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

Abstract

SummaryMonitoring 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.

Distinct CCR7 glycosylation pattern shapes receptor signaling and endocytosis to modulate chemotactic responses

“…To advance further our understanding of CCR7 glycosylation and its regulation, we next exploited a metabolic labeling strategy using inverse electron-demand Diels-Alder reaction between methylcyclopropene tags and tetrazines [47,48]. The feeding of cells with different tagged sugar moieties enabled us to detect sialylated CCR7 (Ac 4 ManNCyoc) and presumed mucin-type O-glycosylation (Ac 4 GalNCyoc) or O-GlcNAcylated CCR7 (Ac 4 GlcNCyoc; Fig.…”

Exploring the Potential of Norbornene‐Modified Mannosamine Derivatives for Metabolic Glycoengineering

“…The hydroxy group of the norbornene allows attachment of the norbornene to the sugar through a carbamate linkage. We and others have previously been able to show that alkenes and alkynes attached through carbamate linkages are accepted by the cells' enzymatic machinery– and can even provide improved reaction kinetics …”

“…Western blot analysis : Western blot analysis was performed in a modified version of the previously described protocol . HEK 293T cells were seeded (1 200 000 cells/10 cm dish), and after 16 h the media was exchanged with media containing labeled sugar [Ac 4 ManNNorboc exo ( 4 ) or Ac 4 ManNNorboc endo ( 5 ), 100 μ m ].…”

Strain‐Promoted Cycloaddition of Cyclopropenes with o‐Quinones: A Rapid Click Reaction

“…1‐Methyl‐3‐substituted cyclopropenes have been recently reported for fast IEDDA cycloadditions with tetrazines . This click reaction has been widely used for glycoprotein conjugation, cell imaging, and so forth . Moreover, the higher stability of substituted cyclopropenes as compared to TCO, an alternative strained alkene, is an additional advantage.…”