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A fluorescent probe targeting mitochondrial thioredoxin reductase and its preparation method and application

A thioredoxin and fluorescent probe technology, applied in the field of fluorescent probes, can solve the problems of low probe sensitivity and selectivity, insufficient for in vivo test research, easy fluorescence quenching, etc., and achieve the effect of fluorescent imaging Good, excellent inhibitory effect, high yield effect

Active Publication Date: 2022-02-25
THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported probes also have the disadvantages of low sensitivity and selectivity, prone to fluorescence quenching, and short emission wavelengths, which are not enough for in vivo research, such as TR-green (Huang, L. et al., A furanylacryl conjugated coumarin as an efficient inhibitor and a highly selective off-on fluorescent probe for covalent labeling of thioredoxin reductase. Chemical Communications 2014,50,6987-90)
In addition, although the reported molecular probes can realize the imaging and diagnosis of thioredoxin reductase in cells, they do not have the function of treatment.

Method used

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  • A fluorescent probe targeting mitochondrial thioredoxin reductase and its preparation method and application
  • A fluorescent probe targeting mitochondrial thioredoxin reductase and its preparation method and application
  • A fluorescent probe targeting mitochondrial thioredoxin reductase and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Preparation of fluorescent probe Cur-fy

[0056] The preparation process of fluorescent probe Cur-fy is as follows:

[0057]

[0058] Step 1: Acetylacetonate (5 g, 50 mmol) is dissolved in ethyl acetate under the protection of boron oxide (2.5 g, 36 mmol, 0.7 eq.), Stirred at 70 degrees for 30 min. 5-methyl-2-furan formaldehyde (1.1 g, 10 mmol), bicrate (2.3 g, 10 mmol) was added for 30 min. EtOAc (EtOAc) ), The dropwise increased to 100 degrees, and the reaction was 5 h. Calculation to room temperature, add 1N HCl to 3, ethyl acetate extraction, dry dry sodium sulfate with anhydrous sodium sulfate, ethyl acetate, solid column chromatography purified yellow solid target compound intermediate A1 1.5 g, production The rate is 81%.

[0059] Intermediate A1: 1 H NMR (400MHz, CDCL 3 Δ15.40 (S, 1H), 7.36-7.29 (m, 1H), 6.52-6.39 (m, 1H), 6.28 (D, J = 15.54 Hz, 1H), 6.10-6.16 (m, 1H), 5.59 (S, 1H), 2.35 (S, 3H), 2.14 (s, 3h) .hrms (TOF) Calculate for C 11 Hide 12 O 3 [M...

Embodiment 2

[0066] Example 2 Determination of ultraviolet absorption spectroscopy and fluorescence emission spectrum of fluorescent probe Cur-fy

[0067] The fluorescent probe Cur-fy ultraviolet maximum feature absorption peak is 440 nm, and the fluorescent emission wavelength is 540 nm, such as image 3 Indicated. In further studies, it was found that after the addition of a sulfur oxygen reductase and a fluorescent probe, fluorescence was significantly enhanced at 540 nm, such as Figure 4 Indicated.

[0068] (1) Determination method of ultraviolet absorption spectrum of fluorescence probe

[0069] Enzyme label, Model: Synergy H1, Biotek, USA. Potassium phosphate buffer (PBS) formulated: sodium chloride (NaCl), 8 g; potassium chloride (KCl), 0.2 g; sodium hydrogen phosphate (NA 2 HPO 4 ), 1.44 g; potassium dihydrogen phosphate (kh 2 PO 4), 0.24 g, adjusting pH 7.4, deposit 1L, high pressure steam sterilization, and preservation room temperature.

[0070] Experimental method: 100 μl of Cur-fy ...

Embodiment 3

[0074] Example 3. Determination of the inhibitory activity of thioxidin reductase

[0075] Fluorescent probe Cur-fy inhibits the activity of sulfur oxygen reductase activity assay with DTNB method, Cur-fy inhibitory activity IC 50 It is 2.48μm ( Figure 5 ), Indicating that Cur-fy has a better inhibitory effect on the sulfur oxygen reductase.

[0076] Experimental method: Preparation of the buffer solution: Take 200 ul of 1 M potassium phosphate solution, 0.2 ml of 500 mM EDTA solution, 20 ul of 20 mg of NADPH, 10 ul 48 mm NADPH, added to 1.73 ml of double steamed water, mix. 35.5 ul Test buffer solution was added to 384-well plates, and a sulfur oxygen reductase (T9698, Sigma) 0.3 ul, 1 ul Cur-Fy (final concentration is 0.3125, 10.625, 1.25, 2.5, 5, 10 μm) was incubated for 5 min, Finally, 3.2 ul 63 mm DTNB, 412 nm reads the absorbance, and records once every 10s, record 2 min, calculated the inhibition of the sulfur oxygen reductase according to the kinetic curve slope.

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Abstract

The invention discloses a thioredoxin reductase fluorescent probe targeting mitochondria, a preparation method and application thereof. Wherein the structure of the fluorescent probe is shown in formula (I) or a pharmaceutically acceptable salt thereof. Cur-Fy of the present invention is covalently bound to selenocysteine ​​at the carbon terminal of thioredoxin reductase through Michael addition reaction, and has good fluorescence imaging effect, high selectivity, high sensitivity, and can be detected qualitatively and quantitatively Thioredoxin reductase and its family enzymes, isoenzymes, and structurally similar enzymes are especially suitable for the qualitative and quantitative detection of thioredoxin reductase in living tumor cells; Excellent inhibitory effect, can be prepared as a thioredoxin reductase inhibitor for application, and has anti-tumor effect; the mitochondrial-targeted thioredoxin reductase fluorescent molecular probe provided by the present invention has fewer synthesis steps and high yield. The cost is low and is suitable for industrialized production.

Description

Technical field [0001] In the field of fluorescent probes in the present invention, more specifically, a fluorescence probe having a targeted mitochondria and a preparation method and application thereof. Background technique [0002] Thioredoxin Reductase (TRXR) belongs to the oxidative enzyme family member of the pyridinucleotide dioxin compound, which is a natural substrate, thioredoxin, TRX, and reduced nicotinoid diblantin phosphate. NICOTINAMIDE ADENINEDINUCLETIDE Phosphate, NADPH, together into a TRX system. The TRX system (main enzyme system for adjusting ROS level) has an important role in resisting cell proliferation, withstanding oxidative stress, maintaining normal oxidative balance between cells, and has an important role in the development of tumors. As the core member of the system, the role of the system is the primary nicotalamide diblantin phosphate phosphate (NADP +) of the mitochondrial respiration to NADPH; the other aspect is to be restored from the electron...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D405/10C09K11/06G01N21/64A61P35/00
CPCC07D405/10C09K11/06G01N21/6428G01N21/6458A61P35/00G01N2021/6417C09K2211/1029C09K2211/1088C09K2211/1007
Inventor 梁宝霞王邦
Owner THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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