Coumarin derivative-based photocaged compound, and preparation method therefor and use thereof

Abstract

The present invention provides a coumarin derivative-based photocaged compound, and a preparation method therefor and a use thereof. The compound can release diol molecules under ultraviolet, visible, and near-infrared light irradiation, has a dissociation ability for intracellular phase separation and high chemical stability, and can achieve high spatiotemporally selective release of the diol molecules by light regulation.

Description

A coumarin derivative light cage compound, its preparation method and application Technical Field

[0001] This invention relates to the field of fine chemical technology, specifically to a coumarin derivative photocage compound and its preparation method and application. Background Technology

[0002] Protein phase separation is a common phenomenon in cellular life activities. A large number of different proteins, nucleic acids, and other biological macromolecules spontaneously form an independent liquid phase in the complex cellular environment, regulating a variety of cellular physiological processes and being crucial for the study of various cellular physiological processes.

[0003] 1,6-Hexanediol (1,6-HD) is a diol molecule with moderate hydrophobic interaction interference capability, which can induce depolymerization during phase separation without disrupting the biological membrane structure, and can be used to regulate protein phase separation within cells. Meanwhile, a series of other diol compounds, such as 2,5-hexanediol (2,5-HD), are also widely used in protein phase separation research.

[0004] In recent years, various methods have been developed for regulating protein phase separation in cells, including photosensitive protein tagging and 1,6-HD molecule treatment. Using 1,6-HD to dissociate protein phases offers advantages such as simplicity, broad applicability, and significant dissociation effects, making it the most widely used method for interfering with intracellular protein phase separation. However, this method suffers from a lack of temporal and spatial selectivity and significant cytotoxicity, making it unable to controllably regulate protein phase separation in specific subcellular regions within the complex cellular environment. Therefore, controllable protein phase separation methods with high subcellular spatiotemporal selectivity urgently need to be developed. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a coumarin derivative photocage compound, its preparation method, and its application.

[0006] To achieve the above objectives, in one respect, the present invention provides a compound having the structure of general formula I:

[0007] In general formula I, R1 is selected from diethylamino, dimethylamino, amino, nitro, hydroxyl, methoxy, ... Any one of the following (R1 together with the connected benzene ring forms this structure);

[0008] R2 is selected from oxygen, Any one of them;

[0009] R3 is selected from oxygen, sulfur, Any one of them;

[0010] R4 is selected from one of the following structural groups:

[0011] R5 is selected from one of the following structural groups:

[0012] According to a specific embodiment of the present invention, the deuterium (R5 group) 2 The content of H) is 0 to 100%.

[0013] According to a specific embodiment of the present invention, preferably, the compound is selected from one of the following compounds:

[0014] R5 is selected from one of the following structural groups:

[0015] According to a specific embodiment of the present invention, preferably, the compound is selected from one of the following compounds:

[0016] On the other hand, the present invention provides a composition comprising the above-described compound or its derivatives;

[0017] The derivatives include those linked to a target group, antibody, or specific protein, which can alter the localization and enrichment of the compound in cells or tissues; the specific protein is a protein with phase-separating ability or a protein that interacts with a phase-separating protein.

[0018] On the other hand, the present invention provides a method for preparing the above-mentioned compound, wherein the preparation method includes:

[0019] S1 and DMA are added to DMF and heated under reflux to react and generate intermediate S2.

[0020] Intermediate S2 reacts with sodium periodate dissolved in a mixed solvent of THF and water to generate intermediate S3.

[0021] Intermediate S3 was produced by adding a diol compound using a Dean Stark apparatus with toluene as the solvent and p-toluenesulfonic acid as the catalyst.

[0022] The reaction route for S1, S2, S3, and their preparation method is shown below:

[0023] In the above preparation method, preferably, the ratio of S1 to DMF is 1-5g:2-100mL, more preferably 1-5g:5-100mL.

[0024] In the above preparation method, preferably, the ratio of DMF to DMA is 1:10 to 10:1.

