Water-soluble tetrazine, tetrazine membrane probe, preparation method thereof, and application

Abstract

The present invention discloses a water-soluble tetrazine, a tetrazine membrane probe and a preparation method thereof. The water-soluble tetrazine is not only easy to synthesize, but also, after being connected with hydrophobic BCy, makes the tetrazine membrane probe an amphiphilic molecule, capable of being connected to the cell membrane surface for fluorescence imaging. Moreover, the tetrazine group can undergo an in-situ bioorthogonal reaction with a dienophile, thereby changing the hydrophilicity and hydrophobicity of the tetrazine membrane probe, so that the tetrazine membrane probe can stay on the cell membrane surface for a longer time or enter the hydrophobic environment inside the cell membrane more quickly, thereby flexibly adjusting the targeting ability of the tetrazine membrane probe to the cell membrane.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent dyes, and in particular to a water-soluble tetrazine, a tetrazine membrane probe prepared based on the water-soluble tetrazine, a preparation method of the tetrazine membrane probe, and application of the tetrazine membrane probe in cell membrane fluorescence imaging. Background Art

[0002] Fluorescence imaging, as a visualization technique, boasts remarkable characteristics such as high sensitivity, non-destructiveness, real-time detection, and high spatiotemporal resolution, and has been widely used in biological sciences and biomedicine. Small molecule organic fluorophores can help visualize gene expression, post-transcriptional modifications, biomolecule concentrations, organelle trafficking, disease detection, drug development, and response assessment. They can also guide tumor surgery through images. Therefore, when modern dye chemistry is combined with modern bioorthogonal chemistry, fluorophores have been endowed with considerable functionality in biological imaging.

[0003] When traditional fluorescent dyes are used to image biologically relevant targets such as cell membranes, the duration and specificity of their imaging are primarily determined by the physicochemical properties of the pre-synthesized compounds. For example, chemical modification of the fluorescent dye's anchoring group, fluorophore, and polar group is required to create biomembrane probes with varying targeting capabilities. However, these pre-synthesized membrane probes lack flexibility during fluorescence imaging due to their fixed targets, requiring multiple imaging cycles, resulting in complex workflows and low imaging efficiency. Summary of the Invention

[0004] An object of the present invention is to provide a water-soluble tetrazine that is easy to synthesize and can utilize its own hydrophilicity to react with a series of hydrophobic borondifluoroindolenine cyanine fluorescent dyes (BCy) covering the far infrared to near infrared to prepare an amphiphilic tetrazine membrane probe.

[0005] Specifically, the above objectives are achieved through the following technical solutions:

[0006] A water-soluble tetrazine precursor having the structural formula shown in Formula I:

[0007]

[0008] In Formula I, R 2 is selected from hydrogen, saturated alkyl, unsaturated alkyl, sulfonic acid, carboxyl, phosphoric acid, ester or quaternary ammonium salt, R 3 Selected from hydroxyl, amino or ammonium salt, x=1-16, y=1-8, z=1-8.

[0009] In this technical solution, a water-soluble tetrazine having the structural formula I is easily synthesized. The preparation method disclosed by the inventors in patent CN112010817A utilizes nitrile compounds (a) and (b) as raw materials, adds a thiol compound and a hydrazine compound as catalysts, and reacts. After the reaction is completed, the mixture is poured into ice water, and an aqueous solution of sodium nitrite is added with stirring. The pH is then adjusted to 2-3 with an acid. Extraction, washing, concentration, and purification are performed to obtain a tetrazine compound represented by Formula IV, wherein the R4 group is a tert-butyloxycarbonylamino group or a hydroxyl group.

[0010] Subsequently, the tetrazine compound shown in Formula IV is subjected to a quaternization reaction to obtain a water-soluble tetrazine, wherein R 2 The group can be hydrogen, saturated alkyl, unsaturated alkyl, sulfonic acid, carboxyl, phosphoric acid, ester or quaternary ammonium salt depending on the reactants. In some embodiments, the tetrazine compound of formula IV is reacted with sultone or halogenated alkyl acid or halogenated alkyl ester under alkaline or non-alkaline conditions to obtain R 2 The hydrophilic tetrazine group is hydrogen, saturated alkyl, unsaturated alkyl, sulfoalkyl, carboxyalkyl, phosphate alkyl, quaternary ammonium, or ester alkyl.

[0011] Among them, for R 4 The water-soluble tetrazine with NHBoc group is deprotected in the presence of acid to obtain R shown in formula I 3 The water-soluble tetrazine with a hydrochloride or trifluoroacetate group is neutralized to obtain R 3 The water-soluble tetrazine with an amino group has the following overall reaction synthesis route:

[0012]

[0013] In the present technical solution, the water-soluble tetrazine is not only easy to synthesize, but can also be used to synthesize tetrazine membrane probes with a series of hydrophobic fluoroboron merocyanine fluorescent dyes represented by formula III.

[0014] As part of the tetrazine membrane probe, in one or more embodiments, the R 2The groups can be hydrophilic groups such as sulfonic acid, carboxyl, phosphate, and quaternary ammonium salts, or hydrophobic groups such as alkyl and lipid groups. However, water-soluble tetrazines possess a high overall hydrophilicity due to the presence of quaternary ammonium cations. When linked to the hydrophobic BCy, the tetrazine membrane probe becomes an amphiphilic molecule, making it difficult to fully enter the hydrophobic environment within the cell membrane. This causes the hydrophobic dye of the tetrazine membrane probe to partially entrap within the cell membrane, while the hydrophilic tetrazine end is exposed on the outer surface of the cell membrane, thereby enabling fluorescence imaging of the cell membrane surface. More importantly, the tetrazine group of the tetrazine membrane probe can undergo in situ bioorthogonal reactions with dienophiles such as trans-cyclooctene on the cell membrane, thereby altering the hydrophilicity and hydrophobicity of the tetrazine membrane probe, allowing the tetrazine membrane probe to remain on the outer surface of the cell membrane for a longer period of time or more easily cross the cell membrane and enter the cytoplasm, thereby flexibly adjusting the tetrazine membrane probe's targeting ability to the cell membrane or intracellular subcellular structures.

[0015] Furthermore, the water-soluble tetrazine is selected from the following compounds:

[0016]

[0017]

[0018] Another object of the present invention is to provide a tetrazine membrane probe, which is prepared by reacting any of the aforementioned water-soluble tetrazines with a fluoroboron merocyanine fluorescent dye. The amphiphilic tetrazine membrane probe can achieve fluorescence imaging of cell membranes and can undergo in situ bioorthogonal reactions with dienophiles to change its own hydrophilicity and hydrophobicity, thereby flexibly changing its targeting ability to the cell membrane and achieving in situ targeted imaging of subcellular structures in living cells.

