Coumarin derivative light cage compound as well as preparation method and application thereof

Abstract

The invention provides a coumarin derivative light cage compound as well as a preparation method and application thereof, and the compound is a light cage molecule which can be controlled to release diol molecules through illumination. The polymer has the capability of efficiently releasing glycol molecules under the illumination of ultraviolet light, visible light and near-infrared light, the universal dissociation capability for intracellular phase separation, the good enrichment capability in living cells, the extremely low cytotoxicity and the high chemical stability, and high space-time selective release of the glycol molecules can be achieved through light regulation and control. The method can be applied to the aspects of biological pathway analysis related to cell protein phase separation, cell activity regulation and control, phenotype sorting and the like.

Description

Technical Field

[0001] The invention relates to the technical field of fine chemical industry, and in particular to a coumarin derivative photocage compound and a preparation method and application thereof. Background Art

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

[0003] 1,6-Hexanediol (1,6-HD) is a diol molecule with moderate hydrophobic interaction interference ability, which can depolymerize phase separation without destroying the biomembrane structure and can be used to regulate protein phase separation in cells. At the same time, a series of other diol compounds such as 2,5-hexanediol (2,5-HD) are also widely used in the study of protein phase separation.

[0004] In recent years, a variety of methods have been developed that can be used to regulate protein phase separation in cells, including photosensitive protein labels and 1,6-HD molecular treatment. The use of 1,6-HD to dissociate protein phase separation has the advantages of simple operation, strong universality, and significant dissociation effect. It is currently the most widely used method to interfere with intracellular protein phase separation. However, this method has problems such as lack of time and space selectivity and high cytotoxicity. It cannot controllably regulate protein phase separation in specific subcellular regions in a complex cellular environment. Therefore, a controllable protein phase separation dissociation method with high spatiotemporal selectivity at the subcellular level is urgently needed to be developed. Summary of the invention

[0005] In order to solve the above problems, the object of the present invention is to provide a coumarin derivative photocage compound and a preparation method and application thereof.

[0006] In order to achieve the above object, on the one hand, the present invention provides a compound having a structure of general formula I:

[0007]

[0008] In the general formula I, R1 is selected from diethylamino, dimethylamino, amino, nitro, hydroxyl, methoxy, (R1 together with the benzene ring to which it is connected forms the structure) any one;

[0009] R2 is selected from oxygen, Any of;

[0010] R3 is selected from oxygen, sulfur, Any of;

[0011] R4 is selected from one of the following structural groups:

[0012]

[0013] R5 is selected from one of the following structural groups:

[0014]

[0015] According to a specific embodiment of the present invention, the deuterium ( 2 H) content is 0 to 100%.

[0016] According to a specific embodiment of the present invention, preferably, the compound is selected from one of the following compounds:

[0017]

[0018] Wherein R5 is selected from one of the following structural groups:

[0019]

[0020] According to a specific embodiment of the present invention, preferably, the compound is selected from one of the following compounds:

[0021]

[0022] In another aspect, the present invention provides a composition comprising the above-mentioned compound or a derivative thereof;

[0023] The derivatives include derivatives in which the compound is connected with a targeting group, an antibody or a specific protein, which can change the localization and enrichment of the compound in cells or tissues; the specific protein is a protein with phase separation ability, or a protein that interacts with a phase separation protein.

[0024] In another aspect, the present invention provides a method for preparing the above-mentioned compound, wherein the preparation method comprises:

[0025] S1 and DMA were added to DMF and heated under reflux to react to form intermediate S2;

[0026] Intermediate S2 is reacted with sodium periodate dissolved in a mixed solvent of THF and water to generate intermediate S3;

[0027] Intermediate S3 uses a Dean Stark apparatus with toluene as solvent and p-toluenesulfonic acid as catalyst, and a diol compound is added to react to generate the compound;

[0028] The S1, S2, S3 and the preparation method are shown in the following reaction scheme:

[0029]

[0030] In the above preparation method, preferably, the ratio of S1 to DMF is 1-5 g:2-100 mL, more preferably 1-5 g:5-100 mL.

[0031] In the above preparation method, preferably, the addition ratio of DMF to DMA is 1:10 to 10:1.

[0032] In the above preparation method, preferably, in the heating reflux reaction to generate the intermediate S2, the reaction temperature is 120 to 160° C., preferably 140° C.; and the reaction time is 12 to 48 h.

