Site-specific labeling of proteins and peptides with trans-cyclooctene containing handles capable of tetrazine ligation

Chem Biol Drug Des. 2014 Aug;84(2):140-7. doi: 10.1111/cbdd.12303. Epub 2014 May 13.

Abstract

There is a growing library of functionalized non-natural substrates for the enzyme protein farnesyltransferase (PFTase). PFTase covalently attaches these functionalized non-natural substrates to proteins ending in the sequence CAAX, where C is a cysteine that becomes alkylated, A represents an aliphatic amino acid, and X is Ser, Met, Ala, or Gln. Reported substrates include a variety of functionalities that allow modified proteins to undergo subsequent bioconjugation reactions. To date the most common strategy used in this approach has been copper catalyzed azide-alkyne cycloaddition (CuAAC). While being fast and bioorthogonal CuAAC has limited use in live cell experiments due to copper's toxicity.(1) Here, we report the synthesis of trans-cyclooctene geranyl diphosphate. This substrate can be synthesized from geraniol in six steps and be enzymatically transferred to peptides and proteins that end in a CAAX sequence. Proteins and peptides site-specially modified with trans-cyclooctene geranyl diphosphate were subsequently targeted for further modification via tetrazine ligation. As tetrazine ligation is bioorthogonal, fast, and is contingent on ring strain rather than the addition of a copper catalyst, this labeling strategy should prove useful for labeling proteins where the presence of copper may hinder solubility or biological reactivity.

Keywords: bioorthogonal; inverse-electron-demand Diels-Alder; protein farnesyltransferase; protein prenylation; tetrazine ligation; trans-cyclooctene.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkyl and Aryl Transferases / metabolism*
  • Amino Acid Sequence
  • Cyclooctanes / chemistry*
  • Cyclooctanes / metabolism
  • Diphosphates / chemistry*
  • Diphosphates / metabolism
  • Diterpenes / chemistry*
  • Diterpenes / metabolism
  • Peptides / chemistry*
  • Peptides / metabolism
  • Protein Prenylation
  • Proteins / chemistry*
  • Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Substrate Specificity

Substances

  • Cyclooctanes
  • Diphosphates
  • Diterpenes
  • Peptides
  • Proteins
  • Recombinant Proteins
  • geranyl diphosphate
  • Alkyl and Aryl Transferases
  • p21(ras) farnesyl-protein transferase