A kind of high brightness, high stability wash-free snap-tag probe and its preparation method and application

A high-stability, high-brightness technology, applied in the field of fluorescent probes, which can solve the problems of fluorescence signal error, fluorescence accompanied by blue shift, and capture of fluorescence signal, etc.

Active Publication Date: 2022-03-18
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is usually a large blue shift in the fluorescence from the aqueous environment to the hydrophobic cavity of the SNAP-tag
In the complex environment of cells, it is difficult to use a suitable wavelength band to capture fluorescent signals, resulting in errors in fluorescent signals

Method used

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  • A kind of high brightness, high stability wash-free snap-tag probe and its preparation method and application
  • A kind of high brightness, high stability wash-free snap-tag probe and its preparation method and application
  • A kind of high brightness, high stability wash-free snap-tag probe and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Synthesis of Intermediate N-(4-Hydroxymethyl)benzyl-4-bromo-5-nitro-1,8-naphthalimide (BA-NBr)

[0045]

[0046] 4-Bromo-5-nitro-1,8-naphthalimide (1.00 g, 3.11 mmol) was dissolved in 50 mL of ethanol, and 4-aminomethylbenzyl alcohol (853 mg, 6.22 mmol) was added thereto. After 10 h at 80°C, the solvent was distilled off under reduced pressure, and the residue was separated through a silica gel column (petroleum ether: dichloromethane = 3:1-dichloromethane: methanol = 200:1, V / V) to obtain 480 mg of off-white solid. Yield 35%. Its nuclear magnetic spectrum hydrogen spectrum data are as follows:

[0047] 1 H NMR (400MHz, DMSO-d 6 )δ8.69(d, J=8.1Hz, 2H), 8.50–8.39(m, 2H), 7.35(d, J=8.1Hz, 2H), 7.25(d, J=7.9Hz, 2H), 5.23( s,2H),5.13(t,J=5.8Hz, 1H),4.45(d,J=5.5Hz,2H).

[0048] Synthesis of BA-DAC

[0049]

[0050] BA-NBr (200 mg, 0.45 mmol) was dissolved in 30 mL of ethylene glycol methyl ether, and 400 mg of 1,2-cyclohexanediamine was added thereto. The reaction...

Embodiment 2

[0063] Synthesis of Intermediate N-(4-Hydroxymethyl)benzyl-4-bromo-5-nitro-1,8-naphthalimide (BA-NBr)

[0064]

[0065] 4-Bromo-5-nitro-1,8-naphthalimide (1.00 g, 3.11 mmol) was dissolved in 20 mL of ethanol, and 4-aminomethylbenzyl alcohol (500 mg, 3.65 mmol) was added thereto. After 10 h at 80°C, the solvent was distilled off under reduced pressure, and the residue was separated through a silica gel column (petroleum ether: dichloromethane=3:1-dichloromethane:methanol=200:1, V / V) to obtain 480 mg of off-white solid. Yield 35%.

[0066] Synthesis of BA-DAC

[0067]

[0068] BA-NBr (200 mg, 0.45 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and 200 mg of 1,2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 110°C and reacted for 12h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=80:1, V / V) to obtain 90 mg of a yellow sol...

Embodiment 3

[0075] Synthesis of Intermediate N-(4-Hydroxymethyl)benzyl-4-bromo-5-nitro-1,8-naphthalimide (BA-NBr)

[0076]

[0077] 4-Bromo-5-nitro-1,8-naphthalimide (1.00 g, 3.11 mmol) was dissolved in 80 mL of ethanol, and 4-aminomethylbenzyl alcohol (2 g, 14.6 mmol) was added thereto. After 10 h at 90°C, the solvent was distilled off under reduced pressure, and the residue was separated through a silica gel column (petroleum ether: dichloromethane = 3:1-dichloromethane: methanol = 200:1, V / V) to obtain 480 mg of off-white solid. Yield 35%.

[0078] Synthesis of BA-DAC

[0079]

[0080] BA-NBr (200 mg, 0.45 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and 600 mg of 1,2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 110°C and reacted for 12h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=80:1, V / V) to obtain 90 mg of a yellow s...

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Abstract

The invention provides a high-brightness, high-stability wash-free SNAP-tag probe and its preparation method and application. The probe is designed and synthesized by introducing a rigid ring of cyclohexanediamine at the 4,5-position of naphthalimide A high-brightness, high-stability wash-free SNAP-tag probe, the structural formula of which is shown in (1). Due to the effect of aggregation-induced fluorescence quenching, the fluorescence brightness of the probe molecules in water is extremely low, but after combining with SNAP-tag, the fluorescence quantum yield can reach more than 0.80, and the fluorescence enhancement is about 28 times. Therefore, the probe can specifically label the target protein fused with SNAP-tag in living cells and realize wash-free fluorescence imaging. In addition, due to the improvement of stability and brightness, the probe realizes super-resolution fluorescence imaging (SIM / STED) with a resolution of 120nm.

Description

technical field [0001] The invention belongs to the field of fluorescent probes, and in particular relates to a high-brightness, high-stability wash-free SNAP-tag probe and a preparation method and application thereof. Background technique [0002] The SNAP-tag tag method has become the most widely used protein labeling technology at present. Through its fusion with the target protein, the target protein can be traced and function monitored. Therefore, with the help of rational design of organic small molecule dyes, multicolor fluorescent labeling of target proteins can be achieved, and real-time monitoring of proteins can be performed in the fields of single-molecule detection and super-resolution fluorescence imaging. This also requires small molecule-based SNAP-tag fluorescent probes to have high stability and a high fluorescence enhancement factor after binding to SNAP-tag to achieve precise imaging and high signal-to-noise ratio. [0003] However, the current commercia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D471/06C07D519/00C09K11/06G01N21/64
CPCC07D471/06C07D519/00C09K11/06G01N21/6428C09K2211/1007C09K2211/1029C09K2211/1044
Inventor 徐兆超乔庆龙
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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