The synthesis of specifically isotope labelled fluorotryptophan and its use in mammalian cell-based protein expression for 19F-NMR applications

19F 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 19F-13C spin pairs into carbonic anhydrase 2 and superoxide dismutase, following an expression protocol utilizing HEK cells.