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Ligand, preparation method thereof, fluorescent probe, and preparation method and application of fluorescent probe

A technology of fluorescent probes and ligands, applied in the fields of fluorescent probes and their preparation, ligands and preparations, can solve problems such as low signal-to-noise ratio and accuracy, achieve good selectivity, improve accuracy and precision , the effect of high measurement sensitivity

Inactive Publication Date: 2019-10-15
湖南艾科瑞生物工程有限公司
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AI Technical Summary

Problems solved by technology

[0004] Although the above-mentioned various determination methods have been well applied in the determination of histidine, because these non-ratiometric probes are affected by autofluorescence, scattered light and excitation light intensity from the biological background, probe concentration and sample The influence of the environment makes these probes have low signal-to-noise ratio and accuracy in the process of detecting histidine in vivo and in vitro

Method used

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  • Ligand, preparation method thereof, fluorescent probe, and preparation method and application of fluorescent probe
  • Ligand, preparation method thereof, fluorescent probe, and preparation method and application of fluorescent probe
  • Ligand, preparation method thereof, fluorescent probe, and preparation method and application of fluorescent probe

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Embodiment 1

[0057] A ligand of the present invention: 2,2', 2", 2"'-(((4'-(4-((bis(pyridin-2-ylmethyl)amino)methyl)-1H- 1,2,3-triazol-1-yl)-[2,2':6',2"-terpyridine]-6,6'-diyl)bis(methylene))bis(azanetri Base)) tetraacetic acid (abbreviated DATP); DATP has the structural formula of following formula I:

[0058]

[0059] A synthetic method of the ligand of this embodiment, the synthetic route is as follows image 3 As shown, the specific process is as follows:

[0060] (1) Synthesis of 4'-azido-2,2':6',2"-biterpyridine-6,6"-dimethylaminetetraacetic acid tert-butyl ester (compound 2), the synthetic route is as follows figure 2 Shown:

[0061] 1.1. In a 100mL round bottom flask, add 20mL DMF, 825mg compound 1 (1.0mmol), 650mgNaN 3 (10mmol), 67.8mg tetrabutylammonium bisulfate (0.5mmol), under the protection of argon, reacted at 105°C for 24 hours to obtain the reaction product 1.

[0062] 1.2. Add a small amount of water to the reaction product 1 in step 1.1, extract with methyl tert...

Embodiment 2

[0090] A kind of fluorescent probe of the present invention: Ni 2+ -DATP-Eu 3+ / Tb 3+ , which has the structural formula of the following formula II:

[0091]

[0092] Ni in this example 2+ -DATP-Eu 3+ / Tb 3+ Prepared by the following method:

[0093] DATP (6.0μmol), EuCl 3 ﹒ 6H 2 O (2.0 μmol) and TbCl 3 ﹒ 6H 2 O (4.0μmol) was dissolved in 6.0mL of 10mM PBS buffer solution and stirred vigorously for 0.5h, then Ni was added 2+ (6.0μmol) after stirring for 0.5h, the probe Ni 2+ -DATP-Eu 3+ / Tb 3+ . Ni 2+ :DATP:Eu 3+ :Tb 3+ =3:3:1:2.

[0094] Fluorescent probe Ni 2+ -DATP-Eu 3+ / Tb 3+ To measure the fluorescence properties:

[0095] (1) Fluorescence spectrum

[0096] Add Ni to the 10mmol / L PBS buffer solution with a pH value of 7.4 2+ -DATP-Eu 3+ / Tb 3+ (Ni 2+ / DATP / Eu 3+ / Tb 3+ =3 / 3 / 1 / 2,C total =10 μM) and histidine concentrations of 0, 0.5, 1, 2, 3, 4, 10, 40, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μM were stirred at 37° C. for ...

Embodiment 3

[0110] A kind of fluorescent probe Ni of embodiment 2 2+ -DATP-Eu 3+ / Tb 3+ For the application of histidine time-resolved fluorescence imaging assay in Hela cells:

[0111] (1) Preparation of AM-DATP-Eu 3+ / Tb 3+ : put Ni 2+ -DATP-Eu 3+ / Tb 3+ DATP-Eu 3+ / Tb 3+ Reaction with bromomethyl acetate to generate AM-DATP-Eu 3+ / Tb 3+ , making it easier for the probe to enter living cells. The specific steps are: dissolve 15.5 μmol DATP in 210 μL anhydrous DMSO, then add 282 μmol (40 μL) triethylamine and 618 μmol (61 μL) bromomethyl acetate, respectively. After stirring at room temperature for 20 hours, centrifuge to remove trace insoluble matter, and then add 5.17 μmol EuCl 3 ·6H 2 O solid and 10.3 μmol TbCl 3 ·6H 2 O, the prepared solution is AM-DATP-Eu 3+ / Tb 3+ , the concentration is about 50mmol / L. Directly diluted with isotonic saline for cell culture.

[0112] (2) will contain 200M AM-DATP-Eu 3+ / Tb 3+ (DATP / Eu 3+ / Tb 3+ =3 / 1 / 2) after culturing Hela cel...

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Abstract

The invention discloses a preparation method of a ligand, a fluorescent probe, and a preparation method and an application of the fluorescent probe. The ligand is DATP, and is obtained by reacting a compound 1, NaN3, tetrabutylammonium hydrogen sulfate, bis(2-pyridylmethyl)amine, propargyl bromide, CuI, and a CH2Cl2 and CF3COOH mixed solvent. The fluorescent probe adopts DATP and Ni<2+> as cores,Eu<3+> and Ni<2+> are linked with the ligand through a Ni-N coordination bond, and Tb<3+> is linked with the ligand trough Tb-O and Tb-N coordination bonds. The fluorescent probe of the invention is prepared with DATP as the core, and a Eu<3+> / Tb<3+> complex can be used in time-resolved fluorescence detection to effectively eliminate interference from samples and short-lived fluorescence such as scattered light, so the prepared fluorescent probe has the advantages of good water solubility, extremely high sensitivity, excellent selectivity to histidine, simple preparation method and mild conditions, and is suitable for detecting histidine in biological systems.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a ligand, a preparation method, a fluorescent probe, a preparation method and an application. Background technique [0002] As an essential amino acid component in many proteins, histidine (His) has attracted much attention in recent years due to its important biological functions. Recent studies have shown that abnormalities in histidine levels are often associated with various diseases. For example, histidine deficiency may lead to rheumatoid arthritis, nerve deafness, liver cirrhosis, and lung disease. On the other hand, high levels of L-histidine in serum and urine may lead to metabolic disorders or histidineemia. Since histidine has such an important physiological role, accurate detection and quantification of histidine has become an important task in the field of biochemistry and clinical applications. [0003] At present, the methods used to detect histidine mainly include ...

Claims

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Application Information

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IPC IPC(8): C07D401/14C07F19/00C09K11/06G01N21/64A61K49/00
CPCA61K49/0021C07D401/14C07F15/045C09K11/06C09K2211/182C09K2211/187G01N21/6428
Inventor 刘宏伟张大平蔡正春
Owner 湖南艾科瑞生物工程有限公司
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