The rapid radiosynthesis of disease-specific tracers from widely available [¹⁸F]FDG is a promising approach to democratize PET imaging in the United States. Our laboratories have recently studied the chemoenzymatic manipulation of [¹⁸F]FDG given the advantages of regioselective and stereoselective glycan construction. Motivated by reported bacterial- and fungi-specific disaccharides, we dimerized [¹⁸F]FDG to form positron-labeled disaccharides for detecting microorganisms in vivo. When [¹⁸F]FDG was reacted with β-D-glucose 1-phosphate in the presence of maltose phosphorylase (MP), the positron-labeled products 2-deoxy-[¹⁸F]-fluoro-maltose (α-1,4 linked; [¹⁸F]FDM, major product ~ 85%) and 2-deoxy-2-[¹⁸F]-fluoro-sakebiose (α-1,3 linked; [¹⁸F]FSK, minor product ~ 15%) were obtained in 20 minutes¹. Here we report the further prolongation of [¹⁸F]FDM to synthesize higher order a-1,4 linked oligomers- that have been of very high interest in PET with tracers derived from N=2², N=3³, and N=6⁴ glucose units all reported.

When HPLC-purified [¹⁸F]FDM was incubated with α-D-glucose 1-phosphate and maltodextrin phosphorylase (also called α-glucan phosphorylase (E.C. 2.4.1.1), PET labeled products from N=2 to N=11 were observed. We first purified [¹⁸F]FDM by HPLC as reported¹; removal of [¹⁸F]FSK was required for the radiosyntheses of pure a-1,4 oligomers. In a 4 mL borosilicate vial containing PTFE stir bar, maltodextrin phosphorylase (0.1 mg, 1 unit) and αGlc1-P (5 mg, 0.016 mmol) were added. A dose of [¹⁸F]FDM (2-10 mCi) in citrate buffer (0.1M, pH=6.5, 0.5 mL) was directly transferred to the vial and the mixture was stirred at 75 °C for 30 min. The mixture was diluted with MeCN before being purified via semi prep HPLC (YMC-Pack Polyamine II, 250 X 10 mm) using mobile phase 65% MeCN/35 % H₂O. New [¹⁸F]-products were isolated in 5-7mL fraction. The fraction was then diluted with MeCN (40 mL) before being passed through Sep-pak Plus NH₂ Cartridge at 4 mL/min to trap each dimer product. After flushing the cartridge with air and N₂ gas, they were eluted using citrate buffer solution for direct formulation before next step. Chemical purity of both [¹⁸F] maltodextrin product was confirmed by analytical HPLC.

We propose that these oligomers can be screened efficiently to identify the most promising bacteria-specific PET tracers. Interestingly, all tracers are [¹⁸F]FDG-derived. We showed that the 2-position ¹⁸F substituted maltose derivative [¹⁸F]FDM appears to have numerous advantages over the 6-position ¹⁸F derivative originally reported by Namavari et al., in terms of stability and microorganism export. Although the 6-position is more chemically accessible, the corresponding 6-[¹⁸F] derivative is both more vulnerable to defluorination, and prohibits 6-position phosphorylation, which is a major mechanism of [¹⁸F]FDG retention (via hexokinase, E.C. 2.7.1.1). Indeed, in terms of in vitro stability, bacterial export, and in vivo performance [¹⁸F]FDM and [¹⁸F]FSK more closely mimic the “second generation” PET tracer 6”-[¹⁸F]-fluoromaltotriose. Our simultaneous chemoenzymatic radiosynthesis of [¹⁸F]-labeled α-1,4 linked oligomers (maltose, maltotriose, maltotetraose, etc.) will therefore guide clinical implementation and will be further studied in our lab. Chemoenzymatic radiosyntheses can be further tailored to provide specific products, or be followed with routine chemical preparations of the most promising PET radiotracers.

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Figure: Simultaneous radiosyntheses of a-1,4 linked oligosaccharides for PET imaging of infection. [¹⁸F]FDG is first reacted with β-glucose-1-phosphate in the presence of maltose phosphorylase to obtain [¹⁸F]FDM via reverse phosphorolysis. [¹⁸F]FDM is subsequently reacted with maltodextrin phosphorylase to make fluorine-labeled oligosaccharides. Depending on the mobile phase employed, resolution of individual oligomers is possible.

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