Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins Via a Pyrrolysyl-tRNA Synthetase/tRNA(CUA) Pair and Click Chemistry

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.