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Rhodamine pH fluorescent probe for monitoring mitochondrial autophagy as well as preparation and application thereof

A technology of mitophagy and fluorescent probes, which is applied in the field of pH fluorescent probes, can solve problems such as strong background fluorescence, large cytotoxic side effects, and unsuitability for cell monitoring, and achieve simple detection methods, easy large-scale production, and good cell membranes. The effect of permeability

Active Publication Date: 2020-04-14
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, many fluorescent probes for detecting lysosomal pH changes have been reported in the literature. However, since the excitation and emission wavelengths of these probes are mostly in the ultraviolet and near-visible regions, they overlap with cell autofluorescence, resulting in strong background fluorescence and low ultraviolet light. Light has great toxic side effects on cells, so it is not suitable for long-term monitoring of cells

Method used

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  • Rhodamine pH fluorescent probe for monitoring mitochondrial autophagy as well as preparation and application thereof
  • Rhodamine pH fluorescent probe for monitoring mitochondrial autophagy as well as preparation and application thereof
  • Rhodamine pH fluorescent probe for monitoring mitochondrial autophagy as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 3',6'-Bis(diethylamino)-2-(2-(aminomorpholine)ethyl)spiro[isoindoline-1,9'-xanthene]-3-one (MSO) Preparation and characterization:

[0032]

[0033] In a round-bottomed flask, dissolve the N-p-toluenesulfonic acid ethyl ester derivative of rhodamine B (170mg, 0.62mmol) and N-aminomorpholine (300mg, 0.93mmol) in dichloromethane, and heat to reflux for 12h to The reaction was complete; the system was cooled to room temperature, and the solvent was removed by rotary evaporation under reduced pressure to obtain a crude product; the crude product was purified by silica gel column (eluent by volume ratio of dichloromethane: anhydrous methanol = 20:1) to obtain a light yellow solid as the target compound for monitoring MSO, a rhodamine-based pH fluorescent probe for mitophagy.

[0034] 1 H NMR (600MHz, CD 3 OD-d 4 , Figure 1a )δ(ppm):1.17-1.19(t,12H,-CH 3 -),2.88-3.01(d,2H,-CH 2 -),3.18-3.22(m,4H,-CH 2 -),3.32-3.33(m,2H,-CH 2 -),3.38-3.42(m,8H,-CH 2 -),4.91(s,4H,-...

Embodiment 2

[0038] Dilute the probe to a final concentration of 5 μM with Britton-Robinson buffer at different pH values, fix the excitation wavelength at 574 nm, and record the fluorescence of the fluorescent probe MSO in the DMSO / BR (1 / 99, v / v) system as it changes with pH The emission spectrum changes. As the pH value decreased from 8.0 to 4.8, the fluorescence intensity at 590nm gradually increased ( image 3 ). At the same time, the fluorescent color of the solution changed from colorless to pink ( Figure 4 ). According to the Singmoidal fitting curve calculation of the fluorescence intensity value of the fluorescent probe MSO at 590nm as a function of pH, the pKa value is 5.42 ( Figure 5 ), the pH response linear range is 5.0-6.0, which is very suitable for the detection of pH changes in the weakly acidic environment of lysosomes.

Embodiment 3

[0040] The concentration of the fluorescent probe MSO in Example 1 was maintained at 5 μM, and the probe was investigated in the presence of common ions and amino acids for H + selectivity. Such as Image 6 As shown, in the DMSO / BR (1 / 99, v / v) system, at pH 8.0 and pH 5.0, the fluorescent probe RML has almost no response to the above substances, which proves that the fluorescent probe RML has no response to the H + Has high selectivity. Image 6 The sequence and concentration of the substances in the sequence are: 1, blank; 2, K + (150mM),3,Na + (150mM),4,Mg 2+ (2mM),5,Ca 2+ (2mM),6,Ba 2+ (0.2mM),7,Cu 2+ (0.2mM),8,Fe 2+ (0.2mM),9,Fe 3+ (0.2mM),10,Ni 2+ (0.2mM),11,Zn 2+ (0.2mM),12,Cl - (10mM),13,SO 4 2- (0.2mM),14,SO 3 2- (0.2mM),15,NO - (0.2mM),16,Ac - (0.2mM),17,Al 3+ (0.2mM),18,Ag + (0.2mM),19,Br - (0.2mM),20,Cd 2+ (0.2mM),21,Cu 2+ (0.2mM),22,Mn 2+ (0.2mM),23,Cys(0.1mM),24,GSH(0.1mM),25,Hcy(0.1mM),26,Ala(0.1mM),27,His(0.1mM),28,Arg(0.1 mM),29,Lys(0.1...

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Abstract

The invention relates to the technical field of pH fluorescent probes, in particular to a rhodamine pH fluorescent probe for monitoring mitochondrial autophagy and preparation and application thereof.The invention aims to solve the problems that many fluorescent probes for detecting lysosome pH change are not suitable for long-time monitoring of cells, and lysosome targeting pH fluorescent probespractically used for monitoring mitochondrial autophagy are few and the like. The invention discloses a preparation method, and the method comprises the following steps: dissolving an N-p-toluenesulfonic acid ethyl ester derivative of rhodamine B and N-aminomorpholine in dichloromethane, carrying out heating reflux, carrying out cooling to room temperature, carrying out reduced pressure rotary evaporation to remove a solvent so as to obtain a crude product, and carrying out silica gel column chromatography purifying on the crude product so as to obtain a faint yellow solid which is the pH fluorescent probe. The fluorescent probe has good cell film permeability, can mark lysosome in a targeted manner, realizes visual monitoring of the mitochondrial autophagy process by detecting the changeof the pH of the lysosome, and has potential application prospects in lysosome pH physiology and pathology research.

Description

technical field [0001] The invention relates to the technical field of pH fluorescent probes, in particular to a rhodamine-based pH fluorescent probe for monitoring mitophagy and its preparation and application. Background technique [0002] Autophagy refers to the formation of autophagosomes (autophagosomes) from the double-membrane-wrapped part of the cytoplasm and organelles and proteins that need to be degraded from the ribosome-free attachment area of ​​the rough endoplasmic reticulum. Autolysosomes fuse to form autolysosomes, which degrade their wrapped contents to meet the metabolic needs of the cell itself and the renewal of certain organelles. Mitochondria will produce reactive oxygen species during biological oxidation and energy conversion, and mitochondrial DNA is more prone to mutation than nuclear DNA, so mitochondria are relatively vulnerable organelles. Timely removal of damaged mitochondria in cells plays an important role in maintaining the normal state of...

Claims

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Application Information

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IPC IPC(8): C07D491/107C09K11/06G01N21/64
CPCC07D491/107C09K11/06C09K2211/1033G01N21/6428
Inventor 樊丽王晓东李峰董川
Owner SHANXI UNIV
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