1H detection and dynamic nuclear polarization-enhanced NMR of Aß1-42 fibrils.

Several publications describing high-resolution structures of amyloid-ß (Aß) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments-namely, 1H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on 13C,15N-enriched, and fully protonated samples of M0Aß1-42 fibrils by high-field 1H-detected NMR at 23.4 T and 18.8 T, and 13C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M0Aß1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than 1H detection. These results suggest that 1H detection and DNP may be the spectroscopic approaches of choice for future studies of Aß and other amyloid systems.

Rational Design of Dinitroxide Polarizing Agents for Dynamic Nuclear Polarization to Enhance Overall NMR Sensitivity.

We evaluate the overall sensitivity gains provided by a series of eighteen nitroxide biradicals for dynamic nuclear polarization (DNP) solid-state NMR at 9.4 T and 100 K, including eight new biradicals. We find that in the best performing group the factors contributing to the overall sensitivity gains, namely the DNP enhancement, the build-up time, and the contribution factor, often compete with each other leading to very similar overall sensitivity across a range of biradicals. NaphPol and HydroPol are found to provide the best overall sensitivity factors, in organic and aqueous solvents respectively. One of the new biradicals, AMUPolCbm, provides high sensitivity for all three solvent formulations measured here, and can be considered to be a "universal" polarizing agent.

Optically Enhanced Solid-State 1H NMR Spectroscopy.

Low sensitivity is the primary limitation to extending nuclear magnetic resonance (NMR) techniques to more advanced chemical and structural studies. Photochemically induced dynamic nuclear polarization (photo-CIDNP) is an NMR hyperpolarization technique where light is used to excite a suitable donor-acceptor system, creating a spin-correlated radical pair whose evolution drives nuclear hyperpolarization. Systems that exhibit photo-CIDNP in solids are not common, and this effect has, up to now, only been observed for 13C and 15N nuclei. However, the low gyromagnetic ratio and natural abundance of these nuclei trap the local hyperpolarization in the vicinity of the chromophore and limit the utility for bulk hyperpolarization. Here, we report the first example of optically enhanced solid-state 1H NMR spectroscopy in the high-field regime. This is achieved via photo-CIDNP of a donor-chromophore-acceptor molecule in a frozen solution at 0.3 T and 85 K, where spontaneous spin diffusion among the abundant strongly coupled 1H nuclei relays polarization through the whole sample, yielding a 16-fold bulk 1H signal enhancement under continuous laser irradiation at 450 nm. These findings enable a new strategy for hyperpolarized NMR beyond the current limits of conventional microwave-driven DNP.

Deuterated TEKPol Biradicals and the Spin-Diffusion Barrier in MAS DNP.

The sensitivity of NMR spectroscopy is considerably enhanced by dynamic nuclear polarization (DNP). In DNP polarization is transferred from unpaired electrons of a polarizing agent to nearby proton spins. In solids, this transfer is followed by the transport of hyperpolarization to the bulk via 1 H-1 H spin diffusion. The efficiency of these steps is critical to obtain high sensitivity gains, but the pathways for polarization transfer in the region near the unpaired electron spins are unclear. Here we report a series of seven deuterated and one fluorinated TEKPol biradicals to probe the effect of deprotonation on MAS DNP at 9.4 T. The experimental results are interpreted with numerical simulations, and our findings support that strong hyperfine couplings to nearby protons determine high transfer rates across the spin diffusion barrier to achieve short build-up times and high enhancements. Specifically, 1 H DNP build-up times increase substantially with TEKPol isotopologues that have fewer hydrogen atoms in the phenyl rings, suggesting that these protons play a crucial role transferring the polarization to the bulk. Based on this new understanding, we have designed a new biradical, NaphPol, which yields significantly increased NMR sensitivity, making it the best performing DNP polarizing agent in organic solvents to date.

Probing the Structural Dynamics of a Bacterial Chaperone in Its Native Environment by Nitroxide-Based EPR Spectroscopy.

