A Two-Armed Probe for In-Cell DEER Measurements on Proteins**

The application of double electron-electron resonance (DEER) with site-directed spin labeling (SDSL) to measure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin-label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances. Here, three two-armed Gd^III complexes, Gd^III-CLaNP13a/b/c were synthesized. Rather than the disulfide linkage of most other CLaNP molecules, a thioether linkage was used to avoid reductive dissociation of the linker. The doubly Gd^III labeled N55C/V57C/K147C/T151C variants of T4Lysozyme were measured by 95 GHz DEER. The constructs were measured in vitro, in cell lysate and in Dictyostelium discoideum cells. Measured distances were 4.5 nm, consistent with results from paramagnetic NMR. A narrow distance distribution and typical modulation depth, also in cell, indicate complete and durable labeling and probe rigidity due to the dual attachment sites.     

Measurement of Angstrom to Nanometer Molecular Distances with 19F Nuclear Spins by EPR/ENDOR Spectroscopy

Spectroscopic and biophysical methods for structural determination at atomic resolution are fundamental in studies of biological function. Here we introduce an approach to measure molecular distances in bio‐macromolecules using ¹⁹F nuclear spins and nitroxide radicals in combination with high‐frequency (94 GHz/3.4 T) electron–nuclear double resonance (ENDOR). The small size and large gyromagnetic ratio of the ¹⁹F label enables to access distances up to about 1.5 nm with an accuracy of 0.1–1 Å. The experiment is not limited by the size of the bio‐macromolecule. Performance is illustrated on synthesized fluorinated model compounds as well as spin‐labelled RNA duplexes. The results demonstrate that our simple but strategic spin‐labelling procedure combined with state‐of‐the‐art spectroscopy accesses a distance range crucial to elucidate active sites of nucleic acids or proteins in the solution state.     

Improving the accuracy of 1H–19F internuclear distance measurement using 2D 1H–19F HOESY

With the rise in fluorinated pharmaceuticals, it is becoming increasingly important to develop new ¹⁹F NMR-based methods to assist in their analysis. Crucially, obtaining information regarding the conformational dynamics of a molecule in solution can aid the design of strongly binding therapeutics. Herein, we report the development of a 2D ¹H–¹⁹F Heteronuclear Overhauser Spectroscopy (HOESY) experiment to measure ¹H–¹⁹F internuclear distances, with accuracies of ~5% when compared with ¹H–¹⁹F internuclear distances calculated by quantum chemical methods. We demonstrate that correcting for cross-relaxation of ¹H, using the diagonal peaks from the 2D ¹H–¹H Nuclear Overhauser Enhancement Spectroscopy (NOESY), is critical in obtaining accurate values for ¹H–¹⁹F internuclear distances. Finally, we show that by using the proposed method to measure ¹H–¹⁹F internuclear distances, we are able to determine the relative stereochemistry of two fluorinated pharmaceuticals.     

Achievement of 1H–19F heteronuclear experiments using the conventional spectrometer with a shared single high band amplifier

The ¹H–¹⁹F heteronuclear NMR experiments were achieved using the conventional spectrometer equipped with a single high band amplifier and a ¹H/¹⁹F/¹³C double‐tuned probe. Although double high band amplifiers are generally required to perform such experiments, a simple modification of pathway in the conventional spectrometer was capable of acquiring various ¹H–¹⁹F heteronuclear spectra. The efficiency of the present technique was demonstrated in an application for ¹⁹F{¹H} and ¹H{¹⁹F} saturation transfer difference experiments. Copyright © 2015 John Wiley & Sons, Ltd.     

