Targeting covalent linkage aggregation protein probe, preparation method and application

Abstract

The invention discloses a targeting covalently-linked aggregation protein probe, a preparation method and application. The targeting covalently-linked aggregation protein probe has a structure as shown in a formula I; r1 is selected from at least one of a dimethylamino group, a diethylamino group, a julolidine group, a C1-C5 alkoxy group, a cyano group, a nitro group, an ester group and hydrogen; r2 is selected from at least one of C1-C5 alkyl groups, C1-C5 alkoxy groups, cyano groups, ester groups and hydrogen. The molecules disclosed by the invention can be selectively combined with aggregation proteins and covalently linked with the aggregation proteins. The covalent fluorescent probe can be used for in-situ imaging of aggregation proteome in living cells. The invention provides a portable and efficient method for detecting the aggregated protein in the living cell progen. # imgabs0 #

Description

Technical Field

[0001] The present application relates to a type of targeted covalently linked aggregated protein probe, a preparation method and an application thereof, and belongs to the field of probe molecule technology. Background Art

[0002] Misfolding and aggregation of proteins in cells can cause a variety of degenerative diseases, including Alzheimer's disease, ALS, Parkinson's disease and other diseases. At present, the pathogenesis of the above diseases is still unclear, mainly due to the lack of imaging detection tools for the aggregated proteome in cells. Therefore, the visualization and component analysis of the aggregated proteome in the cellular environment is of great research significance for improving cell stress and finding potential treatments.

[0003] The current development of intracellular aggregated protein imaging probes mainly focuses on non-covalent probes and fluorescent labeling technology. Compared with non-covalent probes, the development of covalent fluorescent probes is very rare. And the reactive functional groups are limited to maleimide, aldehyde and 1,4-Michael acceptor.

[0004] Therefore, the development of new covalent fluorescent probes for imaging aggregated proteins in cells has important scientific significance and clinical value for studying diseases caused by protein aggregation. Summary of the invention

[0005] According to one aspect of the present application, a class of molecules capable of covalently capturing aggregated proteins is provided. When such molecular proteins are misfolded, denatured, and aggregated, the molecules can target and bind to aggregated proteins and covalently bind. The above-mentioned properties of the fluorescent molecules can be used for in situ imaging and component analysis of aggregated proteomes in living cells. The molecules of the invention can be used for in situ imaging and component analysis of aggregated proteins in living cells by targeting and recognizing aggregated proteins and covalently binding.

[0006] According to one aspect of the present application, a type of targeted covalently linked aggregated protein probe is provided, wherein the targeted covalently linked aggregated protein probe has a structure as shown in Formula I;

[0007]

[0008] R1 is selected from at least one of dimethylamino, diethylamino, julolidine, C1-C5 alkoxy, cyano, nitro, ester, and hydrogen;

[0009] R2 is selected from at least one of a C1-C5 alkyl group, a C1-C5 alkoxy group, a cyano group, an ester group, and hydrogen.

[0010] According to another aspect of the present application, a method for preparing the above-mentioned targeted covalently linked aggregated protein probe is provided, and the preparation method comprises the following steps:

[0011] (1) reacting a mixture containing a compound represented by formula II, p-hydroxybenzaldehyde, solvent I, and Lewis acid to obtain an intermediate product;

[0012] (2) reacting a mixture containing the intermediate product, the compound represented by formula III, solvent II, and an alkaline substance to obtain the targeted covalently linked aggregated protein probe;

[0013]

[0014] R1 is selected from at least one of dimethylamino, diethylamino, julolidine, C1-C5 alkoxy, cyano, nitro, ester, and hydrogen;

[0015] R2 is selected from at least one of a C1-C5 alkyl group, a C1-C5 alkoxy group, a cyano group, an ester group, and hydrogen.

[0016] Optionally, the intermediate product has a structure shown in Formula IV;

[0017]

[0018] R1 is at least one selected from dimethylamino, diethylamino, julolidine, C1-C5 alkoxy, cyano, nitro, ester, and hydrogen.

