Genetic Encoding of 7-Aza-L-tryptophan: Isoelectronic Substitution of a Single CH-Group in a Protein for a Nitrogen Atom for Site-Selective Isotope Labeling

Genetic encoding of a noncanonical amino acid (ncAA) in an in vivo expression system requires an aminoacyl-tRNA synthetase that specifically recognizes the ncAA, while the ncAA must not be recognized by the canonical protein expression machinery. We succeeded in genetically encoding 7-aza-tryptophan (7AW), which is isoelectronic with tryptophan. The system is fully orthogonal to protein expression in Escherichia coli, enabling high-yielding site-selective isotope labeling in vivo. 7AW is readily synthesized from serine and 7-aza-indole using a tryptophan synthetase beta-subunit (TrpB) mutant, affording easy access to isotope-labeled 7AW. Using labeled 7AW produced from N-15/C-13-labeled serine, we produced 7AW mutants of the 25 kDa Zika virus NS2B-NS3 protease. N-15-HSQC spectra display single cross-peaks at chemical shifts near those observed for the wild-type protein labeled with N-15/C-13-tryptophan, confirming the structural integrity of the protein and yielding straightforward NMR resonance assignments for site-specific probing.