A Minimal, Unstrained S-Allyl Handle for Pre-Targeting Diels-Alder Bioorthogonal Labeling in Live Cells

5 pags, 3 figsThe unstrained S-allyl cysteine amino acid was site-specifically installed on apoptosis protein biomarkers and was further used as a chemical handle and ligation partner for 1,2,4,5-tetrazines by means of an inverse-electron-demand Diels-Alder reaction. We demonstrate the utility of this minimal handle for the efficient labeling of apoptotic cells using a fluorogenic tetrazine dye in a pre-targeting approach. The small size, easy chemical installation, and selective reactivity of the S-allyl handle towards tetrazines should be readily extendable to other proteins and biomolecules, which could facilitate their labeling within live cells.We thank China Scholarship Council (PhD studentship to Z.G.), FCT Portugal (FCT Investigator to G.J.L.B.), and the EU (Marie-Sklodowska Curie ITN Protein Conjugates; Marie-Sklodowska Curie IEF to B.L.O. and O.B.), MINECO (CTQ2015-70524-R and RYC-2013-14706 to G.J.O.), and the EPSRC for funding. We also thank Dr. André Neves and Prof. Kevin Brindle for providing the C2Am protein; Beatrice Amgarten for expressing and purifying Annexin V; Dr. Mike Deery and Ms. Julie Howard for help with mass spectrometry analysis; and BiFi (Memento cluster) for computer support. G.J.L.B. is a Royal Society University Research Fellow and the recipient of a European Research Council Starting Grant (TagIt).Peer reviewe