Gadolinium(III)-Based Dual 1H/19F Magnetic Resonance Imaging Probes

We present two novel octadentate cyclen-based ligands bearing one (L¹) or two (L²) phenylacetamide pendants with two CF₃ groups either at positions 3 and 5 (L¹) or 4 (L²). The corresponding Gd³⁺ complexes possess one coordinated water molecule, as confirmed by luminescence lifetime measurements on the Eu^III and Tb^III analogues. A detailed ¹H and ¹⁷O relaxometric characterization has revealed the parameters that govern the relaxivities of these complexes. The water-exchange rate of the mono-amide derivative GdL¹ (k_ex²⁹⁸=1.52×10⁶ s⁻¹) is faster than that determined for the bis-amide complex GdL² (k_ex²⁹⁸=0.73×10⁶ s⁻¹). ¹H and ¹⁹F NMR studies have indicated that the complexes are present in solution almost exclusively as the square-antiprismatic (SAP) isomers. ¹⁹F NMR relaxation studies indicated Gd ⋅⋅⋅ F distances of 7.4±0.1 and 9.1±0.1 Å for GdL¹ and GdL², respectively. Phantom MRI studies revealed the favorable properties of GdL² as a dual ¹H/¹⁹F magnetic resonance imaging (MRI) probe, whereas the shorter Gd ⋅⋅⋅ F distance of GdL¹ reduces the signal-to-noise ratio due to the very short transverse relaxation time of the ¹⁹F NMR signal.     

A Reactive,Rigid GdIII Labeling Tag for In‐Cell EPR Distance Measurements in Proteins

The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in‐cell distance measurements by the double electron–electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed Gd^III spin label, a thiol‐specific DOTA‐derivative (DO3MA‐3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in‐cell distance measurements by the DEER method carried out at W‐band frequencies. The high performance of DO3MA‐3BrPy‐Gd^III is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High‐quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in‐cell protein concentrations as low as 5–10 μm .     

A Reactive,Rigid GdIII Labeling Tag for In‐Cell EPR Distance Measurements in Proteins

The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in‐cell distance measurements by the double electron–electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed Gd^III spin label, a thiol‐specific DOTA‐derivative (DO3MA‐3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in‐cell distance measurements by the DEER method carried out at W‐band frequencies. The high performance of DO3MA‐3BrPy‐Gd^III is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High‐quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in‐cell protein concentrations as low as 5–10 μm .     

Organic Nanoprobes for Fluorescence and 19F Magnetic Resonance Dual‐Modality Imaging

Multimodal imaging techniques have been demonstrated to be greatly advantageous in achieving accurate diagnosis and gained increasing attention in recent decades. Herein, we present a new strategy to integrate the complementary modalities of ¹⁹F magnetic resonance imaging (¹⁹F MRI) and fluorescence imaging (FI) into a polymer nanoprobe composed of hydrophobic fluorescent organic core and hydrophilic fluorinated polymer shell. The alkyne‐terminated fluorinated copolymer (Pn) of 2,2,2‐trifluoroethyl acrylate (TFEA) and poly(ethylene glycol) methyl ether acrylate (PEGA) was first prepared via atom transfer radical polymerization (ATRP). The PEGA plays an important role in both improving ¹⁹F signal and modulating the hydrophilicity of Pn. The alkynyl tail in Pn is readily conjugated with azide modified tetra‐phenylethylene (TPE) through click chemistry to form azo polymer (TPE‐azo‐Pn). The core‐shell nanoprobes (TPE‐P3N) with an average particle size of 57.2 ± 8.8 nm are obtained via self‐assembly with ultrasonication in aqueous solution. These nanoprobes demonstrate high water stability, good biocompatibility, strong fluorescence and good ¹⁹F MRI performance, which present great potentials for simultaneous fluorescence imaging and ¹⁹F–MR imaging.     

