Abstract View
Efficient radiosynthesis of [18F]FSK via a thermostable nigerose phosphoryalse
Category: Fluorine-18 Chemistry
Authors:
Jung Min Kim1, Sang Hee Lee1, Shari Dhaene2, Adolfo Ancona3,4, Jaelim Kim5, Marina López-Álvarez1, Alexandre M. Sorlin1, Joseph Blecha1, Kiel D. Neumann6, Giuseppe Felice Mangiatordi3, Eunsung Lee7, Bernd Nidetky8, Robert R. Flavell1, Youngho Seo1,9,10, Joanne Engel11, Michael A. Ohliger1,12, Tom Desmet2*, David M. Wilson1*
1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, CA 94158, United States. 2Department of Biotechnology, Ghent University, Gent B-9000, Belgium. 3CNR-Istituto di Cristallografia, I-70126 Bari, Italy. 4Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100, Siena, Italy. 5Department of Chemistry, Pohang University of Science and Technology, Pohang 37673, Republic of Korea. 6Department of Radiology, St. Jude Children’s Research Hospital, Memphis, TN 38105, United States. 7Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea. 8Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz 8010, Austria. 9Department of Nuclear Engineering, University of California, Berkeley, California. 10UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, California. 11Department of Medicine, University of California, San Francisco, San Francisco, CA 94158, United States. 12Department of Radiology, Zuckerberg San Francisco General Hospital, San Francisco CA 94110, United States
Introduction: [18F]FDG-derived disaccharides have emerged as promising pathogen-specific PET tracers [1,2]. Among them, we identified [18F]FSK as an optimal 18F-disaccharide for detecting Staphylococcus aureus with high sensitivity and selectivity as well as favorable in vivo characteristics. However, its chemoenzymatic radiosynthesis using maltose phosphorylase (MP) is limited due to the substrate specificity of MP (α-1,4 linkages). We therefore sought to improve the radiochemical yield of [18F]FSK.
Methods: Chemoenzymatic radiosynthesis of [18F]FSK was performed using both maltose phosphorylase (MP) and nigerose phosphorylase (NP) following our established protocol. Briefly, [18F]FDG solutions were added to a vial containing β-glucose-1-phosphate and enzyme, then stirred at 37 °C or 55 °C for 20 min. The resulting mixture was injected onto a Polyamine II column (YMC-Pack Polyamine II 250 x 10.0 mm I.D. S-5um, 72.5% MeCN/water, flow rate: 4.0 mL/min).
Results: Under the previously reported conditions, MP-catalyzed reverse phosphorolysis yielded [18F]FDM (56.2 ± 1.6%) as the major product, while [18F]FSK formed as a minor product (10.3 ± 0.9%). Addition of phosphate significantly altered the product distribution, markedly increasing [18F]FSK formation (37.8 ± 2.0%) and reducing [18F]FDM to 17.2 ± 2.8%.
To achieve selective formation of [18F]FSK, we screened 6 different NPs. The enzymes derived from mesophilic organisms showed near-quantitative conversion of [18F]FDG into [18F]FSK (up to ~96%) at 37 °C, but their reactivities were decreased dramatically at 55 °C (<40%). In contrast, an NP originating from thermostable Spirochaeta thermophila (SpNP) showed high activity at both 37 °C (77.0 ± 2.5%) and 55 °C (97.2 ± 2.6%). Unlike the MP-catalyzed reaction, all NPs exclusively generated [18F]FSK, with no detectable side product.
Conclusions: We successfully improved the synthesis of [18F]FSK using two enzymes (MP and NP). Despite the improved MP-catalyzed approach, the presence of [18F]FDM necessitated HPLC purification to obtain pure [18F]FSK. In contrast, NPs enabled efficient and selective formation of [18F]FSK without byproduct formation. Moreover, the selective formation of [18F]FSK facilitated the development of a rapid cartridge-based purification method, enabling straightforward preparation of an injectable [18F]FSK solution. These findings support the future clinical translation of [18F]FSK as a bacteria-specific PET imaging agent.
References
[1] Sorlin AM, López-Álvarez M, Rabbitt SJ, et al. Chemoenzymatic syntheses of fluorine-18-labeled disaccharides from [18F]FDG yield potent sensors of living bacteria in vivo. J Am Chem Soc. 2023, 14, 17632-42.
[2] Lee SH, Kim JM, López-Álvarez M, et al. Regioselective Glycosylation of Fluorine-18-Labeled Sorbitol for Enhanced Bacterial Detection In Vivo Using PET. JACS Au 2025, 5, 6189-6199.
Figure. (A) Scheme of phosphorylase-mediated chemoenzymatic conversion of [18F]FDG to [18F]FSK. MP indicates maltose phosphorylase. NP indicates nigerose phosphorylase. (B) Representative HPLC profile of the reaction using MP with additive phosphate. Arrows indicate the peak of [18F]FDG (purple), [18F]FDM (black), and [18F]FSK (red), respectively. (C) Representative HPLC profile of the reaction using SpNP at 55 °C.