Linear hexapeptides featuring the asparagine mimetics alanine-β-hydrazide, alanine-β-hydroxylamine, and 1,3-diaminobutanoic acid have been synthesized as oligosaccharyl transferase (OT) substrate mimetics and chemoselectively N-glycosylated to obtain the corresponding neoglycopeptides as OT product mimetics. The effect of glycosylation on the binding of these asparagine surrogates is in stark contrast with the effect of modification of native asparagine. In native N-linked glycosylation, product inhibition is minimal and glycopeptides show very low affinity for OT. In contrast, glycosylation of the substrate mimetics maintains or even improves affinity of the corresponding product mimetic for OT. Conformational considerations suggest that the flexibility of the N-glycosyl linkage in these neoglycopeptides allows them to be accommodated in the OT binding site while the native trans glycosyl amide linkage is rejected. These results provide insight into how OT minimizes product inhibition, thereby ensuring effective substrate turnover.
