Illumination cage-removing probe as well as preparation method and application thereof

Abstract

The invention discloses an illumination cage-removing probe as well as a preparation method and application thereof. The illumination cage-removing probe has a structure as shown in a formula I; the illumination cage removal probe has a very high illumination cage removal rate in a non-polar solvent, and has a very low illumination cage removal rate in a water phase. The solvent polarity-sensitive illumination decaging probe can be used for proximity labeling of lipid droplet surrounding proteins in fatty liver pathological tissue slices. # imgabs0 #

Description

Technical Field

[0001] The present application relates to a type of light-induced uncaged probe and its preparation method and application, belonging to the technical field of polymer materials. Background Art

[0002] Photocaged probes are a class of photodegradable probes that undergo cleavage reactions upon exposure to light, releasing functional groups. Due to the spatiotemporal controllability of photodegradation, they are widely used in fields such as chemistry, biology, and materials science. Research in this field focuses on improving the rate of photodegradation, extending the excitation wavelength, and optimizing biocompatibility. For example, a number of visible-light-activated photocaged probes have been developed based on the BODIPY backbone, as well as near-infrared-light-activated photocaged probes based on heptamethylcyanine dyes.

[0003] These diverse photocaged probes provide a diverse toolkit for precisely manipulating biological systems. Examples include precise control of biomacromolecule function, targeted drug delivery, bioimaging, and activation of functional biomaterials. Furthermore, the release of reactive quinone methide intermediates can be used for proximity labeling of proteins and proteomics studies.

[0004] However, the aforementioned light-caged probes have primarily been used in aqueous environments. Cells, on the other hand, possess distinct physicochemical microenvironments that regulate diverse physiological functions. For example, lipid droplets have low polarity, cell membranes exhibit high tension, and lysosomes are acidic. Furthermore, proteomic analysis of lipid droplets has been challenging, with the only available methods currently available including density gradient centrifugation and proximity labeling based on APEX technology. These methods suffer from high false-positive rates, complex procedures, and are unsuitable for difficult-to-transfect cell systems and pathological tissue sections.

[0005] Existing bioorthogonal photo-uncaging probes mainly undergo photo-uncaging in aqueous environments, and there have been no reports of probes that can selectively photo-uncaging in non-polar solvents. Summary of the Invention

[0006] In response to the problems existing in existing bio-orthogonal photo-detachable cage probes, the development of solvent polarity-sensitive photo-detachable cage probes can provide a new type of photo-cage probe, and can be used for proximity labeling of proteins surrounding lipid droplets in fatty liver pathological tissue sections, which has important scientific significance and clinical value for the study of fatty liver and liver cancer.

[0007] This study designed and synthesized a series of solvent polarity-sensitive photo-decaging probes that exhibit high photo-decaging rates in nonpolar solvents but very low photo-decaging rates in aqueous phases. These solvent polarity-sensitive photo-decaging probes can be used for proximity labeling of proteins surrounding lipid droplets in pathological sections of fatty liver tissue.

[0008] According to one aspect of the present application, a type of light-induced uncaging probe is provided, wherein the light-induced uncaging probe has a structure shown in Formula I:

[0009]

[0010] R1 is derived from one of alkynyl compounds, azide compounds, and vitamin B7;

[0011] R2 is derived from one of Sudan I, Sudan II, Sudan III, Sudan IV, Oil Red O, Nile Red, boron dipyrrole fluoride, and hydrogen;

[0012] n1 represents the number of carbon atoms, and the value of n1 is an integer from 0 to 5;

[0013] n2 represents the number of repeating structural units, and the value of n2 ranges from 0 to 4.

[0014] Optionally, the alkynyl compound is selected from one of acetylene, propyne, butyne and pentyne.

[0015] Optionally, the azide compound is selected from one of alkyl azide, phenyl azide, naphthyl azide and acyl azide.

[0016] Optionally, the value of n1 is 0.

[0017] According to another aspect of the present application, a method for preparing the above-mentioned light-irradiated uncaged probe is provided, the preparation method comprising the following steps:

[0018] (1) reacting a mixture containing a compound represented by Formula II, a compound represented by Formula III, and a solvent I to obtain an intermediate product I;

[0019] (2) reacting a mixture containing the intermediate product I, the compound represented by formula IV, a condensing agent, 4-dimethylaminopyridine, and a solvent II to obtain the photo-detachable probe;

[0020]

[0021]

[0022] n1, n2, and R1 in formula III are consistent with n1, n2, and R1 described above;

[0023] R2 in formula IV is the same as R2 described above.

