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 ¹⁹ F NMR behavior of the difluoromethyl group, the potential application of the difluoromethyl group as a probe in protein NMR studies was explored.