Programmable Coacervate Droplets via Reaction-Coupled Liquid–Liquid Phase Separation (LLPS) and Competitive Inhibition

Membraneless biomolecular condensates formed by liquid-liquid phase separation (LLPS) are crucial for many spatiotemporal biological functions. Designing synthetic mimics to emulate and understand LLPS is an active area of research, which has led to the development of coacervate droplets through elegant bioinspired designs. However, recent interest in this field has shifted toward designing programmable coacervates to impart spatiotemporal control over these liquid phases. Herein, we demonstrate the programming of LLPS in synthetic systems by employing concepts of competitive binding and reaction-coupled assembly involving dynamic covalent bonds. Our results utilize small building blocks that follow a simple coacervation mechanism, distinguishing this approach from previously reported programmable complex coacervates, which often rely on reaction-controlled generation of one of the components. We introduce these concepts using dynamic covalent bonds (boronate esters) and small chromophoric building blocks appended with terminal boronic acid groups. Upon reaction with substrates (monosaccharides), these building blocks form molecular structures resembling "sticker-and-spacer" designs for coacervation, leading to a reaction-driven, temporally controlled LLPS process. The differential reactivity of various monosaccharides, combined with the reversibility of dynamic bonds, enables competitive binding-driven control over the growth, inhibition, and dissolution of the coacervation process, offering new strategies for programmable LLPS that are reminiscent of protein-induced inhibition in biomolecular condensates. Detailed spectroscopic probing and kinetic analyses provide mechanistic insights into the reaction-coupled and autocatalytic growth processes, revealing the glucose-selective nature of this coacervation system. Finally, coupling dynamic covalent reactions with temporal pH modulation results in transient coacervation, which can be visualized by using confocal microscopy. We anticipate that this approach will pave the way for designing coacervate droplets with novel biorelevant emergent properties.