Fluorescent dye molecules that can undergo spontaneous blinking under physiological conditions, and methods of synthesis and use thereof

Abstract

The present application belongs to the technical field of fluorescent dyes, and particularly relates to a kind of fluorescent dye molecules capable of spontaneous blinking under physiological conditions and a synthesis method and application thereof.The spontaneous blinking fluorescent dye molecules of the present application take aniline squarylium cyanine as a mother nucleus;the spontaneous blinking fluorescent molecules can be prepared by Click reaction through intermediate squarylium fluorophore and azide targeting group;the spontaneously blinking fluorescent molecules designed and synthesized by the present application can occur spontaneous blinking behavior under physiological conditions, so that they are very suitable for single molecule localization microscopy, and can be used for cell super-resolution imaging.

Description

Technical Field

[0001] This invention belongs to the field of spontaneously flashing fluorescent dye technology, specifically a fluorescent dye molecule that can spontaneously flash under physiological conditions, its synthesis method, and its application. Background Technology

[0002] Single-molecule localization microscopy has wide applications in various fields of biology and chemistry. As an important component of single-molecule localization microscopy, fluorescent dyes play a crucial role in obtaining super-resolution fluorescence images. Compared with other types of fluorescent dyes, the study of spontaneously scintillation fluorescent dyes has greatly simplified experimental setups and extended imaging duration. In 2014, Urano's group reported the first spontaneously scintillation rhodamine dye. Through the dynamic equilibrium of its own switching ring, it achieved spontaneous scintillation of sparse single-molecule signals, successfully used for dynamic super-resolution imaging of microscopic structures in living cells. However, the synthesis of spontaneously scintillation fluorophores based on rhodamine molecules is difficult, and they are challenging to modify. The photo-switching properties require complex chemical modifications to alter the intramolecular switching ring equilibrium. Currently, the types of available spontaneously scintillation fluorophores remain limited. Therefore, developing a novel, easily synthesized, and optically superior spontaneously scintillation fluorescent dye is particularly important.

[0003] Squamousine dyes are a class of dyes with resonantly stable zwitterionic structures, typically comprising an electron-deficient central four-membered ring and two electron-donating groups in the form of donor-acceptor-donor (DAD) groups. Due to their planar structure and zwitterionic properties, squamousine dyes exhibit strong absorption and emission in the near-infrared region. This is a valuable wavelength region for many types of imaging applications. To date, no spontaneous scintillation fluorescent dyes based on squamousine have been reported. Summary of the Invention

[0004] The purpose of this invention is to provide a fluorescent dye molecule that can spontaneously scintillate under physiological conditions, its synthesis method, and its applications. This invention uses an intermediate squaricine fluorophore and an azide-based targeting group to obtain a class of spontaneously scintillating fluorescent dyes via a Click reaction. The preparation method of this invention is simple and allows for convenient and rapid preparation. This type of dye can spontaneously scintillate under physiological conditions (i.e., the cellular environment that maintains the normal physiological state of the organism), without the need for the addition of an external nucleophilic thiol or strong laser irradiation, thus enabling its use in super-resolution imaging of live cells.

[0005] This invention provides a fluorescent dye molecule that can spontaneously flash under physiological conditions. It is a fluorescent dye molecule based on aniline-based squaricine as its parent core, and its molecular structure is as follows:

[0006]

[0007] Among them: R1, R2, R3, and R4 are independent arbitrary organic groups;

[0008] Preferably, R1, R2, R3, and R4 are independently benzyl groups, benzyl groups with mono- or poly-substituted hydrogen atoms on the benzene ring, and C1-C... 30 Hydrocarbon group, substituted C1-C 30 Hydrocarbon group.

[0009] More preferably, R1, R2, R3, and R4 are independently benzyl, halogen-monosubstituted benzyl, trifluoromethyl-monosubstituted benzyl, methoxycarbonyl-monosubstituted benzyl, or propynoxy C1-C. 30 Alkyl monosubstituted benzyl, C1-C 30 Alkyl or halogen-substituted C1-C 30 Alkyl, trifluoromethyl substituted C1-C 30 Alkyl, C1-C 20 Alkoxy-substituted C1-C 30 Alkyl, propynoxy-substituted C1-C 30 Any alkyl group, or a heterocyclic substituted C1-C 30 Alkyl and aromatic substituted C1-C 30 Alkyl group. More preferably, R1, R2, R3, and R4 are independently benzyl, p-fluorobenzyl, p-bromobenzyl, p-trifluoromethylbenzyl, p-methoxycarbonylbenzyl, p-propynoxyethylbenzyl, propynoxyethyl, or... Any one of them, where R5 is Or a protein molecule with targeting function; when R5 is a protein molecule with targeting function, it is selected from any one of the following: microfilament protein, endoplasmic reticulum protein, lysosomal protein, mitochondrial protein, and microtubule protein.

