Glycinamide‐Driven Coumarin Construction (GDCC): A Mild Strategy for Fluorescent Labeling and Macrocyclization of Peptides

ABSTRACT Peptidic macrocycles have attracted increasing attention due to their well‐defined structures and enhanced biological activities. However, efficient cyclization of residues lacking reactive side chains remains a significant challenge. Herein, we report a glycinamide‐driven coumarin construction (GDCC) reaction that enables the in situ formation of coumarin scaffolds via condensation between C ‐terminal glycine residues and salicylaldehyde analogs under mild, metal‐ and catalyst‐free conditions. While various salicylaldehyde derivatives afford efficient fluorescent labeling, symmetrical salicylaldehyde‐based linkers uniquely promote intramolecular bis ‐glycine cyclization, generating intrinsically fluorescent peptide macrocycles with tunable topology and hydrophobicity. Notably, the optimal peptide 64 exhibits photophysical properties comparable or even superior to those of the coumarin gold standard, 7‐(diethylamino)‐4‐methylcoumarin. The GDCC reaction displays broad substrate scope and excellent compatibility with both solid‐phase and solution‐phase peptide synthesis. Its potential in chemical biology is demonstrated by the streamlined construction of diverse fluorescent cyclic peptides, including RGD‐based probes for live‐cell imaging, peptide–drug conjugates for targeted doxorubicin delivery, and a panel of PD‐1/PD‐L1 interaction inhibitors with tunable activity.