Most of our PET-CT studies to date have used 18F fluoro deoxyglucose (18F-FDG) to image the metabolism of the eukaryotic cells in TB lesions but we are also making attempts to identify the location, abundance and metabolic state of the bacteria in lesions. In an effort to identify small molecules that could be used to specifically label MTb in vivo, we capitalized on the unusually broad substrate tolerance of the MTb antigen 85 enzymes, which transfer mycolates onto structurally diverse sugars to form part of MTbs cell wall. Antigen-85 enzymes are expressed on the exterior of MTbs cell wall and incorporate exogenous trehalose (a nonmammalian disaccharide consisting of a two 1-1 α, α-linked glucose monomers) as either the mono- or dimycolate, even tolerating trehalose molecules containing bulky modifications. We have used this system to incorporate 18F trehalose into bacteria in the lesions of infected rabbits and 18F activity has been detected in lesions of infected rabbits by PET-CT imaging. A series of different positions and methods for attaching the 18F to the sugar are being explored to see which is most efficiently incorporated. Using trehalose should afford an improvement over the currently used 18F-FDG, as glucose is used by mammalian cells as well as bacterial, causing noise in the scans due to increased metabolism or inflammation in the host. Use of trehalose is unique to bacteria;it is not absorbed by mammalian cells, which should limit noise in PET scans. The Davis group has designed three trehalose analogs incorporating fluorine at the 2, epi-4, or 6-position of trehalose for use as PET radiotracers. The 2-fluorotrehalose synthesis is a biomimetic process, inpired by bacterial synthesis of trehalose from glucose. Chemoenzymatic synthesis of 2-fluorotrehalose (2-FTre)occurs as a one-pot cascade reaction in which hexokinase transfers a phosphate from adenosine triphosphate (ATP) to 18F-FDG (normally glucose in bacterial trehalose synthesis). OtsA then transfers the glucose from the donor UDP-glucose to the acceptor phosphorylated 18F-FDG. Dephosphorylation to give the desired product is effected by OtsB. The entire one-pot process is complete in 45 min. The advantage here is that a relatively technically facile manipulation would convert a commercially available radiotracer to a TB-specific one. We have acquired some preliminary PET-CT scan data in rabbits, one healthy, one infected with HN878 MTb. In the infected animal, four lesions were present. Two were not PET-active, and two were, although all four had similar amounts of colony forming units (CFU). Upon necropsy, the two PET-inactive lesions were extremely rigid and thick-walled, implying that uptake is related more to accessibility (i.e. vasculature) than amount of bacteria present. The t1/2 was approximately 40 min, and the radiotracer was clear from blood at 200 min post-injection, but later animals experienced unexpected toxicity within about 24 hours of the procedure. Metabolite analysis was done on a blood sample drawn 30 min post-injection, and showed that 95% of the 2-FTre was unchanged and 5% had been metabolized to 18F-FDG. In the initial procedures, the radiotracer displayed large amounts of white precipitate upon standing, contained trace protein, the pH was 11 or greater, and the saline content was approximately more than three times physiological levels, which was not reasonable for further development for use in primates and humans, so the purification strategy was reworked to produce a more biologically compatible final product. We were able to use a single strong-anion exchange solid phase extraction (SAX SPE) at a pH of 8 to fully remove ADP and UDPfollowed by a PD10 (size-exclusion column) , which allowed us to remove protein to below detection limit in a Bradford assay. The final procedure gave a 75% yield (decay-corrected) over approx. 65 min synthesis time, with near-physiological pH and salinity.. In the rabbit model, lesions were again visualized, but the animals experienced unexpected toxicity within about 24 hours of the procedure. Tests during the development of the synthesis protocol in Oxford had not shown the presence of endotoxin;however, when we analyzed the tracer for endotoxin (also called lipopolysaccharide, LPS) using a quantitative chromogenic assay (the only model currently approved by the FDA for endotoxin determination in radiotracers formulated for human use) to measure LPS levels and found extremely high levels (>6,000 EU/mL in the radiotracer). We then assayed all mixture components and found that both enzymes were contaminated with high levels (>2,000,000 EU/mL) of LPS. It was concluded that while the original high pH purification reduced the LPS contamination, the procedure still had the original drawbacks that would prevent it from use in humans. Efforts to purify either the proteins or the final 2-FTre radiotracer were unsuccessful, so we decided to express OtsA and OtsB in an organism that does not produce endotoxin. We contracted for expression and purification of the two proteins in baculovirus/insect cells and Picchi pastoris to see which system will give functional, soluble protein. The two systems have been shown to be compatible with GMP manufacturing and will allow use in humans. Unfortunately, the P. pastoris system did not provide provide the desired proteins in pilot experiments performed on 50 mL scale. Further attempts at optimization of OtsB expression were unsuccessful. Luckily, the baculovirus system did produce LPS-free functional proteins. The OtsB was fully functional and did not degrade upon storage. A removable His-tag on the N-terminus allowed Ni-NTA purification followed by tag cleavage to give the desired protein in >10 mg/L yield on medium scale (2L). The baculovirus system could also product OtsA which was functional and produced in good yields (>10 mg/L), but the OtsA was not stable to longterm storage conditions. We are exploring the possibility of moving the His-tag from the N-terminus to the C-terminus and maintaining the tag post-purification, as this protein would be identical to the E. coli protein used in preliminary experiments. Additionally, we are exploring immobilizing the proteins on solid support. Initial testing evaluated function the proteins immobilized on cyanogen bromide-activated agarose beads. Gratifyingly, all three proteins maintain function upon immobilization, losing only a small amount of their catalytic efficiency. Hexokinase completes the phosphorylation within 15 min;OtsA completes the glycosylation within 45 min, and OtsB reaches approx. 50% completion for the dephosphorylation at 60 min. All three solid-supported proteins may be used in a one-pot transformation, which allowed for isolation of the 2-FTre by simple syringe filtration of the reaction mixture. We are currently in the process of optimizing parameters such as bead loading, pH, and temperature to facilitate rapid and high-yielding transformations. If we are successful at improving yield, reaction rater, and stability to storage, we could then ship these cartridges to study sites worldwide. Radiopharmacists would pass through commercial 18F-FDG and a solution of ATP and UDP-Glucose through the cartridge to produce the desired radiotracer with no specialized equipment or special training. The procedure would be operationally simple and allow TB-specific imaging to monitor course of treatment. Ideally, we would be able to rapidly assay treatment success or failure in a manner that relies only on abundance and metabolic state of the bacteria.