[0019] The above purpose is achieved through the following technical solutions:

[0020] A tetrazine membrane probe having any of the aforementioned water-soluble tetrazine structures, wherein the tetrazine membrane probe has a structure shown in Formula II:

[0021]

[0022] In formula II, X is selected from CH or N, R 1 Selected from hydrogen, halogen, C1-C4 alkyl, phenyl, carboxyl, sulfonic acid, methoxy, formate, amide, n=1-6, m=0-16.

[0023] In the present technical scheme, tetrazine membrane probe includes water-soluble tetrazine and fluoroboron moiety cyanine fluorescent dye connected by amide bond.Wherein, fluoroboron moiety cyanine fluorescent dye (BCy) not only has excellent photophysical properties, but also is a hydrophobic fluorescent dye.After being connected with water-soluble tetrazine, tetrazine membrane probe becomes amphoteric molecule, when not reacting with dienophile, tetrazine membrane probe can be used for the fluorescence imaging of cell membrane, after in situ bioorthogonal reaction occurs in cell membrane subcellular structure with dienophile, the hydrophilicity of tetrazine membrane probe can be changed, the time when tetrazine membrane probe enters cell membrane is bidirectionally regulated, i.e., according to the difference of dienophile, the time when tetrazine membrane probe stays on cell membrane surface can be extended, tetrazine membrane probe can also be made to more easily cross cell membrane and enter cytoplasm, thereby realizing flexibly adjusting the targeting ability of tetrazine membrane probe to cell membrane or intracellular subcellular structure.

[0024] In some embodiments, R 1 The group is a substituted or unsubstituted chain alkyl group. The chain alkyl group can be either a straight chain alkyl group or a branched chain alkyl group, and the number of carbon atoms in the chain alkyl group is preferably C1 to C2. In one or more embodiments, the chain alkyl group is preferably a methyl group, an ethyl group, or a trifluoromethyl group.

[0025] In some embodiments, R 1 The group may be substituted or unsubstituted phenyl.

[0026] Furthermore, the tetrazine membrane probe is selected from the following compounds:

[0027]

[0028] The present invention also provides a method for preparing any of the aforementioned tetrazine membrane probes, wherein the method uses any of the aforementioned water-soluble tetrazines to prepare any of the aforementioned tetrazine membrane probes. Specifically, the preparation method comprises the following steps:

[0029] The water-soluble tetrazine is mixed with the fluoroboron merocyanine fluorescent dye of formula III and reacted to obtain the tetrazine membrane probe;

[0030] The structure of the fluoroboron merocyanine fluorescent dye is:

[0031]

[0032] In formula III, R 5 Selected from carboxylic acids or active esters of carboxylic acids.

[0033] In this technical solution, the reaction of a water-soluble tetrazine precursor with a fluoroboronic merocyanine fluorescent dye can be carried out under alkaline or non-alkaline conditions. The water-soluble tetrazine precursor is dissolved in a solvent, BCy is added, and the mixture is stirred at room temperature until the reaction is complete. Purification is then performed to obtain a tetrazine membrane probe.

[0034] In this technical solution, for R 5 For BCy, whose group is a carboxylic acid, a certain amount of condensing agent needs to be added during the reaction to form a BCy ester or amide probe derivative. In one or more embodiments, the condensing agent can be 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, HATU (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and its hydrochloride), or DCC (N,N'-dicyclohexylcarbodiimide).

[0035] In some preferred embodiments, the water-soluble tetrazine precursor and base are dissolved in a solvent, and a fluoroboron merocyanine fluorescent dye is added and reacted at room temperature for 0.5 to 24 hours to obtain the tetrazine membrane probe, wherein the base is sodium bicarbonate, potassium bicarbonate, triethylamine, diisopropylethylamine, pyridine, 2,6-lutidine, sodium acetate, potassium acetate, potassium carbonate or sodium carbonate.

[0036] In some embodiments, the solvent is at least one of dichloromethane, dichloroethane, acetone, chloroform, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, toluene, chlorobenzene, 1,4-dioxane, and xylene.

[0037] In the technical solution, the tetrazine membrane probe synthesized by using water-soluble tetrazine and fluoroboron merocyanine fluorescent dye has mild reaction conditions, a short synthesis path, and is easy to scale up for production.

[0038] Furthermore, the fluoroboron merocyanine fluorescent dye is selected from the following compounds:

[0039]

[0040] The present invention also provides a use of any of the aforementioned tetrazine membrane probes in cell membrane fluorescence imaging, specifically, a reagent for cell membrane fluorescence imaging, the reagent comprising the tetrazine membrane probe.

[0041] Furthermore, the fluorescence imaging reagent also includes a dienophile, which is used to undergo a bioorthogonal reaction with the tetrazine membrane probe to change the targeting ability of the tetrazine membrane probe. In some preferred embodiments, the dienophile is at least one of trans-cyclooctene-triphenylphosphine (TCO-TPP), trans-cyclooctene-acid (TCO-acid), bicyclo[6.1.0]non-4-yn-9-yl-lysosome (BCN-lyso), bicyclo[6.1.0]non-4-yn-9-yl-hexylamine (BCN-HA), and trans-cyclooctene-silirhodamine (TCO-SiR). After undergoing a bioorthogonal reaction with the tetrazine membrane probe, different dienophiles can bidirectionally regulate the hydrophilicity and hydrophobicity of the tetrazine membrane probe to prolong or shorten its cell membrane labeling time.

[0042] In the present technical solution, the labeling method of the cell membrane fluorescence imaging reagent can be to label the cell nuclei of the cells in the imaging culture dish, then add the tetrazine membrane probe for staining, without washing, add the dienophile, incubate for a period of time, and then image using a laser confocal microscope without washing. The labeling method can also be to completely react the tetrazine membrane probe with an equal amount or a slightly excess amount of the dienophile in an organic solvent, use the stock solution as a stock solution, directly stain the cells, and then perform fluorescence imaging.

[0043] Furthermore, the molar ratio of the dienophile to the tetrazine membrane probe is 0.01 to 100. Preferably, the concentration of the dienophile is 0.05-100 μM, and the concentration of the tetrazine membrane probe is 0.05-10 μM.

