Tetrazine aggregation-induced luminescence probe precursor, probe, and preparation method and application thereof

Abstract

Tetrazine aggregation-induced emission probe precursor, probe and preparation method and application thereof. The probe precursor is quenched by dual pathways, which not only avoids the problem of fluorescence dispersion caused by the appearance of fluorescence at low concentrations after the probe undergoes a bioorthogonal reaction, but also significantly enhances the tetrazine polyfluorescence in the aggregated state, with the on-multiplier being as high as 1000 times. At the same time, the probe can adjust the target wavelength by adjusting the R1 and / or R4 groups and replacing the dienophile. Compared with the traditional method of connecting the tetrazine AIE probe to the tetrazine group, there is no need to design different synthesis pathways according to the tetrazine AIE probe, and the adjustment of the target wavelength is more flexible and simple.

Description

Technical Field

[0001] The invention relates to the field of fluorescent probes, and in particular to a tetrazine aggregation-induced luminescence probe precursor, a probe, and a preparation method and application thereof. Background Art

[0002] Fluorescence signals, as the output data of an optical detection system, are widely used in detection, in vivo and in vitro imaging, etc. due to their non-destructive, highly sensitive, specific, and real-time monitoring characteristics. However, due to the π-π stacking between molecules, traditional organic fluorescent molecules produce aggregation-induced quenching (ACQ) when they aggregate, resulting in fluorescence quenching. Therefore, they can only be used at very low concentrations, which limits their application.

[0003] Aggregation-induced emission (AIE) is the opposite of aggregation-induced quenching. This type of material exhibits negligible fluorescence in dilute solutions, but as the molecules aggregate, the fluorescence is significantly enhanced, thereby overcoming the problem of fluorescence self-quenching caused by traditional fluorescent probes after aggregation. It is widely used in cell imaging, fluorescence labeling, biosensing, tumor imaging and treatment and other fields.

[0004] In recent years, bioorthogonal activated fluorescent probes based on tetrazine groups have received increasing attention. This type of fluorescent probe connects mature AIE probes with tetrazine groups, uses the tetrazine group to affect intramolecular charge transfer to quench fluorescence, and then removes the quenching effect and restores fluorescence after the tetrazine group undergoes a bioorthogonal reaction with the dienophile. This type of bioorthogonal activated fluorescent probe based on tetrazine can effectively improve the signal-to-noise ratio and reduce fluorescence background interference, and has become a research hotspot for AIE probes.

[0005] In the existing tetrazine AIE probes, the tetrazine group connected to the mature AIE probe only acts as a "switch", so when fluorescence of the target wavelength is required, it is necessary to select an AIE probe with the target wavelength to connect to the tetrazine group. However, the types of mature AIE probes are not yet abundant, and it is not easy to select an AIE probe with an ideal target wavelength. Even if an AIE probe with a suitable target wavelength is confirmed, it is necessary to design a reasonable synthesis route to connect it to the tetrazine group, making the target wavelength adjustment and design of the existing tetrazine AIE probes very inconvenient. Not only that, when marking organelles, the existing tetrazine AIE probes can usually only mark one or two organelles. Therefore, for different organelles, it is also necessary to consider whether the synthesized molecular skeleton can enter the organelle, which limits the application scenarios of the tetrazine AIE probes. Summary of the invention

[0006] An object of the present invention is to provide a tetrazine aggregation-induced emission probe precursor, which can generate a tetrazine aggregation-induced emission probe in situ after undergoing a bioorthogonal reaction with a dienophile in vivo. The adjustment of the target wavelength of the tetrazine aggregation-induced emission probe can be achieved by changing the substituents on the skeleton and / or replacing the dienophile that reacts therewith, without the need to select compounds for the target wavelength and design a synthesis route, thereby significantly improving the flexibility of adjusting the target wavelength.

[0007] The above objectives are achieved through the following scheme:

[0008] The tetrazine aggregation-induced emission probe precursor has the structural formula shown in Formula I:

[0009]

[0010] In Formula I, R1 is selected from Wherein, R2 and R3 are each independently selected from aryl, heteroaryl or alkyl; n = 1 or 2, m = 1 or 2;

[0011] In formula I, R4 is selected from alkyl or hydroxyalkyl.

[0012] In the technical scheme, before the bioorthogonal reaction with the dienophile, as shown in Formula I, the tetrazine aggregation-induced luminescence probe precursor quenches the fluorescence by the cyano group and the tetrazine group on the skeleton. After the tetrazine aggregation-induced luminescence probe precursor reacts with the dienophile in a bioorthogonal manner, the tetrazine aggregation-induced luminescence probe is obtained in situ, at which time the tetrazine group reacts and connects with the dienophile, the n→π* transition is closed, and the tetrazine aggregation-induced luminescence probe in the unaggregated free state relies on the intramolecular rotation of the cyano group to suppress the fluorescence. When the tetrazine aggregation-induced luminescence probe is aggregated, the intramolecular rotation is further restricted, and the fluorescence of the tetrazine aggregation-induced luminescence probe is significantly enhanced.

[0013] In the present technical scheme, the tetrazine aggregation-induced emission probe precursor is quenched by dual pathways, and can remain in a quenched state with relatively dim fluorescence after the tetrazine aggregation-induced emission probe is generated in situ, and the tetrazine aggregation-induced emission probe releases fluorescence only after local aggregation reaches a certain concentration. This not only avoids the problem of fluorescence dispersion caused by the appearance of fluorescence at low concentrations after the probe undergoes a bioorthogonal reaction, but also the fluorescence onset of the tetrazine aggregation-induced emission probe in the aggregated state is significantly enhanced compared to the free state or unreacted precursor.

[0014] In the present technical scheme, the tetrazine group of the probe precursor is not only a switch of fluorescence, but also affects the target wavelength as a part of the probe. Therefore, when the skeleton is determined to be a structure of Formula I, when the target wavelength needs to be adjusted, the target wavelength can be adjusted in two ways: one way is to adjust the R1 and / or R4 groups, and the other way is to select a dienophile with which a bioorthogonal reaction occurs. Both methods can be used alone or simultaneously. Whether it is to adjust the R1 and R4 groups, or to replace the dienophile, when adjusting the target wavelength, the probe precursor no longer needs to select the traditional tetrazine AIE probe to be connected to the tetrazine group, nor does it need to design different synthetic paths according to the tetrazine AIE probe, thereby making the adjustment of the target wavelength more flexible and simple.

[0015] In the present technical solution, the molecular skeleton shown in Formula I has better biocompatibility. Experiments have shown that after adjusting the R1 and R4 groups to have the target wavelength, the same probe can achieve dienophile pre-targeted lysosomes, Golgi bodies, mitochondria, cell membranes, endoplasmic reticulum and other subcellular organelle imaging, which has a wide range of application value.

