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.

Bioorthogonal Radiolabeling of Azide-Modified Bacteria Using [18F]FB-sulfo-DBCO.

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.

Development of CD46 targeted alpha theranostics in prostate cancer using 134Ce/225Ac-Macropa-PEG4-YS5.

Rationale: 225Ac, a long-lived α-emitter with a half-life of 9.92 days, has garnered significant attention as a therapeutic radionuclide when coupled with monoclonal antibodies and other targeting vectors. Nevertheless, its clinical utility has been hampered by potential off-target toxicity, a lack of optimized chelators for 225Ac, and limitations in radiolabeling methods. In a prior study evaluating the effectiveness of CD46-targeted radioimmunotherapy, we found great therapeutic efficacy but also significant toxicity at higher doses. To address these challenges, we have developed a radioimmunoconjugate called 225Ac-Macropa-PEG4-YS5, incorporating a stable PEGylated linker to maximize tumoral uptake and increase tumor-to-background ratios. Our research demonstrates that this conjugate exhibits greater anti-tumor efficacy while minimizing toxicity in prostate cancer 22Rv1 tumors. Methods: We synthesized Macropa.NCS and Macropa-PEG4/8-TFP esters and prepared Macropa-PEG0/4/8-YS5 (with nearly ~1:1 ratio of macropa chelator to antibody YS5) as well as DOTA-YS5 conjugates. These conjugates were then radiolabeled with 225Ac in a 2 M NH4OAc solution at 30 °C, followed by purification using YM30K centrifugal purification. Subsequently, we conducted biodistribution studies and evaluated antitumor activity in nude mice (nu/nu) bearing prostate 22Rv1 xenografts in both single-dose and fractionated dosing studies. Micro-PET imaging studies were performed with 134Ce-Macropa-PEG0/4/8-YS5 in 22Rv1 xenografts for 7 days. Toxicity studies were also performed in healthy athymic nude mice. Results: As expected, we achieved a >95% radiochemical yield when labeling Macropa-PEG0/4/8-YS5 with 225Ac, regardless of the chelator ratios (ranging from 1 to 7.76 per YS5 antibody). The isolated yield exceeded 60% after purification. Such high conversions were not observed with the DOTA-YS5 conjugate, even at a higher ratio of 8.5 chelators per antibody (RCY of 83%, an isolated yield of 40%). Biodistribution analysis at 7 days post-injection revealed higher tumor uptake for the 225Ac-Macropa-PEG4-YS5 (82.82 ± 38.27 %ID/g) compared to other conjugates, namely 225Ac-Macropa-PEG0/8-YS5 (38.2 ± 14.4/36.39 ± 12.4 %ID/g) and 225Ac-DOTA-YS5 (29.35 ± 7.76 %ID/g). The PET Imaging of 134Ce-Macropa-PEG0/4/8-YS5 conjugates resulted in a high tumor uptake, and tumor to background ratios. In terms of antitumor activity, 225Ac-Macropa-PEG4-YS5 exhibited a substantial response, leading to prolonged survival compared to 225Ac-DOTA-YS5, particularly when administered at 4.625 kBq doses, in single or fractionated dose regimens. Chronic toxicity studies observed mild to moderate renal toxicity at 4.625 and 9.25 kBq doses. Conclusions: Our study highlights the promise of 225Ac-Macropa-PEG4-YS5 for targeted alpha particle therapy. The 225Ac-Macropa-PEG4-YS5 conjugate demonstrates improved biodistribution, reduced off-target binding, and enhanced therapeutic efficacy, particularly at lower doses, compared to 225Ac-DOTA-YS5. Incorporating theranostic 134Ce PET imaging further enhances the versatility of macropa-PEG conjugates, offering a more effective and safer approach to cancer treatment. Overall, this methodology has a high potential for broader clinical applications.

Imaging the Bacterial Cell Wall Using N-Acetyl Muramic Acid-Derived Positron Emission Tomography Radiotracers.

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 (t1/2 = 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 (t1/2 = 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.

Evaluating the Performance of Pathogen-Targeted Positron Emission Tomography Radiotracers in a Rat Model of Vertebral Discitis-Osteomyelitis.

BACKGROUND: Vertebral discitis-osteomyelitis (VDO) is a devastating infection of the spine that is challenging to distinguish from noninfectious mimics using computed tomography and magnetic resonance imaging. We and others have developed novel metabolism-targeted positron emission tomography (PET) radiotracers for detecting living Staphylococcus aureus and other bacteria in vivo, but their head-to-head performance in a well-validated VDO animal model has not been reported. METHODS: We compared the performance of several PET radiotracers in a rat model of VDO. [11C]PABA and [18F]FDS were assessed for their ability to distinguish S aureus, the most common non-tuberculous pathogen VDO, from Escherichia coli. RESULTS: In the rat S aureus VDO model, [11C]PABA could detect as few as 103 bacteria and exhibited the highest signal-to-background ratio, with a 20-fold increased signal in VDO compared to uninfected tissues. In a proof-of-concept experiment, detection of bacterial infection and discrimination between S aureus and E coli was possible using a combination of [11C]PABA and [18F]FDS. CONCLUSIONS: Our work reveals that several bacteria-targeted PET radiotracers had sufficient signal to background in a rat model of S aureus VDO to be potentially clinically useful. [11C]PABA was the most promising tracer investigated and warrants further investigation in human VDO.

