Discovery of a Thermostable Nigerose Phosphorylase for the Efficient Chemoenzymatic Radiosynthesis of a <i>S. aureus</i> –Targeted ¹⁸ F-Disaccharide

Staphylococcus aureus is a leading cause of life-threatening infections worldwide. The diagnosis and treatment of S. aureus infections are further complicated by the global rise of antimicrobial resistance. Therefore, rapid detection of active S. aureus remains a critical unmet need to provide effective infection management. In this study, we identified 2-deoxy-2-[18F]-fluorosakebiose ([18F]FSK) as an optimal radiotracer to detect active S. aureus and established its efficient chemoenzymatic radiosynthesis to facilitate clinical translation. Methods: Several [18F]FDG-derived disaccharides were obtained via reverse phosphorolysis: [18F]FSK (α-1,3–linked), 2-deoxy-[18F]-fluoromaltose (α-1,4–linked), 2-deoxy-2-[18F]-fluorolaminaribiose (β-1,3–linked), and 2-deoxy-2-[18F]-fluorocellobiose (β-1,4–linked). These tracers were screened in vitro in multiple S. aureus isolates to identify bacterial incorporation. The lead candidate, [18F]FSK, was further characterized via biodistribution and dosimetry analyses and evaluated in a S. aureus myositis model to assess antimicrobial treatment response. Finally, to promote the clinical translation of [18F]FSK, 2 different radiosynthetic strategies were investigated: reverse phosphorolysis of [18F]FDG using maltose phosphorylase and using newly identified nigerose (also called sakebiose) phosphorylases. Results: Nigerose phosphorylase–derived [18F]FSK was selected as the optimal radiotracer for detecting S. aureus because of its consistent and robust uptake in multiple S. aureus isolates. [18F]FSK demonstrated favorable distribution and elimination over time, with minimal nonspecific signals in uninfected organs. The estimated human effective doses indicated an effective dose comparable to that of [18F]FDG. The radiosynthesis of [18F]FSK, initially obtained as an accidental byproduct of maltose phosphorylase catalysis, was further improved using a nigerose phosphorylase originating from thermostable Spirochaeta thermophila, enabling nearly quantitative conversion of [18F]FDG to [18F]FSK (up to 97%). Conclusion: We demonstrated that [18F]FSK is a potent and robust PET radiotracer for the detection of active S. aureus in vivo and aids in the selection of an appropriate antimicrobial treatment. These findings highlight the potential use of [18F]FSK in promoting the effective management of S. aureus infections in clinical settings.