[0033] In the above preparation method, preferably, in the mixed solvent of THF and water, the volume ratio of THF to H2O is 1:10 to 10:1.

[0034] 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.

[0035] In the above preparation method, preferably, the ratio of S3 to toluene is 0.1-2 g:10-200 mL.

[0036] In the above preparation method, preferably, the mass ratio of S3 to diol molecules is 0.1-2:0.1-20.

[0037] In the above-mentioned preparation method, preferably, in the reaction to generate the compound, the reaction temperature is 120 to 160° C. and the reaction time is 6 to 72 h.

[0038] According to a specific embodiment of the present invention, preferably, the preparation method comprises:

[0039] Coumarin derivative S1 and DMA are added to DMF solvent and heated under reflux at 140°C for 12 to 48 hours (the ratio of S1 to solvent is 1 to 5 g: 2-100 mL, and the ratio of DMA to DMF is 1:10 to 10:1), and the reaction generates intermediate S2;

[0040] S2 and sodium periodate (1-10 e.q.) are dissolved in a mixed solvent of THF and water (THF; H2O=1:10-10:1, the ratio of S2 to solvent is 0.1-2 g:1-50 mL), and reacted at room temperature for 0.1-12 h to generate a coumarin derivative intermediate S3 containing an aldehyde group;

[0041] The intermediate S3 uses a Dean stark apparatus with anhydrous toluene as solvent (the ratio of S3 to solvent is 0.1-2g:10-200mL), p-toluenesulfonic acid as catalyst (1-50mg, cat.amount), adds diol compounds (the ratio of S3 to diol molecules is 0.1-2g:0.1-20g), and heats under reflux at 120-160°C for 6-72h under nitrogen protection to generate the final product, coumarin derivative photocage compound, with diol compounds. The S1, S2, S3 and preparation method are shown in the following reaction scheme:

[0042]

[0043] In a specific embodiment of the present invention, taking the diol compound as 1,6-hexanediol as an example, the specific reaction route is as follows:

[0044]

[0045] In another aspect, the present invention provides a method for controlled release of a diol compound based on the above-mentioned compound, wherein the method comprises the following steps:

[0046] a light source irradiating a sample containing the compound;

[0047] The compound undergoes an acetal hydrolysis reaction after being irradiated by a light source, releasing a diol compound into the reaction system in the sample.

[0048] According to a specific embodiment of the present invention, preferably, the method further comprises:

[0049] The compound is dissolved in a PBS buffer or a cell culture medium, and incubated at 20 to 37° C. for 10 to 20 minutes, and the compound enters the cell by free diffusion. After replacing the PBS buffer or the cell culture medium, a specific area of ​​the sample is irradiated by a light source;

[0050] After being irradiated by a light source, the compound undergoes acetal hydrolysis to release the diol compound into the reaction system. More preferably, the release radius of the diol compound is 0.1 to 50 μm with the compound as the center.

[0051] According to a specific embodiment of the present invention, preferably, the concentration of the photocage molecule dissolved in the PBS buffer cell culture medium is 10 nM to 10 mM.

[0052] According to a specific embodiment of the present invention, preferably, the wavelength of the light source is 350-1500 nm, more preferably 350-600 nm; the irradiation time of the light source is 0.1 s-120 min, more preferably 1 s-60 min.

[0053] According to a specific embodiment of the present invention, preferably, the wavelength of the light source is excited by single photon or multi-photon, and the irradiation power is 1 μW to 100 mW, more preferably 40 μW to 10 mW.

[0054] According to a specific embodiment of the present invention, preferably, the compound is used for the in situ controlled release of diol molecules in in vitro samples, cells, tissues or living bodies by irradiation with a light source, and can be used for the controlled dissociation of protein phase separation in in vitro samples, cells, tissues or living bodies.

[0055] In the present invention, the PBS buffer formula is as follows: NaCl: 137 mM, KCl: 2.7 mM, Na2HPO4: 10 mM, KH2PO4: 1.8 mM.

[0056] On the other hand, the present invention provides an application of the above compound, the above preparation method or the above method for controlled release of diol compounds from the above compound in the preparation of a product for regulating protein phase separation.

[0057] On the other hand, the present invention provides an 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 a product for regulating protein phase separation by in situ controlled release of diol molecules through light source irradiation.

