19F Atoms Research Articles

2'-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions.

Protein-RNA interactions are central to numerous cellular processes. In this work, we present an easy and straightforward NMR-based approach to determine the RNA binding site of RNA binding proteins and to evaluate the binding of pairs of proteins to a single-stranded RNA (ssRNA) under physiological conditions, in this case in nuclear extracts. By incorporation of a 19F atom on the ribose of different nucleotides along the ssRNA sequence, we show that, upon addition of an RNA binding protein, the intensity of the 19F NMR signal changes when the 19F atom is located near the protein binding site. Furthermore, we show that the addition of pairs of proteins to a ssRNA containing two 19F atoms at two different locations informs on their concurrent binding or competition. We demonstrate that such studies can be done in a nuclear extract that mimics the physiological environment in which these protein-ssRNA interactions occur. Finally, we demonstrate that a trifluoromethoxy group (-OCF3) incorporated in the 2'ribose position of ssRNA sequences increases the sensitivity of the NMR signal, leading to decreased measurement times, and reduces the issue of RNA degradation in cellular extracts.

Visualization of drug release in a chemo-immunotherapy nanoplatform via ratiometric 19F magnetic resonance imaging.

Visualization of drug release in vivo is crucial for improving therapeutic efficacy and preventing inappropriate medication dosing, yet, challenging. Herein, we report a pH-activated chemo-immunotherapy nanoplatform with visualization of drug release in vivo by ratiometric 19F magnetic resonance imaging (19F MRI). This nanoplatform consists of ultra-small histamine-modified perfluoro-15-crown-5-ether (PFCE) nanodroplets loaded with doxorubicin (Dox), which are packaged in trifluoromethyl-containing metal-organic assemblies via coordination-driven self-assembly. The chemical shifts of two types of 19F atoms in the nanoplatform are significantly different in 19F nuclear magnetic resonance (NMR) spectra, which facilitates the implementation of ratiometric 19F MRI without any signal crosstalk. In an acidic tumor microenvironment, this nanoplatform gradually degrades, which results in a sustained drug release with a real-time change in the ratiometric 19F MRI signal. Therefore, a linear correlation between the Dox release profile and ratiometric 19F MRI signal is established to visualize Dox release. Moreover, the pH-triggered disassembly of the nanoplatform leads to cell pyroptosis, which evokes immunogenic cell death (ICD), resulting in the regression of the primary tumor and inhibition of distal tumor growth. This study provides the proof-of-concept application of ratiometric 19F MRI to visualize drug release in vivo.

Improving the Sensitivity of Enantioanalysis with Densely Fluorinated NMR Probes.

Nuclear magnetic resonance (NMR) spectroscopy has long been utilized as a classic method for chiral discrimination of enantiomers. However, its sensitivity limitations have hindered the detection of analytes at low concentrations. In this study, we present our efforts to overcome this challenge by employing chiral NMR probes that are labeled with a significant number of chemically equivalent 19F atoms. Specifically, we have designed and synthesized three chiral palladium pincer complexes, all of which are labeled with nonafluoro-tert-butoxy groups to enhance detectability. The recognition of enantiomers with the probe induces distinct changes in microenvironments, resulting in differential perturbations on the chemical shift of the 19F atoms in proximity. This method is applicable to the enantiodifferentiation of various amines, amino alcohols, and amino acid esters. The abundance of 19F atoms enables the detection of chiral analytes at low concentrations, which is otherwise challenging to achieve through traditional 1H NMR-based analysis. Two of the probes are constructed with asymmetric pincer ligands with structurally varied sidearms, allowing for facile manipulation of the chiral binding pocket. The C2 symmetrical probe possesses 36 equivalent 19F atoms, enabling the determination of enantiocomposition of samples with concentrations in the low micromolar range.

