Controlled aqueous nanoassembly-driven fully automated Fmoc solid-phase peptide synthesis (SPPS) with in-water coupling.

Fmoc-based solid-phase peptide synthesis (SPPS) is a cornerstone of modern peptide manufacturing; however, its reliance on dimethylformamide (DMF)-a toxic and environmentally regulated solvent-poses significant sustainability challenges. Herein, we report a controlled aqueous nanoassembly-based strategy that enables fully automated Fmoc-SPPS under water-based coupling conditions. Aqueous nanoassemblies of Fmoc-protected amino acids, generated through a simple two-step preparation protocol, provide a stable and reactive coupling environment compatible with automated synthesis. The methodology was implemented on a commercially available peptide synthesizer and applied to peptides of varying lengths and sequence complexities, affording products of acceptable purity. Importantly, the platform was applied to the automated synthesis of long-chain peptides exceeding 30 residues, including GLP-1 (1-37), demonstrating its applicability to challenging sequences. At the time of writing, this represents, to the best of our knowledge, one of the earliest fully automated Fmoc-SPPS platforms employing water-based coupling cycles without the use of DMF. By eliminating DMF and employing environmentally benign solvents such as water and ethyl acetate, the present methodology provides a practical and sustainable alternative to conventional SPPS, advancing green practices in automated peptide synthesis.