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

## TL;DR

This paper shows how clustered O-glycosylation in MUC5AC peptides makes their backbone stiffer, using a new efficient synthesis method.

## Contribution

A high-efficiency synthesis method enabled the creation of MUC5AC peptides with the highest glycoclustering reported, revealing structural stiffening effects.

## Key findings

- CD measurements showed GalNAcylation shifts peptide conformation to extended polyproline type II helix.
- PELDOR spectroscopy revealed significant stiffening of the MUC5AC backbone with four or six GalNAcylated amino acids per octad repeat.

## 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.

Peptides featuring a previously unattained degree of glycoclustering were obtained by fully automated solid‐phase synthesis in 1,3‐dioxolane/Tween‐20 and diselenide–selenoester ligation. Circular Dichroism (CD) and Pulsed Electron‐Electron Double Resonance (PELDOR) measurements of densely GalNAcylated MUC5AC multi‐tandem repeat peptides provided insights into the correlation between the occupancy of O‐glycosylation sites and stiffening of the peptide backbone.