[0019] Optionally, the molar ratio of the compound represented by formula II to p-hydroxybenzaldehyde is 1:1-10.

[0020] Optionally, the molar ratio of the compound represented by Formula II to p-hydroxybenzaldehyde is selected from any value of 1:1, 1:2, 1:5, 1:7, 1:10 or a range between the above two values.

[0021] Optionally, the molar ratio of the compound represented by formula II to the solvent I is 1:1 to 1:10.

[0022] Optionally, the molar ratio of the compound represented by formula II to the Lewis acid is 1:0.05 to 1:1.

[0023] Optionally, in step (1), the solvent I is selected from at least one of 1,4-dioxane, N,N-dimethylformamide, tetrahydrofuran, and dimethyl sulfoxide.

[0024] Optionally, the Lewis acid is selected from at least one of zinc chloride, aluminum chloride, titanium tetrachloride, iron chloride, boron trifluoride and niobium pentachloride.

[0025] Optionally, in the step (2), the molar ratio of the intermediate product to the compound represented by formula III is 1:1.0 to 2.0.

[0026] Optionally, the molar ratio of the intermediate product to the solvent II is 1:1 to 1:10.

[0027] Optionally, the molar ratio of the intermediate product to the alkaline substance is 1:1 to 1:5.

[0028] Optionally, in step (2), the alkaline substance is selected from at least one of potassium tert-butoxide, sodium tert-butoxide, lithium tert-butoxide, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, and cesium carbonate.

[0029] Optionally, in step (2), the solvent II is selected from at least one of 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran and dimethyl sulfoxide.

[0030] Optionally, in step (1), the temperature of reaction I is 30 to 150° C., and the time of reaction I is 12 to 30 hours.

[0031] Optionally, the temperature of the reaction I is selected from any value of 30°C, 50°C, 80°C, 100°C, 120°C, 150°C or a range therebetween.

[0032] Optionally, the reaction time I is selected from any value among 12h, 15h, 18h, 20h, 24h, 28h, 30h or a range between any two of the above.

[0033] 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.

[0034] Optionally, the temperature of the reaction II is selected from any value of 25°C, 50°C, 60°C, 70°C, 80°C or a range therebetween.

[0035] Optionally, the time of the reaction II is selected from any value of 12h, 15h, 18h, 20h, 24h or a range between any two of the above.

[0036] According to another aspect of the present application, there is provided the use of the above-mentioned targeted covalently linked aggregated protein probe in intracellular aggregated protein imaging.

[0037] The probe of the present application has the characteristic of covalently labeling aggregated proteins in cells and can be used for imaging aggregated proteins in cells, thus making up for the lack of covalent fluorescent probes in the field for studying protein aggregation.

[0038] Most of the existing fluorescent probes used for imaging aggregated proteins in living cells are non-covalent probes. Reports on the use of covalent probes for imaging aggregated proteins in cells are very rare. The present invention designs and synthesizes a series of probes that target covalently linked aggregated proteins. Such molecules can selectively bind to aggregated proteins and covalently link with them. Such covalent fluorescent probes can be used for in situ imaging of aggregated proteomes in living cells. The invention provides a portable and efficient method for detecting aggregated proteins in living cells.

[0039] In the present application, C1 to C5 refer to the number of carbon atoms contained in the group.

[0040] In the present application, the term "alkyl" refers to a group formed by losing any hydrogen atom from an alkane compound molecule.

[0041] In the present application, the terms "dimethylamino", "diethylamino" and "julolidine group" refer to a group formed by losing any hydrogen atom from the molecules of dimethylamine, diethylamine and julolidine compounds.

[0042] The beneficial effects of this application include:

[0043] 1) The probe provided in the present application can undergo a covalent reaction with aggregated proteins and be used to capture aggregated proteins in cells.

[0044] 2) The probe provided in this application can realize in situ imaging of aggregated proteomes in cardiomyocytes.

[0045] 3) The probe provided in this application can be used to analyze the proteomic aggregation in myocardial tissue sections. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The probe molecule in Test Example 1 of the present application covalently labels the aggregated protein.

