Spiro‐epoxyglycosides as Activity‐Based Probes for Glycoside Hydrolase Family 99 Endomannosidase/Endomannanase

Abstract

N‐Glycans direct protein function, stability, folding and targeting, and influence immunogenicity. While most glycosidases that process N‐glycans cleave a single sugar residue at a time, enzymes from glycoside hydrolase family 99 are endo‐acting enzymes that cleave within complex N‐glycans. Eukaryotic Golgi endo‐1,2‐α‐mannosidase cleaves glucose‐substituted mannose within immature glucosylated high‐mannose N‐glycans in the secretory pathway. Certain bacteria within the human gut microbiota produce endo‐1,2‐α‐mannanase, which cleaves related structures within fungal mannan, as part of nutrient acquisition. An unconventional mechanism of catalysis was proposed for enzymes of this family, hinted at by crystal structures of imino/azasugars complexed within the active site. Based on this mechanism, we developed the synthesis of two glycosides bearing a spiro‐epoxide at C‐2 as electrophilic trap, to covalently bind a mechanistically important, conserved GH99 catalytic residue. The spiro‐epoxyglycosides are equipped with a fluorescent tag, and following incubation with recombinant enzyme, allow concentration, time and pH dependent visualization of the bound enzyme using gel electrophoresis.

Glycosylation network mapping and site-specific glycan maturation in vivo

“…Subsequent ER processing intermediates were also found to enter the Man-6-P pathway. Further processing of these structures was possibly linked to the enzymatic activity of the endo-α1,2-mannosidase described to be able to cut out a mannose linked to glucoses residues from the A-branch of glycans ( Kukushkin et al., 2012 ) ( Schroder et al., 2018 ). Furthermore, the kinetics of phosphorylated glycans indicated that once the P-GlcNAc group added, there was no longer a site-specific effect on the kinetic, because all phosphorylated had the same kinetic behavior ( Figure 7 ).…”

Spirocyclic Scaffolds in Medicinal Chemistry

“…An approach for chemically covalent binding probes was published by Schroder et al 147 Glycosidases cleave N-glycans, which are complex oligosaccharides linked to asparagine residues in proteins. Glycoside hydrolase (GH) family 99 endomannodidase/endomannanase cleaves within complex Nglycans.…”

From Phenotypic Hit to Chemical Probe: Chemical Biology Approaches to Elucidate Small Molecule Action in Complex Biological Systems

“…To overcome these limitations, the attention has shifted to methods that use a covalent interaction between the compound and its target. This covalent interaction is either established by the reaction of an intrinsic electrophilic group in the parent molecule (for example: epoxides [33,34], carbamates [35], Michael acceptors [36]) that can react with a nucleophilic residue in the protein target, or by introduction of a photo-reactive group that crosslinks upon irradiation with UV light (Figure 1b). The first category is comprised of covalent inhibitors and activity-based probes, and the second of photo-affinity probes, as discussed in more detail below (Section 4.3).…”