[0044] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0045] 1. The water-soluble tetrazine of the present invention is not only easy to synthesize, but also, after being linked to the hydrophobic BCy, makes the tetrazine membrane probe an amphiphilic molecule that can be attached to the cell membrane surface for fluorescence imaging. Moreover, its tetrazine group can undergo an in situ bioorthogonal reaction with the dienophile, changing the hydrophilicity and hydrophobicity of the tetrazine membrane probe, allowing the tetrazine membrane probe to remain on the cell membrane surface for a longer period of time or enter the hydrophobic environment inside the cell membrane more quickly, thereby flexibly adjusting the tetrazine membrane probe's targeting ability to the cell membrane;

[0046] 2. The tetrazine membrane probe of the present invention not only has excellent photophysical properties, but also, as an amphiphilic molecule, can be used to label cell membranes. After reacting with a dienophile, it can adjust its residence time on the cell membrane surface for fluorescence imaging, thereby flexibly regulating its own imaging of different cell structures.

[0047] 3. The preparation method of the present invention is simple, the reaction conditions are mild, and the synthesis path is short, which is conducive to scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0049] Figure 1 The invention shows a tetrazine film probe, a tetrazine film probe and fluorescence imaging of various dienophiles in a specific embodiment of the invention;

[0050] Figure 2 The chemical structural formulas of some dienophiles used in specific embodiments of the present invention are shown. DETAILED DESCRIPTION

[0051] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with examples and drawings. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention.

[0052] The term "connected" used in the present invention, unless otherwise specified, may refer to direct connection or indirect connection via other groups.

[0053] All raw materials of the present invention are not particularly limited to their source, and can be prepared by commercially purchased or conventional methods well known to those skilled in the art. All raw materials of the present invention are not particularly limited to their purity, and the present invention preferably adopts the purity requirement of analytically pure or fluorescent dye field routine. All raw materials of the present invention, its trade mark and abbreviation all belong to conventional trade mark and abbreviation in this area, and each trade mark and abbreviation are all clear and definite in the field of its related use, and those skilled in the art can, according to trade mark, abbreviation and corresponding purposes, purchase from commercially available or prepare by conventional method.

[0054] The present invention has no particular limitation on the expression of the substituents, and all expressions familiar to those skilled in the art are adopted. Based on common sense, those skilled in the art can correctly understand the meaning of the substituents according to the expressions.

[0055] 1. Preparation of water-soluble tetrazine precursors

[0056]

[0057] By selecting nitrile compounds (a) and (b) with different methylene numbers, tetrazines IV can be obtained, wherein R 4 It is -NHBoc or -OH, y=1~8, z=1~8.

[0058] [Example 1]

[0059]

[0060] Dissolve 2-dimethylaminopropionitrile (2.94 g, 30 mmol), 3-mercaptopropionic acid (1.3 mL, 15 mmol), and tert-butyl 2-cyanomethylcarbamate (2.34 g, 15 mmol) in 5 mL of ethanol, and then add hydrazine hydrate (5.8 mL, 120 mmol). The reaction mixture is stirred vigorously at 40°C for 15 h. After the reaction is complete, cool the reaction solution with ice water, add sodium nitrite (8.4 g, 120 mmol) dissolved in ice water (100 mL), and then slowly add 2M HCl in an ice bath, causing the solution to turn bright red and generate gas. Continue to add 2M HCl until gas precipitation stops, at which point the pH of the reaction system is 2-3. After stirring for 5 minutes, add saturated sodium bicarbonate aqueous solution to adjust the pH to 8. The resulting solution was extracted with dichloromethane (200 mL×5), and the organic phase was dried over anhydrous Na 2 SO 4 and concentrated in vacuo, and then purified by silica gel column chromatography to obtain 1.39 g of pink oily product Tz-1 with a yield of 30%.

[0061] 1 H NMR (400MHz, Chloroform-d) δ5.61 (s, 1H), 4.89 (d, J = 6.0Hz, 2H), 3.42 (t, J = 7.0Hz, 2H), 2.88 (t, J = 7.1Hz, 3H), 2.21 (s, 6H), 1.39 (s, 9H).

[0062] [Example 2]

[0063]

[0064] Dissolve 3-dimethylaminobutyronitrile (3.4 g, 30 mmol), 3-mercaptopropionic acid (1.3 mL, 15 mmol), and tert-butyl 2-cyanomethylcarbamate (2.34 g, 15 mmol) in 5 mL of ethanol, then add hydrazine hydrate (5.8 mL, 120 mmol). The reaction mixture is stirred vigorously at 40°C for 15 h. After the reaction is complete, cool the reaction solution with ice water, add sodium nitrite (8.4 g, 120 mmol) dissolved in ice water (100 mL), and then slowly add 2M HCl in an ice bath, causing the solution to turn bright red and generate gas. Continue adding 2M HCl until gas evolution ceases, at which point the pH of the reaction system is between 2 and 3. After stirring for 5 min, adjust the pH to 8 by adding saturated aqueous sodium bicarbonate. The resulting solution was extracted with dichloromethane (200 mL×5), and the organic phase was dried over anhydrous Na 2 SO 4 and concentrated in vacuo, and then purified by silica gel column chromatography to obtain 1.55 g of pink oily product Tz-2 with a yield of 35%.

[0065] 1H NMR (400MHz, Chloroform-d) δ5.67(s,1H),4.97(d,J=5.9Hz,2H),3.49–3.42(m,2H),3.01–2.94(m,2H),2.62(s,6H),2.40–2.30(m,2H),1.46(s,9H).

[0066] [Example 3]

[0067]

[0068] Dissolve 3-dimethylaminobutyronitrile (3.4 g, 30 mmol), 3-mercaptopropionic acid (1.3 mL, 15 mmol), and tert-butyl 2-cyanoethylcarbamate (2.55 g, 15 mmol) in 5 mL of ethanol, then add hydrazine hydrate (5.8 mL, 120 mmol). The reaction mixture is stirred vigorously at 40°C for 15 h. After the reaction is complete, cool the reaction solution with ice water, add sodium nitrite (8.4 g, 120 mmol) dissolved in ice water (100 mL), and then slowly add 2M HCl in an ice bath, causing the solution to turn bright red and generate gas. Continue adding 2M HCl until gas evolution stops, at which point the pH of the reaction system is 2-3. After stirring for 5 min, add saturated sodium bicarbonate aqueous solution to adjust the pH to 8. The resulting solution was extracted with dichloromethane (200 mL×5), and the organic phase was dried over anhydrous Na 2 SO 4 and concentrated in vacuo, and then purified by silica gel column chromatography to obtain 1.60 g of pink oily product Tz-3 with a yield of 35%.