[0016] In the present technical solution, the R1 group in Formula I can be Wherein, R2 and R3 are each independently selected from aryl, heteroaryl or alkyl.

[0017] In some embodiments, R2 and R3 are each independently selected from substituted or unsubstituted aryl groups, the aryl group can be either a monocyclic aryl group or a condensed aryl group, and the number of carbon atoms of the aryl group is preferably C6 to C 24 , more preferably C6~C 18 , and more preferably C6~C 12 In one or more embodiments, the aryl group is phenyl, tolyl, ethylphenyl, naphthyl, or biphenyl.

[0018] In some embodiments, R2 and R3 are each independently selected from substituted or unsubstituted heteroaryl groups, the heteroaryl group may be a monocyclic or condensed ring aromatic group, the heteroatom of the heteroaryl group may be sulfur, oxygen or nitrogen, and the number of carbon atoms of the heteroaryl group is preferably C4 to C 12 , more preferably C4 to C8, and even more preferably C4 to C6. In one or more embodiments, the heteroaryl group is pyridyl, furyl, thienyl, imidazolyl, quinolyl, pyrazolyl, or pteridinyl.

[0019] In some embodiments, R2 and R3 are each independently selected from a substituted or unsubstituted alkyl group, and the alkyl group may be a chain alkyl group or a cyclic alkyl group. In some embodiments, the chain alkyl group may be a straight chain alkyl group or a branched chain alkyl group, and the number of carbon atoms of the chain alkyl group is preferably C1 to C 36, more preferably C1 to C6, and even more preferably C1 to C4. In one or more embodiments, the chain alkyl group is methyl, ethyl, propyl, isopropyl, butyl or tert-butyl. The number of carbon atoms in the cyclic alkyl group is preferably C4 to C6. 36 , more preferably C4~C 12 , and more preferably C4 to C6. In one or more embodiments, the cyclic alkyl group is cyclobutyl, cyclopentyl or cyclohexyl.

[0020] In the present technical solution, the R1 group in Formula I can also be Wherein, n=1 or 2, m=1 or 2. The values ​​of n and m can be the same or different. In some preferred embodiments, n=1, m=1.

[0021] In the technical scheme, R4 is selected from alkyl or hydroxyalkyl. In some preferred embodiments, the alkyl is preferably a chain alkyl, and in one or more embodiments, R4 is selected from C1-C6 straight chain alkyl, C1-C6 branched chain alkyl. In a more preferred embodiment, R4 is selected from methyl, ethyl, isopropyl, tert-butyl. In some preferred embodiments, the hydroxyalkyl can be C1-C6 hydroxyalkyl, and in one or more embodiments, R4 is selected from C1-C3 hydroxyalkyl. In a more preferred embodiment, R4 is selected from hydroxymethyl or hydroxyethyl.

[0022] Furthermore, the tetrazine aggregation-induced emission probe precursor has any of the following chemical structural formulas:

[0023]

[0024]

[0025] Another object of the present invention is to provide a tetrazine aggregation-induced emission probe, which is prepared by a bioorthogonal reaction between any of the aforementioned tetrazine aggregation-induced emission probe precursors and a dienophile, and the synthesis route is as follows:

[0026]

[0027] According to the different dienophiles, the target wavelength of the in-situ generated probe can be adjusted. Nx represents the reaction product of NBD and the probe precursor, and so on, Tx, hT-x, Bx, dT-x represent the reaction products of TCO, 4E-TCO, BCN, and d-TCO, respectively.

[0028] Further, the dienophile is bicyclo[6,1,0]nonyne (BCN), trans-cyclooctene (TCO) or norbornadiene (NBD). In some embodiments, trans-cyclooctene (TCO) can be TCO, d-TCO or 4E-TCO. In some preferred embodiments, it is found through experiments that BCN can react with tetrazine to directly generate pyridazine, the product is single, and BCN has a more reasonable steric hindrance, which can prevent π-π stacking and is conducive to the AIE effect. Therefore, when the dienophile is BCN, the fluorescence turn-on is enhanced, and the turn-on multiple can reach up to 1033 times.

[0029] The present invention also provides a method for preparing any of the aforementioned tetrazine aggregation-induced emission probe precursors. Specifically, the preparation method comprises the following steps:

[0030] Under an inert atmosphere, dissolving the aldehyde represented by formula II and tetrazine phosphate in a solvent to obtain a mixture;

[0031] After cooling the mixture, lithium diisopropylamide is added, and the reaction is stirred until the reaction is completed, and the tetrazine aggregation-induced emission probe precursor is separated;

[0032]

[0033] In formula II, R1 is selected from Wherein, R2 and R3 are each independently selected from aryl, heteroaryl or alkyl; n=1 or 2, m=1 or 2.

[0034] In the present technical scheme, the inert atmosphere is preferably an argon atmosphere. Under an inert atmosphere, the aldehyde and tetrazine phosphate shown in Formula II are dissolved in a solvent to obtain a mixture. In some preferred embodiments, the solvent is ultra-dry THF. Subsequently, the mixture is cooled to, preferably, -10 to -25°C, and then lithium diisopropylamide (LDA) is added, and then the reaction is stirred. After the reaction is completed by thin layer chromatography monitoring, the probe precursor is separated. In some preferred embodiments, saturated NH4Cl aqueous solution is used for quenching, extracted with ethyl acetate, and then the organic phases are combined, washed with saturated brine, dried with Na2SO4 and the solvent is removed by a rotary evaporator, and the residue is purified by silica gel column chromatography to obtain a probe precursor.

[0035] The present invention also provides the use of any of the aforementioned tetrazine aggregation-induced emission probe precursors. Specifically, the same tetrazine aggregation-induced emission probe precursor can be bioorthogonally reacted with a dienophile to label at least four organelles, wherein the dienophile is bicyclo[6,1,0]nonyne, trans-cyclooctene or norbornadiene, wherein the bicyclo[6,1,0]nonyne has a structural formula shown in Formula III:

[0036]

[0037] Wherein, the R group is selected from any of the following:

[0038]

[0039] In some preferred embodiments, the same tetrazine aggregation-induced luminescence probe precursor can label multiple subcellular organelles such as lysosomes, Golgi bodies, mitochondria, cell membranes, endoplasmic reticulum, etc. Based on its characteristics of easy adjustment of the target wavelength and no need to design the synthesis path multiple times, it can be used to label multiple different subcellular organelles after adjusting the target wavelength, and has a wide range of application value.

