Skip to main content

Site-Specific Modification of Proteins by the Staudinger-Phosphite Reaction

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 794))

Abstract

Chemoselective reactions are important tools for the modification of peptides and proteins. Thereby the modification is desired to be site specific and bioorthogonal. Here we describe the site-specific modification of azido-proteins via a Staudinger-type phosphite ligation. The reaction was carried out in aqueous system on proteins containing p-azido-phenylalanine in a single position introduced by the amber codon technique. A selective introduction of branched polyethylene scaffolds can be achieved with the application of the methodology reported herein.

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Wang L, Schultz PG (2005) Expanding the genetic code. Angew Chemie Int Ed 44, 34–66.

    Article  CAS  Google Scholar 

  2. Wang L, Xie J, Schultz PG (2006) Expanding the genetic code. Ann Rev Biophys Biomol Struc 35, 225–249.

    Article  Google Scholar 

  3. Budisa N (2004) Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire. Ang Chemie Int Ed 116, 6426–6463.

    Article  Google Scholar 

  4. Dougherty DA (2000) Unnatural amino acids as probes of protein structure and function. Curr Opin Chem Biol 4, 645–652.

    Article  PubMed  CAS  Google Scholar 

  5. Xie J, Schultz PG (2005) Adding amino acids to the genetic repertoire Curr Opin Chem Biol 9, 548–554.

    CAS  Google Scholar 

  6. Link AJ, Mock ML, Tirrell DA (2003) Non-canonical amino acids in protein engineering. Curr Opin Biotechn 14, 603–609.

    Article  CAS  Google Scholar 

  7. Gerrits M et al., (2007) In: Cell-Free Protein Expression, Landes Bioscience, Austin, 2007.

    Google Scholar 

  8. Prescher JA, Bertozzi CR (2005) Chemistry in living systems. Nat Chem Biol 1, 13–21.

    Article  PubMed  CAS  Google Scholar 

  9. Rostovtsev VV et al. (2002) A stepwise huisgen cycloaddition process: copper(I)-catalyzed ­regioselective “ligation” of azides and terminal alkynes. Angew Chem Int Ed 2002, 41, 2596–2599.

    Article  CAS  Google Scholar 

  10. Tornøe CW, Christensen C, Meldal M (2002) 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.

    Article  PubMed  Google Scholar 

  11. Codelli JA et al. (2008) Second-generation difluorinated cyclooctynes for copper-free click chemistry. J Am Chem Soc 130, 11486–11493.

    Article  PubMed  CAS  Google Scholar 

  12. Ning X, Guo J, Wolfert MA, Boons G-J (2008) Visualizing metabolically labeled glycoconjugates of living cells by copper-free and fast huisgen cycloadditions. Angew Chem Int Ed 47, 2253–2255.

    Article  CAS  Google Scholar 

  13. Debets MF et al. (2010) Aza-dibenzo-cyclooctynes for fast and efficient enzyme PEGylation via copper-free (3  +  2) cycloaddition. Chem Commun 46, 97–99.

    Article  CAS  Google Scholar 

  14. Saxon E, Bertozzi CR (2000) Cell surface engineering by a modified Staudinger reaction. Science 287, 2007–2010.

    Article  PubMed  CAS  Google Scholar 

  15. Prescher JA, Dube DH, Bertozzi CR (2004) Chemical remodelling of cell surfaces in living animals. Nature 430, 873–877.

    Article  PubMed  CAS  Google Scholar 

  16. Agard NJ et al. (2006) A comparative study of bioorthogonal reactions with azides. ACS Chemical Biology 1, 644–648.

    Article  PubMed  CAS  Google Scholar 

  17. Böhrsch V et al. (2010) Site-specific functionalisation of proteins by a Staudinger-type reaction using unsymmetrical phosphites. Chem Commun 46, 3176–8.

    Article  Google Scholar 

  18. Serwa R et al. (2010) Site-specific PEGylation of proteins by a Staudinger-phosphite reaction. Chemical Science, 596–602.

    Google Scholar 

  19. Serwa R et al. (2009) Chemoselective Staudinger-phosphite reaction of azides for the phosphorylation of proteins. Angew Chem Int Ed 48, 8234–8139.

    Article  CAS  Google Scholar 

  20. Veronese FM (2001) Peptide and protein PEGylation: a review of problems and solutions. Biomaterials 22, 405–417.

    Article  PubMed  CAS  Google Scholar 

  21. Veronese FM, Mero A (2008) The impact of PEGylation on biological therapies. Biodrugs 22, 315–329.

    Article  PubMed  CAS  Google Scholar 

  22. Roberts MJ, Bentley MD, Harris JM (2002) Chemistry for peptide and protein PEGylation. Adv Drug Deliv Rev 54, 459–476.

    Article  PubMed  CAS  Google Scholar 

  23. Caliceti P, Veronese FM (2003) Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates Adv Drug Deliv Rev 55, 1261–1277.

    Google Scholar 

Download references

Acknowledgments

The authors acknowledge financial support from the German Science Foundation (DFG) within the Emmy-Noether program (HA 4468/2-1), the SFB 765, the Fonds der Chemischen Industrie (FCI) and the Böhringer-Ingelheim Foundation (“Plus 3-Perspektiven Programm”).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Christian P. R. Hackenberger .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Majkut, P., Böhrsch, V., Serwa, R., Gerrits, M., Hackenberger, C.P.R. (2012). Site-Specific Modification of Proteins by the Staudinger-Phosphite Reaction. In: Pollegioni, L., Servi, S. (eds) Unnatural Amino Acids. Methods in Molecular Biology, vol 794. Humana Press. https://doi.org/10.1007/978-1-61779-331-8_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-331-8_15

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-330-1

  • Online ISBN: 978-1-61779-331-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics