19F NMR as a tool in chemical biology

We previously reviewed the use of 19F NMR in the broad field of chemical biology [Cobb, S. L.; Murphy, C. D. J. Fluorine Chem. 2009, 130, 132–140] and present here a summary of the literature from the last decade that has the technique as the central method of analysis. The topics covered include the synthesis of new fluorinated probes and their incorporation into macromolecules, the application of 19F NMR to monitor protein–protein interactions, protein–ligand interactions, physiologically relevant ions and in the structural analysis of proteins and nucleic acids. The continued relevance of the technique to investigate biosynthesis and biodegradation of fluorinated organic compounds is also described.


Introduction
Although fluorine is abundant in the environment, it is not a nutrient nor is it a feature of biochemistry for most species [1]. This reflects its low bioavailability, as it is typically found in insoluble minerals, and its physicochemical properties, in particular its high redox potential and the poor reactivity of the fluoride ion in aqueous solution. Despite its near-absence in biology, it is a particularly important element in the broader space of chemical biology [2]. Many commercial and industrial compounds are fluorinated, including refrigerants, degreasers, drugs, pesticides and anti-stick materials, and consequently there is a high degree of interaction between fluorinated compounds and nature [3]. Furthermore, owing to the lack of naturally-occurring fluorinated compounds, fluorine is a useful probe to investigate structure and mechanism of biological molecules. Central to these studies is fluorine-19 nuclear magnetic resonance spectroscopy ( 19 F NMR), which allows the user to readily visualize changes in chemical shift and splitting pattern depending on the changes of the (biological) environment, without substantial purification or processing of samples. In 2009 we published a review describing the application of 19 F NMR in chemical biology [4] and here we present the advances that have been made in this field since then.

Review
Structural analysis of macromolecules using 19

F NMR
In order to elucidate their various functions, it is essential to have a detailed understanding of both the structure of biological molecules and the way in which they interact in their envi- ronment and with one another. With regards to probing both the structure and the interactions between biomolecules in complex settings the analytical tool 19 F NMR has become invaluable. Some key highlights of how 19 F NMR has been employed in this area are given in the following section.
Recent advances in protein 19 F labelling 19 F NMR offers an attractive option for investigating the interactions between proteins and other biomolecules such as nucleic acids. Many of the advantages of 19 F NMR have already been discussed but it is worth highlighting that it is a particularly useful technique to study large proteins that cannot easily be probed by conventional NMR experiments. Given that fluorine atoms (e.g., 19 F labels) are not naturally present in proteins, a key element to establishing 19 F NMR in this area has been the development of methods that can be used to give access to 19 Flabelled proteins.
Methods for the introduction of unnatural amino acids into proteins have been reviewed extensively elsewhere [5][6][7] and here we will only discuss the most recent advances. The chemical structures of a selection of 19 F-labelled amino acid analogues that have been utilized in 19 F NMR studies in chemical biology are shown in Figure 1. In general, relatively small monofluorinated amino acids such as 1-4, can be biosynthetically incorporated directly into proteins by including the fluorinated amino acid in the growth medium of a suitable auxotrophic bacterium [8]. However, a challenge when producing 19 F-labelled proteins using this technique is that the natural amino acid translation process incorporates the specific fluorinated analogue in all the locations where the original amino acid was present, resulting in a "global" labelling of the protein. This can result in a variety of unwanted side effects, including, in some cases, structure disruption and possible 19 F NMR spectral overcrowding [9]. To achieve controlled site-specific amino acid incorporation the methodology pioneered by Schultz et al. [10] is often employed. Here orthogonal amber suppressor tRNA with a paired tRNA synthetase enables the introduction of a broader variety of 19 F-labelled acids at single site-specific positions. However, while these systems can be highly precise for certain proteins, the expression yields are strongly context dependent and can lead to poor levels of fluorinated amino acid incorporation [11,12]. To overcome this issue, Otting and co-workers have recently developed a synthetic strategy, based on a continuous exchange cell-free system (CECF), in which a key chitinbinding mutant of release factor RF1 can be removed under conditions that maintain the full activity of the S30 extract, thereby minimizing the incidence of premature translation termination and improving the expression yields by suppressing the production of truncated proteins [13]. This strategy has proven efficient for the site-specific incorporation of up to four trifluoromethylphenylalanines (5, Figure 1) within West Nile virus NS2B-NS3 protease (WNVpro) and for the incorporation of 5-fluorotryptophan (1) and 6-fluorotryptophan (2) in streptavidin [14].
Parallel to the development of these new strategies for recombinant fluorinated protein production, significant efforts have been made within the last decade to expand the pool of available amino acids that can be used as 19 F NMR reporters. Aromatic amino acids have been preferentially used as 19 F NMR probes both in binding and in conformational studies owing to their ready availability. However, more recently fluoro-aliphatic, and in particular perfluoro-aliphatic amino acids, have become increasingly prominent in 19 F NMR studies owing to their improved spectral properties. Among the first perfluorinated amino acids to be reported was perfluoro-tert-butylhomoserine (8), which was synthesized by Marsh and co-workers and incorporated into a variety of antimicrobial peptides, including MSI-78, for their NMR study on lipid bicelles ( Figure 2) [15]. In contrast to more traditional amino acids, such as 1-4, the perfluorinated tert-butyl moiety in 8 has nine equivalent fluorine atoms and no coupled hydrogens, so it gives rise to a characteristic high intensity 19 F NMR singlet. As shown by the authors, all peptides where 8 was introduced could be detected even at concentrations as low as 5 μM, demonstrating that pFtBSer is a highly sensitive tool to study binding events by way of 19 F NMR chemical shift and nuclear relaxation changes.  19 F NMR spectra showing the changes associated with MSIF9-1, MSIF9-6 and MSIF9-7 upon binding to bicelles. Figure 2 is adapted from [15] In addition to homoserine, perfluorinated analogues of both tyrosine [16] (pFtBTyr, 9) and proline [17] (4R and 4S-pFtBHyp, 10 and 11, Figure 1) have also been utilised as 19 F NMR probes, both showing promise as valuable tools for biomolecular studies. Particularly impressive is that by incorporating pFtBTyr (9) into a model peptide, Tressler and Zondlo were able to detect the presence of a sharp singlet in the 19 F NMR spectrum even at nanomolar peptide concentrations.
It is worth noting here that, as recently reviewed in detail by Koksch [18,19], while some perfluorinated amino acids such as hexafluoroleucine (12, Figure 1) and pentafluorophenylalanine (13, Figure 1) might exhibit, in general, low α-helix propensities, others such as 8 have been found to promote significant levels of α-helix formation [15]. This specific conformational propensity seen in 8 allowed, for example, for its non-disruptive incorporation into the α-helical motif of several estrogen receptor (ER) co-activator peptides and enabled the sensitive detection of their protein-peptide interaction inhibition by the ER antagonist tamoxifen [20]. Significantly, different secondary structure conformational preferences were also found among the diastereomers of perfluoro-tert-butyloxyproline, with the 4R analogue exhibiting a higher propensity for the polypro-  19 F chemical shift (Δδ) of various CF 3 tags as a function of solvent polarity. (c) Schematics showing the enzymatic isotope labelling of glutamine γ-carboxamide groups using transglutaminase (TGase) and 15 N-ammnonium chloride ( 15 NH 4 Cl) or 19 F-2,2,2-trifluoroethylamine hydrochloride (TFEA-HCl). (d) Example of a cyclic peptide hormone, oxytocin, and its perfluoropyridazine "tagged" analogue. Figure 3b was adapted from [28]; Figure 3d was reprinted from [30]. line helix structure than the 4S [17]. This differentiated conformational bias observed highlights the potential application of these amino acids as unique probes for molecular recognition studies.
The synthesis of unnatural amino acids carrying novel fluorinebased functionalities such as -SF 5 has also recently achieved considerable interest. For instance, the work carried out within the Welch group, who reported the synthesis and NMR conformational characterization of the first heptapeptide containing a pentafluorosulfanylated aliphatic amino acid, (E)-2-amino-5-(pentafluorosulfanyl)pent-4-enoic acid (14, Figure 1), SF 5 NVa [21]. Most recently, Cobb et al. [22] reported the synthesis of several pentafluorosulfanyl phenylalanine derivatives with suitable protecting groups to allow incorporation into peptides through common solid-phase peptide synthesis (SPPS) methods (15 and 16, Figure 1).
A second well-established methodology for the 19 F isotopic labelling of protein and peptides involves the post-translational chemical conjugation of an 19 F probe to specific amino acids present within the protein, typically cysteine and lysine residues. This approach is attractive as it can be directly applied to isotopically enrich unlabelled proteins, which may have been obtained previously from natural sources or as recombinant proteins under less demanding cell growth conditions. As shown in Figure 3, these fluorine tags consist, in general, of reactive trifluoromethyl derivatives such as 2,2,2-trifluoroethanethiol (TFET, 17) [23], 3-bromo-1,1,1-trifluoroacetone (BTFA, 18) [24] and fluorinated haloacetamides [25] that can react with nucleophilic side chains on the protein of interest. This approach has been extensively applied to study both soluble proteins [26] and membrane proteins [27]. Fluorine tags such at TFET (17) or BFTA (18) offer a good degree of sensitive and narrow 19 F NMR line widths due to rapid rotation about the trifluoromethyl symmetry axis. Thus, they are very effective tools when studying small conformational changes in proteins and other real-time events, even if relatively low concentrations are employed. In addition, these fluorine tags have proven to be sensitive to very subtle changes in their environment, providing further local-site specific conformational information [28]. Recent advances in this area include the tags based on fluorinated phenylacetamides, such as the 2-bromo-N-(4-(trifluoromethyl)phenyl)acetamide (BTFMA,19) and N-(4-bromo-3-(trifluoromethyl)phenyl)acetamide (3-BTFMA, 20) developed by Manglik et al. to study the structural dynamics of the cytoplasmic domain of the β2-adrenergic receptor (β2AR) [29]. In this work conjugation of the CF 3 group to an aromatic ring was shown to give rise to substantially improved 19 F NMR chemical shift sensitivities over the more traditional thiol-specific trifluoromethyl tags (Figure 3b) [28].
In addition to chemical modification, novel methodologies are being investigated that enable the enzymatic post-translational modification of non-nucleophilic residues, such as glutamate [31]. As recently demonstrated by Kojima and co-workers, recombinant protein transglutaminase (TGase) could be used to catalyse the chemical replacement of the γ-carboxyamide groups in Glu residues by free 2,2,2-trifluoroethylamine (TFEA-HCl) in model binding protein FKB12. This resulted in the site-specific incorporation of CF 3 CH 2 -motifs into these amino acids (Figure 3c). This 19 F-labelling strategy clearly offers new opportunities in the area as it can be easily combined with the aforementioned chemical approaches based on cysteine and lysine modification.
It is worth noting that for small proteins and peptides the chemical incorporation of the desired fluorinated amino acids using SPPS protocols still remains the method of choice, as it enables the site-specific introduction of the 19 F NMR probe. In addition, SPPS offers a powerful technique to access uniquely labelled peptides and proteins when combined with new strategies for peptide stapling and tagging using perfluoroaromatic reagents. Perfluorophenyl, perfluoropyridyl and perfluoropyridazinyl labelled peptides can now all be accessed, expanding even further the tool-box of 19 F NMR probes available for protein studies (Figure 3d) [30,32]. 19 F NMR is a valuable analytical tool to study the binding interactions between a variety of biological substrates and 19 Flabelled proteins. It has also become a widely adopted method for ligand screening, enabling the evaluation of small molecule-protein interactions over a wide range of affinities. In protein-observed fluorine NMR experiments (PrOF) binding events can be readily monitored using simple 1D techniques, and the dissociation binding constant of ligands (K d ) determined from the changes in the chemical shift of the labelled nuclei within the protein. Intuitively, the largest chemical shift perturbations are expected to be seen for residues in the closest proximity to the bound ligand. An additional advantage of this method is that signal acquisition in these experiments is typically rapid, as large signal-to-noise ratios are not needed to obtain precise indication of the positive presence of binding interactions [33].

