Line-Shifting Triarylmethyl Radicals for Imaging of Enzyme Activity Using Overhauser-Enhanced Magnetic Resonance Imaging: Application to Alkaline Phosphatase

Enzyme catalytic activities are critical biomarkers of tissue states under physiological and pathophysiological conditions. However, the direct measurement and imaging of enzyme activity <i>in vivo</i> remains extremely challenging. We report the synthesis and characterization of the first stable triarylmethyl (TAM) radical substrate of alkaline phosphatase (TAM-ALPs). The enzymatic dephosphorylation of TAM-ALPs results in a drastic change in its electron paramagnetic resonance (EPR) spectrum that can be used to image enzyme activity using EPR-based technologies. TAM-ALPs and their enzyme products were fully characterized using EPR and HPLC-MS techniques. A proof of concept of imaging enzyme activity using Overhauser-enhanced magnetic resonance imaging was demonstrated <i>in vitro.</i> This study clearly demonstrates the potential of EPR-based imaging technologies associated with TAM spin probes to map enzyme activity <i>in vivo</i> in future studies.