Chemical synthesis of trehalose-6-phosphate photoaffinity probes

Abstract

This invention belongs to the fields of chemical synthesis and biotechnology, specifically relating to the synthesis of trehalose-6-phosphate photoaffinity probes. Starting from trehalose, trehalose-6-phosphate probes are synthesized through a four-step chemical process. The preparation method and application of the trehalose-6-phosphate photoaffinity probes provided by this invention have the following advantages compared with existing methods: (1) This invention is the first to synthesize trehalose-6-phosphate probes capable of click reactions, which can be used for labeling and omics identification of trehalose-6-phosphate interacting proteins in biological systems, as well as imaging in cells. (2) The probes of this invention have not been found in existing reports, and have high reaction efficiency, few side reactions, and mild reaction conditions. They can capture trehalose-6-phosphate interacting proteins in plants and image them in living cells, which is an important technical support for their research.

Description

Technical Field

[0001] This invention belongs to the fields of chemical synthesis and biotechnology, specifically relating to the chemical synthesis of trehalose-6-phosphate photoaffinity probes. Background Technology

[0002] Trehalose is a non-reducing disaccharide composed of two glucose molecules linked by α,α-1,1-glycosidic bonds, and its metabolic precursor is trehalose-6-phosphate. The plant endogenous metabolite trehalose-6-phosphate can directly participate as a signaling molecule in multiple physiological processes, including seed germination, seedling growth, stomatal opening and closing, flowering and fruiting, and stress response. Literature reports that in vitro administration of trehalose-6-phosphate photoceramide precursors, through interaction with the core kinase SnRK1, regulates sucrose utilization and distribution, increasing crop yield and improving crop drought resistance (Chemical intervention in plant sugarsignalling increases yield and resilience, Nature, 2016, 540, 574-578). Furthermore, literature reports that trehalose-6-phosphate, through SnRK1, acts on phytochromes and plant growth hormones, ensuring normal plant growth and development and improving plant adaptability to the environment.

[0003] Despite the increasing research on trehalose-6-phosphate in recent years, its low abundance in plants and its synergistic regulation of plant physiological functions with multiple signaling molecules make it difficult to study its function in plants independently. To investigate the mechanism of action of trehalose-6-phosphate, it is crucial to elucidate its interaction with proteins and its target signaling pathways. A panoramic identification strategy of bioactive molecular target proteins based on photoaffinity labeling, bioorthogonal chemistry, and chemical proteomics is a cutting-edge direction in studying small molecule-protein interactions. However, because this approach relies on small molecule probes with high sensitivity, specificity, and no impact on the molecule's function, there is currently a lack of trehalose-6-phosphate probes capable of undergoing photoaffinity labeling and bioorthogonal chemistry reactions. Summary of the Invention

[0004] To address the shortcomings of existing methods, the first objective of this invention is to provide a method for preparing a trehalose-6-phosphate probe; the second objective of this invention is to provide applications of the aforementioned trehalose-6-phosphate probe.

[0005] The technical solution adopted by this invention to solve its technical problem is:

[0006] This invention protects the structure of a class of compounds, as shown in structural formula I.

[0007]

[0008] The synthesis route of Equation I above is as follows:

[0009]

[0010] (1) Trehalose reacts with trimethylchlorosilane and then reacts with potassium carbonate to give compound A;

[0011] (2) Compound A, 5-phenyltetrazole and tert-butanol peroxide react to give compound B;

[0012] (3) Compound B reacts with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and 3-(3-butynylazadipropidin-3-yl)propionic acid to give compound C;

[0013] (4) Compound C reacts with Amberlite 732 to give compound I;

[0014] The present invention also protects a molecular probe, wherein the fluorescent probe is a compound of the aforementioned formula I or a pharmaceutically acceptable salt thereof.

[0015] The molecular probe is prepared in the same manner as the aforementioned compound of Formula I or its pharmaceutically acceptable salt.

[0016] This invention also protects the use of the aforementioned compounds in the preparation of photocrosslinked probes for identifying target proteins.

[0017] As a preferred technical solution of this application, the molecular probe is used to identify target proteins.

