Simultaneous dual protein labeling using a triorthogonal reagent

Mohammad Rashidian, Sidath C. Kumarapperuma, Kari Gabrielse, Adrian Fegan, Carston R. Wagner, Mark D. Distefano

Research output: Contribution to journalArticlepeer-review

46 Scopus citations


Construction of heterofunctional proteins is a rapidly emerging area of biotherapeutics. Combining a protein with other moieties, such as a targeting element, a toxic protein or small molecule, and a fluorophore or polyethylene glycol (PEG) group, can improve the specificity, functionality, potency, and pharmacokinetic profile of a protein. Protein farnesyl transferase (PFTase) is able to site-specifically and quantitatively prenylate proteins containing a C-terminal CaaX-box amino acid sequence with various modified isoprenoids. Here, we describe the design, synthesis, and application of a triorthogonal reagent, 1, that can be used to site-specifically incorporate an alkyne and aldehyde group simultaneously into a protein. To illustrate the capabilities of this approach, a protein was enzymatically modified with compound 1 followed by oxime ligation and click reaction to simultaneously incorporate an azido-tetramethylrhodamine (TAMRA) fluorophore and an aminooxy-PEG moiety. This was performed with both a model protein [green fluorescent protein (GFP)] as well as a therapeutically useful protein [ciliary neurotrophic factor (CNTF)]. Next, a protein was enzymatically modified with compound 1 followed by coupling to an azido-bis-methotrexate dimerizer and aminooxy-TAMRA. Incubation of that construct with a dihydrofolate reductase (DHFR)-DHFR-anti-CD3 fusion protein resulted in the self-assembly of nanoring structures that were endocytosed into T-leukemia cells and visualized therein. These results highlight how complex multifunctional protein assemblies can be prepared using this facile triorthogonal approach.

Original languageEnglish (US)
Pages (from-to)16388-16396
Number of pages9
JournalJournal of the American Chemical Society
Issue number44
StatePublished - Nov 6 2013
Externally publishedYes

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


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