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Preparation of the functionalizable methionine surrogate azidohomoalanine via copper-catalyzed diazo transfer

Nature Protocols volume 2, pages 18791883 (2007) | Download Citation

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Abstract

The azide functional group has assumed a prominent role in chemical biology efforts in recent years. Azides may be readily introduced into proteins upon replacement of methionine residues with the non-canonical amino acid azidohomoalanine (AHA). This protocol describes a synthetic route to AHA based on the copper-catalyzed conversion of amines to azides. An alternate protocol for the preparation of AHA is presented in a companion paper. The synthesis and purification of AHA via the route described herein can be completed in 3–4 days.

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References

  1. 1.

    & Chemistry in living systems. Nat. Chem. Biol. 1, 13–21 (2005).

  2. 2.

    & Cell surface engineering by a modified Staudinger reaction. Science 287, 2007–2010 (2000).

  3. 3.

    , & Staudinger ligation: a peptide from a thioester and azide. Org. Lett. 2, 1939–1941 (2000).

  4. 4.

    , & Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40, 2004–2021 (2001).

  5. 5.

    , , & A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective 'ligation' of azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596–2599 (2002).

  6. 6.

    , & 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).

  7. 7.

    , & A strain-promoted [3+2] azide–alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 126, 15046–15047 (2004).

  8. 8.

    et al. Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation. J. Am. Chem. Soc. 124, 14893–14902 (2002).

  9. 9.

    et al. A tagging-via-substrate technology for detection and proteomics of farnesylated proteins. Proc. Natl. Acad. Sci. USA 101, 12479–12484 (2004).

  10. 10.

    , , & Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc. Natl. Acad. Sci. USA 99, 19–24 (2002).

  11. 11.

    & Conversion of DNA methyltransferases into azidonucleosidyl transferases via synthetic cofactors. Nucleic Acids Res. 33, 1644–1652 (2005).

  12. 12.

    et al. Bioconjugation by copper(I)-catalyzed azide-alkyne 3+2 cycloaddition. J. Am. Chem. Soc. 125, 3192–3193 (2003).

  13. 13.

    , & Azidoalanine mutagenicity in Salmonella—effect of homologation and alpha-methyl substitution. Mutat. Res. 216, 27–33 (1989).

  14. 14.

    , , & Effects of deuterium labeling on azido amino-acid mutagenicity in Salmonella-typhimurium. Mutat. Res. 308, 33–42 (1994).

  15. 15.

    , & Presentation and detection of azide functionality in bacterial cell surface proteins. J. Am. Chem. Soc. 126, 10598–10602 (2004).

  16. 16.

    & Cell surface labeling of Escherichia coli via copper(I)-catalyzed 3+2 cycloaddition. J. Am. Chem. Soc. 125, 11164–11165 (2003).

  17. 17.

    et al. Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids. Proc. Natl. Acad. Sci. USA 103, 10180–10185 (2006).

  18. 18.

    , & Expanding the scope of protein biosynthesis by altering the methionyl-tRNA synthetase activity of a bacterial expression host. Angew. Chem. Int. Ed. 39, 2148–2152 (2000).

  19. 19.

    , , , & Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc. Natl. Acad. Sci. USA 103, 9482–9487 (2006).

  20. 20.

    et al. Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging. Nat. Protoc. 2, 532–540 (2007).

  21. 21.

    , & Synthesis of the functionalizable methionine surrogate azidohomoalanine using Boc-homoserine as precursor. Nat. Protoc. 2, 1884–1887 (2007).

  22. 22.

    & Improved solid-phase peptide synthesis method utilizing alpha-azide-protected amino acids. Org. Lett. 3, 781–783 (2001).

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Acknowledgements

We thank Rebecca Connor and Nick Fisk for refinements to this protocol. This work was supported by NIH, by an NSF graduate fellowship to A.J.L. and by a grant from the Netherlands Organization for Scientific Research to M.K.S.V.

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Author notes

    • A James Link
    •  & Mandy K S Vink

    Present address: Department of Chemical Engineering, Princeton University, A207 Engineering Quadrangle, Princeton, New Jersey 08544, USA (A.J.L.). BioCatalytics Europe GmbH, Parkring 18, A-8074, Grambach, Austria (M.K.S.V.).

Affiliations

  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, MC 210-41, Pasadena, California 91106, USA.

    • A James Link
    • , Mandy K S Vink
    •  & David A Tirrell

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to A James Link.

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DOI

https://doi.org/10.1038/nprot.2007.268

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