Protein-observed binding interactions
Pomerantz and co-workers analysed the difference in NMR chemical shift perturbations for the 1 H-(NH) and 19 F nuclei in native tryptophan, and 5-FTrp-labelled bromodomain protein Brd4 against a panel of ligands ( Figure 4) [34]. 1 H NMR was found to be 6-20 times less responsive than fluorine with regards to chemical shift perturbations. Indeed, the high resolution observed in the fluorine spectra enabled the direct calculation of most of the ligand binding constants. The superior capacity of 19 F NMR to monitor binding events was also noted by Richards et al., when studying the interaction of human protein disulphide isomerase (hPDI) to Δ-somastatin [35]. In this work there was improved precision in the analysis of the dissociation constants due to the higher spectrum resolution and greater chemical environment sensitivity of the 19 F nuclei when compared to that offered by the 15 N.
Due to the fact that different regions of a protein give rise to unique NMR resonances, protein-observed experiments using multi-site labelled proteins can be used to provide information on changes in the chemical environment of the protein surface, which can be used to characterize binding-sites and/or conformational changes upon binding. For example, 19 F NMR studies on cysteine-labelled ß2-adrenergic receptor (β2AR) using either a 19 F-BTFA [29] or a 19 F-TFET [36] tag were used to explore the differential long range conformational effects induced by agonists, inverse agonists, and partial agonists on GPCR signalling. As exemplified by Liu et al. [36], in the case of multiply site-specific labelled 19 F-TFET-β2AR, changes in the NMR signals monitored within different helices of the protein revealed that agonist binding primarily shifted the equilibrium towards the G protein-specific active state of helix VI. In contrast, β-arrestin-biased ligands predominantly affected the conformation of specifically helix VII, providing thus insights into the structural plasticity of β2AR receptor.
By taking a novel approach to the use of PrOF NMR, Stadmiller et al. have recently developed a pioneering methodology based on 1D 19 F NMR line shape analysis that can be utilized to simultaneously determine both thermodynamic equilibrium binding constants (K) and kinetic rates of association (k on ) and dissociation (k off ) in protein-ligand binding events [37]. In order to achieve this, they analysed the binding of four different proline-rich peptides to a 5-FTrp-labelled Src homology 3 (SH3) recognition protein domain. By comparing the results obtained using both, the newly proposed 19 F NMR-based analysis and the traditional 2D 15 N, 1 H-HSQC experiments they could demonstrate that simple 19 F NMR line shape analysis achieved comparable high quantitative accuracy in the determination of all binding parameters. In addition, qualitative residue-specific information was also provided, but with a much improved and straightforward post-acquisition analysis procedure. It is clear that this more user-friendly methodology has the potential to have a significant impact in future studies of protein-ligand interactions, protein folding, and potentially small-molecule library screening, as it can broaden the accessibility of quantitative NMR spectroscopy to a wider range of laboratories.

Ligand-observed protein binding interactions
In addition to protein-observed fluorine (PrOF) NMR spectroscopy, ligand-observed fluorine NMR spectroscopy for drug screening has also evolved in the last decade to become a key tool to study the binding of drug candidates to target proteins. 19 F NMR-based screening, which was developed by Dalvit et al., includes fluorine chemical shift anisotropy and exchange for screening (FAXS) [38] which is a binding assay, and fluorine atoms for biochemical screening (n-FABS) methodology which is a functional assay [39,40]. Both of these methods have been successfully used for ligand-based screening, fragment-based functional screening and dynamic library screening, and have been recently reviewed in detail by Dalvit and Vulpetti [41,42]. The main advantages of these methods for lead compound screening is that they offer a platform not only for the rapid high-throughput screening of multiple protein small ligands, but also for the direct screening of functional inhibitors of much larger and complex biomolecules, such as enzymes.
In a recent study exemplifying this application, 19 F NMR has been employed to investigate the metabolism of carnitine. In animals, carnitine is biosynthesised from trimethyllysine in four enzyme-catalysed steps, which involves in the last step the action of the γ-butyrobetaine hydroxylase enzyme (hBBOX) (Figure 5a-c). As the fluorine shift of the metabolised product is distinctively different from the shift of the precursor, by producing a novel fluoromethyl analogue of the γ-butyrobetaine substrate (GBBNF) Rydzik et al. were able to monitor carnitine biosynthesis through hBBOX-catalysed GBBNF hydroxylation, both in vitro and in cell lysates [43]. Moreover, by using a competitive substrate for the enzyme, inhibition experiments could be directly employed to determine the IC 50 values in the basis of fluoride release, and the extent of GBBNF turnover investigated in the presence of cell extracts.
Ojima and collaborators have also employed functional ligandobserved NMR experiments to develop a range of novel 3′-difluorovinyltaxoids, such as BLT-F2 and BLT-S-F6 (Figure 5c), that are active against drug-sensitive and multidrug-resistant (MDR) human cancer cell lines [44]. Here, the incorporation of CF 3 and CF 3 O groups as NMR reporters into the tumour-targeting drug conjugates enabled the direct investigation of the mechanism of the pro-drug metabolic cleavage and the pharmacophore release by real-time 19 F NMR analysis. 19 F NMR was also employed as a convenient technique to study a range of factors that might influence the plasma and metabolic stability of the lead compounds, proving itself as a valuable alternative to more challenging conventional HPLC or 1 H NMR analyses.
It is also worth noting that recent investigations within the Dalvit group have further expanded the applications of n-FABS into the screening and direct IC 50 measurement of bioactive substrates within intact living cells [45]. By studying mammalian cells expressing the membrane protein fatty acid amide hydrolase (FAAH), they showed that it is possible to screen and identify target inhibitors in situ using 19 F NMR spectroscopy ( Figure 6). For this, the activity of the membrane-bound FAAH enzyme was evaluated by monitoring the hydrolysis of a fluorinated anandamide analogue ARN1203, a previously reported FAAH substrate, to arachidonic acid and 1-amino-3-fluoropropanol in the presence and absence of a broad range of known FAAH inhibitors with widely different potencies. The results clearly showed that the proposed n-FABS method was successful in detecting strong inhibitors of FAAH activity in cells. Also, it allowed the accurate quantification of the corresponding IC 50 values, all of which were consistent with those obtained in previous work but measured in cell membrane extracts [46].

Structural analysis of macromolecules using 19 F NMR
The three-dimensional (3D) structure of biomacromolecules and their conformational dynamics are often the starting point for the development of an in-depth understanding of their function and mode of action. Owing to the large size of biomacromolecules, the high complexity of the processes in which they participate and the natural environment in which they occur, an integrated analytical approach is often required to succeed in revealing their functional architecture. Such an approach typically includes the use of a variety of high-resolution proteomic tools, cryo-electron microscopy and X-ray crystallography to achieve full molecular characterization at the atomic level. However, macromolecules such as DNA/RNA and proteins are not static entities, for which also complementary dynamic and  19 F NMR spectra of ARN1203 incubated for 2.5 h in hFAAHeHEK293 intact cells in the presence and absence of an hFAAH activity inhibitor. Figure 6b was reproduced and adapted from [45].
targeted local site-specific conformational information is needed to fulfil our understanding on how these assemblies perform their biological role and function in vivo. It is in this regard that 19 F NMR has proven to be a valuable analytical tool, providing simultaneous access to both structural and dynamic information that would be difficult to obtain using traditional 13

