A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral Ln III tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues.Loaded with Tb III or Tm III ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron-electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the Tb III complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.

A mutant aminoacyl-tRNA synthetase identified by a library selection system affords site-specific incorporation of 7-fluoro-L-tryptophan in response to an amber stop codon. The enzyme allows the production of proteins with a single hydrogen atom replaced by a fluorine atom as a sensitive nuclear magnetic resonance (NMR) probe. The substitution of a single hydrogen atom by another element that is as closely similar in size and hydrophobicity as possible minimizes possible perturbations in the structure, stability, and solubility of the protein. The fluorine atom enables site-selective monitoring of the protein response to ligand binding by 19 F NMR spectroscopy, as demonstrated with the Zika virus NS2B-NS3 protease. KEYWORDS: fluoro-tryptophan, 19 F NMR spectroscopy, ligand binding, genetic encoding, noncanonical amino acids, pyrrolysyl-tRNA synthetase 19 F NMR spectroscopy of proteins labeled with fluorine atoms is becoming increasingly popular for studying protein structure, conformational changes, and protein−ligand interactions. 1−3 19

Efficient syntheses of fluorinated leucines, valines and alanines are described. The synthetic routes provide expedient access to various 13C/15N/D isotopologues requiring solely readily available and inexpensive isotope containing reagents such...