Protein-Observed Fluorine NMR: A Bioorthogonal Approach for Small Molecule Discovery

Abstract

The (19)F isotope is 100% naturally abundant and is the second most sensitive and stable NMR-active nucleus. Unlike the ubiquitous hydrogen atom, fluorine is nearly absent in biological systems, making it a unique bioorthogonal atom for probing molecular interactions in biology. Over 73 fluorinated proteins have been studied by (19)F NMR since the seminal studies of Hull and Sykes in 1974. With advances in cryoprobe production and fluorinated amino acid incorporation strategies, protein-based (19)F NMR offers opportunities to the medicinal chemist for characterizing and ultimately discovering new small molecule protein ligands. This review will highlight new advances using (19)F NMR for characterizing small molecule interactions with both small and large proteins as well as detailing NMR resonance assignment challenges and amino acid incorporation approaches.

Glycation of human serum albumin impairs binding to the glucagon-like peptide-1 analogue liraglutide

“…Results from this identification are shown in Tables 1-2. More modification sites have been reported for G100-HSA (21) and MGO-HSA (26) compared to native albumin G0-HSA (14). Only four additional modification sites were common between both in vitro glycated HSA (R98, R145, K262 and K525), eight sites were specifically modified on G100-HSA and eleven were only found for MGO-HSA.…”

“…19 F NMR is one of the best validated techniques for monitoring ligand-protein interactions (22)(23)(24)(25)(26)(27). This method has already been used to study the binding of various drugs to albumin, either using naturally fluorinated drugs or by competition with fluorinated spy molecules (24,(37)(38)(39)(40)(41)(42).…”

“…In this work, 19 F NMR transverse relaxation time experiments were used to investigate how albumin glycation influences its binding properties to liraglutide. Fluorine NMR is one of the best suited techniques for probing protein-ligand interactions (22)(23)(24)(25)(26)(27). This method requires either the use of a naturally fluorinated ligand or fluorine labelling of the protein (26), ligand or spy molecules (24).…”

“…Fluorine NMR is one of the best suited techniques for probing protein-ligand interactions (22)(23)(24)(25)(26)(27). This method requires either the use of a naturally fluorinated ligand or fluorine labelling of the protein (26), ligand or spy molecules (24). Here, we used a strategy where the peptidic liraglutide ligand was first labelled with a fluorine probe by trifluoroacetylation of the unique N-terminal free amine group using S-ETFA reagent (Fig.1b).…”

“…It differs from endogenous GLP-1 by a one amino acid substitution (Lys34Arg) and by the addition of a palmitic acid grafted to Lys 26 via a glutamate linker (Fig. 1a).…”