[0010] In a further optimized configuration, R1 and R2 are the same, and R3 and R4 are the same.

[0011] A further preferred fluorescent dye molecule that can spontaneously flash under physiological conditions has the following structural formula:

[0012]

[0013] In the formula, R is any one of benzyl, p-fluorobenzyl, p-bromobenzyl, p-trifluoromethylbenzyl, p-methoxycarbonylbenzyl, p-propynoxyethylbenzyl, or propynoxyethyl, and R5 is... Or a protein molecule with a targeting function; wherein: when R5 is a protein molecule with a targeting function, it is selected from any one of the following: microfilament protein, endoplasmic reticulum protein, lysosomal protein, mitochondrial protein, and microtubule protein.

[0014] This invention also provides the application of fluorescent dye molecules that can spontaneously scintillate under the aforementioned physiological conditions in single-molecule localization microscopy. Preferably, it is applied in single-molecule localization super-resolution imaging of cells; during super-resolution imaging, a power density of 100-400 W / cm² is used. 2 Near-infrared light source excitation and irradiation, with or without the addition of thiol molecules (glutathione GSH), using either live or dead cells; more preferably, in applications such as single-molecule localization super-resolution imaging of live cells, a 100-200 W / cm² light source is used. 2 Excitation and irradiation with a near-infrared light source, without the addition of thiol molecules (glutathione GSH).

[0015] Furthermore, this invention provides a method for synthesizing a fluorescent dye molecule that can spontaneously flash under the above-mentioned physiological conditions, the synthetic route being as follows:

[0016]

[0017] The specific steps are as follows:

[0018] Step 1: Add the aniline derivative and squaric acid of the structure shown in formula (I) to the first organic solvent, reflux overnight using a Dean-Stark water separator, and monitor the reaction by TCL; then, cool the mixture to room temperature, remove the solvent, and purify the residue by column chromatography, and obtain the squaric acid cyanine fluorescent molecule of the structure shown in formula (II) after post-processing.

[0019] Step 2: The cubic cyanine fluorescent molecule with the structure shown in formula (II) and the azide-based targeting molecule with the structure shown in formula (III) undergo a Click reaction in a second organic solvent under the action of a catalyst to prepare the spontaneously flashing fluorescent molecule with the targeting properties shown in formula (IV).

[0020] In step one above, the molar ratio of aniline derivative intermediate to squaric acid is 2:1, and the first organic solvent is toluene; in step two, the molar ratio of squaric acid cyanine fluorescent molecule to azide-based target molecule and catalyst is 1:(1.01-1.1):0.2; the second organic solvent is toluene, and the catalyst is cuprous thiophene-2-carboxylic acid (I)CuTC.

[0021] In step two above, the target molecules for azide compounds are selected from any one of the following compounds:

[0022]

[0023] in:

[0024] It can be any of the following structural formulas:

[0025]

[0026] Compared with existing technologies, the advantages of this invention are as follows: Traditional cyanine dyes used for super-resolution imaging require high-power laser irradiation and additives such as thiols to promote photoscintillation. However, high-power laser irradiation or additives may lead to phototoxicity and affect cell physiology. However, the spontaneously scintillated fluorescent molecules designed and synthesized in this invention do not require high-power laser irradiation or additives such as thiols, and are therefore suitable for super-resolution imaging of live cells. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the principle of spontaneous scintillation properties of all dye molecules in this invention and their application in super-resolution imaging.

[0028] Figure 2 The 1H NMR spectrum of sq1 in deuterated chloroform is shown in Example 1.

[0029] Figure 3 The sq1 high-resolution time-of-flight mass spectrometry is shown in Example 1.

[0030] Figure 4 The hydrogen NMR spectrum of SQ-F in deuterated chloroform is shown in Example 1.

[0031] Figure 5 This is the SQ-F high-resolution time-of-flight mass spectrometry of Example 1.

[0032] Figure 6 The absorption and emission spectrum of SQ-F in chloroform is shown in Example 1.

