Synthesis and In Vivo Incorporation of 4-Difluoromethyl-L-Phenylalanine Into Proteins

The difluoromethyl group exhibits unique chemical and nuclear magnetic resonance (NMR) spectroscopic properties. Here, the synthesis and characterization of 4-difluoromethyl-L-phenylalanine is reported. Methods have been developed to incorporate this versatile unnatural amino acid into full-length green fluorescent protein and carbonic anhydrase, using an orthogonal transfer RNA (tRNA)/synthetase pair with standard genetic code expansion techniques. Taking advantage of the characteristic ¹⁹F NMR behavior of the difluoromethyl group, the potential application of the difluoromethyl group as a probe in protein NMR studies was explored.