Tailored Phosphate Leaving Groups Direct Pathway-Dependent Self-Assembly

Phosphate esters and anhydrides are central to biology, storing and transferring chemical energy to sustain processes from metabolism to translation. Among them, acyl phosphates are highly reactive, yet biology channels their activation chemistry almost exclusively through aminoacyl adenylates. This conserved design leaves unexplored how alternative phosphate leaving groups might influence reactivity and structure. Here we show that aminoacyl phosphate esters with varied leaving groups (ethyl, phenyl, naphthyl, dodecyl) direct peptide bond formation and self-assembly through distinct pathways in water. Structural features of the leaving group guide preorganization into spherical aggregates before acyl transfer and influence coassembly with peptides after bond formation, imprinting outcomes that persist beyond activation. Consequently, the leaving group determines not only peptide yields, but also the supramolecular architectures and mechanical properties of assemblies arising from the same peptide sequences. In multicomponent mixtures, aminoacyl phosphates create recognition microenvironments in which aromaticity, hydrophobicity, or charge bias electrophile-nucleophile pairing, thereby transforming them from simple electrophilic reagents into active design elements capable of driving sequence selectivity. Moreover, soluble phosphates undergo phosphoryl exchange with orthophosphate, pyrophosphate, or adenosine monophosphate (AMP) to generate alternative intermediates that divert reactivity, whereas self-assembling phosphates resist exchange and favor amino acid oligomerization. These findings establish the leaving group as a tunable design element that governs reactivity, directs supramolecular organization and regulates pathway dynamics, transforming activation from a synthetic step into an active driver of recognition and assembly.