ABSTRACT
Raising the bar: the efficacy of bioorthogonal reactions for bioconjugation has been thoroughly evaluated in four different biological settings. Powered by the development of new biocompatible ligands, the copper-catalyzed azide-alkyne cycloaddition has brought about unsurpassed bioconjugation efficiency, and thus it holds great promise as a highly potent and adaptive tool for a broader spectrum of biological applications.
Subject(s)
Click Chemistry , Alkynes/chemistry , Animals , Azides/chemistry , Biotin/chemistry , Catalysis , Copper/chemistry , Cycloaddition Reaction , Embryo, Nonmammalian/chemistry , Fluorescent Dyes/chemistry , Ligands , Polysaccharides/chemistry , Streptavidin/chemistry , Succinimides/chemistry , ZebrafishABSTRACT
Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid and N-acetylgalactosamine, in developing zebrafish and in other living organisms.
Subject(s)
Click Chemistry/methods , Polysaccharides/analysis , Zebrafish/metabolism , Alkynes/chemistry , Animals , Azides/chemistry , Embryo, Nonmammalian/chemistry , Embryo, Nonmammalian/metabolism , Fluorescent Dyes/chemistry , Guanosine Diphosphate Fucose/chemistry , Microinjections , Microscopy, Confocal , Polysaccharides/metabolismABSTRACT
The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is the standard method for bioorthogonal conjugation. However, current Cu(I) catalyst formulations are toxic, hindering their use in living systems. Here we report that BTTES, a tris(triazolylmethyl)amine-based ligand for Cu(I), promotes the cycloaddition reaction rapidly in living systems without apparent toxicity. This catalyst allows, for the first time, noninvasive imaging of fucosylated glycans during zebrafish early embryogenesis. We microinjected embryos with alkyne-bearing GDP-fucose at the one-cell stage and detected the metabolically incorporated unnatural sugars using the biocompatible click chemistry. Labeled glycans could be imaged in the enveloping layer of zebrafish embryos between blastula and early larval stages. This new method paves the way for rapid, noninvasive imaging of biomolecules in living organisms.
Subject(s)
Biocompatible Materials/chemistry , Copper/chemistry , Molecular Imaging/methods , Polysaccharides/metabolism , Alkynes/chemistry , Animals , Azides/chemistry , Biocompatible Materials/toxicity , Blastula/metabolism , Catalysis , Cell Line , Cell Survival , Click Chemistry , Embryonic Development , Fucose/metabolism , Humans , Microinjections , Time Factors , Triazoles/chemistry , Zebrafish/embryologyABSTRACT
A combination of recombinant FKP and alpha-(1-->3)-fucosyltransferase allows the facile synthesis of the sialyl Lewis X tetrasaccharide glycan and its derivatives in excellent yield. In this system, the universal fucosyl donor, guanidine 5'-diphosphate-beta-L-fucose (GDP-fucose), or its analogues can be generated in situ by cofactor recycling using pyruvate kinase.