[0040] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0041] 1. The tetrazine aggregation-induced luminescence probe precursor of the present invention is quenched by dual pathways, which not only avoids the problem of fluorescence dispersion caused by the appearance of fluorescence at low concentrations after the probe undergoes a bioorthogonal reaction, but also significantly enhances the fluorescence of the tetrazine in the aggregated state, with the opening multiple being as high as 1000 times;

[0042] 2. The probe of the present invention can adjust the target wavelength by adjusting the R1 and / or R4 groups and replacing the dienophile. Compared with the traditional method of connecting the tetrazine AIE probe to the tetrazine group, there is no need to design different synthesis paths according to the tetrazine AIE probe, and the adjustment of the target wavelength is more flexible and simple.

[0043] 3. The probe of the present invention has better biocompatibility. The same probe can realize the imaging of various subcellular organelles such as lysosomes, Golgi bodies, mitochondria, cell membranes, endoplasmic reticulum, etc. with dienophile pre-targeting, and has wide application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] 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:

[0045] Figures 1 to 6 The relevant spectra of the probe characterization in Examples 9 to 14 are shown, wherein: (a) LC-MS monitors the reaction between the probe precursor and BCN; (b) the relevant mass spectrum of the probe at 3h; (c) the absorption spectra of the probe precursor and the probe (10μM); (d) and (e) are the fluorescence emission spectra of the probe precursor and the probe in H2O (d) and DMSO (e), respectively (10μM);

[0046] Figures 7 to 12The AIE performance test results of each probe in Example 15 are shown, wherein: (a) the fluorescence emission spectrum of the probe (10 μM) in a DMSO / H2O mixed solvent containing different volume fractions of water; (b) a line graph of the relative fluorescence intensity (I / I0) versus the volume fraction of water, wherein I0 is the maximum fluorescence emission intensity when the volume fraction of water is 0; Figure 7 (c) is the result of dynamic light scattering experiment: particle size distribution of probe B-1 (10 μM) in H2O (containing 1% DMSO);

[0047] Fig.13 This is a diagram showing the results of the mitochondrial targeted fluorescence imaging and co-localization experiment of the probe precursor 2 in Example 16;

[0048] Fig.14 This is a diagram showing the lysosome-targeted fluorescence imaging and co-localization experimental results of the probe precursor 2 in Example 17;

[0049] Fig.15 This is a diagram showing the endoplasmic reticulum-targeted fluorescence imaging and co-localization experimental results of the probe precursor 2 in Example 18;

[0050] Fig.16 This is a diagram showing the Golgi apparatus-targeted fluorescence imaging and co-localization experimental results of the probe precursor 2 in Example 19;

[0051] Fig.17 This is a diagram showing the cell membrane targeted fluorescence imaging and co-localization experimental results of the probe precursor 2 in Example 20;

[0052] Fig.18 These are the fluorescence photographs of probe B-1 in Example 21 with different volume fractions of H2O. DETAILED DESCRIPTION

[0053] 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 embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0054] All raw materials of the present invention are not particularly limited to their sources, and can be purchased on the market or prepared according to 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 analytically pure or conventional purity requirements in the field of fluorescent probes. All raw materials of the present invention, their brands and abbreviations belong to conventional brands and abbreviations in this field, and each brand and abbreviation is clear and definite in the field of its related use. Those skilled in the art can purchase them from the market or prepare them by conventional methods according to the brands, abbreviations and corresponding uses.

[0055] 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 meanings according to the expressions.

[0056] The term "connected" used in the present invention, unless otherwise specified, may mean directly connected or indirectly connected via other groups.

[0057] 1. Tetrazine Aggregation-Induced Emission Probe Precursor

[0058] [Example 1]

[0059]

[0060] 1) Synthesis of aldehyde S2

[0061] Under argon protection, ultra-dry DMF (1.9 mL, 24.7 mmol) and POCl3 (2.3 mL, 24.7 mmol) were added to a 100 mL round-bottom flask equipped with a magnetic stirrer. The above solution was stirred at 0°C for 0.5 hours. The reactant S1 (786 mg, 3.1 mmol) was dissolved in 1,2-dichloroethane (30 mL), then added dropwise to the above solution, and the resulting mixture was refluxed and stirred at 95°C for 3 hours. After cooling to room temperature, the mixture was poured into crushed ice and the pH was adjusted to 7 with 6M NaOH. The desired compound was extracted with DCM (100 mL×3), and the combined organic phases were washed with saturated brine (150 mL), dried over anhydrous Na2SO4 and the solvent was removed by rotary evaporator. The residue was purified by silica gel column chromatography to obtain aldehyde S2 (343.2 mg, 39%) as a red solid.

[0062] 1 H NMR (400MHz, CDCl3) δ9.84 (s, 1H), 7.85 (d, J = 8.7Hz, 2H), 7.68 (d, J = 4.0Hz, 1H), 7.41 (s, 1H), 7.32 (d, J = 4.0Hz, 1H), 6.71 (d, J = 8.7Hz, 2H), 3.09 (s, 6H).

[0063] 13 C NMR (101MHz, CDCl3) δ182.5,152.6,150.6,143.8,141.5,137.4,132.3,125.5,120.6,117.7,111.8,97.8,40.2.

[0064] HRMS[M+H]+m / z calcd.for[C16H15N2S]+283.0900,found 283.0894.

[0065] 2) Synthesize probe precursor 1

[0066] Under argon protection, aldehyde S2 (56.5 mg, 0.2 mmol), tetrazine phosphate S3 (64.0 mg, 0.26 mmol) and ultra-dry THF (4 mL) were added to a 10 mL dry reaction tube equipped with a magnetic stirrer. The mixture was cooled to -20 ° C, and then LDA (150 μL 2M) was added dropwise. The above mixture was stirred for 2 hours. After the reaction was completed by thin layer chromatography monitoring, it was quenched with saturated NH4Cl aqueous solution and extracted with ethyl acetate (20 mL×3). The combined organic phases were washed with saturated brine, dried with Na2SO4 and the solvent was removed by rotary evaporator. The residue was purified by silica gel column chromatography to obtain a black solid tetrazine aggregation-induced emission probe precursor 1 (28.7 mg, 38%).

[0067] 1 H NMR (400MHz, CDCl3) δ8.33(d,J=15.9Hz,1H),7.85(d,J=8.6Hz,2H),7.31(s,1H),7.28(d,J=3.8Hz, 1H),7.25(d,J=3.7Hz,1H),7.18(d,J=15.9Hz,1H),6.74(d,J=8.6Hz,2H),3.09(s,6H),3.04(s,3H).

[0068] 13 C NMR (101MHz, CDCl3) δ166.1,164.9,152.2,144.0,141.8,139.4,133.1,132.2,131.8,126.1,121.1,119.7,117.9,111.9,98.8,40.2,21.3.

[0069] HRMS[M+H]+m / z calcd.for[C20H19N6S]+375.1386,found 375.1380.

