Monitoring Dynamic Glycosylation in Vivo Using Supersensitive Click Chemistry

Abstract

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–alkyne 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 20–38-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 30–45 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 (0.75–2 hpf) in an animal model using bioorthogonal chemistry.

CalFluors: A Universal Motif for Fluorogenic Azide Probes across the Visible Spectrum

“…With recent advances in increasing the efficiency of Cu-catalyzed click reactions, these technologies will further enhance the ease and sensitivity of detecting linear alkyne-functionalized biomolecules in biological systems. 47–49 …”

Highly accelerated inverse electron-demand cycloaddition of electron-deficient azides with aliphatic cyclooctynes

“…The latter mode of reactivity enabled the development of highly accelerated SPAAC by means of introduction of electron-withdrawing substituents on the aromatic azide, thereby leading to unprecedented reaction rates (42 M À 1 s À 1 ), up to 30-fold faster than 'traditional' SPAAC. In this respect, use of IED SPAAC also compares favourably with the so-called supersensitive copper-catalysed click chemistry, which can be accelerated only 4-6 times by including picolyl azides instead of regular benzyl azides 34,35 . It must be noted that several research groups reported on a sizable effect of electronegative substituents on aryl azides in cycloaddition with alkenes already 50 years ago 23,36,37 .…”

Click Chemistry in Complex Mixtures: Bioorthogonal Bioconjugation

“…Chelation-assisted copper catalysis is a recent and complementary approach to ligand-based acceleration (Brotherton et al, 2009; Kuang et al, 2011; Kuang et al, 2010), designed to enhance the “weakest link” in the CuAAC mechanism: the association of azide with the metal center (Rodionov et al, 2007a). For example, a bidentate picolyl azide (shown in Figure 4D) was used in combination with BTTAA for site-specific conjugations of proteins on the surface of live cells with metabolically labeled RNA and protein molecules, providing 25-fold enhancement in specific labeling rate relative to conventional non-chelating azides at low copper concentrations (10–100μM) (Jiang et al, 2014; Uttamapinant et al, 2013; Uttamapinant et al, 2012). Azides bearing tighter binding ligands such as tetradentate bis(triazolyl)amino azide, have also exhibited outstanding reactivity with alkynes under dilute conditions in the presence of complex cellular media, and was used for the tracking of paclitaxel inside living cells (Bevilacqua et al, 2014).…”