Intracellular Bismuth Coordination of Peptides and Miniproteins

Abstract Cyclisation of peptides has emerged as a powerful methodology to enhance molecular stability and functional efficacy. Bismuth(III) coordination represents a particularly compact and chemoselective cyclisation modality, yet to date has remained confined to in vitro or phage-based systems. Here we report the first example of bismuth-mediated peptide and minprotein cyclisation occurring within living cells, achieved directly during recombinant expression in E. coli . Supplementation of growth media with bismuth salts enables efficient intracellular coordination of either three or six cysteine residues, yielding bicyclic and tetracyclic architectures with minimal cellular toxicity. Bis-bismuth coordination generated a tetra-cyclic minprotein with exceptional thermal and serum stability, representing a level of intracellular structural reinforcement not previously accessible. This chemistry enables the intracellular constraint of peptides and proteins and is directly compatible with genetically encoded peptide and miniprotein libraries, establishing bismuth coordination as a compact and robust modality for live-cell molecular screening.