Synthesis of fluorinated leucines, valines and alanines for use in protein NMR

Efficient syntheses of fluorinated leucines, valines and alanines are described. The synthetic routes provide expedient access to various ¹³C/¹⁵N/D isotopologues requiring solely readily available and inexpensive isotope containing reagents such as NaBD₄, carbon-¹³C dioxide and sodium azide-1-¹⁵N. The lightly fluorinated leucines and valines were found to be good substrates for cell-free protein expression and even 3-fluoroalanine, which is highly toxic to bacteria in vivo, could be incorporated into proteins this way. ¹⁹F-NMR spectra of the protein GB1 produced with these amino acids showed large chemical shift dispersions. Particularly high incorporation yields and clean ¹⁹F-NMR spectra were obtained for GB1 produced with valine residues, which had been synthesized with a single fluorine substituting a hydrogen stereospecifically in one of the methyl groups.