Stabilizing Impact of N‑Glycosylation on the WW Domain Depends Strongly on the Asn-GlcNAc Linkage

N-glycans play important roles in many cellular processes and can increase protein conformational stability in specific structural contexts. Glycosylation (with a single GlcNAc) of the reverse turn sequence Phe-Yyy-Asn-Xxx-Thr at Asn stabilizes the Pin 1 WW domain by −0.85 ± 0.12 kcal mol^–1. Alternative methods exist for attaching carbohydrates to proteins; some occur naturally (e.g., the O-linkage), whereas others use chemoselective ligation reactions to mimic the natural N- or O-linkages. Here, we assess the energetic consequences of replacing the Asn linkage in the glycosylated WW domain with a Gln linkage, with two natural O-linkages, with two unnatural triazole linkages, and with an unnatural α-mercaptoacetamide linkage. Of these alternatives, only glycosylation of the triazole linkages stabilizes WW, and by a smaller amount than N-glycosylation, highlighting the need for caution when using triazole- or α-mercaptoacetamide-linked carbohydrates to mimic native N-glycans, especially where the impact of glycosylation on protein conformational stability is important

Structural and Energetic Basis of Carbohydrate–Aromatic Packing Interactions in Proteins

Carbohydrate–aromatic interactions mediate many biological processes. However, the structure–energy relationships underpinning direct carbohydrate–aromatic packing interactions in aqueous solution have been difficult to assess experimentally and remain elusive. Here, we determine the structures and folding energetics of chemically synthesized glycoproteins to quantify the contributions of the hydrophobic effect and CH−π interactions to carbohydrate–aromatic packing interactions in proteins. We find that the hydrophobic effect contributes significantly to protein–carbohydrate interactions. Interactions between carbohydrates and aromatic amino acid side chains, however, are supplemented by CH−π interactions. The strengths of experimentally determined carbohydrate CH−π interactions do not correlate with the electrostatic properties of the involved aromatic residues, suggesting that the electrostatic component of CH−π interactions in aqueous solution is small. Thus, tight binding of carbohydrates and aromatic residues is driven by the hydrophobic effect and CH−π interactions featuring a dominating dispersive component

Iminosugar C‑Glycoside Analogues of α‑d‑GlcNAc-1-Phosphate: Synthesis and Bacterial Transglycosylase Inhibition

We herein describe the first synthesis of iminosugar C-glycosides of α-d-GlcNAc-1-phosphate in 10 steps starting from unprotected d-GlcNAc. A diastereoselective intramolecular iodoamination–cyclization as the key step was employed to construct the central piperidine ring of the iminosugar and the C-glycosidic structure of α-d-GlcNAc. Finally, the iminosugar phosphonate and its elongated phosphate analogue were accessed. These phosphorus-containing iminosugars were coupled efficiently with lipophilic monophosphates to give lipid-linked pyrophosphate derivatives, which are lipid II mimetics endowed with potent inhibitory properties toward bacterial transglycosylases (TGase)