Comparative Enzymology of (2S,4R)4-Fluoroglutamine and (2S,4R)4-Fluoroglutamate

Many cancer cells have a strong requirement for glutamine. As an aid for understanding this phenomenon the (18)F-labeled 2S,4R stereoisomer of 4-fluoroglutamine [(2S,4R)4-FGln] was previously developed for in vivo positron emission tomography (PET). In the present work, comparative enzymological studies of unlabeled (2S,4R)4-FGln and its deamidated product (2S,4R)4-FGlu were conducted as an adjunct to these PET studies. Our findings are as follows: Rat kidney preparations catalyze the deamidation of (2S,4R)4-FGln. (2,4R)4-FGln and (2S,4R)4-FGlu are substrates of various aminotransferases. (2S,4R)4-FGlu is a substrate of glutamate dehydrogenase, but not of sheep brain glutamine synthetase. The compound is, however, a strong inhibitor of this enzyme. Rat liver cytosolic fractions catalyze a γ-elimination reaction with (2S,4R)4-FGlu, generating α-ketoglutarate. Coupling of a deamidase reaction with this γ-elimination reaction provides an explanation for the previous detection of (18)F(-) in tumors exposed to [(18)F](2S,4R)4-FGln. One enzyme contributing to this reaction was identified as alanine aminotransferase, which catalyzes competing γ-elimination and aminotransferase reactions with (2S,4R)4-FGlu. This appears to be the first description of an aminotransferase catalyzing a γ-elimination reaction. The present results demonstrate that (2S,4R)4-FGln and (2S,4R)4-FGlu are useful analogues for comparative studies of various glutamine- and glutamate-utilizing enzymes in normal and cancerous mammalian tissues, and suggest that tumors may metabolize (2S,4R)4-FGln in a generally similar fashion to glutamine. In plants, yeast and bacteria a major route for ammonia assimilation involves the consecutive action of glutamate synthase plus glutamine synthetase (glutamate synthase cycle). It is suggested that (2S,4R)4-FGln and (2S,4R)4-FGlu will be useful probes in studies of ammonia assimilation by the glutamate synthase pathway in these organisms. Finally, glutamine transaminases are conserved in mammals, plants and bacteria, and probably serve to close the methionine salvage pathway, thus linking nitrogen metabolism to sulfur metabolism and one-carbon metabolism. It is suggested that (2S,4R)4-FGln may be useful in studies of the methionine salvage pathway in a variety of organisms

Enantioselective Radiosynthesis of Positron Emission Tomography (PET) Tracers Containing [¹⁸F]Fluorohydrins

Herein, we describe an operationally straightforward radiosynthesis of a chiral transition metal fluoride catalyst, [¹⁸F]­(salen)­CoF, and its use for late-stage enantioselective aliphatic radiofluorination. We demonstrate the utility of the method by preparing single enantiomer experimental and clinically validated PET tracers that contain base-sensitive functional groups, epimerizable stereocenters, and nitrogen-rich motifs. Unlike the conventional radiosyntheses of these targets with [¹⁸F]­KF, labeling with (salen)­CoF is possible in the last step and under exceptionally mild conditions. These results constitute a rare example of a nucleophilic radiofluorination using a transition metal fluoride and highlight the potential of such reagents to enhance traditional methods for labeling aliphatic hydrocarbons

Multidentate ¹⁸F-Polypegylated Styrylpyridines As Imaging Agents for Aβ Plaques in Cerebral Amyloid Angiopathy (CAA)

β-Amyloid plaques (Aβ plaques) in the brain are associated with cerebral amyloid angiopathy (CAA). Imaging agents that could target the Aβ plaques in the living human brain would be potentially valuable as biomarkers in patients with CAA. A new series of ¹⁸F styrylpyridine derivatives with high molecular weights for selectively targeting Aβ plaques in the blood vessels of the brain but excluded from the brain parenchyma is reported. The styrylpyridine derivatives, <b>8a</b>–<b>c</b>, display high binding affinities and specificity to Aβ plaques (<i>K</i>_i = 2.87, 3.24, and 7.71 nM, respectively). In vitro autoradiography of [¹⁸F]<b>8a</b> shows labeling of β-amyloid plaques associated with blood vessel walls in human brain sections of subjects with CAA and also in the tissue of AD brain sections. The results suggest that [¹⁸F]<b>8a</b> may be a useful PET imaging agent for selectively detecting Aβ plaques associated with cerebral vessels in the living human brain

