Phase separation of a heterochiral peptide-drug conjugate amplified by ionic interactions

Artificial membraneless organelles offer a platform for understanding biological compartmentalization and advancing biomedical applications, while designing them with precise chemical control remains challenging. Herein, inspired by natural heterochiral peptide systems, we have developed a chiral engineered tripeptide-drug conjugate with alternating D/L residues, which undergoes phase separation through a stereochemical-ionic interplay, enabling cation-tunable liquid–liquid phase separation (LLPS) under physiological conditions. Notably, metastable heterochiral condensates can be stabilized by Na⁺ or K⁺ via cation–tripeptide interactions. Specifically, heterochiral configuration possibly introduces a stereochemical effect that elevates the energy barrier for liquid-to-solid phase transition, redirecting assembly toward LLPS rather than fibrillization, whereas homochiral diastereomers preferentially form β-sheet-rich hydrogels. Further, chirality-controlled phase behavior results in altered drug properties, including cellular uptake and liver metabolism. By altering chirality of a single residue, we present a minimalist stereochemical strategy for synthetically controlling over the energy landscapes of peptide-based condensates, expanding functional versatility through rational design. Authors apply chiral engineering to tripeptide-drug conjugates (PDC), observing liquid-liquid phase separation for some PDCs with alternating L/D amino acids, while others form β-sheet-rich hydrogels. Chirality-controlled phase behavior offers a strategy to control phase transition and material properties in PDCs.