19F NMR Signals Research Articles

Synthesis of Intrinsically Disordered Fluorinated Peptides for Modular Design of High‐Signal 19F MRI Agents

Abstract19F MRI is valuable for in vivo imaging due to the only trace amounts of fluorine in biological systems. Because of the low sensitivity of MRI however, designing new fluorochemicals remains a significant challenge for achieving sufficient 19F signal. Here, we describe a new class of high‐signal, water‐soluble fluorochemicals as 19F MRI imaging agents. A polyamide backbone is used for tuning the proteolytic stability to avoid retention within the body, which is a limitation of current state‐of‐the‐art perfluorochemicals. We show that unstructured peptides containing alternating N‐ϵ‐trifluoroacetyllysine and lysine provide a degenerate 19F NMR signal. 19F MRI phantom images provide sufficient contrast at micromolar concentrations, showing promise for eventual clinical applications. Finally, the degenerate high signal characteristics were retained when conjugated to a large protein, indicating potential for in vivo targeting applications, including molecular imaging and cell tracking.

Orientation and Location of the Cyclotide Kalata B1 in Lipid Bilayers Revealed by Solid-State NMR

Cyclotides are ultra-stable cyclic disulfide-rich peptides from plants. Their biophysical effects and medically interesting activities are related to their membrane-binding properties, with particularly high affinity for phosphatidylethanolamine lipids. In this study we were interested in understanding the molecular details of cyclotide-membrane interactions, specifically with regard to the spatial orientation of the cyclotide kalata B1 from Oldenlandia affinis when embedded in a lipid bilayer. Our experimental approach was based on the use of solid-state 19F-NMR of oriented bilayers in conjunction with the conformationally restricted amino acid L-3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine as an orientation-sensitive 19F-NMR probe. Its rigid connection to the kalata B1 backbone scaffold, together with the well-defined structure of the cyclotide, allowed us to calculate the protein alignment in the membrane directly from the orientation-sensitive 19F-NMR signal. The hydrophobic and polar residues on the surface of kalata B1 form well-separated patches, endowing this cyclotide with a pronounced amphipathicity. The peptide orientation, as determined by NMR, showed that this amphipathic structure matches the polar/apolar interface of the lipid bilayer very well. A location in the amphiphilic headgroup region of the bilayer was supported by 15N-NMR of uniformly labeled protein, and confirmed using solid-state 31P- and 2H-NMR. 31P-NMR relaxation data indicated a change in lipid headgroup dynamics induced by kalata B1. Changes in the 2H-NMR order parameter profile of the acyl chains suggest membrane thinning, as typically observed for amphiphilic peptides embedded near the polar/apolar bilayer interface. Furthermore, from the 19F-NMR analysis two important charged residues, E7 and R28, were found to be positioned equatorially. The observed location thus would be favorable for the postulated binding of E7 to phosphatidylethanolamine lipid headgroups. Furthermore, it may be speculated that this pair of side chains could promote oligomerization of kalata B1 through electrostatic intermolecular contacts via their complementary charges.

Diffusions of β-cyclodextrins in mucus studied by 19F diffusion NMR

β-Cyclodextrin (β-CD) based materials have been widely used as drug carriers for pharmaceutical applications. To understand the diffusion of β-CDs in mucus is important for selecting β-CD based drug carriers for applications targeting mucosal absorption because the surfaces of many biological membranes are covered with a highly viscous aqueous mucus layer which forms relatively effective diffusion barriers for drugs. In this study, 19F self-diffusion NMR technique has been applied to study the self-diffusions of β-CDs in mucus. The 19F NMR signals arose from 1-fluoroadamantane molecules entrapped in the cavities of β-CDs. The diffusive abilities of different β-CDs in mucus were assessed through analyzing the diffusion coefficients using the presented kinetic model, and Ogston’s and Renkin’s diffusion models for hydrogel systems. The kinetic results show that 2-hydroxypropyl-β-CD and 2-Carboxyethyl-β-CD have the smallest binding affinities to bovine submaxillary mucin and human nasal mucin among five tested β-CDs. The mesh sizes of the bovine submaxillary mucus at different concentrations and that of the human nasal mucus were evaluated using the diffusion models. We hope that this 19F diffusion method will be useful to study the diffusion of β-CD based materials in other biological systems.