[0025] In the above preparation method, preferably, in the heating and reflux reaction to generate intermediate S2, the reaction temperature is 120-160℃, preferably 140℃; and the reaction time is 12-48h.

[0026] In the above preparation method, preferably, the volume ratio of THF to H2O in the mixed solvent of THF and water is 1:10 to 10:1.

[0027] In the above preparation method, preferably, the ratio of S2 to the mixed solvent of THF and water is 0.1-2 g: 1-50 mL.

[0028] In the above preparation method, preferably, the ratio of S3 to toluene is 0.1-2 g: 10-200 mL.

[0029] In the above preparation method, preferably, the mass ratio of S3 to diol molecules is 0.1-2:0.1-20.

[0030] In the above preparation method, preferably, the reaction temperature is 120-160°C and the reaction time is 6-72 h in the reaction to generate the compound.

[0031] According to a specific embodiment of the present invention, preferably, the preparation method includes:

[0032] Coumarin derivative S1 and DMA were added to DMF solvent and heated under reflux at 140°C for 12-48 h (the ratio of S1 to solvent was 1-5 g: 2-100 mL, and the ratio of DMA to DMF was 1:10-10:1). The reaction produced intermediate S2.

[0033] S2 is dissolved in sodium periodate (1-10 e.q.) in a mixed solvent of THF and water (THF:H2O = 1:10-10:1, S2 to solvent ratio is 0.1-2 g:1-50 mL), and reacted at room temperature for 0.1-12 h to generate coumarin derivative intermediate S3 containing aldehyde group;

[0034] Intermediate S3 was prepared using a Dean Stark apparatus with anhydrous toluene as solvent (S3 to solvent ratio: 0.1–2 g : 10–200 mL), catalyzed by p-toluenesulfonic acid (1–50 mg, cat.amount), and a diol compound (S3 to diol molecular ratio: 0.1–2 g : 0.1–20 g). The reaction was carried out under nitrogen protection at 120–160 °C under reflux for 6–72 h, reacting with the diol compound to form the final product, a coumarin derivative photocage compound. The reaction route for S1, S2, S3, and the preparation method is shown below:

[0035] In one specific embodiment of the present invention, taking 1,6-hexanediol as an example, the specific reaction route is as follows:

[0036] On the other hand, the present invention provides a method for the controlled release of diol compounds based on the above-mentioned compounds, wherein the steps of the method include:

[0037] A light source illuminates a sample containing the compound;

[0038] The compound undergoes acetal hydrolysis upon irradiation with a light source, releasing diol compounds into the reaction system within the sample.

[0039] According to a specific embodiment of the present invention, preferably, the method further includes the following steps:

[0040] The compound was dissolved in PBS buffer or cell culture medium and incubated at 20–37°C for 10–20 min. The compound entered the cells by free diffusion. After changing the PBS buffer or cell culture medium, a specific area of ​​the sample was irradiated with a light source.

[0041] When the compound is irradiated by a light source, the acetal undergoes hydrolysis, releasing diol compounds into the reaction system. More preferably, the release radius of the diol compounds is 0.1–50 μm with the compound as the center.

[0042] According to a specific embodiment of the present invention, preferably, the concentration of the photocage molecule dissolved in the PBS buffered cell culture medium is 10 nM to 10 mM.

[0043] According to a specific embodiment of the present invention, preferably, the wavelength of the light source is 350-1500nm, more preferably 350-600nm; the irradiation time of the light source is 0.1s-120min, more preferably 1s-60min.

[0044] According to a specific embodiment of the present invention, preferably, the wavelength of the light source is excited by single photon or multiphoton, and the irradiation power is 1μW to 100mW, more preferably 40μW to 10mW.

[0045] According to a specific embodiment of the present invention, preferably, the compound is used to control the in-situ release of diol molecules in in vitro samples, cells, tissues or living organisms by means of light source irradiation, and can also be used for the controlled dissociation of protein phases in in vitro samples, cells, tissues or living organisms.