Partially Fluorinated Copolymers as Oxygen Sensitive 19F MRI Agents

Effective diagnosis of disease and its progression can be aided by ¹⁹F magnetic resonance imaging (MRI) techniques. Specifically, the inherent sensitivity of the spin–lattice relaxation time (T₁) of ¹⁹F nuclei to oxygen partial pressure makes ¹⁹F MRI an attractive non-invasive approach to quantify tissue oxygenation in a spatiotemporal manner. However, there are only few materials with the adequate sensitivity to be used as oxygen-sensitive ¹⁹F MRI agents at clinically relevant field strengths. Motivated by the limitations in current technologies, we report highly fluorinated monomers that provide a platform approach to realize water-soluble, partially fluorinated copolymers as ¹⁹F MRI agents with the required sensitivity to quantify solution oxygenation at clinically relevant magnetic field strengths. The synthesis of a systematic library of partially fluorinated copolymers enabled a comprehensive evaluation of copolymer structure–property relationships relevant to ¹⁹F MRI. The highest-performing material composition demonstrated a signal-to-noise ratio that corresponded to an apparent ¹⁹F density of 220 mm , which surpasses the threshold of 126 mm ¹⁹F required for visualization on a three Tesla clinical MRI. Furthermore, the T₁ of these high performing materials demonstrated a linear relationship with solution oxygenation, with oxygen sensitivity reaching 240×10⁻⁵ mmHg⁻¹s⁻¹. The relationships between material composition and ¹⁹F MRI performance identified herein suggest general structure–property criteria for the further improvement of modular, water-soluble ¹⁹F MRI agents for quantifying oxygenation in environments relevant to medical imaging.     

Sonoluminescence of aqueous solution of gadolinium chloride

A line of the Gd^III ion was detected at 311 nm in the multibubble sonoluminescence spectrum of a concentrated (1 mol L⁻¹) solution of gadolinium chloride. A comparison with the earlier studied sonoluminescence of the Ce^III and Tb^III ions shows that the Gd^III ion is excited in the volume and/or on the surface of cavitation bubbles upon collisions with “hot” particles. The efficiency of excitation of the lanthanide ions via this mechanism depends on the type of electron transition. For the same energy of the excited state, the efficiency of Gd^III excitation (f-f transition) exceeds by at least 50 times that of Ce^III excitation (f-d transition). Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 1341–1344, June, 2005.     

Struktur‐ und magnetochemische Untersuchungen an Ba5Mn3F19 und verwandten Verbindungen AII5MIII3F19

Structural and Magnetochemical Studies of Ba₅Mn₃F₁₉ and Related Compounds A^II₅M^III₃F₁₉ Single crystal structure determinations by X‐ray methods were performed at the following compounds, crystallizing tetragonally body‐centred (Z = 4): Sr₅V₃F₁₉ (a = 1423.4(2), c = 728.9(1) pm), Sr₅Cr₃F₁₉ (a = 1423.5(2), c = 728.1(1) pm), Ba₅Mn₃F₁₉ (a = 1468.9(1), c = 770.3(1) pm, Ba₅Fe₃F₁₉ (a = 1483.5(1), c = 766.7(1) pm), and Ba₅Ga₃F₁₉ (a = 1466.0(2), c = 760.1(2) pm). Only Ba₅Mn₃F₁₉ was refined in space group I4cm (mean distances for elongated octahedra Mn1–F: 185/207 pm equatorial/axial; for compressed octahedra Mn2–F: 199/182 pm), the remaining compounds in space group I4/m. In all cases the octahedral ligand spheres of the M1 atoms showed disorder, the [M1F₆] octahedra being connected into chains in one part of the compounds and into dimers in the other. The magnetic properties of the V, Cr and Mn compounds named above and of Pb₅Mn₃F₁₉ and Sr₅Fe₃F₁₉ as well were studied; the results are discussed in context with the in part problematic structures.     

Elastic Polymer Coated Nanoparticles with Fast Clearance for 19F MR Imaging

¹⁹F magnetic resonance imaging (MRI) is a powerful molecular imaging technique that enables high-resolution imaging of deep tissues without background signal interference. However, the use of nanoparticles (NPs) as ¹⁹F MRI probes has been limited by the immediate trapping and accumulation of stiff NPs, typically of around 100 nm in size, in the mononuclear phagocyte system, particularly in the liver. To address this issue, elastic nanomaterials have emerged as promising candidates for improving delivery efficacy in vivo. Nevertheless, the impact of elasticity on NP elimination has remained unclear due to the lack of suitable probes for real-time and long-term monitoring. In this study, we present the development of perfluorocarbon-encapsulated polymer NPs as a novel ¹⁹F MRI contrast agent, with the aim of suppressing long-term accumulation. The polymer NPs have high elasticity and exhibit robust sensitivity in ¹⁹F MRI imaging. Importantly, our ¹⁹F MRI data demonstrate a gradual decline in the signal intensity of the polymer NPs after administration, which contrasts starkly with the behavior observed for stiff silica NPs. This innovative polymer-coated NP system represents a groundbreaking nanomaterial that successfully overcomes the challenges associated with long-term accumulation, while enabling tracking of biodistribution over extended periods.     

