Fluorescent molecule with light stability and high photon number rate and preparation and application thereof

Abstract

The invention provides a construction method of a light-stable high-photon-number fluorescent probe and application of the light-stable high-photon-number fluorescent probe in single-molecule and super-resolution fluorescence imaging. The probe is efficiently prepared through one-pot multi-component reaction of aldehyde containing cyclooctatetraene, amine containing benzothiazole methyl sulfone, fluorophore carboxylic acid with a click tag and tert-butyl isocyanide. The probe can realize cysteine site specific labeling through nucleophilic aromatic substitution, the cyclooctadiene light stabilizer can effectively quench triplet, photon output and photobleaching resistance are improved synergistically, and the probe and the cyclooctadiene light stabilizer are suitable for super-resolution fluorescence imaging of biomacromolecules such as single molecules and nucleopore proteins. And a high-performance probe is provided for an advanced super-resolution fluorescence microscopy technology.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probes and relates to the preparation methods and applications of labeled precursor tags and fluorescent probes with intramolecular photoprotection function and bio-coupling synergistic enhancement of photon number effect. Background Technology

[0002] In recent years, advanced super-resolution fluorescence microscopy has made continuous breakthroughs, driving the rapid development of life science research at the single-molecule level. In conjunction with this, the development of next-generation organic fluorescent dyes is constantly evolving towards higher brightness, excellent photostability, and tunable optical properties. Especially when combined with highly specific biolabeling strategies, this has greatly enhanced the spatiotemporal resolution of biomolecular interactions and their dynamic processes.

[0003] Nevertheless, the reliability and statistical significance of super-resolution and single-molecule fluorescence imaging data remain highly dependent on the total number of photons emitted by the fluorophore before photobleaching. Currently used fluorescent probes generally suffer from drawbacks such as long triplet lifetimes and susceptibility to photobleaching, limiting their photon output capabilities and making it difficult to meet the high spatiotemporal resolution requirements of super-resolution imaging. Therefore, developing fluorescent probes with higher photon yields and resistance to photobleaching has become a crucial issue that urgently needs to be addressed in this field.

[0004] This study focuses on the synergistic strategy of intramolecular physical photoprotection mechanisms and chemibiolabeling groups to efficiently construct fluorescent probe tags with high photon output capabilities. Through modular molecular design, functional modules such as fluorophores / labels, photostabilizers, and bioconjugation groups are integrated to construct multifunctional linked compounds with intramolecular synergistic photoprotection effects, thereby significantly improving the photostability and photon rate of the fluorophore during single-molecule imaging.

[0005] In terms of photostable module design, cyclooctatetraene derivatives, which are highly matched to the excited-state energy levels of Cyanine dyes, were selected as triplet quenchers. This module can effectively quench the triplet component in the excited state of the fluorophore through intermolecular energy transfer mechanisms, thereby inhibiting the photobleaching reaction associated with the triplet state and improving the photostability of the fluorophore.

[0006] In terms of bioconjugation modules, a benzothiazole methyl sulfone functional group with excellent reactivity was designed and introduced. This structure can undergo efficient covalent coupling reactions with cysteine ​​residues in target biomolecules. Simultaneously, this functional group synergistically enhances the photon yield of the fluorophore by reducing the electron cloud delocalization effect of the sulfur atom in cysteine ​​and suppressing the non-radiative inactivation process caused by free radical intermediates. Based on a "one-step modular" synthesis strategy, this study employed a multi-component reaction method to achieve efficient assembly of the fluorophore tag, photostable agent, and bioconjugation group, significantly simplifying the synthesis process of fluorescent probes and realizing the integrated integration of functional modules. This strategy provides a new approach for constructing high-performance single-molecule fluorescent probes and lays the foundation for subsequent applications in super-resolution imaging, molecular tracking, and other fields. Summary of the Invention

[0007] To enhance the photophysical properties of fluorescent probes and improve their applicability and signal stability in single-molecule bioanalysis, thereby enabling precise observation of conformational changes and interactions in biomolecules at higher temporal resolution, this study proposes a fluorophore structure optimization scheme based on a physicochemical synergistic enhancement strategy. This scheme aims to significantly increase the photon number rate of the probe in single-molecule detection systems. The tag compound provided in this invention exhibits excellent resistance to photobleaching and higher single-molecule photon output efficiency, which can significantly improve the resolution and signal-to-noise ratio of fluorescence imaging, making it particularly suitable for high-precision optical detection fields such as single-molecule fluorescence imaging and super-resolution fluorescence microscopy.

