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Fluorescence-labeled O6-benzyl guanine and preparation and application thereof

A technology of benzylguanine and fluorescent labeling, applied in fluorescence/phosphorescence, biochemical equipment and methods, luminescent materials, etc., can solve the complex preparation process of labeled DNA and labeled oligonucleotides, difficult long-term storage, and membrane permeability gender issues

Inactive Publication Date: 2012-05-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although these two methods have high sensitivity, due to the use of radioactive isotopes, the experimental conditions are relatively high, and must be carried out in professional laboratories; in addition, radioactive isotopes are extremely harmful to the environment; and However, as biomolecules, DNA and oligonucleotides have poor cell membrane permeability, so that labeling experiments can only be carried out in cell homogenate; secondly, the preparation process of labeled DNA and labeled oligonucleotides is complicated, poor in stability, and not easy to carry out for a long time. preservation, these restrict the wide application of the above two methods
[0008] Although the monoclonal antibody method avoids the application of radioactive isotopes, antibodies, as biomolecules, also have the disadvantages of poor cell membrane permeability and poor stability; more importantly, antibodies bind to proteins by recognizing epitopes, so the results do not reflect protein functional status of
[0009] The above three detection methods are all limited in application in living cells due to the shortcomings of large volume of detection reagents and poor membrane permeability.

Method used

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  • Fluorescence-labeled O6-benzyl guanine and preparation and application thereof
  • Fluorescence-labeled O6-benzyl guanine and preparation and application thereof
  • Fluorescence-labeled O6-benzyl guanine and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of ethyl 4-((2-(2-(2-azidoethoxy) ethoxy) ethoxy) methyl) benzoate (IIc)

[0041] Weigh 2-(2-(2-azidoethoxy)ethoxy)ethanol (1.060 g, 6 mmol), dissolve in dry tetrahydrofuran (25 mL), add 1 equivalent of NaH, stir at room temperature for 5 minutes, add 1 Equivalent of ethyl p-chloromethylbenzoate (1.200 g, 6 mmol), continued to react at room temperature for 3 hours, quenched with water (10 mL), the crude product was extracted with ethyl acetate (50 mL), and the organic phase was Dry over anhydrous sodium sulfate for 10 minutes, concentrate, and purify by silica gel column chromatography, eluting with petroleum ether / ethyl acetate (3:1), to obtain a colorless oil (0.950 g, 2.8 mmol), with a yield of 47% .

[0042] δ H (500 MHz, CDCl 3 ) 8.02 (2 H, d, J =8.0), 7.41 (2 H, d, J =8.0), 4.63 (2 H, s), 4.37 (2 H, q, J =7.1), 3.73 – 3.64 (10 H, m), 3.38 (2 H, t, J =5.0), 1.39 (3 H, t, J =7.1).

Embodiment 2

[0043] Example 2: Preparation of ethyl 3-((2-(2-(2-azidoethoxy)ethoxy)ethoxy)methyl)benzoate (IIh)

[0044] Weigh 2-(2-(2-azidoethoxy)ethoxy)ethanol (1.260 g, 7.2 mmol), dissolve in dry tetrahydrofuran (35 mL), add 1 equivalent of NaH, stir at room temperature for 5 minutes, add 1 equivalent of ethyl m-bromomethylbenzoate (1.750 g, 7.2 mmol), continued to react at room temperature for 3 hours, quenched with water (10 mL), the crude product was extracted with ethyl acetate (50 mL), and the organic phase After drying over anhydrous sodium sulfate for 10 minutes, it was concentrated and purified by silica gel column chromatography, eluting with petroleum ether / ethyl acetate (3:1), to obtain a colorless oil (1.400 g, 4.1 mmol), with a yield of 58 %.

[0045] δ H (500 MHz, CDCl 3 ) 8.01 (1 H, s), 7.96 (1 H, d, J =7.7), 7.56 (1 H, d, J =7.7), 7.42 (1 H, t, J =7.7), 4.61 (2 H, s), 4.38 (2 H, q, J= 7.1), 3.72 – 3.65 (10 H, m), 3.38 (2 H, t, J =5.1), 1.40 (3 H, t, J =...

Embodiment 3

[0046] Example 3: Preparation of 4-(2-(2-(2-(trifluoroacetamido)ethoxy)ethoxy)ethoxy)methyl-benzyl alcohol (IIIc)

[0047] Weigh 4-((2-(2-(2-azidoethoxy)ethoxy)ethoxy)methyl)ethyl benzoate (0.950 g, 2.8 mmol), dissolve in dry tetrahydrofuran (40 ml ), add 1.5 equivalents of LiAlH 4 (0.160 g, 4.2 mmol), the system was stirred and reacted under reflux for 3 hours, then the heating was stopped, and after it was naturally cooled to room temperature, it was quenched by adding water (5 mL), and the product was extracted with ethyl acetate (50 mL), acetic acid The ethyl ester phase was washed once with saturated brine (10 mL), dried by adding anhydrous sodium sulfate for 10 minutes, filtered, and the filtrate was concentrated under reduced pressure to obtain a light yellow oil. The oil was then dissolved in absolute ethanol (20 mL), and 1 equivalent of triethylamine (0.280 g, 2.8 mmol) and 1 equivalent of ethyl trifluoroacetate (0.400 g, 2.8 mmol) were added successively, and the r...

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Abstract

The invention provides a fluorescence-labeled O6-benzyl guanine. A 1,3,5,7-tetramethyl-8-butyryl-BODIPY fluorescent group is introduced into a para-position or a meta-position of benzyl of O6-benzyl guanine through ether coupling chains of different lengths. The invention provides a small molecular fluorescent probe for marking functional MGMT (O6-Methylguanine-DNA Methyltransferase) proteins in living cells. Probe molecules are covalent inhibitors designed according to the catalyzing mechanism of MGMT, have high cell membrane permeability, can be used for inhibiting MGMT in cells and performing fluorescence labeling on the MGMT, and can be applied to preparation of probe molecules for marking living MGMT in living cells. A structural general formula is shown in the specifications.

Description

technical field [0001] The invention belongs to chemical medicine, and relates to a small molecular fluorescent probe for marking functional MGMT protein in living cells, a preparation method thereof, and a marking method using the same. This type of probe is O 6 -Benzylguanine compound, which has good cell membrane permeability and selectivity to functional MGMT protein. In living cells, they can be combined with the functional MGMT protein, and the MGMT protein then catalyzes the covalent binding of the BODIPY-benzyl moiety in the molecular structure of the probe to the sulfhydryl group of cysteine ​​145 in MGMT, resulting in a functional state. MGMT is inactivated while being labeled due to the acquisition of the BODIPY fluorophore and can be detected qualitatively and quantitatively. Background technique [0002] In recent years, the incidence of malignant tumors has continued to increase, and it has become one of the biggest killers that seriously threaten human life....

Claims

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Application Information

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IPC IPC(8): C07F5/02C09K11/06C12Q1/02G01N21/64
Inventor 李新胡永洲杨波何俏军钱石静
Owner ZHEJIANG UNIV
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