[0046] In this invention, the PBS buffer formulation is as follows: NaCl: 137mM, KCl: 2.7mM, Na2HPO4: 10mM, KH2PO4: 1.8mM.

[0047] On the other hand, the present invention provides the application of the above-mentioned compound, the above-mentioned preparation method, or the above-mentioned method for controlled release of diol compounds in the preparation of products for regulating protein phase separation.

[0048] On the other hand, the present invention provides the application of the above-mentioned compound, the above-mentioned preparation method, or the above-mentioned method for controlled release of diol compounds in the preparation of products for controlling protein phase separation by in-situ controlled release of diol molecules through light source irradiation.

[0049] According to a specific embodiment of the present invention, preferably, the protein phase separation includes protein phase separation in in vitro samples, cells, tissues, or living organisms.

[0050] To address the lack of temporal and spatial selectivity in regulating protein phase separation using existing diol compounds, this invention provides a method for synthesizing and applying coumarin derivative photocage compounds based on existing diol compounds. Using coumarin derivatives as the main photocage component, the photocage compound is modified with functional groups to controllably release diol compounds under ultraviolet, visible, and near-infrared light irradiation. This allows for the controllable release of diol molecules in subcellular regions within the cellular environment via light irradiation, further regulating protein phase separation droplets in these subcellular regions. Furthermore, the autofluorescence of the photocage compound can be used to pinpoint its intracellular enrichment location.

[0051] In summary, the present invention has the following advantages:

[0052] (1) The photocage compound of the present invention forms an acetal by modifying coumarin derivatives with different functional groups and diol molecules. After being tested in vitro or enriched in cells, tissues or living organisms, it releases diol molecules under visible light and near-infrared light, thereby achieving the controllable release of diol molecules regulated by light.

[0053] (2) The photocage compound described in this invention is easy to synthesize.

[0054] (3) The photocage molecules described in this invention have good chemical stability, can be used for excitation by conventional laser confocal microscopes, and have high photochemical conversion efficiency.

[0055] (4) The photocage molecules described in this invention can be used for in situ controlled release in vitro, in cells, tissues or living organisms, and the fluorescence of the coumarin derivative photocage compounds themselves can indicate the location of the photocage molecules.

[0056] (5) In terms of protein phase separation regulation, compared with the traditional diol molecule treatment method, the photocage molecule described in this invention can achieve in-situ light-controlled release in living cells with extremely low cytotoxicity, dissociate protein phase separation in specific subcellular regions, and has universality for different protein phase separation systems, which helps to precisely regulate the protein phase separation behavior and cell activity of living cells. Attached Figure Description

[0057] Figure 1 is the proton NMR spectrum of the photocage molecule pc-16HD prepared in Example 1 of the present invention;

[0058] Figure 2 is the carbon NMR spectrum of the photocage molecule pc-16HD prepared in Example 1 of the present invention;

[0059] Figure 3 is a high-resolution mass spectrum of the photocage molecule pc-16HD prepared in Example 1 of the present invention;

[0060] Figure 4 shows the ultraviolet-visible absorption spectra of the photocage molecule pc-16HD and its pyrolysis products prepared in Example 1 of the present invention.

[0061] Figure 5 shows the fluorescence emission spectra of the photocage molecule pc-16HD and its pyrolysis products prepared in Example 1 of the present invention;

[0062] Figure 6 shows the concentration distribution of the photocage molecule pc-16HD prepared in Example 1 of the present invention in living cells;

[0063] Figure 7 shows the quantitative results of intracellular light-activated concentration of the light cage molecule pc-16HD prepared in Example 1 of the present invention.

[0064] Figure 8 shows the experimental results of protein phase separation and dissolution control group of the light cage molecule pc-16HD prepared in Example 1 of the present invention in living cells under light-free conditions.