[0070] [Example 2]

[0071]

[0072] Under argon protection, aldehyde S2 (56.5 mg, 0.2 mmol), tetrazine phosphate S4 (101.5 mg, 0.26 mmol) and ultra-dry THF (4 mL) were added to a 10 mL dry reaction tube equipped with a magnetic stirrer. The mixture was cooled to -20 °C, and then LDA (150 μL 2M) was added dropwise. The above mixture was stirred for 2 hours. After the reaction was completed by thin layer chromatography monitoring, the reaction was quenched with saturated NH4Cl aqueous solution, and the solvent was removed by rotary evaporation. 0.2 M hydrochloric acid solution (4.5 mL) was added to a solution of THF (11.0 mL) and methanol (4.5 mL) of the residue. After stirring at room temperature for 1 hour, the above solution was diluted with 50 mL EtOAc and saturated NaHCO3 aqueous solution (1.0 mL, 0.2 M). The organic phase was separated, and the aqueous phase was further extracted with EtOAc (30 mL×3). The combined organic phase was washed with saturated brine, dried with Na2SO4, and then rotary evaporated to remove the solvent. The residue was purified by silica gel column chromatography to obtain the tetrazine aggregation-induced emission probe precursor 2 (33.9 mg, 42%) as a black solid.

[0073] 1 H NMR (400MHz, CDCl3) δ8.34(d,J=15.8Hz,1H),7.86(d,J=8.9Hz,2H),7.32(s,1H),7.29(d,J=3.9Hz,1H),7.25(s ,1H),7.18(d,J=15.8Hz,1H),6.74(d,J=9.0Hz,2H),4.27(t,J=5.8Hz,2H),3.57(t,J=5.8Hz,2H),3.09(s,6H).

[0074] 13 C NMR (101MHz, CDCl3) δ167.1,165.3,152.2,144.3,141.9,139.3,133.6,132.5,131.8,126.1,121.0,119.4,117.9,111.9,98.6,60.2,40.2,37.6.

[0075] HRMS[M+H]+m / z calcd.for[C21H21N6OS]+405.1492,found 405.1483.

[0076] [Example 3]

[0077]

[0078] Under argon protection, aldehyde S2 (56.5 mg, 0.2 mmol), tetrazine phosphate S5 (75.0 mg, 0.26 mmol) and ultra-dry THF (4 mL) were added to a 10 mL dry reaction tube equipped with a magnetic stirrer. The mixture was cooled to -20 ° C, and then LDA (150 μL 2M) was added. The above mixture was stirred for 2 hours. After the reaction was completed by thin layer chromatography monitoring, it was quenched with saturated NH4Cl aqueous solution and extracted with ethyl acetate (20 mL×3). The combined organic phases were washed with saturated brine, dried with Na2SO4, and then rotary evaporated to remove the solvent. The residue was purified by silica gel column chromatography to obtain a black solid tetrazine aggregation-induced emission probe precursor 3 (66.5 mg, 80%).

[0079] 1 H NMR (400MHz, CDCl3) δ8.32(d,J=15.9Hz,1H),7.85(d,J=8.9Hz,2H),7.31(s,1H),7.26(d ,J=3.6Hz,1H),7.24(d,J=3.9Hz,1H),7.19(d,J=15.9Hz,1H),3.08(s,6H),1.59(s,9H).

[0080] 13 C NMR (101MHz, CDCl3) δ174.5,164.3,152.2,143.9,141.8,139.5,133.0,132.1,131.8,126.1,121.1,119.8,117.9,111.9,98.7,40.2,38.0,29.3.

[0081] HRMS[M+H] + m / z calcd.for[C 23 H 25 N6S] + 417.1817, found 417.1846.

[0082] [Example 4]

[0083]

[0084] 1) Synthesis of Thiophene S8

[0085] 9-aldehyde julolidine S6 (2 g, 10 mmol), 2-thiopheneacetonitrile S7 (1.2 g, 10 mmol) and 30 mL of ultra-dry ethanol were added to a 250 mL round-bottom flask. 6 M NaOH (0.1 mL) was added to the reaction mixture at room temperature, and the resulting mixture was refluxed for 2 hours. The mixture was then poured into water and extracted with CHCl3. The combined organic phases were dried over Na2SO4. The solvent was removed using a rotary evaporator, and the residue was purified by silica gel column chromatography to give thiophene S8 (2.4 g, 77%) as an orange solid.

[0086] 1 H NMR (400MHz, CDCl3) δ7.38(s,2H),7.22(d,J=3.6Hz,1H),7.17(d,J=5.0Hz,1H),7.13(s,1H), 7.02(dd,J=5.1,3.7Hz,1H),3.26(t,J=5.8Hz,4H),2.76(t,J=6.4Hz,4H),2.01-1.93(m,4H).

[0087] 13 C NMR (101MHz, CDCl3) δ145.1,141.2,140.9,128.9,128.0,124.7,124.1,121.0,120.3,118.7,97.8,50.1,27.8,21.6.

[0088] HRMS[M+H]+m / z calcd.for[C19H19N2S]+307.1263,found 307.1256.

[0089] 2) Synthesis of aldehyde S9

[0090] Under argon protection, ultra-dry DMF (1.2 mL, 15.0 mmol) and POCl3 (1.4 mL, 15.0 mol) were added to a 100 mL round-bottom flask equipped with a magnetic stirrer. The above solution was stirred at 0°C for 0.5 hours. Thiophene S8 (786 mg, 3.1 mmol) dissolved in 1,2-dichloroethane (15 mL) was added dropwise to the above solution, and the resulting mixture was stirred at 95°C for 1 hour. After cooling to room temperature, the mixture was poured into crushed ice and alkalized with 6M NaOH. Extracted with DCM (100 mL × 3), the combined organic phases were washed with saturated brine (150 mL), dried over anhydrous Na2SO4 and the solvent was removed by rotary evaporator. The residue was purified by silica gel column chromatography to obtain aldehyde S9 (275.3 mg, 27%) as a red solid.

[0091] 1H NMR (400MHz, CDCl3) δ9.80 (s, 1H), 7.64 (d, J = 4.0Hz, 1H), 7.41 (s, 2H), 7.24 (d, J=3.8Hz,1H),3.28(t,J=5.8Hz,4H),2.74(t,J=6.3Hz,4H),1.98-1.92(m,4H).

[0092] 13 C NMR (101MHz, CDCl3) δ182.4,151.5,146.4,143.9,141.0,137.6,130.0,124.9,121.1,119.6,118.1,95.8,50.2,27.8,21.4.

[0093] HRMS[M+H]+m / z calcd.for[C20H19N2OS]+335.1213,found 335.1208.

