title: Simultaneous dual protein labeling using a triorthogonal reagent.
abstract: 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.
authors.affiliation: Department of Chemistry, and §Department of Medicinal Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Rashidian
authors.forename: Sidath C
authors.initials: SC
authors.lastname: Kumarapperuma
authors.forename: Kari
authors.initials: K
authors.lastname: Gabrielse
authors.forename: Adrian
authors.initials: A
authors.lastname: Fegan
authors.forename: Carston R
authors.initials: CR
authors.lastname: Wagner
authors.forename: Mark D
authors.initials: MD
authors.lastname: Distefano
mesh_terms: D020934:Ciliary Neurotrophic Factor / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D011106:Polyisoprenyl Phosphates / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D012717:Sesquiterpenes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D020934:Ciliary Neurotrophic Factor; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D049452:Green Fluorescent Proteins; C004808:farnesyl pyrophosphate