[0065] Figure 9 shows the results of the highly spatially and temporally selectively controllable dissociation of the light cage molecule pc-16HD prepared in Example 1 of the present invention for the phase separation of EGFP-FUS protein in living cells. Detailed Implementation

[0066] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention will now be described in detail with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of application of this invention. In the embodiments, all original reagent materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0067] Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art in the relevant field.

[0068] The term "Dean Stark apparatus" as used in this invention refers to a common glass instrument for chemical reactions, used to continuously remove water or other components generated in a reaction system at reflux temperature, thereby shifting the reaction equilibrium to the forward direction.

[0069] The instruments and equipment used in the embodiments are as follows:

[0070] In the detection of compounds, the following methods were used in this invention: nuclear magnetic resonance (NMR) was measured using a Bruker Avance III 400M NMR spectrometer; high-resolution mass spectrometry (HDMS) was measured using a Shimadzu LCMS-IT / TOF HDMS spectrometer; ultraviolet-visible (UV-Vis) spectroscopy was measured using an Agilent Cary 60 UV-Vis spectrophotometer; fluorescence spectroscopy was measured using a Hitachi F-7000 fluorescence spectrometer; LC-MS was measured using a Waters SQ Detector 2 ultra-high performance liquid chromatography-tandem mass spectrometry (ULLC-MS); and cell fluorescence imaging results were obtained using an Olympus FV-3000 laser confocal fluorescence microscope.

[0071] All cell lines used in this invention were purchased from the ATCC website.

[0072] The following detailed description of the coumarin derivative photocage compounds represented by general formula I is provided in conjunction with the embodiments.

[0073] Example 1

[0074] Example 1 provides a coumarin derivative photocage molecule that can controllably release 1,6-hexanediol. The specific reaction route is shown below:

[0075] Its synthesis method includes the following steps:

[0076] S2: Under nitrogen protection, compound S1 (2.0 g, 8.8 mmol) and N,N-dimethylacetamide (DMA, 1.8 g, 15.0 mmol) were added to anhydrous N,N-dimethylformamide (DMF, 5.0 mL), and the mixture was heated under reflux at 140 °C with stirring for 24 h. After the reaction was completed, the reaction mixture was cooled to room temperature, and the solvent was removed by rotary evaporation under reduced pressure. The remaining solid was thoroughly ground and suspended in cyclohexane (10 mL) and filtered under reduced pressure. The filter cake was resuspended in acetone (25 mL) and filtered. After washing with acetone (10 mL × 2), a pale yellow solid S2 (1.8 g, 6.3 mmol, 72%) was obtained.

[0077] S3: Compound S2 (530 mg, 1.8 mmol) was added to tetrahydrofuran-water (1:1, 20 mL), and sodium periodate (1.2 g, 5.5 mmol) was added under stirring at room temperature. The mixture was stirred for 2 h at room temperature. After the reaction was complete, the reaction mixture was filtered to remove the remaining solid fraction. The filter cake was washed with ethyl acetate (10 mL × 2) and all the filtrate was collected. The organic layer fraction of the filtrate was washed with saturated sodium bicarbonate solution (20 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was evaporated under reduced pressure using a rotary evaporator to obtain a reddish-black oily compound S3 (450 mg, 1.8 mmol, 100%).

[0078] pc-16HD: Dehydrated compound S3 (450 mg, 1.8 mmol), 1,6-hexanediol (920 mg, 7.8 mmol), p-toluenesulfonic acid (5 mg, cat.amount), and anhydrous toluene (30 mL) were added to a round-bottom flask. The mixture was heated and refluxed at 135 °C for 18 h under nitrogen protection using a Dean Stark apparatus. During the reaction, a small amount of water produced was separated using a Dean Stark apparatus. After the reaction was complete, ethyl acetate (30 mL) and distilled water (30 mL) were added to the reaction mixture, and the organic layer was extracted and separated. The organic layer was further washed with saturated sodium bicarbonate aqueous solution (50 mL × 2), deionized water (50 mL), and saturated sodium chloride aqueous solution (50 mL), and dried over anhydrous sodium sulfate. The crude product was separated by alkaline alumina column chromatography, with the mobile phase being petroleum ether-ethyl acetate (4:1), finally yielding the pale yellow compound pc-16HD (6.7 mg, 20 μmol, 1.1%).

