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Fluorescent probe for intracellular protein labelling as well as synthesis method and application of fluorescent probe

A technology of fluorescent probe and synthesis method, which is applied to the fluorescent probe for intracellular protein labeling and its synthesis and application fields, can solve the problems of inability to achieve satisfactory results, reduced cell permeability, long incubation time, etc., and achieves important goals. The effect of application value, good photostability and simple steps

Active Publication Date: 2018-05-25
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, most probes can only moderately enhance the fluorescence during the labeling process and cannot achieve satisfactory results.
In addition, FRET-based fluorescent probes are generally complex to synthesize, and the larger molecular size reduces cell permeability resulting in longer incubation times

Method used

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  • Fluorescent probe for intracellular protein labelling as well as synthesis method and application of fluorescent probe
  • Fluorescent probe for intracellular protein labelling as well as synthesis method and application of fluorescent probe
  • Fluorescent probe for intracellular protein labelling as well as synthesis method and application of fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Synthesis of novel fluorescent probes for protein labeling.

[0049] Synthesis of intermediate 4-azido-1,8-naphthalene anhydride:

[0050] 4-Bromo-1,8-naphthalene anhydride (2.5 g, 9 mmol) was placed in a 100 mL one-necked flask, and 20 mL of N,N-dimethylformamide was added. Sodium azide (1.8g, 27.7mmol) was dissolved in 3.5mL of water and added dropwise to the reaction solution, heated to 100°C for 6h, cooled, poured into ice water for suction filtration, and dried in vacuo to obtain 2g of a dark yellow solid, yielding The rate is 92%.

[0051]Synthesis of intermediate 4-amino-1,8-naphthalene anhydride:

[0052] 4-Azido-1,8-naphthalene anhydride (1.8 g, 7.6 mmol) was dissolved in 100 mL of acetonitrile, and sodium sulfide nonahydrate (6.5 g, 41 mmol) was added. Heating to 60° C. for 10 h, cooling, pouring into ice water, suction filtration, and vacuum drying gave 1.3 g of a yellow solid with a yield of 80%.

[0053] Synthesis of intermediate 4-(2-chloroa...

Embodiment 2

[0060] Example 2: MALDI-TOF mass spectrometry before and after the probe reacts with the SNAP protein

[0061] Add 7.5 μL of probe solution (the mother solution is 2 mM DMSO solution) to 1 mL of SNAP protein containing 5 μM, 20 mM, pH=7.4 in PBS solution, stir at 37 ° C, and use an 8000 Da dialysis bag at 20 mM, pH=7.4 after 2 hours Dialyze in PBS solution to remove excess probes, take 10 μL of reaction solution and 10 μL SNAP protein for MALDI-TOF-MS respectively.

[0062] Figure 5 It shows that the molecular weight of SNAP protein is 21634.03, and a single peak appears in MALDI-TOF-MS after reacting with the fluorescent probe, which proves that the SNAP protein reacts completely, and the molecular weight is 22187.52, which is the same molecular weight as the reaction product of SNAP protein and probe in theory. Demonstrate that the SNAP protein covalently reacts with the probe.

Embodiment 3

[0063] Example 3: Fluorescence spectra before and after the probe reacts with the SNAP protein

[0064] Add 1 μL probe mother solution to 5 μM, 2 mL SNAP protein PBS solution, react at 37 ° C for 1 h, and then detect its fluorescence spectrum, and use the fluorescence spectrum of unreacted 1 μM probe as a control.

[0065] Figure 6 The concentration of the fluorescent probes in the medium was 1 μM, and after the probes reacted with the SNAP protein, the ～ The fluorescence intensity at 510nm was significantly enhanced, about 10 times.

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Abstract

The invention provides a fluorescent probe for intracellular protein labelling as well as a synthesis method and an application of the fluorescent probe. The provided probe is synthesized in simple steps and has good light stability. Compared with existing fluorescent probes for cell labeling, the probe can be specifically bound with SNAP labels in a complicated system and label any protein in cells. The probe can be applied to detection of copper ions in the cells, fluorescence gradually disappears with increase of the concentration of the copper ions, and the copper ion detection function isrealized. The probe realizes labeling of any protein and detection of the copper ions in the complicated environment and has very important application value in the biological and medical fields.

Description

technical field [0001] The invention relates to a fluorescent probe for intracellular protein labeling, its synthesis method and application. Background technique [0002] Using small molecule fluorescent probes to label proteins to study the location, function, and communication between proteins in living cells is an advanced technology that has been widely studied. Compared with fluorescent proteins, protein labeling technology shows the superior photophysical properties of fluorescent probes, and the precise control of the position and time of labeling. However, in order to achieve higher signal intensity and improve signal-to-noise ratio, the step of washing out unreacted probe molecules is essential, which limits its continuous observation of cells. [0003] Fluorescence-enhanced fluorescent probes that label proteins by labeling them with a protein tag not only reduce the background signal, but also greatly improve the signal-to-noise ratio. Nevertheless, most of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D473/18C09K11/06G01N21/64
CPCC07D473/18C09K11/06C09K2211/1044G01N21/6486
Inventor 徐兆超苗露赵秒冷双
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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