Glycosylation of proteins is a posttranslational modification that is characterized by the transfer of multiple monosaccharides (glycan structure) e.g. to serine/threonine residues (O-linked glycosylation), asparagine residues (N-linked glycosylation) or tryptophan residues (C-linked glycosylation). The dynamics of global glycan synthesis is an essential parameter to characterize the cellular response under various physiological and pathological conditions however, metabolic labeling studies of the cellular glycan level still primarily rely on classical radioactive labeling with 3H-monosaccharides.
CLICKable tetraacetylated derivatives of naturally occurring monosaccharides N-Acetylmannosamine (ManNAc), N-Acetylglucosamine (GlcNAc), N-Acetylgalactosamine (GalNAc) provide a non-radioactive alternative for metabolic glycan labeling[2-5] (Fig. 1).
Azide-functionalized monosaccharides (Ac4ManNAz, Ac4GlcNAz, Ac4GalNAz) facilitate the subsequent detection of the correspondingly functionalized proteins via Cu(I)-catalyzed Azide-terminal Alkyne or Cu(I)-free Azide-strained Alkyne click chemistry , respectively. Cyclopropene-functionalized monosaccharides (Ac4ManNCyoc, Ac4GlcNCyoc) are detected via Cyclopropene-Tetrazine reaction.
Azide- and Cyclopropene-functionalizd monosaccharides are ideally suited for dual-labeling approaches.[4,5]
Figure 1: CLICKable monosaccharides are metabolically processed and subsequently incorporated instead of their natural counterparts into glycan structures of proteins (modified according to ).
Step 1: Intracelluar uptake of tetraacetylated CLICKable monosaccharide. Step 2: Deacetylation by endogenous esterases. Step 3: Metabolisation and incorporation into cellular glycan structures. Step 4: Detection of CLICK-functionalized proteins.