[0079] The pc-16HD structure has been confirmed to be correct. 1H NMR (400MHz, Chloroform-d) δ (ppm): 7.68 (d, J = 9.1Hz, 1H), 6.55 (dd, J = 9.1, 2.7Hz, 1H), 6.50 (d, J = 2.6Hz, 1H), 6.43 (d,J=0.9Hz,1H),5.71(s,1H),3.83(m,2H),3.66(m,2H),3.40(q,J=7.1Hz,4H),1.72(m,8H),1.20(t,J=7.1Hz,6H).

[0080] 13 C NMR(400MHz,Chloroform-d)δ(ppm):162.48,156.73,151.21,150.46,126.3 6,108.69,108.40,106.35,98.48,97.77,67.72,44.81,29.10,25.32,12.62.

[0081] HRMS(ESI) calculated m / z for C 20 H 28 NO4 + [M+H] + 346.2013, found 346.2018.

[0082] The photodissociation reaction of the photocage molecule pc-16HD prepared in this embodiment can release the target diol molecule through hydrolysis under 405nm light irradiation. The reaction formula of the photodissociation reaction of pc-16HD is shown below:

[0083] Figure 1 shows the proton NMR spectrum of the photocage molecule pc-16HD prepared in Example 1 of this invention.

[0084] Figure 2 shows the carbon NMR spectrum of the photocage molecule pc-16HD prepared in Example 1 of this invention.

[0085] Figure 3 shows the high-resolution mass spectrometry characterization results of the photocage molecule pc-16HD prepared in Example 1 of the present invention.

[0086] Figure 4 shows the UV-Vis absorption spectra of the photocage molecule pc-16HD and its photolysis products prepared in Example 1 of this invention. As can be seen from the figure, the maximum absorption wavelength of pc-16HD in aqueous solution is 381 nm, which can be excited by a 405 nm light source. Under irradiation with a handheld 405 nm LED light source (6W), the UV-Vis absorption spectrum of this compound changes, reaching stability within 90 minutes. The maximum absorption wavelength blue-shifts to 360 nm, and the absorption coefficient decreases to 25% of its initial value, demonstrating the excellent photoresponse capability of this photocage molecule. As a control, the photocage molecule remains stable in aqueous solution under no-light conditions.

[0087] Figure 5 shows the fluorescence emission (405nm excitation) spectra of the photocage molecule pc-16HD and its photolysis products prepared in Example 1 of this invention. Under irradiation with a handheld 405nm LED light source, the fluorescence emission signal of this compound excited at 405nm is significantly reduced, which can be used for the localization and concentration quantification of this compound in cells.

[0088] Application examples

[0089] Figure 6 shows the concentration distribution of the photocage molecule pc-16HD prepared in Example 1 of this invention in living cells. The experiment used the U-2 OS cell line. The coumarin derivative photocage compound pc-16HD from Example 1 was dissolved in complete cell culture medium with 0.1% Pluronic F127 as a solubilizer, incubated at 37°C for 20 min, and then washed once with PBS buffer before fluorescence imaging at 405 nm. The results showed that pc-16HD was mainly enriched in the endoplasmic reticulum (ER). A fluorescence intensity quantitative standard curve was plotted, revealing an average concentration of 10 mM of pc-16HD enriched in the ER.

[0090] Figure 7 shows the quantification of intracellular photoactivated concentration of pc-16HD prepared in Example 1 of this invention. This experiment used a 405nm laser source to irradiate subcellular local areas at different powers and times. The photoactivated release amount was quantified by the change in fluorescence intensity of pc-16HD before and after irradiation. The release amount was found to be 1–6 mM under irradiation conditions of 40–120 μW 405nm laser power for 1–10 s, and the release amount was directly proportional to the irradiation time and intensity. Under fluorescence imaging power conditions (4 μW 405nm), the photocage molecule pc-16HD remained stable.