The sensitivity of magnetic particle imaging and fluorine-19 magnetic resonance imaging for cell tracking

Magnetic particle imaging (MPI) and fluorine-19 (19F) MRI produce images which allow for quantification of labeled cells. MPI is an emerging instrument for cell tracking, which is expected to have superior sensitivity compared to 19F MRI. Our objective is to assess the cellular sensitivity of MPI and 19F MRI for detection of mesenchymal stem cells (MSC) and breast cancer cells. Cells were labeled with ferucarbotran or perfluoropolyether, for imaging on a preclinical MPI system or 3 Tesla clinical MRI, respectively. Using the same imaging time, as few as 4000 MSC (76 ng iron) and 8000 breast cancer cells (74 ng iron) were reliably detected with MPI, and 256,000 MSC (9.01 × 1016 19F atoms) were detected with 19F MRI, with SNR > 5. MPI has the potential to be more sensitive than 19F MRI for cell tracking. In vivo sensitivity with MPI and 19F MRI was evaluated by imaging MSC that were administered by different routes. In vivo imaging revealed reduced sensitivity compared to ex vivo cell pellets of the same cell number. We attribute reduced MPI and 19F MRI cell detection in vivo to the effect of cell dispersion among other factors, which are described.

Resolution of chemical shift anisotropy in 19F ENDOR spectroscopy at 263 GHz/9.4 T

Pulsed 19F ENDOR spectroscopy provides a selective method for measuring angstrom to nanometer distances in structural biology. Here, the performance of 19F ENDOR at fields of 3.4 T and 9.4 T is compared using model compounds containing one to three 19F atoms. CF3 groups are included in two compounds, for which the possible occurrence of uniaxial rotation might affect the distance distribution. At 9.4 T, pronounced asymmetric features are observed in many of the presented 19F ENDOR spectra. Data analysis by spectral simulations shows that these features arise from the chemical shift anisotropy (CSA) of the 19F nuclei. This asymmetry is also observed at 3.4 T, albeit to a much smaller extent, confirming the physical origin of the effect. The CSA parameters are well consistent with DFT predicted values and can be extracted from simulation of the experimental data in favourable cases, thereby providing additional information about the geometrical and electronic structure of the spin system. The feasibility of resolving the CSA at 9.4 T provides important information for the interpretation of line broadening in ENDOR spectra also at lower fields, which is relevant for developing methods to extract distance distributions from 19F ENDOR spectra.

Fluorinated ZnFeIII Hollow Metal-Organic Framework as a 19F NMR Probe for Highly Sensitive and Selective Detection of Hydrogen Sulfide.

Hydrogen sulfide (H2S) is considered as a highly toxic environmental pollutant and an important signal transmitter in physiological processes, and the selective and reliable detection of H2S is of great concern and remains challenging. Herein, we report a smart sensitive “off–on” 19F NMR sensor for H2S by partially introducing a fluorinated ligand to construct a hollow dual metal–organic framework (MOF) nanosystem, F-ZnFeIII hMOF, in which the fluorinated ligand acts as the 19F signal source but is initially quenched due to the strong paramagnetic relaxation enhancement (PRE) effect from neighboring Fe3+ nodes. Upon exposure to sulfide ions, reduction of Fe3+ to Fe2+ is specifically triggered, which attenuates PRE efficiency, thus turning on the 19F NMR signal. The unique hollow MOF architecture benefits the mobility of 19F atoms, thereby improving the response sensitivity. Meanwhile, the desirable H2S-sorption feature and appropriate redox potential of Fe3+/Fe2+ account for the favorable selectivity. The increase in the 19F signal is linear with the concentration of sulfide in the range of 20 to 150 μM with a detection limit of 2.8 μM. The probe is well demonstrated by analyzing H2S in complex matrixes such as biological and foodstuff samples.