[0094] 3) Synthesis of probe precursor 4

[0095] Under argon protection, aldehyde S9 (66.9 mg, 0.2 mmol), tetrazine phosphate S3 (64.0 mg, 0.26 mmol) and ultra-dry THF (4 mL) were added to a 10 mL dry reaction tube equipped with a magnetic stirring bar. The mixture was cooled to -20 ° C, and then LDA (150 μL 2M) was added. The above mixture was stirred for 2 hours. After the reaction was completed by thin layer chromatography monitoring, it was quenched with saturated NH4Cl aqueous solution and extracted with ethyl acetate (20 mL×3). The combined organic phases were washed with saturated brine, dried with Na2SO4, and then rotary evaporated to remove the solvent. The residue was purified by silica gel column chromatography to obtain a black solid tetrazine aggregation-induced emission probe precursor 4 (56.4 mg, 66%).

[0096] 1 H NMR (400MHz, CDCl3) δ8.31(d,J=15.9Hz,1H),7.42(s,2H),7.25(d,J=3.9Hz,1H),7.19(d,J=3.9Hz,1H),7.17 (s,1H),7.13(d,J=15.8Hz,1H),3.29(t,J=5.8Hz,4H),3.03(s,3H),2.77(t,J=6.4Hz,4H),2.00-1.94(m,4H).

[0097] 13C NMR (101MHz, CDCl3) δ166.0,164.9,145.8,144.7,142.0,138.9,133.2,132.4, 129.5,125.5,121.1,120.0,119.2,118.2,96.8,50.2,29.8,27.8,21.5,21.3.

[0098] HRMS[M+H]+m / z calcd.for[C24H23N6S]+427.1699,found 427.1694.

[0099] [Example 5]

[0100]

[0101] Under argon protection, aldehyde S9 (66.9 mg, 0.2 mmol), tetrazine phosphate S4 (101.5 mg, 0.26 mmol) and ultra-dry THF (4 mL) were added to a 10 mL dry reaction tube equipped with a magnetic stirrer. The mixture was cooled to -20 ° C, and then LDA (150 μL 2M) was added dropwise. The above mixture was stirred for 2 hours. After the reaction was completed by thin layer chromatography monitoring, the reaction was quenched with saturated NH4Cl aqueous solution, and the solvent was removed by rotary evaporation. 0.2 M hydrochloric acid solution (4.5 mL) was added to a solution of THF (11.0 mL) and methanol (4.5 mL) of the residue. After stirring at room temperature for 1 hour, the above solution was diluted with 50 mL EtOAc and saturated NaHCO3 aqueous solution (1.0 mL, 0.2 M). The organic phase was separated, and the aqueous phase was further extracted with EtOAc (30 mL×3). The combined organic phase was washed with saturated brine, dried with Na2SO4, and then rotary evaporated to remove the solvent. The residue was purified by silica gel column chromatography to obtain tetrazine aggregation-induced emission probe precursor 5 (39.3 mg, 43%) as a black solid.

[0102] 1 H NMR (400MHz, CDCl3) δ8.31(d,J=15.8Hz,1H),7.41(s,2H),7.25(s,1H),7.19(d,J=3.9Hz,1H),7.17(s,1H),7.13(d,J=15 .8Hz,1H),4.26(t,J=5.8Hz,2H),3.56(t,J=5.8Hz,2H),3.29(t,J=5.8Hz,4H),2.77(t,J=6.3Hz,4H),2.00–1.94(m,4H).

[0103] 13C NMR (101MHz, CDCl3) δ167.0,165.4,145.8,145.0,142.1,138.7,133.7,132.6, 129.5,125.5,121.1,120.0,118.9,118.2,96.7,60.2,50.2,37.6,27.8,21.5.

[0104] HRMS[M+H]+m / z calcd.for[C25H25N6OS]+457.1805,found 457.1797.

[0105] [Example 6]

[0106]

[0107] Under argon protection, aldehyde S9 (66.9 mg, 0.2 mmol), tetrazine phosphate S5 (75.0 mg, 0.26 mmol) and ultra-dry THF (4 mL) were added to a 10 mL dry reaction tube equipped with a magnetic stirring bar. The mixture was cooled to -20 ° C, and then LDA (150 μL 2M) was added. The above mixture was stirred for 2 hours. After the reaction was completed by thin layer chromatography monitoring, it was quenched with saturated NH4Cl aqueous solution and extracted with ethyl acetate (20 mL×3). The combined organic phases were washed with saturated brine, dried with Na2SO4, and then rotary evaporated to remove the solvent. The residue was purified by silica gel column chromatography to obtain a black solid tetrazine aggregation-induced emission probe precursor 6 (56.4 mg, 66%).

[0108] 1 H NMR (400MHz, CDCl3) δ8.31(d,J=15.9Hz,1H),7.42(s,2H),7.25(d,J=3.9Hz,1H),7.19(d,J=3.9Hz,1H),7.18(s ,1H),7.15(d,J=15.8Hz,1H),3.29(t,J=5.8Hz,4H),2.77(t,J=6.4Hz,4H),1.97(p,J=6.2Hz,2H),1.59(s,8H).

[0109] 13 C NMR (101MHz, CDCl3) δ174.4,164.4,145.7,144.6,142.0,138.9,133.1,132.3, 129.5,125.4,121.1,120.0,119.4,118.2,96.9,50.2,38.0,29.3,27.8,21.5.

[0110] HRMS[M+H]+ m / z calcd.for[C 27 H 29 N6OS] + 469.2130, found 469.2119.

[0111] [Example 7]

[0112]

[0113] Under argon protection, aldehyde S10 (81.3 mg, 0.2 mmol), tetrazine phosphate S3 (64.0 mg, 0.26 mmol) and ultra-dry THF (4 mL) were added to a 10 mL dry reaction tube equipped with a magnetic stirring bar. The mixture was cooled to -20 ° C, and then LDA (150 μL 2M) was added. The above mixture was stirred for 2 hours. After the reaction was completed by thin layer chromatography monitoring, it was quenched with saturated NH4Cl aqueous solution and extracted with ethyl acetate (20 mL×3). The combined organic phases were washed with saturated brine, dried with Na2SO4, and then rotary evaporated to remove the solvent. The residue was purified by silica gel column chromatography to obtain a black solid tetrazine aggregation-induced emission probe precursor 7 (32.9 mg, 33%).

[0114] 1 H NMR(400MHz, CDCl3)δ8.32(d,J=15.9Hz,1H),7.76(d,J=8.6Hz,2H),7.36-7.3 1(m,5H),7.29(s,1H),7.24-7.11(m,8H),7.03(d,J=8.6Hz,2H),3.04(s,3H).

