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Citrulline probe compound and application thereof

A compound, citrulline technology, applied in the field of citrulline probe compounds, can solve problems such as inability to apply cells, poor citrulline detection effect, and hindering probe application.

Active Publication Date: 2020-04-07
GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these studies have greatly helped us understand citrullinated proteins, the strong acidic reaction conditions are still a huge problem, which greatly hinders the application of these probes.
The main problems brought about by strong acidic conditions are: 1) Since the labeling reaction must be carried out under strong acidic conditions, it can only be used for a single protein or cell lysate, and cannot be applied to cells; 2) A considerable part of the fluorescence The group is pH-sensitive, and the strong acidic reaction conditions make the combination of phenylglyoxal compounds and fluorescent groups in the detection of citrulline poor.

Method used

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  • Citrulline probe compound and application thereof
  • Citrulline probe compound and application thereof
  • Citrulline probe compound and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Synthesis of compound 2-GBA

[0049]

[0050] Dissolve 2-acetylphenylboronic acid (100 mg, 0.6 mmol) in 10 mL DMSO / H 2 In a mixed solvent of O (1:1), iodine (228mg, 0.9mmol) was then added, and heated to 100°C for 3 hours. Stirring was stopped, the reaction was removed, quenched with 0.2M aqueous sodium thiosulfate solution, extracted three times with 40 mL ether, the organic phases were combined, washed once with saturated aqueous NaCl solution, and dried over anhydrous sodium sulfate. The solvent was spin-dried on a rotary evaporator to obtain a crude yellow solid. Recrystallized from water to obtain 56 mg of pure yellow product with a yield of 51%. NMR: 1 H NMR (400MHz,D 2 O / CD 3 CN=5:1)δ / ppm:7.97-7.95(d,J=7.6Hz,1H),7.86-7.85(d,J=7.2Hz,1H),7.80-7.76(m,1H),7.71-7.67 (m,1H),5.62(s,1H); 1 H NMR (400MHz,d 6 -DMSO)δ / ppm: 9.23 (s, 1H), 7.91-7.87 (m, 2H), 7.79-7.69 (m, 2H), 7.64-7.62 (d, J=7.6Hz, 1H), 5.42-5.40 ( d,J=7.6Hz,1H); 1 H NMR (400MHz, CD 3 OD)δ / ppm: ...

Embodiment 2

[0053] The synthetic route of the compound shown in 2-GBA-Biotin is as follows:

[0054]

[0055] The preparation method of the compound shown in 2-GBA-Biotin comprises the following steps:

[0056] (1) Preparation of compound 2:

[0057] Add 2-4 dihydroxyacetophenone (500mg, 3.28mmol), anhydrous potassium carbonate (681mg, 4.92mmol) and 1,2-dibromoethane (924mg, 4.92mmol) into 25mL of acetonitrile and heat to 40 °C, stirred for 12 hours. Stop heating, let it cool to room temperature, add 50 mL of water, and then extract with dichloromethane (50 mL×3), collect and combine the organic phases. followed by H 2 O. Wash once with saturated NaCl aqueous solution, then add anhydrous NaCl 2 SO 4 After drying, filtering and rotary evaporation under reduced pressure, the crude product was obtained as a white powdery solid. Separation and purification by 200-300 mesh silica gel column chromatography [eluent: V (petroleum ether): V (ethyl acetate) = 15: 1] to obtain 463 mg of pur...

Embodiment 3

[0073] 1. The reaction of the compound shown in 2-GBA with citrulline:

[0074] The compound shown in 2-GBA or phenylglyoxal and L-citrulline are reacted under neutral conditions, and the reactivity is tested, and the reaction conditions are: 2-GBA or phenylglyoxal (1.1 equivalents) and L-citrulline (1.0 equiv) in 1X PBS and CH 3 Mixed solution of CN (PBS and CH 3 The volume ratio of CN is: 3:1), reacted at 37°C for 3h, and tested the 1H NMR spectra of the reaction products (2-GBA+L-citrulline and phenylglyoxal+L-citrulline), and the 1H NMR spectrum of pure L-citrulline, see Figure 5 . from Figure 5 It can be seen that in 2-GBA+L-citrulline, the signal peak of L-citrulline is gradually disappearing, and two new peaks appear at the same time, suggesting that a chemical reaction occurs. And under the same conditions, there is no change in the 1H NMR comparison spectrum of phenylglyoxal and citrulline reaction, which is also consistent with existing reports, indicating tha...

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Abstract

The invention discloses a compound which is shown as a formula 2-GBA, 2-GBA-1, 2-GBA-N3, 2-GBA-N3-1, 2-GBA-Biotin or 2-GBA-Biotin-1. The compound shown as 2-GBA can react with citrulline under a neutral condition, namely, the compound can remove protein of citrulline like an antibody. Azido and alkynyl substitution are carried out on a parent nucleus of the compound shown as 2-GBA, biotin and fluorescein are introduced through click reaction, and the prepared compound shown as 2-GBA-Biotin can be used for labeling and detecting citrullinated protein. The invention also discloses application ofthe compound.

Description

technical field [0001] The invention relates to the technical field of chemical and biological detection, in particular to a citrulline probe compound and its application. Background technique [0002] Protein post-translational modification is an epigenetic regulation of protein function, which plays an important role in the life activities of cells. Among them, arginine is an important amino acid, and there are mainly three known post-translational modifications: methylation modification, demethylation modification and citrullination modification. Such as figure 1 As shown, the citrullination modification of arginine residues on proteins mainly refers to the conversion of a positively charged guanidine group to The conversion of functional groups to electrically neutral urea functional groups is a process by which arginine residues are converted to citrulline residues. [0003] At present, it is generally believed that the abnormality of protein citrullination level is ...

Claims

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Application Information

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IPC IPC(8): C07F5/02C09K11/06G01N21/64
CPCG01N21/6428C07F5/02C09K11/06G01N2021/6432
Inventor 张静
Owner GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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