Line-Shifting Triarylmethyl\nRadicals for Imaging of\nEnzyme Activity Using Overhauser-Enhanced Magnetic Resonance Imaging:\nApplication to Alkaline Phosphatase

Enzyme catalytic activities are critical biomarkers of\ntissue states\nunder physiological and pathophysiological conditions. However, the\ndirect measurement and imaging of enzyme activity <i>in vivo</i> remains extremely challenging. We report the synthesis and characterization\nof the first stable triarylmethyl (TAM) radical substrate of alkaline\nphosphatase (TAM-ALPs). The enzymatic dephosphorylation of TAM-ALPs\nresults in a drastic change in its electron paramagnetic resonance\n(EPR) spectrum that can be used to image enzyme activity using EPR-based\ntechnologies. TAM-ALPs and their enzyme products were fully characterized\nusing EPR and HPLC-MS techniques. A proof of concept of imaging enzyme\nactivity using Overhauser-enhanced magnetic resonance imaging was\ndemonstrated <i>in vitro.</i> This study clearly demonstrates\nthe potential of EPR-based imaging technologies associated with TAM\nspin probes to map enzyme activity <i>in vivo</i> in future\nstudies.