Protein folding
From the late 1960s and early 1970s 19 F NMR has firmly established itself as a highly versatile and powerful analytical tool for studying both protein structure and conformational changes within proteins [47]. For additional information on the development of the field of 19 F NMR protein studies readers are encouraged to consult the excellent earlier reviews written by Gerig [48] and Danielson and Falke [49].
However, over the last 20 years the application of 19 F NMR in this area has shifted from the mere study of proteins and peptides as static entities, to the detailed analysis of the much more sophisticated protein transient conformational states that are produced upon substrate interaction, folding or binding. Thus, it is possible to accurately track the complete chain of events that take place within the molecule upon interaction and/ or folding. Given that the intermediates involved in protein folding and binding are difficult to observe owing to the shortlived nature of the states and their marginal concentration, high sensitivity 19 F NMR has been employed to offer unique opportunities for their sampling and characterization in situ [50].
Prosser et al. demonstrated the utility of this approach when employing a combination of variable temperature (VT) 19 F NMR analysis and CD spectroscopy to study the nearnative thermal folding intermediate structure of calmodulin (CAM) (Figure 7) [51]. To this end, CAM was biosynthetically enriched with 3-fluorophenylalanine (3-FPhe) and the 19 F NMR signals corresponding to each one of the eight 3-FPhe residues present resolved and assigned in the spectra for the native state. Changes in the 19 F NMR signals produced upon increasing the temperature were then used to map the evolution of the protein along the heat-induced denaturalization curve and to confirm the presence of an intermediate transient structure at 67 °C. Notably, single Lorentzian lines were observed for all 3F-Phe labelled sites regardless of the temperature employed, signifying either a single state at low temperatures or fast exchange between the native and near-native states at higher temperatures (40-70 °C). The NMR data obtained also provided evidence of the rapid ring flipping of the 3-FPhe probes, inherent to the side-chain dynamics within the protein hydrophobic interior [52][53][54]. 19 F NMR has also been used to characterize in more detail the topography of the CAM conformational changes by using molecular contrast agents. 19 F NMR solvent-induced isotope shift experiments, resulting from replacement of H₂O with D₂O, provide a robust measure of the relative degree that a specific labelled position is exposed to the solvent [55]. Complementary to this technique, the use of dissolved O 2 as a paramagnetic agent, having a relatively short electronic relaxation time, gives rise to paramagnetic shifts and spin-lattice relaxation rate enhancements that can be directly correlated with its accessibility to the 19 F-spin of interest [55,56]. This way, the combined ratio derived from both experiments was employed as a convenient measure of the solvent exposure and local hydrophobicity changes experienced at each individual fluorinated residue upon heating. Ultimately, 19 F NMR enables profiling of Figure 7: (a) X-ray crystal structure of CAM highlighting the location the phenylalanine residues replaced by 3-FPhe and the corresponding deconvoluted 19 F NMR spectrum of the fractionally labelled peptide (PDB file 3CLN). (b) 19 F NMR spectra of 70% 3-FPhe fractionally labelled CAM as a function of temperature. Adapted with permission from [51]. © 2013 American Chemical Society.
the specific position and nature of the conformational perturbations that the protein undergoes as it progresses from its native state along the heat-denaturation pathway [51,57].
In a much broader context, 19 F NMR paramagnetic relaxation enhancement experiments (PREs), have been shown to constitute a versatile tool for extracting quantitative structural distance information in selectively 19 F-labelled proteins. As a proof of concept, Matei et al. [58] demonstrated the applicability of this approach in studying the HIV-inactivating lectin cyanovirin-N protein as a model system ( S52 CCV-N). Single fluorine atoms were introduced at the 4-, 5-, 6-or 7-positions of Trp49 and the 4-position of Phe4, Phe54, and Phe80 ( Figure 8). Simultaneously, the paramagnetic nitroxide spin label was chemically attached to the protein by using two available Cys residues at positions 50 or 52. As the presence of an unpaired electron in the tag increases the nuclear relaxation rates of the residues that are in spatial proximity, it resulted in their spectral signal attenuation. Due to this effect being inversely correlated with the distance between the probes, the transverse 19 F-PRE rate ( 19 F-Γ2) was directly employed for the determination of the average distance between the 19 F nucleus and the paramagnetic centres, within a range of 12-24 Å. Overall, the work showed the potential applicability of 19 F NMR PREs as excellent alternative parameters for the quantitative analysis of site-specific intramolecular distances, with a more particular significant value in the exploration of large proteins and macromolecular complexes where substrate spectral crowding and/or background incompatibility prevents the use of other nuclei such as 1 H or 15 N PREs. 19 F NMR has also been applied to study the conformational heterogeneity and dynamics of a broad range of proteins and peptides upon their interaction with model lipid vesicles [59], micelles [60] and bicelles [61]. It has enabled the quantification and mechanistic characterization of intermolecular dynamic processes such as protein dimerization, oligomerization and fibrillation, even within large and especially complex systems [62,63].
In a recent study, Aramini et al. employed 19 F NMR to investigate the conformational dynamics within the interacting inter-  could be directly employed as a reliable reporter to monitor the monomer/dimer exchange dynamics. Moreover, it could also be employed to characterize the ED helix-helix dimer interface in more detail by means of additional 1D 19 F T1, T2, and CPMG relaxation dispersion experiments and solvent-induced isotope shift effects. This study also highlighted the particular advantages of employing 19 F NMR fluorine probes strategically labelled in the second ring of the Trp indole moiety to directly measure conformational exchange phenomena at protein-protein interfaces mediated by such residues [65].
Correct folding is essential for normal biological function of proteins [66]. The formation and accumulation of misfolded proteins has been shown to be a common pathological feature in a significant number of human disorders, including neurodegenerative Alzheimer's and Parkinson's diseases and several metabolic type II diabetes conditions. So far, the application of realtime NMR to characterize amyloid formation has been very limited [67,68], largely due to the spectral overcrowding of the 1D 1 H NMR spectra and the difficulties associated with performing multidimensional experiments at a fast enough rate to follow the aggregation process accurately. Despite these aforementioned challenges the Marsh laboratory has recently demonstrated the utility of 19 F NMR to access direct, sensitive and real-time observation of amyloid fibril formation, enabling the elucidation of the underlying mechanics of peptide fibrillation.
To study the aggregation mechanism of the model Aβ-peptide, a well-known amyloidogenic peptide related to Alzheimer's disease, Marsh and collaborators synthesized a 19 F-labelled Aβ1−40 peptide version in which methionine at position 35 was replaced by tfmMet (Aβ1−40-tfmMet35) [9,62]. The aggregation process was then monitored by 19 F NMR over a period of several weeks [62]. Upon analysis, formation of at least six spectroscopically distinguishable intermediates could be detected during fiber formation ( Figure 10). Each independent intermediate was characterized on the basis of its 19 F NMR chemical shift and the kinetics by which each species formed or decayed were evaluated in real-time. By combining the 19 F NMR data with that provided by complementary analytical techniques, mass spectrometry (electrospray ionization and ion mobility), CD spectroscopy and atomic force microscopy, the authors were able to obtain detailed information about the size and secondary structure associated with some of the soluble intermediates. These included: large β-sheet oligomers formed immediately after solubilization (oligomer O1); a small oligomer that forms transiently during the early stages of the lag phase (oligomer O2) and 4 spectroscopically distinct forms of oligomers that appear during the later stages of aggregation and apparently coexist with the proto-fibrillar species (oligomers O3-O6, Figure 10).
It is worth noting that the ability to follow the formation and decay of multiple intermediate species using a single 19 F NMR 1D experiment provides a straightforward route for the comparative analysis of different complex aggregation behaviours. For example, another study by the Marsh group carried out using amyloidogenic tfmPhe-labelled islet amyloid polypeptide hormone (tfmF-IAPP), showed that fibril formation can indeed proceed through well differentiated and distinguishable alternative pathways [69]. In this case, the consumption of 19 F-labelled monomeric tfmF-IAPP was monitored in real-time by 19 F NMR but significantly no new 19 F resonances were produced during the time course of aggregation. This suggests that, unlike what is observed in the case of amyloidogenic Aβ-peptide, no soluble intermediates accumulate in the aggregation pathway of IAPP.
Conformational polymorphism is also particularly important for the infectious amyloid particles known as prions [70] and this area has been recently investigated using a range of 19 F NMR approaches [63]. By selectively introducing 3-FPhe labels into the human prion protein segment PrP(90−231) β via induced auxotrophy [71], Prosser and co-workers have been able to characterize and quantify the relative populations of monomeric, octameric, and higher oligomer species that populate the pathway from the native PrP state to fibril formation. The equilibrium constants between states were also analysed as a function of both temperature and pressure, allowing to determine the enthalpic and entropic contributions to their transitions. In addition, 19 F NMR saturation transfer experiments allowed a convenient estimate of the kinetic rates at which the various species interconvert. This study by Prosser et al. nicely illustrates how quantitative and detailed thermodynamic and kinetic information for complex systems can be obtained by 19 F NMR.
Another rapidly expanding and particularly challenging area of research where 19 F NMR has found new applications is in the field of intrinsically disordered proteins (IDPs). IDPs play a key role in cell signalling and regulation processes and represent a distinct class of proteins that exhibit no stable 3D structure [72]. It is broadly understood that, unlike folded proteins, IDPs exist as an average combination of interconverting conformers. However, when bound to a substrate, IDPs may acquire a defined secondary structure [73]. The innate flexibility of IDPs imposes tremendous technical challenges to standard NMR analysis, as pronounced conformational averaging gives rise to narrow signal dispersion and low signal-to-noise ratios. Indeed, unless more sophisticated NMR techniques are employed, the combination of these two effects prevents the characterization of low-populated and short-lived states that might be critical for the peptide biological function [74].
The Pielak group carried out an early study on the conformational dynamics of the model IDP, α-synuclein (αSyn) using 19 F NMR. In this work 19 F NMR was employed to study several properties of a specifically 3-FTyr-labelled α-synuclein analogue, including its native conformation and the conformational changes induced by urea, spermine and sodium dodecyl sulfate (SDS) [75]. Subsequently, α-synuclein interaction with SDS micelles and model membranes have also been investigated in detail [59,76], as well as the kinetics of α-Syn oligomerization and fibril formation both in vitro [60,75] and in vivo [77,78]. In these more recent studies, incorporation of 4-tfmF residues using amber-suppressing codons at various positions was shown to be more advantageous as a 19 F reporting strategy, owing to the higher sensitivity and improved NMR relaxation properties of the CF 3 -group. The results from these studies, summarized in Figure 11, show that while α-Syn remains disordered in solution, it acquires partial helical secondary structure in the presence of SDS and membrane-like environments. Specifically, the N-terminal region of the peptide, which includes the first 4-tfmF-labelled amino acid, was shown to be involved in the most important membrane-binding interactions and conformational changes observed, allowing the protein to adopt a metastable aggregation-prone state that is apt to stabilize further intermolecular interactions and progress to the fibril state. The C-terminus non-amyloid component region, as monitored at position 133, was in comparison found to keep largely dynamically disordered in all species formed. Moreover, results from these studies revealed that the fibril-forming path of α-Syn might occur without accumulation of soluble low molecular weight intermediates, as no new 19 F NMR signals were observed during the time-course of the aggregation process. 19 F NMR spectroscopy has also now been employed to analyse the conformational dynamics of IDPs upon tertiary and quaternary complex formation [79]. The Myc-Max heterodimer is a well-known oncogenic transcription factor complex able to bind to enhancer box (E-box) regions (5'-CACGTG-3') of DNA with low-nanomolar affinity, what triggers its biological function as a transcriptional regulator [80]. Previous studies have shown that in the monomeric state the two helical domains of Myc display unstable structural features, similar to those of small IDPs, existing rather as a highly flexible pair of transient α-helices [81]. Based on this, Konrat and co-workers explored a combination of 19 F tagging and PRE NMR spectroscopy to probe and trace the conformational changes experienced by disordered Myc upon Myc-Max heterodimerization. Going beyond, they also employed 19 F NMR to interrogate the changes induced in the Myc-Max heterodimer structure upon full quaternary complex formation in the presence of DNA, and another intrinsically disordered binding partner, breast cancer antigen 1 (BRCA1) (Figure 12). In this work the strategy employed involved the introduction of a perfluorinated [ 19 F]3,5-bis(trifluoromethyl)benzyl-based tag into the single cysteine residue of Myc. This modification boosted the overall spectral sensitivity even when minimal protein concentrations were employed. The structural dynamics of the Myc-Max dimer formation were then evaluated by using intermolecular PREs between 19 F-Myc and three differently paramagnetic spin labelled MTSL ([(1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3methyl) methanethiosulfonate]) tagged Max mutants, and novel insights revealed regarding the differential structural dynamics of Myc-Max bound to DNA and the tumour suppressor BRCA1. Given its ease of implementation, future applications of this strategy to new structural biology targets and inhibitor screening can be expected. In the field of 19 F NMR protein conformational studies another area that has seen increasing attention is that of membrane proteins (MPs). Despite their physiological importance, analysis of the structural biology of MPs is significantly limited due to difficulties associated with protein expression, purification, stability, solubility and structural heterogeneity [82][83][84]. In particular, the application of solution NMR techniques to the study of MPs has proven exceptionally challenging due to the slower tumbling rates that MPs experience within membrane mimetics environments, which results in rapidly decaying signals and generally broad linewidths [85,86]. However, technical improvements, such as cryogenically-cooled probe-heads, along with the development of new high sensitivity 19 F NMR probes and novel strategies to produce recombinant proteins with optimal isotope labelling have greatly opened up this field of study [87]. For example, by using highly sensitive 2,2,2-trifluoroethanethiol (TFET) 19 F probes, Bondarenko et al. have developed a novel TFET/MTSL ((1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate) orthogonal labelling scheme that enables the accurate determination of inter-subunit distances in pentameric ligand-gated ion channels (pLGICs) by means of solution 19 F PRE NMR experiments in micelles ( Figure 13) [88]. To ensure a uniform 19 F PRE signal from the adjacent paramagnetic labels, the 19 F NMR TFET probe was tagged to a selected cysteine residue in the channel protein, L253C, and the paramagnetic probe, MTSL, was then used to label the rest of available equivalent cysteine sites in a molar ratio of 1 TFET:4 MTSL. The experimental distances, that were calculated on the basis of the corresponding 19 F NMR signal decay profiles, were in good agreement to those predicted for modelled MTSL-TFET pairs in adjacent subunits in the X-ray structure, showing only small discrepancies. Overall, the results of this work clearly demonstrated the value of solution 19 F NMR for quaternary structure determination and as an alternative approach for generating distance restraints for ion channels and other protein complexes that would be difficult to be defined by using other analytical tools.
DNA and RNA secondary and tertiary structure 19 F NMR spectroscopy also represents a useful analytical approach to study the structure, function and molecular dynamics of nucleic acids [89]. Following early advances by Micura and co-workers in the late 2000s that investigated the suitability of 19 F NMR for the conformational analysis of single and double helix RNA strands [90][91][92][93] 19 F NMR has found a particularly rich niche of applications in the conformational analysis of higher-ordered DNA and RNA G-quadruplex structures. G-quadruplexes are four-stranded nucleic acid secondary struc- Figure 14: (a) General structure of a selection of recently developed 19 F-labelled nucleotides for their use as 19 F NMR reporters. (b) Concept for the detection different RNA structures by using 19 F labels and its application to the study of telomer RNA structure at different concentrations and temperatures. Red and green spots indicate formation of dimer and two-subunits stacked G-quadruplex. The peaks of single strand RNA are marked with black spots. Temperatures are indicated on the right. Figure 14 is adapted from [102].
tures formed in specific guanine-rich sequences showing, in general, highly polymorphic structures and various folding topologies. These structures have been suggested to play an important role in key biological processes such as gene expression and regulation [94,95], telomere length maintenance [96][97][98], transcription and DNA replication [99,100]. Investigation of the specific G-quadruplex structures associated with these biological events is therefore essential to understand their functions. However, because the formation of a stabilized G-quadruplex causes an overall reduction in the tumbling rates of the molecule, and thus a great decay of NMR signal sensitivity, analysis of these supramolecular structures by common NMR techniques has proved challenging.
To overcome the aforementioned issues, Virta and co-workers have explored the application of trifluoromethyl analogues of guanosine, cytidine and uridine based in 2 ' -O-[(4-trifluoromethyltriazol-1-yl)methyl] reporter groups as 19 F NMR probes for the detection of RNA secondary structures ( Figure 14). As shown by Granqvist et al. [101], the 19 F NMR signals observed when employing these probes to evaluate the thermal denaturation of a range of RNA hairpins were indeed found to be sensitive enough to allow the monitoring of their secondary structural changes with relatively wide shift dispersion. It also enabled to characterize by 19 F NMR spectroscopic methods an RNA triple helix for the first time.
Following on from this a new range of even more sensitive 19 F NMR probes, based in the use of 3,5-bis(trifluoromethyl)benzene moieties have recently been reported (Figure 14a) [103]. Here, the presence of six equivalent 19 F atoms within the probe results key in providing a much more superior spectral enhancement, proving successful in enabling to monitor the conformational changes experienced within the DNA/RNA strains upon G-quadruplex formation both in vitro and in vivo. In addition, these novel probes have  Figure 15 was reprinted from [106]. the extra advantage that can be easily incorporated either as internally fluorinated nucleobases or as external 19 F-labelled terminal tags in longer oligonucleotides (Figure 14b). As a proof of concept, Bao et al. demonstrated the utility of these tags for the direct observation and quantitative thermodynamic characterization of dimeric and two-subunits stacked telomeric RNA and DNA G-quadruplexes within in living cells [99,102,104]. Moreover, by using these new reporters, 19 F NMR analysis has also been applied as an efficient strategy to probe and characterize the binding interactions of fluorolabelled RNA-and DNA-based G-quadruplex complexes with different ligand molecules. This was exemplified by the Xu group who investigated the interaction of a RNA G-quadruplex and the telomeric protein TRF2 [103] and the interaction of the DNA thrombin binding aptamer (TBA) G-quadruplex with thrombin [100]. Overall, these examples demonstrate that 19 F NMR offers a suitable and non-perturbing approach by which to access detailed structural information of complex DNA and RNA folding topologies and sophisticated supramolecular assemblies.

