Click or Acyl transfer? Pathway Selection Driven by Acyl Phosphates and Amino Acid Side Chains

Covalent transformations in biology follow precise temporal sequences and orchestrated by enzymes to regulate processes such as selective side-chain modifications, including acylation and phosphorylation. Achieving such temporal control in synthetic systems remains challenging due to overlapping reaction pathways and limited selection mechanisms. Herein, we present an abiotic aqueous system in which aminoacyl phosphate esters bearing alkyne groups undergo a defined sequence of covalent reactions governed by pep-tide-based nucleophiles. In the presence of phenolic groups (e.g., tyrosine), copper-catalyzed azide-alkyne cycloaddition (CuAAC) proceeds rapidly, generating mixtures of esters and triazole products. In contrast, cysteine-containing peptides first form selectively thioesters, suppressing early CuAAC and enabling temporal resolution. Monitoring the reaction over time reveals that thiol-copper coordination controls early steps, while self-assembly prolongs intermediate lifetimes, introducing a second layer of regulation. Embedding both nucleophiles in one peptide (Ac-CY) yields a three-step sequence: thioester, diester, followed by CuAAC. The chemical structure of azides further tunes product selectivity, be-yond acyl transfer. The transient acylated intermediates and their subsequent conversion into stable triazoles exemplify how post-acyl transfer modifications shaped by supramolecular context, can direct irreversible covalent pathways. Control over these transformations is achieved through the interplay of reactivity and self-assembly, offering a blueprint for constructing chemical reaction networks with intrinsic temporal order.