Monitoring Dynamic Glycosylation in Vivo Using Supersensitive Click Chemistry

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• 作者：Hao Jiang ; Tianqing Zheng ; Aime Lopez-Aguilar ; Lei Feng ; Felix Kopp ; Florence L. Marlow ; Peng Wu
• 刊名：Bioconjugate Chemistry
• 出版年：2014
• 出版时间：April 16, 2014
• 年：2014
• 卷：25
• 期：4
• 页码：698-706
• 全文大小：490K
• 年卷期：v.25,no.4(April 16, 2014)
• ISSN：1520-4812

文摘

To monitor the kinetics of biological processes that take place within the minute time scale, simple and fast analytical methods are required. In this article, we present our discovery of an azide with an internal Cu(I)-chelating motif that enabled the development of the fastest protocol for Cu(I)-catalyzed azide鈥揳lkyne cycloaddition (CuAAC) to date, and its application toward following the dynamic process of glycan biosynthesis. We discovered that an electron-donating picolyl azide boosted the efficiency of the ligand-accelerated CuAAC 20鈥?8-fold in living systems with no apparent toxicity. With a combination of this azide and BTTPS, a tris(triazolylmethyl)amine-based ligand for Cu(I), we were able to detect newly synthesized cell-surface glycans by flow cytometry using as low as 1 nM of a metabolic precursor. This supersensitive chemistry enabled us to monitor the dynamic glycan biosynthesis in mammalian cells and in early zebrafish embryogenesis. In live mammalian cells, we discovered that it takes approximately 30鈥?5 min for a monosaccharide building block to be metabolized and incorporated into cell-surface glycoconjugates. In zebrafish embryos, the labeled glycans could be detected as early as the two-cell stage. To our knowledge, this was the first time that newly synthesized glycans were detected at the cleavage period (0.75鈥? hpf) in an animal model using bioorthogonal chemistry.