[0115] 13 C NMR (101MHz, CDCl3) δ166.2,164.8,150.6,146.5,143.1,140.7,140.2,132.9,13 2.1,131.1,129.8,127.1,126.1,125.7,124.9,120.6,120.3,117.2,101.7,21.3.

[0116] HRMS[M+H]+m / z calcd.for[C30H23N6S]+499.1699,found 499.1693.

[0117] [Example 8]

[0118]

[0119] Under argon protection, aldehyde S10 (81.3 mg, 0.2 mmol), tetrazine phosphate S4 (101.5 mg, 0.26 mmol) and ultra-dry THF (4 mL) were added to a 10 mL dry reaction tube equipped with a magnetic stirrer. The mixture was cooled to -20 °C, and then LDA (150 μL 2M) was added dropwise. The above mixture was stirred for 2 hours. After the reaction was completed by thin layer chromatography monitoring, the reaction was quenched with saturated NH4Cl aqueous solution, and the solvent was removed by rotary evaporation. 0.2 M hydrochloric acid solution (4.5 mL) was added to a solution of THF (11.0 mL) and methanol (4.5 mL) of the residue. After stirring at room temperature for 1 hour, the above solution was diluted with 50 mL EtOAc and saturated NaHCO3 aqueous solution (1.0 mL, 0.2 M). The organic phase was separated, and the aqueous phase was further extracted with EtOAc (30 mL×3). The combined organic phase was washed with saturated brine, dried with Na2SO4, and then rotary evaporated to remove the solvent. The residue was purified by silica gel column chromatography to obtain tetrazine aggregation-induced emission probe precursor 8 (54.9 mg, 52%) as a black solid.

[0120] 1 H NMR (400MHz, CDCl3) δ8.33(d,J=15.9Hz,1H),7.76(d,J=8.9Hz,2H),7.35-7.29(m,7H),7.22-7 .12(m,7H),7.03(d,J=8.9Hz,2H),4.27(t,J=7.6Hz,2H),3.57(t,J=5.8Hz,2H),2.46(brs,1H).

[0121] 13 C NMR (101MHz, CDCl3) δ167.2,165.2,150.7,146.5,143.3,140.8,140.1,133.4,132.3 ,131.1,129.8,127.1,126.1,125.6,124.9,120.6,120.0,117.2,101.6,60.2,37.7.

[0122] HRMS[M+H]+m / z calcd.for[C31H25N6OS]+529.1805,found 529.1801.

[0123] 2. Performance test of tetrazine aggregation-induced emission probe precursor

[0124] [Example 9 to Example 14]

[0125] The dienophile was BCN, the probe precursor was prepared with DMSO to a 10mM mother solution, and exo-BCN was prepared with DMSO to a 100mM mother solution. The above mother solution was prepared with DMSO to prepare a reaction solution containing a probe concentration of 1mM and a BCN concentration of 10mM. The probe and BCN were reacted at 37°C for 3 hours, and the reaction was monitored by LC-MS. The relevant molecular weight displayed by the mass spectrometer determined that the probe was completely oxidized to generate the probe, and the absorption and emission spectra of the probe precursor and the probe were tested.

[0126] Embodiment 9:

[0127]

[0128] like Figure 1 As shown, probe 1 reacts with BCN to obtain probe B-1, and the maximum absorption wavelength (λ abs ) is 454nm, the maximum emission wavelength (λ em ) is 640nm, the fluorescence turn-on is 622 times, the maximum absorption wavelength in DMSO is 448nm, the maximum emission wavelength is 591nm, the fluorescence emission is blue-shifted, and the fluorescence turn-on is 34 times.

[0129] Embodiment 10:

[0130]

[0131] like Figure 2 As shown, probe 2 reacts with BCN to obtain probe B-2. The λ of probe B-2 in H2O (containing 1% DMSO) is abs is 445nm, λ em The turn-on is 1033 times higher than the λ in DMSO. abs is 447nm, λ em The wavelength is 596 nm, the fluorescence emission is blue-shifted, and the fluorescence turn-on factor is 53 times.

[0132] Embodiment 11:

[0133]

[0134] like Figure 3 As shown, probe 4 reacts with BCN to obtain probe B-4. The λ of probe B-4 in H2O (containing 1% DMSO) is abs is 491nm, λ em The turn-on is 361 times higher than the λ in DMSO. abs is 481nm, λ em The wavelength is 619 nm, the fluorescence emission is blue-shifted, and the fluorescence turn-on factor is 28 times.

[0135] Embodiment 12:

[0136]

[0137] like Figure 4 As shown, probe 5 reacts with BCN to obtain probe B-5. The λ of probe B-5 in H2O (containing 1% DMSO) is abs is 478nm, λ em 665nm, Turn-on up to 201 times, λ in DMSO abs is 482nm, λ em The wavelength is 621 nm, the fluorescence emission is blue-shifted, and the fluorescence turn-on factor is 44 times.

[0138] Embodiment 13:

[0139]

[0140] like Figure 5 As shown, probe 7 reacts with BCN to obtain probe B-7. The λ of probe B-7 in H2O (containing 1% DMSO) is abs is 457nm, λ em 605nm, Turn-on up to 103 times, λ in DMSO abs is 441nm, λ em The wavelength is 603nm, the fluorescence emission is blue-shifted, and the fluorescence turn-on factor is 23 times.

[0141] Embodiment 14:

[0142]

[0143] like Figure 6 As shown, probe 8 reacts with BCN to obtain probe B-8. The λ of probe B-8 in H2O (containing 1% DMSO) is abs is 458nm, λ em 605nm, Turn-on up to 201 times, λ in DMSO abs is 434nm, λ em The wavelength is 601nm, the fluorescence emission is blue-shifted, and the fluorescence turn-on factor is 36 times.

[0144] [Example 15]

[0145] The above reaction solution was prepared into a set of test solutions with different H2O volume fractions and a probe precursor concentration of 10 μM using dd-H2O and DMSO. The water volume fraction settings include: 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 0%. The maximum emission wavelength of 99% of the test solution was used as the excitation wavelength to collect the fluorescence emission spectra of a set of test solutions. A set of fluorescence emission intensity spectra and relative fluorescence intensity graphs of the probe were plotted to determine the AIE properties after the reaction of the probe precursor and BCN.

[0146] For probe B-1, Figure 7 As shown in (a) and (b), with the increase of the volume fraction of H2O, the fluorescence emission intensity of B-1 (10μM) gradually increases and red-shifts. When the volume fraction of H2O in the DMSO / H2O mixed solvent is greater than 70%, the fluorescence emission is significantly enhanced. This change indicates that B-1 has a typical AIE effect. Figure 7 (c) Dynamic light scattering test confirmed that it aggregated in H2O (containing 1% DMSO) to form nanoparticles with an average particle size of 209 nm, indicating that probe B-1 has AIE properties.

