells and exosomes, and the lack of in-situ labeling capabilities for drug-target interactions in existing technologies, making it difficult to achieve high-resolution ultrasensitive analysis, the first objective of this invention is to propose a fluorescence-enhancing anchoring agent based on fluoroboron dipyrrole-tetraazine dye to achieve dual fluorescence enhancement.

[0042] The second objective of this invention is to propose a drug probe targeting EGFR, which uses the non-covalent drug gefitinib as the core of the drug probe and modifies it with a BCN click handle to achieve in-situ reaction and fluorescence imaging of the drug probe with the tetrazine fluorescent reporter group while maintaining drug activity.

[0043] A third objective of this invention is to provide a method for preparing the fluorescence enhancer and the drug probe.

[0044] The fourth objective of this invention is to propose an application of the aforementioned fluorescence-enhancing anchor in dilatational microscopy, which isotropically dilatates cells by 7-8 times, breaking through the optical diffraction limit (280nm) of fluorescence microscopy to improve resolution and achieve super-resolution imaging of non-covalent drug action.

[0045] Compound 1 in the examples was prepared by synthetic optimization based on the synthetic 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 in the examples was prepared by reference (Kim, D., H. Son and SBPark, 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] I. Preparation of fluorophores of formula (I) and formula (II) of the present invention

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

[0049] (1) 3-Cyanobenzaldehyde (328 mg) was added to a flask and dissolved in 25 mL of dry dichloromethane. 0.55 mL of 2,4-dimethylpyrrole (516.6 mg) was slowly added dropwise. Under nitrogen protection, 3 drops of trifluoroacetic acid were added at -40 °C, and the reaction was allowed to proceed for 2 hours. The reaction was monitored by TCL until complete. 20 mL of water was added, and the mixture was extracted twice with dichloromethane. The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. Separation was performed by silica gel column chromatography (ethyl acetate: petroleum ether = 7%, iodine staining) to give 687 mg of a 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) Compound (1-1) (1.15 g) was added to a flask and dissolved in 3.97 mL of acetonitrile (3.12 g) and 3 mL of ethanol. Under ice bath conditions, 331 μL of 3-mercaptopropionic acid (403.3 mg) was slowly added dropwise. After 5 minutes, 2.96 mL of hydrazine hydrate (3.04 g) was slowly added dropwise. The reaction was carried out overnight at 45 °C for 18 hours under nitrogen protection, and the reaction was complete as monitored by TCL. For post-treatment, 5 mL of saturated ammonium chloride solution was added, followed by 20 mL of water and 20 mL of ethyl acetate. The mixture was extracted twice, and the combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 1.28 g of a reddish-brown solid compound (1-2). Compounds (1-2) were transferred to a 100 mL flask and dissolved in 20 mL of tetrahydrofuran. A solution of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (1.29 g) prepared with 5 mL of tetrahydrofuran was added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 hours. After the intermediate was completely oxidized, 5.28 mL of triethylamine (3.845 g) was slowly added dropwise under ice bath conditions. After 10 minutes, 7.19 mL of boron trifluoride diethyl ether (8.09 g) was slowly added dropwise. The reaction was then carried out at 40 °C for 2 hours under TCL monitoring. Once the reaction was complete, 30 mL of water and 30 mL of ethyl acetate were added, and the mixture was extracted twice. The combined organic phases were washed with saturated ammonium chloride water, dried over anhydrous sodium sulfate, filtered, and concentrated. Separation by silica gel column chromatography (ethyl acetate: petroleum ether = 7%, iodine staining) yielded 450 mg of red solid compound 1, with a yield of 28.3%.

[0051] The optimized synthetic route for compound 1 is as follows:

[0052]

[0053] The specific steps for preparing the fluorophores of formula (Ⅰ) and formula (Ⅱ) are as follows:

[0054] The fluorophore of formula (I) was prepared by dissolving compound 1 (100 mg) and 4-aminobenzaldehyde (115.8 mg) in 4.9 mL toluene and 0.4 mL DMF (to promote the dissolution of 4-aminobenzaldehyde) in a flask. Then, 283 μL piperidine (244.2 mg) and 164 μL acetic acid (172.1 mg) were added dropwise. The reaction was carried out at 120 °C for 5 minutes under nitrogen protection. After cooling to room temperature, 10 mL and 15 mL dichloromethane were added, and the mixture was extracted twice. The combined organic phases were washed with saturated ammonium chloride water, dried over anhydrous sodium sulfate, filtered, and concentrated. Separation by silica gel column chromatography (ethyl acetate: petroleum ether = 1:2, UV) yielded 25 mg of a black solid, the fluorophore of formula (I), with a yield of 20.1%. The 1H NMR spectrum is shown below. 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) was prepared by dissolving compound 1 (100 mg) and 4-aminobenzaldehyde (173.7 mg) in 5 mL of DMF in a flask. Then, 283 μL of piperidine (244.2 mg) and 164 μL of acetic acid (172.1 mg) were added dropwise. The reaction was carried out at 120 °C for 10 minutes under nitrogen protection. After cooling to room temperature, 10 mL and 15 mL of dichloromethane were added, and the mixture was extracted twice. The combined organic phases were washed with saturated ammonium chloride water, dried over anhydrous sodium sulfate, filtered, and concentrated. Separation by silica gel column chromatography (methanol:dichloromethane = 5%, UV) yielded 23 mg of a black solid, which is the fluorophore of formula (II); yield 15.5%. The 1H NMR spectrum is shown below. 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 for the preparation methods of fluorophores of formula (I) and formula (II) are as follows:

[0057]

[0058] II. 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) described in this invention are as follows:

[0060] Compound 2 (54 mg) and 4-aminobenzaldehyde (58.7 mg) were added to a flask and dissolved in 4.9 mL toluene and 0.4 mL DMF (to promote the dissolution of 4-aminobenzaldehyde). Then, 94 μL of piperidine (81.2 mg) and 55 μL of acetic acid (57.3 mg) were added dropwise. The reaction was carried out at 120 °C for 5 minutes under nitrogen protection. After cooling to room temperature, 10 mL and 15 mL of dichloromethane were added, and the mixture was extracted twice. The combined organic phases were washed with saturated ammonium chloride water, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by RP-HPLC (reversed-phase preparative high-performance liquid chromatography) to obtain a black solid, namely the fluorophore of formula (III), with a yield of 24.8%. The 1H NMR spectrum is shown below. 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 for the preparation method of the fluorophore of formula (III) is as follows:

[0062]

[0063] Example 2: Preparation of drug probes

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

[0065] (1) Pyridine hydrochloride (5.2 g) was added to a flask and heated to 150 °C until completely melted. Then, Gefitinib (530 mg) solid was added, and the reaction was allowed to proceed for 3 hours under TCL monitoring until complete. After cooling to room temperature, the substrate was dissolved in 25 mL of 5 M NaOH solution, followed by extraction twice with 60 mL of ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Separation by silica gel column chromatography (methanol:dichloromethane = 8%, PMA) yielded 410 mg of a yellow-green solid (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) In a flask, add formula (Ⅳ-1) (285 mg), N-Boc-3-aminopropyl bromide (238 mg), and K2CO3 (276 mg), dissolve in 7 mL of acetonitrile, and react overnight at 75 °C. The reaction was monitored by TCL until 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 by silica gel column chromatography (methanol:dichloromethane = 5%, PMA) to obtain 304 mg of clear solid formula (Ⅳ-2), yield 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 in 2 mL of anhydrous dichloromethane, add 300 μL of trifluoroacetic acid dropwise under nitrogen protection and ice bath, react at room temperature for 1 hour, and monitor with TCL until the reaction is complete. For post-treatment, first remove dichloromethane under reduced pressure, then add 5 mL of trichloromethane to dissolve completely and evaporate to dryness 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) dropwise under nitrogen protection and ice bath until the pH is around 9. After 10 minutes, add (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimide carbonate (58 mg) dissolved in 1 mL of DMF. After 15 minutes of addition, transfer to room temperature and react overnight. Monitor with TCL until the reaction is complete. Add 15 mL of water and 20 mL of ethyl acetate, wash twice with water, wash the organic phase with saturated saline solution, dry with anhydrous sodium sulfate, filter, and concentrate. Separate by silica gel column chromatography (methanol:dichloromethane = 4%, PMA) to obtain 113 mg of a transparent solid, namely the drug probe of formula (IV), with a yield of 85.4%. The 1H NMR spectrum is shown below. 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 for the preparation method of the drug probe of formula (IV) is as follows:

[0069]

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

[0071] The fluorophores of formulas (I), (II), and (III) were added to 5 equivalents of BCN-NHs and reacted in acetonitrile for 5 hours. After dilution to 10 μM, their emission spectra were measured using a Hitachi fluorescence spectrophotometer F-7100. The excitation and emission wavelengths of the three fluorophores, as well as the emission intensity ratios (fluorescence on-off factors) at the emission wavelengths, were obtained. 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) Fold-on 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 dyes can be quenched by resonant energy transfer through tetrazine functionalization, and then the quenching effect can be effectively removed by reaction with bicyclic [6,1,0]nonyne (BCN) or trans-cyclooctene (TCO), thereby turning on the fluorescence.