[0033] Figure 7 The GSH response of the squaricine dye in solution and single-molecule imaging of SQ-F in Example 1 are shown. (a) Addition of GSH (6 × 10⁻⁶) -4 (a) An aqueous solution of SQ1 (7.7 mmol) was added, followed by the addition of GSH + HCl (7.7 mmol), and the absorption spectra of SQ1 were observed before and after different time points. Time 0 minutes is the initial moment after the addition of HCl to the mixture. (b) A graph of the normalized SQ1 absorbance at 647 nm as a function of GSH, and fitted curves for Kd and GSH. (ch) Single-molecule images at 5 different time points and a stacked image of 4000 frames. Power density: 100 W cm⁻¹ -2 (i) SQ-F at 400W / cm 2 Typical intensity time trajectories are shown. The blue line represents the change in brightness over time, and the red line represents the bright state of the molecule, determined by the Hidden Markov Model (HMM). (j) The cumulative localization number versus time with or without GSH (pH 7.4 PBS buffer containing 5.0 mM GSH) at different power densities. (k) Localization accuracy distribution of SQ-F at different power densities, with a median of 100 W cm⁻¹.-2 6.0nm, 200W cm -2 5.8nm, 400Wcm -2 5.5nm. (l) 100W cm⁻¹ with or without GSH. -2 The on-time distribution is shown below, and the time constant is obtained by double exponential fitting. (m,n) represents the number of flashes (m) and duty cycle (n) of SQ-F with or without GSH at different power densities.

[0034] Figure 8 SQ1 in Example 1 at 400 W / cm 2 Typical intensity time trajectory under the following conditions.

[0035] Figure 9 SQ2 in Example 1 at 400 W / cm 2 Typical intensity time trajectory under the following conditions.

[0036] Figure 10 SQ3 in Example 1 at 400 W / cm 2 Typical intensity time trajectory under the following conditions.

[0037] Figure 11 SQ4 in Example 1 at 400 W / cm 2 Typical intensity time trajectory under the following conditions.

[0038] Figure 12 SQ5 in Example 1 at 400W / cm 2 Typical intensity time trajectory under the following conditions.

[0039] Figure 13 SQ6 in Example 1 at 400 W / cm 2 Typical intensity time trajectory under the following conditions.

[0040] Figure 14 SQ7 in Example 1 at 400W / cm 2 Typical intensity time trajectory under the following conditions.

[0041] Figure 15 SQ8 in Example 1 at 400W / cm 2 Typical intensity time trajectory under the following conditions.

[0042] Figure 16 SQ-Ly in Example 1 at 400 W / cm 2 Typical intensity time trajectory under the following conditions.

[0043] Figure 17 SQ-Mito in Example 1 at 400W / cm 2 Typical intensity time trajectory under the following conditions.

[0044] Figure 18 SQ-TAR-Protein in Example 1 at 400 W / cm 2 Typical intensity time trajectory under the following conditions.

[0045] Figure 19 The squaricine dye SF-Q from Example 1 was used for super-resolution imaging of live cell membranes. (a) Super-resolution and diffraction-limited images of HeLa cell membranes. (b) Intensity distribution of the cross-section of neutron region 2, with black and red dots fitted by a single Gaussian function corresponding to the diffraction-limited and super-resolution images, respectively. (c) Enlarged images of the diffraction-limited (left) and super-resolution (right) images of neutron region 1. (d) Intensity distribution of the cross-section of neutron region 1, with black bars corresponding to the diffraction-limited image and red dots fitted by two Gaussian functions corresponding to the super-resolution image. (e, f) Distribution of single-molecule brightness (e) and positioning accuracy (f) during the entire imaging process with an exposure time of 40 ms. (g) FRC analysis of the super-resolution image resolution, with correlation = 1 / 7 selected as the threshold. The power density of live cell super-resolution imaging was 300 W / cm². 2 . Detailed Implementation

[0046] Figure 1 This diagram illustrates the principle of the self-scintillation property of the dye molecule of the present invention and its application in super-resolution imaging; the structural formula of the dye molecule is as follows:

[0047]

[0048] In the formula: R1, R2, R3, and R4 are independent organic groups.

