Purpose

Detection of bacteria-specific metabolism via positron emission tomography (PET) is an emerging strategy to image human pathogens, with dramatic implications for clinical practice. In silico and in vitro screening tools have recently been applied to this problem, with several monosaccharides including l-arabinose showing rapid accumulation in Escherichia coli and other organisms. Our goal for this study was to evaluate several synthetically viable arabinofuranose-derived ¹⁸ F analogs for their incorporation into pathogenic bacteria.

Procedures

We synthesized four radiolabeled arabinofuranose-derived sugars: 2-deoxy-2-[¹⁸ F]fluoro-arabinofuranoses (d-2-¹⁸ F-AF and l-2-¹⁸ F-AF) and 5-deoxy-5-[¹⁸ F]fluoro-arabinofuranoses (d-5-¹⁸ F-AF and l-5-¹⁸ F-AF). The arabinofuranoses were synthesized from ¹⁸ F^- via triflated, peracetylated precursors analogous to the most common radiosynthesis of 2-deoxy-2-[¹⁸ F]fluoro-d-glucose ([¹⁸ F]FDG). These radiotracers were screened for their uptake into E. coli and Staphylococcus aureus. Subsequently, the sensitivity of d-2-¹⁸ F-AF and l-2-¹⁸ F-AF to key human pathogens was investigated in vitro.

Results

All ¹⁸ F radiotracer targets were synthesized in high radiochemical purity. In the screening study, d-2-¹⁸ F-AF and l-2-¹⁸ F-AF showed greater accumulation in E. coli than in S. aureus. When evaluated in a panel of pathologic microorganisms, both d-2-¹⁸ F-AF and l-2-¹⁸ F-AF demonstrated sensitivity to most gram-positive and gram-negative bacteria.

Conclusions

Arabinofuranose-derived ¹⁸ F PET radiotracers can be synthesized with high radiochemical purity. Our study showed absence of bacterial accumulation for 5-substitued analogs, a finding that may have mechanistic implications for related tracers. Both d-2-¹⁸ F-AF and l-2-¹⁸ F-AF showed sensitivity to most gram-negative and gram-positive organisms. Future in vivo studies will evaluate the diagnostic accuracy of these radiotracers in animal models of infection.

Currently, there exists no accurate, noninvasive clinical imaging method to detect living bacteria in vivo. Our goal is to provide a positron emission tomography (PET) method to image infection by targeting bacteria-specific metabolism. Standard of care methodologies detect morphologic changes, image immunologic response to infection, or employ invasive tissue sampling with associated patient morbidity. These strategies, however, are not specific for living bacteria and are often inadequate to detect bacterial infection during fever workup. As such, there is an unmet clinical need to identify and validate new imaging tools suitable for noninvasive, in vivo (PET) imaging of living bacteria. We have shown that d-[methyl-¹¹C]methionine (d-[¹¹C]Met) can distinguish active bacterial infection from sterile inflammation in a murine infection model and is sensitive to both Gram-positive and Gram-negative bacteria. Here, we report an automated and >99% enantiomeric excess (ee) synthesis of d-[¹¹C]Met from a linear d-homocysteine precursor, a significant improvement over the previously reported synthesis utilizing a d-homocysteine thiolactone hydrochloride precursor with approximately 75-85% ee. Furthermore, we took additional steps toward applying d-[¹¹C]Met to infected patients. d-[¹¹C]Met was subject to a panel of clinically relevant bacterial strains and demonstrated promising sensitivity to these pathogens. Finally, we performed radiation dosimetry in a normal murine cohort to set the stage for translation to humans in the near future.

Incorporation of d-amino acids into peptidoglycan is a unique metabolic feature of bacteria. Since d-amino acids are not metabolic substrates in most mammalian tissues, this difference can be exploited to detect living bacteria in vivo. Given the prevalence of d-alanine in peptidoglycan muropeptides, as well as its role in several antibiotic mechanisms, we targeted this amino acid for positron emission tomography (PET) radiotracer development. d-[3-¹¹C]Alanine and the dipeptide d-[3-¹¹C]alanyl-d-alanine were synthesized via asymmetric alkylation of glycine-derived Schiff-base precursors with [¹¹C]methyl iodide in the presence of a cinchonidinium phase-transfer catalyst. In cell experiments, both tracers showed accumulation by a wide variety of both Gram-positive and Gram-negative pathogens including Staphylococcus aureus and Pseudomonas aeruginosa. In a mouse model of acute bacterial myositis, d-[3-¹¹C]alanine was accumulated by living microorganisms but was not taken up in areas of sterile inflammation. When compared to existing clinical nuclear imaging tools, specifically 2-deoxy-2-[¹⁸F]fluoro-d-glucose and a gallium citrate radiotracer, d-alanine showed more bacteria-specific uptake. Decreased d-[3-¹¹C]alanine uptake was also observed in antibiotic-sensitive microbes after antimicrobial therapy, when compared to that in resistant organisms. Finally, prominent uptake of d-[3-¹¹C]alanine uptake was seen in rodent models of discitis-osteomyelitis and P. aeruginosa pneumonia. These data provide strong justification for clinical translation of d-[3-¹¹C]alanine to address a number of important human infections.