Next‐Generation Metabolic Glycosylation Reporters Enable Detection of Protein O−GlcNAcylation in Living Cells without S‐Glyco Modification

Norbornene-modified glycosyl phosphates with suitable protecting groups enable labeling of O−GlcNAcylated proteins by metabolic glycoengineering without artificial modification of cysteine residues. A subsequent inverse-electron-demand Diels–Alder reaction is used to introduce a fluorescent tetramethylrhodamine (TAMRA) label at O−GlcNAc sites allowing the imaging of protein-specific O−GlcNAcylation within living cells.

Abstract

Protein O−GlcNAcylation is a ubiquitous posttranslational modification of cytosolic and nuclear proteins involved in numerous fundamental regulation processes. Investigation of O−GlcNAcylation by metabolic glycoengineering (MGE) has been carried out for two decades with peracetylated N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine derivatives modified with varying reporter groups. Recently, it has been shown that these derivatives can result in non-specific protein labeling termed S-glyco modification. Here, we report norbornene-modified GlcNAc derivatives with a protected phosphate at the anomeric position and their application in MGE. These derivatives overcome two limitations of previously used O−GlcNAc reporters. They do not lead to detectable S-glyco modification, and they efficiently react in the inverse-electron-demand Diels–Alder (IEDDA) reaction, which can be carried out even within living cells. Using a derivative with an S-acetyl-2-thioethyl-protected phosphate, we demonstrate the protein-specific detection of O−GlcNAcylation of several proteins and the protein-specific imaging of O−GlcNAcylation inside living cells by Förster resonance energy transfer (FRET) visualized by confocal fluorescence lifetime imaging microscopy (FLIM).

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