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. 2020 Sep;74(8-9):365-379.
doi: 10.1007/s10858-020-00331-z. Epub 2020 Jul 10.

The precious fluorine on the ring: fluorine NMR for biological systems

Andras Boeszoermenyi  1   2 , Barbara Ogórek  3 , Akshay Jain  4 , Haribabu Arthanari  4   5 , Gerhard Wagner  6
Affiliations

The precious fluorine on the ring: fluorine NMR for biological systems

Andras Boeszoermenyi et al. J Biomol NMR. 2020 Sep.

Abstract

The fluorine-19 nucleus was recognized early to harbor exceptional properties for NMR spectroscopy. With 100% natural abundance, a high gyromagnetic ratio (83% sensitivity compared to 1H), a chemical shift that is extremely sensitive to its surroundings and near total absence in biological systems, it was destined to become a favored NMR probe, decorating small and large molecules. However, after early excitement, where uptake of fluorinated aromatic amino acids was explored in a series of animal studies, 19F-NMR lost popularity, especially in large molecular weight systems, due to chemical shift anisotropy (CSA) induced line broadening at high magnetic fields. Recently, two orthogonal approaches, (i) CF3 labeling and (ii) aromatic 19F-13C labeling leveraging the TROSY (Transverse Relaxation Optimized Spectroscopy) effect have been successfully applied to study large biomolecular systems. In this perspective, we will discuss the fascinating early work with fluorinated aromatic amino acids, which reveals the enormous potential of these non-natural amino acids in biological NMR and the potential of 19F-NMR to characterize protein and nucleic acid structure, function and dynamics in the light of recent developments. Finally, we explore how fluorine NMR might be exploited to implement small molecule or fragment screens that resemble physiological conditions and discuss the opportunity to follow the fate of small molecules in living cells.

Keywords: 4-fluorophenylalanine; Drug discovery; Fluorine NMR; Nucleic acids; Proteins; TROSY.

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Conflict of interest statement

Conflicts of interest/Competing interests: The authors declare no conflicts or competing financial interests.

Figures

Fig. 1
Fig. 1
The 180-kDa α7 single-ring of the 20S proteasome CP from T. acidophilum interacts with the antimalaria drug chloroquine (CQ). An overlay of 19F-13C TROSYs of 1mM apo proteasome α7 single-ring in absence (blue) and in presence of 2 mM CQ is shown (brown). Both experiments were recorded at 45 °C
Fig. 2
Fig. 2
4-fluorophenylalanine replaces phenylalanine but not tyrosine. (a) 1D 19F NMR spectra of GB1 grown with 4-fluorophenylalanine as the source of phenylalanine (blue, sample-1) and tyrosine (red, sample-2), respectively. One of the two 4-fluorophenylalanine signals in the spectrum of sample-1 is split into two resonances of similar height, indicating slow exchange on the NMR timescale. The two resonances in the spectrum of sample-2 appear to result from 4-fluorophenylalanine incorporation into phenylalanine positions and not tyrosine. Peak heights are reported next to the corresponding peak positions. (b) The two phenylalanines in the GB1 sequence are highlighted in blue on a solution structure of GB1 (PDB ID: 2j52).
Fig. 3
Fig. 3
Illustration of fluorine systems NMR. (a) Schematic of complex formation between fluorine substituted small molecules, a 3-19F-13C-tyrosine labeled protein and a 5-19F-13C-uracil labeled RNA molecule. (b) Hypothetical 1D spectra of a pool of fluorine substituted small molecules are shown in absence (black) and in presence (green) of binding partners. Resonance frequencies of two molecules shift as a consequence of binding to RNA or protein. (c) Schematic of a 19F-13C TROSY of the 5-19F-13C labeled uracil. 2D resonance frequencies are represented as blue triangles and arrows with dotted lines connect ligand free RNA resonances to small molecule bound frequencies (green hexagons). A broader triangle represents a line broadened resonance frequency and is correlated to its invisible protein-bound counterpart (dotted lines) with a 2D 19F-13C CEST experiment. In this experiment, the large dip (inverted peak) corresponds to saturation at the resonance frequency of the ground (free) state and the smaller dip corresponds to saturation at the resonance frequency of the invisible (bound) state. (d) Schematic of a 19F-13C TROSY of the 3-19F-13C tyrosine labeled protein. Dotted lines with arrows connect the free 2D resonance frequencies (red squares) to the small molecule bound frequencies (green hexagons). A 2D 19F-13C CEST experiment correlates the line broadened free and invisible bound resonances (dotted lines) involved in RNA binding. Broad light red rectangles represent line broadened cross-peaks due to microsecond timescale conformational exchange. The kinetics of the conformational exchange and the resonance frequencies corresponding to the invisible state are detected with 2D 19F-13C RD experiments.
Fig. 4
Fig. 4
Fluorine NMR of atorvastatin and fluoxetine in DMEM. (a) Schematic representation of atorvastatin (Lipitor) and (b) 1D fluorine NMR spectra of atorvastatin in absence (blue) and in presence of 10% FBS (purple), respectively. (c) Schematic representation of fluoxetine (Prozac) and (d) 1D fluorine NMR spectra of fluoxetine in absence (red) and in presence of 10% FBS (orange), respectively.
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