[0069] The tetrazine compound IV is further quaternized to obtain a water-soluble tetrazine, wherein R 2 The group can be hydrogen, saturated alkyl, unsaturated alkyl, sulfonic acid, carboxyl, phosphoric acid, ester or quaternary ammonium salt depending on the reactants. In some embodiments, the tetrazine compound of formula IV is reacted with sultone or halogenated alkyl acid or halogenated alkyl ester under alkaline or non-alkaline conditions to obtain R 2 The hydrophilic tetrazine group is hydrogen, saturated alkyl, unsaturated alkyl, sulfoalkyl, carboxyalkyl, phosphate alkyl, quaternary ammonium, or ester alkyl.

[0070] Among them, for R 4 The water-soluble tetrazine with NHBoc group is deprotected in the presence of acid to obtain R shown in formula I 3 The water-soluble tetrazine with an amino group has the following overall reaction synthesis route:

[0071]

[0072] [Example 4]

[0073] R 2 For example, if the group is a sulfonic acid group, the tetrazine compound of Formula IV is reacted with a sultone or a haloalkylsulfonic acid. The solvent can be at least one of dichloromethane, dichloroethane, chloroform, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, toluene, chlorobenzene, 1,4-dioxane, and xylene. The reaction can be carried out in an alkaline or non-alkaline environment, and the base used can be sodium bicarbonate, potassium bicarbonate, sodium acetate, potassium acetate, potassium carbonate, sodium carbonate, etc.

[0074]

[0075] Tz-2 (148.2 mg, 0.5 mmol) was dissolved in 1 mL of DMF, and 4-butane sultone (131 μL, 1.5 mmol) was added. The reaction was stirred at 60°C for 10 h. After dilution with 2 mL of dichloromethane, the product was precipitated in 30 mL of MBTE at 4°C overnight. The precipitate was purified by reverse-phase silica gel column chromatography using acetonitrile and water containing 0.1% formic acid as eluents to obtain 104.8 mg of product Tz-4 in a 50% yield.

[0076] 1 H NMR (400MHz, Methanol-d4) δ3.57 (dq, J=10.7, 6.5, 5.8Hz, 4H), 3.46 (t, J=7.2Hz, 2H), 3.16(s,6H),2.87(t,J=6.8Hz,2H),2.57–2.46(m,2H),2.28–2.18(m,2H),1.45(s,9H).

[0077]

[0078] Tz-4 (161.7 mg, 0.4 mmol) was dissolved in 3 mL of dichloromethane, and a 4M HCl solution (0.3 mL, 1.2 mmol) in 1,4-dioxane was added. The solution was heated to room temperature for 60 minutes. After evaporation of the solvent, the solution was washed with methanol / chloroform / methyl tert-butyl ether (1:1) to afford approximately 125 mg of Tz-11 as a pink solid, with a yield of 89%.

[0079] 1 H NMR (400MHz, Methanol-d4) δ3.54(m,6H),3.16(s,6H),2.83(t,J=6.9Hz,2H),2.56–2.45(m,2H),2.10(q,J=8.5,7.9Hz,2H).

[0080] [Example 5]

[0081]

[0082] Tz-3 (155.2 mg, 0.5 mmol) and NaHCO (42 mg, 0.5 mmol) were dissolved in 1 mL of DMF, and 4-butane sultone (131 μL, 1.5 mmol) was added. The mixture was stirred at 60°C for 8 h. After dilution with 3 mL of dichloromethane, the mixture was precipitated in 30 mL of ether at 4°C overnight. Filtering, washing with ether, and drying the filter cake gave 186.2 mg of the product, Tz-5, in an 86% yield.

[0083] 1 H NMR (400MHz, CD3OD) δ3.66–3.53(m,6H),3.48–3.39(m,4H),3.16(s,6H),2.88(t,J=6.8Hz,2H),2.57–2.45(m,2H),2.28–2.18(m,2H),1.36(s,9H).

[0084] 13 C NMR (101MHz, CD3OD) δ168.48,168.30,156.92,78.72,62.98,62.70,50.03,38.53,35.29,30.56,27.32,19.74,18.54.

[0085]

[0086] Tz-5 (172.8 mg, 0.4 mmol) was dissolved in 5 mL of methylene chloride. 4 M HCl (2 mmol, 0.5 mL) in 1,4-dioxane was added at 0°C. The solution was allowed to react at room temperature for 60 min. After evaporation of the solvent, the product was washed with methyl tert-butyl ether and dried to yield 128 mg of the product, Tz-6, as a red solid (87% yield).

[0087] 1 H NMR(400MHz,Methanol-d4)δ3.75–3.69(m,2H),3.66–3.60(m,2H),3.60–3.52(m,4H),3.48 (t,J=6.8Hz,2H),3.16(s,6H),2.84(t,J=6.8Hz,2H),2.55–2.43(m,2H),2.17–2.10(m,2H).

[0088] High resolution mass spectrometry (HRMS) identification: [M+Na] + m / z theoretical molecular formula and molecular weight [C 12 H 24 N6NaO3S] +:355.1523; actual molecular weight:355.1526

[0089] [Example 6]

[0090]

[0091] 3-Bromopropyltrimethylammonium bromide (50.6 mg, 0.2 mmol) and Tz-3 (31 mg, 0.1 mmol) were dissolved in dimethylformamide (0.5 mL) and reacted at 80°C for 6 h. Purification by reverse phase column gave 30.4 mg of the product Tz-7 with a yield of 74%.

[0092] 1 H NMR (400MHz, Methanol-d4) δ3.69–3.59(m,4H),3.58–3.42(m,8H),3.28(s,9H),3.27(s,6H),2.61–2.51(m,2H),2.50–2.40(m,2H),1.37(s,9H).

[0093]

[0094] 4M HCl (0.125 mL, 0.5 mmol) dissolved in 1,4-dioxane was added to a 1 mL dichloromethane solution of Tz-7 (20.5 mg, 0.05 mmol). The solution was then reacted at room temperature for 60 min. After the solvent was evaporated, 1 mL of methanol was added. After dissolution, 20 mL of acetonitrile was added to precipitate the product. After filtration, 15.4 mg of a pink solid, i.e., the product Tz-8, was obtained with a yield of 89%.

[0095] 1 H NMR(400MHz,Methanol-d4)δ3.75–3.69(m,2H),3.66–3.60(m,2H),3.60–3.52(m,4H),3.48 (t,J=6.8Hz,2H),3.16(s,6H),2.84(t,J=6.8Hz,2H),2.55–2.43(m,2H),2.17–2.10(m,2H).

[0096] [Example 7]

[0097]

[0098] tert-Butyl 4-bromobutyrate (44 mg, 0.2 mmol) and Tz-3 (31 mg, 0.1 mmol) were dissolved in acetonitrile (0.3 mL). Sodium bicarbonate (8.4 mg, 0.1 mmol) was added and the mixture was reacted at 80°C for 8 h. Column chromatography afforded 41 mg of the product, Tz-9, with a yield of 77%.