[0075] Example 4: Preparation of hydrogel

[0076] 1. Hydrogel manufacturing process

[0077] (1) All monomer stock solutions were prepared with deionized water (Note: When preparing, g / 100mL should be the total volume after adding water of 100mL, and the solute volume effect should not be ignored), as shown in Table 2:

[0078] Table 2 Preparation of hydrogel monomer stock solution

[0079] ST (g / 100 mL) DMAA (N,N-dimethylacrylamide) ～ SA (sodium acrylate) 45 AA (acrylamide) 65 Bis (N,N'-methylene bisacrylamide) 2 NaCl (sodium chloride) 30 PBS 10× APS (ammonium persulfate) 10 TEMED (tetramethyl ethylenediamine) 10 4-HT (nitroxide piperidinol) 1

[0080] (2) Mix the hydrogel monomer stock solution together in a certain proportion to ensure that the final concentration of PBS 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 it is not strictly required (the prepared gel can be stored at 4℃ for 1 month and at -20℃ for 6 months).

[0081] (3) Transfer the required 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) Hydrogel cavity fabrication: Place three drops of 4μL water on a glass slide (25.4×76.2mm), place three coverslips (20×20mm) side by side on the glass slide to form the first layer, and then place two more coverslips on the second layer to form a rectangular cavity of 10×20mm.

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

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

[0085] (7) Take out the sample, remove the coverslip with a blade, cut the hydrogel into a rectangle, record the size before expansion, place it in deionized water to absorb water and expand, 15min×3, until it no longer expands, and record the size after expansion to obtain a physical expansion factor.

[0086] 2. Hydrogel formulation

[0087] To test the effect of varying monomer content in the gel formulation on the hydrogel swelling ratio, this invention, while keeping the total amount of SA and AA constant (total 35g / 100mL) and other components constant, varied the SA:AA ratio and observed the swelling ratio at different Bis crosslinking agent concentrations. Figure 6 As shown in Table 3, the vertical axis represents the physical expansion factor before and after expansion, and the horizontal axis represents the total concentration of the cross-linking agent. Therefore, it can be seen that as the total concentration of the Bis cross-linking agent increases, the physical expansion factor of the hydrogel gradually decreases. To further improve the resolution in expansion microscopy imaging, the hydrogel formulation was determined as shown in Table 3.

[0088] Table 3 Hydrogel Formulations

[0089] Final concentration (g / 100 mL) DMAA (N,N-dimethylacrylamide) 3 SA (sodium acrylate) 23 AA (acrylamide) 12 Bis (N,N'-methylene bisacrylamide) 0.01 NaCl (sodium chloride) 3.024 PBS 1× APS (ammonium persulfate) 0.25 TEMED (tetramethyl ethylenediamine) 0.2 4-HT (nitroxide piperidinol) 0.02

[0090] Example 5: Microscopic Imaging

[0091] Expanded microscopy imaging was performed based on the hydrogel formulation determined in Example 4. Human non-small cell lung cancer A549 cells cultured to a density of 50%-60% were incubated in imaging dishes with a Gefitinib-BCN probe for 1 hour, followed by incubation with compounds 1, 2, and the fluorophores of formulas (I) and (III) for 0.5 hours. Cells were washed with PBS, fixed with 4% paraformaldehyde, stained with Hoechst 33342 for 15 minutes, and washed with PBST. Slides were mounted at room temperature. Figure 7 As shown, this is a fluorescence image of A549 cells after co-incubation with EGFR probes and labeled with different BODIPY anchoring agents (scale bar: 20 μm). All images were obtained on different detection channels of the Leica-DMi8 confocal microscope system.