Metabolism studies Biotransformation of fluorinated xenobiotics
Fluorine is present in a large number of anthropogenic compounds, in particular pharmaceuticals, agrochemicals and antistain/anti-stick compounds (per-and poly-fluorinated alkyl substances, PFAS). When these compounds come into contact with microorganisms, there is a high likelihood that they will be biotransformed to some degree. In our previous review [4], we highlighted 19 F NMR's usefulness in following the biodegradation of compounds such as fluorophenols and fluorobenzoates. The technique has since been applied to monitor the biotransformation/biodegradation of fluorinated drugs such as flurbiprofen [105], and the pesticide cyhalothrin [106] by the fungus Cunninghamella elegans. In the former, 19 F NMR demonstrated the appearance of phase 1 (oxidative) and phase 2 (conjugative) metabolites, and in the latter, it was possible to monitor the migration of the pesticide into the biomass in the first 24 h after its introduction before being biotransformed to new trifluoromethyl-containing metabolites ( Figure 15). 19 F NMR was also employed to determine the degree of Figure 16: Following the biodegradation of emerging fluorinated pollutants by 19 F NMR. The spectra are from culture supernatants of different bacteria incubated with SF 5 -aminophenol. Figure 16 was reprinted from [110].
biotransformation of drug-like fluorophenylpyridine carboxylic acids in the same fungus [107]. However, aside from these few examples, the technique is not widely used by those investigating the biodegradation of fluorinated xenobiotics, with researchers more commonly relying on liquid or gas chromatography-mass spectrometry methods. Objectively, these techniques are much more sensitive than 19 F NMR, provide more structural information and are probably more accessible; however, it is possible that important biotransformation products may not to be detected using these techniques. For example, the fluorometabolites detected upon fungal degradation of cyhalothrin were not observed by GC-MS of culture supernatants.
When the nature of the degradation products is unknown, 19 F NMR is particularly useful, since even minor changes to a substrate's structure can lead to a chemical shift change. Notably, compounds with new fluorinated groups, for which there is no precedent in the literature, pose challenges for analy-sis of biotransformation products. For instance, the pentafluorosulfanyl (-SF 5 ) group is proposed as a replacement for trifluoromethyl (-CF 3 ) and has been incorporated into numerous biologically active compounds already [108,109]. Intuitively, investigating the biodegradation of these compounds in the absence of any reference compounds is complicated. Saccomanno et al. [110] investigated the bacterial degradation of pentafluorosulfanyl (SF 5 )-substituted aminophenols and via 19 F NMR analysis of culture extracts was able to determine the production of new fluorinated metabolites ( Figure 16). Subsequent analysis by GC-MS was required to further characterize the products, but only one could be detected (SF 5 -catechol) despite the 19 F NMR analysis showing the presence of multiple fluorometabolites.