[0024] Optionally, in step (1), the molar ratio of the compound represented by formula II to the compound represented by formula III is 1:1-10.

[0025] Optionally, in step (1), the molar ratio of the compound represented by formula II to the compound represented by formula III is independently selected from any value of 1:1, 1:2, 1:3, 1:5, 1:7, 1:8, 1:10 or a range between any two of the above.

[0026] Optionally, in step (2), the molar ratio of the intermediate product I to the compound represented by formula IV is 1:1.0-3.0.

[0027] Optionally, in step (2), the molar ratio of the intermediate product I to the compound represented by formula IV is independently selected from any value of 1:1, 1:1.5, 1:2, 1:2.5, 1:3, or a range between any two of the above values.

[0028] Optionally, the molar ratio of the intermediate product I to the 4-dimethylaminopyridine is 1:1.0-3.0.

[0029] Optionally, in step (2), the molar ratio of the intermediate product I to the condensing agent is 1:1 to 3.0.

[0030] Optionally, in step (2), the molar ratio of the intermediate product I to the condensing agent is independently selected from any value of 1:1, 1:1.5, 1:2, 1:2.5, 1:3 or a range between any two of the above.

[0031] Optionally, the condensing agent is selected from at least one of dicyclohexylcarbodiimide, diisopropylcarbodiimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

[0032] Optionally, in step (1), the solvent I is selected from at least one of dichloromethane, chloroform, carbon tetrachloride, toluene, and acetonitrile.

[0033] Optionally, the molar volume ratio of the compound represented by formula II to the solvent I is 1:0.2-5 mol / L.

[0034] Optionally, in step (2), the solvent II is selected from at least one of dichloromethane, chloroform, carbon tetrachloride, and N,N-dimethylformamide.

[0035] Optionally, the molar volume ratio of the intermediate product I to the solvent II is 1:15-22 mol / L.

[0036] Optionally, in step (1), the temperature of reaction I is 20 to 80° C., and the time of reaction I is 12 to 30 hours.

[0037] Optionally, the temperature of the reaction I is independently selected from any value of 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C or a range between any two of the above values.

[0038] Optionally, the reaction time I is independently selected from any value among 12h, 15h, 18h, 20h, 24h, 28h, 30h or a range between any two of the above.

[0039] Optionally, in step (2), the temperature of reaction II is 25 to 80° C., and the time of reaction II is 12 to 24 hours.

[0040] Optionally, the temperature of the reaction II is independently selected from any value among 25°C, 35°C, 45°C, 50°C, 65°C, 70°C, 80°C or a range between any two of the above values.

[0041] Optionally, the time of the reaction II is independently selected from any value among 12h, 16h, 18h, 20h, 24h or a range between any two of the above.

[0042] According to another aspect of the present application, there is provided an application of the above-mentioned light-irradiated uncaged probe in cells and fatty liver pathological tissue sections.

[0043] The core of this application is a class of probe molecules that can be selectively uncaged by light in low-polarity solvents. This class of molecules can achieve proximity labeling of proteins surrounding lipid droplets in fatty liver pathological tissue sections.

[0044] The beneficial effects of this application include:

[0045] 1) The light-induced uncaging probe provided in this application can be selectively uncaged by light in low-polarity solvents;

[0046] 2) The light-elevated uncaged probe provided in this application can label proteins surrounding lipid droplets;

[0047] 3) The light-elevated uncaged probe provided in this application can be used to reveal proteins surrounding lipid droplets in pathological tissue sections of fatty liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The light detachment rate of the probe molecule in aqueous phase and 1,4-dioxane in Test Example 1 of this application;

[0049] Figure 2 The probe molecule in Test Example 2 of this application is covalently labeled with the lipid droplet periprotein;

[0050] Figure 3 The probe molecule in Test Example 3 of this application is used to reveal the components of proteins surrounding lipid droplets in pathological tissue sections of fatty liver. DETAILED DESCRIPTION

[0051] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0052] Unless otherwise specified, the raw materials and reagents in the examples of this application were purchased from commercial sources and used directly without treatment. The instruments and equipment used adopted the protocols and parameters recommended by the manufacturers.

