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Rhodamine fluorescent probe with pseudo nucleic acid base as recognition site and preparation thereof and application to nucleotide image

A technology of identifying sites and nucleobases, applied in the field of fluorescent probes, can solve the problems of large light damage to cells, strong background signals, and it is difficult to accurately examine the physiological functions of cells, and achieve good cell condition and sensitive imaging effects. , the effect of good selectivity

Active Publication Date: 2014-01-29
DALIAN UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The excitation wavelength and emission wavelength of the above-mentioned fluorescent probes for nucleotide detection are mostly in the short-wave region, and the background signal is strong in fluorescence imaging, and short-wave excitation and emission cause great damage to cells, making it difficult to accurately investigate the normal physiological functions of cells

Method used

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  • Rhodamine fluorescent probe with pseudo nucleic acid base as recognition site and preparation thereof and application to nucleotide image
  • Rhodamine fluorescent probe with pseudo nucleic acid base as recognition site and preparation thereof and application to nucleotide image
  • Rhodamine fluorescent probe with pseudo nucleic acid base as recognition site and preparation thereof and application to nucleotide image

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Effect test

Embodiment 1

[0022] Embodiment 1 (preparation of probe RBS)

[0023] Dissolve 3.0g (27.5mM) 2,6-diaminopyridine in the first reaction kettle filled with 35mL concentrated phosphoric acid, heat to 90°C in an argon atmosphere, and weigh 3.70g (27.5mM) 4,4- Dimethoxy-2-butanone was placed in a constant pressure separatory funnel and added dropwise to the phosphoric acid solution of 2,6-diaminopyridine, heated to 115°C, stirred and reacted for 5 hours, cooled to room temperature, and added with 15% Phosphoric acid in the reaction solution was neutralized with ammonia water to pH=8, extracted 5 times with chloroform, the extracted chloroform solution was washed 3 times with saturated sodium chloride solution, dried with anhydrous magnesium sulfate, and the chloroform was rotary evaporated to obtain a black-red solid substance , recrystallized from toluene to obtain 2.30 g (52%) of light yellow powder intermediate 2-amino-methyl-1,8-naphthyridine (AMND); 1H NMR (CDCl 3 ,ppm):7.82(d,1H,J=4.0Hz),...

Embodiment 2

[0024] Example 2 (Measurement of Selective Response of Probes to Nucleotides)

[0025] Weigh 30mg of the probe, and make it into a 10mM standard stock solution of acetonitrile, and make 10mM stock solution of nucleotides ADP, ATP, AMP, CDP, CTP, CMP, GDP, GTP, GMP, UDP, UTP, UMP respectively. Take 2 μL of the probe stock solution and add it to 2 mL of TRIS-HCl buffer solution with a concentration of 50 mM and a pH of 6.04, shake well and measure its fluorescence intensity (F 0), then add 30 μL of nucleotides, measure the fluorescence intensity (F) under the same conditions, and calculate the relative change value of the fluorescence intensity, the results are as follows figure 1 shown. The probes have different responses to all nucleotides, among which CDP, ATP, ADP and CTP cause obvious relative changes in fluorescence intensity, and CDP can form a strong complementary nucleotide-like base with naphthyridine in the probe. The triple hydrogen bond has the strongest effect, s...

Embodiment 3

[0026] Embodiment 3 (probe fluorescence intensity and absorbance change with CDP concentration)

[0027] Weigh 10 mg of the probe, prepare a standard stock solution of 10 mM water (1), and then prepare a stock solution of 10 mM CDP (2). Measure 2 μL of the stock solution (1) and add it to 2 mL of TRIS-HCl buffer solution with a concentration of 50 mM and a pH of 6.04, shake well, add the calculated amount of stock solution (2), and prepare a standard test solution. The fluorescence intensity and absorbance were measured respectively, and the test results were as follows: image 3 shown. With the increase of CDP concentration, the fluorescence intensity and absorbance value of the probe gradually increased. Among them, Figure a is a graph of fluorescence change; Figure b is a graph of absorbance change, and the direction of the arrow in the figure indicates the direction of image intensity increase.

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Abstract

The invention relates to a fluorescent probe, which belongs to the technical field of fine chemical engineering, and discloses a rhodamine fluorescent probe with a pseudo nucleic acid base as a recognition site and preparation thereof and application to a nucleotide image. The ring opening of rhodamine lactone is induced between amino naphthyridine and nucleic acid base through a base analogue complementary hydrogen bond effect, and the selective response of specific nucleotide is realized by using the weakness of electrostatic interaction between imine positive ions generated by a ring opening product and phosphoric acid link negative ions with proper length. The probe shows good selectivity to cytidine diphosphate (CDP) and adenosine triphosphoric acid (ATP). Hela cell copolymerization focal imaging experiments show that the probe can produce a sensitive imaging effect on the nucleotide in the cell, the cell state is good after and before imaging, and the probe has small cell toxicity. Since the probe has good bioavailability and a high-sensitivity imaging effect, the probe is widely applied to medical fluorescent imaging.

Description

technical field [0001] The invention relates to a fluorescent probe, in particular to a rhodamine fluorescent probe whose pseudo-nucleic acid base is a recognition site, its preparation and its application in nucleotide imaging. Background technique [0002] As an important class of small biological molecules, nucleotides play an extremely important role in many life processes such as human DNA synthesis, cell signal transmission, transmembrane transport of various substances, energy transfer, and gene expression. And when many physiological processes are abnormal, or when the body has a disease, it is accompanied by the abnormal expression of a certain nucleotide. Therefore, realizing real-time, in situ, and non-damaging high-resolution images of intracellular nucleotides provides an intuitive and effective way for people to study many physiological functions of cells. Designing and developing molecular probes that respond specifically to nucleotides is one of the most eff...

Claims

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Application Information

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IPC IPC(8): C09K11/06C07D491/107G01N33/52G01N21/64
Inventor 段春迎孔继川何成张奉禄
Owner DALIAN UNIV OF TECH
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