title: Hydrophilic trans-Cyclooctenylated Noncanonical Amino Acids for Fast Intracellular Protein Labeling.
abstract: Introduction of bioorthogonal functionalities (e.g., trans-cyclooctene-TCO) into a protein of interest by site-specific genetic encoding of non-canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine-functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours-long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO-ncAAs. One derivative, DOTCO-lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy.
authors.affiliation: Hungarian Academy of Sciences, Research Centre for Natural Sciences, Institute of Organic Chemistry, Magyar tudósok krt. 2, 1117, Budapest, Hungary.
authors.forename: Eszter
authors.initials: E
authors.lastname: Kozma
authors.affiliation: Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
authors.forename: Ivana
authors.initials: I
authors.lastname: Nikić
authors.affiliation: Hungarian Academy of Sciences, Research Centre for Natural Sciences, Institute of Organic Chemistry, Magyar tudósok krt. 2, 1117, Budapest, Hungary.
authors.forename: Balázs R
authors.initials: BR
authors.lastname: Varga
authors.affiliation: Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
authors.forename: Iker Valle
authors.initials: IV
authors.lastname: Aramburu
authors.affiliation: Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany.
authors.forename: Jun Hee
authors.initials: JH
authors.lastname: Kang
authors.affiliation: Center of Infectious Diseases, Integrative Virology, University of Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany.
authors.forename: Oliver T
authors.initials: OT
authors.lastname: Fackler
authors.affiliation: Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany. lemke@embl.de.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Lemke
authors.affiliation: Hungarian Academy of Sciences, Research Centre for Natural Sciences, Institute of Organic Chemistry, Magyar tudósok krt. 2, 1117, Budapest, Hungary. kele.peter@ttk.mta.hu.
authors.forename: Péter
authors.initials: P
authors.lastname: Kele
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D034242:Cyclooctanes / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D011506:Proteins / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D034242:Cyclooctanes; D005456:Fluorescent Dyes; D011506:Proteins
keywords: amino acids; click chemistry; fluorescence; hydrophilicity; protein engineering