Rhodamine B targeted lysosome pH fluorescent probe with cysteine ethyl ester structure and application of rhodamine B targeted lysosome pH fluorescent probe

A cystine ethyl ester, fluorescent probe technology, used in pH fluorescent probes and application fields, can solve the problems of affecting monitoring results and increasing lysosome pH, and achieve rapid response, good reversibility, high sensitivity and high sensitivity. selective effect

Inactive Publication Date: 2013-09-25
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some reported rhodamine-based pH probes have been used to monitor the pH in lysosomes, these probes often cause "alkaline effects", that is, after long-term incubation, the pH in lysosomes will be induced. increase, affecting the monitoring results

Method used

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  • Rhodamine B targeted lysosome pH fluorescent probe with cysteine ethyl ester structure and application of rhodamine B targeted lysosome pH fluorescent probe
  • Rhodamine B targeted lysosome pH fluorescent probe with cysteine ethyl ester structure and application of rhodamine B targeted lysosome pH fluorescent probe
  • Rhodamine B targeted lysosome pH fluorescent probe with cysteine ethyl ester structure and application of rhodamine B targeted lysosome pH fluorescent probe

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Experimental program
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Effect test

Embodiment 1

[0038] Synthesis of rhodamine B class-targeted lysosome pH fluorescent probe containing cysteine ​​ethyl ester structure

[0039]

[0040] 1g (2.1mmol) Rhodamine B, 400mg (2.6mmol) POCl 3 Add 15mL of dry 1,2-dichloroethane into a 50mL reaction flask, heat to reflux, and react for 4h. After cooling, the solvent was distilled off under reduced pressure to obtain rhodamine B acid chloride, which was directly used in the next reaction.

[0041] Add 430 mg (2.3 mmol) of L-cysteine ​​ethyl ester hydrochloride to 10 mL of dichloromethane, add 2 mL of triethylamine, stir at room temperature for 30 min, and filter to obtain a dichloromethane solution of cysteine ​​ethyl ester. Cool in an ice bath. The crude rhodamine B acid chloride obtained above was dissolved in 10 mL of dichloromethane, dropped into the dichloromethane solution of ethyl cysteine, stirred for 4 hours under ice bath, and then reacted overnight at room temperature. Concentrate the reaction solution under reduced ...

Embodiment 2

[0047] Prepare 10mL solutions of probe RCE (10μM) at different pH values ​​(ethanol / Britton-Robinson buffer solution, 40mM acetic acid, phosphoric acid, boric acid, 1:4, volume ratio), and perform UV-visible spectrophotometry and fluorescence spectrometry respectively. Photometric test, and draw the curve of the fluorescence intensity at 584nm as a function of the pH value.

[0048] The results show that the probe RCE has a good response to the pH value. After the pH value changes from 7.51 to 3.53, the solution changes from colorless to red and produces fluorescence. The fluorescence intensity is about 150 times stronger, which has a good fluorescence enhancement effect. Can improve signal sensitivity and accuracy ( figure 1 , 2 , 3, 4).

Embodiment 3

[0050] After adding various metal ions to the solution of probe RCE (10μM) with a pH value of 7.2 (ethanol / Britton-Robinson buffer solution, 40mM acetic acid, phosphoric acid, boric acid, 1:4, volume ratio), test its 584nm changes in fluorescence intensity. (excitation wavelength: 563nm; K + and Na + 1mM, Ca 2+ and Mg 2+ 0.1mM, 50μM for other ions)〖 Figure 5 (a)〗.

[0051] After adding various metal ions and bio-related substances to the solution of probe RCE (10μM) with a pH value of 5.4 (ethanol / Britton-Robinson buffer solution, 40mM acetic acid, phosphoric acid, boric acid, 1:4, volume ratio), Test its fluorescence intensity change at 584nm. (excitation wavelength: 563nm; K + and Na + 1mM, Ca 2+ and Mg 2+ 0.1mM, other ions 50μM, Histidine: 0.2mM, Tyrosine: 0.2mM, Arginine: 0.2mM, Glutamic acid: 0.2mM, Lysine: 0.2mM, Threonine: 0.2mM, Glycine: 0.2mM, Glucose: 1mM, H 2 o 2 : 2mM)〖 Figure 5 (b)〗.

[0052] The results show that the response of the probe RCE to t...

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Abstract

The invention discloses a rhodamine B targeted lysosome pH fluorescent probe with a cysteine ethyl ester structure, wherein the rhodamine B targeted lysosome pH fluorescent probe has the structure as shown in a formula (I). Meanwhile, the invention discloses an application of the probe as a living cell lysosome pH fluorescent probe. Experiments show that the probe provided by the invention does not generate fluorescence under the neutral and alkaline conditions, the fluorescence intensity is rapidly enhanced along with the reduction of the pH value of the solution, is up to the maximum value when the pH value is about 4.0 and is enhanced by about 150 times when the pH value ranges from 7.51 to 3.53, and the probe has favorable antijamming capability and reversibility in the presence of various metal ions. An intracellular colocalization experiment and an interlysosome pH regulation experiment prove that the probe can specially mark a lysosome and sensitively monitor the small change of the interlysosome pH value. A cell survival rate experiment shows that the probe is nontoxic to cells, which indicates that the probe disclosed by the invention has an important application value in the aspects of imaging the cells and monitoring the change of the interlysosome pH value.

Description

technical field [0001] The present invention relates to a pH fluorescent probe and its application, in particular to a rhodamine B class targeting lysosome pH fluorescent probe containing cysteine ​​ethyl ester structure and its use as a fluorescent probe for lysosome pH in living cells. needle application. Background technique [0002] Human intracellular pH plays a role in cellular, enzyme and tissue activities such as cell metabolism, signal transduction, cell growth, cell proliferation, apoptosis, autophagy, multidrug resistance, endocytosis and muscle contraction very crucial role. Monitoring pH changes in living cells is also important for studying cellular internalization pathways such as phagocytosis. Abnormal intracellular pH can cause cellular dysfunction and affect the physiological processes of the human body, leading to some serious diseases such as cancer and Alzheimer's disease. [0003] The pH value in human cells is not evenly distributed. The cytoplasm ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C07D491/107G01N33/52G01N21/64
Inventor 赵宝祥苗俊英吕洪水黄淑亚
Owner SHANDONG UNIV
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