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Peroxynitrite near infrared fluorescent probe ONP and preparation method and application thereof

A peroxynitrite, a technology for peroxynitrite, applied in the field of biochemistry, can solve problems such as lack of in vivo imaging methods, and achieve the effects of high sensitivity, excellent concentration linear relationship, and good stability

Active Publication Date: 2019-06-28
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many fluorescent probes have been reported for ONOO in cells or tissues - Imaging, but still lacking for ONOO in the brain - In vivo imaging methods for assays, including brain imaging in epileptic disease states
In addition, there is a shortage of imaging probes that can be used to build screening platforms for rapid screening of antiepileptic drugs
To achieve these goals, there are several challenges: (1) The main challenge in developing probes is whether the probes can efficiently cross the blood-brain barrier (BBB) ​​to enable imaging of brain regions; The probe is advantageous for deeper tissue penetration, less photodamage and less background fluorescence interference; (3) Effective monitoring of ONOO in a real physiological environment - Probes requiring high selectivity and sensitivity

Method used

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  • Peroxynitrite near infrared fluorescent probe ONP and preparation method and application thereof
  • Peroxynitrite near infrared fluorescent probe ONP and preparation method and application thereof
  • Peroxynitrite near infrared fluorescent probe ONP and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0067] A kind of preparation method of near-infrared fluorescent probe ONP for peroxynitrite, its preparation process is as follows:

[0068]

[0069] Under argon protection, methylene blue (374mg, 1mmol), 10mL DCM and 10mL water were added to a 50mL round bottom flask, and stirred evenly. Na 2 S 2 o 4 (525mg, 1.5mmol) and NaHCO 3 (168mg, 2mmol) was slowly added to the above mixture. The mixture was then stirred for 20 minutes until the aqueous phase turned yellow. Aqueous layer The aqueous phase was extracted with dichloromethane (2 x 5 mL), and the organic layer was separated. The organic phases were combined and dried over anhydrous sodium sulfate. Under argon protection, the dry organic phase was quickly poured into a round bottom flask containing triethylamine (TEA, 170 μL, 1.2 mmol). 1 mL of DCM containing triphosgene (TPG, 120 mg, 0.32 mmol) was slowly added to the reaction mixture. After the addition was complete, the reaction was stirred at room temperature...

Embodiment 2

[0072] Synthesis and preliminary evaluation of ONP

[0073] The final structure of the near-infrared fluorescent probe ONP prepared in Example 1 is passed 1 H and 13 C NMR spectroscopy and mass spectroscopy fully confirmed. Then, preliminary in vitro tests were performed in PBS buffer (pH 7.4, 5% MeCN). ONP alone exhibits very weak UV absorption and fluorescence because the fluorescence of MB (methylene blue) is locked in the interrupted LMB form ( figure 1 B and C), however, it provides significant NIR excitation (640nm) and emission (650-850nm with a maximum at 692nm). Correspondingly, ONP (10 μ M, PBS buffer (pH7.4, 5% MeCN)) and ONOO - (100 μM, aqueous solution) after incubation at 37°C for 30 minutes, a significant increase in absorbance and fluorescence intensity at 665 nm and 692 nm, respectively, was observed. In order to study the stability and reliability of ONP to different pH conditions, the fluorescence changes in PBS buffer in different pH ranges (3-11) were...

Embodiment 3

[0075] ONP spectral properties and selectivity

[0076] To study ONP vs ONOO in more detail - The response of ONOO was evaluated by recording the change of fluorescence intensity at 692nm - Time-dependent fluorescence response in presence. Discover ONP and ONOO - The reaction between 10μM ONP and 100μM ONOO - at 5% CH 3 After co-incubating in CN's PBS system for 1 minute, a significant increase in fluorescence intensity can be clearly detected at 692 nm using a fluorescence spectrophotometer, and the reaction is completed within 15 minutes, ONOO - After activation, ONP is efficiently converted to MB ( image 3 A). Furthermore, when 10 μM ONP was combined with increasing concentrations of ONOO - (0-100μM) when incubated together, a concentration-dependent fluorescence enhancement was observed ( image 3 B), indicating that ONP is not only for ONOO - Fast response to low concentration of ONOO - Also highly sensitive, and the fluorescence intensity at 692 nm is comparab...

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Abstract

The invention discloses a peroxynitrite near infrared fluorescent probe ONP and a preparation method and an application thereof. The near infrared fluorescent probe ONP is obtained by combining boratewith a methylene blue framework. The structure is as shown in a formula I. The near infrared fluorescent probe ONP can trace an endogenous ONOO-signal in KA-induced epilepsy. The probe ONP can imageONOO- effectively and selectively in vitro and in vivo, can penetrate blood brain Barrier (BBB) effectively, and has a brain targeting characteristic. By means of the characteristics of the probe, up-regulation of ONOO- in a KA-induced epilepsy period is observed directly in vivo and in vitro. In addition, the invention discloses a method of screening an anti-epilepsy inhibitor by combining high connotation analysis and ONP, thereby providing simple and effective product and method for researching ONOO- in a biological system and screening an anti-epilepsy drug.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and relates to a peroxynitrite near-infrared fluorescent probe ONP and its preparation method and application, especially the design and synthesis of the near-infrared fluorescent probe ONP for peroxynitrite and its application in Tracer imaging in epilepsy. Background technique [0002] Epilepsy is a chronic neurodegenerative disorder characterized by recurrent and unpredictable seizures that affects approximately 0.7% of people worldwide. Despite the increasing availability of antiepileptic drugs over the past two decades, more than 30% of patients are medically refractory to or do not respond effectively to them. These drugs currently developed for antiepileptic use only provide basic symptomatic treatment, and have not successfully addressed the problem of drug resistance and the prevention of epilepsy and the treatment of status epilepticus. Accumulating evidence indicates that epilep...

Claims

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Application Information

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IPC IPC(8): C07F5/02C09K11/06G01N21/64A61K49/00
Inventor 钱勇邵晨雯胡炯圣刘红科
Owner NANJING NORMAL UNIVERSITY
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