Aromatic 19F–13C TROSY—[19F, 13C]-Pyrimidine Labeling for NMR Spectroscopy of RNA

We present the access to [5-¹⁹F, 5-¹³C]-uridine and -cytidine phosphoramidites for the production of site-specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5-¹⁹F, 5-¹³C]-pyrimidine labels into five RNAs—the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ϵ (hHBV ϵ) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre-microRNA (miRNA) 21 and the 59 nt full length pre-miRNA 21. The main stimulus to introduce the aromatic ¹⁹F–¹³C-spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole-dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic ¹³C–¹⁹F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5-¹⁹F, 5-¹³C]/[5-¹⁹F] pyrimidine labeling. For the 61 nt hHBV ϵ we find a beneficial ¹⁹F–¹³C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [¹⁹F, ¹³C]-labeling of the SAM VI aptamer domain and the pre-miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting.     

A new series of lanthanoid containing Keggin-type germanotungstates with acetate chelators: [{Ln(CH3COO)GeW11O39(H2O)}2] {Ln=Eu, Gd, Tb, Dy, Ho, Er, Tm, and Yb}

A series of head-on complexes of lanthanoid containing germanotungstates was isolated from a one pot reaction in an acetate buffer at pH 4.5. This convenient approach brought forward the [{Ln(CH₃COO)GeW₁₁O₃₉(H₂O)}₂]¹²⁻ (Ln=Eu^III, Gd^III, Tb^III, Dy^III, Ho^III, Er^III, Tm^III, and Yb^III) family with acetate chelators in the rarely observed μ₂: η²-η¹ mode. All compounds were structurally characterized using various solid state analytics, such as single crystal X-ray diffraction, FT-IR spectroscopy, and thermogravimetric analysis. The isostructural polyanions crystallize in the monoclinic system (S.G. P2₁/c). Temperature-dependent magnetic susceptibility measurements were performed on the Gd^III-complex which exhibits near perfect Curie-type behavior.     

Synthesis of gemini surfactants with twelve symmetric fluorine atoms and one singlet F MR signal as novel F MRI agents

A family of fluorinated gemini surfactants derived from perfluoropinacol has been synthesized as novel ¹⁹F magnetic resonance imaging (¹⁹F MRI) agents. These fluorinated surfactants with 12 symmetric fluorine atoms and one singlet ¹⁹F MR peak can be conveniently prepared from perfluoropinacol and oligo(ethylene glycols) on multi-gram scales. Solubility, hydrophilicity (log P), and critical micelle concentration (CMC) measurements of these fluorinated surfactants indicated that high aqueous solubility can be achieved by introducing oligo(ethylene glycols) with appropriate length into perfluoropinacol, i.e., manipulating the fluorine content (F%). One of these fluorinated surfactants with high aqueous solubility and excellent ¹⁹F MR properties has been identified by ¹⁹F MRI phantom experiments as a promising ¹⁹F MRI agent.     

Preparation of Highly Efficient MRI Contrast Agents through Complexation of Cationic GdIII‐Containing Metallosurfactant with Biocompatible Polyelectrolytes

Novel contrast agents were developed through assembling of Gd^III‐containing metallosurfactant (MS) with biocompatible polyelectrolytes sodium hyaluronate (HA), heparinsodium (HS) and dextran sulfate sodium (DSS). The formed polyelectrolyte–surfactant complexes showed different structural patterns as the charge ratio increased, including spherical aggregates, rod‐like aggregates and network patterns in monovalent HA system, while spherical structures emerged in multivalent HS and DSS systems. Energy dispersive spectroscopy analysis and scanning electron microscopy mapping showed the presence of Gd^III in these complexes. Inductively coupled plasma atomic emission spectrometry was further used to quantify the contents of Gd^III in the assemblies. T1 magnetic resonance imaging showed that these Gd^III‐loaded complexes exhibited relaxivity of up to 63.81 mM ^?1 s^?1, much higher than that of Ominiscan (4.64 mM ^?1 s^?1). The cytotoxicity test in vitro demonstrated the excellent biocompatibility of these complexes, which is essential for clinical application.     

