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

Abstract

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.