Chitosan-coated microvesicles: Effect of polysaccharide-phospholipid affinity on decafluorobutane dissolution

The stability of perfluorinated microvesicles is mainly determined by the presence of interfacial materials and their ability to hinder the gas component diffusibility into the bloodstream. The goal of this study is to increase the persistence of the gaseous-core by introducing chitosan-coated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) microvesicles, reducing gas diffusion from microvesicles, and increasing for a long time ultrasonic signals. Our hypothesis was based on the irreversible adhesion of chitosan towards DSPC head groups observed in thin-films models. This affinity enhanced the stabilization of gaseous-core microvesicles, in which the polysaccharide effectively reduced the phospholipid phase transition enthalpy from 383±5.5Jmg−1 for plain to 150±9.7Jmg−1 for chitosan-coated microvesicles, providing a more stable structure that diminished the gaseous component lost and provided the persistence of intense 19F-NMR signals after 48h, twice as long compared to plain samples. As a result, stronger and long-lasting ultrasonic signals were produced by the more stable chitosan-containing microvesicles, thus, presenting great potential to increase the diagnostic and therapeutic applications of perfluorocarbon carries.

Comparative analysis of the interaction of capecitabine and gefitinib with human serum albumin using 19F nuclear magnetic resonance-based approach

Monitoring the interaction between drugs and proteins is critical to understanding drug transport and metabolism underlying pharmacodynamics. The binding capacities to human serum albumin of two anticancer drugs, capecitabine and gefitinib, were compared via an approach combining 19F NMR, 1H saturation transfer difference (STD) NMR, circular dichroism and docking simulations. Results showed that the two drugs interaction with human serum albumin caused 19F NMR signal shifted to different directions. Capecitabine had accurate binding site and higher binding affinity than gefitinib. This study provided fresh insights into ligand–protein interaction and the strength of 19F NMR approach in biomedical research was well illustrated in this case.

The role of nonbonding interactions and the presence of fluoride on the conformational isomerism of 1,2-ethanediol

This work reports the analysis of the effects ruling the conformational preference of 1,2-ethanediol (1,2-ED) using theoretical calculations, since there is no general consensus about the role of intramolecular hydrogen bond on the conformational isomerism of 1,2-ED. While the predominance of the gauche conformers along with the OCCO fragment relative to the trans ones was found to be mainly due to hyperconjugation, the orientation of the hydroxyl groups is better described by a balance between low steric hindrance and high stabilization from hyperconjugation than by intramolecular hydrogen bond. Nevertheless, the presence of a fluoride anion induces a conformational change in 1,2-ED that maximizes hydrogen bonds between the fluoride and the hydroxyl groups. This effect was observed experimentally by the shift of 1H(O) and 19F NMR signals upon complexation, then suggesting that compounds containing the 1,2-ED moiety can be possible anion transporters.

Modified polysaccharides as potential 19F magnetic resonance contrast agents

The introduction of 3-aminobenzotrifluoride into partially oxidized alginic acid, dextran, and polygalacturonic acid (10–100 kDa) by means of the imine formation and a subsequent reduction resulted in water-soluble materials containing 1–14% of fluorine. They showed a single or split 19F NMR signal in a narrow range of −63 to −63.5 ppm. The observed T1 and T2 were approximately 1 and 0.2 s at 400 or 500 MHz instruments, respectively. The samples showed low toxicity and uptake toward the HeLa cells similar to native polysaccharides and were preferentially localized in lysosomes. A tail intravenous injection of 5 mg of modified dextran containing 1% of fluorine revealed that the probe was not trapped in liver, spleen or kidneys, but was quickly cleared with urine. The proposed materials can be used for imaging of the gastrointestinal tract or the genitourinary system and act as a material for more complex 19F MRI agent synthesis.

A CoII complex for 19F MRI-based detection of reactive oxygen species.

