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A fluorescent probe for detecting protein sulfhydrylation and its preparation method and application

A technique of fluorescent probes and groups, which is applied in the field of fluorescent probes for detecting protein thiolation and their preparation, can solve the problems of limited sensitivity, high false positive rate, complicated operation steps, etc., and achieves fast reaction speed and stability. Good, high sensitivity effect

Active Publication Date: 2019-08-27
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have certain shortcomings, including high false positive rate, cumbersome operation steps, limited sensitivity, and inability to realize real-time monitoring in vivo.

Method used

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  • A fluorescent probe for detecting protein sulfhydrylation and its preparation method and application
  • A fluorescent probe for detecting protein sulfhydrylation and its preparation method and application
  • A fluorescent probe for detecting protein sulfhydrylation and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Synthesis of Compound I-2

[0038]

[0039] Dissolve IR780 (1g, 1.5mmol) and 2g sodium acetate in 20ml DMF and react at 80°C for 6 hours. The reaction solution is extracted with ethyl acetate and washed three times with water. Methane / methanol = 20:1 was used as eluent to pass through the column to obtain 600 mg of red solid I-2 with metallic luster, and the yield was 60%. 1 H NMR (400MHz, CDCl 3 )δ8.17(d, J=13.2Hz, 2H), 7.17(dd, J=7.0,5.6Hz, 4H), 6.90(t, J=7.4Hz, 2H), 6.67(d, J=8.0Hz, 2H), 5.46(d, J=13.2Hz, 2H), 3.64(t, J=7.4Hz, 4H), 2.61(t, J=5.7Hz, 4H), 1.88(dd, J=11.9, 6.1Hz, 2H), 1.76(dd, J=14.7, 7.4Hz, 4H), 1.67(s, 12H), 1.00(t, J=7.4Hz, 6H); 13 CNMR (100MHz, CDCl 3 )δ186.4, 162.4, 144.4, 139.7, 132.9, 127.6, 126.5, 121.7, 120.4, 106.7, 92.5, 77.4, 77.0, 76.7, 46.5, 44.1, 28.8, 25.9, 22.6, 19.8, 11.7; ESI-HRMS (m / z ):[M+H] + calculated for HQO(C 36 h 45 N 2 O, M + ):521.3532, found 521.3388.

[0040] Preparation of fluorescent probe I-1

[0041] ...

Embodiment 2

[0043] Embodiment 2, Fluorescence before and after the reaction of fluorescent 1 probe I-1 with different concentrations of sulfur-thiolated papain Variety

[0044] A small amount of fluorescent probe was dissolved in DMSO, and different concentrations of sulfur-sulfhydryl-modified papain were added. The final concentration of the probe was 20 μM. After reacting for 10 minutes, use 520nm excitation to record the solution at the maximum emission wavelength (640nm). Fluorescence intensity, record its fluorescence spectrum, such as figure 1 shown by figure 1 It can be seen that the fluorescent probe I-1 can react with the sulfur-thiolated protein and emit fluorescence.

Embodiment 3

[0045] Embodiment 3, the selectivity of fluorescent probe I-1 to sulfur-thiolated protein

[0046] The fluorescent probes dissolved in DMSO were added to different solutions containing or not containing sulfhydryl-modified papain, from 1 to 21 were only blank group, only sulfhydryl-modified papain, SCN - , SO 3 2- , S 2 o 4 2- , S 2 o 5 2- , S 2 o 3 2- , S-nitroso glutathione, O 2 - 、H 2 o 2 , OCl - , tert-BuOOH, Ala, Arg, His, Me, Ser, Thr, Trp, Tyr, Val. Record the fluorescence intensity of their solutions respectively, such as figure 2 shown by figure 2 It can be seen that the fluorescent probe I-1 is selective for sulfhydrylated proteins.

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Abstract

The invention provides a fluorescent probe for detecting sulfur-thiolation of protein as well as a preparation method and application of the fluorescent probe. The probe takes a heptamethine cyanine type fluorescent dyestuff as a parent body and a chemical structure general formula is shown as a formula (I). The fluorescent probe for detecting the sulfur-thiolation of the protein in mitochondria is synthesized for the first time; the fluorescent probe provided by the invention has good stability and is simple to synthesize and convenient to use; the fluorescent probe only reacts with sulfur-thiolated protein and is not interfered by other compounds containing thiol / sulfur or biological macromolecules; the probe has a subcellular targeting property and can be used for specifically detectingthe sulfur-thiolated protein in the mitochondria. The probe has a wide application prospect in life science and medical researches; the formula (I) is shown in the description.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a fluorescent probe for detecting protein sulfhydrylation, a preparation method and application thereof. Background technique [0002] S-sulfhydration of proteins is a process that depends on hydrogen sulfide (H 2 S) and persulfur compounds (H 2 S n ) reversible protein post-translational modification. h 2 S and H 2 S n It can modify the specific cysteine ​​sulfhydryl group in the protein to undergo sulfhydrylation, change the protein structure, and regulate protein function. It is a new signal transduction regulation method similar to phosphorylation and acetylation. Protein sulfhydryl modification plays a key role in many physiological and pathological processes, including autophagy, oxidative stress, nerve conduction, apoptosis, inflammation, etc. In addition, previous studies have shown that excessive activation or inhibition of protein sulfhydryl modification is clos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D209/12C09K11/06G01N21/64A61K49/00
Inventor 孟文琪肖凯沈璐张浩孙铭学徐庆强赵杰岑金凤陈永春师文文冯雍炜
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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