Unveiling the Liquid-Liquid Phase Separation of Benzene-1,3,5-Tricarboxamide in Water

The intricate interplay between self-assembly and phase separation orchestrates biomolecular organization inside cells, thereby dictating the formation of vital structures such as protein assemblies and membraneless organelles (MLOs). However, in the context of supramolecular polymerization, these fundamental processes have traditionally been studied separately. This study reevaluates the supramolecular polymerization process to unveil the presence of phase-separated droplet state. Utilizing the well-studied benzene-1,3,5-tricarboxamide (BTA) supramolecular motif, we explore its thermally driven liquid-liquid phase separation (LLPS). Thermodynamic and kinetic analysis, employing temperature-dependent spectroscopic and microscopic techniques, elucidates the distinct BTA states and their evolution towards the thermodynamic fiber state. This research sheds light on the existence of hidden phases of supramolecular monomers, emphasizing the delicate balance of non-covalent interactions among monomers and with solvents in governing self-assembly vs. phase separation. This is particularly important in comprehending phase separation in the biological realm such as in MLOs, and for applications such as condensate-modifying therapeutics.