Abstract
Enzyme catalytic activities are critical biomarkers of tissue states under physiological and pathophysiological conditions. However, the direct measurement and imaging of enzyme activity in vivo 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 in vitro. This study clearly demonstrates the potential of EPR-based imaging technologies associated with TAM spin probes to map enzyme activity in vivo in future studies.