Aqueous-Phase Peptide Synthesis via Stepwise Amino Acid Elongation Catalyzed by Aqualigase.

Peptides have broad applications in fields such as food, medicine, and functional materials. Conventional solid-phase peptide synthesis (SPPS) constructs diverse peptide sequences through stepwise amino acid elongation but relies on stoichiometric coupling reagents and organic solvents, thereby generating considerable chemical waste. Aqualigase, an engineered subtilisin, offers a green alternative that directly condenses free carboxylic acids and amines in pure aqueous media. However, whether this system can support stepwise elongation using amino acids as building blocks remains unclear. Here, using the Aqualigase-catalyzed ligation of Ala-Ala-Pro-Phe and Ala-Phe as a model reaction, hydrogen-bond analysis revealed that a stable interaction between the P1' residue of the acyl acceptor and Asn155 in the substrate-binding pocket is critical for effective binding of the acyl acceptor. Guided by this finding, we demonstrated that Aqualigase ligates peptides ranging from dipeptides to pentapeptides with amino acids as acyl acceptors, expanding its catalytic mode from peptide-fragment ligation to fragment-amino acid ligation. RMSF analysis further revealed pronounced flexibility in the loops surrounding the catalytic pocket, particularly residues 95 to 106 and 155 to 164, providing a structural basis for substrate accommodation. Substrate scope analysis showed that, under Aqualigase catalysis, amino acids are more broadly compatible as acyl acceptors than as acyl donors, supporting an N-to-C-terminal stepwise elongation strategy. Using this approach, the functional tripeptide Gly-Leu-Phe was synthesized in pure aqueous media through sequential amino acid elongation. This study establishes an engineered subtilisin-catalyzed strategy for stepwise peptide synthesis in pure aqueous media, providing a green route for flexible peptide construction.