[0099] 1 H NMR(400MHz,Chloroform-d)δ5.49(s,1H),3.87(d,J=8.6Hz,2H),3.72(d,J=6.2Hz,2H),3.55(dq,J=25.3,7.0, 5.9Hz,6H),3.37(s,6H),2.60–2.49(m,2H),2.42(t,J=6.5Hz,2H),2.11–1.97(m,2H),1.44(s,9H),1.38(s,9H).

[0100] 13 C NMR (101MHz, CDCl3) δ171.33,168.75,167.99,166.62,155.99,81.49,79. 31,63.08,62.85,51.25,38.61,35.63,30.91,28.40,28.08,20.05,18.04.

[0101]

[0102] Tz-9 (41 mg) was dissolved in dichloromethane (1 mL), and 0.5 mL of trifluoroacetic acid was added. The mixture was reacted at room temperature for 2 h, and then dried to obtain the product Tz-10 with a yield of 95% and 39 mg.

[0103] 1 H NMR (400MHz, Methanol-d4) δ3.72(t,J=6.8Hz,2H),3.63(t,J=6.9Hz,3H),3.61–3.52(m,2H),3.47(t,J=7.2Hz ,3H),3.46–3.37(m,3H),3.16(s,6H),2.57–2.46(m,3H),2.46(t,J=6.5Hz,3H),2.06(dq,J=14.3,6.9Hz,3H).

[0104] 2. Preparation of difluoroborondolenine electron acceptor

[0105] [Example 8]

[0106]

[0107] 2,3,3-Trimethyl-3H-indolenine (5.0 g, 31.4 mmol) was added to 50 mL of acetic anhydride, and boron trifluoride etherate (5 mL, 41 mmol) was added under argon. The reaction was stirred at 120°C for 6 hours. The solvent was removed in vacuo using a rotary evaporator, and the product was purified by silica gel column chromatography to obtain 2.8 g of the pale yellow product BFI 1 in a 35% yield.

[0108] 1 H NMR (400MHz, CDCl3) δ7.65 (d, J = 7.9Hz, 1H), 7.39–7.30 (m, 2H), 7.26–7.21 (m, 1H), 5.71 (s, 1H), 2.28 (s, 3H), 1.43 (s, 6H).

[0109] 13 C NMR (101MHz, CDCl3) δ181.44,179.57,143.00,140.66,128.68,125.88,121.84,116.21,116.19,116.17,91.17,91.13,49.64,24.49,23.59.

[0110] [Example 9]

[0111]

[0112] 2,3,3-Trimethyl-3H-indole-5-carboxylic acid (6.1 g, 30 mmol) was added to 60 mL of acetic anhydride, and boron trifluoride etherate (4.5 mL, 36 mmol) was added under argon. The reaction was stirred at 120°C for 8 hours. The solvent was removed in vacuo using a rotary evaporator, and the product was purified by silica gel column chromatography to obtain 3.3 g of the pale yellow product BFI 2 in a 37% yield.

[0113] 1 H NMR (400 MHz, dimethyl sulfoxide-d6) δ 13.05 (s, 1H), 8.19 (d, J = 1.6 Hz, 1H), 8.04 (d, J = 8.3 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 2.31 (s, 3H), 1.49 (s, 6H).

[0114] 13 C NMR (101 MHz, dimethyl sulfoxide) δ 183.98, 181.88, 167.32, 146.41, 141.81, 131.08, 128.65, 124.42, 115.11, 92.83, 49.98, 24.12, 23.81.

[0115] [Example 10]

[0116]

[0117] 1,1,2-Trimethyl-1H-benz[e]indole (5.0 g, 24 mmol) was added to 35 mL of acetic anhydride. Boron trifluoride etherate (3.8 mL, 31 mmol) was added under argon. The reaction was stirred at 130°C for 10 hours. The solvent was removed in vacuo on a rotary evaporator and purified by silica gel column chromatography to obtain 2.39 g of the pale yellow product, BFI 3, in a 33% yield.

[0118] 1 H NMR (400MHz, CDCl3) δ7.98(d,J=8.2Hz,1H),7.95(d,J=8.1Hz,1H),7.93–7.86(m,2H),7.58(ddd, J=8.4,6.9,1.4Hz,1H),7.48(ddd,J=8.2,6.8,1.2Hz,1H),5.81(s,1H),2.32(s,3H),1.69(s,6H).

[0119] 13 C NMR (101MHz, CDCl3) δ182.66,179.10,140.69,133.96,132.36,130.21,129 .93,128.47,127.14,125.13,122.17,115.58,91.06,51.25,24.06,23.66.

[0120] [Example 11]

[0121]

[0122] 5-Chloro-2,3,3-trimethylindole (2.5 g, 13 mmol) was added to 20 mL of acetic anhydride, followed by the addition of boron trifluoride etherate (2 mL, 16 mmol) under argon. The reaction was stirred at 120°C for 8 hours. The solvent was removed in vacuo using a rotary evaporator, and the mixture was purified by silica gel column chromatography to obtain 1.65 g of the pale yellow product, BFI 4, in a 45% yield.

[0123] 1 H NMR (400MHz, Chloroform-d) δ7.56(d,J=8.2Hz,1H),7.40–7.30(m,2H),5.72(d,J=2.4Hz,1H),2.30(s,3H),1.45(s,6H).

[0124] 13C NMR (101MHz, CDCl3) δ181.37,180.43,142.43,141.58,131.63,128.87,122.54,117.09,117.07,117.05,91.25,49.81,24.45,23.71.

[0125] [Example 12]

[0126]

[0127] BFI 1 (750 mg, 3 mmol) and N-chlorosuccinimide (NCS, 479.7 mg, 3.6 mmol) were added to a reaction flask, followed by 15 mL of chloroform and dimethyl sulfoxide (45 μL, 0.6 mmol). The reaction system was stirred at room temperature overnight. The solvent was removed in vacuo using a rotary evaporator and purified by silica gel column chromatography to obtain 245 mg of the white solid product BFI 5 with a yield of 87%.

[0128] 1 H NMR (400MHz, CDCl3) δ7.66 (d, J = 7.8Hz, 1H), 7.45–7.23 (m, 3H), 2.43 (s, 3H), 1.71 (s, 6H).

[0129] 13 C NMR (101MHz, CDCl3) δ176.63,176.22,142.03,141.65,128.81,126.72,121.53,116.62,52.58,22.10,21.67.