[0047] Figure 2 This is a graph showing the probe molecule in Test Example 2 of the present application being used for imaging and analyzing the aggregated protein composition of cardiomyocytes under the stimulation of doxorubicin.

[0048] Figure 3 This is an imaging analysis diagram of myocardial tissue sections of mice after administration of doxorubicin using the probe molecule in Test Example 3 of this application. DETAILED DESCRIPTION

[0049] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0050] Unless otherwise specified, the raw materials in the examples of the present application were purchased through commercial channels and used directly without treatment, and the instruments and equipment used adopted the schemes and parameters recommended by the manufacturers.

[0051] The nuclear magnetic resonance data in the examples of this application were obtained using a Bruker AVANCE III 700MHz nuclear magnetic resonance spectrometer;

[0052] The confocal fluorescence microscope used in this application example is Olympus FV1000 FluoView TM Confocal microscope.

[0053] The mass spectrometer model used in the examples of the present application is Orbitrap Exploris 480mass spectrometer.

[0054] The targeted covalently linked aggregated protein probe of the present application is prepared by the following preparation method:

[0055] (1) adding compound III and solvent I to compound II, reacting under the action of Lewis acid to obtain intermediate product I;

[0056] The compound II has a structure shown in formula II;

[0057]

[0058] wherein R1 is selected from at least one of dimethylamino, diethylamino, julolidine, C1-C5 alkoxy, cyano, nitro, ester, and hydrogen;

[0059] The compound III has a structure shown in formula III;

[0060]

[0061] (2) adding compound IV, a base and solvent II to the intermediate product I, reacting, quenching, extracting, and separating by column chromatography to obtain the target product;

[0062] The intermediate product I has the structure shown in IV;

[0063]

[0064] The compound IV has a structure shown in formula V;

[0065]

[0066] Among them, R2 is selected from at least one of C1~C5 alkyl, C1~C5 alkoxy, cyano, ester and hydrogen.

[0067] Example 1

[0068] Compound II (1.07 g, 3.6 mmol) and compound III (2.2 g, 18 mmol) were dissolved in 1,4-dioxane (10 mL), followed by the addition of zinc chloride (360 μL, 0.36 mmol). The mixture was heated to 90°C and stirred overnight. Dichloromethane was added for extraction, the organic phase components were collected and concentrated, and finally purified by column chromatography to obtain intermediate I (883.7 mg, yield: 61%).

[0069] The synthetic route of the present embodiment is as follows:

[0070]

[0071] Example 2

[0072] Intermediate I (200 mg, 0.5 mmol) and cesium carbonate (289 mg, 0.74 mmol) were dissolved in N,N-dimethylformamide (5 mL), and then compound V (39 μL, 0.5 mmol) was added and heated to 75°C with stirring for 3 h. After the reaction was completed, ethyl acetate was added for extraction, the organic phase components were collected and concentrated, and finally purified by column chromatography to obtain the final product P1 (77.9 mg, yield: 34%).

[0073] P1 was tested using a nuclear magnetic resonance spectrometer, and liquid chromatography high-resolution time-of-flight mass spectrometry Q-TOF 6540 was used for structural characterization and purity determination. The results are as follows: 1 H-NMR (700MHz, CDCl3) δ8.17(d,J=8.4Hz,2H),8.01(d,J=15.6Hz,1H),7.61-7.56(m,2H),7.13(s,1H),6.96(d,J=8.7Hz,2H),6.77-6.73( m,3H),4.29(m,1H),4.00(m,1H),3.91(t,J=6.0Hz,2H),3.61(t,J=5.9Hz,2H),3.40-3.36(m,1H),3.08(s,6H),2.93(m,1H),2.78(m,1H). 13 C-NMR (176MHz, DMSO-d6) δ170.8,160.0,156.0,151.8,139.2,135.1,134.8,129.6,129.2,129.1 ,123.0,115.2,112.0,111.5,110.9,69.0,50.4,50.2,44.8,40.2,39.7.HRMS(m / z)Anal.Calc'd for C 25 H 26 N6O3(M+H) +:459.2139,Found(M+H) + :459.2154.

