N-Oxide-Driven Heme-Activatable Biomolecule Labeling for Visualization of Labile Heme in Living Cells and Mouse Brain

Abstract

Labile heme, a complex comprising protoporphyrin IX coordinated with an iron ion, functions as a critical biological mediator in physiological and pathological processes across diverse organisms. Despite its fundamental importance, both the subcellular dynamics and the systemic trafficking of heme remain inadequately characterized, primarily due to a scarcity of chemical tools capable of detecting labile heme in living systems. Herein, we report the development of innovative chemical probes that enable the detection of labile heme in live cells, fixed specimens, and in vivo models through a novel N-oxide-driven heme-activatable biomolecule labeling strategy. These probes exhibit exceptional selectivity, becoming activated exclusively upon heme interaction to form covalent bonds with neighboring proteins. We demonstrate that the tertiary amine N-oxide moiety triggers heme activation, followed by self-oxidation to generate reactive quinone intermediates responsible for covalent bond formation. This reaction proceeds effectively in living cells and within the brains of live mice. Our probe enables fluorescence imaging of living cells and 3D tissue visualization of mouse brains for labile heme detection, while facilitating multiomics analysis, including proteomics and single-cell RNA sequencing. This comprehensive approach provides unprecedented opportunities for investigating the physiological and pathological roles of labile heme in complex living systems.