Detection and biosynthesis of natural organofluorine compounds
As naturally-occurring organofluorine compounds are so rare, it is possible to easily detect them in a crude, complex mixture, such as a culture medium, using 19 F NMR. The earliest exam-  Figure 17 was reprinted from [113].
ple of the technique being applied in this was in the discovery of fluoroacetate and 4-fluorothreonine in cultures of the bacterium Streptomyces cattleya [111]. 19 F NMR became a cornerstone for investigating the biosynthesis of these fluorometabolites, and was instrumental in the discovery of the very first fluorinase, which catalyses the production of 5'-fluoro-5'deoxyadenosine (5-FDA) from fluoride ion and S-adenosylmethionine (SAM) [1].
While cumbersome as a tool for screening for new fluorometabolites in multiple strains, 19 F NMR continues to be successfully applied to the discovery of such compounds, typically after genome sequencing and scanning for genes with homology to those initially discovered in S. cattleya. For example, Huang et al. [112] analysed the genome sequence of the bacterium S. xinghaiensis NRRL-B24674, which revealed the presence of a fluorinase gene, suggesting that the microorganism could produce fluorinated compounds from fluoride ion. This was confirmed by 19 F NMR of culture supernatant after the bacterium was grown in medium containing 2 mM fluoride ion. Furthermore, because of the large shift changes due to minor structural differences, the observation of new fluorinated compounds in crude culture supernatants is possible. 19 F NMR analysis of cultures of the bacterium Streptomyces sp. MA37, which was isolated from a Ghanian soil sample, revealed several new resonances in addition to those known for fluoroacetate and 4-fluorothreonine ( Figure 17) [113]. (2R,3S,4S)-5-Fluoro-2,3,4trihydroxypentanoic acid and 5-fluoro-5-deoxy-ᴅ-ribulose were confirmed as two of the new metabolites [114]. Most recently 19 F NMR was the key analytical technique to assess in vivo production of 5'-FDA in an engineered E. coli, which, in addition to expressing the fluorinase, had its fluoride efflux protein, CrcB, deleted and a gene coding for a SAM transport protein heterologously expressed, to ensure an adequate supply of substrates [115].
Nucleocidin was famously isolated from S. calvus in the 1950s as it had significant anti-trypanosomal properties [116]; however, the presence of fluorine in its structure was not discovered until a decade later [117], by which time the original strains had lost their biosynthetic capability, frustrating attempts to investigate its biosynthesis. It took over 50 years to determine the reason for the loss of nucleocidin production, which was due to the presence of a point mutation in the bldA gene [118] that codes for a rare Leu-tRNA (UUA). Upon complementation with a functional gene, S. calvus produced nucleocidin once again, which was detected by 19 F NMR. Subsequent biosynthetic investigations, again relying on 19 F NMR, have revealed that the elaboration of this unusual fluorometabolite involves the production of glucosylated precursors [119], which are detectable in the culture supernatant ( Figure 18). These are possibly inactive forms of nucleocidin, generated in the first instance so that the producing organism is protected from its deleterious effects, and can be activated outside the cell by glucosidases.

