Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins via a Pyrrolysyl-tRNA Synthetase/tRNA_CUA Pair and Click Chemistry

Abstract

We demonstrate that an orthogonal Methanosarcina barkeri MS pyrrolysyl-tRNA synthetase/tRNA(CUA) pair directs the efficient, site-specific incorporation of N6-[(2-propynyloxy)carbonyl]-L-lysine, containing a carbon-carbon triple bond, and N6-[(2-azidoethoxy)carbonyl]-L-lysine, containing an azido group, into recombinant proteins in Escherichia coli. Proteins containing the alkyne functional group are labeled with an azido biotin and an azido fluorophore, via copper catalyzed [3+2] cycloaddition reactions, to produce the corresponding triazoles in good yield. The methods reported are useful for the site-specific labeling of recombinant proteins and may be combined with mutually orthogonal methods of introducing unnatural amino acids into proteins as well as with chemically orthogonal methods of protein labeling. This should allow the site specific incorporation of multiple distinct probes into proteins and the control of protein topology and structure by intramolecular orthogonal conjugation reactions.

Fluorescent Amino Acids: Modular Building Blocks for the Assembly of New Tools for Chemical Biology

“…77,78 To accomplish this, they employed a pyrrolysine Methanosarcina bakeri/mazei tRNA/pylRS pair evolved by Chin and coworkers. 79 Lemke evolved the pair to accommodate the extra bulk of the cyclooctyne amino acid and used it to incorporate the cyclooctyne moiety into the mCherry protein ( Figure 9). Labeling with the fluorogenic coumarin azide confirmed incorporation of the cyclooctyne amino acid, suggesting the potential to tag any protein with a variety of cargo in vivo.…”

Genetisch kodierte kupferfreie Klick‐Chemie

“…In der Tat wurde bereits eine Vielzahl von Aziden mithilfe von beispielsweise optimierten Methanococcus-jannaschii-tRNA Tyr /RS-, E.-coli-tRNA Leu /RS-und Methanosarcina-bakeri/mazei-tRNA pyl /pylRS-Paaren (Tyr = Tyrosyl, Leu = Leucyl, pyl = Pyrrolysyl) genetisch kodiert. [15,16] Chromophore reagieren könnten, bei denen durch die Azidgruppe die Fluoreszenz zunächst weitgehend gelöscht wird und erst mit erfolgreicher Klick-Reaktion die Fluoreszenz stark ansteigt (Schema 1). [17] Mit einem fluorogenen Cumarinazidderivat wurden z.…”

“…Zudem wurde kürzlich gezeigt, dass die Carbamatbindung der Lysinseitenkette ein wichtiger Diskriminator für den erfolgreichen Einbau von UASs ist. [16,19,20] Daher synthetisierten wir, wie in Schema S1 (siehe Hintergrundinformationen) dargestellt, das Lysinderivat 1 in sechs Stufen aus kommerziell erhältlichem Cyclohepten. Der letzte Reaktionsschritt, das Abspalten der tert-Butoxycarbonyl-Schutzgruppe mit 70-proz.…”

Recombinant thiopeptides containing noncanonical amino acids

“…1B), which provide bioorthogonal alkene and alkyne moieties for postbiosynthetic modifications, respectively (29,30). In addition, to test the tolerance of ncAAs at different sites, we also examined substitutions at positions T4, V6, T8, and T13 with all three ncAAs.…”