Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Preparation of 18F-labeled peptides using the copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition

Abstract

An optimized procedure for preparing fluorine-18 (18F)-labeled peptides by the copper-catalyzed azide-alkyne 1,3-dipolar cyloaddition (CuAAC) is presented here. The two-step radiosynthesis begins with the microwave-assisted nucleophilic 18F-fluorination of a precursor containing a terminal p-toluenesulfonyl, terminal azide and polyethylene glycol backbone. The resulting 18F-fluorinated azide-containing building block is coupled to an alkyne-decorated peptide by the CuAAC. The reaction is accelerated by the copper(I)-stabilizing ligand bathophenanthroline disulfonate and can be performed in either reducing or nonreducing conditions (e.g., to preserve disulfide bonds). After an HPLC purification, 18F-labeled peptide can be obtained with a 31 ± 6% radiochemical yield (n = 4, decay-corrected from 18F-fluoride elution) and a specific activity of 39.0 ± 12.4 Ci μmol−1 within 77 ± 4 min.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1
Figure 2
Figure 3
Figure 4: Deconvoluted mass spectrum of crude 4.
Figure 5
Figure 6: Deconvoluted mass spectrum demonstrating the enrichment of 4 from 1.

References

  1. Kolb, H.C., Finn, M.G. & Sharpless, K.B. Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. Engl. 40, 2004–2021 (2001).

    CAS  Article  Google Scholar 

  2. Rostovtsev, V.V., Green, L.G., Fokin, V.V. & Sharpless, K.B. A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective 'ligation' of azides and terminal alkynes. Angew. Chem. Int. Ed. Engl. 41, 2596–2599 (2002).

    CAS  Article  Google Scholar 

  3. Tornoe, C.W., Christensen, C. & Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67, 3057–3064 (2002).

    CAS  Article  Google Scholar 

  4. Kolb, H.C. & Sharpless, K.B. The growing impact of click chemistry on drug discovery. Drug Discovery Today 8, 1128–1137 (2003).

    CAS  Article  Google Scholar 

  5. Meldal, M. & Tornoe, C.W. Cu-catalyzed azide-alkyne cycloaddition. Chem. Rev. 108, 2952–3015 (2008).

    CAS  Article  Google Scholar 

  6. Glaser, M. & Robins, E.G. 'Click labelling' in PET radiochemistry. J. Label. Compd. Radiopharm. 52, 407–414 (2009).

    CAS  Article  Google Scholar 

  7. Sirion, U. et al. An efficient F-18 labeling method for PET study: Huisgen 1,3-dipolar cycloaddition of bioactive substances and F-18-labeled compounds. Tetrahedron Lett. 48, 3953–3957 (2007).

    CAS  Article  Google Scholar 

  8. Nguyen, Q.D. et al. Positron emission tomography imaging of drug-induced tumor apoptosis with a caspase-3/7 specific [18F]-labeled isatin sulfonamide. Proc. Natl. Acad. Sci. USA 106, 16375–16380 (2009).

    CAS  Article  Google Scholar 

  9. Gill, H.S. et al. A modular platform for the rapid site-specific radiolabeling of proteins with 18F exemplified by quantitative positron emission tomography of human epidermal growth factor receptor 2. J. Med. Chem. 52, 5816–5825 (2009).

    CAS  Article  Google Scholar 

  10. Ramenda, T., Kniess, T., Bergmann, R., Steinbach, J. & Wuest, F. Radiolabelling of proteins with fluorine-18 via click chemistry. Chem. Commun. (Camb), 7521–7523 (2009).

  11. Marik, J. & Sutcliffe, J.L. Click for PET: rapid preparation of [18F]fluoropeptides using Cu(I) catalyzed 1,3-dipolar cycloaddition. Tetrahedron Lett. 47, 6681–6684 (2006).

    CAS  Article  Google Scholar 

  12. Glaser, M. & Arstad, E. 'Click labeling' with 2-[18F]fluoroethylazide for positron emission tomography. Bioconjug. Chem. 18, 989–993 (2007).

    CAS  Article  Google Scholar 

  13. Inkster, J.A.H., Guerin, B., Ruth, T.J. & Adam, M.J. Radiosynthesis and bioconjugation of [18F]FPy5yne, a prosthetic group for the 18F labeling of bioactive peptides. J. Label. Compd. Radiopharm. 51, 444–452 (2008).

    CAS  Article  Google Scholar 

  14. Hausner, S.H., Marik, J., Gagnon, M.K. & Sutcliffe, J.L. In vivo positron emission tomography (PET) imaging with an alphavbeta6 specific peptide radiolabeled using 18F-'click' chemistry: evaluation and comparison with the corresponding 4-[18F]fluorobenzoyl- and 2-[18F]fluoropropionyl-peptides. J. Med. Chem. 51, 5901–5904 (2008).