[0008] The technical solution of this invention is as follows: a class of high-photon-count fluorescent molecules, prepared from 4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)aniline as the starting material, wherein the compound structural formulas of Click fluorescent tag I, fluorescent probe I, and fluorescent probe II are shown below.

[0009] The compound structure shown in Click fluorescent label I is as follows:

[0010]

[0011] The compound structure shown by fluorescent probe I is as follows:

[0012]

[0013]

[0014] The method for preparing the aforementioned high-photon-count fluorescent molecule involves a multi-component reaction using 4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)aniline as the starting material, along with an aldehyde compound containing a photoprotectant cyclooctatetraene structure, a carboxylic acid component with a Click group or fluorophore, and tert-butylisocyanate. The synthesis of the Click fluorescent tag I is as follows:

[0015]

[0016] The synthesis of the fluorescent probe I is as follows:

[0017]

[0018] The synthesis of the fluorescent probe II is as follows:

[0019]

[0020] The preparation method of the aforementioned click fluorescent tag I includes the following steps:

[0021] Compounds 4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)aniline and 1-formylcyclooctatetraene were dissolved in a first solvent and ultrasonically dispersed. Then, compounds 4-pentynic acid and tert-butyl isocyanate were added. The aqueous layers were combined and then extracted with a second solvent. The resulting organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and purified by column chromatography to finally obtain the click fluorescent tag I.

[0022] The preparation method of the fluorescent probes I and II includes the following steps:

[0023] Compound 4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)aniline and 1-formylcyclooctatetraene or paraformaldehyde were dissolved in a first solvent and ultrasonically dispersed. Then, compound sulfonic acid-Cy5 carboxylic acid and tert-butyl isocyanate were added, and the reaction was stirred at room temperature. After the reaction was completed, the solvent was removed by vacuum concentration, and the solution was purified by column chromatography to finally obtain fluorescent probe I.

[0024] In the steps described, the first organic solvent is methanol, the second solvent is ethyl acetate, and the column chromatography uses petroleum ether and ethyl acetate in a volume ratio of 1:1.

[0025] The reaction temperature was room temperature, and the reaction time was 48–72 hours.

[0026] The application of the high-photon-count fluorescent tag I and fluorescent probes I and II in single-molecule total internal reflection fluorescence imaging.

[0027] The application of the high-photon-count fluorescent probe I in the preparation of DNA-PAINT super-resolution fluorescent imaging contrast agents for nucleoporin.

[0028] Beneficial effects:

[0029] (1) This invention belongs to the field of fluorophore molecular design, aiming to enhance its photon output capability in single-molecule and super-resolution fluorescence imaging. The probe is prepared through a multi-component reaction involving an aldehyde compound containing a photoprotectant cyclooctatetraene structure, an amine compound containing a benzothiazole methyl sulfone functional group, a carboxylic acid component of a fluorescent tag or fluorophore with a Click group, and tert-butylisocyanate. The resulting product is a fluorescent dye tag or probe with high photon yield. By constructing a fluorescent tag and probe system with intramolecular conjugated photoprotection function and bio-coupling synergistic enhancement effect, the stability of the fluorescence signal and imaging performance are significantly improved.

[0030] (2) Based on the intramolecular conjugation design of multi-component reactions, this invention successfully constructs fluorescent dye tags or probe molecules with high photon yield. The one-pot synthesis method of fluorescent tags and probes used in this invention does not require the use of catalysts or additives, and has the advantages of being green and environmentally friendly, economical and efficient, simple to operate and synthesize, and highly efficient and stable, making it suitable for large-scale preparation and application.

[0031] (3) The fluorescent probe obtained in this invention can significantly improve the anti-photobleaching ability and total photon output in single-molecule TIRF imaging, and is suitable for super-resolution fluorescence imaging of biomacromolecules such as nucleoporins. The fluorescent probe prepared in this invention has excellent photostability and high photon yield, and can be used as an ideal labeling tool for long-term single-molecule fluorescence imaging, suitable for real-time observation of dynamic processes.

[0032] (4) This fluorescent probe can also be used as an imaging agent in super-resolution fluorescence imaging techniques such as DNA-PAINT targeting nucleoporins. It has good targeting and resolution performance and is suitable for high-precision organelle localization and structural analysis. Attached Figure Description

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0034] Figure 1 The image shows the 1H NMR spectrum of the click fluorescent tag I prepared in Example 1.