One of the greatest current challenges in structural biology is to study protein dynamics over a wide range of timescales in complex environments, such as the cell. Among magnetic resonances suitable for this approach, electron paramagnetic resonance spectroscopy coupled to site-directed spin labeling (SDSL-EPR) has emerged as a promising tool to study protein local dynamics and conformational ensembles. In this work, we exploit the sensitivity of nitroxide labels to report protein local dynamics at room temperature. We demonstrate that such studies can be performed while preserving both the integrity of the cells and the activity of the protein under investigation. Using this approach, we studied the structural dynamics of the chaperone NarJ in its natural host, Escherichia coli. We established that spin-labeled NarJ is active inside the cell. We showed that the cellular medium affects NarJ structural dynamics in a site-specific way, while the structural flexibility of the protein is maintained. Finally, we present and discuss data on the time-resolved dynamics of NarJ in cellular context.

Trehalose matrices for high temperature dynamic nuclear polarization enhanced solid state NMR.

Dynamic nuclear polarization (DNP) at cryogenic temperatures has proved to be a valuable technique to enhance the sensitivity of solid-state NMR spectroscopy. Over the years, sample formulations have been optimized for experiments at cryogenic temperatures. At 9.4 T, the best performing polarizing agents are dinitroxides such as AMUPol and TEKPol that lead to enhancement factors of around 250 at 100 K. However, the performance of these radicals plummets at higher temperatures. Here we introduce trehalose-based DNP polarizing matrices, suitable to embed biomolecular assemblies. Several formulation protocols are investigated, in combination with various polarizing agents, including a new biradical structure chemically tethered to a trehalose molecule. The DNP efficiency of these new polarizing media is screened as a function of the radical concentration, the hydration level of the matrix and the protein content. Sizeable enhancement factors are reported at 100 K and 9.4 T. More importantly, we show that the DNP performance of these new polarizing media outperform the conventionally used water/glycerol mixture at temperatures above 180 K. This study establishes trehalose matrices as a promising DNP medium for experiments at temperatures >150 K where conventional water-based formulations soften and are no longer viable, thus opening new avenues for DNP enhanced solid-state NMR spectroscopy at temperatures close to ambient temperature.

EPR Investigations to Study the Impact of Mito-Metformin on the Mitochondrial Function of Prostate Cancer Cells.

BACKGROUND: Mito-metformin10 (MM10), synthesized by attaching a triphenylphosphonium cationic moiety via a 10-carbon aliphatic side chain to metformin, is a mitochondria-targeted analog of metformin that was recently demonstrated to alter mitochondrial function and proliferation in pancreatic ductal adenocarcinoma. Here, we hypothesized that this compound may decrease the oxygen consumption rate (OCR) in prostate cancer cells, increase the level of mitochondrial ROS, alleviate tumor hypoxia, and radiosensitize tumors. METHODS: OCR and mitochondrial superoxide production were assessed by EPR (9 GHz) in vitro in PC-3 and DU-145 prostate cancer cells. Reduced and oxidized glutathione were assessed before and after MM10 exposure. Tumor oxygenation was measured in vivo using 1 GHz EPR oximetry in PC-3 tumor model. Tumors were irradiated at the time of maximal reoxygenation. RESULTS: 24-hours exposure to MM10 significantly decreased the OCR of PC-3 and DU-145 cancer cells. An increase in mitochondrial superoxide levels was observed in PC-3 but not in DU-145 cancer cells, an observation consistent with the differences observed in glutathione levels in both cancer cell lines. In vivo, the tumor oxygenation significantly increased in the PC-3 model (daily injection of 2 mg/kg MM10) 48 and 72 h after initiation of the treatment. Despite the significant effect on tumor hypoxia, MM10 combined to irradiation did not increase the tumor growth delay compared to the irradiation alone. CONCLUSIONS: MM10 altered the OCR in prostate cancer cells. The effect of MM10 on the superoxide level was dependent on the antioxidant capacity of cell line. In vivo, MM10 alleviated tumor hypoxia, yet without consequence in terms of response to irradiation.