[0058] According to a specific embodiment of the present invention, preferably, the protein phase separation comprises protein phase separation in an in vitro sample, cell, tissue or living body.

[0059] In view of the problem that existing diol compounds have no time and space selectivity in regulating protein phase separation, the present invention provides a synthesis method and application of a coumarin derivative photocage compound constructed on the basis of existing diol compounds, using coumarin derivatives as the main part of the photocage, and modifying the functional groups so that the photocage compound can controllably release the diol compound under ultraviolet, visible and near-infrared light irradiation, which can not only realize the controllable release of diol molecules in subcellular regions by light in a cellular environment and further regulate the protein phase separation droplets in the subcellular region, but also locate the enrichment position in the cell through the autofluorescence of the photocage compound.

[0060] In summary, the present invention has the following advantages:

[0061] (1) The photocage compound described in the present invention forms acetals through coumarin derivatives modified with different functional groups and diol molecules. After in vitro experiments or enrichment in cells, tissues or living bodies, the diol molecules are released under visible light and near-infrared light, thereby achieving controllable release of diol molecules regulated by light.

[0062] (2) The photocage compound described in the present invention is easy to synthesize.

[0063] (3) The photocage molecules of the present invention have good chemical stability, can be applied to the light source excitation of a conventional laser confocal microscope, and have a high photochemical conversion rate.

[0064] (4) The photocage molecules of the present invention can be used for in situ controlled release in vitro, in cells, tissues or in vivo, and the fluorescence of the coumarin derivative photocage compounds themselves can indicate the location of the photocage molecules.

[0065] (5) Application in the regulation of protein phase separation. Compared with the traditional diol molecule treatment method, the photocage molecule of the present invention can achieve in situ light-controlled controlled release in living cells, has extremely low cytotoxicity, and can dissociate protein phase separation in specific subcellular regions. It is universal for different protein phase separation systems and helps to accurately regulate the protein phase separation behavior and cell activity of living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the photocage molecule pc-16HD prepared in Example 1 of the present invention;

[0067] Figure 2 This is the carbon NMR spectrum of the photocage molecule pc-16HD prepared in Example 1 of the present invention;

[0068] Figure 3 This is a high-resolution mass spectrum of the photocage molecule pc-16HD prepared in Example 1 of the present invention;

[0069] Figure 4 The UV-visible absorption spectrum of the photocage molecule pc-16HD and its cleavage product prepared in Example 1 of the present invention;

[0070] Figure 5 The fluorescence emission spectrum of the photocage molecule pc-16HD and its cleavage product prepared in Example 1 of the present invention;

[0071] Figure 6 This is a graph showing the concentration distribution of the photocage molecule pc-16HD prepared in Example 1 of the present invention in living cells;

[0072] Figure 7 This is a quantitative result diagram of the intracellular light-activated concentration of the photocage molecule pc-16HD prepared in Example 1 of the present invention;

[0073] Figure 8 This is a graph showing the experimental results of the control group of the protein phase separation and dissolution of the photocage molecule pc-16HD prepared in Example 1 of the present invention in living cells without light illumination;

[0074] Fig. 9This is a diagram showing the results of the highly spatially and temporally selective controllable dissociation of the photocage molecule pc-16HD prepared in Example 1 of the present invention for the phase separation aggregates of EGFP-FUS proteins in living cells. DETAILED DESCRIPTION

[0075] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical scheme of the present invention is now described in detail in conjunction with specific embodiments, and it should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of use of the present invention. In the embodiments, each raw reagent material can be obtained commercially, and the experimental method without specifying the specific conditions is a conventional method and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0076] Unless specifically defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the relevant art.

[0077] The term "Dean Stark apparatus" used in the present invention refers to a common chemical reaction glass apparatus, which is used to continuously discharge water or other components generated in the reaction system at reflux temperature to promote the positive movement of the reaction equilibrium.

[0078] The instruments and equipment used in the examples are:

[0079] In the process of detecting the compound of the present invention, nuclear magnetic resonance is measured by a Bruker Avance III 400M nuclear magnetic resonance instrument; high-resolution mass spectrum is measured by a Shimadzu LCMS-IT / TOF high-resolution mass spectrometer; ultraviolet visible spectrum is measured by an Agilent Cary 60 ultraviolet visible spectrophotometer; fluorescence spectrum is measured by a Hitachi F-7000 fluorescence spectrometer; LC-MS is measured by a Waters SQ Detector 2 ultra-high performance liquid chromatography tandem mass spectrometer; and cell fluorescence imaging results are measured by an Olympus FV-3000 laser confocal fluorescence microscope.