[0018] The present invention also protects a detection kit for identifying target proteins, the kit containing a compound of formula I or a pharmaceutically acceptable salt thereof.

[0019] This invention also protects the use of the aforementioned Formula I compound or its pharmaceutically acceptable salt, the aforementioned molecular probe, and the aforementioned kit in the identification of target proteins.

[0020] (1) Arabidopsis thaliana sampling

[0021] Full seedlings grown under long-day conditions for 10 days were sampled on ZT16 on day 10. Upon sampling, the seedlings were carefully removed from the culture medium, placed in clean water to maintain moisture, then dried with absorbent paper, weighed, wrapped in aluminum foil, dispensed, labeled, flash-frozen in liquid nitrogen, and stored at -80℃ for later use.

[0022] (2) Grinding & Protein Extraction

[0023] Grind the sample in a mortar and pestle with liquid nitrogen. After grinding, aliquot each sample into 50mL centrifuge tubes for later use. Note: Liquid nitrogen should be continuously added during grinding to prevent the sample from melting. After removing the sample from the liquid nitrogen, cool it slightly but do not allow it to melt. Then add the lysis buffer (50mM Tris-HCl, pH 8.0, 150mM NaCl, 0.2% Triton-X 100, cocktail) to the sample at a volume / mass ratio of 1:1 (1g sample to 1mL lysis buffer). Incubate on ice for 30min, stirring constantly to promote lysis. After lysis, centrifuge at 20000g for 20min at 4°C. After centrifugation, place on ice and aspirate the supernatant to determine the protein concentration using BCA. Dilute the protein concentration to 2mg / mL with the lysis buffer.

[0024] (3) Trehalose-6-phosphate probe incubation & photocrosslinking

[0025] Take 1.1 mL of sample and add 11 μL of 100 mM T6P probe solution. Incubate at 37°C with shaking for 1 h at 700 rpm. Transfer the sample to a 6-well plate and irradiate with UV light for 5 min (1500 energy) on ice. Transfer the sample to a 15 mL centrifuge tube, add 5.5 mL of methanol / chloroform (4:1) and 3.3 mL of water, mix well, and centrifuge at 15000 g for 15 min at 4°C. Discard the waste liquid. Take 1.1 mL of 0.4% SDS / PBS and add it to an EP tube. Sonicate to disperse the protein clumps evenly. (3) Click reaction

[0026] Take 100 μL of sample and add 10 μL of Click reaction solution A. Click reaction solution A is a mixed solution of 1×TBTA (60 μL, 1.7 mM), CuSO4 (20 μL, 50 mM), TECP (20 μL, 50 mM), and N3-TAMRA (6 μL, 20 mM). After mixing thoroughly, incubate at 29°C with a shaker for 1 h. Add 27.5 μL of protein loading buffer (5×) and incubate at 95°C for 10 min. Run a protein gel.

[0027] Beneficial technical effects

[0028] The present invention provides a method for preparing and applying a trehalose-6-phosphate photoaffinity probe, which has the following advantages compared with the prior art: The probe (compound I) of this application has not been found in the existing reports of related probes, and the synthesis method has high reaction efficiency, few side reactions, mild reaction conditions, good selectivity and stability, and can directly capture and identify interacting proteins in the lysis buffer, which is an important technical support for the functional study of sugar metabolites. Attached Figure Description

[0029] Figure 1The labeling effect of compound I in Arabidopsis thaliana. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to examples. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market.

[0031] The chemicals and solvents were purchased from Bailingwei Company, McLean Company, Bid Company, Annegi Company and Tongguang Company.

[0032] Example 1

[0033] The preparation scheme of compound I provided in this embodiment is as follows:

[0034]

[0035]

[0036] (1) Trehalose (1 g, 2.92 mmol) was dissolved in anhydrous pyridine (6.4 mL) under nitrogen atmosphere, and trimethylchlorosilane (4.6 mL, 29.2 mmol) was slowly added under ice bath conditions. The reaction system was reacted at room temperature for 17 h, and the reaction was detected by thin-layer chromatography. When the starting material was completely eliminated, hexane (50 mL) and ice water (50 mL) were added to the reaction system in sequence. The organic phase was extracted three times with saturated sodium bicarbonate solution (50 mL), and then extracted three times with saturated brine (50 mL). The solution was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The solution was dissolved in methanol (12.6 mL) and dichloromethane (4.2 mL), and potassium carbonate (43.8 mg, 0.35 mmol) was added under ice bath conditions. The reaction was carried out at room temperature for 6 h, and the reaction was quenched by adding 0.075 mL of acetic acid. The solvent was removed by rotary evaporation to obtain compound A (1.58 g, yield 70%). 1 H NMR(400MHz,Chloroform-d)δ4.93(d,J=2.98Hz,2H),3.96–3.84(m,4H),3.78–3.66 (m,4H),3.54–3.42(m,4H),3.61(m,1H),0.19(s,18H),0.17(s,18H),0.15(s,18H).

[0037]

[0038] (2) Compound A (50 mg, 0.06 mmol) and tetrazolium (19.9 mg, 0.13 mmol) were dissolved in anhydrous dichloromethane (0.3 mL) under nitrogen atmosphere. N,N-diethylphosphonamide di-tert-butyl ester (14.4 μL, 0.05 mmol) was slowly added under ice bath conditions, and the reaction was carried out at room temperature for 9 h. Subsequently, 70% peroxytert-butanol aqueous solution (26.8 μL, 0.19 mmol) was added under ice bath conditions, and the reaction was continued under ice bath conditions for 3 h. Thin-layer chromatography was performed. When the starting material had completely disappeared, saturated sodium thiosulfate (5 mL) and saturated sodium bicarbonate (5 mL) were added, and the mixture was stirred at room temperature for 30 min. The organic phase was extracted three times with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The product B (46.7 mg, 75% yield) was purified by silica gel column chromatography. 1 H NMR(400MHz,Chloroform-d)δ4.95(d,J=3.11Hz,1H),4.90(d,J=3.05Hz,1H),4.16–4.07(m,1H),4.07-3.98(m,1H),3.96–3.85(m,4H ),3.74–3.69(m,2H),3.57–3.40(m,4H),1.51(s,9H),1.50(s,9H),0.20(s,9H),0.19(s,9H),0.17(s,18H),0.15(s,9H),0.14(s,9H).

[0039]

[0040] (3) Compound B (32 mg, 0.03 mmol) was added with 4-dimethylaminopyridine and 3-(3-butynylazadipropidin-3-yl)propionic acid (8.2 mg, 0.045 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (9.5 mg, 0.045 mmol) and 4-dimethylaminopyridine (0.8 mg, 0.006 mmol), dissolved in dichloromethane (0.2 mL) under nitrogen protection, and reacted at room temperature for 11.5 h. The reaction was detected by thin-layer chromatography. When the starting material was completely gone, the mixture was filtered, the solvent was removed by rotary evaporation, and the mixture was purified by silica gel column chromatography to obtain compound C (22.8 mg, 62%). 1H NMR(400MHz,Chloroform-d)δ4.94(d,J=3.05Hz,1H),4.89(d,J=3.02Hz,1H),4.35–4.27 (m,1H),4.13-3.98(m,4H),3.91(td,J=9.0,1.9Hz,3H),3.58–3.39(m,4H),2.24–2.14(m ,2H),2.08–2.00(m,3H),1.87–1.80(m,2H),1.71–1.64(t,J=7.3Hz,2H),1.50(s,9H),1. 49(s,9H),0.19(s,9H),0.17(s,9H),0.16(s,9H),0.15(s,9H),0.14(s,9H),0.13(s,9H).

