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SNAP-tag protein tag fluorescence probe with quick specific marking ability

A fluorescent probe and labeling technology, applied in the field of fluorescence imaging, can solve the problems of fast reaction speed, slow reaction speed, uncontrollable quantity and position, etc., and achieve the effect of easy purification and low-cost synthetic raw materials

Active Publication Date: 2019-03-01
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, organic small molecule dyes are exogenous species, and the problem is that they cannot be derived from cells like fluorescent proteins, so the number and position in cells cannot be controlled
Commercial SNAP-tag fluorescent substrates mainly connect fluorophores (such as rhodamine, cyanine dyes, etc.) to benzylguanine through longer chains, and the labeling reaction speed is fast. No significant changes, unresponsive or inaccurately localized fluorescent substrates must be eluted when imaging cells
It has been reported that several fluorescent probes can cause significant enhancement of fluorescence after being connected with SNAP-tag, which can be used for wash-free cell imaging, but the disadvantage of these probes is that the reaction speed is slow, which is slow compared with commercial products. 1-2 orders of magnitude

Method used

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  • SNAP-tag protein tag fluorescence probe with quick specific marking ability
  • SNAP-tag protein tag fluorescence probe with quick specific marking ability
  • SNAP-tag protein tag fluorescence probe with quick specific marking ability

Examples

Experimental program
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Effect test

Embodiment 1

[0029] A method for synthesizing a SNAP-tag protein-labeled fluorescent probe with specific and rapid labeling ability.

[0030] (1) Synthesis of intermediate 4-amino-N-(4-hydroxymethylbenzyl) 1,8-naphthalimide:

[0031] Put 4-bromo-N-(4-hydroxymethylbenzyl)1,8-naphthalimide (200mg, 0.50mmol) in a 100mL one-necked bottle, and add 10mL of N,N-dimethylformamide. Sodium azide (100 mg, 1.50 mmol) was dissolved in 1 mL of water and added to the mixture, and the reaction solution was heated to 90°C. After 6h, the heating was stopped, and the reaction solution was poured into ice water to settle and suction filtered to obtain a dark yellow solid. The resulting solid was dissolved in 50 mL of acetonitrile, and sodium sulfide nonahydrate (720 mg, 3.00 mmol) was added. The reaction solution was heated to 60°C for 12h. Silica gel column separation (200-300 mesh) and dichloromethane and methanol (200:1-100:1) were used as developing solvents to obtain 45 mg of a yellow solid with a yie...

Embodiment 2

[0040] A method for synthesizing a SNAP-tag protein-labeled fluorescent probe with specific and rapid labeling ability.

[0041] (1) Synthesis of intermediate 4-amino-N-(4-hydroxymethylbenzyl) 1,8-naphthalimide:

[0042] Put 4-bromo-N-(4-hydroxymethylbenzyl)1,8-naphthalimide (500mg, 1.25mmol) in a 100mL one-necked bottle, and add 50mL of N,N-dimethylformamide. Sodium azide (500 mg, 7.50 mmol) was dissolved in 2 mL of water and added to the mixture, and the reaction solution was heated to 100°C. After 8 hours, the heating was stopped, and the reaction solution was poured into ice water to settle and suction filtered to obtain a dark yellow solid. The resulting solid was dissolved in 80 mL of acetonitrile, and sodium sulfide nonahydrate (2500 mg, 10.4 mmol) was added. The reaction solution was heated to 70°C for 15h. Silica gel column separation (200-300 mesh) and dichloromethane and methanol (200:1-100:1) were used as developing solvents to obtain 200 mg of a yellow solid wi...

Embodiment 3

[0046] A method for synthesizing a SNAP-tag protein-labeled fluorescent probe with specific and rapid labeling ability.

[0047] (1) Synthesis of intermediate 4-amino-N-(4-hydroxymethylbenzyl) 1,8-naphthalimide:

[0048] Put 4-bromo-N-(4-hydroxymethylbenzyl)1,8-naphthalimide (1000mg, 2.50mmol) in a 100mL one-necked bottle, and add 75mL of N,N-dimethylformamide. Sodium azide (500 mg, 7.50 mmol) was dissolved in 2 mL of water and added to the mixture, and the reaction solution was heated to 110°C. After 10 h, the heating was stopped, and the reaction solution was poured into ice water to settle and suction filtered to obtain a dark yellow solid. The resulting solid was dissolved in 80 mL of acetonitrile, and sodium sulfide nonahydrate (3600 mg, 15.00 mmol) was added. The reaction solution was heated to 80°C for 18h. Silica gel column separation (200-300 mesh) and dichloromethane and methanol (200:1-100:1) were used as developing solvents to obtain 300 mg of a yellow solid wit...

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Abstract

The invention relates to an SNAP-tag protein tag fluorescence probe with quick specific marking ability. The fluorescence probe takes 4-amino-1,8-naphthalimide as a fluorophore and benzyloxy as a binding site. A structure of the fluorescence probe is shown as formula (1) as shown in the specification. The probe is derived from environment sensitive fluorophore naphthalimide, and can specifically react with an SNAP-tag protein; and the fluorescence intensity is increased by 12 times after a reaction. A reaction rate of the fluorescence enhanced SNAP-tag fluorescence probe reaches 13247-15625M<-1>s<-1>; the speed of the fluorescence probe is equivalent to that of a commercial fluorescence substrate requiring a cell elution step; and the fluorescence probe has the highest marking speed in fluorescence enhanced SNAP-tag fluorescence probes reported at present. The probe can achieve specific marking of a target protein fused with an SNAP-tag in a living cell in short time to achieve elution-free fluorescence imaging. The probe can be widely applied in the fields of protein marking, protein identification, interaction between the protein and micromolecules / macromolecules, cell fluorescence imaging and the like.

Description

technical field [0001] The invention belongs to the technical field of fluorescence imaging, in particular to a SNAP-tag protein label fluorescent probe with specific and fast labeling ability. Background technique [0002] Fluorescence imaging technology has gradually become a powerful tool for studying protein function from the cellular level to the individual level. Due to the advantages of small size, broad fluorescence emission spectrum, and a variety of fluorescent colors, organic small molecule fluorescent dyes have gradually become a substitute for fluorescent proteins in the field of protein labeling. However, organic small molecule dyes are exogenous species, and the problem is that they cannot be derived from cells like fluorescent proteins, so the number and position in cells cannot be controlled. In order to solve this problem, chemists have developed a variety of bioorthogonal methods to covalently attach small molecule dyes to target proteins, so that the loc...

Claims

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Application Information

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IPC IPC(8): C07D473/18C09K11/06G01N21/64
CPCC07D473/18C09K11/06C09K2211/1044G01N21/6428G01N2021/6432
Inventor 徐兆超乔庆龙苗露尹文婷
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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