Hydrophilic fluorinated molecules for spectral 19F MRI

Fluorine-19 (19F) Magnetic Resonance Imaging (MRI) is an emerging modality for molecular imaging and cell tracking. The hydrophobicity of current exogenous probes, perfluorocarbons (PFCs) and perfluoropolyethers (PFPEs), limits the formulation options available for in vivo applications. Hydrophilic probes permit more formulation flexibility. Further, the broad Nuclear Magnetic Resonance (NMR) chemical shift range of organofluorine species enables multiple probes with unique 19F MR signatures for simultaneous interrogation of distinct molecular targets in vivo. We report herein a flexible approach to stable liposomal formulations of hydrophilic fluorinated molecules (each bearing numerous magnetically equivalent 19F atoms), with 19F encapsulation of up to 22.7 mg/mL and a per particle load of 3.6 × 106 19F atoms. Using a combination of such probes, we demonstrate, with no chemical shift artifacts, the simultaneous imaging of multiple targets within a given target volume by spectral 19F MRI.

Ligand Binding Studies of a Plasmid Encoded Dihydrofolate Reductase by 19F NMR

Plasmid encoded-R67 dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. R67 DHFR is a homotetramer with a single active site pore and two tryptophans per monomer (W38 and W45). W38 and its symmetry-related partners occur at the dimer-dimer interfaces while the W45 residues occur at the monomer-monomer interfaces. We have labeled these tryptophans using 19F-labeled indole with the 19F atoms at different positions (4-, 5-, 6- or 7-) of the indole ring. In vitro ligand binding studies of NADP+ to the apoprotein or a ternary complex with NADP+ and DHF showed characteristic spectra for each complex. The apoprotein gave rise to a sharp and a broad peak. Upon addition of NADP+, the sharp peak for W38 shows line broadening while the broad peak for W45 remains unchanged. In the ternary complex, the sharp peak in the apo and binary complex splits into three peaks while the broad peak remains unchanged. The appearance of three new resonances can be explained by how NADPH and DHF bind in the active site pore. Two symmetry related lysine 32s at the edge of one side of the pore constrain the position of NADPH by forming ionic interactions with the phosphate groups. However, on the other side of the pore, the glutamate tail of DHF is disordered and switches between ion pairs with the K32s on that side of the pore. This results in two different environments for the nearby W38 residue. Our NMR results are consistent with computational simulations of the glutamate-tail interacting with symmetry related lysine 32 residues at the edge of the pore. This approach will be valuable for determining the binding affinity of DHF towards R67 DHFR by in-cell NMR.

Electron shakeoff following theβ+decay ofNe+19andAr+35trapped ions

The electron shakeoff of 19F and 35Cl atoms resulting from the \b{eta}+ decay of 19Ne+ and 35Ar+ ions has been investigated using a Paul trap coupled to a time of flight recoil-ion spectrometer. The charge-state distributions of the recoiling daughter nuclei were compared to theoretical calculations based on the sudden approximation and accounting for subsequent Auger processes. The excellent agreement obtained for 35Cl is not reproduced in 19F. The shortcoming is attributed to the inaccuracy of the independent particle model employed to calculate the primary shakeoff probabilities in systems with rather low atomic numbers. This calls for more elaborate calculations, including explicitly the electron-electron correlations.

A unique 19F MRI agent for the tracking of non phagocytic cells in vivo

There is currently intense interest in new methods for understanding the fate of therapeutically-relevant cells, such as mesenchymal stem cells (MSCs). The absence of a confounding background signal and consequent unequivocal assignment makes 19F MRI one of the most attractive modalities for the tracking of injected cells in vivo. We describe here the synthesis of novel partly-fluorinated polymeric nanoparticles with small size and high fluorine content as MRI agents. The polymers, constructed from perfluoropolyether methacrylate (PFPEMA) and oligo(ethylene glycol) methacrylate (OEGMA) have favourable cell uptake profiles and excellent MRI performance. To facilitate cell studies the polymer was further conjugated with a fluorescent dye creating a dual-modal imaging agent. The efficacy of labelling of MSCs was assessed using 19F NMR, flow cytometry and confocal microscopy. The labelling efficiency of 2.6 ± 0.1 × 1012 19F atoms per cell, and viability of >90% demonstrates high uptake and good tolerance by the cells. This loading translates to a minimum 19F MRI detection sensitivity of ∼7.4 × 103 cells per voxel. Importantly, preliminary in vivo data demonstrate that labelled cells can be readily detected within a short acquisition scan period (12 minutes). Hence, these copolymers show outstanding potential for 19F MRI cellular tracking and for quantification of non-phagocytic and therapeutically-relevant cells in vivo.