    CAS  Article  Google Scholar 

  15. Li, Z.B., Wu, Z., Chen, K., Chin, F.T. & Chen, X. Click chemistry for 18F-labeling of RGD peptides and microPET imaging of tumor integrin alphavbeta3 expression. Bioconjug. Chem. 18, 1987–1994 (2007).

    CAS  Article  Google Scholar 

  16. Rodionov, V.O., Presolski, S.I., Diaz, D.D., Fokin, V.V. & Finn, M.G. Ligand-accelerated Cu-catalyzed azide-alkyne cycloaddition: a mechanistic report. J. Am. Chem. Soc. 129, 12705–12712 (2007).

    CAS  Article  Google Scholar 

  17. Rodionov, V.O., Presolski, S.I., Gardinier, S., Lim, Y.H. & Finn, M.G. Benzimidazole and related ligands for Cu-catalyzed azide-alkyne cycloaddition. J. Am. Chem. Soc. 129, 12696–12704 (2007).

    CAS  Article  Google Scholar 

  18. Campbell-Verduyn, L.S., Mirfeizi, L., Dierckx, R.A., Elsinga, P.H. & Feringa, B.L. Phosphoramidite accelerated copper(I)-catalyzed [3 + 2] cycloadditions of azides and alkynes. Chem. Commun. (Camb), 2139–2141 (2009).

  19. Lewis, W.G., Magallon, F.G., Fokin, V.V. & Finn, M.G. Discovery and characterization of catalysts for azide-alkyne cycloaddition by fluorescence quenching. J. Am. Chem. Soc. 126, 9152–9153 (2004).

    CAS  Article  Google Scholar 

  20. Chan, T.R., Hilgraf, R., Sharpless, K.B. & Fokin, V.V. Polytriazoles as copper(I)-stabilizing ligands in catalysis. Org. Lett. 6, 2853–2855 (2004).

    CAS  Article  Google Scholar 

  21. Soriano Del Amo, D. et al. Biocompatible copper(I) catalysts for in vivo imaging of glycans. J. Am. Chem. Soc. 132, 16893–16899.

  22. Sen Gupta, S. et al. Accelerated bioorthogonal conjugation: a practical method for the ligation of diverse functional molecules to a polyvalent virus scaffold. Bioconjug. Chem. 16, 1572–1579 (2005).

    CAS  Article  Google Scholar 

  23. Sutcliffe-Goulden, J.L., O'Doherty, M.J. & Bansal, S.S. Solid phase synthesis of [18F]labelled peptides for positron emission tomography. Bioorg. Med. Chem. Lett. 10, 1501–1503 (2000).

    CAS  Article  Google Scholar 

  24. Sutcliffe-Goulden, J.L. et al. Rapid solid phase synthesis and biodistribution of 18F-labelled linear peptides. Eur. J. Nucl. Med. Mol. Imaging 29, 754–759 (2002).

    CAS  Article  Google Scholar 

  25. Marik, J., Hausner, S.H., Fix, L.A., Gagnon, M.K. & Sutcliffe, J.L. Solid-phase synthesis of 2-[18F]fluoropropionyl peptides. Bioconjug. Chem. 17, 1017–1021 (2006).

    CAS  Article  Google Scholar 

  26. Berndt, M., Pietzsch, J. & Wuest, F. Labeling of low-density lipoproteins using the 18F-labeled thiol-reactive reagent N-[6-(4-[18F]fluorobenzylidene)aminooxyhexyl]maleimide. Nucl. Med. Biol. 34, 5–15 (2007).

    CAS  Article  Google Scholar 

  27. Cai, W., Zhang, X., Wu, Y. & Chen, X. A thiol-reactive 18F-labeling agent, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide, and synthesis of RGD peptide-based tracer for PET imaging of alpha v beta 3 integrin expression. J. Nucl. Med. 47, 1172–1180 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Toyokuni, T. et al. Synthesis of a new heterobifunctional linker, N-[4-(aminooxy)butyl]maleimide, for facile access to a thiol-reactive 18F-labeling agent. Bioconjug. Chem. 14, 1253–1259 (2003).

    CAS  Article  Google Scholar 

  29. Wuest, F., Kohler, L., Berndt, M. & Pietzsch, J. Systematic comparison of two novel, thiol-reactive prosthetic groups for 18F labeling of peptides and proteins with the acylation agent succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). Amino Acids 36, 283–295 (2009).

    CAS  Article  Google Scholar 

  30. Glaser, M. et al. Methods for 18F-labeling of RGD peptides: comparison of aminooxy [18F]fluorobenzaldehyde condensation with 'click labeling' using 2-[18F]fluoroethylazide, and S-alkylation with [18F]fluoropropanethiol. Amino Acids 37, 717–724 (2009).

