Fluorescence probe for detecting nitroreductase and application thereof

A technology of nitroreductase and fluorescent probe, applied in the field of fluorescent probe

Inactive Publication Date: 2017-10-24
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to our literature survey, a fluorescent probe for detecting nitroreductase that is completely unresponsive to hydrogen sulfide has not been reported yet.

Method used

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  • Fluorescence probe for detecting nitroreductase and application thereof
  • Fluorescence probe for detecting nitroreductase and application thereof
  • Fluorescence probe for detecting nitroreductase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1 (synthesis of fluorescent probe A):

[0020] The synthesis process of fluorescent probe A is as follows: figure 2 shown. Add N-morpholinoethylamino-4-hydroxyl-1,8-naphthalimide (0.33g, 1mmol), potassium carbonate (1.00g), p-nitrobenzyl bromide (0.43g, 2mmol) into a 250mL three-necked flask ), after 3 times of vacuum / argon replacement, add 150ml of anhydrous acetonitrile, heat up to acetonitrile reflux under the protection of argon, heat and stir for 4 hours. The reaction solution was evaporated under reduced pressure, and the obtained solid was purified by column chromatography to obtain the target compound fluorescent probe A. 1 HNMR (400MHz, CDCl 3 )δ(ppm)8.63(d,J=8.0Hz,2H),8.54(d,J=8.0Hz,1H),8.33(d,J=8.4Hz,2H),7.78–7.72(m,3H), 7.10(d, J=8.0Hz, 1H), 5.49(s, 1H), 4.34(t, J=7.0Hz, 2H), 3.70(t, J=4.2Hz, 4H), 2.73(t, J=6.8 Hz,2H),2.63(s,4H). 13 CNMR(100MHz,CDCl3)δ(ppm):14.14,22.70,29.71,31.93,37.03,53.77,56.17,66.94,69.46,106.42,115.93,122.55,124.15,12...

Embodiment 2

[0021] Embodiment 2 (absorption spectrum and emission spectrum response of fluorescent probe A to nitroreductase):

[0022] Add different volumes of nitroreductase solution (1mg / mL). After reacting at 37° C. for 60 min, the absorption spectrum and fluorescence emission spectrum of the working solution were measured, and the excitation wavelength was 445 nm.

[0023] The experimental results of the absorption spectrum and emission spectrum response of fluorescent probe A to nitroreductase are as follows: image 3 and Figure 4 shown. From image 3 It can be seen from the figure that as the amount of nitroreductase added increases (from 0 to 30 μg / mL), the absorbance of the solution system at 445 nm increases gradually, and a new absorption peak appears, which is contributed by product B. Figure 4 a shows that with the increase of the amount of nitroreductase added, the fluorescence of the system is significantly enhanced, and the new fluorescence peak at 550nm is contrib...

Embodiment 3

[0024] Embodiment 3 (kinetic experiment of nitroreductase catalyzed fluorescent probe A reaction)

[0025] Add different concentrations of fluorescent probe A to the phosphate buffer solution (pH=7.2, 10 mM) containing nitroreductase (25 μg / mL) and reduced coenzyme I (0.5 mM) volume 5%), measure the fluorescence intensity of the solution at 550 nm at intervals of 1 min at 37° C. with a microplate reader, and the excitation wavelength is 445 nm.

[0026] In addition, solutions containing different known concentrations of molecule B (synthesized according to literature) were prepared under the same conditions to obtain a standard curve representing the fluorescence intensity and concentration of molecule B at 550 nm.

[0027] The kinetic experiment results of nitroreductase catalyzing the reaction of fluorescent probe A are as follows: Figure 5 As shown, the abscissa indicates the reaction time (s), and the ordinate indicates the fluorescence intensity at 550nm, the concentrat...

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Abstract

The invention provides a fluorescence probe for detecting nitroreductase and application of the fluorescence probe. The structural formula of the fluorescence probe is shown in the description. The fluorescence probe detects the activity of the nitroreductase through the fluorescence difference of a reactant and a product.

Description

technical field [0001] The invention provides a fluorescent probe which can be used for selective detection of nitroreductase, and realizes the detection of nitroreductase activity by using the fluorescence difference between reactants and products. Background technique [0002] Nitroreductase can be synthesized by Escherichia coli and other bacteria, and it will also be expressed in large quantities when certain types of human cells are in a state of hypoxia. In the presence of reduced coenzyme Ⅰ (NADH) or Ⅱ (NADPH), nitroreductase can Metabolize aromatic nitro compounds; on the other hand, nitroreductase can be used in biochemical synthesis, anti-tumor drug development and other fields (Wan Qiongqiong et al., Journal of Analytical Science, 2014, Vol. 30, pp. 755-759). Therefore, it is of great significance to evaluate the activity of nitroreductase. [0003] Fluorescence method is one of the means to effectively detect the activity of nitroreductase. A fluorescent probe ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C07D211/88C12Q1/26
CPCC09K11/06C07D211/88C09K2211/1029C09K2211/1033C12Q1/26
Inventor 曲宗金韩克利李鹏贾燕
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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