A Rapid Manual Solid Phase Peptide Synthesis Method for High‐Throughput Peptide Production

Solid phase peptide synthesis (SPPS) techniques are critical for developing and using peptides in various biomedical applications. However, typical synthesis routes used in SPPS are either resource-intensive (e.g., with automated synthesis or commercial services) or time-consuming (e.g., with manual benchtop synthesis). Here, a rapid manual synthesis method was developed to produce up to 8 peptides with fast cycle times simultaneously. Peptides synthesized manually were of equivalent or superior quality to those produced by in-house microwave-assisted automated peptide synthesis, with higher average crude purity of 70% compared to 50%. The method significantly reduced synthesis time, enabling the parallel coupling of up to 8 amino acids simultaneously in 15-20 min, as opposed to traditional benchtop peptide synthesis, which requires 80-150 min per amino acid. This approach offers an intermediate throughput between milligram-scale libraries and gram-scale single peptide synthesis, enabling rapid iteration for novel peptide designs without the need for expensive automated systems. As a result, peptide modifications, including incorporation of unnatural amino acids, can be explored, accelerating the development of peptides for a wide range of applications.