A Sterically Overcrowded, Isopropyl-Substituted, Lanthanide-Chelating Tag for Protein Pseudocontact Shift NMR Spectroscopy: Synthesis of its Macrocyclic Scaffold and Benchmarking on Ubiquitin S57 C and hCA II S166 C.

A sterically overcrowded lanthanide-chelating tag has been synthesized in order to investigate the influence on the obtained pseudocontact shifts and the anisotropic part of the magnetic susceptibility tensor compared to those of its predecessor DOTA-M8-(4R,4S)-SSPy. For the first time, a concise synthetic route is presented for isopropyl-substituted cyclen, the macrocyclic scaffold of the lanthanide-chelating tag, delivering the macrocycle in an overall yield of 6 % over 11 steps. The geometry of the lutetium complex has been assigned by ROESY experiments, adopting exclusively a Λ(δδδδ) conformation, and DFT calculations have confirmed a stabilization of 32.6 kJ mol compared to the Δ(δδδδ) conformer. The highly rigidified lanthanide-chelating tag induces strong pseudocontact shifts of up to 6.5 ppm on ubiquitin S57 C, shows significantly improved tensor properties compared to those of its predecessor, and constitutes a highly promising starting point for the further development of lanthanide-chelating tags.