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Small molecule fluorescent probe and preparation method for specific detection and activation of pkm2 protein

A fluorescent probe and specific technology, applied in chemical instruments and methods, fluorescence/phosphorescence, material excitation analysis, etc., to achieve high selectivity, intuitive and stable detection, and high sensitivity

Active Publication Date: 2022-03-08
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no invention of fluorescent probes for PKM2 protein

Method used

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  • Small molecule fluorescent probe and preparation method for specific detection and activation of pkm2 protein
  • Small molecule fluorescent probe and preparation method for specific detection and activation of pkm2 protein
  • Small molecule fluorescent probe and preparation method for specific detection and activation of pkm2 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the synthesis of TEPC466

[0029] EDC (46mg, 0.24mmol), HOBt (36.48mg, 0.24mmol), a mixture of coumarin-3-carboxylic acid (38mg, 0.2mmol) was dissolved in DMF (10mL), stirred at room temperature for about half an hour, and then TEPP46 (74.4 mg, 0.2 mmol) was added and stirred for 4 hours. The mixture was poured into 50 mL of water, and extracted with EtOAc (20 mL×3). The combined organic layers were first washed with saturated NH 4 Cl (20mL×2) washed, followed by brine (20mL), washed with anhydrous Na 2 SO 4 Dry, filter and concentrate. It was purified by silica gel column chromatography to obtain TEPC466 as a yellow solid.

[0030]

Embodiment 2

[0031] Example 2: AIE characteristics of TEPC466

[0032] As attached to the manual figure 1 As shown, under the ultraviolet light with an excitation wavelength of 365nm, we found that solid TEPC466 emits strong yellow fluorescence, but TEPP46 does not. The low solubility of TEPC466 can lead to the precipitation of fluorescent features via excited-state intramolecular proton transfer. As a compound with AIE properties, TEPC466 can emit strong fluorescence in the solid state, probably because of the restricted rotation of the carbon-carbon bond.

[0033] In order to further prove that TEPC466 has AIE characteristics, we prepared TEPC466 stock solution with dimethyl sulfoxide (DMSO) at a concentration of 10 mM, and studied the fluorescence emission spectrum of TEPC466 (10 μM) in tetrahydrofuran (THF) / H2O system. As attached to the manual figure 2 As shown, using a Shimadzu RF6000 fluorescence spectrophotometer, at the maximum excitation wavelength of 310nm, set the excitatio...

Embodiment 3

[0034] Example 3: His-Tagged-PKM2 plasmid transfection and protein purification

[0035] at 37°C with 5% CO 2 293T cells (Cell Bank, Chinese Academy of Sciences) were cultured in DMEM (Gibco) medium containing 10% fetal bovine serum (Biological Industries) and 1% penicillin / streptomycin (Gibco) in a cell culture incubator. When the cell coverage in the Petri dish reaches about 70%, use Lipofectamine according to the manufacturer's instructions TM 3000 transfection reagent (Invitrogen), the His-Tagged-PKM2 plasmid (EX-Z7438-M01, GeneCopoeia) was transfected into 293T cells in antibiotic-free DMEM medium. 24 hours after the 293T cells were transfected with the plasmid, the His-Tagged-PKM2 protein was purified using the His-Tagged Protein Nickel Column Purification Kit (TaKaRa), and then the purified His-Tagged-PKM2 protein was purified with the BCA protein concentration assay kit (Beyotime). The concentration of PKM2 protein was determined, and the protein was stored in a -80°C...

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Abstract

The invention discloses a small molecule fluorescent probe for specifically detecting and activating PKM2 protein and a preparation method thereof. Dissolve the mixture of EDC, HOBt, and coumarin-3-carboxylic acid in DMF, add TEPP46 and stir, and obtain solid TEPC466 after dehydration condensation reaction. The fluorescent probe has the property of aggregation-induced luminescence. After specifically binding to the PKM2 protein, it induces PKM2 to form a tetramer to aggregate the dispersed probes. The photochemical effect is significantly enhanced, and the sensitivity is high. It is not susceptible to other components in the biological system during the detection process. Interfering with high selectivity for PKM2 protein. The probe has low cytotoxicity and good biocompatibility, and can be used for imaging detection and specific targeting of PKM2 protein in cells. As the quantitative and qualitative detection and agonist of PKM2 protein target, the probe provides a valuable method for the integration of clinical diagnosis and treatment.

Description

technical field [0001] The invention relates to a chemical probe and a preparation method, in particular to a small molecular fluorescent probe and a preparation method for specifically detecting and activating PKM2 protein. Background technique [0002] Pyruvate kinase (PK) is the key rate-limiting enzyme that catalyzes the last step in the glycolytic pathway, which catalyzes the transfer of phosphate groups from phosphoenolpyruvate (PEP) to adenosine diphosphate (Adenosine diphosphate). diphosphate, ADP) to generate adenosine triphosphate (ATP) and pyruvate. [0003] PK has four isozyme forms, namely PKM1, PKM2, PKL and PKR. Each isozyme has unique tissue expression specificity. PKL, PKR, and PKM1 all exist as stable tetramers with high metabolic enzyme activity, whereas PKM2 subunits can form tetramers and dimers. Dimeric PKM2 has a higher Km value for the substrate PEP than the tetrameric form of PKM2 and is inactive at physiological concentrations of PEP. High expre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D495/14C09K11/06G01N21/64
CPCC07D495/14C09K11/06G01N21/6428C09K2211/1066C09K2211/1088C09K2211/1007
Inventor 郝海平王东李春朦徐小为
Owner CHINA PHARM UNIV
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