Structural and Functional Role of Methylene Linkers in Self-Coacevate-Mediated Membrane Interaction and Antiviral Defense

Self and complex coacervates are intriguing model compartments that are involved in a plethora of cellular processes and biological applications. While the fundamental principles governing the self‐coacervation of intrinsically disordered proteins and complex coacervation are well studied, self-coacervation of small molecules (MW ≤ 1500) at low micromolar concentrations remains poorly understood. The design, tuning the dynamic properties, and low critical coacervate concentration (CCC) of small molecules hold great significance in cellular organization, the regulation of biological events, and drug delivery. Herein, we designed a novel class of pKa tunable polyionic lipidated flavonoids (LFs), where the aromatic core of flavonoid and the hydrophobic interaction of acyl chains serve as stickers, bringing monomers together, while hydrophilic polyanionic moieties interact via multivalent COO‾‧‧‧‧HOOC hydrogen bond formation, leading to coacervation at low concentrations (10 µM). Inspired by nature’s selection of amino acids (glutamic acid vs 2-amino hexane dioic acid) with the profound role of methylene linkers, our comprehensive design was screened on a library of LFs to explore the influence of methylene linker length (n = 0 to 3). Fluorescence recovery after photobleaching (FRAP) studies suggest that shorter linkers (n = 0, 1) produce gel‐like coacervates, whereas longer linkers (n = 2, 3) yield more fluidic droplets. The self-coacervates exhibited strong membrane association and resulted in the formation of 2D domains and/or nano-coacervate-like domains in the lipid bilayer. The domains of LFs (particularly MFEA with n=2) are mem-brane active and modulate the surface potential, interfacial pH, and the membrane ordering to inhibit the membrane fusion. MFEA also protected the cells from SARS-CoV-2 and influenza infections by inhibiting the membrane fusion, high-lighting its potential as a broad-spectrum antiviral. Interestingly, the binding of MFEA coacervates to viral proteins (Spike protein RBD) may further inhibit virus attachment to fine-tune the antiviral efficacy. Our design of amphiphilic self-coacervates with low CCC offers a versatile new platform for engineering functional materials and highlights the role of methylene linker length for tuning coacervate mechanics and bioactivity.