Small Fluorogenic Amino Acids for Peptide‐Guided Background‐Free Imaging

The multiple applications of super‐resolution microscopy have prompted the need for minimally invasive labeling strategies for peptide‐guided fluorescence imaging. Many fluorescent reporters display limitations (e.g., large and charged scaffolds, non‐specific binding) as building blocks for the construction of fluorogenic peptides. Herein we have built a library of benzodiazole amino acids and systematically examined them as reporters for background‐free fluorescence microscopy. We have identified amine‐derivatized benzoselenadiazoles as scalable and photostable amino acids for the straightforward solid‐phase synthesis of fluorescent peptides. Benzodiazole amino acids retain the binding capabilities of bioactive peptides and display excellent signal‐to‐background ratios. Furthermore, we have demonstrated their application in peptide‐PAINT imaging of postsynaptic density protein‐95 nanoclusters in the synaptosomes from mouse brain tissues. Benzodiazole amino acids are excellent small building blocks for the construction of background‐free peptide probes for fluorescence imaging. We demonstrate their robustness and versatility for solid‐phase peptide synthesis, their minimally invasive character, and their compatibility with different optical imaging modalities, including super‐resolution peptide‐PAINT imaging.