Blood‐Brain‐Barrier‐Crossing Fluorinated Luciferase Prosubstrates Enable Sensitive Monitoring of D‐Cysteine Levels in Brain Diseases

Abstract

ABSTRACT D‐Cysteine (D‐Cys) is critically involved in regulating redox homeostasis within the brain, and its dysregulation has been closely related to the pathogenesis of various neurological disorders. However, the precise mapping of cerebral D‐Cys levels in situ remains a formidable challenge due to the lack of specific detection tools. Herein, we report a new class of fluorinated firefly luciferase prosubstrates for sensitive and noninvasive detection of D‐Cys in the brain via nitrile‐aminothiol bioorthogonal reaction. The lipophilic 7'F‐CBT exhibits enhanced blood‐brain barrier (BBB) permeability and reacts with endogenous D‐Cys to form a luciferin analog in situ, generating prolonged and intense bioluminescence. It sensitively detected D‐Cys in vitro, achieving a detection limit of 1.37 nM, and successfully imaged endogenous D‐Cys in the mouse brain with a high signal‐to‐noise ratio (60:1) after intravenous administration. Furthermore, 7'F‐CBT enabled unprecedented video‐rate visualization of D‐Cys in freely moving glioblastoma‐bearing mice. More importantly, by leveraging BBB‐permeable 7'F‐CBT , we revealed a precipitous reduction of brain D‐Cys levels in a drug‐induced Parkinson's disease (PD) mouse model for the first time. This study paves the way for diagnosing D‐Cys‐related brain diseases and provides a novel diagnostic tool.