Metabolic glycoengineering - exploring glycosylation with bioorthogonal chemistry.

Glycans are involved in numerous biological recognition events. Being secondary gene products, their labeling by genetic methods - comparable to GFP labeling of proteins - is not possible. To overcome this limitation, metabolic glycoengineering (MGE, also known as metabolic oligosaccharide engineering, MOE) has been developed. In this approach, cells or organisms are treated with synthetic carbohydrate derivatives that are modified with a chemical reporter group. In the cytosol, the compounds are metabolized and incorporated into newly synthesized glycoconjugates. Subsequently, the reporter groups can be further derivatized in a bioorthogonal ligation reaction. In this way, glycans can be visualized or isolated. Furthermore, diverse targeting strategies have been developed to direct drugs, nanoparticles, or whole cells to a desired location. This review summarizes research in the field of MGE carried out in recent years. After an introduction to the bioorthogonal ligation reactions that have been used in in connection with MGE, an overview on carbohydrate derivatives for MGE is given. The last part of the review focuses on the many applications of MGE starting from mammalian cells to experiments with animals and other organisms.

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

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).

Temporally Controlled Photouncaged Epidermal Growth Factor Influences Cell Fate in Hydrogels.

Hydrogels are powerful materials that more accurately mimic the cellular microenvironment over static two-dimensional culture. Photochemical strategies enable dynamic complexity to be achieved within hydrogels to better mimic the extracellular matrix; however, many photochemical systems to pattern proteins within hydrogels are complicated by long reaction times to immobilize these proteins wherein the protein can lose activity. As proof-of-concept, we demonstrate an elegant method where photocaged proteins are immobilized in hydrogels and then directly photoactivated. Specifically, we immobilized streptavidin-ortho-nitrobenzyl-modified epidermal growth factor (EGF) to cross-linked hyaluronan hydrogels and cultured two EGF-responsive cancer cells of breast and lung therein. We used light to temporally uncage and control EGF activation, thereby inducing cell death in breast cancer cells and proliferation in lung cancer cells. These results show how temporal, photochemical, protein activation influences cellular response and lays the foundation for further advances in manipulating the in vitro environment to control cell fate.

Application of the Inverse-Electron-Demand Diels-Alder Reaction for Metabolic Glycoengineering.

The inverse electron-demand Diels-Alder (IEDDA or DAinv) reaction is an emerging bioorthogonal ligation reaction that finds application in all areas of chemistry and chemical biology. In this review we highlight its application in metabolic glycoengineering (MGE). MGE is a versatile tool to introduce unnatural sugar derivatives that are modified with a chemical reporter group into cellular glycans. The IEDDA reaction can then be used to modify the chemical reporter group allowing, for instance, the visualization or isolation of glycoconjugates. During the last years, many different sugar derivatives as well as reporter groups have been published. These probes are summarized, and their chemical and biological properties are discussed. Furthermore, we discuss examples of MGE and subsequent IEDDA reaction that highlight its suitability for application within living systems.