Design of Highly Fluorinated Peptides for Cell-based ¹⁹F NMR

The design of imaging agents with high fluorine content is essential for overcoming the challenges associated with signal detection limits in ¹⁹F MRI-based molecular imaging. In addition to perfluorocarbon and fluorinated polymers, fluorinated peptides offer an additional strategy for creating sequence-defined ¹⁹F magnetic resonance imaging (MRI) imaging agents with a high fluorine signal. Our previously reported unstructured trifluoroacetyllysine-based peptides possessed good physiochemical properties and could be imaged at high magnetic field strength. However, the low detection limit motivated further improvements in the fluorine content of the peptides as well as removal of nonspecific cellular interactions. This research characterizes several new highly fluorinated synthetic peptides composed of highly fluorinated amino acids. ¹⁹F NMR analysis of peptides TB-1 and TB-9 led to highly overlapping, intense fluorine resonances and acceptable aqueous solubility. Flow cytometry analysis and fluorescence microscopy further showed nonspecific binding could be removed in the case of TB-9. As a preliminary experiment toward developing molecular imaging agents, a fluorinated EGFR-targeting peptide (KKKKK-A-YHWYGYTPENVI) and an EGFR-targeting protein complex E₁-DD bioconjugated to TB-9 were prepared. Both bioconjugates maintained good ¹⁹F NMR performance in aqueous solution. While the E₁-DD-based imaging agent will require further engineering, the success of cell-based ¹⁹F NMR of the EGFR-targeting peptide in A431 cells supports the potential use of fluorinated peptides for molecular imaging.