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Two-photon fluorescence probe and application thereof in detecting anoxic-zone nitroreductase

A two-photon fluorescence, anoxic region technology, applied in the field of fluorescent probes and organic small molecule fluorescent probes, can solve the problems that have not been widely reported and the two-photon fluorescent probes of nitroreductase are scarce.

Inactive Publication Date: 2016-07-06
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the search found that two-photon fluorescent probes for the detection of nitroreductase in hypoxic regions are still scarce and have not been widely reported.

Method used

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  • Two-photon fluorescence probe and application thereof in detecting anoxic-zone nitroreductase
  • Two-photon fluorescence probe and application thereof in detecting anoxic-zone nitroreductase
  • Two-photon fluorescence probe and application thereof in detecting anoxic-zone nitroreductase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The synthesis process of the two-photon fluorescent probe is as follows:

[0034] 1) Synthesis of compound 6-(4-hydroxyloxy)-2,3,4,4a-tetrahydro-xanthene-1-one (3):

[0035] In a 100mL round bottom flask, add 10mmol of 2,4-dihydroxybenzaldehyde (1), 15mmol of cyclohexenone (2) and 25mmol of triethylenediamine, then add water / 1,4-dioxane mixed solvent 60mL (V / V=1:1), under nitrogen atmosphere, heated to reflux for two days, distilled off the solvent under reduced pressure, and separated the residue by column chromatography to obtain light yellow solid 6-(4-hydroxyloxy)-2, 3,4,4a-tetrahydro-xanthene-1-one (3), referred to as GCTPOC. Yield: 15%.

[0036] 1 HNMR (400MHz, DMSO-d 6 ):10.17(s,1H),7.34(d,J=2.0Hz,1H),7.34(d,J=8.4Hz,1H),6.41(dd,J 1 =8.0Hz,J 2 =2.0Hz,1H),6.29(d,J=2.4Hz,1H),4.94(m,1H),2.36(m,3H),1.91(m,2H),1.67(m,1H);HR-MS calculated for C 13 h 12 o 3 [M-H] - m / z215.0703,found215.0747.

[0037] Synthesis of compound 6-((4-nitrobenzyl)oxy)-2,3,4,4a-tetrah...

Embodiment 2

[0043] Absorption Spectrum Changes of Compound GCTPOC-HY Two-Photon Hypoxic Fluorescent Probe

[0044] Prepare 1 part of 5mL 1mM dimethyl sulfoxide (DMSO) solution of the two-photon fluorescent probe GCTPOC-HY prepared by the present invention in advance, then take 25 μL and add them to two identical 5mL volumetric flasks, add 225 μL of DMSO solution, and then Both added nicotinamide adenine dinucleotide (NADH) 500μM. Nitroreductase phosphate buffer solution (10 μg / mL, 500 μL) was added to one of them, and both volumetric flasks were adjusted to 5 mL with phosphate buffer solution (PBS, pH=7.4), and then acted at 37°C for 50 minutes, and then the absorption spectrum test was performed. The absorption of a group of compounds with nitroreductase becomes stronger, the results are shown in figure 1 , where (A) is the absorption curve of the probe reacting with 1.0 μg / mL nitroreductase for 50 min; (B) is the absorption curve of the probe itself.

Embodiment 3

[0046] The Selectivity of Compound GCTPOC-HY Two-Photon Hypoxic Fluorescent Probe to Different Molecules or Ions

[0047] Prepare 5mL of PBS aqueous solution of various conventional ions and amino acids with a concentration of 40mM and a two-photon fluorescent probe with a concentration of 1mM as the mother solution; Dilute the volume of phosphate buffer to 5mL, shake well and perform fluorescence detection (λ ex =450nm,λ em =511nm), establish the curve of fluorescence intensity and each ion (or amino acid) (results see figure 2 ). After 50min, detect the change of the fluorescence emission spectrum of the solution, by figure 2 It can be found that other ions (or amino acids) have almost no effect on the fluorescence of compound GCTPOC-HY, but the addition of nitroreductase can significantly enhance the fluorescence of compound GCTPOC-HY. figure 2 Among them, the ions added in 1-21 are: probe, Na 2 SO 3 、NaNO 2 、NaNO 3 , NaClO, CaCl 2 、Na 2 S, FeSO 4 , KBr, H 2 ...

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Abstract

The invention discloses a novel dimolecular fluorescence probe of which the chemical name is 6-((4-nitrobenzyl)oxy)-2,3,4,4a-tetrahydro-xanthene-1-one. The invention also discloses application of the two-photon fluorescence probe in detecting nitroreductase in cells. The fluorescence intensity of the probe obviously increases as the nitroreductase content increases. The probe disclosed by the invention can be used for detecting the nitroreductase content in the tumor anoxic zone by a fluorescent imaging technique, thereby evaluating and researching the anoxic level of the tumor anoxic zone.

Description

technical field [0001] The invention relates to a fluorescent probe and its application in detecting nitroreductase (NTR) in anoxic region, belonging to the field of organic small molecule fluorescent probes. Background technique [0002] During the growth of malignant tumors, there will be a state of unlimited and crazy growth, resulting in insufficient oxygen provided by the blood in the blood vessels to support the growth of cells, thereby forming a highly hypoxic area, which is the tumor hypoxic area. Hypoxia is an important feature of the solid tumor microenvironment. Under normal circumstances, the oxygen content in most solid tumors is about 5%, and the oxygen content in some tumor areas can even drop to about 1%. The formation of hypoxic regions makes tumor microvasculature abnormal, making it difficult for cancer chemotherapy drugs to enter this region, thereby reducing the efficacy of anticancer drugs. Based on this, assessing the degree of hypoxia in hypoxic reg...

Claims

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Application Information

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IPC IPC(8): C07D311/82C09K11/06G01N21/64
CPCC07D311/82C09K11/06C09K2211/1088G01N21/6428G01N21/6456G01N21/6458
Inventor 林伟英刘展榕徐安唐永和
Owner UNIV OF JINAN
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