    CAS  Article  Google Scholar 

  31. Glaser, M. et al. Radiosynthesis and biodistribution of cyclic RGD peptides conjugated with novel [18F]fluorinated aldehyde-containing prosthetic groups. Bioconjug. Chem. 19, 951–957 (2008).

    CAS  Article  Google Scholar 

  32. Namavari, M. et al. Direct site-specific radiolabeling of an Affibody protein with 4-[18F]fluorobenzaldehyde via oxime chemistry. Mol. Imaging Biol. 10, 177–181 (2008).

    Article  Google Scholar 

  33. Namavari, M. et al. A novel method for direct site-specific radiolabeling of peptides using [18F]FDG. Bioconjug. Chem. 20, 432–436 (2009).

    CAS  Article  Google Scholar 

  34. Bruus-Jensen, K. et al. Chemoselective hydrazone formation between HYNIC-functionalized peptides and 18F-fluorinated aldehydes. Nucl. Med. Biol. 33, 173–183 (2006).

    CAS  Article  Google Scholar 

  35. Olberg, D.E. et al. A novel prosthetic group for site-selective labeling of peptides for positron emission tomography. Bioconjug. Chem. 19, 1301–1308 (2008).

    CAS  Article  Google Scholar 

  36. Olberg, D.E. et al. Site-specific addition of an 18F-N-methylaminooxy-containing prosthetic group to a vinylsulfone modified peptide. J. Label. Compd. Radiopharm. 52, 571–575 (2009).

    CAS  Google Scholar 

  37. Schirrmacher, E. et al. Synthesis of p-(di-tert-butyl[18F]fluorosilyl)benzaldehyde ([18F]SiFA-A) with high specific activity by isotopic exchange: a convenient labeling synthon for the 18F-labeling of N-amino-oxy derivatized peptides. Bioconjug. Chem. 18, 2085–2089 (2007).

    CAS  Article  Google Scholar 

  38. Schirrmacher, R. et al. 18F-labeling of peptides by means of an organosilicon-based fluoride acceptor. Angew. Chem. Int. Ed. Engl. 45, 6047–6050 (2006).

    CAS  Article  Google Scholar 

  39. McBride, W.J. et al. A novel method of 18F radiolabeling for PET. J. Nucl. Med. 50, 991–998 (2009).

    CAS  Article  Google Scholar 

  40. Laverman, P. et al. A novel facile method of labeling octreotide with 18F-fluorine. J. Nucl. Med. 51, 454–461 (2010).

    CAS  Article  Google Scholar 

  41. McBride, W.J. et al. Improved 18F labeling of peptides with a fluoride-aluminum-chelate complex. Bioconjug. Chem. 21, 1331–1340 (2010).

    CAS  Article  Google Scholar 

  42. Hong, V., Presolski, S.I., Ma, C. & Finn, M.G. Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation. Angew. Chem. Int. Ed. Engl. 48, 9879–9883 (2009).

    CAS  Article  Google Scholar 

  43. Berridge, M.S., Apana, S.M. & Hersh, J.M. Teflon radiolysis as the major source of carrier in fluorine-18. J. Label. Compd. Radiopharm. 52, 543–548 (2009).

    CAS  Article  Google Scholar 

  44. Fuchtner, F., Preusche, S., Mading, P., Zessin, J. & Steinbach, J. Factors affecting the specific activity of [18F]fluoride from a [18O]water target. Nuklearmedizin 47, 116–119 (2008).

    CAS  Article  Google Scholar 

  45. Kaiser, E., Colescott, R.L., Bossinger, C.D. & Cook, P.I. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595–598 (1970).

    CAS  Article  Google Scholar 

  46. Patgiri, A., Menzenski, M.Z., Mahon, A.B. & Arora, P.S. Solid-phase synthesis of short α-helices stabilized by the hydrogen bond surrogate approach. Nat. Protoc. 5, 1857–1865 (2010).

    CAS  Article  Google Scholar 

  47. Chan, W.C. & White, P.D. (eds.) Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Oxford University Press, 2000).

Download references

Acknowledgements

We thank S. Williams and J.L. Sutcliffe for critical reading of the manuscript and their valuable comments; C. Quan and J. Tom for the peptide synthesis; and J. Tinianow for general assistance with radiolabeling and product characterization.

Author information

Authors and Affiliations

Authors

Contributions

H.S.G. and J.M. performed the work and wrote the manuscript.

Corresponding author

Correspondence to Jan Marik.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Solid phase extraction (SPE) as a replacement for the HPLC purification of 18F-labeled peptides (DOC 33 kb)

Supplementary Table 1

The ascorbate-based and ascorbate-free CuAAC were each tested in varied reaction conditions. (DOC 152 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gill, H., Marik, J. Preparation of 18F-labeled peptides using the copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition. Nat Protoc 6, 1718–1725 (2011). https://doi.org/10.1038/nprot.2011.390

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.390

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing