Thermo Scientific Pierce Azido-Sugars (GlcNAz, GalNAz and ManNAz) are azide-labeled sugars that provide a highly specific approach for studying glycoproteins through in vivo metabolic labeling and chemoselective ligation.
These sugars are azide-derivatives of naturally occurring monosaccharides that cells use to glycosylate proteins using post-translational modification biochemical pathways. The azide functional group is small and nonreactive with endogenous molecules. When supplied to cells, these compounds become incorporated by glycosylation events to effectively "tag" glycoproteins with the azide group. The azide group then can be specifically targeted for detection or conjugation using alkyne-activated reagents ("click" chemistry) or phosphine-activated reagents (Staudinger ligation).
- Bioorthogonal – the azido group is small, nonreactive and absent from living systems; as such the azido-sugar compounds do not interfere with endogenous cellular pathways and substitute for their naturally occurring analogs
- Compatible – reaction chemistry with phosphine compounds occurs effectively in simple buffer conditions; requires no accessory reagents such as copper or reducing agents
- Chemoselective – azide and phosphine groups do not react or interfere with components of biological samples but conjugate to one another with high efficiency
- Versatile – azide tag can be targeted for detection, immobilization, conjugation or affinity purification depending on which phosphine-activated compound it is reacted with
|Chemical structures of Thermo Scientific Azido-Sugars for metabolic labeling. All three compounds have the same molecular weight (MW 430.37). Once these compounds are incorporated into molecular structures, they can be detected using phosphine-activated molecules via the Staudinger ligation reaction chemistry.
When used in combination with phosphine-activated fluorescent dyes, biotin reagents, and or other compounds, these azido-modified sugars facilitate the investigation of cellular pathways involving glycosylation.
There are several classes of glycoproteins grouped by the type of carbohydrate and amino acid linkage site. N-linked glycosylation is a modification of asparagine amines, whereas O-linked glycosylation occurs through the hydroxyl of serine and threonine residues. The azido-modified sugars are metabolic substitutes for endogenous amino sugars. ManNAz is converted by cells to an azido sialic acid derivative that is used for N-linked glycosylation of cell surface proteins. GlcNAz and GalNAz are predominantly used to label the O-linked glycosylation (O-GlcNAc and O-GalNAc).
|Detection of metabolic labeling with azido-sugars in different cell types using azide-reactive Thermo Scientific DyLight Dyes. A549, U2OS and HK-2 cells were incubated with 40µM azido-acetylmannosamine in cell culture media for 72 hours and then incubated with 100µM of DyLight 550-Phosphine (yellow). The cells were washed, fixed with 4% paraformaldehyde and counterstained with Hoechst 33342 (blue).
|Comparison of fluorescent detection reagents for metabolically labeled sugars. Cell extracts were prepared from the A549 incubated with azido-sugar N-azidoacetylgalactosamine (ManNAz), N-azidoacetylglucosamine (GlcNAz) or N-azidoacetylmannosamine (GalNAz). The cell extracts were incubated with either Thermo Scientific DyLight 650-Phosphine or with Click-iT Alexa Fluor 647 DIBO Alkyne and analyzed by SDS-PAGE. The samples incubated with DyLight 650-Phosphine show a different labeling pattern for each of the three incorporated azido-sugar, demonstrating that different types of glycosylation (i.e., one sugar vs. another) can be detected specifically by using the DyLight Phosphine-activated detection reagents.
Azide-Phosphine Reagent Guide
Review of Metabolic Labeling for Chemoselective Ligation
Review of Staudinger Ligation Reaction Chemistry
Superiority of Azide-Phosphine Chemistry
NHS-Azide Staudinger Ligation Reagents
NHS-Phosphine Staudinger Ligation Reagents
Phosphine-activated DyLight Fluors