Peptidoglycan-Targeted [¹⁸F]3,3,3-Trifluoro-d-alanine Tracer for Imaging Bacterial Infection

Imaging is increasingly used to detect and monitor bacterial infection. Both anatomic (X-rays, computed tomography, ultrasound, and MRI) and nuclear medicine ([¹¹¹In]-WBC SPECT, [¹⁸F]FDG PET) techniques are used in clinical practice but lack specificity for the causative microorganisms themselves. To meet this challenge, many groups have developed imaging methods that target pathogen-specific metabolism, including PET tracers integrated into the bacterial cell wall. We have previously reported the d-amino acid derived PET radiotracers d-methyl-[¹¹C]-methionine, d-[3-¹¹C]-alanine, and d-[3-¹¹C]-alanine-d-alanine, which showed robust bacterial accumulation in vitro and in vivo. Given the clinical importance of radionuclide half-life, in the current study, we developed [¹⁸F]3,3,3-trifluoro-d-alanine (d-[¹⁸F]-CF₃-ala), a fluorine-18 labeled tracer. We tested the hypothesis that d-[¹⁸F]-CF₃-ala would be incorporated into bacterial peptidoglycan given its structural similarity to d-alanine itself. NMR analysis showed that the fluorine-19 parent amino acid d-[¹⁹F]-CF₃-ala was stable in human and mouse serum. d-[¹⁹F]-CF₃-ala was also a poor substrate for d-amino acid oxidase, the enzyme largely responsible for mammalian d-amino acid metabolism and a likely contributor to background signals using d-amino acid derived PET tracers. In addition, d-[¹⁹F]-CF₃-ala showed robust incorporation into Escherichia coli peptidoglycan, as detected by HPLC/mass spectrometry. Based on these promising results, we developed a radiosynthesis of d-[¹⁸F]-CF₃-ala via displacement of a bromo-precursor with [¹⁸F]fluoride followed by chiral stationary phase HPLC. Unexpectedly, the accumulation of d-[¹⁸F]-CF₃-ala by bacteria in vitro was highest for Gram-negative pathogens in particular E. coli. In a murine model of acute bacterial infection, d-[¹⁸F]-CF₃-ala could distinguish live from heat-killed E. coli, with low background signals. These results indicate the viability of [¹⁸F]-modified d-amino acids for infection imaging and indicate that improved specificity for bacterial metabolism can improve tracer performance.