19F-MRI for monitoring human NK cells in vivo

ABSTRACTThe availability of clinical-grade cytokines and artificial antigen-presenting cells has accelerated interest in using natural killer (NK) cells as adoptive cellular therapy (ACT) for cancer. One of the technological shortcomings of translating therapies from animal models to clinical application is the inability to effectively and non-invasively track these cells after infusion in patients. We have optimized the nonradioactive isotope fluorine-19 (19F) as a means to label and track NK cells in preclinical models using magnetic resonance imaging (MRI). Human NK cells were expanded with interleukin (IL)-2 and labeled in vitro with increasing concentrations of 19F. Doses as low as 2 mg/mL 19F were detected by MRI. NK cell viability was only decreased at 8 mg/mL 19F. No effects on NK cell cytotoxicity against K562 leukemia cells were observed with 2, 4 or 8 mg/mL 19F. Higher doses of 19F, 4 mg/mL and 8 mg/mL, led to an improved 19F signal by MRI with 3 × 1011 19F atoms per NK cell. The 4 mg/mL 19F labeling had no effect on NK cell function via secretion of granzyme B or interferon gamma (IFNγ), compared to NK cells exposed to vehicle alone. 19F-labeled NK cells were detectable immediately by MRI after intratumoral injection in NSG mice and up to day 8. When 19F-labeled NK cells were injected subcutaneously, we observed a loss of signal through time at the site of injection suggesting NK cell migration to distant organs. The 19F perfluorocarbon is a safe and effective reagent for monitoring the persistence and trafficking of NK cell infusions in vivo, and may have potential for developing novel imaging techniques to monitor ACT for cancer.

Determination of Intermolecular Interactions Using Polarization Compensated Heteronuclear Overhauser Effect of Hyperpolarized Spins.

The nuclear Overhauser effect (NOE) has long been used as a selective indicator for intermolecular interactions. Due to relatively small changes of signal intensity, often on the order of several percent, quantitative NOE measurements can be challenging. Hyperpolarization of nuclear spins can dramatically increase the NOE intensity by increasing population differences, but poses its own challenge in quantifying the original polarization level. Here, we demonstrate a method for the accurate measurement of intermolecular heteronuclear cross-relaxation rates by simultaneous acquisition of signals from both nuclei. Using this method, we measure cross-relaxation rates between water protons and (19)F of trifluoroacetic acid at concentrations ranging from 23 to 72 mM. A concentration-independent value of 2.46 × 10(-4) ± 1.02 × 10(-5) s(-1) M(-1) is obtained at a temperature of 301 K and validated using a nonhyperpolarized measurement. In a broader context, accurate measurement of heteronuclear cross-relaxation rates may enable the study of intermolecular interactions including those involving macromolecules where (19)F atoms can be introduced as site-selective labels.

Wettability control of BeO surfaces by alpha-irradiation-induced nuclear transmutation

A simple method to control the wettability of BeO surfaces is developed on the base of alpha irradiation. In this research, BeO disks were irradiated with an alpha beam under conditions of ∼25MeV in alpha particle energy and ∼315nA/cm2 in beam current density. After the alpha irradiation, changes in morphology and chemical composition of BeO surfaces were analyzed using a field emission scanning electron microscopy and X-ray photoelectron spectroscopy. The wettability of pristine and alpha-irradiated BeO surfaces was analyzed by measuring water contact angles (CAs). The result of analysis indicated that C and F atoms were produced by the alpha irradiation. 12C atoms were mainly produced by (α, n) nuclear reaction when 9Be atoms collide with energetic alpha particles. On the other hand, 19F atoms were mostly created by (α, n) reaction and following β+ decay when 16O atoms collide with alpha particles. Moreover, CF2 functional groups, which provide hydrophobic property, were formed by the combination of produced C and F atoms. The amount of CF2 functional groups produced on the surface increased as the fluence increased while no significant change in the surface roughness was observed. Accordingly, the CA of alpha-irradiated BeO surfaces gradually increased as the fluence increased. In conclusion, the wettability of BeO surfaces could be easily and precisely controlled by the alpha irradiation, from hydrophilicity to hydrophobicity.