[0035] Figure 2 The image shows the carbon NMR spectrum of the click fluorescent tag I prepared in Example 1.

[0036] Figure 3 The mass spectrum of the click fluorescent tag I prepared in Example 1 is shown.

[0037] Figure 4 The chromatogram is the preparation chromatogram of fluorescent probe I prepared in Example 2.

[0038] Figure 5 The mass spectrum of fluorescent probe I prepared in Example 2 is shown.

[0039] Figure 6 The chromatogram is the preparation chromatogram of fluorescent probe II prepared in Example 2.

[0040] Figure 7 The mass spectrum of fluorescent probe II prepared in Example 3 is shown.

[0041] Figure 8 The image shows the 1H NMR spectrum of the click fluorescent tag II prepared in Example 3.

[0042] Figure 9 The mass spectrum of the click fluorescent tag II prepared in Example 3.

[0043] Figure 10 This is a comparison of the TIRFM single-molecule time trajectories of click fluorescent tag I, fluorescent probe II, and Sulfo-Cyanine5-labeled DNA.

[0044] Figure 11 The photophysical properties of TIRFM single-molecule fluorescence of click fluorescent tag I, fluorescent probe II, and click fluorescent tag II with Sulfo-Cyanine5 labeled DNA.

[0045] Figure 12 DNA-PAINT super-resolution fluorescence imaging study of U2OS cell nuclear porin Nup96 labeled with fluorescent probe I.

[0046] Figure 13 The NMR spectrum is for 1-formylcyclooctatetraene.

[0047] Figure 14 This is the mass spectrum of 1-formylcyclooctatetraene. Detailed Implementation

[0048] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0049] This strategy starts with molecular structure design and focuses on dual optimization in the following two key directions:

[0050] (1) Introduction of intramolecular conjugated light stabilizers: Through molecular engineering, the light stabilizer structure is conjugated with the fluorophore to achieve intramolecular energy level regulation. This design physically suppresses the lifetime of the triplet state (T1) in the excited state of the fluorophore, significantly reducing the photobleaching reaction and scintillation events associated with the triplet state, thereby improving the stability of the fluorescence signal and the total photon output.

[0051] (2) Based on S N Optimization of chemical coupling sites for Ar reactions: Electron-deficient aromatic heterocyclic structures (such as benzothiazole methyl sulfone groups) are used to replace traditional cysteine ​​labeling sites. By dispersing the electron density of sulfur atoms, a more stable π-conjugated system is constructed, which effectively reduces the quenching effect of sulfur atoms on fluorophores and further improves the photon yield of fluorophores.

[0052] In terms of probe construction, this study uses a highly efficient and mild multi-component reaction as the core chemical bonding platform. This reaction rapidly generates an α-acylaminoamide skeleton with good stability and functional integration through a one-step reaction of four components: amine, aldehyde, carboxylic acid, and isonitrile under mild conditions. This provides an ideal synthetic route for the modular and efficient assembly of fluorophores (F), photoprotective modules (P), and biocoupler groups (B). The innovation of this strategy is mainly reflected in the following two aspects: (1) Synergistic integration of physical and chemical modules: by combining triplet quenching photoprotective agents with S N Ar-reactive benzothiazole methyl sulfone functional groups are synergistically integrated into the same probe backbone, thereby improving the probe photon yield while minimizing interference with the conformation and function of the target protein, and enhancing the probe's compatibility and applicability in the biological environment. (2) Modular and diverse probe programmability: The high structural diversity and compatibility of organic multi-component reactions enable the probe construction process to have excellent modular characteristics. Researchers can flexibly replace the type of fluorophore, photostable module and biorecognition unit according to different experimental needs, thereby achieving precise control and customized design of fluorescence performance (such as excitation / emission wavelength, photon number rate, photostable stability, etc.).

[0053] Invention concept: This application provides the concept and synthesis method of click fluorescent tags and probes with intramolecular photoprotection and bio-coupling synergistic enhancement of photon number effect.

[0054] This invention discloses a click fluorescent tag (I) and a fluorescent probe (I) that enhances the photon count. The compound structural formula of the fluorescent probe is shown in the figure below.

