Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A preparation method and application of a bioluminescent probe for detecting pyroglutamate aminopeptidase

A bioluminescence and probe technology, applied in chemiluminescence/bioluminescence, luminescent materials, chemical instruments and methods, etc., can solve the problems of harsh conditions, poor optical probe selectivity, low sensitivity, etc., to achieve low cost, specificity The effect of strong performance and simple preparation method

Active Publication Date: 2022-04-15
WEST CHINA HOSPITAL SICHUAN UNIV
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a bioluminescent probe for detecting pyroglutamate aminopeptidase in organisms and its preparation method and application, so as to solve the problem of poor selectivity, low sensitivity and harsh conditions of existing pyroglutamate aminopeptidase optical probes In order to achieve qualitative and quantitative analysis of pyroglutamate aminopeptidase with high selectivity and high sensitivity

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A preparation method and application of a bioluminescent probe for detecting pyroglutamate aminopeptidase
  • A preparation method and application of a bioluminescent probe for detecting pyroglutamate aminopeptidase
  • A preparation method and application of a bioluminescent probe for detecting pyroglutamate aminopeptidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of bioluminescence probe CX-1 of the present invention

[0038] Bioluminescent probe CX-1 was prepared according to the following synthetic route:

[0039]

[0040] (1) Dissolve Boc-L-pyroglutamic acid (3.0mmol, 500mg) in 10mL of anhydrous acetonitrile, and under the protection of nitrogen, add dropwise 5mL of anhydrous acetonitrile ), isobutyl chloroformate (3.0mmol, 285μL), 2-cyano-6-aminobenzothiazole (3.0mmol, 405mg) were reacted at 25°C for 5h, then spin-dried, extracted, and separated by column chromatography (elution The agent is petroleum ether and ethyl acetate with a volume ratio of 1:5) to obtain solid powder intermediate 3, 1 H NMR (400MHz, DMSO) δ10.81 (s, 1H), 8.76 (d, J = 1.8Hz, 1H), 8.23 ​​(d, J = 9.0Hz, 1H), 7.78 (dd, J = 9.0, 2.0Hz ,1H),4.73(dd,J=9.0,3.5Hz,1H),2.50–2.26(m,3H),1.96(ddd,J=17.4,8.4,4.3Hz,1H),1.36(s,9H); 13 C NMR (101MHz, DMSO) δ174.02, 171.11, 149.40, 148.30, 139.70, 137.23, 135.81, 125.40, 121.22, 114.01, 112....

experiment example 1

[0045] In Vitro Detection of Experimental Example 1 Probe CX-1 Activity

[0046] In a black 96-well plate, add 100 μM probe CX-1 of the present invention, pyroglutamate aminopeptidase solution with different concentration gradients (see details for specific concentrations) image 3 ), with a volume of 100uL each, incubated at 37°C for 30min, then added 50μL of ATP-containing luciferase mixed solution (ATP1.1mg / ml, luciferase 20ug / ml), and each concentration was replicated three times. The holes were then imaged under an intravital imager.

[0047] The result is as image 3 As shown, with the increase of the concentration of pyroglutamate aminopeptidase, the intensity of bioluminescence gradually increased. In the in vitro detection, the bioluminescence intensity and pyroglutamate aminopeptidase showed a good linear relationship within a certain concentration range.

[0048] The above results show that the probe CX-1 of the present invention has good detection sensitivity an...

experiment example 2

[0050] Selective detection of experimental example 2 probes

[0051] In a black 96-well plate, add 100 uL of 100 μM probe CX-1 solution and different analyte solutions (Blank, KCl, ZnCl 2 , CaCl 2 , MgCl 2 , CuCl 2 , NaClO, Glucose, Cys, GSH, Try, Ala, Thr, Lys, Ser, Asp, Vitamin C, Glu, Esterase, Trypsin, Thrombin, V8, PGP (pyroglutamate aminopeptidase); each volume 100uL, 100μM ) were incubated at 37°C for 30 min, 50 μL of ATP-containing luciferase solution (ATP 1.1 mg / ml, luciferase 20 ug / ml) was added to each well, and immediately imaged under an in vivo imager.

[0052] The result is as Figure 4 As shown, among various in vivo enzymes and ionic compounds, only pyroglutamate aminopeptidase caused strong bioluminescence, and other in vivo enzymes and ionic compounds hardly produced obvious bioluminescent signals.

[0053] The experimental results show that the probe CX-1 can specifically select and detect pyroglutamate aminopeptidase without being interfered by other ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a compound represented by formula I. The invention also discloses the use of the compound represented by formula I in preparing bioluminescent probes, especially bioluminescent probes for detecting pyroglutamate aminopeptidase. The probe provided by the invention shows good sensitivity, selectivity and biocompatibility to the target product. The probe provided by the invention can not only detect pyroglutamate aminopeptidase with good linearity and semi-quantitatively in an in vitro environment, but also has the ability to visually detect endogenous pyroglutamate aminopeptidase at the living level. The preparation method of the probe of the present invention is simple, high in yield, low in cost, high in efficiency, strong in specificity, fast and sensitive in substrate detection, and can realize qualitative and semi-quantitative detection and analysis of pyroglutamate aminopeptide at the same time Enzymes are easy to popularize and apply.

Description

technical field [0001] The invention specifically relates to a preparation method and application of a bioluminescent probe for detecting pyroglutamate aminopeptidase. Background technique [0002] Bioluminescence is a kind of chemiluminescence that is ubiquitous in nature and depends on the normal life activities of organisms. Its essence is the interaction between enzymes and substrates, through a series of biological reactions to produce chemical energy, which is released in the form of light energy. Bioluminescence imaging (Bioluminescence imaging) is based on the principle that photons are generated by chemical reactions catalyzed by enzymes in living organisms. The most common bioluminescence system is the firefly luciferase (Luciferase)-luciferin (Luciferin) system, the essence of which is that firefly luciferase catalyzes the substrate luciferin under the condition of energy (ATP) and oxygen. An electronic transition occurs, and the molecule generates photons when ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C07D417/14C09K11/06G01N21/76
CPCC07D417/14C09K11/06G01N21/763C09K2211/1037C09K2211/1029
Inventor 柯博文李敏勇陈新新胡世龙康婷
Owner WEST CHINA HOSPITAL SICHUAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com