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Permeable membrane fluorescent small-molecule probe focused on proteases in cysteine proteinase family C1, method for synthesizing and purifying permeable membrane fluorescent small-molecule probe and application of permeable membrane fluorescent small-molecule probe

A technology of cysteine ​​protease and small molecule probe, which is applied in the field of biomolecules, can solve the problems of small range of use, slow membrane penetration speed, and limited detection spectrum, and achieves reasonable structure, simple synthesis method, and low cost. low effect

Active Publication Date: 2011-09-07
SINGULPROBE NANJING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the only fluorescent probes that can realize cathepsin labeling in living cells (in Vivo) are boron fluorescein (BODIPY) dyes or Cyanine dye series (CY3, CY5) probes, but their membrane penetration speed is slow and cannot It can realize real-time imaging, and at the same time, due to the limitation of dye types, the detection spectrum is limited, the range of use is small, and the price of Cyanine 5 dye is very expensive, which seriously limits its popularization and application
In addition, because this probe cannot carry markers such as biotin, the enrichment of target proteins and proteomics research can be realized.

Method used

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  • Permeable membrane fluorescent small-molecule probe focused on proteases in cysteine proteinase family C1, method for synthesizing and purifying permeable membrane fluorescent small-molecule probe and application of permeable membrane fluorescent small-molecule probe
  • Permeable membrane fluorescent small-molecule probe focused on proteases in cysteine proteinase family C1, method for synthesizing and purifying permeable membrane fluorescent small-molecule probe and application of permeable membrane fluorescent small-molecule probe
  • Permeable membrane fluorescent small-molecule probe focused on proteases in cysteine proteinase family C1, method for synthesizing and purifying permeable membrane fluorescent small-molecule probe and application of permeable membrane fluorescent small-molecule probe

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The structure of the transmembrane fluorescent small molecule probe I for proteases of the cysteine ​​protease C1 family is:

[0040]

[0041] R is rhodamine, and the chemical reaction formula of its synthetic route is:

[0042]

[0043] The MS detection report of the transmembrane fluorescent small molecule probe I targeting cysteine ​​protease C1 family proteases is as follows figure 1 shown.

[0044] Co-localization experiment of transmembrane fluorescent small molecule probe I and lysosomal dyes targeting cysteine ​​protease C1 family proteases:

[0045] HeLa cells (2x10 4 cells / well) were seeded in Lab-Tek I 8-well plates. 37°C, 5% CO 2 Incubate overnight. During the experiment, the culture medium was aspirated, and the cells were washed twice with PBS solution. Prepare 300 μL of serum-free DMEM high glucose solution containing red transmembrane fluorescent small molecule probe (10 μM), and add the mixed medium into the Lab-Tek I 8-well plate. 37°C, 5...

Embodiment 2

[0049] The structure of the transmembrane fluorescent small molecule probe II for proteases of the cysteine ​​protease C1 family is:

[0050]

[0051] R is 5-carboxyfluorescein, and the chemical reaction formula of its synthetic route is:

[0052]

[0053] The MS detection report of the transmembrane fluorescent small molecule probe II targeting cysteine ​​protease C1 family proteases is as follows figure 2 shown.

[0054] Time-series experiment of laser confocal scanning imaging of cells incubated with transmembrane fluorescent small molecule probe II targeting proteases of cysteine ​​protease C1 family:

[0055] HeLa cells (2x10 4 cells / well) were seeded in Lab-Tek I 8-well plates. 37°C, 5% CO 2 Incubate overnight. During the experiment, the culture medium was aspirated, and the cells were washed twice with PBS solution. 300 μL of serum-free DMEM high glucose solution containing transmembrane fluorescent small molecule probe (10 μM) was prepared, and the mixed m...

Embodiment 3

[0066] The structure of the transmembrane fluorescent small molecule probe III for caspase C1 family protease is:

[0067]

[0068] The chemical reaction formula of its synthetic route is:

[0069]

[0070] The MS detection report of the transmembrane fluorescent small molecule probe III targeting cysteine ​​protease C1 family proteases is as follows image 3 shown.

[0071] Live cell protein labeling experiments:

[0072] About 1.6*107 cells of RAW264.7 were washed twice with PBS, added serum-free DMEM dilution of transmembrane fluorescent small molecule probe (final probe concentration was 10 μM), incubated at 37°C and 5% CO2 for 1 hour, and aspirated Probe solution, add DMEM solution containing 10% fetal bovine serum. The cells were cultured at 37°C and 5% CO2 for 5 hours, washed twice with PBS, and trypsin was added to enrich the cells. After the enriched cells were washed with PBS, RIPA lysate (Beyotime) lysate containing Complete Protease Inhibitor (Roche cockt...

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Abstract

The invention discloses synthesis, purification and application of a permeable membrane fluorescent small-molecule probe focused on proteases in cysteine proteinase family C1, and belongs to the field of biomolecular science. The permeable membrane fluorescent small-molecule probe can mark and image proteases in the cysteine proteinase family C1 in living cells in real time, and can be used for proteomics research on a target proteinase.

Description

technical field [0001] The invention belongs to the field of biomolecules. Background technique [0002] Activity-based probes (activity-based probes, ABPs) are a new generation of functional protein detection probes that have emerged in recent years. They mainly use organic chemistry methods to artificially synthesize targeted small-molecule probes and bind to specific enzyme proteins. , enabling the identification and study of specific enzyme families. These small molecule probes usually consist of three parts: a reactive group that can be covalently cross-linked with the enzyme; a detectable labeling group; and a linker that can link the reactive group and the labeling group together. group. The working principle of ABPs is: small molecule probes with reactive groups and labeling groups interact with the proteome to be studied, and the reactive groups in the small molecule probes can specifically and covalently bind to a certain class of proteins in the proteome. Chara...

Claims

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Application Information

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IPC IPC(8): C09K11/06C07D493/10C07D407/12G01N21/64
CPCY02P20/55
Inventor 张玉慧骆清铭樊锋凯
Owner SINGULPROBE NANJING BIOTECH CO LTD
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