Rapid Labeling of Metabolically Engineered Cell-Surface Glycoconjugates with a Carbamate-Linked Cyclopropene Reporter

Metabolic oligosaccharide engineering is a valuable tool to monitor cellular carbohydrates. Here, we report the synthesis of a novel N-acyl-mannosamine derivative bearing a methylcyclopropene tag that is attached to the sugar via a carbamate moiety. This derivative undergoes rapid Diels–Alder reaction with inverse electron demand. We demonstrate that the cell’s biosynthetic machinery incorporates this non-natural mannosamine derivative into glycoconjugates that can, subsequently, be labeled within less than 10 min with a new sulfo-Cy3–tetrazine conjugate. Using this tetrazine-dye conjugate for the detection of the methylcyclopropene-tagged mannosamine derivative, we could achieve dual labeling of two different metabolically incorporated sugars combining a Diels–Alder reaction with inverse electron demand and a strain-promoted azide–alkyne cycloaddition which are carried out simultaneously in a single step

Expanding the scope of cyclopropene reporters for the detection of metabolically engineered glycoproteins by Diels-Alder reactions

Monitoring glycoconjugates has been tremendously facilitated by the development of metabolic oligosaccharide engineering. Recently, the inverse-electron-demand Diels–Alder reaction between methylcyclopropene tags and tetrazines has become a popular ligation reaction due to the small size and high reactivity of cyclopropene tags. Attaching the cyclopropene tag to mannosamine via a carbamate linkage has made the reaction even more efficient. Here, we expand the application of cyclopropene tags to N-acylgalactosamine and N-acylglucosamine derivatives enabling the visualization of mucin-type O-glycoproteins and O-GlcNAcylated proteins through Diels–Alder chemistry. Whereas the previously reported cyclopropene-labeled N-acylmannosamine derivative leads to significantly higher fluorescence staining of cell-surface glycoconjugates, the glucosamine derivative gave higher labeling efficiency with protein preparations containing also intracellular proteins

Triple Orthogonal Labeling of Glycans by Applying Photoclick Chemistry

Bioorthogonal labeling of multiple biomolecules is of current interest in chemical biology. Metabolic glycoengineering (MGE) has been shown to be an appropriate approach to visualizing carbohydrates. Here, we report that the nitrile imine–alkene cycloaddition (photoclick reaction) is a suitable ligation reaction in MGE. Using a mannosamine derivative with an acrylamide reporter group that is efficiently metabolized by cells and that quickly reacts in the photoclick reaction, we labeled sialic acids on the surface of living cells. Screening of several alkenes showed that a previously reported carbamate‐linked methylcyclopropene reporter that is well suited for the inverse‐electron‐demand Diels–Alder (DAinv) reaction has a surprisingly low reactivity in the photoclick reaction. Thus, for the first time, we were able to triply label glycans by a combination of DAinv, photoclick, and copper‐free click chemistry.publishe

Rapid Labeling of Metabolically Engineered Cell-Surface Glycoconjugates with a Carbamate-Linked Cyclopropene Reporter

Metabolic oligosaccharide engineering is a valuable tool to monitor cellular carbohydrates. Here, we report the synthesis of a novel <i>N</i>-acyl-mannosamine derivative bearing a methylcyclopropene tag that is attached to the sugar via a carbamate moiety. This derivative undergoes rapid Diels–Alder reaction with inverse electron demand. We demonstrate that the cell’s biosynthetic machinery incorporates this non-natural mannosamine derivative into glycoconjugates that can, subsequently, be labeled within less than 10 min with a new sulfo-Cy3–tetrazine conjugate. Using this tetrazine-dye conjugate for the detection of the methylcyclopropene-tagged mannosamine derivative, we could achieve dual labeling of two different metabolically incorporated sugars combining a Diels–Alder reaction with inverse electron demand and a strain-promoted azide–alkyne cycloaddition which are carried out simultaneously in a single step