Guest Exchange by a Partial Energy Ratchet in Water.

Molecular machines are ubiquitous in nature and function away from equilibrium by consuming fuels to produce appropriate work. Chemists have recently excelled at mimicking the fantastic job performed by natural molecular machines with synthetic systems soluble in organic solvents. In efforts toward analogous systems working in water, we show that guest molecules can be exchanged in the synthetic macrocycle cucurbit[7]uril by involving kinetic traps, and in such a way as modulating energy wells and kinetic barriers using pH, light, and redox stimuli. Ditolyl-viologen can also be exchanged using the best kinetic trap and interfaced with alginate, thus affording pH-responsive blue, fluorescent hydrogels. With tunable rate and binding constants toward relevant guests, cucurbiturils may become excellent ring molecules for the construction of advanced molecular machines working in water.

Structural Analysis of an Antigen Chemically Coupled on Virus-Like Particles in Vaccine Formulation.

Structure determination of adjuvant-coupled antigens is essential for rational vaccine development but has so far been hampered by the relatively low antigen content in vaccine formulations and by their heterogeneous composition. Here we show that magic-angle spinning (MAS) solid-state NMR can be used to assess the structure of the influenza virus hemagglutinin stalk long alpha helix antigen, both in its free, unformulated form and once chemically coupled to the surface of large virus-like particles (VLPs). The sensitivity boost provided by high-field dynamic nuclear polarization (DNP) and proton detection at fast MAS rates allows to overcome the penalty associated with the antigen dilution. Comparison of the MAS NMR fingerprints between the free and VLP-coupled forms of the antigen provides structural evidence of the conservation of its native fold upon bioconjugation. This work demonstrates that high-sensitivity MAS NMR is ripe to play a major role in vaccine design, formulation studies, and manufacturing process development.

Open and Closed Radicals: Local Geometry around Unpaired Electrons Governs Magic-Angle Spinning Dynamic Nuclear Polarization Performance.

The development of magic-angle spinning dynamic nuclear polarization (MAS DNP) has allowed atomic-level characterization of materials for which conventional solid-state NMR is impractical due to the lack of sensitivity. The rapid progress of MAS DNP has been largely enabled through the understanding of rational design concepts for more efficient polarizing agents (PAs). Here, we identify a new design principle which has so far been overlooked. We find that the local geometry around the unpaired electron can change the DNP enhancement by an order of magnitude for two otherwise identical conformers. We present a set of 13 new stable mono- and dinitroxide PAs for MAS DNP NMR where this principle is demonstrated. The radicals are divided into two groups of isomers, named open (O-) and closed (C-), based on the ring conformations in the vicinity of the N-O bond. In all cases, the open conformers exhibit dramatically improved DNP performance as compared to the closed counterparts. In particular, a new urea-based biradical named HydrOPol and a mononitroxide O-MbPyTol yield enhancements of 330 ± 60 and 119 ± 25, respectively, at 9.4 T and 100 K, which are the highest enhancements reported so far in the aqueous solvents used here. We find that while the conformational changes do not significantly affect electron spin-spin distances, they do affect the distribution of the exchange couplings in these biradicals. Electron spin echo envelope modulation (ESEEM) experiments suggest that the improved performance of the open conformers is correlated with higher solvent accessibility.

In-Cell EPR: Progress towards Structural Studies Inside Cells.

Exploring the structure and dynamics of biomolecules in the context of their intracellular environment has become the ultimate challenge for structural biology. As the cellular environment is barely reproducible in vitro, investigation of biomolecules directly inside cells has attracted a growing interest. Among magnetic resonance approaches, site-directed spin labeling (SDSL) coupled to electron paramagnetic resonance (EPR) spectroscopy provides competitive and advantageous features to capture protein structure and dynamics inside cells. To date, several in-cell EPR approaches have been successfully applied to both bacterial and eukaryotic cells. In this review, the major advances of in-cell EPR spectroscopy are summarized, as well as the challenges this approach still poses.

