Here, we will focus on applications utilizing NMR-active unnatural amino acids (Fig. 1).

Subsequent technological advances have enabled the in vivo incorporation of many different unnatural amino acids and in a proof-of-concept study, ¹⁵N-labeled p-methoxy-phenylalanine (p-OMePhe, Fig. 1a) was incorporated at modest yields into myoglobin (Deiters et al.

This was confirmed for three different unnatural amino acids: p-trifluoromethoxy-phenylalanine (p-OCF₃Phe, Fig. 1a), ¹³C/¹⁵N-labeled p-OMePhe (Fig. 1a), and ¹⁵N-labeled o-nitrobenzyl-tyrosine (o-NBTyr, Fig. 1b) that were individually incorporated at 11 different sites around the active site using only 8–25 mg of the unnatural amino acid per 50 ml culture (Cellitti et al.

For example, ¹³C-labeled p-OMePhe (Fig. 1a) has recently been used to study local ligand-induced conformational heterogeneity in cytochrome P450 CYP119 (Lampe et al.

Two different fluorinated unnatural amino acids (Fig. 1a) have recently been introduced site-specifically into proteins and have been used to monitor ligand binding and structural transitions by ¹⁹F-NMR (Hammill et al.

To confirm this, the study was repeated o-nitrobenzyl-tyrosine (o-NBTyr, Fig. 1b) incorporated at the same site: The photocaged form of o-NBTyr also prevented binding of the tool compound binding.

In a first application, the system was used to evolve an aminoacyl-tRNA synthetase for photocaged lysine, o-nitrobenzyl-oxycarbonyl-N^ε-lysine (o-NBLys, Fig. 1b) (Chen et al.

2007) (Fig. 1b) are currently available for incorporation in yeast and mammalian cells..

Similarly, photocaged serine (DMNBSer, Fig. 1b) has recently been used in cell culture experiments to spatially and temporally control phosphorylation of a transcription factor (Lemke et al.

So far two metal chelating unnatural amino acids have been developed (Fig. 1c).