Chemoenzymatic Syntheses of Fluorine-18-Labeled Disaccharides from [18F] FDG Yield Potent Sensors of Living Bacteria In Vivo.

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[18F]-fluoro-d-glucose ([18F]FDG), the most common tracer used in clinical imaging, to form [18F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [18F]FDG was reacted with ß-d-glucose-1-phosphate in the presence of maltose phosphorylase, the α-1,4- and α-1,3-linked products 2-deoxy-[18F]-fluoro-maltose ([18F]FDM) and 2-deoxy-2-[18F]-fluoro-sakebiose ([18F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (ß-1,3), and cellobiose (ß-1,4) phosphorylases to synthesize 2-deoxy-2-[18F]fluoro-trehalose ([18F]FDT), 2-deoxy-2-[18F]fluoro-laminaribiose ([18F]FDL), and 2-deoxy-2-[18F]fluoro-cellobiose ([18F]FDC). We subsequently tested [18F]FDM and [18F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. Both [18F]FDM and [18F]FSK were stable in human serum with high accumulation in preclinical infection models. The synthetic ease and high sensitivity of [18F]FDM and [18F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of these tracers to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [18F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.

Chemoenzymatic syntheses of fluorine-18-labeled disaccharides from [ 18 F]FDG yield potent sensors of living bacteria in vivo.

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach, that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[ 18 F]-fluoro-D-glucose ([ 18 F]FDG), the most common tracer used in clinical imaging, to form [ 18 F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [ 18 F]FDG was reacted with ß-D-glucose-1-phosphate in the presence of maltose phosphorylase, both the α-1,4 and α-1,3-linked products 2-deoxy-[ 18 F]-fluoro-maltose ([ 18 F]FDM) and 2-deoxy-2-[ 18 F]-fluoro-sakebiose ([ 18 F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (ß-1,3), and cellobiose (ß-1,4) phosphorylases to synthesize 2-deoxy-2-[ 18 F]fluoro-trehalose ([ 18 F]FDT), 2-deoxy-2-[ 18 F]fluoro-laminaribiose ([ 18 F]FDL), and 2-deoxy-2-[ 18 F]fluoro-cellobiose ([ 18 F]FDC). We subsequently tested [ 18 F]FDM and [ 18 F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. The lead sakebiose-derived tracer [ 18 F]FSK was stable in human serum and showed high uptake in preclinical models of myositis and vertebral discitis-osteomyelitis. Both the synthetic ease, and high sensitivity of [ 18 F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of this tracer to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [ 18 F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.

The Role of Trichloroacetimidate To Enable Iridium-Catalyzed Regio- and Enantioselective Allylic Fluorination: A Combined Experimental and Computational Study.

Asymmetric allylic fluorination has proven to be a robust and efficient methodology with potential applications for the development of pharmaceuticals and practical synthesis for 18F-radiolabeling. A combined computational (dispersion-corrected DFT) and experimental approach was taken to interrogate the mechanism of the diene-ligated, iridium-catalyzed regio- and enantioselective allylic fluorination. Our group has shown that, in the presence of an iridium(I) catalyst and nucleophilic fluoride source (Et3N·3HF), allylic trichloroacetimidates undergo rapid fluoride substitution to generate allylic fluoride products with excellent levels of branched-to-linear ratios. Mechanistic studies reveal the crucial role of the trichloroacetimidate as a potent leaving group and ligand to enable conversion of racemic allylic trichloroacetimidates to the corresponding enantioenriched allylic fluorides, via a dynamic kinetic asymmetric transformation (DYKAT), in the presence of the chiral bicyclo[3.3.0]octadiene-ligated iridium catalyst.

Iridium-Catalyzed Radiosynthesis of Branched Allylic [18F]Fluorides.

The rapid and operationally simple radiosynthesis of branched allylic [18F]fluorides bearing a variety of functional groups, via iridium-catalyzed nucleophilic substitution reaction utilizing allylic trichloroacetimidates and [18F]KF·Kryptofix[2.2.2] complex in 5-15 min at room temperature, is reported. The versatility of the allyl functional group of the resulting radiofluorinated products offers the benefit of being subsequently available for further functionalization.