Electronic structure and magnetic properties of endohedral metallofullerenes based on mixed metal cluster within fullerene cage with trifluoromethyl groups

Endohedral metallofullerenes based on encapsulated nitride scandium cluster within C80 fullerene cage with trifluoromethyl group of CF3 were characterized as a candidate model for controlling nuclear spin gate in NMR quantum computers. Effects of yttrium in nitride scandium cluster of Sc3N within the fullerene cage with trifluoromethyl group on electronic structures and magnetic properties were investigated. Electronic structure and chemical shifts in 45Sc, 89Y, 19F, 13C and 14N atoms were calculated by quantum calculation using density functional theory. Addition of yttrium in nitride scandium cluster of Sc3N within the fullerene cage provided a different position of chemical shift in 45Sc, 89Y, 19F, 13C and 14N atoms. This result was originated in magnetic interaction with spin-local interaction, dipole-dipole interaction and nuclear quadrupole interaction based on nuclear quadrupole moment and asymmetry of electron field gradient with a bias value of charge and spin density distribution of spd-hybrid orbital by inductive effect.

Design and evaluation of theranostic perfluorocarbon particles for simultaneous antigen-loading and 19F-MRI tracking of dendritic cells

Perfluorocarbon (PFC) particles are currently on the rise as cell labeling agents for 19F-MRI tracking of dendritic cell (DC)-based vaccines. In this work, we design theranostic PFC particles for single-step loading of DCs with both antigenic protein and with a liquid PFC for 19F-MRI detection of the antigen-loaded cells. Upon addition to DCs in vitro, the antigen-loaded PFC particles are efficiently internalized, resulting in intracellular presence of up to 40pmol 19F atoms per cell. At the same time, the DCs become loaded with antigenic proteins, that can be efficiently processed, without important effects on cell viability or altering the DC's phenotype and the cell's capacity to respond to danger signals. In addition, antigen-loaded PFC particle containing DCs are capable of inducing extensive proliferation of antigen-specific CD8+ T cells in vitro. Importantly, the antigen-coated PFC particles allow in vitro19F-MRI-based detection of the antigen-containing DCs with detection limits as low as 103cellsμl−1. The dual-modality characteristics of the designed particles could assure that only those DCs that have taken up the antigen, and hence are responsible for an immune response, are traceable via 19F-MRI. Taken together, these novel dual-modality particles represent an interesting strategy in the development of a traceable DC vaccine.

Multilayered core–shell structure of polyol-stabilized calcium fluoride nanoparticles characterized by NMR

Diethyleneglycol (DEG)-stabilized calcium fluoride (CaF2) nanoparticles with a mean diameter of about 20nm consist of an inorganic crystalline core, covered by a roughly monomolecular layer of organic material according to standard physico-chemical analysis. Multinuclear NMR experiments under magic angle spinning were used to identify the constituent 13C, 1H and 19F atoms. All resonances were assigned (DEG, EtOH, H2O, H3O+, OH−, F−, CaF2), and different populations could be discriminated in terms of their mobilities and correlations with neighboring atoms. We observed several different layers: mobile EtOH on the particle surface, an “interphase” containing immobile DEG molecules and fluoride ions, plus the single-crystalline CaF2 core. It was thus possible for the first time to characterize by NMR all layered components of such core–shell particles, and the thickness of the newly discovered interphase could be estimated from the NMR intensities.

