Synthesis and Evaluation of Novel Ring-Strained Noncanonical Amino Acids for Residue-Specific Bioorthogonal Reactions in Living Cells

Christopher D. Reinkemeier, Christine Koehler, Paul F. Sauter, Nataliia V. Shymanska, Cecile Echalier, Anna Rutkowska, David W. Will, Carsten Schultz, Edward A. Lemke

Research output: Contribution to journalArticlepeer-review

Abstract

Bioorthogonal reactions are ideally suited to selectively modify proteins in complex environments, even in vivo. Kinetics and product stability of these reactions are crucial parameters to evaluate their usefulness for specific applications. Strain promoted inverse electron demand Diels–Alder cycloadditions (SPIEDAC) between tetrazines and strained alkenes or alkynes are particularly popular, as they allow ultrafast labeling inside cells. In combination with genetic code expansion (GCE)-a method that allows to incorporate noncanonical amino acids (ncAAs) site-specifically into proteins in vivo. These reactions enable residue-specific fluorophore attachment to proteins in living mammalian cells. Several SPIEDAC capable ncAAs have been presented and studied under diverse conditions, revealing different instabilities ranging from educt decomposition to product loss due to β-elimination. To identify which compounds yield the best labeling inside living mammalian cells has frequently been difficult. In this study we present a) the synthesis of four new SPIEDAC reactive ncAAs that cannot undergo β-elimination and b) a fluorescence flow cytometry based FRET-assay to measure reaction kinetics inside living cells. Our results, which at first sight can be seen conflicting with some other studies, capture GCE-specific experimental conditions, such as long-term exposure of the ring-strained ncAA to living cells, that are not taken into account in other assays.

Original languageEnglish (US)
Pages (from-to)6094-6099
Number of pages6
JournalChemistry - A European Journal
Volume27
Issue number19
DOIs
StatePublished - Apr 1 2021

Keywords

  • click chemistry
  • kinetics
  • live-cell labeling
  • protein engineering
  • unnatural amino acids

ASJC Scopus subject areas

  • Catalysis
  • Organic Chemistry

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