Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

The Fluoro renaissance  A rebirth of techniques and applications for the genetic incorporation of fluorinated non-canonical amino acids (#153)

Edan I Habel 1 , Haocheng Qianzhu 1 , Elwy H Abdelkader 2 , Gottfried Otting 2 , Thomas Huber 1
  1. Research School of Chemistry, The Australian National University, Acton, ACT, Australia
  2. ARC Centre of Excellence for Innovations in Peptide & Protein Science, The Australian National University, Acton, ACT, Australia

Recent advances in the field of genetic incorporation of non-canonical amino acids has led to a rapid expansion of fluorinated groups that can be site-specifically incorporated into a protein. Consequently, full exploration into the physical properties of fluorine in a biomolecular context has only just begun.

Fluorine has very similar steric properties to hydrogen: it can only form a single bond, has a short bond length (C-F = 1.35 Å, C-H = 1.10 Å), and the second smallest van der Waals radius (rWF = 1.47 Å, rWH = 1.20 Å). Although the electrostatic properties of fluorine differ significantly from those of hydrogen, local fluorination of an amino acid typically has a negligible effect on the tertiary structure of proteins. Fluorine is naturally absent in biomolecules and therefore its analysis has no background signal.  This means fluorine atoms – introduced into the spatial context of a protein with precision – are excellent structural probes for the analysis of proteins using a smorgasbord of experimental techniques and offer new opportunities to design function in proteins. 

In this presentation we will focus on the applications of fluorinated aromatic amino acids and their site-specific incorporation into proteins. We introduce the unique absorbance properties of fluorinated indole sidechains, show how 4-, 5-, 6- and 7-fluorotryptophan can be incorporated site-specifically and in high yields, and demonstrate how fluorotryptophan can be used as antenna to excite rare-earth metal ions in lanthanide luminescence studies. In 19F NMR, 2D spectra provide a structural restraint that act as a facile experimental validation of computationally generated models.  We further look at the application of fluorinated phenylalanines; pentafluoro-phenylalanine has a reversed aromatic quadrupole observed in the canonical phenylalanine side chain. We demonstrate how we take advantage of this effect by engineering specific protein-protein interactions in vivo through the genetic encoding of phenylalanine and pentafluorophenylalanine.

  1. Orton, H. W.; Qianzhu, H.; Abdelkader, E. H.; Habel, E. I.; Tan, Y. J.; Frkic, R. L.; Jackson, C. J.; Huber, T.; Otting, G. Through-Space Scalar 19F-19F Couplings between Fluorinated Noncanonical Amino Acids for the Detection of Specific Contacts in Proteins. J. Am. Chem. Soc. 2021, 143, 19587– 19598, DOI: 10.1021/jacs.1c10104