Resonant acoustic mixing enables solvent-less amide coupling in solid-phase peptide synthesis

Abstract

Solid-phase peptide synthesis (SPPS) is the backbone of modern peptide production. However, it relies heavily on relatively toxic solvents and generates significant waste, limiting its sustainability and scalability. To address these limitations, we report the first fully solvent-less peptide coupling protocol for SPPS enabled by Resonant Acoustic Mixing (RAM), representing a step toward greener peptide manufacturing. This method eliminates bulk solvent use, reagent pre-dissolution, and pre-activation during coupling by using mechanical agitation to drive efficient amide bond formation. Optimized conditions (95g acceleration, 5 min, 1.5 equiv. Fmoc-amino acids) afford rapid and clear reactions with high conversion and purity. Notably, no external solvent is added during coupling; instead, residual solvent retained from resin pre-swelling creates a localized microenvironment sufficient for in situ activation. Compared to conventional SPPS, this protocol significantly reduces solvent and reagent use, reaction time, and waste. Process Mass Intensity (PMI) calculations show clear improvements, highlighting the method's environmental and economic benefits. This approach was validated by synthesizing two bioactive peptides (IKVAV and Angiotensin 1–7) in high yield and purity, and further demonstrated excellent scalability in a tenfold scale-up.