[0049] Figure 1 The invention describes dyes that can be reversibly attacked by biological nucleophiles such as hydroxyl or thiols, leading to fluorescence quenching and recovery. Under physiological conditions, this type of dye exhibits spontaneous scintillation behavior in its single-molecule state and has been successfully used for super-resolution imaging of live cells, achieving a significant improvement in imaging resolution compared to traditional optical microscopy.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0051] Example 1: Preparation of SQ-F

[0052]

[0053] Add 0.114 g (1 mmol) of squaric acid (0.566 g, 2 mmol) of aniline derivative to 16 mL of dry benzene and 8 mL of n-butanol solution. Reflux overnight at 100 °C using a water separator. Wash the mixture three times with water, extract with dichloromethane, and dry with anhydrous Na₂SO₄. Remove the solvent under vacuum. Purify the crude product by column chromatography using dichloromethane and methanol (v / v = 30:1) as eluent. Yield: 15%. The product undergoes... 1 Confirmed by H NMR and MS: 1 H NMR (400MHz, CDCl3) δ8.37(d,J=9.1Hz,4H),7.14(dd,J=8.6,5.2Hz,4H),7.03(t,J=8.6Hz,4H),6.82(d,J=9 .3Hz,4H),4.78(s,4H),4.16(d,J=2.4Hz,4H),3.80(s,8H),2.43ppm(t,J=2.4Hz,2H).HRMS(MALDI-TOF)m / z calcd.for[M]+:644.2492,found:644.2490.( Figure 2 ,3)

[0054]

[0055] CuTC (7.7 mg, 0.02 mmol) was dissolved in toluene (1.5 mL) and stirred for 5 minutes. A solution of sql (64.4 mg, 0.1 mmol) and dodecylbenzenesulfonylazide (38.6 mg, 0.11 mmol) in toluene (3 mL) was added to this solution. After addition, the mixture was stirred at 30 °C for 12 hours. The reaction was quenched with saturated NH4Cl aqueous solution and extracted with dichloromethane. The organic layer was dried over anhydrous Na2SO4. The solvent was removed under vacuum. The crude product was purified by column chromatography using dichloromethane and methanol (v / v = 20:1) as eluents. Yield: 30%. The product underwent... 1 Confirmed by H NMR and MS: 1H NMR (400MHz, CDCl3) δ8.38(d,J=8.9Hz,4H),8.08–7.94(m,6H),7.37(dd,J=13.4,8.4Hz,4H),7.11(dd,J=8.2,5.3Hz,4H),7.00(t,J=8 .5Hz,4H),6.81(d,J=8.9Hz,4H),4.74(s,4H),4.62(s,4H),3.80(dd,J=10.2,3.9Hz,8H),1.55–0.98ppm(m,50H).HRMS(MALDI-TOF)m / z calcd.for[M]+:1347.6487,found:1347.5725.( Figure 4 5) Prepare a 1 μM SQ-F CHCl3 solution, and measure its absorption spectrum using a UV-Vis absorption spectrometer. The maximum absorption peak is at 624 nm. Measure its emission spectrum using a fluorescence spectrometer. The maximum emission wavelength is at 643 nm. Figure 6 )

[0056] In addition to compounds SQ-9 and SQ-F, the inventors also used structural formulas... The aniline derivatives and squaric acid were added to benzene, refluxed overnight using a Dean-Stark water separator, and the reaction was monitored by TCL. Afterward, the mixture was cooled to room temperature, the solvent was removed, and the residue was purified by column chromatography. The post-processing yielded squaric acid cyanine fluorescent molecules with the following structures: R1=R2, R3=R4.

[0057]

[0058]

[0059] Example 2: Single-molecule imaging of SQ-F

[0060] Cover slips and slides were assembled into several 2-4 μL microchambers. Before single-molecule measurements, the SQ-F dye was diluted to ~200 pM with PBS buffer, and then 10 μL of 0.1% polylysine solution was injected into the chamber. After 3 minutes, the chamber was rinsed three times with 200 μL PBS. Next, 10 μL of SQ-F dye solution was injected into the chamber. After 3 minutes, the chamber was rinsed five times with 200 μL PBS buffer. After completing these steps, the sample was placed on a fluorescence microscope stage for imaging.