[0130] [Example 13]

[0131]

[0132] To a mixture of BFI 2 (596 mg, 2 mmol), CHCl 3 (12 mL), and N-methyl-2-pyrrolidone (0.6 mL) were added N-chlorosuccinimide (400 mg, 3 mmol) and dimethyl sulfoxide (38 μL, 0.5 mmol). The reaction was stirred at room temperature for 8 hours. The solvent was removed in vacuo using a rotary evaporator and the mixture was purified by silica gel column chromatography to obtain 536.3 mg of the product BFI 6 as a white solid in an 82% yield.

[0133] 1H NMR (400 MHz, dimethyl sulfoxide-d6) δ 13.11 (s, 1H), 8.21 (s, 1H), 8.06 (d, J = 9.9 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H), 2.47 (s, 3H), 1.73 (s, 6H).

[0134] 13 C NMR (101 MHz, dimethyl sulfoxide) δ 178.95, 178.47, 167.16, 145.30, 142.43, 131.21, 129.56, 124.18, 115.64, 102.10, 52.82, 22.27, 21.77.

[0135] [Example 14]

[0136]

[0137] To a mixture of BFI 3 (449 mg, 1.5 mmol) and N-chlorosuccinimide (300 mg, 2.25 mmol) was added dimethyl sulfoxide (32 L, 0.45 mmol) and CHCl₃ (8 mL) to dissolve the mixture, followed by stirring at room temperature for 5 hours. Upon complete conversion, the solvent was removed in vacuo on a rotary evaporator, and the mixture was purified by silica gel column chromatography to afford 413.6 mg of the yellow solid product, BFI 7, in an 83% yield.

[0138] 1 H NMR(400MHz,Chloroform-d)δ8.02(d,J=8.0Hz,1H),7.97(d,J=8.2Hz,1H),7.90(q,J =8.8Hz,2H),7.62(t,J=8.3Hz,1H),7.52(t,J=8.1Hz,1H),2.46(s,3H),1.97(s,6H).

[0139] 13 C NMR (101MHz, CDCl3) δ177.69,175.68,139.50,132.88,130.42,130.05,127.72,127.30,125.48,122.34,115.54,101.46,54.18,21.70,20.72.

[0140] 3. Preparation of fluoroboron merocyanine fluorescent dye

[0141] [Example 15]

[0142]

[0143] BFI 2 (43.9 mg, 0.15 mmol) and 1,3,3-trimethyl-2-(formylmethylene)indoline (42 mg, 0.2 mmol) were dissolved in a mixture of toluene (2 mL), piperidine (40 μL), and acetic acid (20 μL) under argon. The mixture was then stirred at 90°C for 3 hours. After removing the solvent under reduced pressure, most impurities were removed by silica gel column chromatography to yield BCy1.

[0144] BCy 1 (approximately 0.15 mmol) and N,N'-disuccinimidyl carbonate (76.8 mg, 0.3 mmol) were dissolved in anhydrous dichloromethane (2 mL), and diisopropylethylamine (49 μL, 0.3 mmol) was added. The mixture was stirred at room temperature for 5 hours. The solvent was removed in vacuo using a rotary evaporator, and the mixture was purified by silica gel column chromatography to obtain 62.7 mg of the blue product BCy 2 (75% yield).

[0145] 1 H NMR(400MHz,Chloroform-d)δ8.26(t,J=13.4Hz,1H),8.15(dd,J=8.4,1.8Hz,1H),8.00(d,J=1.7Hz,1H),7.62(d,J=8.3Hz,1H),7.29–7.22(m,3H),7.0 4(t,J=7.3Hz,1H),6.85–6.80(m,1H),5.89(d,J=13.6Hz,1H),5.62(d,J=13 .0Hz,1H),5.60(s,1H),3.30(s,3H),2.92(s,4H),1.67(s,6H),1.46(s,6H).

[0146] 13 C NMR (101MHz, CDCl3) δ178.95,174.45,169.44,167.82,161.77,150.12,143.66,143.33,140.80,139.87,132.48,128. 04,124.04,122.34,121.96,119.61,114.62,114.15,107.82,96.75,91.36,48.29,47.55,29.70,28.71,25.81,25.72.

[0147] [Example 16]

[0148]

[0149] BFI 6 (65.4 mg, 0.2 mmol) and 1,3,3-trimethyl-2-(formylmethylene)indoline (52.3 mg, 0.26 mmol) were dissolved in a mixture of toluene (3 mL), piperidine (60 μL), and acetic acid (30 μL) under argon. The mixture was then stirred at 60°C for 5 hours. After the solvent was removed under reduced pressure, most impurities were removed by silica gel column chromatography to yield BCy3.

[0150] BCy 3 (approximately 0.2 mmol) and N,N'-disuccinimidyl carbonate (76.8 mg, 0.3 mmol) were dissolved in anhydrous dichloromethane (2 mL), followed by the addition of diisopropylethylamine (49 μL, 0.3 mmol). The resulting mixture was stirred at room temperature for 5 hours, after which the solvent was removed. Purification by silica gel column chromatography afforded 103.9 mg of the blue product, BCy 4, in an 85% yield.

[0151] 1 H NMR (400MHz, CDCl3) δ8.33(t,J=13.4Hz,1H),8.04(dd,J=8.4,1.7Hz,1H),7.89(d,J=1.7Hz,1H),7.50(d,J=8.3Hz,1H),7.27–7.20(m,2H),7.0 3(t,J=7.4Hz,1H),6.83(d,J=8.1Hz,1H),6.41(d,J=13.4Hz,1H),5.76(d,J=13.3Hz,1H),3.31(s,3H),2.84(s,4H),1.67(s,6H),1.61(s,6H).

[0152] 13 C NMR (101MHz, CDCl3) δ172.61,170.20,170.13,169.41,161.67,149.43,146.74,143.26,141.37,140.21,132.41,128.2 0,123.67,123.25,122.07,119.78,114.51,109.62,108.51,100.11,98.59,51.08,48.14,30.01,28.71,25.71,22.94.

[0153] [Example 17]

[0154]

[0155] BFI 6 (65.4 mg, 0.2 mmol) and Fischer's aldehyde (59.0 mg, 0.26 mmol) were dissolved in a mixture of toluene (3 mL), piperidine (60 μL), and acetic acid (30 μL) under argon. The mixture was then stirred at 50°C for 2 hours. After the solvent was removed under reduced pressure, most impurities were removed by silica gel column chromatography to yield BCy 5.

[0156] BCy 5 (approximately 0.2 mmol) and N,N'-disuccinimidyl carbonate (76.8 mg, 0.3 mmol) were dissolved in anhydrous dichloromethane (2 mL), followed by the addition of diisopropylethylamine (49 μL, 0.3 mmol). The mixture was then stirred at room temperature for 5 hours. After removal of the solvent, the mixture was purified by silica gel column chromatography to yield 98.7 mg of the blue product, BCy 6, in a 78% yield.

