Design and Synthesis of Metabolic Chemical Reporters for the Visualization and Identification of Glycoproteins

Abstract

Glycosylation events play an invaluable role in regulating cellular processes including enzymatic activity, immune recognition, protein stability, and cell-cell interactions. However, researchers have yet to realize the full range of glycan mediated biological functions due to a lack of appropriate chemical tools. Fortunately, the past 25 years has seen the emergence of modified sugar analogs, termed metabolic chemical reporters (MCRs), which are metabolized by endogenous enzymes to label complex glycan structures. Here, we review the major reporters for each class of glycosylation and highlight recent applications that have made a tremendous impact on the field of glycobiology.

Fig. 1. (A) Common mammalian monosaccharide building blocks and their geometric codes. (B) Core structures for O-linked mucin, N-linked, GAG, and O-GlcNAc glycosylation.

Fig. 2. MCRs take advantage of the endogenous sugar salvage pathways that exist for most monosaccharides. (1) Per-acetylated MCRs passively diffuse across the cell membrane. (2) Promiscuous deacetylase enzymes remove acyl groups revealing hydroxides. (3) MCRs are metabolically transformed into high energy sugar donors via covalent bonds to NDPs. These sugar donors can then be added on to protein substrates to generate proteoglycans (4), O- or N-linked glycoproteins (5), or intracellular O-GlcNAc modified proteins (6).

Fig. 3. Bioorthogonal labeling of glycoproteins is a two-step process. (1) Proteins are labeled with metabolically transformed sugar analogs. (2) Probes undergo orthogonal reactions with functionalized tags allowing for downstream processing.

Fig. 4. Bioorthogonal reactions commonly used for labeling glycans.

Fig. 5. Examples of first generation metabolic chemical reporters for different glycosylation events.

Fig. 6. Second generation MCRs expand their applications. (A) Encapsulating reporters in liposomes allows for targeting of a specific class of cells of tissues. (B) One-step labeling eliminates secondary incubation with tags allowed for direct detection of reporters on protein substrates. (C) The diverse range of bioorthogonal reactions allows for multi-labeling experiments where two bioorthogonal reactions can be performed concurrently without risking cross-labeling.

Fig. 7. Mechanistic insights into S-glycosylation. (top) Proposed mechanism for non-enzymatic S-glycosylation. (1) Per-acetylated sugars are deacetylated at the anomeric position. (2) The resulting hemiacetal exists in equilibrium between its open and closed confirmations. (3) Open sugars can undergo β-elimination reaction with a proximal bases. (4) Acyl migration between C4 and C5 position generates two isomers. (5) α,β-Unsaturated aldehydes are susceptible to Michael-addition with endogenous thiols. (6) Ring closure generates 3-thiol furanose and pyranose adducts. (bottom) Structures of next partially acetylated MCRs.

Fig. 8. In a given glycoprotein, glycans can vary in their structure and linkages (microheterogeneity) and presence (macroheterogeneity) complicating their detection and identification.