First demonstration of biocatalytic production of MRI contrast agent

Motivation: Despite the importance of 5-MDHT as an excellent CEST-MRI probe for imaging the HSV1-TK reporter gene expression in vivo, efficient biosynthesis methods are needed. Goal(s): We aimed to produce 5-MDHT enzymatically and test it in cells overexpressing the HSV1-TK. Approach: We identified the enzymes and substrates involved in the 5-MDHT biosynthesis. We validated 5-MDHT biosynthesis and phosphorylation using LC-MS, NMR and CEST-MRI. Results: hPNPase catalysis showed promising results for synthesizing the 5-MDHT in a single-step reaction. Also, we found that transfected HEK-293FT cells with hPNPase are active and able to produce the 5-MDHT. Impact: Simplifying the production of imaging probes will significantly advance and redirect our understanding of biological mechanisms, diagnosis, and treatment of human diseases. Once established, this new approach could be used to produce many novel imaging probes for specific medical conditions.