A fluorinated, air-stable cobalt(ii) complex serves as a turn-on 19F magnetic resonance imaging (MRI) tracer for reactive oxygen species including H2O2. Upon oxidation with H2O2, the complex converts from paramagnetic high spin CoII to diamagnetic low spin CoIII resulting in a chemical shift change and enhancement in 19F NMR signal. Further, the oxidation can be reversed in the presence of reductant Na2S2O4. The turn-on response is demonstrated by 19F MRI, characterized by a ∼2-3 fold enhancement in signal.

Sensitivity of magnetic resonance imaging based on the detection of 19F NMR signals

The evaluation of the sensitivity of magnetic resonance tomography with a magnetic field of 7 T based on the detection of 19 F NMR signals was performed with the use of the solutions of trifluoroacetic acid, 1,1,1,3,3,3-hexafluoroisopropanol and sym -nonafluoroneopentanol, whose 19 F NMR spectra consist of single line signals. The scanning of a 12×12 cm zone with a resolution of 1.2 mm and a cut thickness of 4 mm for 10 min gave reliable images if the local concentration of 19 F atoms in the test zone exceeded 0.3–0.6%.

Control of the radiofrequency field in fluorine (19F) magnetic resonance imaging

Ways of recording 19F NMR signals using different configurations of transmitting and receiving coils, including the version with a nonresonant transmitting coil and one of the receiving coils serving as an RF amplifier, are considered.

Activatable Dendritic 19F Probes for Enzyme Detection.

We describe a novel activatable probe for fluorine-19 NMR based on self-assembling amphiphilic dendrons. The dendron probe has been designed to be spectroscopically silent due to the formation of large aggregates. Upon exposure to the specific target enzyme, the aggregates disassemble to give rise to a sharp 19F NMR signal. The probe is capable of detecting enzyme concentrations in the low nanomolar range. Response time of the probe was found to be affected by the hydrophilic–lipophilic balance of dendrons. Understanding the structural factors that underlie this design principle provides the pathway for using this strategy for a broad range of enzyme-based imaging.

Synthesis and characterization of [S2MoS2Cu(n-SPhF)]2 −(n = o, m, p) clusters: Potential 19F-NMR structural probes for Orange Protein

Three fluorinated Mo–Cu–thiolate isomers, [Ph4P]2[S2MoS2Cu(n-SPhF)], [n-SPhF=2-fluorothiophenol (1a)], 3-fluorothiophenol (1b), and 4-fluorothiophenol (1c)] were synthesized and spectroscopically characterized. The 19F-NMR signal of the fluorine atom in the benzene has different chemical shift for each isomer, which is highly influenced by the local environment that can be manipulated by different solvents and solutes. The fluorine-19 chemical shift is an advantageous NMR structural probe in alternative to 1H-NMR [B.K. Maiti, T. Avilés, M. Matzapetakis, I. Moura, S.R. Pauleta, J.J.G. Moura, Eur. J. Inorg. Chem. (2012) 4159.], that can be used to provide local information on the pocket of the metal cluster in the Orange Protein (ORP).

The Competition of σ-Hole···Cl– and π-Hole···Cl– Bonds between C6F5X (X = F, Cl, Br, I) and the Chloride Anion and Its Potential Application in Separation Science

On the basis of the varying amplitude and patterns of the (19)F NMR chemical shift of C6F5X (X = F, Cl, Br, I) in the presence of chloride anions, bonding models of C6F5X·Cl(-) complexes were tentatively established, and the relevant binding constants were obtained. Interaction models were also simulated using computational chemistry. The theoretical computations were found to be highly consistent with the results of the experiments. The results show that C6F5Br/C6F5I and Cl(-) were prone to forming C-I/Br···Cl(-) σ-hole bonding complexes with the (19)F NMR signal shifting to higher fields, and the interaction strength of the C6F5I···Cl(-) σ-hole bond was larger than that of C6F5Br···Cl(-); C6F6/C6F5Cl and Cl(-) formed π-hole···Cl(-) bonding complexes with the signal shifting to lower fields, and the interaction strength of C6F6 was larger than that of C6F5Cl. The binding constant of the C6F5I···Cl(-) σ-hole bonding complex is 38.0 M(-1), which is nearly 165- to 345-fold larger than that of the other C6F5X·Cl(-) complexes. On the basis of the above results, solid phase extraction experiments were designed, and the results demonstrated the potential applicability of the C-I···Cl(-) σ-hole bond in separation science.

