Abstract View

Objectives: Glutaminolysis is a key metabolic pathway in cancer, particularly in triple-negative breast cancer (TNBC), where tumor growth depends strongly on glutamine utilization [1]. Fluoroglutamine-based PET tracers have been developed to image this pathway; however, stereochemistry can significantly influence radiolabeling efficiency, precursor stability, and biological performance. [18F] (2S,4R)-4-Fluoroglutamine (4R-FGln), utilized in numerous preclinical and clinical studies, has shown limited metabolic turnover, while the [18F] (2S,4S)-4-Fluoroglutamine (4S-FGln) may better reflect glutamine metabolism due to higher enzymatic processing [2-4]. This project focuses on the radiochemical synthesis and comparison of both stereoisomers, emphasizing nucleophilic [¹⁸F]fluorination, precursor development, and evaluation of their suitability for imaging glutaminolysis in TNBC.

Methods: Cold (non-radioactive) synthesis of (2S,4R)-4-fluoroglutamine and (2S,4S)-4-fluoroglutamine were performed using established stereoselective methods, and tosylate precursors were prepared for radiolabeling studies [5]. Semi-automated radiosynthesis of 4R-[¹⁸F]fluoroglutamine was successfully achieved via nucleophilic fluorination, followed by purification to obtain a radiochemically pure product. Radiochemical yield, purity, molar activity, and in vitro stability were assessed.

Results: The cold (non-radioactive) synthesis of 4R-fluoroglutamine was successfully completed. We synthesized (2S,4S)-4- and (2S,4R)-4-tosylate precursors, along with the corresponding nonradiolabeled reference standard, using a modified literature method in which a streamlined one-step Dess-Martin oxidation replaced the reported five-step sequence. We successfully synthesized [¹⁸F] (2S4R)-4-fluoroglutamine, and after purification we obtained a radiochemical yield (RCY) of ~12% with radiochemical purity (RCP) >95%, as determined by analytical chiral HPLC (Chirex 3126 (D)-penicillamine, 250 × 4.6 mm, 2 mM CuSO₄, 1 mL/min, UV 210 nm and radio-detection). The retention time for the (2S,4R) isomer was ~13 min.

Treatment with the glutaminase inhibitor CB-839 increased [19F] (2S,4R)-4-Fluoroglutamine levels in both HCC-1806 and MCF-7 cells. At the same time, [19F] (2S,4R)-4-Fluoroglutamate levels decreased, consistent with reduced conversion of glutamine to glutamate by glutaminase. Overall, these results support the use of ¹⁹F stable isotope tracing to study glutamine metabolism and help interpret PET tracer behavior (Fig 1 a,b).

Initial cell uptake studies revealed that 1806 cells exhibited high uptake of [¹⁸F] (2S,4R)-4-fluoroglutamine after 2 h, with a noticeable effect of CB-839 treatment observed at 1 h. CB-839 significantly increased [¹⁸F] (2S,4R)-4-fluoroglutamine CPM/mg in 1806 cells, an effect of significantly decreased magnitude in MCF7 cells, as previously published [2]. 

Conclusion: Using the described methods, we successfully synthesized both non-radioactive (2S,4R)-4- and (2S,4S)-4-fluoroglutamine standards and achieved the radiosynthesis of [18F] (2S,4R)-4-Fluoroglutamine through nucleophilic fluorination. Preliminary biological studies suggest sensitivity to changes in glutamine pathway activity. Ongoing and future work includes synthesis of [18F] (2S,4S)-4-Fluoroglutamine, followed by direct comparative evolution of both tracers in cell-based assays and in vivo PET imaging to assess their utility for imaging glutamine metabolism in breast cancer. Complementary metabolic studies using ¹³C-glutamine, ¹⁹F analogs, and glutamine pathway inhibitors (CB-839 and DRP-104) will further characterize glutamine flux and metabolic dependency in breast cancer models.

Acknowledgments:

Funding:

This work is currently funded by the 2025-2027 SNMMI Molecular Imaging Research Grant for Junior Academic Faculty with Christopher Hensley as principal investigator, as well as IMPACT award funding from the UAB School of Medicine with matching funds from the UAB Department of Radiology and UAB O’Neal Comprehensive Cancer Center to Christopher Hensley.

Radiological Society of North America Research Scholar Grant RSCH25-187, 2025-2027.