[0157] 1 H NMR(400MHz, CDCl3)δ8.12(dd,J=8.4,1.7Hz,1H),8.01–7.89(m,2H),7.61( d,J=8.4Hz,1H),7.55(t,J=13.1Hz,2H),7.30–7.21(m,3H),7.03(t,J=7.5Hz ,1H),6.81(d,J=7.8Hz,1H),6.54(d,J=13.9Hz,1H),6.34(t,J=12.0Hz,1H), 5.63(d,J=12.8Hz,1H),3.29(s,3H),2.92(s,4H),1.75(s,7H),1.64(s,6H).

[0158] 13 C NMR (101 MHz, deuterated dimethyl sulfoxide) δ 168.86, 165.28, 164.61, 161.10, 156.84, 146.57, 144.31, 141.61, 138.99, 136.86, 134.99, 127.63, 123.35, 118.97, 118.63, 117.49, 117.16, 115.61, 110.18, 107.32, 103.02, 96.25, 93.78, 46.67, 42.54, 24.94, 23.50, 20.96, 18.05.

[0159] [Example 18]

[0160]

[0161] Under argon, diisopropylethylamine (59 μL, 0.33 mmol) was added to a mixed solution of BFI 6 (49 mg, 0.15 mmol) and hemicyanine (99 mg, 0.2 mmol) in dichloromethane (2 mL). The mixture was then stirred at 50°C for 90 minutes. After cooling to room temperature, N,N'-disuccinimidyl carbonate (76 mg, 0.3 mmol) and diisopropylethylamine (33 μL, 0.2 mmol) were added. The mixture was stirred at room temperature for 12 hours, after which the solvent was removed. Purification by silica gel column chromatography afforded 45.5 mg of the product, BCy 7, in a 46% yield.

[0162] 1 H NMR(400MHz,Chloroform-d)δ8.14(dd,J=8.4,1.7Hz,1H),8.00(d,J=1.7Hz,1H),7.83(dd,J=14 .1,12.1Hz,1H),7.65(d,J=8.3Hz,1H),7.25–7.16(m,3H),7.01(dd,J=13.9,11.6Hz,1H),6.95(t ,J=7.4Hz,1H),6.73(d,J=7.8Hz,1H),6.65(d,J=14.1Hz,1H),6.41(t,J=13.0Hz,1H),6.22(dd,J =13.7,11.7Hz,1H),5.53(d,J=12.5Hz,1H),3.22(s,3H),2.92(s,4H),1.75(s,6H),1.62(s,6H).

[0163] 13 C NMR (101MHz, CDCl3) δ174.71,169.85,169.34,162.54,161.53,149.78,149.36,148.75,144.23,141.85,140.10,139.35,132.37,1 27.98,126.13,124.63,123.76,121.76,121.23,120.93,115.38,115.12,101.63,98.04,51.73,46.62,29.43,28.32,25.72,22.65.

[0164] 4. Preparation of Tetrazine Membrane Probes

[0165] [Example 19]

[0166]

[0167] Tz-6 (9.2 mg, 0.025 mmol) and diisopropylethylamine (12 μL, 0.075 mmol) were dissolved in 0.3 mL of dimethyl sulfoxide, and BCy2 (11.5 mg, 0.02 mmol) was added. The mixture was stirred at 25°C for 2 h. After completion of the reaction, the product was purified by reverse-phase silica gel column chromatography using MeCN and H2O containing 0.1% formic acid as eluents to obtain MemTz-1.

[0168] 1 H NMR (400MHz, CD3OD) δ8.22(t,J=13.4Hz,1H),7.80(d,J=1.9Hz,1H),7.72(d,J=8.3Hz,1H),7.43(d,J =8.1Hz,1H),7.30–7.22(m,2H),7.02(t,J=7.4Hz,1H),6.90(dd,J=8.0,2.7Hz,1H),5.96(d,J=12.3Hz ,1H),5.77–5.67(m,2H),3.97(t,J=6.3Hz,2H),3.68–3.50(m,6H),3.45(t,J=7.0Hz,2H),3.33(s,3H) ,3.14(s,6H),2.88(t,J=6.7Hz,2H),2.54–2.44(m,2H),2.27–2.14(m,2H),1.66(s,6H),1.47(s,6H).

[0169] 13 C NMR (101MHz, CD3OD) δ179.08,173.60,168.52,168.21,167.51,146.80,143.73,142.60,140.81,139.63,129.46,127.96,127.86,122.07,121. 63,121.27,113.73,113.53,107.88,96.60,90.65,63.00,62.78,50.41 ,47.23,47.17,38.35,34.93,30.61,29.19,28.09,25.09,19.78,18.67.

[0170] [Example 20]

[0171]

[0172] Tz-6 (9.2 mg, 0.025 mmol) and diisopropylethylamine (12 μL, 0.075 mmol) were dissolved in 0.3 mL of dimethyl sulfoxide, and BCy 4 (12.2 mg, 0.02 mmol) was added. The mixture was stirred at 25°C for 2 h. After completion of the reaction, the product was purified by reverse-phase silica gel column chromatography using MeCN and H 2 O containing 0.1% formic acid as eluents to obtain MemTz-2.

[0173] 1 HNMR(400MHz,DMSO-d6)δ8.64(t,J=5.8Hz,1H),8.29–8.21(m,1H),7.91(d,J=1.7Hz,1H),7.76(dd,J=8.3,1 .7Hz,1H),7.53–7.50(m,1H),7.38–7.32(m,1H),7.26(d,J=6.1Hz,2H),7.15(t,J=7.4Hz,1H),6.33(dd,J=8 9.4,13.4Hz,1H),3.85–3.79(m,2H),3.53(t,J=7.0Hz,2H),3.49(s,3H),3.47–3.43(m,4H),3.37(s,2H),3. 05(s,6H),2.49(t,J=5.7Hz,2H),2.39–2.30(m,2H),2.00(dq,J=11.6,7.1Hz,2H),1.69(s,6H),1.61(s,6H).

[0174] 13 C NMR (101MHz, DMSO-d6) δ171.94,168.71,168.49,168.16,166.55,146.20,145.64,143.64,141.04,140.42,130.33,128.73,128.61,123. 88,122.56,121.69,113.19,110.34,98.40,63.07,62.41,51.19,50.58,48.25,48.10,38.59,35.17,31.24,28.51,23.08,20.41,19.37.

