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Using 5-deoxy-5-[18F]fluororibose to glycosylate peptides for positron emission tomography

Nature Protocols volume 9, pages 138–145 (2014)Cite this article

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Abstract

So far seven peptide-based 18F-radiopharmaceuticals for diagnostic applications with positron emission tomography (PET) have entered into clinical trials. Three candidates out of these seven are glycosylated peptides, which may be explained by the beneficial influence of glycosylation on in vivo pharmacokinetics of peptide tracers. This protocol describes the method for labeling peptides with 5-deoxy-5-[18F]fluororibose ([18F]FDR) as a prosthetic group. The synthesis of [18F]FDR is effected by a nucleophilic fluorination step by using dried Kryptofix 2.2.2-K2CO3-K18F complex and a subsequent HCl-catalyzed hydrolysis. The conjugation of [18F]FDR to the N-terminus aminooxy (-ONH2)-functionalized peptides is carried out in anilinium buffer at pH 4.6 and at room temperature (RT, 21–23 °C), with the concentration of peptide precursors being 0.3 mM. The procedure takes about 120 min and includes two cartridge isolation steps and two reversed-phase (RP) HPLC purification steps. The quaternary methyl amine (QMA) anion exchange cartridge and the hydrophilic-lipophilic balanced (HLB) cartridge are used for the isolation of 18F-fluoride and [18F]FDR-conjugated peptides, respectively. The first HPLC purification provides the 18F-fluorinated precursor of [18F]FDR and the second HPLC purification is to separate labeled peptides from their unlabeled precursors. The final product is formulated in PBS ready for injection, with a radiochemical purity of >98% and a radiochemical yield (RCY) of 27–37% starting from the end of bombardment (EOB). The carbohydrate nature of [18F]FDR and the operational convenience of this protocol should facilitate its general use.

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Figure 1
Figure 2
Figure 3: HPLC and radio-TLC analyses of [18F]FDR-7.

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Acknowledgements

The Academy of Finland is acknowledged for financial support (grant nos. 133127, 136805 and 140965). The work was conducted within the Finnish Centre of Excellence in Molecular Imaging in Cardiovascular and Metabolic Research supported by the Academy of Finland, the University of Turku, Turku University Hospital and Åbo Akademi University. R.M. Badeau is gratefully acknowledged for the English-language revision of this manuscript.

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Authors and Affiliations

  1. Turku Positron Emission Tomography (PET) Centre, University of Turku and Turku University Hospital, Turku, Finland

    Xiang-Guo Li, Anne Roivainen & Juhani Knuuti

  2. Department of Chemistry, Laboratory of Radiochemistry, University of Helsinki, Helsinki, Finland

    Kerttuli Helariutta & Anu J Airaksinen

  3. Turku Centre for Disease Modeling, University of Turku, Turku, Finland

    Anne Roivainen

  4. MediCity Research Laboratory and Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland

    Sirpa Jalkanen

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All authors contributed to the finalization of the project and the preparation of the paper.

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Integrated supplementary information

Supplementary Figure 1 The device setup for the radiosynthesis.

The radiosynthesis device was constructed by DM Automation, Sweden. V1-V18 denote remote-controlled solenoid valves. RA and RB denote the reaction vial A and vial B, respectively. Cyl 1–Cyl 5 denote needle lifters. All the valves, needle lifters, heating and cooling of reaction vessels are controlled by a touch panel which is installed outside of the hot cell. Three radioactivity detectors (PIN diode semi-conductor detectors) are placed at the QMA cartridge, the reaction vessel RA and the HPLC outlet, respectively. All waste lines are connected to a waste bottle, which is connected to a decay coil. Ascarite is placed between the waste bottle and the decay coil.

Supplementary Figure 2 Photo and preparation of the ascarite cartridge.

Ascarite (4 ml) is added in between the two pads of cotton in a 5 ml plastic syringe. An adapter is used for connecting this cartridge to the vent system of the radiosynthesis unit. Ascarite is commercially available, e.g. from Aldrich (cat. no. 223921). This cartridge is changed once a week.

Supplementary information

Supplementary Figure 1

The device setup for the radiosynthesis. (PDF 97 kb)

Supplementary Figure 2

Photo and preparation of the ascarite cartridge. (PDF 117 kb)

Supplementary Method 1

Preparation of [19F]FDR-7 as a reference compound. (PDF 99 kb)

Supplementary Method 2

Determination of the specific radioactivity of 18F-labeled peptides. (PDF 54 kb)

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Li, XG., Helariutta, K., Roivainen, A. et al. Using 5-deoxy-5-[18F]fluororibose to glycosylate peptides for positron emission tomography. Nat Protoc 9, 138–145 (2014). https://doi.org/10.1038/nprot.2013.170

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