Paramagnetic labels have emerged as a powerful tool for structural studies of proteins and protein-ligand complexes. In particular, pseudocontact shifts (PCS) induced by paramagnetic lanthanide ions are easy to measure in NMR spectra and provide long-range structural information up to about 40 Å.¹² To generate useful PCS data, however, the lanthanide tag must be attached to the protein site-specifically and with minimal mobility of the metal ion.

While most of the traditional lanthanide tags rely on single cysteine residues for site-specific attachment to proteins, a more generally applicable method employs unnatural amino acids. We used an orthogonal suppressor-tRNA/aminoacyl-tRNA synthetase pair developed by the group of P. G. Schultz for site-specific incorporation of p-azido-phenylalanine (AzF) into different target proteins.³ Subsequently, the AzF residue was reacted with a lanthanide-binding tag in a Cu(I)-catalyzed cycloaddition reaction.⁴ We show that sizeable PCSs can be generated in this way using lanthanide complexes of an alkyne derivative of nitrilotriacetic acid, which is a relatively small tag.