A two-photon ratiometric fluorescent probe compound for aminopeptidase n detection and its preparation method

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, failure to provide real-time information, poor spatial resolution, etc., and achieve a wide range of applications and strong anti-interference ability. , the effect of a wide range of applications

Active Publication Date: 2021-07-09
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A two-photon ratiometric fluorescent probe compound for aminopeptidase n detection and its preparation method
  • A two-photon ratiometric fluorescent probe compound for aminopeptidase n detection and its preparation method
  • A two-photon ratiometric fluorescent probe compound for aminopeptidase n detection and its preparation method

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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 (90mmol, 3 eq), 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 =375 nm), 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 for accurately measuring aminopeptidase N in tumor cells with a two-photon ratio type fluorescent compound. The structure of the fluorescent compound is shown in formula I. The probe compound is designed based on the principle of fluorescence resonance energy transfer. Two-photon naphthalene derivatives are selected as energy donors, rhodol fluorophores are used as energy acceptors, and alanine is introduced as aminopeptidase N-specific recognition units. The ability of aminopeptidase N to preferentially hydrolyze the N-terminal alanyl group results in a proportional change in the fluorescence emission signal. The advantage is that it has high sensitivity and high selectivity, large emission shift, and can quickly detect aminopeptidase N. Two-photon fluorescence imaging can be successfully applied in living cells and tissues, providing a potential tool for clinical detection of kidney injury.

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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Patent Type & Authority Patents(China)
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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