Design of Photocaged Puromycin for Nascent Polypeptide Release and Spatiotemporal Monitoring of Translation

• Published in: Angewandte Chemie International Edition Vol. 54; no. 12; pp. 3717 - 3721
• Main Authors: Buhr, Florian, Kohl-Landgraf, Jörg, tom Dieck, Susanne, Hanus, Cyril, Chatterjee, Deep, Hegelein, Andreas, Schuman, Erin M., Wachtveitl, Josef, Schwalbe, Harald
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 16.03.2015; WILEY‐VCH Verlag; Wiley; Wiley Subscription Services, Inc
• Edition: International ed. in English
• Subjects: Animals; Antibiotics; Benzaldehydes - chemistry; Biological; Cell Survival - drug effects; Chemistry; Chemistry, Multidisciplinary; Derivatives; HEK293 Cells; Hippocampus - metabolism; Humans; Illumination; Inhibitors; Microscopy, Fluorescence; Peptides - chemistry; Peptides - metabolism; photoactivation; Photolysis - radiation effects; Physical Sciences; Polypeptides; protecting groups; Protein Biosynthesis - drug effects; protein expression; Proteins; Puromycin - chemistry; Puromycin - toxicity; Rats; Science & Technology; Translations; Ultraviolet Rays; inhibitors; REQUIREMENTS; MECHANISM; COMPLEXES; PEPTIDE-BOND FORMATION; CHAIN; protecting groups; photoactivation; INHIBITION; SELECTION; antibiotics; protein expression; EXPRESSION; RIBOSOME
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201410940

The antibiotic puromycin, which inhibits protein translation, is used in a broad range of biochemical applications. The synthesis, characterization, and biological applications of NVOC‐puromycin, a photocaged derivative that is activated by UV illumination, are presented. The caged compound had no effect either on prokaryotic or eukaryotic translation or on the viability of HEK 293 cells. Furthermore, no significant release of ribosome‐bound polypeptide chains was detected in vitro. Upon illumination, cytotoxic activity, in vitro translation inhibition, and polypeptide release triggered by the uncaging of NVOC‐puromycin were equivalent to those of the commercial compound. The quantum yield of photolysis was determined to be 1.1±0.2 % and the NVOC‐puromycin was applied to the detection of newly translated proteins with remarkable spatiotemporal resolution by using two‐photon laser excitation, puromycin immunohistochemistry, and imaging in rat hippocampal neurons. On like a light: The antibiotic puromycin (green) is a translation inhibitor that triggers the release of the nascent polypeptide chain (red) from the ribosome (yellow) and it is used in a number of applications. A photocaged puromycin derivative, NVOC‐puromycin, was synthesized and characterized. Both functional recovery upon UV illumination and biological inactivity in vitro and in vivo were demonstrated.