Synthesis and Evaluation of Novel Ring‐Strained Noncanonical Amino Acids for Residue‐Specific Bioorthogonal Reactions in Living Cells. Issue 19 (4th March 2021)

• Record Type: Journal Article
• Authors: Reinkemeier, Christopher D.; Koehler, Christine; Sauter, Paul F.; Shymanska, Nataliia V.; Echalier, Cecile; Rutkowska, Anna; Will, David W.; Schultz, Carsten; Lemke, Edward A.
• Is Part Of: Chemistry. Volume 27:Issue 19(2021)
• Page Start: 6094
• Page End: 6099
• Publication Date: 2021-03-04
• Subjects: click chemistry -- kinetics -- live-cell labeling -- protein engineering -- unnatural amino acids; Chemistry -- Periodicals; 540
• DOI: 10.1002/chem.202100322
• Languages: English
• ISSNs: 0947-6539

Abstract

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. Abstract : How do they click? A high throughput fluorescence flow cytometry‐based FRET assay enables us to directly measure bioorthogonal reaction kinetics in living cells. Using this assay, we test several new compounds and identify well suited strained dienophiles for genetic code expansion experiments to site‐specifically label proteins in vivo .