Dynamic imaging of proteins in live cells is routinely performed by using genetically encoded reporters, an approach that cannot be extended to other classes of biomolecules such as glycans and lipids. Here, we report a Cu-free variant of click chemistry that can label these biomolecules rapidly and selectively in living systems, overcoming the intrinsic toxicity of the canonical Cu-catalyzed reaction. The critical reagent, a substituted cyclooctyne, possesses ring strain and electron-withdrawing fluorine substituents that together promote the [3 ؉ 2] dipolar cycloaddition with azides installed metabolically into biomolecules. This Cu-free click reaction possesses comparable kinetics to the Cu-catalyzed reaction and proceeds within minutes on live cells with no apparent toxicity. With this technique, we studied the dynamics of glycan trafficking and identified a population of sialoglycoconjugates with unexpectedly rapid internalization kinetics.azide ͉ bioorthogonal reaction ͉ cyclooctyne glycan trafficking ͉ molecular imaging

Glycans are attractive targets for molecular imaging but have been inaccessible because of their incompatibility with genetically encoded reporters. We demonstrated the noninvasive imaging of glycans in live developing zebrafish, using a chemical reporter strategy. Zebrafish embryos were treated with an unnatural sugar to metabolically label their cell-surface glycans with azides. Subsequently, the embryos were reacted with fluorophore conjugates by means of copper-free click chemistry, enabling the visualization of glycans in vivo at subcellular resolution during development. At 60 hours after fertilization, we observed an increase in de novo glycan biosynthesis in the jaw region, pectoral fins, and olfactory organs. Using a multicolor detection strategy, we performed a spatiotemporal analysis of glycan expression and trafficking and identified patterns that would be undetectable with conventional molecular imaging approaches.The cell-surface glycome is a rich source of information that reports on the cell's physiological state. For example, changes in glycan structures serve as markers of altered gene expression during development (1) and disease progression (2). The dynamics of glycans at the plasma membrane reflect the activity of the cell's secretory machinery (3), and their relative abundances report on flux in metabolic pathways inside the cell (4). Glycans are therefore attractive targets for in vivo imaging but have been inaccessible because of their incompatibility with genetically encoded reporters (5).To image glycans in vivo, we employed a strategy in which an azide is introduced into target biomolecules, priming them for selective covalent reaction with fluorescent probes (5). The azide is small, stable in biological systems, and selectively reactive with phosphines or activated alkynes. Previously, the Staudinger ligation (6,7) or copper-catalyzed click chemistry (8,9) have been used to detect azide-labeled biomolecules on cells ex vivo. However, in vivo †To whom correspondence should be addressed. E-mail: E-mail: crb@berkeley.edu. * These authors contributed equally to this work. imaging of dynamic biological processes using these chemistries could be complicated by slow reaction kinetics or reagent toxicity. The copper-free click reaction of azides with difluorinated cyclooctyne (DIFO) reagents (10) overcomes these limitations, suggesting its potential application to in vivo imaging.We chose zebrafish as a model organism because of their well-defined developmental program (11), emerging disease models (12), and amenability to optical imaging. The metabolic substrate peracetylated N-azidoacetylgalactosamine (Ac 4 GalNAz) was selected on the basis of its known incorporation into mucin-type O-linked glycoproteins in mammalian cells and mice via the N-acetylgalactosamine (GalNAc) salvage pathway (13,14) ( fig. S1). We envisioned an imaging experiment ( Before performing imaging experiments, we confirmed that the zebrafish glycan biosynthetic enzymes are permissive of the unnatural sugar. The...

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Metabolic labeling of glycans with a bioorthogonal chemical reporter such as the azide enables their visualization in cells and organisms as well as the enrichment of specific glycoprotein types for proteomic analysis. This process involves two steps. Azido sugars are fed to cells or organisms and integrated by the glycan biosynthetic machinery into various glycoconjugates. The azido sugars are then covalently tagged with imaging probes or epitope tags, either ex vivo or in vivo, using an azide-specific reaction. This protocol details the syntheses of the azido sugars N-azidoacetylmannosamine (ManNAz), N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz) and 6-azidofucose (6AzFuc), and the detection reagents phosphine-FLAG and phosphine-FLAG-His6. Applications to the visualization of cellular glycans and enrichment of glycoproteins for proteomic analysis are described. The synthesis of the azido sugars (ManNAz, GalNAz, GlcNAz or 6AzFuc) or detection reagents (phosphine-FLAG or phosphine-FLAG-His6) can be completed in approximately 1 week. A cell metabolic labeling experiment can be completed in approximately 4 d.

Developmental events can be monitored at the cellular and molecular levels by using noninvasive imaging techniques. Among the biomolecules that might be targeted for imaging analysis, glycans occupy a privileged position by virtue of their primary location on the cell surface. We previously described a chemical method to image glycans during zebrafish larval development; however, we were unable to detect glycans during the first 24 hours of embryogenesis, a very dynamic period in development. Here we report an approach to the imaging of glycans that enables their visualization in the enveloping layer during the early stages of zebrafish embryogenesis. We microinjected embryos with azidosugars at the one-cell stage, allowed the zebrafish to develop, and detected the metabolically labeled glycans with copper-free click chemistry. Mucin-type O-glycans could be imaged as early as 7 hours postfertilization, during the gastrula stage of development. Additionally, we used a nonmetabolic approach to label sialylated glycans with an independent chemistry, enabling the simultaneous imaging of these two distinct classes of glycans. Imaging analysis of glycan trafficking revealed dramatic reorganization of glycans on the second time scale, including rapid migration to the cleavage furrow of mitotic cells. These studies yield insight into the biosynthesis and dynamics of glycans in the enveloping layer during embryogenesis and provide a platform for imaging other biomolecular targets by microinjection of appropriately functionalized biosynthetic precursors.azide | cyclooctyne | bioorthogonal | oxime | cytokinesis

The nematode Caenorhabditis elegans is an excellent model organism for studies of glycan dynamics, a goal that requires tools for imaging glycans in vivo. Here we applied the bioorthogonal chemical reporter technique for the molecular imaging of mucin-type O-glycans in live C. elegans. We treated worms with azidosugar variants of N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and N-acetylmannosamine (ManNAc), resulting in the metabolic labeling of their cell-surface glycans with azides. Subsequently, the worms were reacted via copper-free click reaction with fluorophore-conjugated difluorinated cyclooctyne (DIFO) reagents. We identified prominent localization of mucins in the pharynx of all four larval stages, in the adult hermaphrodite pharynx, vulva and anus, and in the tail of the adult male. Using a multicolor, time-resolved imaging strategy, we found that the distribution and dynamics of the glycans varied anatomically and with respect to developmental stage.

Fucosylated glycoproteins are involved in many cell-cell recognition events and are markers of embryonic and malignant tissue. Here we report a method for rapid profiling of fucosylated glycoproteins from human cells using 6-azido fucose as a metabolic label.

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