Chromatographic conditions for the separation of fluorinated amino acids and oligopeptides from their non-fluorinated counterparts were explored. The separation of six pairs of analytes, including both aromatic and aliphatic fluorocarbons, was investigated at various temperatures using both hydrocarbon and fluorocarbon columns and eluents. Our results show that when hydrocarbon eluents are used, fluorocarbon column provides better separation of fluorinated amino acids or oligopeptides from their non-fluorinated counterparts; when fluorocarbon eluents are used, hydrocarbon column provides better separation of fluorinated amino acids or oligopeptides from their non-fluorinated counterparts. These chromatographic behaviors reflect the fluorophilicity possessed by fluorinated amino acids and oligopeptides.

An efficient method for the enantioselective synthesis of (2R, 3S)-and (2S, 3R)-4,4,4-trifluoro-NFmoc-O-tert-butyl-threonine (tfT) on multi-gram scales was developed. Absolute configurations of the two stereoisomers were ascertained by X-ray crystallography. Racemization-free coupling conditions for the incorporation of tfT into oligopeptides were then explored. For solution-phase synthesis, tfT racemization was not an issue under conventional coupling conditions. For solid-phase synthesis, the following conditions were identified to achieve racemization-free synthesis: if tfT (3.0 eq.) was not the first amino acid to be linked to the resin (1.0 eq.), the condition is: 2.7 eq. DIC/3.0 eq. HOBt as the coupling reagent at 0 °C for 20 h; if tfT (3.0 eq.) was the first amino acid to be linked to the resin (1.0 eq.), then 1.0 eq. of CuCl 2 needs to be added to the coupling reagent.