Gd‐DTPA‐Dopamine‐Bisphytanyl Amphiphile: Synthesis,Characterisation and Relaxation Parameters of the Nanoassemblies and Their Potential as MRI Contrast Agents

Here, a new amphiphilic magnetic resonance imaging (MRI) contrast agent, a Gd^III‐chelated diethylenetriaminepentaacetic acid conjugated to two branched alkyl chains via a dopamine spacer, Gd‐DTPA‐dopamine‐bisphytanyl (Gd‐DTPA‐Dop‐Phy), which is readily capable of self‐assembling into liposomal nanoassemblies upon dispersion in an aqueous solution, is reported. In vitro relaxivities of the dispersions were found to be much higher than Magnevist, a commercially available contrast agent, at 0.47 T but comparable at 9.40 T. Analysis of variable temperature ¹⁷O NMR transverse relaxation measurements revealed the water exchange of the nanoassemblies to be faster than that previously reported for paramagnetic liposomes. Molecular reorientation dynamics were probed by ¹H NMRD profiles using a classical inner and outer sphere relaxation model and a Lipari–Szabo “model‐free” approach. High payloads of Gd^III ions in the liposomal nanoassemblies made solely from the Gd‐DTPA‐Dop‐Phy amphiphiles, in combination with slow molecular reorientation and fast water exchange makes this novel amphiphile a suitable candidate to be investigated as an advanced MRI contrast agent.     

Through‐space 19F–19F spin–spin coupling in ortho‐fluoro Z‐azobenzene

We report through‐space (TS) ¹⁹F–¹⁹F coupling for ortho‐fluoro‐substituted Z ‐azobenzenes. The magnitude of the TS‐coupling constant (^TSJ_FF) ranged from 2.2–5.9 Hz. Using empirical formulas reported in the literature, these coupling constants correspond to non‐bonded F–F distances (d_FF) of 3.0–3.5 Å. These non‐bonded distances are significantly smaller than those determined by X‐ray crystallography or density functional theory, which argues that simple models of ¹⁹F–¹⁹F TS spin–spin coupling solely based d_FF are not applicable. ¹H, ¹³C and ¹⁹F data are reported for both the E and Z isomers of ten fluorinated azobenzenes. Density functional theory [B3YLP/6‐311++G(d,p)] was used to calculate ¹⁹F chemical shifts, and the calculated values deviated 0.3–10.0 ppm compared with experimental values. Copyright © 2015 John Wiley & Sons, Ltd.     

C-F?M interaction in anionic α-fluorovinyl rhenium oxycarbene complexes and their β-fluoroenolate analogs

The C-F?M⁺ interaction in anionic σ-(α-fluorovinyl)rhenium oxycarbene complexes, [RCFCFReC(O)R′(CO)₄]M (1-6), M = Na, Li, K is studied by ¹⁹F NMR in THF and Et₂O. The coordination of α-F to M⁺ results in an upfield shift of the corresponding ¹⁹F NMR signal and a decrease of ¹J_CF. The maximum shift is found for the Li salt of complex 4 in Et₂O (Δδ_Fα = 36.4 ppm), in which case a ⁷Li-¹⁹F spin-spin coupling is also observed (J_LiF = 40 Hz). The ΔE of C-F?M⁺ interaction and its effect on ¹⁹F shielding was further studied by DFT calculations using β-fluoroenolates as models, which confirmed a strong impact of CF-bond environment on the coordination ability of fluorine in these F,O-chelates. A compound with a β-fluoroenolate backbone but without rhenium, o-(α-fluorovinyl)phenolate 12, was prepared and studied by ¹⁹F NMR, and similarly showed indications of C-F?M⁺ interaction in THF solution. It is concluded that the donor ability of fluorine in the studied system is enhanced because of the conjugation of α-fluorovinyl group with the enolate π-system and back donation from the transition metal.