Biodegradable core crosslinked star polymer nanoparticles as19F MRI contrast agents for selective imaging

With the aim of developing stimuli-responsive imaging agents, we report here the synthesis of core crosslinked star (CCS) polymers and their evaluation as pH-sensitive 19F magnetic resonance imaging (19F MRI) contrast agents. Block copolymers consisting of poly(ethylene glycol)methyl ether methacrylate (PPEGMA) as the first block and a copolymer of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and 2,2,2-trifluoroethyl methacrylate (TFEMA) as the second block were synthesised using RAFT polymerisation. The polymerisation kinetics were studied in detail. The block copolymers were then used as arm precursors for the arm-first synthesis of CCS polymers through RAFT dispersion polymerisation. The synthetic conditions were investigated and optimised. CCS polymers with a degradable core were also synthesised and evaluated as 19F MRI contrast agents. The degradation of the core was confirmed by treatment with various reducing agents. The particle size, 19F NMR signal and relaxation times as well as 19F MRI imaging performance of the CCS polymers were studied at a range of value of solution pH. Significant enhancement of the image intensity was observed when the pH was decreased from 8 to 5, indicating that the CCS nanoparticles could be used as 19F MRI contrast agents for the detection of the acidic environment within tumour tissue.

First Fluorinated Zwitterionic Micelle with Unusually Slow Exchange in an Ionic Liquid

The micellization of a fluorinated zwitterionic surfactant in ethylammonium nitrate (EAN) was investigated. The freeze-fracture transmission electron microscope (FF-TEM) observations confirm the formation of spherical micelles with the average diameter 25.45 ± 3.74 nm. The micellization is an entropy-driven process at low temperature but an enthalpy-driven process at high temperature. Two sets of (19)F NMR signals above the critical micelle concentration (cmc) indicate that the unusually slow exchange between micelles and monomers exists in ionic liquid; meanwhile, surfactant molecules are more inclined to stay in micelle states instead of monomer states at higher concentration. Through the analysis of the half line width (Δν1/2), we can obtain the kinetic information of fluorinated zwitterionic micellization in an ionic liquid.

Efficient NMR enantiodiscrimination of bridge fluorinated paracyclophanes using lanthanide tris β-diketonate complexes

A selection of amino-substituted 1,1,2,2,9,9,10,10 octafluoro[2.2]paracyclophanes were tested for enantiodiscrimination by 1H and 19F NMR spectroscopy via their interaction with different lanthanide tris β-diketonate chiral shift reagents. The amino-, and the pseudo-ortho di-amino substituted octafluoro[2.2]paracyclophanes, both of which exhibit planar chirality, revealed significant shifts and splittings of various 1H and 19F NMR signals upon the addition of the chiral shift reagents, which allowed the easy determination of the enantiomeric purity. When the chiral shift reagent was added to an inseparable mixture of the (chiral) pseudo-meta, and (achiral) pseudo-para diamino analogues, both the chiral and achiral molecules revealed NMR doubling. In the case of the achiral molecule, this NMR behavior is due to the meso nature of the pseudo-para species.

Cover Picture: Probing DNA Mismatched and Bulged Structures by Using19F NMR Spectroscopy and Oligodeoxynucleotides with an19F-Labeled Nucleobase (Chem. Eur. J. 45/2013)

19F-Labeled oligonucleotides for monitoring DNA structures were prepared, and their signal changes in 19F NMR spectra, triggered by the formation of matched, mismatched, and bulged duplexes, were characterized. 19F NMR signals attributed to each duplex as well as single strands were identified. In addition, dissociation of mixed duplexes possessing several local structures could be monitored with increasing temperature. Thus, the motions of individual duplexes could be quantitatively tracked even in the mixture of the duplexes by the measurement of 19F NMR spectra. For more information see the Full Paper by K. Tanabe et al. on page 15133 ff.