[0055] The structural formula of the compound represented by Formula I is as follows:

[0056]

[0057] The compound represented by Formula II has the following structural formula:

[0058]

[0059] Furthermore, the present invention provides a method for preparing the above-mentioned fluorescent tag and probe, comprising the following steps:

[0060] (1) Compound 4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)aniline (200 mg, 0.84 mmol) and 1-formylcyclooctatetraene (110 mg, 0.84 mmol) were dissolved in methanol (2.0 mL) and ultrasonically dispersed. Then, compound 4-pentyneic acid (82 mg, 0.84 mmol) and tert-butylisocyanate (70 mg, 0.84 mmol) were added. The aqueous layers were combined and then extracted with ethyl acetate (2 × 10 mL). The resulting organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to finally obtain the click fluorescent tag I;

[0061] (2) 4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)aniline (24 mg, 0.1 mmol) and 1-formylcyclooctatetraene (13 mg, 0.1 mmol) were dissolved in 3.0 mL of methanol, followed by the sequential addition of sulfonic acid-Cy5 carboxylic acid (5.0 mg) and tert-butyl isocyanate (8.0 mg, 0.1 mmol). The mixture was stirred at room temperature for 48 h. After the reaction was complete, the solvent was removed by vacuum concentration. The mixture was then purified using preparative HPLC. R =12.5min, freeze-dry to obtain fluorescent probe I;

[0062] In some embodiments, the organic solvent is methanol, which is used to dissolve or disperse the raw material evenly.

[0063] In some embodiments, in step (1), the molar ratio of the aldehyde compound to the amino compound, the carboxylic acid compound, and the tert-butylisocyanate is 1.0:1.0:1.0:1.0.

[0064] In some embodiments, the reaction time in step (1) is 72 hours.

[0065] In some embodiments, preferably, the reaction time is 48 hours.

[0066] The application of the aforementioned fluorescent probes in single-molecule fluorescence is also within the scope of protection of this invention.

[0067] The application of the aforementioned fluorescent probes in super-resolution fluorescence imaging of nucleoporins is also within the scope of protection of this invention.

[0068] The performance of this high photon number probe stems from the following two intramolecular cooperative mechanisms:

[0069] (1) The benzothiazole methyl sulfone functional group introduced into the probe can react with biothiols (such as cysteine) via S N Ar (nucleophilic aromatic substitution) reaction enables rapid, site-specific labeling, while simultaneously enhancing the photon output capability of conjugated fluorophores by reducing the electron density of the mercapto sulfur atom and suppressing the nonradiative quenching process.

[0070] (2) The cyclooctatetraene photoprotective structure contained in the probe can effectively quench the triplet component in the excited state of the fluorophore, reduce the photobleaching process caused by the triplet state, and thus further improve the photon number performance.

[0071] The two types of structures mentioned above are integrated into the same probe molecule through a one-step "framework conjugation" strategy, achieving intramolecular synergistic enhancement of high photon number output characteristics, providing a new option of high-performance fluorescent probes for high-resolution applications such as single-molecule detection and super-resolution imaging.

[0072] Synthesis of starting material 1-formylcyclooctatetraene (COT-CHO):

[0073]

[0074] The steps for synthesizing COT-Br are as follows:

[0075] Under a nitrogen atmosphere, cyclooctatetraene (COT) (1 g, 9.6 mmol, 1 eq.) was dissolved in 20 mL of anhydrous DCM. The solution was stirred at -78 °C for about 10 min, and liquid Br2 (1.5 g, 9.4 mmol, 1 eq.) was slowly added dropwise. After 1 h, potassium tert-butoxide (13.4 mL, 1.0 M in THF, 13.4 mmol, 1.4 eq.) was added dropwise. The solution was stirred at -50 °C for 4 h, and the solution was poured into ice water. The solution was extracted with diethyl ether, dried, filtered, and purified by column chromatography (petroleum ether) to give 1.5 g of a yellow oily substance, with a yield of 80%.

[0076] The steps for synthesizing COT-CHO:

[0077] Under a nitrogen atmosphere, butyllithium (9 mL, 14.4 mmol, 1.2 eq., 1.6 M concentration) was slowly added to 40 mL of anhydrous THF in a COT-Br solution (2.2 g, 12.0 mmol, 1 eq.). The resulting mixture was stirred at -78 °C for 30 min. DMF (877 mg, 1 eq.) was added, and the reaction mixture was naturally warmed to room temperature and stirred for 2 h. A saturated NH4Cl solution was added, and the mixture was extracted with ethyl acetate, dried, filtered, and purified by column chromatography (petroleum ether:ethyl acetate = 10:1) to give 763 mg of a yellow oil, with a yield of 48%. Characterization is shown in [reference needed]. Figure 13 and14 .

