title: Site-specific labeling of proteins and peptides with trans-cyclooctene containing handles capable of tetrazine ligation.
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.
authors.affiliation: Department of Chemistry and Biochemistry, St. Catherine University, 2004 Randolph Avenue, Saint Paul, MN, 55105, USA.
authors.forename: James W
authors.initials: JW
authors.lastname: Wollack
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Monson
authors.forename: Jonathan K
authors.initials: JK
authors.lastname: Dozier
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Dalluge
authors.forename: Kristina
authors.initials: K
authors.lastname: Poss
authors.forename: Scott A
authors.initials: SA
authors.lastname: Hilderbrand
authors.forename: Mark D
authors.initials: MD
authors.lastname: Distefano
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D034242:Cyclooctanes / Q000737:chemistry* / Q000378:metabolism; D011756:Diphosphates / Q000737:chemistry* / Q000378:metabolism; D004224:Diterpenes / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D017368:Protein Prenylation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D034242:Cyclooctanes; D011756:Diphosphates; D004224:Diterpenes; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; C511282:geranyl diphosphate; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase
keywords: bioorthogonal; inverse-electron-demand Diels-Alder; protein farnesyltransferase; protein prenylation; tetrazine ligation; trans-cyclooctene