Chemical approaches to perturb, profile, and perceive glycans

Abstract

Glycosylation is an essential form of post-translational modification that regulates intracellular and extracellular processes. Regrettably, conventional biochemical and genetic methods often fall short for the study of glycans, because their structures are often not precisely defined at the genetic level. To address this deficiency, chemists have developed technologies to perturb glycan biosynthesis, profile their presentation at the systems level, and perceive their spatial distribution. These tools have identified potential disease biomarkers and ways to monitor dynamic changes to the glycome in living organisms. Still, glycosylation remains the underexplored frontier of many biological systems. In this Account, we focus on research in our laboratory that seeks to transform the study of glycan function from a challenge to routine practice. In studies of proteins and nucleic acids, functional studies have often relied on genetic manipulations to perturb structure. Though not directly subject to mutation, we can determine glycan structure-function relationships by synthesizing defined glycoconjugates or by altering natural glycosylation pathways. Chemical syntheses of uniform glycoproteins and polymeric glycoprotein mimics have facilitated the study of individual glycoconjugates in the absence of glycan microheterogeneity. Alternatively, selective inhibition or activation of glycosyltransferases or glycosidases can define the biological roles of the corresponding glycans. Investigators have developed tools including small molecule inhibitors, decoy substrates, and engineered proteins to modify cellular glycans. Current approaches offer a precision approaching that of genetic control. Genomic and proteomic profiling form a basis for biological discovery. Glycans also present a rich matrix of information that adapts rapidly to changing environs. Glycomic and glycoproteomic analyses via microarrays and mass spectrometry are beginning to characterize alterations in glycans that correlate with disease. These approaches have already identified several cancer biomarkers. Metabolic labeling can identify recently synthesized glycans and thus directly track glycan dynamics. This approach can highlight changes in physiology or environment and may be more informative than steady-state analyses. Together, glycomic and metabolic labeling techniques provide a comprehensive description of glycosylation as a foundation for hypothesis generation. Direct visualization of proteins via the green fluorescent protein (GFP) and its congeners has revolutionized the field of protein dynamics. Similarly, the ability to perceive the spatial organization of glycans could transform our understanding of their role in development, infection, and disease progression. Fluorescent tagging in cultured cells and developing organisms has revealed important insights into the dynamics of these structures during growth and development. These results have highlighted the need for additional imaging probes.

Figure 1

Examples of glycoconjugates. Many proteins are glycosylated at asparagine (N-linked) or serine/threonine residues (mucin-type O-linked and O-GlcNAc are shown). Lipids, secondary metabolites, and tRNA are examples of other biomolecules found in glycosylated form.

Figure 2

Structures of mucin-type O-linked glycosylation and the structure and synthesis of a mucin mimetic. (A) Mucin-type O-linked glycosylation is characterized by densely functionalized arrays of glycans on a polypeptide backbone. (B) A derivatized polyvinylketone backbone can approximate endogenous mucins. (C) The synthesis of mucin mimics proceeds by condensing aminooxy-GalNAc with polyvinyl ketone. Extended sugars can also be incorporated.

Figure 3

Perturbing glycosylation with synthetic sugars. (A) Glycosyltransferases add activated glycan donors onto proteins in the secretory pathway to biosynthesize cell-surface glycoproteins. (B) Primers of glycosylation divert the action of glycosyltransferases away from their endogenous substrates. Instead, the primers are glycosylated at the expense of cellular glycoproteins, and elaborated primers are secreted. (C) Unnatural monosaccharides can be metabolically incorporated into glycans, wherein they prevent further elongation.

Figure 4

Engineering glycosyltransferases for small molecule control. (A) In the absence of rapamycin, a “chemical inducer of dimerization”, catalytic and localization domains remain separate and the glycosyltransferase cannot engage its substrates. (B) In the presence of rapamycin, the glycosyltransferase domains associate and the enzyme regains activity.

Figure 5

The bioorthogonal chemical reporter strategy for profiling and visualizing glycans. (A) Metabolic labeling of cell-surface glycans with an azidosugar. The azide and alkyne functionalities can be covalently reacted with a probe that bears the functionality shown in panel C. (B) Azidosugars and alkynyl sugars that have been employed as metabolic labels. The unnatural monosaccharides are typically used in peracetylated form to facilitate cellular uptake. (C) Reagents for covalent labeling of azides (i, iii) and alkynes (ii): (i) phosphines for the Staudinger ligation; (ii) terminal alkynes or azides and copper(I) for copper-mediated azide−alkyne cylcoaddition, “click chemistry”; (iii) difluorinated cyclooctyne (DIFO) for strain-promoted cycloadditions.

Figure 6

The quench−label−tag sequence for visualizing dynamic glycosylation. Zebrafish embryos labeled with GalNAz can be tagged with fluorescently labeled DIFO reagents to visualize total O-linked glycosylation. A dynamic picture of glycosylation can be obtained by reducing residual azides with TCEP, incubating briefly with GalNAz, and labeling the newly synthesized glycans with a blue-shifted DIFO conjugate.