[0091] Figure 8 shows the protein phase separation and dissolution control experiment of pc-16HD prepared in Example 1 of this invention in live cells under light-free conditions. The experiment used the U-2 OS cell line transiently transfected with EGFP-FUS protein, which formed obvious EGFP-FUS protein phase separation condensates in live cells after treatment with sodium arsenite. As shown in Figure 8, adding the photocage molecule pc-16HD prepared in Example 1 alone did not affect the EGFP-FUS protein phase separation condensates in live cells, proving that pc-16HD molecules have no dissolving ability for protein phase separation condensates under light-free conditions.

[0092] Figure 9 shows the results of the highly spatially and temporally selectively controllable dissociation of pc-16HD for EGFP-FUS protein phase separation condensates in living cells, prepared according to Example 1 of this invention. The experiment used the U-2 OS cell line with transient EGFP-FUS protein transfection. After treatment with sodium arsenite, obvious EGFP-FUS protein phase separation condensates were formed within the living cells. After treatment with this photocage molecule, the controlled release of diol molecules by irradiating the subcellular local region with a 405nm light source effectively dissociated the EGFP-FUS protein phase separation condensates in living cells, achieving photo-controlled dissociation of the phase separation condensates in living cells. This demonstrates the high spatial and temporal resolution controllable dissociation capability of the pc-16HD molecule for intracellular phase separation.

Claims

1. A compound, wherein, The compound has the structure of general formula I: In general formula I, R1 is selected from diethylamino, dimethylamino, amino, nitro, hydroxyl, methoxy, ... Any one of them; R2 is selected from oxygen, Any one of them; R3 is selected from oxygen, sulfur, Any one of them; R4 is selected from one of the following structural groups: R5 is selected from one of the following structural groups:

2. The compound according to claim 1, wherein, The deuterium content of the R5 group is 0 to 100%.

3. The compound according to claim 1, wherein, The compound is selected from one of the following compounds: R5 is selected from one of the following structural groups:

4. The compound according to claim 1, wherein, The compound is selected from one of the following compounds:

5. A composition comprising the compound or a derivative thereof as described in any one of claims 1-4; The derivatives include compounds linked to a target group, antibody, or protein.

6. A method for preparing a compound according to any one of claims 1-4, wherein, The preparation method includes: S1 and DMA are added to DMF and heated under reflux to react and generate intermediate S2. Intermediate S2 reacts with sodium periodate dissolved in a mixed solvent of THF and water to generate intermediate S3. Intermediate S3 was produced by adding a diol compound using a Dean Stark apparatus with toluene as the solvent and p-toluenesulfonic acid as the catalyst. The reaction route for S1, S2, S3, and their preparation method is shown below:

7. A method for the controlled release of diol compounds based on any one of claims 1-4, wherein, The steps of the method include: A light source illuminates a sample containing the compound; The compound undergoes acetal hydrolysis upon irradiation with a light source, releasing diol compounds into the reaction system within the sample.

8. The method according to claim 7, wherein, The wavelength of the light source is 350–1500 nm, and the irradiation time is 0.1 s–120 min.

9. The use of the compound of any one of claims 1-4, the composition of claim 5, the preparation method of claim 6, or the method for controlled release of diol compounds of the compound of claim 7 or 8 in the preparation of products for regulating protein phase separation.

10. The use of the compound of any one of claims 1-4, the composition of claim 5, the preparation method of claim 6, or the method for controlled release of diol compounds of the compound of claim 7 or 8 in the preparation of a product for controlling protein phase separation by in-situ controlled release of diol molecules via light source irradiation; Preferably, the protein phase separation includes protein phase separation in in vitro samples, cells, tissues, or living organisms.

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