Trifluoroacetic\nAcid as a Molecular Probe for the\nDense Phase in Liquid–Liquid Phase-Separating Peptide Systems

Although trifluoroacetic acid (TFA) is not typically\nconsidered\na Hofmeister reagent, it has been demonstrated to modulate biocoacervation.\nWe show that TFA can be employed to probe specific interactions in\ncoacervating bioinspired peptide phenylalanine (Phe) <sup>19</sup>F-labeled at a single site, altering its liquid–liquid phase\nseparation (LLPS) behavior. Solid-state nuclear magnetic resonance\n(NMR) spectroscopy revealed two dynamically distinct binding modes\nof TFA with Phe, resulting in a structured, dipolar-ordered complex\nand a more dynamic complex, highlighting the proximity between TFA\nand Phe. Quantum chemistry modeling of <sup>19</sup>F chemical shift\ndifferences indicates that the structured complex is formed by the\nintercalation of one TFA molecule between two stacked Phe aromatic\nrings, possibly contributing to the stabilization of the condensed\ndense phase. Thus, we propose that TFA can be used as a convenient\nmolecular probe in <sup>19</sup>F NMR-based studies of the structure\nand dynamics of the dense phase in LLPS peptide systems.