[0078] Example 1: Synthesis of Click fluorescent tag I

[0079]

[0080] Compounds 4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)aniline (200 mg, 0.84 mmol) and 1-formylcyclooctatetraene (110 mg, 0.84 mmol) were dissolved in methanol (2.0 mL) and ultrasonically dispersed. Then, compounds 4-pentynic acid (82 mg, 0.84 mmol) and tert-butylisocyanate (70 mg, 0.84 mmol) were added. After stirring the reaction mixture at room temperature for 72 hours, ethyl acetate (100 mL) was added. The organic phase was washed with water (3 × 10 mL), and the aqueous layers were combined and extracted with ethyl acetate (2 × 10 mL). All organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to give fluorescent label I as a creamy white solid (50 mg, yield 11%).

[0081] Fluorescent tag I 1 H-NMR and 13 C-NMR and LC-MS mass spectrometry characterization were performed. See the specific 1H NMR spectrum for details. Figure 1 The carbon NMR spectrum is shown below. Figure 2 LC-MS mass spectra are shown below. Figure 3 .

[0082] Example 2: Synthesis of Fluorescent Probe I

[0083]

[0084] 4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)aniline (24 mg, 0.1 mmol) and 1-formylcyclooctatetraene (13 mg, 0.1 mmol) were dissolved in 3.0 mL of methanol, followed by the sequential addition of sulfonic acid-Cy5 carboxylic acid (5.0 mg) and tert-butyl isocyanate (8.0 mg, 0.1 mmol). The reaction mixture was stirred at room temperature for 48 h. After the reaction was complete, the solvent was removed by vacuum concentration. The mixture was then purified using preparative HPLC. R =12.5 min, lyophilized to obtain fluorescent probe I, yield 5.1 mg (61.2%).

[0085] The fluorescent probe I product was characterized by analytical liquid chromatography (HPLC) and high-resolution mass spectrometry (HR-MS). The specific HPLC chromatogram is shown in Figure 4, and the HR-MS chromatogram is shown in [Figure number missing]. Figure 5 .

[0086] Example 3: Synthesis of Fluorescent Probe II

[0087]

[0088] 4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)aniline (24 mg, 0.1 mmol) and paraformaldehyde (5.0 mg) were dissolved in 3.0 mL of methanol, followed by the sequential addition of sulfonic acid-Cy5 carboxylic acid (5.0 mg) and tert-butyl isocyanate (8.0 mg, 0.1 mmol). The reaction was stirred at room temperature for 48 h. After the reaction was complete, the solvent was removed by vacuum concentration. The mixture was then purified using preparative HPLC. R =12.1 min, freeze-dried to obtain product 2, yield 4.5 mg, yield 59.6%.

[0089] The fluorescent probe II product was characterized by analytical liquid chromatography (HPLC) and high-resolution mass spectrometry (HR-MS). The specific HPLC chromatogram is shown in Figure 6, and the HR-MS chromatogram is shown in [Figure number missing]. Figure 7 .

[0090] Comparative Example: Synthesis of Click Fluorescent Tag II [Reference: Zhang et al., Angew. Chem. Int. Ed. 2022, 61, e202112959]

[0091]

[0092] The synthesis steps of intermediate I are as follows:

[0093] The compound propyneamine (319 mg, 1 eq.) was dissolved in 20 mL of methanol. Cyclooctetral (1 eq.) was added first, followed by N-tert-butoxycarbonyl-beta-alanine (1 eq.) and 2 (1 eq.). After stirring at room temperature for 72 h, the mixture was extracted with ethyl acetate, dried, filtered, and purified by column chromatography (petroleum ether: ethyl acetate = 3:1) to give intermediate I, a white solid of 1.7 g, with a yield of 62%.

[0094] Synthesis steps of intermediate II:

[0095] Intermediate I (1.1 g, 1 eq.) was dissolved in dichloromethane (60 mL), and trifluoroacetic acid (40 eq.) was slowly added. The mixture was stirred at room temperature for 3 h, the solvent was evaporated, and the mixture was washed with dichloromethane. This process was repeated three times without further purification to obtain intermediate II.