[0147] For probe B-2, Figure 8 As shown in (a) and (b), the fluorescence emission intensity of probe B-2 (10 μM) gradually increases with the volume fraction of H2O and undergoes a red shift; when the volume fraction of H2O in the DMSO / H2O mixed solvent is greater than 80%, the fluorescence emission is significantly enhanced.

[0148] For probe B-4, Fig. 9 As shown in (a) and (b), the fluorescence emission intensity of probe B-4 (10 μM) gradually increases with the volume fraction of H2O and undergoes a red shift; when the volume fraction of H2O in the DMSO / H2O mixed solvent is greater than 60%, the fluorescence emission is significantly enhanced.

[0149] For probe B-5, Fig.10 As shown in (a) and (b), the fluorescence emission intensity of probe B-5 (10 μM) gradually increases with the volume fraction of H2O and undergoes a red shift; when the volume fraction of H2O in the DMSO / H2O mixed solvent is greater than 60%, the fluorescence emission is significantly enhanced.

[0150] For probe B-7, Fig.11 As shown in (a) and (b), the fluorescence emission intensity of probe B-7 (10 μM) gradually increases with the volume fraction of H2O and undergoes a red shift; when the volume fraction of H2O in the DMSO / H2O mixed solvent is greater than 50%, the fluorescence emission is significantly enhanced.

[0151] For probe B-8, Fig.12 As shown in (a) and (b), the fluorescence emission intensity of probe B-8 (10 μM) gradually increases with the volume fraction of H2O and undergoes a red shift; when the volume fraction of H2O in the DMSO / H2O mixed solvent is greater than 50%, the fluorescence emission is significantly enhanced.

[0152] [Example 16 to Example 20] Organelle Imaging

[0153] SKOV3 ovarian cancer cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution. The cells were seeded on a 100 mm culture dish and cultured in a cell culture incubator at 5% carbon dioxide and 37°C.

[0154] For live cell imaging, cells were seeded on 35 mm glass-bottom culture dishes and grown to a density of 60-70%. Different targeted BCNs (50 μM) were added to the culture medium and treated for 3-30 min, then washed 3 times with PBS and finally incubated with cell imaging solution containing probes (20 μM) for 20-60 min. Commercial probes for different organelles were used as positioning references.

[0155] The cells in the cell imaging solution were imaged without washing using a confocal fluorescence microscope with an excitation wavelength of 488 nm and an emission wavelength of 600-700 nm.

[0156] The different targeted BCN structures are as follows:

[0157]

[0158] Embodiment 16:

[0159] Mitochondria-targeted fluorescence imaging and colocalization experiments based on tetrazine AIE probe precursor 2.

[0160] SKOV3 cells were pre-incubated with or without BCN-Mito (50 μM) at 37°C for 30 minutes, and mitochondria were labeled with Mito-TrackerGreen (50 nM; green; Ex: 488 nm; Em: 510-550 nm), followed by the addition of probe precursor 2 (20 μM; red; Ex: 488 nm; Em: BP 428-480 + LP 605 nm), and imaged without washing 1 hour later (Scale bar: 5 μm). The signal-to-noise ratio of the target area reached 131 times (Scale bar: 100 pixels).

[0161] The experimental results are as follows Fig.13As shown, in the experimental group (above), when the probe precursor 2 was added to the SKOV3 cells pre-incubated with BCN-Mito for 1 hour, confocal imaging showed obvious red fluorescence signals; at the same time, the commercial probe Mito-Tracker Green showed obvious green fluorescence signals. In the white dashed area, the fluorescence signals generated by the probe precursor 2 and Mito-Tracker Green overlap significantly. The colocalization coefficient obtained by the software Image J was 0.96. In the control group (below), there was no obvious red fluorescence signal, and only the green fluorescence signal from the commercial probe Mito-Tracker Green was observed.

[0162] It can be seen that the colocalization experiment confirmed that the probe precursor 2 signal and the fluorescence signal of the commercial probe Mito-TrackerGreen overlapped well, with a colocalization coefficient of up to 0.96, indicating that the AIE probe B-2 generated in situ by the probe precursor 2 reaction was localized in the mitochondria of SKOV3 cells; at the same time, through the BCN-Mito pre-targeting method, the probe precursor 2 was able to generate AIE probe B-2 in situ on the mitochondria of SKOV3 cells, achieving a signal-to-noise ratio of up to 131 times for wash-free live cell mitochondrial fluorescence labeling.

[0163] Embodiment 17:

[0164] Lysosome-targeted fluorescence imaging and colocalization experiments based on tetrazine AIE probe precursor 2.

[0165] SKOV3 cells were pre-incubated with or without BCN-Lyso (50 μM) at 37°C for 30 minutes, and lysosomes were labeled with Lyso-TrackerGreen (50 nM; green; Ex: 488 nm; Em: 510-550 nm), followed by the addition of probe precursor 2 (20 μM; red; Ex: 488 nm; Em: BP 428-480 + LP 605 nm), and imaged without washing 1 hour later (Scale bar: 5 μm). The signal-to-noise ratio of the target area reached 42 times (Scale bar: 100 pixels).

[0166] The experimental results are as follows Fig.14As shown, in the experimental group (above), when the probe precursor 2 was added to the SKOV3 cells pre-incubated with BCN-Lyso for 1 hour, confocal imaging showed obvious red fluorescence signals; at the same time, the commercial probe Lyso-Tracker Green showed obvious green fluorescence signals. In the white dashed area, the fluorescence signals generated by the probe precursor 2 and Lyso-Tracker Green overlap significantly. The colocalization coefficient obtained by the software Image J was 0.90. In the control group (below), there was no obvious red fluorescence signal, and only the green fluorescence signal from the commercial probe Lyso-Tracker Green was observed.

[0167] The above experiments show that the AIE probe B-2 generated in situ by the probe precursor 2 reaction is localized in the lysosomes of SKOV3 cells; at the same time, through the BCN-Lyso pre-targeting method, the probe precursor 2 can generate AIE probe B-2 in situ on the lysosomes of SKOV3 cells, achieving a wash-free living cell lysosome fluorescence labeling with a signal-to-noise ratio of 42 times.

[0168] Embodiment 18:

[0169] Endoplasmic reticulum fluorescence targeted imaging and colocalization experiments based on tetrazine AIE probe precursor-2.

[0170] SKOV3 cells were pre-incubated with or without BCN-ER (50 μM) at 37°C for 30 minutes, and the endoplasmic reticulum was labeled with ER-TrackerGreen (1 μM; green; Ex: 488 nm; Em: 510-550 nm), followed by the addition of probe precursor 2 (20 μM; red; Ex: 488 nm; Em: 600-700 nm), and imaged without washing 1 hour later (Scale bar: 10 μm). The signal-to-noise ratio of the target area reached 63 times (Scale bar: 100 pixels).