A carbonate-fluoride defect model for carbonate-rich fluorapatite

We propose a microscopic model of the dominant carbonate for phosphate substitution in fluorapatite. A well-crystallized sedimentary fluorapatite sample containing ~2.3 ± 0.8 wt% of carbonate was investigated using Fourier transform infrared spectroscopy (FTIR) and 13C and 19F magic angle spinning nuclear magnetic resonance (MAS NMR). About 75% of the carbonate groups replace the phosphate group (“B-site”), whereas a lesser contribution from carbonate groups located in the structural channels (“A-site”) is observed. Beside the dominant 19F NMR signal of channel ions at ~ -102 ppm, an additional signal corresponding to ~8% of fluoride ions is observed at -88 ppm. 19F double quantum-single quantum (DQ-SQ) MAS NMR and 13C{19F} frequency-selective Rotational Echo DOuble Resonance (REDOR) experiments prove that this additional signal corresponds to isolated fluoride ions in the apatite structure, located in close proximity of substituted carbonate groups. Density functional theory (DFT) calculations allow us to propose a composite carbonate-fluoride tetrahedron defect model accounting for these experimental observations. The planar carbonate ion lies in the sloping face of the tetrahedron opposite a fluoride ion occupying the remaining vertex, together replacing the tetrahedral phosphate ion. This “francolite-type” defect leads to a diagnostic narrow IR absorption band at 864 cm-1 that could be used as a guide to, e.g., detect the incipient transformation of fossil bone and teeth samples.

Heat-initiated detection for reduced glutathione with 19F NMR probes based on modified gold nanoparticles

For detecting reduced glutathione (GSH) with a 19F NMR spectroscopy with time-specificity, we developed the probes based on gold nanoparticles modified with the fluorinated groups via the thermally-cleavable linkers. Before the heating treatment with the probe, the maleimide moiety as a binding site with GSH in the probe is inactivated by cycloaddition of furan. At this silent state, the magnitude of 19F NMR signals from the fluorinated groups was suppressed. By heating for the activation of the probe, the maleimide moiety was produced via retro Diels–Alder reaction, and 19F NMR signals were observed. From this moment, GSH started the reaction with the probe via Michael addition to the maleimide moiety, leading to the observation of the new peak in 19F NMR spectra. Finally, the amounts of GSH were determined from the increase of the magnitude of 19F NMR signals.

Solid-state NMR and wide-angle X-ray diffraction study of hydrofluoroether/β-cyclodextrin inclusion complex

An inclusion complex (IC) composed of a hydrofluoroether (HFE) guest and a β-cyclodextrin (β-CD) host was newly prepared, and the crystalline structure and the thermal stability of the IC were examined using several analytical methods, including wide-angle X-ray diffraction (WAXD), solid-state NMR, thermogravimetric analysis (TGA), TG–mass spectrometry (TG–MS), and quantum chemical calculation. The WAXD patterns and elemental analysis identified that the IC of an HFE/β-CD form of a channel-type structure, in which one HFE molecule is included in a common cavity of two β-CD molecules. TGA and TG–MS analysis indicated that the HFE molecules included in β-CD are hardly evaporated or degraded up to the decomposition temperature of the β-CD host. Solid-state 13C NMR indicated that the β-CD ring structure was deformed by including an HFE molecule in it, and that the 19F NMR signals of the HFE guest were significantly shifted to higher frequencies by the inclusion due to the dielectric media effect in the cavity of β-CD. Moreover, the 19F NMR signals of HFE included in IC were further shifted after annealing at 150 °C, which reflected structural changes in HFE/β-CD IC caused at elevated temperatures. The WAXD patterns also confirmed that the packing structure along the crystalline b-direction of HFE/β-CDs, which penetrates the cavities of β-CDs, was compressed by annealing and transformed to a more stable structure.