[0074] The synthetic route of the present embodiment is as follows:

[0075]

[0076] Test Example 1

[0077] P1 is used to covalently label aggregated proteins in vitro

[0078] Mut-DHFR (50uM) and P1 (20uM) were incubated under the following three different conditions:

[0079] Condition 1: Incubate in PBS buffer at 25°C for 10 min;

[0080] Condition 2: incubation at 37°C for 2 h in pH 4.4 buffer;

[0081] Condition 3: Place the rotor in a pH 6.23 buffer and incubate at 800 rpm at 25°C for 2 h;

[0082] After the incubation time, add an equal amount of click reagent (1300uL 1.7mM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), 440uL 50mM copper sulfate, 440uL 50mM tris(2-carbonylethyl)phosphine hydrochloride, 10.8uL 50mM tetramethylrhodamine-azide) to the mixture and incubate on a shaker for 1h, then add 4 times the volume of acetone and incubate overnight at 4°C. After the incubation is completed, centrifuge at 13000rpm to remove the supernatant, and resuspend the precipitate with 4% sodium dodecyl sulfate (SDS) solution to the volume before adding the click reagent. Add 1 / 5 volume of 5x loading buffer solution and heat at 95°C for 5min. Add the heated samples into 12% SDS glue in turn, run the glue at 200V for 45 minutes, and stop when the samples are 1cm away from the bottom edge of the glue plate, and turn off the power.

[0083] After the electrophoresis, remove the SDS gel from the gel plate and place it on a Tanon 2599BR gel imager for fluorescence imaging at 308nm. After imaging, place it in Coomassie Brilliant Blue staining solution (each 100mL contains 0.25g Coomassie Brilliant Blue R250, 45mL methanol, 10mL acetic acid, and 45mL deionized water). Place it on a shaker at room temperature and shake at low speed for 6 hours to fully stain. Take out the SDS gel stained with Coomassie Brilliant Blue and place it in a destaining solution (200mL ethanol, 400mL acetic acid, and dilute to 4L with deionized water). Place it on a shaker at room temperature and shake at low speed for 24 hours. After destaining is complete, place the destained SDS gel in a Tanon 2599BR gel imager for imaging. Figure 1 As shown, from Figure 1 It can be seen that the P1 probe does not covalently react with correctly folded proteins, but selectively covalently reacts with aggregated proteins.

[0084] Test Example 2

[0085] P1 is used for imaging of aggregated proteomes in cardiomyocytes stimulated by doxorubicin

[0086] H9C2 cells were cultured until the cell density reached 80%. Experimental group: Doxorubicin (1 μM) and P1 (10 μM) were added to the culture medium and cultured for 24 hours. Control group: P1 (10 μM) probe was added and cultured for 24 hours. Nuclear staining reagent Hoechst33342 was added half an hour before imaging. The morphology and location of aggregated proteins in living cells were identified by confocal fluorescence microscopy. Figure 2 As shown, from Figure 2 It can be seen that cardiomyocytes form aggregated proteomes under the stimulation of doxorubicin, and the P1 probe can be used for intracellular imaging.

[0087] Test Example 3

[0088] P1 is used for imaging analysis of myocardial tissue slices after administration of doxorubicin in mice

[0089] Mouse type: 6-8 weeks old male Balb / C mice. The mice were divided into control group and experimental group. The experimental group: a single intraperitoneal injection of 15 mg / kg doxorubicin; the control group: an injection of an equal amount of PBS solution. No other treatment was performed during the period and the mice were ensured to have sufficient food and water. 72 hours after the injection, the mice were anesthetized by inhalation of sevoflurane and the eyeballs were removed to collect blood. After the blood was collected, the mice were killed by dislocation of the neck and the hearts were removed.