[0171] The experimental results are as follows Fig.15 As shown, in the experimental group (top), when the probe precursor 2 was added to the SKOV3 cells pre-incubated with BCN-ER for 1 hour, confocal imaging showed obvious red fluorescence signals; at the same time, the commercial probe ER-TrackerGreen showed obvious green fluorescence signals. In the white dashed area, the fluorescence signals generated by the probe precursor 2 and ER-Tracker Green overlapped significantly. The colocalization coefficient was obtained by the software Image J and was 0.78. In the control group (bottom), there was no obvious red fluorescence signal, and only the green fluorescence signal from the commercial probe ER-Tracker Green was observed.

[0172] It can be seen that the AIE probe generated in situ by the reaction of probe precursor 2 is localized in the endoplasmic reticulum of SKOV3 cells; through the BCN-ER pre-targeting method, probe precursor 2 can generate AIE probe B-2 in situ on the endoplasmic reticulum of SKOV3 cells, achieving wash-free living cell endoplasmic reticulum fluorescence labeling with a signal-to-noise ratio of 63 times.

[0173] Embodiment 19:

[0174] Golgi-targeted fluorescence imaging and colocalization experiments based on tetrazine AIE probe precursor 2.

[0175] SKOV3 cells were pre-incubated with or without BCN-ER (50 μM) at 37°C for 30 minutes, Golgi-Tracker Green (333 μg / mL; green; Ex: 488 nm; Em: 510-550 nm) was used to label the Golgi apparatus, followed by the addition of probe precursor 2 (20 μM; red; Ex: 488 nm; Em: 600-700 nm), and imaged without washing 1 hour later (Scale bar: 10 μm). The signal-to-noise ratio of the target area reached 66 times (Scale bar: 100 pixels).

[0176] The experimental results are as follows Fig.16 As shown, in the experimental group (above), when the probe precursor 2 was added to the SKOV3 cells pre-incubated with BCN-Golgi for 1 hour, confocal imaging showed obvious red fluorescence signals; at the same time, the commercial probe Golgi-Tracker Green showed obvious green fluorescence signals. In the white dashed area, the fluorescence signals generated by the probe precursor 2 and Golgi-Tracker Green overlap significantly. The colocalization coefficient obtained by the software Image J was 0.78. In the control group (below), there was no obvious red fluorescence signal, and only the green fluorescence signal from the commercial probe Golgi-Tracker Green was observed.

[0177] It can be seen that the AIE probe generated in situ by the reaction of probe precursor 2 is localized in the Golgi apparatus of SKOV3 cells; through the BCN-Golgi pre-targeting method, probe precursor 2 can generate AIE probe B-2 in situ on the Golgi apparatus of SKOV3 cells, achieving a wash-free living cell Golgi fluorescence labeling with a signal-to-noise ratio of 66 times.

[0178] Embodiment 20:

[0179] Cell membrane fluorescence imaging based on tetrazine AIE probe 2. SKOV3 cells were pre-incubated with BCN-Mem (50μM) at 37°C for 3 minutes to label the cell membrane, followed by the addition of probe 2 (20μM; red; Ex: 488nm; Em: 600-680nm), and imaging was performed without washing after 20 minutes. (Scale bar: 5μm)

[0180] The experimental results are as follows Fig.17 As shown, when the probe precursor 2 was added to the SKOV3 cells pre-incubated with BCN-Mem for 20 minutes, confocal imaging showed an obvious red fluorescence signal, and the fluorescence signal coincided with the cell membrane in the bright field; through the BCN-Mem pre-targeting method, the probe precursor 2 was able to generate AIE probe B-2 in situ on the cell membrane of SKOV3 cells, realizing the fluorescent labeling of the cell membrane of living cells without washing.

[0181] It can be seen from Examples 16 to 20 that the probe provided by the present invention has excellent biocompatibility, and the same probe can achieve dienophile pre-targeted lysosomes, Golgi bodies, mitochondria, cell membranes, endoplasmic reticulum and other subcellular organelle imaging, and has wide application value.

[0182] Embodiment 21:

[0183] The reaction solution of B-1 was prepared with dd-H2O and DMSO into a set of test solutions with different H2O volume fractions and a probe concentration of 10 μM. The water volume fraction settings include: 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 0%. Fluorescence photos were taken under 365nm portable UV light, showing obvious AIE performance.

[0184] 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 aggregation-induced emission probe precursor, characterized in that: It has the structural formula shown in Formula I: In Formula I, R1 is selected from Wherein, R2 and R3 are each independently selected from C6~C 12 Aryl, C1-C4 straight-chain alkyl or C1-C4 branched-chain alkyl; n=1, m=1; In formula I, R4 is selected from C1-C6 straight chain alkyl, C1-C6 branched chain alkyl or C1-C3 hydroxyalkyl.

2. The tetrazine aggregation-induced emission probe precursor according to claim 1, characterized in that The tetrazine aggregation-induced emission probe precursor has any of the following chemical structural formulas:

3. A tetrazine aggregation-induced emission probe, characterized in that: The tetrazine aggregation-induced emission probe is prepared by bioorthogonal reaction between the tetrazine aggregation-induced emission probe precursor according to claim 1 or 2 and bicyclo[6,1,0]nonyne.

4. A method for preparing a tetrazine aggregation-induced emission probe precursor, characterized in that: For preparing the tetrazine aggregation-induced emission probe precursor according to claim 1 or 2, the preparation method comprises the following steps: Under an inert atmosphere, dissolving the aldehyde represented by formula II and tetrazine phosphate in a solvent to obtain a mixture; After cooling the mixture, lithium diisopropylamide is added, and the reaction is stirred until the reaction is completed, and the tetrazine aggregation-induced emission probe precursor is separated; In formula II, R1 is selected from Wherein, R2 and R3 are each independently selected from C6~C 12 Aryl, C1-C4 straight-chain alkyl or C1-C4 branched-chain alkyl; n=1, m=1.

5. The method for preparing a tetrazine aggregation-induced emission probe precursor according to claim 4, characterized in that: After the mixture is cooled to -10 to -25°C, lithium diisopropylamide is added.

6. Use of the tetrazine aggregation-induced emission probe precursor according to claim 1 or 2 in the preparation of a tetrazine aggregation-induced emission probe, characterized in that: The tetrazine aggregation-induced emission probe precursor reacts with a dienophile to generate a tetrazine aggregation-induced emission probe, wherein the dienophile is a bicyclo[6,1,0]nonyne, wherein the bicyclo[6,1,0]nonyne has a structural formula shown in Formula III: Wherein, the R group is selected from any of the following:

Images