The structure of fluoride-containing bioactive glasses: new insights from first-principles calculations and solid state NMR spectroscopy

Fluoride-containing bioactive glasses are attracting particular interest in many fields of dentistry and orthopedics because they combine the bone-bonding ability of bioactive glasses with the anticariogenic protection provided by fluoride ions. Since the biomedical applications of these materials critically depend on the release of ionic species in the surrounding physiological environment, a deep knowledge of their environments is required. In this paper, density functional theory calculations and spin effective Hamiltonians have been employed to analyse the NMR signatures of the various environments of 19F, 29Si, 31P and 23Na atoms in fluorinated bioglasses structural models previously generated by Car–Parrinello molecular dynamics simulations. Comparison with experimental spectra expressly recorded in this work shows a good agreement and allows the enlightenment of some longstanding issues about the atomic structure of fluorinated bioglasses, such as the presence of Si–F and Si–O–P bonds. In particular, it is shown that Si–F bonds cannot be resolved by using MAS NMR experiments only, and 29Si{19F} REDOR experiments, that probes directly spatial proximities among atoms, must be employed. Our results show that F is coordinated entirely to the modifier ions Na and Ca, and that no Si–F bonds are present in the real glass structure. Thus, the addition of fluorine to the 45S5 Bioglass® increases the polymerization of the silicate network by removing modifiers from the siliceous matrix and reducing its reactivity. Finally, the computed isotropic chemical shifts of the various environments of phosphorus show that, if present, Si–O–P bonds should be clearly noticeable in the 31P static NMR experimental spectrum. Instead, the latter show that P is present as isolated orthophosphate units and does not enter into the siliceous matrix by forming Si–O–P bonds as conjectured by molecular dynamics simulations.

ChemInform Abstract: Design, Synthesis and Biological Application of Chemical Probes for Bio‐Imaging

AbstractReview: 29 refs.

Study on the compounds containing 19F and 10B atoms in a single molecule for the application to MRI and BNCT

Magnetic resonance imaging (MRI) and boron-neutron capture therapy (BNCT) are quite attractive techniques for diagnosis and treatment of cancer, respectively. In order to progress the study on both MRI and BNCT, the novel compounds containing 19F and 10B atoms in a single molecule were designed and synthesized. In the present paper, the syntheses and the internalization rates into tumor cells of these compounds are elucidated.

Synthese, Strukturen, EPR‐ und ENDOR‐spektroskopische Untersuchungen an Übergangsmetallkomplexen von N, N‐Diisobutyl‐N′‐(2, 6‐difluor)benzoylselenoharnstoff

AbstractSynthesis, Structures, EPR and ENDOR Investigations on Transition Metal Complexes of N, N‐diisobutyl‐N′‐(2, 6‐difluoro)benzoyl selenoureaThe synthesis and the structures of the NiII and PdII complexes of the ligand N, N‐diisobutyl‐N′‐(2, 6‐difluoro)benzoylselenourea HBui2dfbsu are reported. The ligands coordinate bidentately forming bis‐chelates. The structure of the ligand could not be obtained, however, the structure of its O‐ethyl ester will be reported. Attempts to prepare the CuII complex result only in the formation of oily products. However, the CuII complex could be incorporated into the corresponding NiII and PdII compounds. From this diamagnetically diluted powder and single‐crystal samples were obtained being suitable for EPR‐ENDOR measurements. We report X‐ and Q‐band EPR investigations on the systems [Cu/Ni(Bui2dfbsu)2] and [Cu/Pd(Bui2dfbsu)2] as well as a single‐crystal X‐band EPR study for [Cu/Ni(Bui2dfbsu)2]. The obtained 63, 65Cu and 77Se hyperfine structure tensors allow a determination of the spin‐density distribution within the first coordination sphere. In addition, orientation selective 19F Q‐band pulse ENDOR investigations on powder‐samples of [Cu/Ni(Bui2dfbsu)2] have been performed. The hyperfine structure tensors of two intramolecular 19F atoms could be determined. According to the small 19F couplings only a vanishingly small spin‐density of < 1 % was obtained for these 19F atoms.