Fluorinated natural products via precursor-directed biosynthesis
The modification of known antibiotics and other natural products is an important tactic in the battle against antimicrobial resistance. In addition to fluorinated metabolites that are produced de novo from fluoride ions, it is possible to modify other secondary metabolites to incorporate fluorine by including fluorinated precursors in the culture medium. 19 F NMR has been applied to the detection of the new compounds in the complex supernatants of organisms producing non-ribosomal peptides and polyketides. It has been especially useful for monitoring the Figure 18: Application of 19 F NMR to investigate the biosynthesis of nucleocidin. The spectra are from culture supernatants of S. calvus recorded at different times during growth, showing the production of two glycosylated metabolites (I and II) that precede nucleocidin appearance in the culture. Figure 18 was reprinted from [119].
incorporation of fluorinated amino acids into lipopeptides produced by Bacillus sp. CS93. This bacterial strain was originally isolated from the Mayan fermented food Pozol and was found to produce the dipeptide antibiotic bacilysin and the lipopeptides iturin A, surfactin and fengycin [120]. Precursor-directed biosynthesis experiments with various fluorinated amino acids were conducted and it was observed that in iturin A and fengycin, 3-fluoro-ʟ-tyrosine could replace tyrosine in the peptide chain [121][122][123]. 19 F NMR analysis of culture supernatants revealed that when this fluoro-amino acid was incubated with CS93, three different fluorofengycin species were biosynthesised ( Figure 19), with fluorotyrosine replacing either or both tyrosine residues in the peptide ring.
Although not strictly precursor-directed biosynthesis, Piasecki and Keatinge-Clay [124] employed 19 F NMR to monitor biocatalytic reactions of trifluorinated surrogate substrates of different polyketide synthase activities in cell lysates of recombinant E. coli. Substrate and lysate were incubated in an NMR tube to enable continuous monitoring of reactions catalysed by erythromycin thioesterase (trifluoropropionyl-SNAC substrate) and various ketoreductases (3-oxo-5,5,5-and 2-methyl-3-oxotrifluoromethylpentanoyl-SNAC substrates). 19 F NMR successfully resolved the diastereomers produced by the different ketoreductases assayed.

