Fluorophore-Conjugated Diphenylalanine Peptides Reveal Self-Sorting in a Multicomponent Supramolecular System

The interplay between distinct molecular building blocks in multicomponent supramolecular systems can lead to co-assembly or self-sorting, yet distinguishing between these modes of organization remains challenging. Here, we developed two intrinsically fluorescent derivatives of the self-assembling dipeptide diphenylalanine (FF): nitrobenzoxadiazole-conjugated FF (NBD-FF) and a coumarin-conjugated tert-butyloxycarbonyl-protected FF derivative (BocFF-DAC). Both derivatives formed fibrillar assemblies while exhibiting spectrally distinct fluorescence, enabling their direct visualization by confocal microscopy. Single-crystal X-ray diffraction of NBD-FF revealed an extended hydrogen-bonded lattice in which the NBD moiety participates directly in intermolecular interactions, including aromatic, hydrophobic, and hydrogen-bonding contacts. This finding demonstrates that the fluorophore acts as an active structural element rather than a passive label. When NBD-FF and BocFF-DAC were assembled together, confocal microscopy revealed predominantly spatially distinct fibrillar populations with limited overlap, consistent with preferential self-sorting rather than uniform co-assembly. These findings establish fluorophore-conjugated short peptides as intrinsically traceable building blocks for investigating multicomponent supramolecular organization, while highlighting that fluorophore conjugation can simultaneously enable visualization and influence the assembly.