Molecular assembly type fluorescent probe as well as preparation method and application thereof

A fluorescent probe and molecular technology, applied in the field of biochemistry, can solve the problems of affecting the analysis sensitivity, reducing the hydrogen bond force of amino acid residues, and the difficulty in identifying methylated peptides, achieving fast detection speed and high detection accuracy Effect

Active Publication Date: 2021-07-23
WUHAN TEXTILE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it is still difficult to identify methylation sites at the peptide level, mainly because the introduction of methyl groups in the peptide chain increases the steric hindrance of methylated amino acid residues and reduces the hydrogen bond force of amino acid residues. In addition, it does not significantly change the net charge or isoelectric point of amino acid residues, which makes the identification of methylated peptides more difficult and greatly affects the sensitivity of the analysis.

Method used

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  • Molecular assembly type fluorescent probe as well as preparation method and application thereof
  • Molecular assembly type fluorescent probe as well as preparation method and application thereof
  • Molecular assembly type fluorescent probe as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Preparation method of molecular assembly fluorescent probe

[0050] First, prepare cucurbituril CB[n]:

[0051] Weigh 80.0 g of urea, stir to dissolve it in 250 mL of deionized water, and use concentrated sulfuric acid to adjust the pH value of the solution to 1-2. Under stirring, slowly add 62ml of glyoxal (40% aqueous solution) dropwise to the solution with a dropping funnel, at a rate of about 3 seconds per drop, control the temperature of the oil bath to be lower than 70°C, and raise the temperature to 75°C after the addition is completed About 6h, the reaction is over. The reaction solution was filtered under reduced pressure, washed with water and acetone three times in sequence, and dried in vacuum at 70° C. for 24 hours to obtain 55.8 g of glycoside urea. Weigh 50.0g of glycoside urea and dissolve it in 150mL of concentrated hydrochloric acid. Stir at room temperature and place in an ultrasonic disperser, take it out after 10 minutes to ensure that the glycos...

Embodiment 2

[0053] The application of the molecular assembly fluorescent probe in the recognition of trimethyl-substituted Fmoc-protected lysine, the specific operation steps are as follows:

[0054] 1) First configure 3 parts of fluorescent probe aqueous solution with a concentration of 3.0 μM, measure its emitted fluorescence spectrum at an excitation wavelength of 361 nm, and record the maximum emitted fluorescence intensity I at 400-650 nm for each part 0A , I 0B , I 0C ;

[0055] 2) Add Lys3 dropwise to any of the solutions and mix evenly to ensure that the molar ratio of the added Lys3 to the fluorescent probe is 0.3, measure its emitted fluorescence spectrum at an excitation wavelength of 361nm, and record it at 400 nm. Maximum emission fluorescence intensity I at -650nm 1A ;

[0056] 3) continue to drip Lys3 solution in this solution, the amount of dripping is identical with step 2), same record its maximum emission fluorescence intensity I at 400-650nm place 2A , repeated n ...

Embodiment 3-4

[0059] According to Example 2, the guest molecule Lys3 in step 2) was replaced by Lys2 and Lys respectively, and the fluorescence spectrum was measured after adding Lys2 and Lys in different molar ratios to the fluorescent probe while other conditions remained unchanged. The fluorescence spectrum of Lys is as follows Figure 6 shown. The obtained fluorescence response curve is as follows Figure 7 shown. Obviously, the variation trend of Lys3 curve is the largest, by calculation, I nA / I 0A = 0.44. Therefore, the trimethyl-substituted Fmoc-protected lysine can be accurately and effectively identified.

[0060] It can be understood that the guest molecule in Example 2, 3 or 4 is replaced by an unknown type of lysine, and its I n / I 0 value, if the value is less than or equal to 0.55, it can be judged that Lys3 is included in the solution.

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Abstract

The invention relates to a molecular assembly type fluorescent probe as well as a preparation method and an application thereof. The fluorescent probe is formed by molecular assembly of a Hurst fluorescent dye 33258 and supramolecular cucurbituril. The specific preparation method comprises the following steps: performing condensation polymerization on glycoluril and paraformaldehyde under an acidic condition to prepare CB[n], and separating and purifying according to different solubility of CB [n] in water and hydrochloric acid with different concentrations to obtain respective corresponding cucurbituril pure products; dissolving H33258 in water, adding CB[n] to enable the molar ratio of H33258 to CB[n] to be 1: (1-2), stirring, carrying out rotary evaporation to concentrate a solution, and then freeze-drying to obtain the product. The fluorescent probe can quickly and accurately recognize Lys3 and P-Lys3, provides a brand-new detection means with high sensitivity and high specificity for methylated lysine and methylated peptide, and has great significance for deeply researching the regulation effect of protein methylation modification in the physiological process.

Description

technical field [0001] The invention relates to the technical field of biochemistry, in particular to a molecular assembly fluorescent probe and a preparation method and application thereof. Background technique [0002] Lysine methylation is the transfer of one to three methyl groups from S-adenosylmethionine (SAM) to lysine ε under the catalysis of lysine methyltransferases (KMTs) - Amine side chains form monomethylation (Me1), dimethylation (Me2), and trimethylation (Me3), respectively, and are one of the most common post-translational modifications of proteins. Lysine methylation plays an important role in the regulation of many physiological processes. Studies have shown that lysine methylation modification in proteins can regulate the intramolecular or intermolecular interactions of target proteins, affect their affinity with RNA, and thus affect various cellular processes, such as transcription regulation, cell localization, ribosome Assembly, RNA processing, matura...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06G01N21/64
CPCC09K11/06G01N21/6428G01N2021/6417C09K2211/1044C09K2211/1007C09K2211/1074Y02P20/55
Inventor 卿光焱陈缘愿朱志超
Owner WUHAN TEXTILE UNIV
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