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.

Purpose: This study was motivated by the need for better positron emission tomography (PET)-compatible tools to image bacterial infection. Our previous efforts have targeted bacteria-specific metabolism via assimilation of carbon-11 labeled d-amino acids into the bacterial cell wall. Since the chemical determinants of this incorporation are not fully understood, we sought a high-throughput method to label d-amino acid derived structures with fluorine-18. Our strategy employed a chemical biology approach, whereby an azide (-N₃) bearing d-amino acid is incorporated into peptidoglycan muropeptides, with subsequent "click" cycloaddition with an ¹⁸F-labeled strained cyclooctyne partner. Procedures: A water-soluble, ¹⁸F-labeled and dibenzocyclooctyne (DBCO)-derived radiotracer ([¹⁸F]FB-sulfo-DBCO) was synthesized. This tracer was incubated with pathogenic bacteria treated with azide-bearing d-amino acids, and incorporated ¹⁸F was determined via gamma counting. In vitro uptake in bacteria previously treated with azide-modified d-amino acids was compared to that in cultures treated with amino acid controls. The biodistribution of [¹⁸F]FB-sulfo-DBCO was studied in a cohort of healthy mice with implications for future in vivo imaging. Results: The new strain-promoted azide-alkyne cycloaddition (SPAAC) radiotracer [¹⁸F]FB-sulfo-DBCO was synthesized with high radiochemical yield and purity via N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). Accumulation of [¹⁸F]FB-sulfo-DBCO was significantly higher in several bacteria treated with azide-modified d-amino acids than in controls; for example, we observed 7 times greater [¹⁸F]FB-sulfo-DBCO ligation in Staphylococcus aureus cultures incubated with 3-azido-d-alanine versus those incubated with d-alanine. Conclusions: The SPAAC radiotracer [¹⁸F]FB-sulfo-DBCO was validated in vitro via metabolic labeling of azide-bearing peptidoglycan muropeptides. d-Amino acid-derived PET radiotracers may be more efficiently screened via [¹⁸F]FB-sulfo-DBCO modification.

Imaging infections in patients is challenging using conventional methods, motivating the development of positron emission tomography (PET) radiotracers targeting bacteria-specific metabolic pathways. Numerous techniques have focused on the bacterial cell wall, although peptidoglycan-targeted PET tracers have been generally limited to the short-lived carbon-11 radioisotope (t_1/2 = 20.4 min). In this article, we developed and tested new tools for infection imaging using an amino sugar component of peptidoglycan, namely, derivatives of N-acetyl muramic acid (NAM) labeled with the longer-lived fluorine-18 (t_1/2 = 109.6 min) radioisotope. Muramic acid was reacted directly with 4-nitrophenyl 2-[¹⁸F]fluoropropionate ([¹⁸F]NFP) to afford the enantiomeric NAM derivatives (S)-[¹⁸F]FMA and (R)-[¹⁸F]FMA. Both diastereomers were easily isolated and showed robust accumulation by human pathogens in vitro and in vivo, including Staphylococcus aureus. These results form the basis for future clinical studies using fluorine-18-labeled NAM-derived PET radiotracers.