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.