[0092] Cell crawling slides were placed in 24-well plates and cervical cancer HeLa cells were cultured to a density of 50%-60%. The cells were incubated with the Gefitinib-BCN probe for 1 hour, followed by incubation with the formula (III) fluorophore for 0.5 hours. The cells were washed with PBS, then fixed with 3% paraformaldehyde + 0.1% (v / v) glutaraldehyde for 10 minutes. The aldehyde groups were then reduced with 0.1% (w / v) sodium borohydride (PBS) for 7 minutes, followed by treatment with 100mM glycine (PBS) for 10 minutes. Finally, the cells were anchored with 0.25% (v / v) glutaraldehyde (PBS) for 10 minutes before gelation. After completing the above steps, the cell slides were removed and placed into the cavity containing the hydrogel. The gel was then formed at 37°C for 1 hour, followed by cutting into rectangular pieces. These were then treated at 37°C for 2 hours in a digestion solution (50 mM Tris (pH=8), 1 Mm EDTA, 0.1% Triton X-100, 0.8 M Guanidine HCl, and 8 units / mL ProK), and subsequently incubated in 5 μg / mL Hoechst 33342 (PBS) for 1 hour to facilitate subsequent confocal microscopy for cell location. Finally, dilatational microscopy was performed using a Leica-DMi8 confocal microscope (all images were taken at 20× magnification, some images are magnified). The results are as follows: Figure 8 As shown, the scale bar before expansion is 11μm, the scale bar after expansion is 11μm, the actual physical size is 90μm, and the expansion ratio is 8.18 times.

[0093] The specific embodiments of this application have been described above, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A fluorescence-enhancing anchoring agent based on fluoroboron dipyrrole-tetraazine dye, characterized in that, The fluorophore of the fluorescence-enhancing anchoring agent is shown in formula (I), formula (II), or formula (III): The tetrazine group carried by the fluorescence-enhancing anchoring agent relieves fluorescence quenching through a bioorthogonal reaction with the drug probe, and the drug probe is shown in formula (IV): 。 2. A method for preparing the fluorescence-enhancing anchoring agent as described in 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): Compound 1 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 carry out the reaction. The mixture was then extracted with dichloromethane and separated by silica gel chromatography to obtain black solid 1, which is the fluorophore of formula (I). A2. The fluorophore of formula (II): Compound 1 and 4-aminobenzaldehyde were dissolved in N,N-dimethylformamide DMF, and then piperidine and acetic acid were added dropwise under nitrogen protection to react. The mixture was then extracted with dichloromethane and separated by silica gel chromatography to obtain black solid 2, which is the fluorophore of formula (II). 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. The mixture was then extracted with dichloromethane and purified by reversed-phase preparative high performance liquid chromatography (RP-HPLC) to obtain black solid 3, which is the fluorophore of formula (III). The preparation method of the drug probe of formula (IV) is as follows: C1. Gefitinib EGFR reacts with molten pyridine hydrochloride, is then dissolved in sodium hydroxide solution, extracted with ethyl acetate, and separated by silica gel column chromatography to obtain a yellow-green solid, namely formula (Ⅳ-1). C2. Dissolve the obtained formula (Ⅳ-1), N-Boc-3-aminopropyl bromide and K2CO3 in acetonitrile ACN, then extract with ethyl acetate, and separate by silica gel column to obtain transparent solid 1, namely formula (Ⅳ-2). C3. Dissolve the C2 obtained formula (Ⅳ-2) in anhydrous dichloromethane, add trifluoroacetic acid (TFA) dropwise to react, and treat under reduced pressure to obtain transparent solid 2. Dissolve the transparent solid 2 in N,N-dimethylformamide (DMF), and then add N,N-diisopropylethylamine (DIEA) and (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimide carbonate (BCN-NHs) dissolved in N,N-dimethylformamide (DMF) dropwise in sequence. Separate the mixture by silica gel column to obtain transparent solid 3, which is the drug probe of formula (Ⅳ). The reaction route 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 in A2 to 4-aminobenzaldehyde, piperidine, and acetic acid 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 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).

6. The method for preparing the fluorescence-enhancing anchoring agent according to claim 2, characterized in that, The mass ratio of pyridine hydrochloride to 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).

7. A method for applying the fluorescence-enhancing anchoring agent as described in claim 1 in dilatational microscopy, characterized in that, The application method involves the tetrazine group carried by the fluorescence-enhancing anchoring agent reacting with the drug probe via a bioorthogonal reaction to relieve fluorescence quenching.

8. The method for applying the fluorescence-enhancing anchoring agent in dilatational microscopy according to claim 7, characterized in that, The application method further includes the reaction of the amino groups carried by the fluorescence-enhancing anchor with glutaraldehyde to further relieve fluorescence quenching, and the fluorescence-enhancing anchor is covalently linked to the hydrogel mesh.

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