Impact of Glycoclustering on Stiffening of MUC5AC Peptides Revealed by High‐Efficiency Synthesis

Abstract

Abstract Clustered O‐glycosylation of long tandem repeat regions is the hallmark of secreted mucins such as MUC5AC. Glycosylation is thought to play a key role in rigidifying the peptide backbone. The synthesis of peptides containing extended O‐glycosylation clusters has proven challenging, thus limiting studies on the influence of glycoclustering on peptide structure. Here, we report an efficient glyco‐economic synthesis of peptides featuring a previously unattained degree of glycoclustering. The method is based on a fully automated, DMF‐free solid‐phase synthesis employing the solvent 1,3‐dioxolane (DOL) in all steps. The addition of Tween‐20 enabled fast couplings of and to GalNAcylated amino acids by using only 0.5 excess equivalents at room temperature. Five tandem repeats long MUC5AC glycopeptides containing up to 30 GalNAc residues (100% occupancy of potential glycosylation sites) were accessed by Diselenide–Selenoester Ligation and selective deselenization in the presence of terminal cysteine residues. Circular Dichroism (CD) measurements showed that progressive GalNAcylation shifts the conformational equilibrium from the random coil to the extended polyproline type II helix conformation. Pulsed Electron–Electron Double Resonance (PELDOR) spectroscopy measurements revealed a significant stiffening of the MUC5AC peptide backbone upon GalNAcylation of four or six amino acids in each octad repeat.