Enhanced Intersystem Crossing and Transient Electron Spin Polarization in a Photoexcited Pentacene-Trityl Radical.

Identifying and characterizing systems that generate well-defined states with large electron spin polarization is of high interest for applications in molecular spintronics, high-energy physics, and magnetic resonance spectroscopy. The generation of electron spin polarization on free-radical substituents tethered to pentacene derivatives has recently gained a great deal of interest for its applications in molecular electronics. After photoexcitation of the chromophore, pentacene-radical derivatives can rapidly form spin-polarized triplet excited states through enhanced intersystem crossing. Under the right conditions, the triplet spin polarization, arising from mS-selective intersystem crossing rates, can be transferred to the tethered stable radical. The efficiency of this spin polarization transfer depends on many factors: local magnetic and electric fields, excited-state energetics, molecular geometry, and spin-spin coupling. Here, we present transient electron paramagnetic resonance (EPR) measurements on three pentacene derivatives tethered to Finland trityl, BDPA, or TEMPO radicals to explore the influence of the nature of the radical on the spin polarization transfer. We observe efficient polarization transfer between the pentacene excited triplet and the trityl radical but do not observe the same for the BDPA and TEMPO derivatives. The polarization transfer behavior in the pentacene-trityl system is also investigated in different glassy matrices and is found to depend markedly on the solvent used. The EPR results are rationalized with the help of femtosecond and nanosecond transient absorption measurements, yielding complementary information on the excited-state dynamics of the three pentacene derivatives. Notably, we observe a 2 orders of magnitude difference in the time scale of triplet formation between the pentacene-trityl system and the pentacene systems tethered with the BDPA and TEMPO radicals.

EPR Spectroscopy: A Powerful Tool to Analyze Supramolecular Host•Guest Complexes of Stable Radicals with Cucurbiturils.

Stable organic free radicals are increasingly studied compounds due to the multiple and unusual properties imparted by the single electron(s). However, being paramagnetic, classical methods such as NMR spectroscopy can hardly be used due to relaxation and line broadening effects. EPR spectroscopy is thus better suited to get information about the immediate surroundings of the single electrons. EPR has enabled obtaining useful data in the context of host•guest chemistry, and a classical example is reported here for the stable (2,2,6,6-tetramethyl-4-oxo-piperidin-1-yl)oxyl or 4-oxo-TEMPO nitroxide (TEMPONE) inside the macrocycle host cucurbit[7]uril (CB[7]). Generally and also observed here, a contraction of the spectrum is observed as a result of the reduced nitrogen coupling constant due to inclusion complexation in the hydrophobic cavity of the host. Simulations of EPR spectra allowed determining the corresponding binding constant pointing to a weaker affinity for CB[7], compared to TEMPO with CB[7]. We complement this work by the results of EPR spectroscopy of a biradical: bis-TEMPO-bis-ketal (bTbk) with cucurbit[8]uril (CB[8]). Initial investigations pointed to very weak effects on the spectrum of the guest and incorrectly led us to conclude an absence of binding. However, simulations of EPR spectra combined with NMR data of reduced bTbk allowed showing inclusion complexation. EPR titrations were performed, and the corresponding binding constant was determined. 1H NMR spectra with reduced bTbk suggested a shuttle mechanism, at nearly one equivalent of CB[8], for which the host moves rapidly between two stations.

Detection of mitochondria-generated reactive oxygen species in cells using multiple probes and methods: Potentials, pitfalls, and the future.

