JACS: biorthogonal reaction of isonitrile chloroxime
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Last Update: 2019-12-07
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Source: Internet
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Author: User
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Biorthogonal reaction refers to the chemical reaction that can be carried out in living cells or tissues without interfering with biological biochemical reaction It was proposed by Carolyn R bertozzi in 2003 and can be used as a method of selectively labeling natural products and proteins for imaging Biomolecules with special functional groups are combined with the functional groups of probes to selectively label, visualize and / or separate cell components, such as sugars, lipids, nucleic acids and proteins The key point of the reaction is that the reaction is rapid and has chemical selectivity at low concentration, and is not affected by electrophilic and nucleophilic reagents in biological molecules The reactants and products are stable under physiological conditions, and the product volume is small and has chemical inertia, so as to avoid interfering with the activity of the target biological fraction Recently, Professor helma wennemers of Swiss Federal Institute of technology proposed a biorthogonal reaction based on isocyanide and chloroxime (Figure 1) The reactant has small size, fast reaction rate and good stability The orthogonality of strain promoted azido alkyne cycloaddition reaction (spaac) was confirmed, and the marker of cell membrane metabolized glycan was realized Relevant achievements were published on J am Chem SOC Under the title of "the biological isonitrile − chlorooxime lithium" (DOI: 10.1021 / JACS 9b07632) (source: J am Chem SOC.) isonitrile is a frequent guest of biorthogonal reactions It has a small structure and good pH stability It is often used in biorthogonal reactions with tetrazine molecules The author chooses the reaction marker of isonitrile and chloroxime to produce α - hydroxyiminamide The author speculates that under physiological conditions, chloroxime spontaneously forms the intermediate nitrile oxide I, which reacts with isonitrile to form nitrile ion II, and finally forms the required product (eq.1) under the action of water (source: J am Chem SOC.) firstly, the author evaluated the reactivity of isonitrile and chloroxime by the reaction of isonitrile 1, a lysine derivative, with n-hydroxybenzoyl chloride 2 In THF solution, the reaction did not take place; with the addition of water (PBS solution with pH 7.4), the reaction was completed within 2 hours, and the 4.8mm reactant was rapidly converted into α - hydroxyiminamide (Table 1, entries 1, 2) The results show the importance of water for the reaction Then, the concentration of reactants was reduced to micromol level, and the reaction was still carried out with high conversion (Table 1, entries 3, 4); under physiological temperature (37 ℃), the reaction speed was accelerated (Table 1, entry 5) Then, the author added 1 equivalent carboxylic acid, amine, amide, imidazole, indole, alcohol and olefin respectively to explore their chemical selectivity Surprisingly, the addition of these substances did not affect the occurrence of the reaction (Table 1, entries 6-12) However, mercaptans such as glutathione (GSH) interfere with the reaction, but recover at pH 5 (Table 1, entries 13, 14) (source: j.am Chem SOC.) then, the author determined the rate constant of the reaction by real-time monitoring the formation of α - hydroxyiminamide 3 by HPLC (Figure 2) The analysis of reaction kinetics shows that the ratio of the concentration of isonitrile 1 to the reaction rate and the concentration of chlorooxime 2 (rate / [2]) is linear, which is the second-order kinetic law (Figure 2b) At 37 ℃, the second-order kinetic constant k = 1.04 M-1 s-1 The two order law of dynamics shows that isocyanide and chloroxime participate in the speed limiting step, so the formation of C-C bond is the speed limiting factor Therefore, the author speculates that nitrile II is a highly reactive intermediate, and hydration occurs rapidly, which is consistent with the necessity of water as a solvent (source: J am Chem SOC.) in order to further determine the feasibility of isonitrile chloroxime reaction in complex cell environment, the researchers selected N-acetylmannosamine derivatives to label cell surface glycans for metabolic labeling test Peracetylation can enhance the absorption of N-acetylmannosamine, which is converted into labeled neuraminic acid under the action of cellolactase and aldolase and integrated into glycoconjugate Therefore, the author has synthesized the peracetylated n-isocyanopropyl mannosamine 4 (AC 4 mannc), whose isonitrile part can form a complementary probe (Figure 3a) with the biotin sulfonated chloroxime 5 Different concentrations of mannosamine 4 were added to the medium of chok1 cells and incubated with probe 5 (10 μ m, 5 min) after 3 days Compared with the control group without 4 co incubation (the smallest background fluorescence signal), the cell membrane of the experimental group showed strong green fluorescence (Figure 3B) The quantitative analysis of fluorescence intensity by flow cytometry showed that probe 5 had the strongest fluorescence signal after being treated with 300 μ M 4, and showed concentration dependence (Figure 3C) The cell activity experiment showed that the concentration of 300 μ m had little toxicity to cells (Figure 3D) All the above results show that the reaction of isocyanide and chloroxime is valuable in living cell imaging (source: J am Chem SOC.) finally, the author evaluated the compatibility of isonitrile chloroxime reaction with other chemical selective reactions We synthesized azide N-acetylmannosamine 4a (AC 4 mann3) and incubated chok1 cells with isonitrile 4 and azide 4a Three days later, Dibo with red fluorescence group alexafluor 647, chloroxime 5 with biotin sulfonation and avidin with green fluorescence group alexafluor 488 were added to test whether 4A and 4 were fused in the cell membrane It is expected that both green and red fluorescent clusters mark the cell membrane (Figure 4), demonstrating the importance of the double labeling produced by the isonitrile chloroxime linkage (source: J am Chem SOC.) in a word, the coupling of isonitrile and chloroxime is a significant biorthogonal reaction, which has the advantages of high conversion rate, good chemical selectivity, small structure and stability This reaction realized the marker imaging of metabolic glycan on cell membrane, and provided a certain basis for the application of isonitrile in chemical biology such as biological double marker.
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