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Bacteria nitroreductase detection kit and special-purpose fluorescence probe thereof

A technology of nitroreductase and reductase, which is applied in the fields of fluorescence/phosphorescence, luminescent materials, organic chemistry, etc., to achieve the effects of high sensitivity, fast reaction speed, and easy promotion and application

Active Publication Date: 2013-08-14
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few reports on the methods used to detect bacterial nitroreductase

Method used

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  • Bacteria nitroreductase detection kit and special-purpose fluorescence probe thereof
  • Bacteria nitroreductase detection kit and special-purpose fluorescence probe thereof
  • Bacteria nitroreductase detection kit and special-purpose fluorescence probe thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1, preparation of 7-[(5-nitrothiophen-2-yl)methoxy]-3H-phenoxazin-3-one shown in formula I

[0043] according to figure 1 The chemical reaction scheme shown in the preparation was carried out.

[0044] The operation steps are as follows: Dissolve 7-hydroxyphenoxazinone sodium salt (235mg, 1mmol) in DMF (10mL), add 2-bromomethyl-5-nitrothiophene (223mg, 1mmol), catalyst potassium carbonate (210mg , 1 mmol) in the reaction solution, and reacted at 20°C for 6 hours. After completion of the reaction, after cooling, the solvent was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography using n-hexane / ethyl acetate (2:1, v / v) as eluent to obtain 180 mg of the product.

[0045] The structural characterization data results of the product are as follows:

[0046] 1 H NMR (600MHz, 298K, CF 3COOD): δ8.40(d, J=9.4Hz, 1H), 8.36(d, J=9.4Hz, 1H), 7.92(d, J=4.2Hz, 1H), 7.69(t, J=2.8Hz, 1H), 7.67(t, J=2.8Hz, 1H), 7.56(d, ...

Embodiment 2

[0050] Embodiment 2: Spectral properties of reagent 1 (i.e. compound shown in formula I) reacting with different concentrations of nitroreductase

[0051] Weigh 3.38 mg of reagent 1 and make 10 ml of dimethyl sulfoxide solution as mother solution (1 mM).

[0052] Add 50 μl of the above mother solution dropwise to a certain amount of 10 mM phosphate buffer solution containing 50 μM coenzyme (nicotinamide adenine dinucleotide), then add different concentrations of nitroreductase solutions, and then use 10 mM phosphate buffer The solution was adjusted to 10ml. After reacting at 37° C. for 20 min, measure its UV-Vis absorption spectrum and fluorescence emission spectrum. When measuring the fluorescence emission spectrum, de-excite at 550nm; the slit width for excitation and emission is 10nm; the voltage is 400V.

[0053] figure 2 It is the fluorescence spectrum of reagent 1 reacting with 0-1 μg / ml nitroreductase.

[0054] figure 2 The results show that reagent 1 in the pres...

Embodiment 3

[0058] Embodiment 3: Reagent 1 (compound shown in formula I) reacts with other species (selectivity research)

[0059] Add various substances to the 5μM reagent 1 solution: potassium chloride (150mM), calcium chloride (2.5mM), magnesium chloride (2.5mM), glucose (10mM), vitamin C (1mM), vitamin B 6 (1mM), human serum albumin (100μM), hydrogen peroxide (10μM), hydroxyl radical (10μM), glutamic acid (1mM), arginine (1mM), serine (1mM), glutathione (5mM) , cysteine ​​(1 mM), homocysteine ​​(1 mM), dithiothreitol (1 mM) and nitroreductase (0.5 μg / mL). After reacting at 37°C for 20 min, the fluorescence emission spectrum was measured. When measuring the fluorescence emission spectrum, de-excite at 550nm; the slit width for excitation and emission is 10nm; the voltage is 400V.

[0060] 50 μl of reagent 1 stock solution (1 mM) was added to 10 ml of the solution mixed with the above-mentioned substances, and nitroreductase was added to make the final concentration 0.5 μg / mL.

[006...

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Abstract

The invention discloses a bacteria nitroreductase detection kit and a special-purpose fluorescence probe thereof. The structural formula of the fluorescence probe is shown in the formula I. The bacteria nitroreductase detection kit comprises a reagent stock solution 1 and a reagent stock solution 2, wherein the reagent stock solution 1 is a buffer solution containing coenzyme; the reagent stock solution 2 is a solution of the fluorescence probe. Experiments prove that the fluorescence intensity of the probe is very weak, and the probe has 1,6-rearrangment elimination reaction after nitroreductase is added, so that resorufin fluorescence parent is released, the solution fluorescence is remarkably strengthened and the color is changed into purple red from colorless, and therefore, the method can be used for detecting nitroreductase. The bacteria nitroreductase detection kit is simple to operate, low in cost, quick, efficient, sensitive and the like, is easy to popularize and apply, and has great application prospect in the field of medicine and the like. The structural formula is shown in the description.

Description

technical field [0001] The invention relates to a bacterial nitroreductase detection kit and a special fluorescent probe thereof. Background technique [0002] Bacterial nitroreductase (nitroreductase, NTR) belongs to the flavin mononucleotide (FMN)-dependent nitroreductase superfamily, and usually exists in the form of dimers. They can use reduced nicotinamide adenine dinucleotide (phosphate) (reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H) as an electron donor to catalyze a variety of exogenous nitroaromatic, nitroheterocyclic , the reduction reaction of quinones and flavin compounds, the nitro group is a common functional group in biomedicine, antibacterial agents and pesticides, and its reduction is a key step to achieve the degradation of nitroaryl pollutants. It also plays an important role in activation and detoxification mechanisms. Currently, there are few reports on methods for detecting bacterial nitroreductase. Contents of the invention [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D413/12C09K11/06G01N21/64
Inventor 马会民李照
Owner INST OF CHEM CHINESE ACAD OF SCI
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