Reactive oxygen and nitrogen species (ROS/RNS) such as superoxide (O2̇̄), hydrogen peroxide, lipid hydroperoxides, peroxynitrite, and hypochlorous and hypobromous acids play a key role in many pathophysiological processes. Recent studies have focused on mitochondrial ROS as redox signaling species responsible for promoting cell division, modulating and regulating kinases and phosphatases, and activating transcription factors. Many ROS also stimulate cell death and senescence. The extent to which these processes occur is attributed to ROS levels (low or high) in cells. However, the exact nature of ROS remains unknown. Investigators have used redox-active probes that, upon oxidation by ROS, yield products exhibiting fluorescence, chemiluminescence, or bioluminescence. Mitochondria-targeted probes can be used to detect ROS generated in mitochondria. However, because most of these redox-active probes (untargeted and mitochondria-targeted) are oxidized by several ROS species, attributing redox probe oxidation to specific ROS species is difficult. It is conceivable that redox-active probes are oxidized in common one-electron oxidation pathways, resulting in a radical intermediate that either reacts with another oxidant (including oxygen to produce O2̇̄) and forms a stable fluorescent product or reacts with O2̇̄ to form a fluorescent marker product. Here, we propose the use of multiple probes and complementary techniques (HPLC, LC-MS, redox blotting, and EPR) and the measurement of intracellular probe uptake and specific marker products to identify specific ROS generated in cells. The low-temperature EPR technique developed to investigate cellular/mitochondrial oxidants can easily be extended to animal and human tissues.

Triangular Regulation of Cucurbit[8]uril 1:1 Complexes.

Triangular shapes have inspired scientists over time and are common in nature, such as the flower petals of oxalis triangularis, the triangular faces of tetrahedrite crystals, and the icosahedron faces of virus capsids. Supramolecular chemistry has enabled the construction of triangular assemblies, many of which possess functional features. Among these structures, cucurbiturils have been used to build supramolecular triangles, and we recently reported paramagnetic cucurbit[8]uril (CB[8]) triangles, but the reasons for their formation remain unclear. Several parameters have now been identified to explain their formation. At first sight, the radical nature of the guest was of prime importance in obtaining the triangles, and we focused on extending this concept to biradicals to get supramolecular hexaradicals. Two sodium ions were systematically observed by ESI-MS in trimer structures, and the presence of Na+ triggered or strengthened the triangulation of CB[8]/guest 1:1 complexes in solution. X-ray crystallography and molecular modeling have allowed the proposal of two plausible sites of residence for the two sodium cations. We then found that a diamagnetic guest with an H-bond acceptor function is equally good at forming CB[8] triangles. Hence, a guest molecule containing a ketone function has been precisely triangulated thanks to CB[8] and sodium cations as determined by DOSY-NMR and DLS. A binding constant for the triangulation of 1:1 to 3:3 complexes is proposed. This concept has finally been extended to the triangulation of ditopic guests toward network formation by the reticulation of CB[8] triangles using dinitroxide biradicals.

A Cucurbit[8]uril 2:2 Complex with a Negative pKa Shift.

A viologen derivative carrying a benzimidazole group (V-P-I 2+ ; viologen-phenylene-imidazole V-P-I) can be dimerized in water using cucurbit[8]uril (CB[8]) in the form of a 2:2 complex resulting in a negative shift of the guest pKa , by more than 1 pH unit, contrasting with the positive pKa shift usually observed for CB-based complexes. Whereas 2:2 complex protonation is unclear by NMR, silver cations have been used for probing the accessibility of the imidazole groups of the 2:2 complexes. The protonation capacity of the buried imidazole groups is reduced, suggesting that CB[8] could trigger proton release upon 2:2 complex formation. The addition of CB[8] to a solution containing V-P- I3+ indeed released protons as monitored by pH-metry and visualized by a coloured indicator. This property was used to induce a host/guest swapping, accompanied by a proton transfer, between V-P-I 3+ ⋅CB[7] and a CB[8] complex of 1-methyl-4-(4-pyridyl)pyridinium. The origin of this negative pKa shift is proposed to stand in an ideal charge state, and in the position of the two pH-responsive fragments inside the two CB[8] which, alike residues engulfed in proteins, favour the deprotonated form of the guest molecules. Such proton release triggered by a recognition event is reminiscent of several biological processes and may open new avenues toward bioinspired enzyme mimics catalyzing proton transfer or chemical reactions.

