A kind of molecular assembly type fluorescent probe and its preparation method and application

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: 2022-07-05
WUHAN TEXTILE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A kind of molecular assembly type fluorescent probe and its preparation method and application
  • A kind of molecular assembly type fluorescent probe and its preparation method and application
  • A kind of molecular assembly type fluorescent probe and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Preparation method of molecular assembly type 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 of the solution to 1-2. Under stirring, slowly add 62ml of glyoxal (40% aqueous solution) dropwise to the solution with a dropping funnel. The rate of addition is about 3 seconds, and the temperature of the oil bath is controlled to be lower than 70°C. About 6h, the reaction ends. The reaction solution was filtered under reduced pressure, washed with water and acetone three times in turn, and vacuum-dried at 70° C. for 24 hours to obtain 55.8 g of glycoside urea. Weigh out 50.0 g of glycoside urea and dissolve it in 150 mL of concentrated hydrochloric acid. Stir at room temperature, place it in an ultrasonic disperser, take it out after 10 minutes to ensure that the glycoside urea is completely dissolved, then add 23.0 g of parafo...

Embodiment 2

[0053] The application of the molecular assembly-type fluorescent probe in identifying 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 the fluorescence spectrum of its emission under the condition of excitation wavelength of 361 nm, and record the maximum emission fluorescence intensity I of each part at 400-650 nm 0A , I 0B , I 0C ;

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

[0056] 3) Continue to add Lys3 solution dropwise to this solution, and the amount of dropwise addition is the same as step 2), and similarly record its maximum emi...

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 spectra were measured after adding different molar ratios of Lys2 and Lys to the fluorescent probe with other conditions unchanged. The fluorescence spectrum of Lys is shown in Image 6 shown. The obtained fluorescence response curve is as Figure 7 shown. Obviously, the Lys3 curve has the largest change trend. By calculation, I nA / I 0A =0.44. Thus, trimethyl-substituted Fmoc-protected lysines can be accurately and efficiently identified.

[0060] It is understandable that the guest molecule in Example 2, 3 or 4 was replaced with an unknown type of lysine, and its 1 n / I 0 If the value is less than or equal to 0.55, it can be judged that the solution contains Lys3.

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Abstract

The invention relates to a molecular assembly type fluorescent probe and a preparation method and application thereof. The fluorescent probe is formed by molecular assembly of Hoechst fluorescent dye 33258 and supramolecular cucurbituril. The specific preparation method is: use glycoluril and paraformaldehyde to polycondensate under acidic conditions to prepare CB[n], and then separate and purify the corresponding cucurbituril pure products according to their different solubility in water and hydrochloric acid of different concentrations. . Dissolve H33258 with water, add CB[n], make the molar ratio of H33258 and CB[n] be 1:1-2, stir, concentrate the solution by rotary evaporation, and then freeze-dry to obtain. The fluorescent probe can quickly and accurately identify Lys3 and P-Lys3, providing a new method for the detection of methylated lysine and methylated peptides with high sensitivity and high specificity, which is useful for in-depth study of protein methylation. The regulation of basalization in physiological processes is of great significance.

Description

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

Claims

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Application Information

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Patent Type & Authority Patents(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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