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

  • Paul Majkut
  • Verena Böhrsch
  • Remigiusz Serwa
  • Michael Gerrits
  • Christian P. R. HackenbergerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 794)


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.

Key words

Site-specific protein modification Azido-proteins Bioorthogonal Staudinger-phosphite ligation Protein PEGylation 



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”).


  1. 1.
    Wang L, Schultz PG (2005) Expanding the genetic code. Angew Chemie Int Ed 44, 34–66.CrossRefGoogle Scholar
  2. 2.
    Wang L, Xie J, Schultz PG (2006) Expanding the genetic code. Ann Rev Biophys Biomol Struc 35, 225–249.CrossRefGoogle Scholar
  3. 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.CrossRefGoogle Scholar
  4. 4.
    Dougherty DA (2000) Unnatural amino acids as probes of protein structure and function. Curr Opin Chem Biol 4, 645–652.PubMedCrossRefGoogle Scholar
  5. 5.
    Xie J, Schultz PG (2005) Adding amino acids to the genetic repertoire Curr Opin Chem Biol 9, 548–554.Google Scholar
  6. 6.
    Link AJ, Mock ML, Tirrell DA (2003) Non-canonical amino acids in protein engineering. Curr Opin Biotechn 14, 603–609.CrossRefGoogle Scholar
  7. 7.
    Gerrits M et al., (2007) In: Cell-Free Protein Expression, Landes Bioscience, Austin, 2007.Google Scholar
  8. 8.
    Prescher JA, Bertozzi CR (2005) Chemistry in living systems. Nat Chem Biol 1, 13–21.PubMedCrossRefGoogle Scholar
  9. 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.CrossRefGoogle Scholar
  10. 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.PubMedCrossRefGoogle Scholar
  11. 11.
    Codelli JA et al. (2008) Second-generation difluorinated cyclooctynes for copper-free click chemistry. J Am Chem Soc 130, 11486–11493.PubMedCrossRefGoogle Scholar
  12. 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.CrossRefGoogle Scholar
  13. 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.CrossRefGoogle Scholar
  14. 14.
    Saxon E, Bertozzi CR (2000) Cell surface engineering by a modified Staudinger reaction. Science 287, 2007–2010.PubMedCrossRefGoogle Scholar
  15. 15.
    Prescher JA, Dube DH, Bertozzi CR (2004) Chemical remodelling of cell surfaces in living animals. Nature 430, 873–877.PubMedCrossRefGoogle Scholar
  16. 16.
    Agard NJ et al. (2006) A comparative study of bioorthogonal reactions with azides. ACS Chemical Biology 1, 644–648.PubMedCrossRefGoogle Scholar
  17. 17.
    Böhrsch V et al. (2010) Site-specific functionalisation of proteins by a Staudinger-type reaction using unsymmetrical phosphites. Chem Commun 46, 3176–8.CrossRefGoogle Scholar
  18. 18.
    Serwa R et al. (2010) Site-specific PEGylation of proteins by a Staudinger-phosphite reaction. Chemical Science, 596–602.Google Scholar
  19. 19.
    Serwa R et al. (2009) Chemoselective Staudinger-phosphite reaction of azides for the phosphorylation of proteins. Angew Chem Int Ed 48, 8234–8139.CrossRefGoogle Scholar
  20. 20.
    Veronese FM (2001) Peptide and protein PEGylation: a review of problems and solutions. Biomaterials 22, 405–417.PubMedCrossRefGoogle Scholar
  21. 21.
    Veronese FM, Mero A (2008) The impact of PEGylation on biological therapies. Biodrugs 22, 315–329.PubMedCrossRefGoogle Scholar
  22. 22.
    Roberts MJ, Bentley MD, Harris JM (2002) Chemistry for peptide and protein PEGylation. Adv Drug Deliv Rev 54, 459–476.PubMedCrossRefGoogle Scholar
  23. 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

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Paul Majkut
    • 1
  • Verena Böhrsch
    • 1
    • 2
  • Remigiusz Serwa
    • 1
  • Michael Gerrits
    • 3
  • Christian P. R. Hackenberger
    • 1
    Email author
  1. 1.Institut für Chemie und BiochemieFreie Universität BerlinBerlinGermany
  2. 2.Labor fur BiochemieBeuth-Hochschule für TechnikBerlinGermany
  3. 3.RiNA GmbHBerlinGermany

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