[0053] The nuclear magnetic resonance data in the examples of the present application were obtained using a Bruker AVANCE III 700 MHz nuclear magnetic resonance spectrometer.

[0054] The confocal fluorescence microscope used in this application example is an Olympus FV1000 FluoView TM Confocal microscope.

[0055] The mass spectrometer model used in the examples of this application is Orbitrap Exploris 480mass spectrometer.

[0056] As an optional embodiment, the present application prepares the light-released uncaged probe by the following preparation method:

[0057] (1) Compound III and solvent I are added to compound II, and after reaction, the mixture is quenched, extracted, and separated by column chromatography to obtain intermediate product I;

[0058] The compound II is selected from the compounds having the chemical formula shown in formula II:

[0059]

[0060] The compound III is selected from at least one compound having the chemical formula shown in formula III:

[0061]

[0062] Wherein, n1 represents the number of carbon atoms, which can be selected from one of 0 to 5; n2 represents the number of carbon atoms, which can be selected from one of 0 to 4; R1 is selected from at least one of alkynyl, azide, and biotin;.

[0063] Preferably, n1 is 0, n2 is 0, and R1 represents an alkynyl group or an azide group.

[0064] (2) adding compound IV, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, and solvent II to the intermediate product I, reacting, quenching, extracting, and separating by column chromatography to obtain the target product;

[0065] The compound IV is selected from at least one compound having the chemical formula shown in Formula IV:

[0066]

[0067] Example 1

[0068] Compound II (342 mg, 2.1 mmol) and Compound III (606 mg, 2.5 mmol) were dissolved in dichloromethane (5 mL). After stirring at room temperature overnight, the mixture was extracted with dichloromethane. The organic phase was collected, concentrated, and purified by column chromatography to obtain Intermediate I (323 mg, yield: 38%).

[0069] Intermediate I was tested using a nuclear magnetic resonance spectrometer, and the results were as follows: 1 H-NMR (700MHz, CDCl3) δ6.24(m,1H),4.51(m,2H),4.21(m,2H),3.90(m,1H),3.82(m,1H),3.78(m,1H),3.70(s,2H),3.70–3.66(m,4H), 3.59(t,J=5.3Hz,2H),3.49(t,J=5.2Hz,2H),3.27(m,1H),2.87–2.83(m,1H),2.46(m,1H),2.16–2.14(m,1H),2.02(s,3H),1.98(s,3H).

[0070] The synthetic route of the present embodiment is as follows:

[0071]

[0072] Example 2

[0073] Intermediate I (100 mg, 0.26 mmol), compound IV (78 mg, 0.3 mmol), and 4-dimethylaminopyridine (173 mg, 0.75 mmol) were dissolved in dichloromethane (5 mL) and cooled to 0°C. Dicyclohexylcarbodiimide (155 mg, 0.75 mmol) was then added, and the mixture was allowed to warm to room temperature and stirred overnight. After the reaction, dichloromethane was added for extraction. The organic phase was collected, concentrated, and finally purified by column chromatography to obtain a brown final product, P1 (83 mg, yield: 39%).

[0074] P1 was tested using a nuclear magnetic resonance spectrometer and a liquid chromatography high-resolution time-of-flight mass spectrometer Q-TOF 6540 for structural characterization and purity determination. The results are as follows: 1H-NMR (700MHz, CDCl3) δ8.58(d,J=8.6Hz,1H),7.92(s,1H),7.89–7.84(m,3H),7.83(d,J=8.2Hz,1H),7.68(m ,1H),7.55(m,1H),7.44(m,1H),7.41–7.36(m,3H),7.29(m,1H),6.18(s,1H),5.05–4.97(m,2H),4.26(t,J=6. 1Hz,2H),4.19(m,2H),3.82(m,1H),3.72(m,1H),3.69–3.63(m,6H),3.57(m,2H),3.47(m,2H),2.83(s,3H),2 .78(s,3H),2.56(m,2H),2.44(m,1H),2.17–2.14(m,2H),2.09(m,2H),2.05(m,1H),1.99(s,3H),1.97(s,3H). 13 C-NMR (176MHz, CDCl3) δ184.5,172.9,172.3,156.9,153.2,151.0,149.2,148.72,141.57,13 8.89,138.65,136.93,136.84,131.90,131.47,131.31,129.48,129.03,128.07,126.6,126.2 ,124.8,123.7,121.2,116.4,116.3,115.6,115.2,79.6,74.9,70.2,69.8,69.2,58.6,57.9,5 5.4,52.3,49.3,44.8,39.4,34.1,25.8,25.1,18.1,17.8,13.1,12.2.HRMS(m / z)Anal.Calc'd for C 49 H 53 N6O8(M+H) + :853.3919,Found(M+H) + :853.3910.

[0075] The synthetic route of the present embodiment is as follows:

[0076]

[0077] Example 3

[0078] Intermediate I (100 mg, 0.26 mmol), 4-dimethylaminopyridine (47.6 mg, 0.39 mmol), and triethylamine (39.5 mg, 0.39 mmol) were dissolved in dichloromethane (5 mL), cooled to 0°C, and stirred for 20 minutes. Benzoyl chloride (54.8 mg, 0.39 mmol) was then added dropwise, and the mixture was allowed to warm to room temperature and stirred overnight. After the reaction, dichloromethane was added for extraction. The organic phase was collected, concentrated, and finally purified by column chromatography to obtain the purple final product P2 (62 mg, yield: 49%).

[0079] P2 was tested using a nuclear magnetic resonance spectrometer and a liquid chromatography high-resolution time-of-flight mass spectrometer Q-TOF 6540 for structural characterization and purity determination. The results are as follows: 1 H-NMR(700MHz, CDCl3)δ8.02–7.98(m,2H),7.53(m,1H),7.43–7.38(m,2H),6.26(m,1H),5.27(m,2H),4.20(m,2H),3.87(m,1H),3.80(m,1H),3 .76(m,1H),3.74–3.70(m,1H),3.70–3.68(m,2H),3.66(m,2H),3.58(m ,2H),2.86(m,1H),2.45(m,1H),2.14(m,2H),2.10(s,3H),2.05(s,3H). 13 C-NMR (176MHz, CDCl3) δ187.1,184.5,172.4,166.4,149.4,141.8,137.2,133.2,129.9,129.9,128.5,115.3, 79.5,75.0,70.2,69.8,69.2,58.6,58.3,55.5,52.4,44.8,39.4,29.8,13.2,12.3.HRMS(m / z)Anal.Calc'dfor C 28 H 33 N2O7(M+H) + :509.2282,Found(M+H) + :509.2278.

[0080] The synthetic route of the present embodiment is as follows:

[0081]

[0082] Test Example 1: Solvent Polarity Selectivity of P2 Probe Illuminated Decaging

[0083] P2 (50 μM) prepared in Example 3 was dissolved in 1,4-dioxane or water and irradiated with 530 nm green light. The absorption was measured using a microplate reader at 0, 5, 10, 15, and 20 minutes to estimate the rate of light detachment. Figure 1 As shown in Figure 3, the detachment rate of P2 in the low-polarity 1,4-dioxane is much higher than that in the aqueous phase.

[0084] Test Example 2P1 is used to covalently label proteins surrounding lipid droplets

[0085] The frozen sections were rewarmed, washed three times with PBS, and the target area was selected with a PAP pen. Then, it was infiltrated with 0.3% TritonX-100 at room temperature for 10 minutes. Then, it was washed three times with PBS, and then fixed with 4% formaldehyde at room temperature for 30 minutes. After washing three times with PBS, 5μM P1 probe was added and incubated in the dark for 30 minutes. Then, it was washed twice with PBS and once with 20% ethanol. Irradiate with 530nm light for 7 minutes and let it stand for 5 minutes. Then, β-mercaptoethanol (1μM) was added and let it stand for 10 minutes. Wash once with PBS and once with 40% ethanol. Then, click chemistry reaction (BODIPY-N3) was performed, washed three times with PBS and once with 40% ethanol. Then, BODIPY (5μM) was added for staining for 1 hour. Wash three times with PBS. Stain with Hoechst33342 (2μg / mL) for 15 minutes, wash once with PBS, and then seal the slide. Confocal imaging was then performed, and the results are as follows: Figure 2 The blue signal represents the cell nucleus, and the red signal represents the lipid droplet periprotein covalently labeled with the probe.

[0086] Test Example 3P1 was used to reveal the composition of proteins surrounding lipid droplets in pathological tissue sections of fatty liver;

[0087] Frozen sections were thawed, washed three times with PBS, and the target area was selected using a PAP pen. 5 μM P1 probe was added and incubated in the dark for 30 minutes. The sections were then washed twice with PBS and once with 20% ethanol. Irradiation was performed at 530 nm for 7 minutes and allowed to stand for 5 minutes. β-Mercaptoethanol (1 μM) was then added and allowed to stand for 10 minutes. The sections were washed once with PBS and once with 40% ethanol. The tissues were collected, placed in EP tubes, and ultrasonically disrupted. Click chemistry (Biotin-N3) was then performed at room temperature for 2 hours. The sections were precipitated with acetone overnight, centrifuged, and the supernatant discarded. The precipitate was re-dissolved with 8 M urea and diluted to 1 M with 50 mM ammonium bicarbonate solution. Streptavidin-containing magnetic beads were added and incubated on a shaker at 4°C overnight. The sections were then washed twice with 8 M urea and twice with 2 M sodium chloride. Denatured at 95°C, cooled, and then 10 μg of trypsin was added and digested at 37°C for 16 hours. The supernatant was collected and desalted, and the sample was freeze-dried and loaded into the mass spectrometer for data analysis. Figure 3As shown, proteins related to mitochondrial oxidative phosphorylation were abnormally increased in samples of liver cancer patients with fatty liver.

[0088] The above descriptions are merely a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application discloses the preferred embodiments as above, they are not intended to limit the present application. Any technical personnel familiar with the present profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A type of light-induced detachment probe, characterized in that: The light-induced uncaging probe has a structure shown in Formula I: R1 is derived from one of alkynyl compounds, azide compounds, and vitamin B7; R2 is derived from one of Sudan I, Sudan II, Sudan III, Sudan IV, Oil Red O, Nile Red, boron dipyrrole fluoride, and hydrogen; n1 represents the number of carbon atoms, and the value of n1 is an integer from 0 to 5; n2 represents the number of repeating structural units, and the value of n2 ranges from 0 to 4.

2. The light detaching probe according to claim 1, characterized in that The alkynyl compound is selected from one of acetylene, propyne, butyne and pentyne; Preferably, the azide compound is selected from one of alkyl azide, phenyl azide, naphthyl azide and acyl azide; Preferably, the value of n1 is 0.

3. The method for preparing the light-induced uncaged probe according to any one of claims 1 to 2, characterized in that: The preparation method comprises the following steps: (1) reacting a mixture containing a compound represented by Formula II, a compound represented by Formula III, and a solvent I to obtain an intermediate product I; (2) reacting a mixture containing the intermediate product I, the compound represented by formula IV, a condensing agent, 4-dimethylaminopyridine, and a solvent II to obtain the photo-detachable probe; n1, n2, and R1 in formula III are consistent with n1, n2, and R1 in claim 1; R2 in formula IV is identical to R2 in claim 1.

4. The preparation method according to claim 3, characterized in that In the step (1), the molar ratio of the compound represented by formula II to the compound represented by formula III is 1:1-10.

5. The preparation method according to claim 3, characterized in that In the step (2), the molar ratio of the intermediate product I to the compound represented by formula IV is 1:1.0-3.0; Preferably, the molar ratio of the intermediate product I to the 4-dimethylaminopyridine is 1:1.0-3.

0.

6. The preparation method according to claim 3, characterized in that In the step (2), the molar ratio of the intermediate product I to the condensing agent is 1:1.0-3.0; Preferably, the condensing agent is at least one selected from dicyclohexylcarbodiimide, diisopropylcarbodiimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

7. The preparation method according to claim 3, characterized in that In the step (1), the solvent I is selected from at least one of dichloromethane, chloroform, carbon tetrachloride, toluene, and acetonitrile; Preferably, the molar volume ratio of the compound represented by formula II to the solvent I is 1:0.2-5 mol / L.

8. The preparation method according to claim 3, characterized in that In the step (2), the solvent II is selected from at least one of dichloromethane, chloroform, carbon tetrachloride, and N,N-dimethylformamide; Preferably, the molar volume ratio of the intermediate product I to the solvent II is 1:15 to 22 mol / L.

9. The preparation method according to claim 3, characterized in that In the step (1), the temperature of the reaction I is 20 to 80° C., and the reaction time is 12 to 30 hours; Preferably, in step (2), the temperature of reaction II is 25 to 80° C., and the time of reaction II is 12 to 24 hours.

10. Use of the light-elevated uncaged probe according to claim 1 in cells and fatty liver pathological tissue sections.

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