PROJECT SUMMARY: The on-demand synthesis of microbe-specific PET radiotracers represents a significant challenge to their clinical translation. To be useful, these tracers must be available to clinicians in the acute care setting, when patients present with signs and symptoms of bacterial infection. In this proposal, we develop new chemoenzymatic methods to synthesize infection-targeted PET tracers from the common oncologic tracer 2- deoxy-[18F]-fluoro-D-glucose ([18F]FDG), using a simple phosphorylase-based approach designed to increase access to new PET imaging tools in clinical practice. We will leverage these new methods for the detection of S. aureus, a major culprit in vertebral discitis-osteomyelitis and other musculoskeletal infections. Perhaps surprisingly, chemoenzymatic syntheses are infrequently applied to PET despite the advantages of regioselective and stereoselective glycan construction. Based on reported microbe-specific disaccharides, we developed a method to dimerize [18F]FDG to form positron-labeled disaccharides for detecting microorganisms in vivo. As reported in our preliminary data, when [18F]FDG was reacted with D-glucose 1-phosphate (α/β depending on the enzyme) in the presence of a disaccharide phosphorylase, the positron-labeled products 2- deoxy-[18F]-fluoro-maltose (α-1,4 linked; [18F]FDM), 2-deoxy-2-[18F]-fluoro-sakebiose (α-1,3 linked; [18F]FSK) 2- deoxy-2-[18F]fluoro-trehalose (α-1,1 linked; [18F]FDT), 2-deoxy-2-[18F]fluoro-laminaribiose (β-1,3 linked; [18F]FDL), and 2-deoxy-2-[18F]fluoro-cellobiose (β-1,4 linked; [18F]FDC) were separately obtained in 20 minutes. Of the α-linked products, the sakebiose-derived tracer [18F]FSK showed the best performance in S. aureus, based on high accumulation in vitro, stability in human serum, and specific detection of living bacteria in vivo. Both synthetic ease, and high sensitivity of [18F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of this tracer to infected patients. Initially the α-1,3 linked [18F]FSK was best obtained as the minor product of maltose phosphorylase, but recently we were able to obtain [18F]FSK as the sole product of reverse phosphorolysis using a L. phytofermentans-derived sakebiose phosphorylase, with rapid purification via solid-phase extraction. In the proposed work, we will first expand methods for the phosphorylase-mediated radiosynthesis of [18F]FSK (Specific Aim 1). We will improve techniques to make [18F]FSK, including developing new tools to assist clinical production- for example via phosphorylase immobilization and enzyme engineering to enhance thermal stability. We will then further validate these new tools in preclinical models (Specific Aim 2), and study [18F]FSK in patients suffering from spinal infection (Specific Aim 3). This project is focused on the on-demand radiosyntheses of S. aureus-targeted PET sensors, but the methods developed will have wide applicability to other human patho...