[0080] All cell lines used in the present invention were purchased from the ATCC official website.

[0081] The coumarin derivative photocage compound represented by the general formula I is described in detail below with reference to the examples.

[0082] Example 1

[0083] This embodiment 1 provides a coumarin derivative photocage molecule capable of controlled release of 1,6-hexanediol. The specific reaction route is as follows:

[0084]

[0085] Its synthesis method comprises the following steps:

[0086] S2: Under nitrogen environment 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 heated to reflux at 140°C for 24 h with stirring. After the reaction was completed, the reaction mixture was cooled to room temperature, and the reaction solvent was removed by decompression on a rotary evaporator. The remaining solid was fully ground and suspended in cyclohexane (10 mL) and filtered under reduced pressure. The filter cake was suspended in acetone (25 mL) again and filtered, and washed with acetone (10 mL×2) to obtain a light yellow solid S2 (1.8 g, 6.3 mmol, 72%).

[0087] 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, and stirred at room temperature for 2 h. After the reaction was completed, the reaction mixture was filtered to remove the remaining solid part, and the filter cake was washed with ethyl acetate (10 mL × 2) and all the filtrate was collected. The organic layer of the filtrate was washed with saturated sodium bicarbonate solution (20 mL × 2), dried over anhydrous sodium sulfate, and the solvent was dried under reduced pressure on a rotary evaporator to obtain a red-black oily compound S3 (450 mg, 1.8 mmol, 100%).

[0088] pc-16HD: 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 stirred at 135 ° C under a nitrogen atmosphere using a Dean Stark device for reflux reaction for 18 h. During the reaction, a small amount of water produced by the reaction was separated by a Dean Stark device. After the reaction, 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 with anhydrous sodium sulfate. The crude product was separated by basic alumina column chromatography, in which the mobile phase was separated using petroleum ether-ethyl acetate (4:1), and finally a light yellow compound pc-16HD (6.7 mg, 20 μmol, 1.1%) was obtained.

[0089] It has been confirmed that the pc-16HD structure is 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).

[0090] 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.

[0091] HRMS(ESI)calculated m / z for C 20 H 28 NO4 + [M+H] + 346.2013, found 346.2018.

[0092] The photodissociation reaction of the photocage molecule pc-16HD prepared in this example can be hydrolyzed to release the target diol molecule under 405nm light conditions. The reaction formula of the photodissociation reaction of pc-16HD is as follows:

[0093]

[0094] like Figure 1 Shown is the hydrogen nuclear magnetic resonance spectrum of the photocage molecule pc-16HD prepared in Example 1 of the present invention.

[0095] like Figure 2 Shown is the carbon nuclear magnetic resonance spectrum of the photocage molecule pc-16HD prepared in Example 1 of the present invention.

[0096] like Figure 3 Shown is a graph showing the high-resolution mass spectrometry characterization results of the photocage molecule pc-16HD prepared in Example 1 of the present invention.

[0097] like Figure 4The UV-visible absorption spectra of the photocage molecule pc-16HD prepared in Example 1 of the present invention and its light cleavage product are shown. As can be seen from the figure, the maximum absorption wavelength of pc-16HD in aqueous solution is 381nm, which can be excited by a 405nm light source. Under the irradiation of a handheld 405nm LED light source (6W), the UV-visible absorption spectrum of the compound changes and reaches stability in 90min, the maximum absorption wavelength blue-shifts to 360nm, and the absorption coefficient decreases to 25% of the original, showing the excellent light response ability of the photocage molecule. As a control, the photocage molecule remains stable in aqueous solution under no light conditions.

[0098] like Figure 5 The fluorescence emission (405nm excitation) spectrum of the photocage molecule pc-16HD and its light cleavage product prepared in Example 1 of the present invention is shown. Under the irradiation of a handheld 405nm LED light source, the fluorescence emission signal of the compound excited at 405nm is significantly reduced, which can be used for the localization and concentration quantification of the compound in cells.