[0096] Synthesis steps of Click fluorescent tag II:

[0097] Under a nitrogen atmosphere, 1.1 g (1 eq.) of 3-maleimide propionic acid was dissolved in dichloromethane (68 mL), and 7.74 g (10 eq.) of thionyl chloride was added. The mixture was stirred at room temperature for about 3 h, and the solvent was removed. No further purification was required before use. Intermediate II (477 mg (1.0 eq.)) was dissolved in dichloromethane (20 mL), and N,N-diisopropylethylamine (10 eq.) was slowly added under an ice-water bath. Then, freshly prepared 3-maleimide propionic acid (1.0 eq.) was added, and the mixture was stirred at room temperature for 72 h. After the reaction was complete, the mixture was extracted with DCM, dried over anhydrous magnesium sulfate, filtered, concentrated under vacuum, and purified by column chromatography (ethyl acetate) to give Click fluorescent tag II as a white solid, 120 mg, with a yield of 17%.

[0098] Click fluorescent tag II for 1 H-NMR and 13 C-NMR and LC-MS mass spectrometry characterization were performed. See the specific 1H NMR spectrum for details. Figure 8 LC-MS mass spectra are shown below. Figure 9 .

[0099] Example 5: Time-tracking and photophysical characterization of TIRFM single-molecule fluorescence imaging based on probes I and II labeled DNA

[0100] Experimental Procedure: 1) Sample Cleaning: First, a standard glass slide with dimensions of 76×26mm and a thickness of 0.17mm was selected as the sample slide. After rinsing the slide with ultrapure water, it was ultrasonically cleaned in 2% Hellmanex cleaning solution for 20 minutes to remove surface impurities and organic contaminants. After removing the slide, it was ultrasonically cleaned in ultrapure water for 5 minutes, then in acetone for 15 minutes, and rinsed twice with ultrapure water to ensure that there was no residual organic solvent. Subsequently, the slide was transferred to a 1.0M potassium hydroxide solution and ultrasonically cleaned for 20 minutes to further enhance surface activity. After removal, it was rinsed twice with ultrapure water, then ultrasonically cleaned in ethanol for 20 minutes, and finally rinsed twice with ultrapure water and dried with nitrogen. The dried slide was then surface activated using a plasma cleaner with a cleaning cycle time of 600 seconds. After treatment, the cleaned surface was immediately marked as a modifiable surface for subsequent surface modification steps.

[0101] 2) Surface modification of single-molecule slides: 3.0 mg of biotin-PEG-Silane was dissolved in 300 μL of spectroscopic-grade toluene to prepare a stock solution. Then, 300 μL of the PEG-Silane solution was mixed with 250 μL of the biotin-PEG-Silane stock solution and added to 60 mL of toluene. The mixture was thoroughly mixed to obtain the working solution. The working solution was poured into a glass jar containing the slides to be modified, filled with nitrogen, sealed, and incubated in a 55°C oven for 3 hours. After the first modification, the toluene solution was replaced, and the jar was incubated overnight at 55°C to improve the surface modification density and stability. After modification, the slides were removed from the solution, rinsed twice with ethanol, and sonicated for 5 minutes to remove non-specific binding substances. They were then rinsed once with ultrapure water and finally thoroughly dried with nitrogen. The treated slides were stored in a vacuum desiccator for subsequent single-molecule TIRFM imaging experiments.

[0102] 3) Sample Preparation: The single-molecule multi-well positioning plate was attached to the front side of the surface-modified single-molecule slide, and gently pressed to ensure tight adhesion. The wells were rinsed three times with PBS buffer (pH 7.4) (approximately 160 mL per well) to remove non-specific adsorbates. Streptavidin solution was then added to the wells and incubated at room temperature for 5 minutes to allow binding with biotin on the slide surface. The wells were then rinsed three more times with PBS. Next, DNA solution labeled with fluorescent probes was added, incubated at room temperature for 1 minute, and rinsed three more times to remove unbound probe molecules. Finally, each single-molecule well was filled with deoxygenated imaging buffer and sealed with sealant for single-molecule imaging assays.

