A general strategy towards multicolour fluorogenic peptides for wash-free bioassays.

Peptide-based fluorescent probes are valuable tools for live-cell imaging, but conventional labelling approaches often require washing or long incubation, limiting their signal-to-noise ratios. Fluorogenic amino acids (FgAAs) can overcome these limitations, yet their rational design remains challenging owing to the lack of generalizable quenching principles and synthetic routes compatible with diverse fluorophores. Here we developed a general strategy to convert constitutively bright fluorophores into turn-on FgAAs through intramolecular tryptophan-induced quenching. Computational analysis showed that quenching is governed by photoinduced electron transfer or twisted intramolecular charge transfer mechanisms, with adaptability across ultraviolet-to-near-infrared fluorophore scaffolds. We synthesized a large library of multicolour FgAAs and integrated them into solid-phase peptide synthesis to generate turn-on probes for real-time microscopy in live cells. We then designed fluorogenic peptides targeting programmed cell death protein 1 (PD-1), enabling T cell targeted drug screening and identifying avasimibe as a small-molecule modulator of PD-1 activity. This work establishes a rational strategy for designing multicolour FgAAs for peptide engineering, imaging and drug discovery.