Enzyme substrate promiscuity can be used to label proteins with non-natural functionality. The enzyme used in this work, Protein Farnesyltransferase (PFTase), normally catalyzes the addition of a farnesyl group to a cysteine near the C terminus of proteins with a “CAAX” motif. In this motif, “A” represents an aliphatic amino acid and X is a variable amino acid. PFTase has also been shown to transfer a wide variety of non-natural isoprenoids to “CAAX” proteins including several with bioorthogonal functionality. Ras proteins are an important class of “CAAX” proteins that are mutated in up to 30% of human cancers. PFTase has been successful in transferring non-natural substrates with bioorthogonal functionality to Ras proteins using copper catalyzed click chemistry. Unfortunately, due to copper’s cytotoxicity clickable isoprenoids cannot be targeted in vivo using this process. Presented here is the synthesis of a bioorthogonal PFTase substrate that does not require a copper catalyst for subsequent bioconjugation reactions on enzymatically modified Ras proteins. This synthesized isoprenoid chain contains a terminal alkene which can undergo an inverse electron demand Diels-Alder cycloaddition with a fluorescent BODIPY-tetrazine probe. The terminal alkene reacts with the tetrazine and imparts fluorescent properties onto the Ras protein. This bioconjugation strategy can be used to locate and quantify the amount of Ras proteins in living cells.

Synthesis of a PFTase substrate containing a terminal olefin capable of tetrazine ligation

253rd National Meeting (2017)

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