Measuring gene expression
The lacZ gene, which is part of the lac operon in bacteria such as E. coli, codes for β-galactosidase and is used extensively as a reporter of gene expression. The measurement of β-galactosidase activity is possible using a range of techniques, including 19 F NMR. Yu et al. [125] developed a bimodal fluorinated probe, 1-O-(β-ᴅ-galactopyranosyl)-3-fluorocatechol, to measure the enzyme's activity in transfected cancer cells. The principle relies on the excess Fe 3+ ions characteristically present in tumour cells, which are scavenged by the catechol after it is re- Figure 19: Detection of new fluorofengycins (indicated by arrows) in culture supernatants of Bacillus sp. CS93 incubated with 3-fluoro-ʟ-tyrosine with 19 F NMR. Figure 19 was reprinted from [121].
leased from the sugar via the action of β-galactosidase. The resulting complex has a different chemical shift to the original compound in the 19 F NMR spectrum ( Figure 20). The change could also be measured by 1 H MRI (magnetic resonance imaging) allowing improved precision and reliability of the assay. 19 F MRI can monitor gene expression in living cells by utilising cell-surface displayed β-lactamase (β-lac) and a specifically designed 19 F MRI probe [126]. The probe comprised a trifluoromethoxylated β-lactamase substrate, cephalosporin, attached to a Gd 3+ complex, but it cannot cross the cell membrane. To avoid the need for membrane permeabilization, β-lac was fused to an extracellular region of epidermal growth factor receptor (EGFR). The non-permeable probe can bind the reporter protein on the cell surface, allowing measurement of intracellular gene expression. Hydrolysis of the probe by β-lac released the Gd 3+ complex causing the T 2 of the 19 F-containing group to increase owing to a loss in the intramolecular paramagnetic relaxation enhancement (PRE). The T 2 values can affect the 19 F MRI signal intensity, which can be visually identified and a reduction in the thickness of peaks in Figure 20: Measurement of β-galactosidase activity in MCF7 cancer cells expressing lacZ using 19 F NMR. The deglycosylated FCAT probe binds the Fe 3+ ions present resulting in a complex that gives a new resonance in the spectrum. Figure 20 was reprinted from [125].  19 F iCEST spectra in the presence of Zn 2+ (red) or Fe 2+ (green); (b) Detection of K + upon complexation with trifluorinated thrombin aptamer forming a G-quadruplex.; (c) Structure of fluorinated Zn 2+ -dipicolylamine co-ordination complex and its application to the detection of phosphate released from ATP by apyrase. Figure 21a is reproduced with permission from [129], Copyright © 2014 American Chemical Society, https://pubs.acs.org/doi/10.1021/ja511313k. Further permissions related to the material excerpted should be directed to the ACS. Figure 21b is reprinted with permission from [130], © 2011 The Chemical Society of Japan. Figure 21c was reprinted from [131]. the 19 F NMR spectra can also be observed. Therefore, enzymatic degradation of the Gd-FC-lac probe would lead to the improvement of the 19 F MRI signal, confirming gene expression in living cells.

Physiological measurement Detection of ions
Metal ions play an important role in biological systems and deviations in their levels can be associated with the onset of disease. The real-time detection and tracking of metal ions in vivo are of great interest, which is not possible using optical dyes. One potential way to overcome this is to employ 19 Flabelled chelators and sensors, whose chemical shifts are observed by 19 F NMR upon detection or binding to the metal ion of interest. Early work by Smith et al. [127] employed symmetrically difluorinated 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'tetraacetic acid (BAPTA) to determine intracellular Ca 2+ concentration from the areas of the resonances of free and complexed forms of the chelating reagent. To improve sensitivity of the 5,5'-difluoro-BAPTA (5F-BAPTA) for 19 F MRI Bar-Shir et al.
[128] used ion chemical exchange saturation transfer (iCEST). This approach is an extension of CEST that is used in MRI where a dynamic exchange between radiofrequency (RF) labelled protons and bulk water yields enhanced contrast. In iCEST, RF labelling at the 19 F frequency of Ca 2+ -5F-BAPTA and detection of the label transfer to the frequency of free 5F-BAPTA produced a 100-fold improvement in sensitivity. A tetra-fluorine (5,5',6,6'-tetrafluoro-) BAPTA (TF-BAPTA) ion receptor was designed and successful in the detection of Zn 2+ and Fe 2+ due to slower exchange rates of the free and bound probe observed for both metal ions (Figure 21a) [129].
The conformational changes of biomolecules caused by metal ions has been exploited in the design of 19 F NMR-based ion sensors. For example, in the presence of K + , the thrombin aptamer (5'-GGTTGGTGTGGTTGG-3') undergoes an intramolecular conformational change to promote the formation of the G quadruplex. A K + sensor was designed by introducing a 3,5-bis(trifluoromethyl)benzene moiety at the 5' terminal of the aptamer ( 19 F-TBA) [130]. When KCl was added, a new 19 F NMR signal was observed suggesting the complexation of  Figure 22 was reprinted from [135]. K + and a conformational change had occurred (Figure 21b). The sensor displayed excellent sensitivity as no new 19 F NMR signals and no chemical shift changes were observed upon addition of other metal ions such as Li + , Na + , Mg 2+ and Ca 2+ .
Anions, in particular phosphate, are also important physiological markers, and 19 F NMR can be employed for their detection. Gan et al. [131] reported the chemical shift changes upon phosphate anions binding to a Zn 2+ -dipicolylamine co-ordination complex and used it to follow ATP hydrolysis by apyrase (Figure 21c).

Detection of biological reactive oxygen species
Reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ), superoxide (O 2 − ), and hydroxyl radical (•OH), are produced as part of mitochondrial oxidative metabolism and as a response to cellular invasion by cytokines, xenobiotics, and bacteria [132]. H 2 O 2 is considered to be one of the most important ROS and play a role in homeostatic regulation as well as in healthy physiological signalling pathways such as cell proliferation, differentiation and migration. Fluorescent probes are typically used to detect H 2 O 2 in living organisms and although highly sensitive, the excitation and emission light are unable to penetrate deep sites in the body.
Arylboronic acids are oxidised by H 2 O 2 , allowing for the design of H 2 O 2 -responsive sensors with these substrates as reactive moieties [133]. 19 F-labelled phenylboronic acid was used as a substrate in the design on a 19 F MRI probe to detect H 2 O 2 [134]. The fluorinated phenylboronic acid and the H 2 O 2 interact to form a corresponding phenol, resulting in a large 19 F chemical shift change due to the large electron density change around the 19 F nucleus.
Peroxynitrite (ONOO − ) is a highly reactive nitrogen oxide species formed in vivo from the rapid interaction of O 2 − and nitric oxide radicals. •NO and O 2 − are endogenously formed to moderate cell signalling, whereas ONOO − production is thought to be harmful with increased levels observed in cancer and agerelated pathologies. Detection ONOO − in biological samples is difficult owing to its short lifetime, strong competition from endogenous ROS scavengers and high background noises from other ROS. Two 19 F magnetic resonance probes, 5-fluoroisatin and 6-fluoroisatin, were developed for the detection of ONOO − based on oxidative carbonylation chemistry [135]. Both probes are highly selective for ONOO − , resulting in a chemical shift change in 19 F NMR, which was not observed when the probes were incubated with other reactive sulphur, oxygen and nitrogen species (Figure 22).

Conclusion
In the decade since our previous review, 19 F NMR has continued to prove its versatility in several areas of chemical biology, but predominantly in the structural analysis of protein and nucleic acid, and their interactions with other biomolecules. Advances in the methods for the synthesis of fluorinated amino acids and nucleotides have been the driver for the new applications, coupled with improved sensitivity of the instrumentation. The recognition of the importance of structural biology in disease states is likely to amplify the application of 19 F NMR in this field. Undoubtedly, the technique will continue to be employed in all of the areas highlighted in this review, and new applications identified since fluorine's importance across a range of industries is undiminished.