Biocatalytic Construction of a Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Nucleoside Probe: Synthesis and Evaluation of 5‐Methyl‐5,6‐Dihydrothymidine

Magnetic resonance imaging (MRI) is a cornerstone of modern clinical diagnostics, often enhanced by contrast agents. Traditionally, these agents are chemically synthesized, which can involve complex, costly, and environmentally unfriendly processes. Here, we report a novel biocatalytic approach for the efficient, safe, and eco‐friendly synthesis of 5‐methyl‐5,6‐dihydrothymidine (5‐MDHT), a potent chemical exchange saturation transfer (CEST)‐MRI probe for imaging in vivo expression of the Herpes Simplex Virus Type‐1 Thymidine Kinase (HSV1‐TK) reporter gene. We demonstrate that 5‐MDHT can be biosynthesized via one‐ or two‐step enzymatic reactions using human purine nucleoside phosphorylase (hPNPase) and the SgvM VAV SAM‐dependent methyltransferase. hPNPase catalyzed the base‐exchange reaction with catalytic efficiencies ( k cat /K M ) between 138 and 316 s −1 ·M −1 , while SgvM VAV methylation of 5,6‐dihydrothymidine yielded 5‐MDHT with a catalytic efficiency of 26 s −1 ·M −1 . Molecular dynamics simulations supported the enzymatic binding and selectivity observed experimentally. The resulting 5‐MDHT was validated using CEST‐MRI, showing a distinct exchangeable imino proton signal at 5.3 ppm. These findings highlight the chemo‐ and regioselectivity of the biocatalysts and establish biocatalysis as a viable platform for producing clinically relevant MRI contrast agents.