[0103] 4) TIRFM Microscopic Imaging Test: Single-molecule fluorescence imaging was performed using a Nikon Total Internal Reflection Fluorescence Microscopy (TIRFM) system. The system includes: an inverted fluorescence microscope body, a 100×NA 1.49 TIRF-specific high numerical aperture objective lens, a manual focusing system, multi-wavelength lasers (including 561nm, 640nm, etc.), fiber coupling and laser modulation system, a high-sensitivity sCMOS detector camera, image acquisition and control software (Andor SOLIS), excitation and emission filters, a precision sample stage, and a vibration-resistant platform. Objective parameters were set to 23℃ and 0.17mm thickness. The 640nm laser was turned on, and the laser power was adjusted to 75W / cm². 2Select the emission filter corresponding to the wavelength. Manually adjust the incident angle to the critical angle to form an excitation thin layer with a thickness of approximately 100-200 nm. Place the prepared monomolecule sheet on the sample stage, add a drop of special impregnation oil to the objective lens, and slowly rotate the coarse focusing knob to raise the objective lens until it is in close contact with the bottom of the slide. Observe whether a complete and transparent oil film has formed without bubbles. Then, use the fine focusing knob to find the signal focal plane and adjust it until the signal is clear.

[0104] The imaging parameters were set as follows: In Andor SOLIS software, the EM Gain Level was set to the maximum value, the exposure time was set to 0.1 seconds, the frame rate was 10 frames / second, and the total imaging time was 10 minutes. Each group underwent dozens of imaging experiments under the same imaging parameters. The image storage format was set to TIFF, the resolution was 256×256 pixels, and the data save path was specified. This data will be used for subsequent quantitative studies such as single-molecule fluorescence trajectory extraction, signal intensity, and photobleaching analysis. The data can be found in [link to relevant documentation]. Figure 10 and Figure 11 . Figure 10 Representative images of single-molecule TIRF fluorescence imaging over time for DNA labeled with four molecules (A: first fluorescent tag I followed by click sulfo-Cyanine5 dye (CAS: 2242791-82-6), B: fluorescent probe II, C: first fluorescent tag II followed by click sulfo-Cyanine5 dye, D: sulfo-Cyanine5 dye) are shown. The time-series results from 0 s to 400 s indicate that AC maintains a clear and uniform fluorescence signal, demonstrating significantly better photostability than sulfo-Cyanine5 dye. In particular, the first-pass fluorescent tag I followed by click sulfo-Cyanine5 dye still shows a clear single-molecule fluorescent spot at 400 s, while sulfo-Cyanine5 essentially loses its fluorescence within 40 s, indicating its poorer resistance to photobleaching. Figure 11 Quantitative analysis further validated this trend: Photobleaching time: Fluorescent tag I had the longest photobleaching lifetime, approaching 600 s, approximately 6 times (~100 s) longer than sulfo-Cyanine5, highlighting its superior photostability. Signal-to-noise ratio: The SNR of both fluorescent tags I and II was higher than 3, which is crucial for the accuracy of single-molecule localization imaging. Count rate: The average photon count rate of the first three labeled probes reached 50–80 kHz, while sulfo-Cyanine5 only reached about 20 kHz, indicating that they have higher brightness at the single-molecule level. Total photon count: The total photon output after passing through fluorescent tag I and then through the clicked sulfo-Cyanine5 dye both exceeded 3 × 10⁻⁶. 6The increase in photon budget is reflected in the single photon count. In summary, the synergistic design of the cyclooctatetraene triplet quenching module and the benzothiazole methyl sulfone site-specific labeling module in the fluorescently labeled probe achieves significant improvements in photophysical performance, including extended photobleaching lifetime, increased signal-to-noise ratio, and enhanced total photon output. These advantages will effectively extend the observation window for single-molecule imaging, improve positioning accuracy, and provide an important tool for high-resolution imaging of dynamic biological processes.

[0105] Example 6: DNA-PAINT super-resolution fluorescence imaging study of U2OS cell nuclear porin Nup96 labeled with probe I