[⁶⁸Ga]Ga-HBED-CC-FAPI Derivatives with Improved Radiolabeling and Specific Tumor Uptake

Fibroblast activation protein (FAP) is selectively expressed in tumors and highly important for maintaining the microenvironment in malignant tumors. Radioisotope-labeled FAP inhibitors (FAPIs) were proven to be useful for diagnosis and radionuclide therapy of cancer and are under active clinical investigations. Ga-HBED complex displays a higher in vivo stability constant (log KGaL: 38.5), compared to that of Ga-DOTA (log KGaL: 21.3). Such advantage in stability constant suggests that it may be useful for development of alternative FAPI imaging agents. In this study, previously reported [68Ga]Ga-DOTA-FAPI-02 and -04 were converted to the corresponding [68Ga]Ga-HBED-CC-FAPI-02 and -04 derivatives ([68Ga]Ga-4, [68Ga]Ga-5, [68Ga]Ga-6, and [68Ga]Ga-7). It was found that substituting the DOTA chelating group with HBED-CC led to several unique and desirable tumor-targeting properties: (1) robust, fast, and high yield labelingreadily adaptable to a kit formulation; (2) high stabilities in vitro; (3) excellent FAP binding affinities (IC50 ranging between 4 and 7 nM) and improved cell uptake and retention (in HT1080 (FAP+) cells); and (4) excellent selective in vivo tumor uptake in nude mice bearing U87MG tumor. It appeared that Ga(III) chelation with HBED-CC improved the in vivo kinetics favoring higher tumor uptake and retention compared to the corresponding Ga-DOTA complex. Out of the four tested ligands the new [68Ga]Ga-HBED-CC-FAPI dimer, [68Ga]Ga-6, displayed the best tumor localization properties, and further studies are warranted to demonstrate that it is an alternative FAP imaging agent for cancer patients

[⁶⁸Ga]Ga-HBED-CC-FAPI Derivatives with Improved Radiolabeling and Specific Tumor Uptake

Fibroblast activation protein (FAP) is selectively expressed in tumors and highly important for maintaining the microenvironment in malignant tumors. Radioisotope-labeled FAP inhibitors (FAPIs) were proven to be useful for diagnosis and radionuclide therapy of cancer and are under active clinical investigations. Ga-HBED complex displays a higher in vivo stability constant (log KGaL: 38.5), compared to that of Ga-DOTA (log KGaL: 21.3). Such advantage in stability constant suggests that it may be useful for development of alternative FAPI imaging agents. In this study, previously reported [68Ga]Ga-DOTA-FAPI-02 and -04 were converted to the corresponding [68Ga]Ga-HBED-CC-FAPI-02 and -04 derivatives ([68Ga]Ga-4, [68Ga]Ga-5, [68Ga]Ga-6, and [68Ga]Ga-7). It was found that substituting the DOTA chelating group with HBED-CC led to several unique and desirable tumor-targeting properties: (1) robust, fast, and high yield labelingreadily adaptable to a kit formulation; (2) high stabilities in vitro; (3) excellent FAP binding affinities (IC50 ranging between 4 and 7 nM) and improved cell uptake and retention (in HT1080 (FAP+) cells); and (4) excellent selective in vivo tumor uptake in nude mice bearing U87MG tumor. It appeared that Ga(III) chelation with HBED-CC improved the in vivo kinetics favoring higher tumor uptake and retention compared to the corresponding Ga-DOTA complex. Out of the four tested ligands the new [68Ga]Ga-HBED-CC-FAPI dimer, [68Ga]Ga-6, displayed the best tumor localization properties, and further studies are warranted to demonstrate that it is an alternative FAP imaging agent for cancer patients