[0061] Using a 638nm laser (Cobolt 06-DPL, The laser was used to excite the sample prepared in the previous step. The sample was excited by a 100× oil objective (NA 1.49, Nikon), and the emission from the sample was received by an EMCCD (iXon 897, Andor) connected to the microscope via the same objective. An emission filter (ZET532 / 640, Chroma) was installed in the optical path to filter the excitation light. Measurements were performed at three different power densities: 100 W cm⁻¹. -2 200Wcm -2 400W cm -2 The camera's exposure time was fixed at 50 ms, and 4000-8000 frames were captured under different conditions (with or without 5 mM GSH). The scintillation performance of SQ-F was evaluated through single-molecule imaging experiments. SQ-F exhibited good single-molecule spontaneous scintillation behavior. At 400 W / cm²... 2 At this level, an impressive emissivity of up to 56,840 photons per second and a superior positioning accuracy of 5.5 nm were achieved. Compared to commonly used SMLM probes, the SQ-F has approximately four times the brightness of Cy5 and exhibits excellent positioning accuracy. (100-400 W / cm²) 2 Under laser irradiation, the duty cycle remained between 0.1% and 0.4%. In short, SQ-F exhibited excellent brightness and desirable spontaneous scintillation behavior at low power densities in physiological buffer solutions, suggesting its great potential for single-molecule localization-based super-resolution imaging in live cells. Figure 7 )

[0062] Figures 8-18 Typical intensity-time trajectories of SQ1-SQ8, SQ-Ly, SQ-Mito, and SQ-TAR-Protein (specifically SQ-α-Tubulin Mouse Monoclonal Antibody, which targets tubulin) were disclosed. The figures show that the dyes have multiple alternating bright and dark states, with the bright state duration being relatively shorter than the dark state duration. These compounds also exhibit excellent brightness and ideal spontaneous scintillation behavior at low power densities in physiological buffer solutions, indicating that they also have great potential for single-molecule localization-based super-resolution imaging in living cells.

[0063] Example 3: Super-resolution imaging of SQ-F

[0064] Cells were cultured at 37°C in a humid atmosphere with 5% CO2, and in glass-bottomed culture dishes (BeyoGold). TMCells were grown on 35mm confocal culture dishes for 12 hours to reach a density of 80-90% before use. Live cells were stained with 10μM SQ-F (diluted from 1mM DFM concentrate to DMEM to obtain the final concentration) and incubated in the dark at 37°C in a humid atmosphere of 5% CO2 for 10 minutes. Cells were washed three times with PBS before imaging.

[0065] STORM imaging of dye-labeled HeLa cells was performed using a homemade fluorescence microscope. A 638nm laser (Cobolt 06-DPL) was used. The sample was excited by a photonics laser, and the emission signal from the sample was captured by an EMCCD (iXon897, Andor) connected to the microscope via an emission filter (ZET532 / 640, Chroma). The laser power density was set to 300 W / cm². -2 To avoid significant phototoxicity to living cells, the camera exposure time was set to 40 milliseconds, with 20,000 frames acquired per field of view. Data was analyzed using ThunderSTORM to obtain localized information for each PSF. Finally, the localization information was imported into MATLAB software, and drift correction was performed using an autocorrelation algorithm. Super-resolution images were obtained using corrected localization. SQ-F exhibited strong spontaneous scintillation behavior in a living cell environment. The dynamic behavior was very similar to the scintillation patterns observed in previous single-molecule experiments. In this example, 20,000 consecutive images were recorded and used to reconstruct super-resolution images. The resulting reconstructed images revealed intricate details of the cell membrane structure, providing unparalleled clarity compared to conventional epifluorescence images. This resulted in a cross-sectional full width at half maximum (FWHM) of 112 nm for the super-resolution membrane, a significant improvement over the 1170 nm FWHM of conventional projected images. This advancement in spatial resolution effectively surpasses the optical diffraction limit, validating the application of SQ-F in living cell super-resolution imaging. Figure 19 ).

Claims

1. The application of a fluorescent dye molecule that can spontaneously scintillate under physiological conditions in single-molecule localization microscopy imaging, characterized in that, The structural formula of the fluorescent dye molecule is as follows: , In the formula, R is any one of benzyl, p-fluorobenzyl, p-bromobenzyl, p-trifluoromethylbenzyl, p-methoxycarbonylbenzyl, p-propynoxyethylbenzyl, or propynoxyethyl, and R5 is... , , Or protein molecules with targeting functions.

2. The application according to claim 1, characterized in that, Fluorescent dye molecules are used in single-molecule localization super-resolution imaging of live cells.

3. The application according to claim 1, characterized in that, When applying, use 100-200W / cm 2 It is excited and irradiated by a near-infrared light source without adding any additional thiol molecules.

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