[0106] Experimental Procedure: U2OS cells expressing CRISPR-Nup96-mEGFP were fixed in PBS with 2.4% paraformaldehyde (PFA) for 30 minutes at room temperature. After fixation, the cells were washed three times with PBS and treated with 0.1M NH4Cl (prepared in PBS) for 5 minutes to block residual aldehyde groups. Subsequently, the cells were permeabilized by treatment with 0.25% Triton X-100 for 5 minutes and blocked in blocking buffer for 1 hour. Afterward, the cells were incubated with anti-GFP nanobody in blocking buffer to a final concentration of approximately 50 nM. The cells were then washed with PBS to remove unbound antibody. For super-resolution imaging, cell samples were pretreated using a standard operating procedure optimized for GFP tagging. Cells were first blocked in antibody and docking DNA strand incubation buffer for approximately 30 minutes, followed by dilution of the nanobody in the same buffer and incubation at room temperature for 30–60 minutes. The cells were then washed three times with 1× wash buffer to remove free antibody. The image was then rinsed once with imaging buffer, and an imaging solution containing the imaging DNA strand labeled with fluorescent probe I was added. Super-resolution imaging was performed using the following parameters: exposure time set within 30 milliseconds; laser intensity of 640 nm and laser power of approximately 50 mW; total imaging time of 15 minutes. Data can be found... Figure 12 . Figure 12This study presents the imaging results of nuclear porin Nup96 in U2OS cells using fluorescent probe I via DNA-PAINT technology. Compared with conventional diffraction-limited imaging, the DNA-PAINT image shows a significant resolution improvement. In the low-power field (left column), DNA-PAINT can clearly distinguish more single-point signals, revealing a finer distribution of nuclear pores. In the magnified region (right column), the localization accuracy of DNA-PAINT is significantly better than that of diffraction-limited imaging, capable of resolving single-molecule signals spaced sub-hundred nanometers apart, while the diffraction-limited image presents blurred, contiguous fluorescent spots. Notably, the sharp and dense aggregation pattern of signals in the DNA-PAINT image is highly consistent with the known geometric arrangement of nuclear porins. This verifies the high specificity of the probe design and the sufficiency of photon output, enabling it to support long-term, high-frame-rate imaging accumulation and achieve super-resolution reconstruction. Furthermore, as seen from the 500nm scale bar in the Zoomin region, DNA-PAINT technology achieves a localization accuracy of <20nm, significantly exceeding the resolution limit of traditional fluorescence microscopy (~200–250nm). In summary, the high-photon fluorescence probe I developed in this study, combined with DNA-PAINT imaging, can effectively achieve super-resolution localization and structural analysis of large intracellular protein complexes (such as the Nup96 nuclear pore), providing a powerful tool for revealing subcellular protein distribution and its dynamic changes.

Claims

1. A class of photostable biocoupled fluorescent molecules that synergistically enhance high photon numbers, characterized in that, The fluorescent molecule is prepared using 4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)aniline as the starting material. The compound structural formulas of the Click fluorescent tag I, fluorescent probe I, and fluorescent probe II are shown below. The compound structure shown in Click fluorescent label I is as follows: The compound structure shown by fluorescent probe I is as follows:

2. A method for preparing a class of high-photon-count fluorescent molecules as described in claim 1, characterized in that, It is prepared by a multi-component reaction involving 4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)aniline as the starting material, an aldehyde compound containing the photoprotectant cyclooctatetraene structure, a carboxylic acid component with a fluorescent tag or fluorophore containing a Click group, and tert-butylisocyanate. The synthesis of the Click fluorescent tag I is as follows: The synthesis of the fluorescent probe I is as follows: The synthesis of the fluorescent probe II is as follows:

3. The method according to claim 2, characterized in that, The preparation method of the aforementioned click fluorescent tag I includes the following steps: Compounds 4-(5-(methanesulfonyl)-1,3,4-oxadiazol-2-yl)aniline and 1-formylcyclooctatetraene were dissolved in a first solvent and ultrasonically dispersed. Then, compounds 4-pentynic acid and tert-butyl isocyanate were added. The aqueous layers were combined and then extracted with a second solvent. The resulting organic layers were combined, dried over anhydrous magnesium sulfate, filtered, and purified by column chromatography to finally obtain the click fluorescent tag I.

4. The method according to claim 2, characterized in that, The preparation method of the fluorescent probes I and II includes the following steps: Compound 4-(5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)aniline and 1-formylcyclooctatetraene or paraformaldehyde were dissolved in a first solvent and ultrasonically dispersed. Then, compound sulfonic acid-Cy5 carboxylic acid and tert-butyl isocyanate were added. After reacting for 48 h, the first solvent was removed by vacuum concentration and purified by column chromatography to finally obtain fluorescent probe I.

5. The method according to claim 3 or 4, characterized in that, In the steps described, the first solvent for the reaction is methanol, the second solvent is ethyl acetate, and the column chromatography uses petroleum ether and ethyl acetate in a volume ratio of 1:

1.

6. The method according to claim 2, characterized in that, The reaction temperature was room temperature, and the reaction time was 48–72 hours.

7. The application of the high-photon-count fluorescent molecule of claim 1 in single-molecule fluorescence imaging.

8. The application of the high-photon-count fluorescent probe I according to claim 1 in the preparation of DNA-PAINT super-resolution fluorescent imaging contrast agents for nucleoporin.

Images