[0099] Application Examples

[0100] like Figure 6 The concentration distribution of the photocage molecule pc-16HD prepared in Example 1 of the present invention in living cells is shown. The experiment used the U-2OS cell line, and the coumarin derivative photocage compound pc-16HD of Example 1 was dissolved in complete cell culture medium with the aid of 0.1% PluronicF127, incubated in a 37°C incubator for 20 minutes, and then washed once with PBS buffer using 405nm fluorescence imaging. The results showed that pc-16HD was mainly enriched in the endoplasmic reticulum in the cells. By drawing a fluorescence intensity quantitative standard curve, the average concentration of pc-16HD enriched in the endoplasmic reticulum was obtained to be 10mM.

[0101] like Figure 7 The figure shows the quantification of the intracellular light-activated concentration of pc-16HD prepared in Example 1 of the present invention. The experiment irradiates the local subcellular area with a 405nm laser light source at different powers and times, and quantifies its light-activated release by the change in the fluorescence intensity of pc-16HD before and after irradiation. The release amount under the irradiation conditions of 40-120μW 405nm laser power for 1-10s is 1-6mM, and the release amount is proportional to the illumination time and illumination intensity. Under the fluorescence imaging power conditions (4μW 405nm), the light cage molecule pc-16HD remains stable.

[0102] like Figure 8The figure shows the control experiment of protein phase separation and dissolution of pc-16HD in living cells without light conditions prepared in Example 1 of the present invention. The experiment used the U-2OS cell line transiently transfected with EGFP-FUS protein, and after sodium arsenite treatment, obvious EGFP-FUS protein phase separation aggregates were formed in living cells. Figure 8 As shown, the addition of the light cage molecule pc-16HD prepared in Example 1 alone has no effect on the phase separation aggregates of the EGFP-FUS protein in living cells, proving that the pc-16HD molecule has no solubility ability for the protein phase separation aggregates under the condition of no light.

[0103] like Fig. 9 The results of the highly spatially and temporally selective controllable dissociation of EGFP-FUS protein phase separation aggregates in living cells prepared by pc-16HD in Example 1 of the present invention are shown. The experiment used a U-2OS cell line transiently transfected with EGFP-FUS protein, and after sodium arsenite treatment, obvious EGFP-FUS protein phase separation aggregates were formed in living cells. After adding the photocage molecule treatment, the subcellular local area was irradiated with a 405nm light source to controllably release diol molecules, which can effectively dissociate EGFP-FUS protein phase separation aggregates in living cells, achieving light-controlled dissociation of phase separation aggregates in living cells, demonstrating the high spatial and temporal resolution controllable dissociation ability of pc-16HD molecules for intracellular phase separation.

Claims

1. A compound, wherein The compound has the structure of general formula I: In the general formula I, R1 is selected from diethylamino, dimethylamino, amino, nitro, hydroxyl, methoxy, Any of; R2 is selected from oxygen, Any of; R3 is selected from oxygen, sulfur, Any of; 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 from 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 according to any one of claims 1 to 4; The derivatives include derivatives in which the compound is linked to a targeting group, an antibody or a protein.

6. A method for preparing the compound according to any one of claims 1 to 4, wherein: The preparation method comprises: adding S1 and DMA to DMF and heating under reflux to react and generate an intermediate S2; Intermediate S2 is reacted with sodium periodate dissolved in a mixed solvent of THF and water to generate intermediate S3; Intermediate S3 uses a Dean stark apparatus with toluene as solvent, p-toluenesulfonic acid as catalyst, and a diol compound to react to generate the compound; The S1, S2, S3 and the preparation method are shown in the following reaction scheme:

7. A method for controlled release of a diol compound based on a compound according to any one of claims 1 to 4, wherein: The steps of the method include: A light source illuminates a sample containing the compound; The compound undergoes an acetal hydrolysis reaction after being irradiated by a light source, releasing a diol compound into the reaction system in 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. Use of the compound according to any one of claims 1 to 4, the composition according to claim 5, the preparation method according to claim 6, or the method for controlled release of diol compounds according to claim 7 or 8 in the preparation of a product for regulating protein phase separation.

10. Use of the compound according to any one of claims 1 to 4, the composition according to claim 5, the preparation method according to claim 6, or the method for controlled release of diol compounds according to claim 7 or 8 in the preparation of a product for in situ controlled release of diol molecules to regulate protein phase separation by irradiation with a light source; Preferably, the protein phase separation comprises protein phase separation in an in vitro sample, cell, tissue or living body.

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