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Deoxyfluorination of phenols for chemoselective 18F-labeling of peptides

Nature Protocols volume 18, pages 3614–3651 (2023)Cite this article

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Abstract

The challenge of forming C–18F bonds is often a bottleneck in the development of new 18F-labeled tracer molecules for noninvasive functional imaging studies using positron emission tomography (PET). Nucleophilic aromatic substitution is the most widely employed reaction to functionalize aromatic substrates with the radioactive fluorine-18 but its scope is restricted to arenes containing electron-withdrawing substituents. Furthermore, many protic functional groups are incompatible with basic fluoride anions. Peptide substrates, which are highly desirable targets for PET molecular imaging, are particularly challenging to label with fluorine-18 because they are densely functionalized and sensitive to high temperatures and basic conditions. To expand the utility of nucleophilic aromatic substitution with fluorine-18, we describe two complementary procedures for the radiodeoxyfluorination of bench-stable and easy-to-access phenols that ensure rapid access to densely functionalized electron-rich and electron-poor 18F–aryl fluorides. The first procedure details the synthesis of an 18F–synthon and its subsequent ligation to the cysteine residue of Arg–Gly–Asp–Cys in 10.5 h from commercially available starting materials (189-min radiosynthesis). The second procedure describes the incorporation of commercially available CpRu(Fmoc–tyrosine)OTf into a fully protected peptide Lys–Met–Glu–(CpRu–Tyr)–Leu via solid-phase peptide synthesis and subsequent ruthenium-mediated uronium deoxyfluorination with fluorine-18 followed by deprotection, accomplished within 7 d (116-min radiosynthesis). Both radiolabeling methods are highly chemoselective and have conveniently been automated using commercially available radiosynthesis equipment so that the procedures described can be employed for the synthesis of peptide-based PET probes for in vivo imaging studies according to as low as reasonably achievable (ALARA) principles.

Key points

  • The procedures detail the chemoselective radiolabeling of peptides with fluorine-18 on different amino acids in the peptide sequence via either metal-free uronium deoxyfluorination or ruthenium-assisted uronium deoxyfluorination.

  • The approach facilitates the radiosynthesis of original molecules for noninvasive functional imaging studies using positron emission tomography.

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Fig. 1: Radiolabeling strategies for peptides.
Fig. 2: Metal-free uronium deoxyfluorination with fluorine-18.
Fig. 3: The equilibrium constant for the conversion of uronium fluoride 4 to tetrahedral adduct 5 changes with the nature of the arene substituents.
Fig. 4: Ruthenium-mediated radiodeoxyfluorination of phenols.
Fig. 5: General scheme for 18F-deoxyfluorination of peptides.
Fig. 6: Uronium elution obviates the need for time-consuming dry down procedure.
Fig. 7: Reactions involved in metal-free radiodeoxyfluorination described in Procedure 1.
Fig. 8: General workflow for ruthenium-mediated radiodeoxyfluorination.
Fig. 9: Photographs of the ELIXYS radiochemical synthesis module setup.
Fig. 10: Labeled photograph of the ELIXYS module set up in the hot cell.
Fig. 11: Photographs of the general preparation of the ELIXYS radiosynthesizer.
Fig. 12: Photographs showing Steps 14, 15 and 17.
Fig. 13: Photographs showing Steps 18 and 21.
Fig. 14: Photographs showing Steps 25, 29 and 30.
Fig. 15: Photographs of the synthesis of the peptide precursor.
Fig. 16: Photographs of the synthesis of the peptide precursor.
Fig. 17: Synthesis of labeling precursor 13 from peptide 15.
Fig. 18: Schematic diagram of the ELIXYS radiosynthesizer setup used.
Fig. 19: Photographs of the 18F-deoxyfluorination on ELIXYS radiosynthesizer.

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Data availability

All data are available in the extended data section or the accompanying Supplementary Information.

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Acknowledgements

The research presented in Procedure 1 was supported by the Crump Institute for Molecular Imaging and the American Cancer Society (132467-RSG-18-149-01-CCE). Funding to support Procedure 2 of this work was provided by the Max-Planck-Institut für Kohlenforschung. We thank the UCLA Biomedical Cyclotron staff and Jeffrey Collins for providing [18F]fluoride for the studies in Procedure 1 and for helpful discussion. We thank F. Köhler for mass spectrometry analysis, M. Leutzsch and J. Lingnau for NMR spectroscopy analysis, C. Heidgen, N. Sauerborn and P. Münstermann for liquid chromatographic analysis, and R. Petzold for serving as the radiation safety officer supporting the work presented in Procedure 2 (all from Max-Planck-Institut für Kohlenforschung).

