4-fluorophenylglycine as a label for 19F NMR structure analysis of membrane-associated peptides

The non-natural amino acid 4-fluorophenylglycine (4F-Phg) was incorporated into several representative membrane-associated peptides for dual purpose. The (19)F-substituted ring is directly attached to the peptide backbone, so it not only provides a well-defined label for highly sensitive (19)F NMR studies but, in addition, the D and L enantiomers of the stiff side chain may serve as reporter groups on the transient peptide conformation during the biological function. Besides peptide synthesis, which is accompanied by racemisation of 4F-Phg, we also describe separation of the epimers by HPLC and removal of trifluoroacetic acid. As a first example, 18 different analogues of the fusogenic peptide "B18" were prepared and tested for induction of vesicle fusion; the results confirmed that hydrophobic sites tolerated 4F-Phg labelling. Similar fusion activities within each pair of epimers suggest that the peptide is less structured in the fusogenic transition state than in the helical ground state. In a second example, five doubly labelled analogues of the antimicrobial peptide gramicidin S were compared by using bacterial growth inhibition assays. This cyclic beta-sheet peptide could accommodate both L and D substituents on its hydrophobic face. As a third example, we tested six analogues of the antimicrobial peptide PGLa. The presence of d-4F-Phg reduced the biological activity of the peptide by interfering with its amphiphilic alpha-helical fold. Finally, to illustrate the numerous uses of l-4F-Phg in (19)F NMR spectroscopy, we characterised the interaction of labelled PGLa with uncharged and negatively charged membranes. Observing the signal of the free peptide in an aqueous suspension of unilamellar vesicles, we found a linear saturation behaviour that was dominated by electrostatic attraction of the cationic PGLa. Once the peptide is bound to the membrane, however, solid-state (19)F NMR spectroscopy of macroscopically oriented samples revealed that the charge density has virtually no further influence on the structure, alignment or mobility of the peptide.