[0175] [Example 21]

[0176]

[0177] Tz-6 (9.6 mg, 0.026 mmol) and diisopropylethylamine (15 μL, 0.09 mmol) were dissolved in 0.3 mL of dimethyl sulfoxide, and then BCy 6 (12.7 mg, 0.02 mmol) was added. The mixture was stirred at 25° C. for 30 min and purified by carbon 18 (C 18 ) was purified by reverse-phase silica gel column chromatography to obtain 13.4 mg of the product with a yield of 79%.

[0178] 1 H NMR (400MHz, DMSO-d6) δ8.69(t,J=5.8Hz,1H),8.06–7.87(m,3H),7.79(d,J=8.3Hz,1H),7.42(d,J=7.3H z,1H),7.31(d,J=7.7Hz,1H),7.27(t,J=7.7Hz,1H),7.11(d,J=8.0Hz,1H),7.04(t,J=7.4Hz,1H),6.51–6 .34(m,2H),5.88(d,J=13.0Hz,1H),3.83(q,J=6.5Hz,2H),3.56–3.42(m,8H),3.40(s,3H),3.35(t,J=7.1 Hz,2H),3.06(s,6H),2.53–2.48(m,2H),2.45–2.28(m,2H),2.09–1.96(m,2H),1.69(s,6H),1.59(s,6H).

[0179] 13 C NMR (101MHz, DMSO-d6) δ172.44,168.70,168.48,167.91,167.82,166.50,151. 19,148.88,145.47,143.94,141.29,140.44,130.61,128.68,128.48,124.03, 122.91,122.43,121.74,113.51,109.93,109.41,100.29,99.36,63.03,62.44,51.42,50.57,48.11,47.73,38.61,35.15,31.24,27.87,22.96,20.42,19.36.

[0180] 5. Cell membrane fluorescence imaging experiment

[0181] A549 cells were stained with Hoechst 33342 for 5 minutes to label the nuclei, washed, and then stained with 200 nM MemTz-2 for 5 to 30 minutes before direct fluorescence imaging. In some experimental groups, the dienophiles TCO-acid (final concentration 2 μM), TCO-TPP (final concentration 2 μM), BCN-Lyso (final concentration 10 μM), BCN-HA (final concentration 10 μM), or TCO-SiR (final concentration 2 μM) were added after the addition of MemTz-2. The cells were stained for 0 to 360 minutes and imaged using confocal laser scanning microscopy.

[0182] The experimental results are as follows Figure 1 As shown in the fourth set of fluorescence images, cells stained only with MemTz-2 exhibited fluorescence exclusively on the cell membrane within 60 minutes, and after 120 minutes, some organelles also showed fluorescence. In contrast, in the first, second, third, fifth, and sixth sets of fluorescence images, in which dienophiles were added, the duration of fluorescence on the cell membrane was bidirectionally modulated. In the first set of fluorescence images, in which TCO-TPP was added, and in the second set of fluorescence images, in which TCO-acid was added, the tetrazine membrane probe consistently labeled the cell membrane for 6 hours, with very little fluorescence within the cell membrane. In the third set of fluorescence images, in which BCN-Lyso was added, only a small amount of fluorescence entered the cell membrane within 2 hours. This indicates that some dienophiles, such as TCO-TPP, TCO-acid, and BCN-Lyso, can prolong cell membrane labeling after bioorthogonal reactions with the tetrazine membrane probe. On the contrary, it can be seen from the 5th and 6th experimental groups that after the addition of the dienophile BCN-HA or TCO-SiR, the fluorescence of the cell membrane quickly disappeared and the fluorescence in the cytoplasm was enhanced, indicating that the bioorthogonal reaction between dienophiles such as BCN-HA or TCO-SiR and the tetrazine membrane probe can enable the fluorescent probe to quickly enter the cell membrane, thereby realizing the flexible regulation of the tetrazine membrane probe on the imaging of different cell structures.

[0183] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A tetrazine membrane probe, characterized in that The tetrazine membrane probe has a structure shown in Formula II: In formula II, X is selected from CH or N, R 1 is selected from hydrogen, halogen; R 2 Selected from -SO3 - , x=1~16, y=1~8, z=1~8, n=1~6, m=0~16.

2. A tetrazine membrane probe according to claim 1, characterized in that: The tetrazine membrane probe is selected from the following compounds:

3. A water-soluble tetrazine, characterized in that The water-soluble tetrazine is selected from the following compounds:

4. The method for preparing a tetrazine membrane probe according to claim 1, wherein: The preparation method comprises the following steps: The water-soluble tetrazine of formula I and the fluoroboron merocyanine fluorescent dye of formula III are mixed and reacted to obtain the tetrazine membrane probe; The structure of the fluoroboron merocyanine fluorescent dye is: In formula III, R 5 Selected from carboxylic acid or carboxylic acid active ester, R 1 is selected from hydrogen and halogen, X is selected from CH or N, n=1-6, m=0-16; The structure of the water-soluble tetrazine is: In Formula I, R 3 Selected from amino or ammonium salt, x, y, z, R 2 Same as defined in claim 1.

5. The method for preparing a tetrazine membrane probe according to claim 4, wherein: The fluoroboron merocyanine fluorescent dye is selected from the following compounds:

6. The method for preparing a tetrazine membrane probe according to claim 5, wherein: The water-soluble tetrazine and a base are dissolved in a solvent, and a fluoroboron merocyanine fluorescent dye is added and reacted at room temperature for 0.5 to 24 hours to prepare the tetrazine membrane probe, wherein the base is sodium bicarbonate, potassium bicarbonate, triethylamine, diisopropylethylamine, pyridine, 2,6-lutidine, sodium acetate, potassium acetate, potassium carbonate or sodium carbonate, and the solvent is at least one of dichloromethane, dichloroethane, acetone, chloroform, tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, toluene, chlorobenzene, 1,4-dioxane and xylene.

7. A reagent for cell membrane fluorescence imaging, characterized in that The invention comprises the tetrazine membrane probe according to claim 1 or 2.

8. A reagent for cell membrane fluorescence imaging according to claim 7, characterized in that, It also includes a dienophile, which is used to undergo a bioorthogonal reaction with the tetrazine membrane probe to change the targeting ability of the tetrazine membrane probe, wherein the dienophile is at least one of trans-cyclooctene-triphenylphosphine, trans-cyclooctene-acid, bicyclo[6.1.0]non-4-yn-9-yl-lysosome, bicyclo[6.1.0]non-4-yn-9-yl-hexylamine, and trans-cyclooctene-silirhodamine.

9. A reagent for cell membrane fluorescence imaging according to claim 8, characterized in that, The molar ratio of the dienophile to the tetrazine membrane probe is 0.01-100.

Images