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Two-photon ratiometric fluorescent probe compound for detecting aminopeptidase N, and preparation method thereof

A technology for fluorescent probes and compounds, applied in the field of fluorescent sensor probe compounds, can solve the problems of lack of specificity and sensitivity, poor spatial resolution, application limitations, etc., and achieve a wide range of applications, broad application prospects, and a wide range of applications Effect

Active Publication Date: 2019-10-15
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of sufficient specificity and sensitivity for low-abundance enzymes in living cells, poor spatial resolution (1-2mm), and failure to provide real-time information on enzymes in living cells, these methods Applications are extremely limited

Method used

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  • Two-photon ratiometric fluorescent probe compound for detecting aminopeptidase N, and preparation method thereof
  • Two-photon ratiometric fluorescent probe compound for detecting aminopeptidase N, and preparation method thereof
  • Two-photon ratiometric fluorescent probe compound for detecting aminopeptidase N, and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] A method for preparing a fluorescent probe compound for aminopeptidase N detection, the steps comprising:

[0040] 1) Synthesis of Compound 1:

[0041] Dissolve 6-amino-2-naphthoic acid (30 mmol, 1 eq) in 15 mL of glacial acetic acid and 100 mL of 37% aqueous formaldehyde, then add cyanide dissolved in methanol solution to the mixture under stirring in an ice-water bath Sodium borohydride (90 mmol, 3eq), stirred at room temperature for 3 h, the methanol solution was evaporated, the remaining mixture was diluted with brine, and the pH was adjusted to 6. It was extracted with ethyl acetate, dried over anhydrous sodium sulfate, evaporated under reduced pressure, and recrystallized to obtain compound 1 as a light brown solid with a yield of 80%.

[0042] 2) Synthesis of Compound 2:

[0043] To 25 mL of 3-aminophenol (10 mmol, 1 eq) in ethylene glycol was added bis(2-chloroethyl)amine hydrochloride (12 mmol, 1.2 eq), and the reaction mixture was stirred at 125 °C overnight...

Embodiment 2

[0055] The I of probe compound FTP-N with different APN concentrations (0-90 ng / mL) 540 nm / I 452 nm Ratiometric Fluorescence Emission Changes

[0056] Take the FTP-N synthesized in Example 1 to prepare a 5 μM probe solution, add different amounts (0-90 ng / mL) of APN standard solution, and measure its fluorescence properties. Using 375 nm as the excitation light, as the amount of APN increases, the fluorescence intensity at 452 nm decreases sharply, and a red-shifted fluorescence peak appears at 540 nm, providing a visualized ratiometric fluorescence signal (I 540 nm / I 452 nm ).

Embodiment 3

[0058] Measurement of Fluorescent Linear Range of Compound FTP-N Fluorescent Probe

[0059] Take the fluorescent probe solution (5 μM) in Example 2, add APN (0-5 ng / mL) respectively, and perform fluorescence detection (λ ex =375nm), indicating that the probe has a linear relationship in the range of APN concentration 0-5 ng / mL, and the linear correlation coefficient is R 2 =0.99823.

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Abstract

The invention discloses a preparation method of a two-photon ratiometric fluorescent probe compound for detecting aminopeptidase N. The structure of the fluorescent compound is represented by formulaI. The probe compound is designed based on the principle of fluorescence resonance energy transfer, a two-photon naphthalene derivative is selected as an energy donor, a p-methylaminophenol fluorophore is used as an energy acceptor, and alanine is introduced as an aminopeptidase N specific recognition unit. The aminopeptidase N is used to preferentially hydrolyze an N-terminal alanyl group, whichcauses the proportional change in a fluorescence emission signal. The compound has the advantages of high sensitivity, high selectivity, large emission shift, and quickness in detection of the aminopeptidase N, can be successfully applied to two-photon fluorescence imaging in living cells and tissues, and provides a potential tool for clinical detection of kidney injuries.

Description

technical field [0001] The present invention relates to a small molecule fluorescent probe compound, more specifically a fluorescent probe for the detection of aminopeptidase N (APN), an important marker of kidney damage, based on two-photon ratio compounds based on fluorescence resonance energy transfer. The invention belongs to the field of fluorescent sensor probe compounds. Background technique [0002] Aminopeptidase N (APN), also known as alanine aminopeptidase. It is widely present in various organs, tissues and cells (endothelial cells, epithelial cells, fibroblasts, leukocytes) in mammals and participates in many physiological and pathological processes, especially in inflammatory diseases and cancers (solid and blood tumors) overexpression. Studies have shown that when urinary microglobulin remains at a normal level, APN in urine can indicate early kidney damage and serve as an early biomarker of glomerulonephritis. At the same time, modern medical research show...

Claims

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Application Information

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IPC IPC(8): C07D493/10C09K11/06G01N21/64
CPCC07D493/10C09K11/06C09K2211/1011C09K2211/1044C09K2211/1088G01N21/6428G01N21/643
Inventor 颜梅卫先哲张晶王建荣张乐淘武虹乐
Owner UNIV OF JINAN
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