A compound and a monoamines oxidase activity fluorescence detection method adopting the compound

A technology for monoamine oxidase activity and compounds, which is applied in chemical instruments and methods, fluorescence/phosphorescence, luminescent materials, etc., can solve the problems of low sensitivity and dangerous operation, and achieve simple operation process, improved accuracy and sensitivity, and stable properties. Effect

Active Publication Date: 2014-10-22
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sensitivity of the first two methods is not high, while the radioactive method has high sensitivity, but it requires radioactively labeled compounds, and there is a certain risk in operation.

Method used

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  • A compound and a monoamines oxidase activity fluorescence detection method adopting the compound
  • A compound and a monoamines oxidase activity fluorescence detection method adopting the compound
  • A compound and a monoamines oxidase activity fluorescence detection method adopting the compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Preparation of 2-(3-methoxy-6-(1-methyl-1,2,3,6-tetrahydropyridine-4-oxyl)-9H-oxanthene-9-yl) -4(5)-1-(2-(CY3-carboxamide)carbamoyl)benzoic acid

[0049]

[0050] Weigh resorcinol (2.86g, 2.6mmol) in a 50mL round bottom flask, add 15mL methanesulfonic acid, after the resorcinol is completely dissolved, add trimellitic anhydride (2.5g, 1.3mmol), react at 85°C, TLC The reaction was tracked. After the reaction was completed, it was cooled to room temperature, and the mixture was poured into 7 times the volume of crushed ice, and an orange-yellow precipitate was formed overnight. Suction filtration, water, CH 2 Cl 2 Wash and dry in an oven at 70°C to obtain a red solid. It was dissolved in an aqueous solution of 4M NaOH, and concentrated hydrochloric acid was carefully added dropwise to obtain 4 g of substance A, with a yield of 81.8%; ESI-MS m / z 377.3 (M+1) + .

[0051] Dissolve A (3.455g, 9.2mmol) in 50mL DMF, add K 2 CO 3 (5.71g, 41.4mmol), after sti...

Embodiment 2

[0059] In order to determine the effect of enzyme concentration on the detection results, a set of gradient experiments was designed, three groups of parallel groups were added to the 96-well plate with appropriate amount of boric acid buffer (concentration 50mM, pH=8.4), and then 10μL, 25μL, 40μL , 55 μL of enzyme solution (MAO-B was dissolved in PBS buffer with a concentration of 50 mM and a pH value of 7.4 to obtain an enzyme solution with a concentration of 3 μg / mL), so that the final concentration of the enzyme was 0.030 mg / mL, 0.075 mg / mL, 0.15 mg / mL, 0.16 mg / mL, and finally add 2 μL of the probe prepared in Example 1 in DMSO solution (concentration 10 mM). After reacting at 37℃ for 2h, at λ ex / λ em =475 / 570nm, the detection of the fluorescence emission spectrum is carried out with a fluorescent microplate reader, and the influence of the enzyme concentration on the detection result is obtained.

[0060] Experiments have proved that as long as there is a trace amount ...

Embodiment 3

[0061] Embodiment 3: selectivity test

[0062] In order to study the selection specificity of the probe to monoamine oxidase, three groups of parallel experiments were designed, and 143 μL of borate buffer (concentration 50 mM, pH=8.4) was added to a 96-well plate, followed by 55 μL of enzyme solution (MAO-A or MAO- B is dissolved in PBS buffer with a concentration of 50mM and a pH value of 7.4 to obtain an enzyme solution with a concentration of 3 μg / mL), add 2 μL of the DMSO solution of the probe prepared in Example 1, and make the fluorescent probe in the reaction solution The final concentration is 100μM, after reacting at 37℃ for 2h, at λ em / λ ex = 475 / 570nm with a fluorescent microplate reader for full band scanning. From figure 1 From the test results, the probes prepared in Example 1 have a certain detection effect on monoamine oxidase, and the detection effect on MAO-B is better than that of MAO-A, indicating that the probe is selective to MAO-B.

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Abstract

The invention discloses a compound shown as a formula (i1) or a formula (i2) and a preparing method thereof. The compound can be adopted as a fluorescence probe and applied for monoamines oxidase activity fluorescence detection. The compound has characteristics of simple preparation equipment, simple operation processes, commercialized and easily available raw materials, stable properties of the compound as the fluorescence probe for the monoamines oxidase, and high purity. When the fluorescence probe is applied for the monoamines oxidase activity fluorescence detection method, the accuracy and the sensitivity are largely improved than those of basic methods used in the prior art for detection the monoamines oxidase activity.

Description

(1) Technical field [0001] The invention relates to a new compound, which can be used as a fluorescent probe in the fluorescence detection method of monoamine oxidase activity. (2) Background technology [0002] Monoamine oxidase (EC1.4.3.4; amine-oxygen oxidoreductases) is an important enzyme in the oxidative metabolism of dopamine, which plays an important role in the metabolism of neurotransmitters. Transmitters dopamine, norepinephrine, serotonin, tyramine, 2-phenylethylamine, and neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), and oxidize Deamination produces H 2 o 2 . [0003] Monoamine oxidase B mainly metabolizes amines without hydroxyl, such as phenethylamine and benzylamine. Abnormalities in monoamine oxidase B have been found to contribute to a range of psychiatric disorders, such as Parkinson's disease, Alzheimer's disease, alcoholism, and neurodegeneration. Parkinson's disease (PD) is a common psychiatric disorder characterized by loss of dopamine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D405/14C09K11/06G01N21/64
CPCC07D405/14C09K11/06C09K2211/1088G01N21/6428
Inventor 朱勍李雪峰向雨秘
Owner ZHEJIANG UNIV OF TECH
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