https://www.selleckchem.com/products/azd4547.html 19F NMR spectroscopy provides the ability to quantitatively analyze single species in complex solutions but is often limited by the modest sensitivity inherent to NMR. 4R- and 4S-Perfluoro-tert-buyl hydroxyproline contain 9 equivalent fluorines, in amino acids with strong conformational preferences. In order to test the ability to use these amino acids as sensitive probes of protein modifications, the perfluoro-tert-buyl hydroxyprolines were incorporated into substrate peptides of the protein kinases PKA and Akt. Peptides containing each diastereomeric proline were rapidly phosphorylated by each protein kinase and exhibited 19F chemical shift changes as a result of phosphorylation. The sensitivity of the perfluoro-tert-butyl group allowed quantitative analysis of the kinetics of phosphorylation over three half-lives at single-digit micromolar concentrations of each species. The distinct conformational preferences of these amino acids allowed the optimization of the substrate with a conformationally matched amino acid, in order to maximize the rate of phosphorylation. PKA preferred the 4R-amino acid at the -1 position, whereas the closely related AGC kinase Akt preferred the 4S-amino acid. These data, combined with analysis of structures of the Michaelis complexes of these kinases in the PDB, suggest that PKA recognizes the PPII conformation at the P-1 position relative to the phosphorylation site, while Akt/PKB recognizes an extended conformation at this position. These results suggest that conformational targeting may be employed to increase specificity in recognition by protein kinases. Perfluoro-tert-butyl hydroxyprolines were applied to the real-time detection and quantification of PKA activity and inhibition of PKA activity in HeLa cell extracts via 19F NMR spectroscopy. The coupling of proline ring pucker with main chain conformation suggests broad application of perfluoro-tert-butyl hydroxyprolines in molecula