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

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.