Pathway selection between click and acyl transfer reactions driven by aminoacyl phosphates.

Covalent transformations in biology follow defined temporal sequences that regulate processes such as acylation and phosphorylation, yet achieving comparable temporal control in synthetic systems remains challenging. Here, we report an abiotic aqueous reaction network in which aminoacyl phosphate esters bearing alkyne groups undergo a programmed sequence of covalent transformations governed by peptide-based nucleophiles. Phenolic nucleophiles promote rapid copper-catalyzed azide-alkyne cycloaddition (CuAAC), whereas cysteine-containing peptides transiently coordinate copper via their thiol groups, delaying CuAAC and favoring thioester formation. Kinetic analysis reveals that thiol-copper coordination controls early pathway selection, while self-assembly prolongs intermediate lifetimes and enables subsequent transformations. Combining both nucleophiles within a single peptide yields a three-step cascade comprising thioester formation, diester generation, and CuAAC. Variation of the azide structure further tunes product selectivity beyond acyl transfer. Together, these results demonstrate how the interplay of reactivity and supramolecular organization can encode intrinsic temporal order into chemically driven reaction networks.