An affordable and low-waste protocol for rapid and robust solid-phase peptide synthesis at elevated temperatures

Automated solid-phase peptide synthesis (SPPS) is the premier methodology for producing high-purity peptides across a broad range of applications. However, despite its efficiency, automated SPPS often requires expensive instrumentation and generates significant chemical waste. To overcome these limitations, we present a robust SPPS protocol that competes the thermal acceleration of microwave-assisted systems using standard laboratory equipment, offering a highly accessible and cost-effective alternative. The methodology enables the rapid assembly of a diverse array of linear and cyclic peptides (10–100 mg scale), including two biochemical active sequences, with reaction times reduced to minutes and a decrease in chemical waste. Given that elevated temperatures could compromise stereochemical integrity, we performed an in-depth investigation into epimerization risks using a multi-modal analytical suite, including high-performance liquid chromatography, nuclear magnetic resonance, and ion mobility mass spectrometry. Our results demonstrate that this instrumental approach not only provides satisfactory crude yields and purities but also maintains high stereochemical fidelity and native biochemical function, offering a robust alternative to high-cost automated systems.