The synthesis of specifically isotope labelled fluorotryptophan and its use in mammalian cell-based protein expression for F-NMR applications

19 F nuclei serve as versatile sensors for detecting protein interactions and dynamics in biomolecular NMR spectroscopy. Although various methods have been developed to incorporate fluorine-containing aromatic residues into proteins using E. coli or cell-free expression techniques, similar approaches for protein production in mammalian cell lines remain limited. Here, we present a cost-effective synthetic route to obtain selectively deuterated, carbon-13 labeled fluorotryptophan and demonstrate its use in introducing 19 F- 13 C spin pairs into carbonic anhydrase 2 and superoxide dismutase, following an expression protocol utilizing HEK cells. Combining multistep organic synthesis with mammalian cell based protein overexpression leads to isolated 13 C- 19 F spin systems in tryptophan side chains, which represent ideal sensors to probe protein interaction and dynamics using NMR spectroscopy.