Peptides on Tap: Automated Flow Synthesis With Standard HPLC

Abstract

Peptide synthesis is central to biomedical research, yet automated flow synthesis typically requires custom-built instrumentation, limiting accessibility beyond specialized laboratories. Here we demonstrate that a standard analytical HPLC can be repurposed for fully automated flow-based peptide synthesis. By packing a short guard column with resin and programming reagent injections through the autosampler, we achieved efficient coupling and deprotection cycles under temperature-controlled flow, using TBEC/Oxyma activation. This setup exploits the precise flow control, heating, and in-line UV detection intrinsic to HPLC systems, enabling real-time monitoring of Fmoc deprotection. Using this approach, we synthesized peptides up to 30 residues, including GLP-1 and bivalirudin, with comparable yields and purity to conventional solid-phase synthesis. No significant epimerization was observed under optimized conditions, and crude bivalirudin displayed thrombin inhibition activity identical to purified reference material. The method was also used to obtain a 58-residue miniprotein in under 7 h on mg scale. This work introduces a simple, safe, and economical platform for automated peptide synthesis with the advent of high temperature flow accessible to any laboratory equipped with an HPLC.