Acetylation ofShort Glycopeptides Enables Phase Separation

Liquid–liquid phase separation (LLPS) of biomacromolecules is crucial for regulating cellular functions. To explore their molecular mechanisms, peptide-based coacervates mimicking natural proteins have been developed, but the role of side chain modifications such as glycosylation remains underexplored. Here, we demonstrate that acetylation of short glycopeptides can induce pH- and concentration-dependent phase separation, while removing acetyl groups abolishes this behavior. Circular dichroism spectroscopy revealed a strong link between peptide structural ordering and the phase separation propensity. Peptides capable of forming liquid droplets displayed a significant ellipticity change at 205 nm upon changing solution pH. Moreover, these peptide coacervates can interact with cells and enhance the antiproliferative property of doxorubicin. Therefore, this work highlights the critical role of O-acetylation in LLPS and provides a valuable tool for studying the parameters regulating LLPS and its implications in cellular processes.