Author information

Author notes

  1. Gaoyuan Ma

    Present address: Strateos Inc., San Diego, CA, USA

  2. These authors contributed equally: Riya Halder, Gaoyuan Ma.

Authors and Affiliations

  1. Department of Organic Synthesis, Max-Planck-Institut für Kohlenforschung, Mülheim an der Ruhr, Germany

    Riya Halder, Jens Rickmeier, Roland Petzold & Tobias Ritter

  2. Institute of Organic Chemistry, RWTH Aachen University, Aachen, Germany

    Riya Halder

  3. Department of Molecular and Medical Pharmacology and Crump Institute for Molecular Imaging, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA

    Gaoyuan Ma & Jennifer M. Murphy

  4. Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA, USA

    James W. McDaniel

  5. Department of Heterogeneous Catalysis, Max-Planck-Institut für Kohlenforschung, Mülheim an der Ruhr, Germany

    Constanze N. Neumann

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Contributions

C.N.N., T.R., J.M.M. and J.R. designed the conceptual approach to this work. R.H., G.M., J.W.M. and R.P. carried out the experimental work and analyzed the experimental data with input from J.M.M., C.N.N. and T.R. C.N.N., J.M.M., R.H. and T.R. wrote the manuscript. T.R., C.N.N. and J.M.M. directed the project.

Corresponding authors

Correspondence to Constanze N. Neumann, Jennifer M. Murphy or Tobias Ritter.

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Competing interests

T.R. may benefit financially from PhenoFluor/PhenoFluorMix and [CpRu(Fmoc-tyrosine)]CF3CO2 sales. The other authors declare no competing interests.

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Peer review information

Nature Protocols thanks Anna Kaarina Kirjavainen, Andre Luxen, Jan Marik and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Ma, G. et al. Org. Lett. 23, 530–534 (2021): https://doi.org/10.1021/acs.orglett.0c04054

Neumann, C. N. et al. Nature 534, 369–373 (2016): https://doi.org/10.1038/nature17667

Beyzavi, H. et al. ACS Cent. Sci. 3, 944–948 (2017): https://doi.org/10.1021/acscentsci.7b00195

Rickmeier, J. & Ritter, T. Angew. Chem. Int. Ed. 57, 14207–14211 (2018): https://doi.org/10.1002/anie.201807983

Extended data

Extended Data Fig. 1 Fluid diagram and cassette setup for the ELIXYS radiosynthesis module.

Disposable cassettes (represented by the gray rectangles) contain the necessary internal components (i.e., stopcock valves, transfer dip tubes, fluid connection ports, etc.) to conduct the radiosynthesis protocol; All cassettes are identical, but details are omitted from Cassette 2 for simplicity.

Extended Data Fig. 2 Analytical HPLC chromatograms obtained for 18F-11.

a, analytical HPLC chromatogram obtained for HPLC purified 18F-11. b, co-injection of the reaction mixture containing 18F-11 and an aliquot of reference standard on an analytical HPLC column; γ-trace (lower) and 254 nm UV trace (upper).

Extended Data Fig. 3 Prep HPLC trace of 15.

YMC Triat C-18 column, 5.0 μm, 150 × 4.6 mm, flow rate = 1.0 mL·min−1 by an isocratic elution with 25:75 (0.1% TFA in H2O:MeOH, v-v).

Extended Data Fig. 4 Prep HPLC trace of 13.

YMC Triat C-18 column, 5.0 μm, 150 × 4.6 mm, flow rate = 1.0 mL·min−1 by an isocratic elution with 30:70 (0.1% TFA in H2O:MeOH, v-v).

Extended Data Fig. 5 HPLC-trace of preparative HPLC purification of radiolabeled peptide 18F-14.

Hypersil Gold (250 ✕ 10 mm, 5 μm, flow rate = 4 mL min−1) column with an isocratic mixture of 15:85 (MeCN:water, 0.1% TFA, vol:vol) for 2 min, followed by a linear gradient to 45:55 (MeCN:water, 0.1% TFA, vol:vol) within 18 min.

Extended Data Fig. 6 Analytical HPLC chromatograms obtained for the 18F-labeled L-glutathione conjugate.

a, analytical HPLC chromatogram obtained for HPLC purified 18F-labeled L-glutathione conjugate. b, co-injection of the pure 18F-labeled L-glutathione conjugate and an aliquot of reference standard on an analytical HPLC column; γ-trace (lower) and 254 nm UV trace (upper).

Extended Data Fig. 7 Radiotrace of the reaction mixture obtained by manual synthesis (column 1).

Top: reaction mixture prior to the cleavage step. Bottom: reaction mixture after the cleavage step.

Extended Data Fig. 8 Comparison of HPLC radiotrace of H-Leu-Phe(4-[18F]F)-Glu-Met-Lys-NH2 obtained by manual synthesis with UV-trace (290 nm) of authentic reference (column 2).

Upper: radiotrace of 18F-14 obtained by manual synthesis (radiochemical purity = 96%), lower: UV-trace of authentic reference standard 19F-14 monitored at 290 nm, The spatial separation between the diode array detector and the radioactivity detector introduces a delay that results in an offset of ~0.5 minutes between corresponding peaks in the radiochromatogram and UV-chromatogram.

Extended Data Fig. 9 HPLC radiotrace of H-Leu-Phe(4-[18F]F)-Glu-Met-Lys-NH2 isolated from automated synthesis (column 2, gradient 1).

Radiotrace of 18F-14 obtained by automated radiosynthesis (radiochemical purity = 96%).

Supplementary information

Supplementary Information

Supplementary Tutorials 1−7.

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Halder, R., Ma, G., Rickmeier, J. et al. Deoxyfluorination of phenols for chemoselective 18F-labeling of peptides. Nat Protoc 18, 3614–3651 (2023). https://doi.org/10.1038/s41596-023-00890-z

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