Amino Acids for Diels–Alder Reactions in Living Cells

Abstract

Under tension: A set of genetically encoded unnatural amino acids can be used for biocompatible site‐specific labeling of proteins with fluorogenic dyes. The new compounds have norbornene and trans‐cyclooctene units that react with tetrazine derivatives in an inverse‐electron‐demand Diels–Alder cycloaddition (left in picture). The technique offers fast labeling that is orthogonal to labeling through azide–cyclooctyne click reaction (right).

A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins

“…To further expand the applications of SiRcarboxyl, we evaluated its coupling to intracellular proteins via the incorporation of UAAs in the proteins of interest and their subsequent specific fluorescence labelling [35][36][37] . Specifically, we investigated if a SiR-carboxyl derivative permits the fluorescence labelling of intracellular proteins that bear an appropriate UAA in Escherichia coli.…”

“…6) were incorporated by amber (TAG) stop-codon suppression according to the expanded genetic code concept 38 . We used the previously reported tRNA Pyl /PylRS AF pair and GFP 39TAG (TAG codon in position 39) in our study to express GFP TAG UAA and so genetically encode the respective UAAs 36,39 . The TCO derivative permits fast labelling of probes that carry tetrazines, whereas the SCO derivative serves as a control.…”

Selective chemical protein modification

“…To generally enable IEDDA protein reactions, key reactive UAAs (tetrazine 105 , norbornene [106][107][108] , cyclooctene 98,107 and biscyclononene 98 ) have been incorporated into proteins by amber-stop codon suppression (see Box 2). Such strain-promoted cycloadditions offer intriguing and exciting possibilities for future protein labelling where the speed of labelling is vital, and recent developments suggest that the cyclooctyne-azide and cyclooctenetetrazine reactions may have a degree of mutual compatibility, thereby allowing multi-site labelling 109,110 .…”

Antibody–Drug Conjugates for Tumor Targeting—Novel Conjugation Chemistries and the Promise of non-IgG Binding Proteins

“…To make these reactions applicable to proteins, they must first be charged with aromatic azides, BCN or quinones in a stoichiometrically defined, site-specific manner. This no longer possible with a simple metstrain of E. coli, but requires amber-suppression technology for site-specific incorporation of BCN-lysine or other non-natural amino acids carrying functional groups like quinones or norbonenes [65][66][67][68][69][70][71][72] . So far, this technology was only used for imaging agents, as it shows unfavorably low expression yields 66 .…”