[0090] Mouse heart tissue treated with doxorubicin was fixed in OCT embedding medium. Using a LEICACM1950 cryostat, the OCT-embedded heart tissue was cut into 6 μm thick tissue sections at -20°C. The cut tissue sections were placed in a Coplin jar containing acetone in a 4°C refrigerator for 10 minutes. After 10 minutes, the slides were removed from the acetone and placed in a slide box and stored at -20°C. The slides to be stained were taken from -20°C to 4°C, rewarmed for 30 minutes, and then placed at room temperature to completely rewarm for 30 minutes. The rewarmed tissue sections were soaked in PBS for 5 minutes, and repeated 3 times. Using a PAP pen, circle the range of the tissue section. Use a small amount of P1 (10 μM) solution to stain the tissue area and stain it in the dark for 1 hour. Soak the tissue sections in PBS for 5 minutes. Use 2 μg / mL Hoechst 33342, drop it on the tissue, and stain the nucleus for 15 minutes. Soak the tissue sections in PBS for 5 minutes. Fluorescence imaging of tissue sections using the Olympus FV1000FluoView TM Confocal microscopy was used to collect the images. The nuclear staining fluorescence was observed with a violet laser (405 nm). The fluorescence of P2 staining in the cell environment was observed with a green laser (488 nm). Figure 3 As shown, from Figure 3 It can be seen that aggregated proteomes can be observed in tissue sections of mice treated with doxorubicin, and the P1 probe can specifically identify them.

[0091] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the 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 targeted covalently linked aggregated protein probe, characterized in that: The targeted covalently linked aggregated protein probe has a structure shown in Formula I; R1 is selected from at least one of dimethylamino, diethylamino, julolidine, C1-C5 alkoxy, cyano, nitro, ester, and hydrogen; R2 is selected from at least one of a C1-C5 alkyl group, a C1-C5 alkoxy group, a cyano group, an ester group, and hydrogen.

2. The method for preparing the targeted covalently linked aggregated protein probe according to claim 1, characterized in that: The preparation method comprises the following steps: (1) reacting a mixture containing a compound represented by formula II, p-hydroxybenzaldehyde, solvent I, and Lewis acid to obtain an intermediate product; (2) reacting a mixture containing the intermediate product, the compound represented by formula III, solvent II, and an alkaline substance to obtain the targeted covalently linked aggregated protein probe; R1 is selected from at least one of dimethylamino, diethylamino, julolidine, C1-C5 alkoxy, cyano, nitro, ester, and hydrogen; R2 is selected from at least one of a C1-C5 alkyl group, a C1-C5 alkoxy group, a cyano group, an ester group, and hydrogen.

3. The preparation method according to claim 2, characterized in that: The molar ratio of the compound represented by formula II to p-hydroxybenzaldehyde is 1:1 to 10; Preferably, the molar ratio of the compound represented by formula II to the solvent I is 1:1 to 1:10; Preferably, the molar ratio of the compound represented by formula II to the Lewis acid is 1:0.05 to 1:

1.

4. The preparation method according to claim 2, characterized in that: In the step (1), the solvent I is selected from at least one of 1,4-dioxane, N,N-dimethylformamide, tetrahydrofuran, and dimethyl sulfoxide; Preferably, the Lewis acid is selected from at least one of zinc chloride, aluminum chloride, titanium tetrachloride, iron chloride, boron trifluoride and niobium pentachloride.

5. The preparation method according to claim 2, characterized in that: In the step (2), the molar ratio of the intermediate product to the compound represented by formula III is 1:1.0-2.0; Preferably, the molar ratio of the intermediate product to the solvent II is 1:1 to 1:10; Preferably, the molar ratio of the intermediate product to the alkaline substance is 1:1 to 1:

5.

6. The preparation method according to claim 2, characterized in that: In the step (2), the alkaline substance is selected from at least one of potassium tert-butoxide, sodium tert-butoxide, lithium tert-butoxide, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, and cesium carbonate.

7. The preparation method according to claim 2, characterized in that: In the step (2), the solvent II is selected from at least one of 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, and dimethyl sulfoxide.

8. The preparation method according to claim 2, characterized in that: In the step (1), the temperature of the reaction I is 30 to 150° C., and the time of the reaction I is 12 to 30 hours.

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

10. Use of the targeted covalently linked aggregated protein probe according to claim 1 in intracellular aggregated protein imaging.

Images