[0041]

[0042] (4) Compound C (10 mg, 3.77 mmol) was dissolved in pure water (1 mL), and 200 mg of Amberlite 732 resin was added. The mixture was reacted at 55 °C for 6 h, and the reaction was detected by thin-layer chromatography. When the starting material was completely eliminated, the mixture was filtered, and the solvent was removed by rotary evaporation to obtain product I. (4.9 mg, 96.5%). 1 H NMR(400MHz,Deuterium Oxide)δ5.22- 5.05(m,2H),4.41–3.93(m,5H),3.91–3.74(m,3H),3.64–3.57(m,2H),3.53–3.30(m,2H),2.33(s,1 H), 2.26 (t, J = 7.37Hz, 2H), 1.99 (t, J = 7.09Hz, 2H), 1.77 (t, J = 7.37Hz, 2H), 1.64 (t, J = 7.48Hz, 2H).

[0043] Example 2

[0044] The resulting compound IV was applied to chemical proteomics experiments to identify target proteins.

[0045] 10^6 cells were placed in six-well plates and cultured for 12 h, followed by incubation with 5 mM compound IV for 2 h. The culture medium was aspirated, the cells were washed three times with PBS, and 1 mL of PBS was added. The six-well plates were placed on ice and irradiated with UV energy of 1500 J for 5 min. The cells were scraped off with a cell scraper and collected into 1.5 mL centrifuge tubes. The cells were centrifuged at 1000 G for 3 min at 4 °C, the waste liquid was discarded, and the cells were flash-frozen in liquid nitrogen.

[0046] Add 100 μL of 0.1% Triton / PBS to each cell tube and place on ice. Sonicate to 30% and sonicate until cells are clear and transparent. Centrifuge at 20000G for 30 min at 4℃. After centrifugation, do not shake the EP tube vigorously; place on ice and aspirate the supernatant for BCA assay. Prepare a standard concentration gradient (0, 0.4, 0.8, 1.2, 1.6, 2.0 mg / mL) according to the BCA assay kit. Incubate at 37℃ for 20 min, then measure the absorbance (595 nm) using a microplate reader and calculate the sample protein concentration. Standardize the protein concentration to 2.0 mg / mL, 100 μL. Prepare the reaction solution: 60 μL of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (1.7 mM), 20 μL of copper sulfate (50 mM), 20 μL of tris(2-carboxyethyl)phosphine (50 mM), and 3 μL of N3-TAMRA (20 mM). Add 10 μL of the reaction solution to each sample, vortex briefly, and react at 29 °C for 1 h using a constant temperature shaker. After the reaction is complete, briefly centrifuge. Add 550 μL of methyl chloride and 330 μL of ddH2O to the protein sample at a protein:methanol:chloroform:water ratio of 1:4:1:3. Centrifuge at 15000 g, 20 °C, for 15 min. Remove the liquid, keeping the protein block intact. Then add 100 μL of 0.4% SDS / PBS and sonicate at 20% power to dissolve the protein. Add 25 μL of 5x loading buffer and boil at 95-333℃ for 5 min. Prepare a 10% gel and run at 90V for 15 min; then run at 120V for 60 min.

[0047] The results of labeling Arabidopsis thaliana lysate with compound I are shown in the appendix. Figure 1 Compound I can bind to interacting proteins and connect to fluorescent groups via a click reaction, and the corresponding bands are detected by fluorescence.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, 2. The chemical preparation method of the compound according to claim 1, characterized in that, (1) Trehalose reacts with trimethylchlorosilane and then reacts with potassium carbonate to give compound A; (2) Compound A, 5-phenyltetrazole and tert-butanol peroxide react to give compound B; (3) Compound B reacts with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4-dimethylaminopyridine and 3-(3-butynylazadipropidin-3-yl)propionic acid to give compound C; (4) Compound C reacts with Amberlite 732 to give compound I; 3. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a photoaffinity probe for identifying the target protein of the endogenous metabolite trehalose-6-phosphate.

4. A molecular probe, characterized in that, The molecular probe is the compound described in claim 1.

5. The molecular probe according to claim 4, characterized in that, The molecular probe is used to identify target proteins of endogenous metabolites.

6. A detection kit for identifying target proteins, characterized in that, The kit contains the compound of any one of claims 1-2 or a pharmaceutically acceptable salt thereof.

7. The use of the compound of claim 1 or a pharmaceutically acceptable salt thereof, the molecular probe of claim 4, and the kit of claim 6 in the preparation of products for identifying target proteins.

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