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A specific two-photon fluorescent probe for monoamine oxidase a and its preparation method and application

A technology of monoamine oxidase and two-photon fluorescence, which is applied in the field of organic fluorescent probes, can solve the problems of tissue imaging and achieve good fluorescence properties, reduce biological background fluorescence, and the synthesis process is simple and easy

Active Publication Date: 2020-06-30
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report of a specific two-photon fluorescent probe for monoamine oxidase A. Only the fluorescent probe for monoamine oxidase A that has been reported is single-photon, which cannot be used for deep tissue imaging.

Method used

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  • A specific two-photon fluorescent probe for monoamine oxidase a and its preparation method and application
  • A specific two-photon fluorescent probe for monoamine oxidase a and its preparation method and application
  • A specific two-photon fluorescent probe for monoamine oxidase a and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0035] The chemical synthesis method of embodiment 1 F1

[0036] The reaction formula is as follows:

[0037]

[0038] The first step: 6-bromo-N, N-dimethylnaphthalene-2-amine

[0039] A mixture of dimethylamine solution (2 mL, 30.16 mmol), 6-bromo-2-naphthol (1.0 g, 4, 48 mmol), Na2S2O5 (1.78 g, 9.37 mmol) and H2O (15 mL) in a microwave tube was heated at 160 Stir at °C for 7 hours. The product was washed with sodium chloride solution, the aqueous phase was extracted with ethyl acetate, then purified by flash column chromatography, and spin-dried to obtain 6-bromo-2-dimethylaminonaphthalene as a white solid, namely compound 1, with a yield of 0.71 g, 63%. 1 H NMR (500MHz, CDCl 3 )δ7.84(s,1H),7.84(s,1H),7.62(d,J=9.1Hz,1H),7.58(dd,J=25.5,8.9Hz,2H),7.53(d,J=8.7 Hz,1H),7.43(d,J=8.7Hz,1H),7.43(d,J=8.7Hz,1H),7.33–7.09(m,2H),7.19(d,J=9.8Hz,1H), 6.93(s,1H),6.93(s,1H),3.06(s,6H),3.06(s,7H). 13 C NMR (75MHz, CDCl 3 )δ=151.30, 136.07, 131.99, 130.47, 130.44, 119.69, 117.72, 1...

Embodiment 2

[0048] Embodiment 2 monoamine oxidase concentration curve

[0049] (1) Prepare 200 μL of monoamine oxidase reaction system in advance, including PBS buffer with pH=7.4, 0.4% Triton X-100, monoamine oxidase A / B (0-10 μg / mL), F1 (1 μM), at 37 ° C Shake pre-incubation for 1 hour.

[0050] (2) Fluorescence scanning detection (Ex=430nm) is carried out after reacting for 1 hour, and the fluorescence intensity increases with the concentration of monoamine oxidase A, see Figure 4 .

Embodiment 3

[0051] Activity detection of monoamine oxidase A in the cell lysate of embodiment 3

[0052] (1) SH-SY5Y / HepG-2 cells were inoculated in a 10 cm culture dish, and the culture medium was Dulbecco's modified Eagle medium (DMEM), containing 10% fetal bovine serum (FBS), 100.0 mg / L streptomycin and 100IU / mL penicillin. When the cell density is about 90%, pour out the culture medium in the culture dish, wash once with PBS buffer (pH=7.4), then add 1 mL of PBS buffer (pH=7.4) with 0.4% Triton X-100, The cells were broken with a cell scraper, and then the cell lysate was collected, centrifuged at 4° C. and 12000 rpm for 10 minutes, and the supernatant was taken to obtain a clarified cell lysate.

[0053] (2) Determination of total protein concentration in lysate

[0054] The total protein concentration of the lysate was determined using the BCA protein concentration assay kit.

[0055] (3) Prepare 200 μL of cell lysate reaction system, including PBS buffer with pH=7.4, 0.4% Triton...

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Abstract

The invention relates to a specific two-photon fluorescent probe of monoamine oxidase A, and a preparation method and an application thereof, belonging to the field of organic fluorescent probes. Thespecific two-photon fluorescent probe comprises a compound F1 with a general formula (I) which is described in the specification; and the probe is designed by utilizing the spatial selectivity of enzyme and combining the idea of constructing a conjugated structure. The probe F1 successfully and specifically detects the activity of MAO-A in different biological samples (pure proteins, cell lysates,viable cells, mouse brain tissues and tumor tissues). The probe has high sensitivity and low cost, and is applicable to industrialization.

Description

technical field [0001] The invention belongs to the field of organic fluorescent probes, and in particular relates to a specific two-photon fluorescent probe for monoamine oxidase A and its preparation method and application. Background technique [0002] Monoamine oxidase is a protein expressed on the outer membrane of mitochondria, which catalyzes and transforms various monoamines in organisms, including neurotransmitters serotonin, dopamine, phenylethylamine and exogenous tyrosine. According to their specificity and cellular distribution to natural substrates or inhibitors, monoamine oxidases are divided into two subtypes—MAO-A and MAO-B. MAO-A has a strong affinity for serotonin and norepinephrine, and its specific inhibitors are clorgiline, etc., while MAO-B mainly metabolizes benzylamine and phenethylamine, and its specific inhibitors are rasagiline, etc. MAOs have been proven to be the key proteins to maintain the homeostasis of neurotransmitters in the body. Overexp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D211/70C09K11/06G01N21/64
CPCC07D211/70C09K11/06C09K2211/1011C09K2211/1029G01N21/6428
Inventor 李林方海啸陈鼎张承武余昌敏吴琼杨乃娣黄维
Owner NANJING TECH UNIV
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