Highly Fluorinated Peptide Probes with Enhanced In Vivo Stability for 19 F-MRI

Abstract

A labeling strategy for in vivo ¹⁹ F-MRI (magnetic resonance imaging) based on highly fluorinated, short hydrophilic peptide probes, is developed. As dual-purpose probes, they are functionalized further by a fluorophore and an alkyne moiety for bioconjugation. High fluorination is achieved by three perfluoro-tert-butyl groups, introduced into asparagine analogues by chemically stable amide bond linkages. d-amino acids and β-alanine in the sequences endow the peptide probes with low cytotoxicity and high serum stability. This design also yielded unstructured peptides, rendering all 27 ¹⁹ F substitutions chemically equivalent, giving rise to a single ¹⁹ F-NMR resonance with <10 Hz linewidth. The resulting performance in ¹⁹ F-MRI is demonstrated for six different peptide probes. Using fluorescence microscopy, these probes are found to exhibit high stability and long circulation times in living zebrafish embryos. Furthermore, the probes can be conjugated to bovine serum albumin with only amoderate increase in ¹⁹ F-NMR linewidth to ≈30 Hz. Overall, these peptide probes are hence suitable for in vivo ¹⁹ F-MRI applications.

Keywords: 19F-MRI; bioconjugation; fluorescence imaging; highly fluorinated peptides; perfluoro-tert-butyl amino acid; zebrafish embryos.