Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells

Abstract

Developing tools for investigating the cellular activity of glycans will help to delineate the molecular basis for aberrant glycosylation in pathological processes such as cancer. Metabolic oligosaccharide engineering, which inserts sugar-reporting groups into cellular glycoconjugates, represents a powerful method for imaging the localization, trafficking, and dynamics of glycans and isolating them for glyco-proteomic analysis. Herein, we show that the alkyne-reporting group can be incorporated into cellular glycans. The alkyne group is a small, inert, bio-orthogonal handle that can be chemoselectively labeled by using the Cu(I) catalyzed [3 ؉ 2] azide-alkyne cycloaddition, or click chemistry. Alkynyl sugar monomers, based on fucose (Fuc) and N-acetylmannosamine (ManNAc), were incorporated into fucosylated and sialylated glycans in several cancer cell lines, allowing for cell surface and intracellular visualization of glycoconjugates, as well as, observation of alkyne-bearing glycoproteins. Similarly to our previous results with an azido Fuc/alkynyl probe system, we demonstrated that click-activated fluorogenic probes are practical tools for efficiently and selectively labeling alkynyl-modified glycans. Because Fuc and sialic acid are terminal glycan residues with a notably increased presence in many tumors, we hope that our method will provide useful information about their roles in cancer and ultimately can be used for diagnostic and therapeutic purposes.click chemistry ͉ fluorescent imaging ͉ fucose ͉ sialic acid

Tissue-based metabolic labeling of polysialic acids in living primary hippocampal neurons

“…On the other hand, Bertozzi and coworkers (19)(20)(21)(22) metabolically introduced monosaccharidebased chemical reporters-N-acyl derivatives of ManNAc, N-acetyl-D-glucosamine, or N-acetyl-D-galactosamine containing a ketone or azide group-onto cellular surface glycans followed by bioorthogonal, chemoselective coupling with a fluorescent dye or an affinity tag bearing hydrazide/aminooxy (for ketones) or phosphine/alkyne (for azides) group. This elegant methodology, combining metabolic engineering and bioorthogonal reactions, enabled in situ imaging or proteomic enrichment of one glycan type (19)(20)(21)(22) and has been applied to many different cell lines (e.g., Jurkat, HeLa, CHO, and neuron-like blastoma cells) and organisms (e.g., zebrafish, mice, and microbes) for the selective labeling of sialic acid (23)(24)(25), N-acetyl-D-galactosamine residue (in mucin-type O-linked glycan) (26)(27)(28), fucose residue (29)(30)(31)(32), and LPSs/O-antigen (33,34). Although there are innumerable previous reports, the metabolic labeling and the imaging of glycan structures in primary neurons have yet to be achieved, although the surface glycans, especially PSA-NCAMs, play important roles in neuronal development.…”

Copper-free click chemistry for dynamic in vivo imaging

“…1A). Click chemistry has prevailed in applications where toxicity is irrelevant, such as in probing enzyme activities in cell lysates (11) or visualizing biomolecules in fixed cells (12,13). However, dynamic processes in living systems are inaccessible to click chemistry because the reaction requires a cytotoxic Cu catalyst.…”

Construction of Polymer–Protein Bioconjugates with Varying Chain Topologies: Polymer Molecular Weight and Steric Hindrance Effects

“…Previously, Hsu et al reported the synthesis of biotin azide by utilizing o-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) as the catalyst, but the yield was only about 40 % after purification. [25] We slightly modified the experimental procedures by activating biotin with CDI for 3 hours and the yield dramatically increased to 90.7 %. The 1 H NMR spectrum of biotin azide is shown in Figure 1 a and the signal integral ratios confirmed the successful preparation of biotin azide.…”