Biocatalytic Construction of a CEST MRI 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 138-316 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.