Fluoroaromatics as ¹⁹F NMR probes for chemical biology

Abstract

Fluorine is almost entirely absent from nature but due to the high environmental sensitivity of the nucleus, coupled with its similar atomic radius to hydrogen, fluorine can be incorporated into biomolecules as a ¹⁹F NMR probe to study proteins with no background signal. Fluorine NMR can be used to study peptide and protein conformational changes, which play a role in most biological functions, including potentially malign processes including protein aggregation that can lead to neurodegenerative diseases such as Parkinson’s disease (PD), the cause of which remains mostly elusive. This thesis studied the use of ¹⁹F NMR for conformational analysis of peptide models as well as recombinant proteins. This work explored the two most convenient methods for incorporation of fluorine into peptides and proteins, firstly Chapter 2 explored the use of fluorinated amino acids such as 4-fluorophenylalanine, and then Chapters 3 and 4 investigated post-translational indirect fluorination using small covalently-reactive ‘fluoro-tags’.

In Chapter 2, a simple conformational switching event was studied by examining proline cis-trans isomerism – often the overall rate-determining step in protein folding – in over 60 simple X-Pro-Z pentapeptide models using distal ¹⁹F NMR reporters. Here, the ratio of cis- and trans-conformers was quantified in a variety of biologically relevant conditions (aqueous buffer, variable pH) that would not be possible using ¹H NMR due to the inherent background water and peptide proton signals. This work also confirmed that a) ¹⁹F NMR measurements of %cisPro in these simple models mirrored the reported populations in similar literature models, b) that the nature of the amino acid on the N-terminal side of proline had a larger impact upon %cisPro than the C-terminal residue, c) that aromatic amino acids afforded the greatest cisPro populations, and that the Pro conformational populations as dictated by X-Pro-Z sequences were largely found to be translatable between different peptide sequences.

In Chapter 3, the indirect fluorination of proteins was explored using small, fluorinated electrophilic compounds, mostly commercially available, that yielded the identification of two novel fluoro-tags for protein labelling. These compounds were initially used in a version of the above X-Pro-Z model peptide system to modify cysteine residues to probe a) the sensitivity of the tag and its fluorine environment to proline conformation, and b) the effect it has on %cisPro. The fluoro-tags proved to be non-biased reporters of proline cis-trans status as they measured the ratio of the conformers, from a distal position, in accordance with values expected from the literature and Chapter 2 of this work. The compounds were also successfully and chemoselectively used to conjugate to a mutant of alpha-synuclein (A100C), the protein linked to PD, with evidence of the fluoro-tags showing sensitivity to aggregation of the protein in a ¹⁹F NMR study.

Following on from this, in Chapter 4, a series novel aryl-DABCOnium reagents, quaternary ammonium salts based on 1,4-diazabicyclo[2.2.2]octane, were developed as water-soluble, highly fluorinated tags to give improved sensitivity in protein-observed ¹⁹F NMR protein conformational studies, but also with potential applications of the warhead beyond the scope of this work, such as for cell-penetrating covalent drugs. One of these reagents was used to selectively modify cysteine mutants of a) the steroid carrier protein type 2 like (SCP-2L) domain of human multifunctional enzyme 2 (MFE-2) to explore protein-observed ligand binding, and b) the A100C mutant of alpha-synuclein to observe oligomerization, both by ¹⁹F NMR. This revealed that the fluoro-tag was sensitive to the structural perturbations of SCP-2L during ligand binding by ¹⁹F NMR, suggesting a potential application as a sensor of protein-ligand interactions, and the tag was also sensitive to the oligomerization of alpha-synuclein, a process not well understood, potentially identifying different types of oligomers.

Finally, in Chapter 5, a fluorine tag was developed that could undergo a double-tagging to cross-link a pair of cysteine residues artificially introduced into the α-helix of a bacterially derived peptide PSMα-3, to conformationally constrain the peptide and introduce a fluorine reporter. This ‘fluoro-stapling’ method enabled the ¹⁹F NMR ligand-observed study of the binding of the peptide to fibrils of alpha-synuclein, with the potential application for a quantitative detection method for these malign protein structures. Overall, this work illustrated the power of fluorine tagging to study biomolecules and their conformational behaviour in absence of any background signals.