Selective Incorporation of 19F‐Labeled Trp Side Chains for NMR‐Spectroscopy‐Based Ligand–Protein Interaction Studies

In the last few years NMR spectroscopy has increasingly been used in pharmaceutical drug discovery and development as a result of its ability to provide structural and dynamic information at atomic resolution. However, the attainment of structural information on protein ± ligand complexes by NMR spectroscopy is limited to small to medium size proteins (MW 30 kDa) and by the lengthy nature of the protein resonance assignment process. Nonetheless it is known that protein binding sites often contain only a small subset of residues–mainly tryptophan (Trp), tyrosine (Tyr), and arginine (Arg). This fact has led to a growing interest in the search for new techniques for the selective isotope labeling of these residues. For example, a new labeling method to selectively incorporate C at position 2 or 4 of Trp side chains has very recently been reported and has been successfully applied to the study of protein ± ligand interactions by use of 2D [C,H] correlation spectra. Herein, we report a simple and effective method to selectively label Trp side chains with F at position 5 or 6. Until now, F-Trp has generally been introduced into proteins by using Escherichia coli strains auxotrophic for Trp and by including the amino acid analogue in the growth medium. This method requires the timeconsuming extra steps of cloning the protein of interest into these strains, which may result in a loss in yield and poor incorporation levels. Instead of employing auxotrophic strains, we find that 3-indoleacrilic acid (IAA) (Figure 1a) can be used as an inhibitor of tryptophan biosynthesis. Addition of IAA (50 mgL ) to minimal media results in the complete inhibition of E. coli cell growth and the inhibition is reversed by L-Trp (50 mgL ) (Figure 1b). This observation is in agreement with the accumulation of indole observed in bacterial culture media Figure 1. a) Chemical structures of 5F-Trp and IAA. b) Inhibitory effect of IAA on E. coli cell growth.