Embedding cyclic nitrone in mesoporous silica particles for EPR spin trapping of superoxide and other radicals.

The generation of superoxide radical anion in biological systems is one of the major initiating events in the redox biology of NADPH oxidases and mitochondrial redox signalling. However, the pallette of chemical tools for superoxide detection is very limited, hampering progress in understanding the chemical biology of superoxide. Although EPR spin trapping is regarded as the most rigorous technique for superoxide detection, rapid reduction of the EPR-active superoxide spin adducts to EPR-silent hydroxylamines, or to hydroxyl radical adducts by bioreductants, significantly limits the applicability of this technique in biological systems. To overcome these limitations, in this work, we report the synthesis and characterization of a new mesoporous silica functionalized with a phosphorylated cyclic spin trap (DIPPMPO nitrone). The DIPPMPO-grafted silica is a versatile spin-trap agent enabling the identification of a wide range of carbon or oxygen-centered transient radicals in organic and in aqueous media. Moreover, superoxide was efficiently trapped under in vitro conditions in both cell-free and cellular systems. The generated superoxide adduct exhibited an exceptional half-life of 3.5 h and a resistance toward bioreductant agents such as glutathione for several hours.

Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications.

Mitochondria are recognized as one of the most important targets for new drug design in cancer, cardiovascular, and neurological diseases. Currently, the most effective way to deliver drugs specifically to mitochondria is by covalent linking a lipophilic cation such as an alkyltriphenylphosphonium moiety to a pharmacophore of interest. Other delocalized lipophilic cations, such as rhodamine, natural and synthetic mitochondria-targeting peptides, and nanoparticle vehicles, have also been used for mitochondrial delivery of small molecules. Depending on the approach used, and the cell and mitochondrial membrane potentials, more than 1000-fold higher mitochondrial concentration can be achieved. Mitochondrial targeting has been developed to study mitochondrial physiology and dysfunction and the interaction between mitochondria and other subcellular organelles and for treatment of a variety of diseases such as neurodegeneration and cancer. In this Review, we discuss efforts to target small-molecule compounds to mitochondria for probing mitochondria function, as diagnostic tools and potential therapeutics. We describe the physicochemical basis for mitochondrial accumulation of lipophilic cations, synthetic chemistry strategies to target compounds to mitochondria, mitochondrial probes, and sensors, and examples of mitochondrial targeting of bioactive compounds. Finally, we review published attempts to apply mitochondria-targeted agents for the treatment of cancer and neurodegenerative diseases.

Dynamic Nuclear Polarization / solid-state NMR of membranes. Thermal effects and sample geometry.

Whereas specially designed dinitroxide biradicals, reconstitution protocols, oriented sample geometries and NMR probes have helped to much increase the DNP enhancement factors of membrane samples they still lag considerably behind those obtained from glasses made of protein solutions. Here we show that not only the MAS rotor material but also the distribution of the membrane samples within the NMR rotor have a pronounced effect on the DNP enhancement. These observations are rationalized with the cooling efficiency and the internal properties of the sample, monitored by their T1 relaxation, microwave ON versus OFF signal intensities and DNP effect. The data are suggestive that for membranes the speed of cooling has a pronounced effect on the membrane properties and concomitantly the distribution of biradicals within the sample.

19 F Magic Angle Spinning Dynamic Nuclear Polarization Enhanced NMR Spectroscopy.

The introduction of high-frequency, high-power microwave sources, tailored biradicals, and low-temperature magic angle spinning (MAS) probes has led to a rapid development of hyperpolarization strategies for solids and frozen solutions, leading to large gains in NMR sensitivity. Here, we introduce a protocol for efficient hyperpolarization of 19 F nuclei in MAS DNP enhanced NMR spectroscopy. We identified trifluoroethanol-d3 as a versatile glassy matrix and show that 12 mm AMUPol (with microcrystalline KBr) provides direct 19 F DNP enhancements of over 100 at 9.4 T. We applied this protocol to obtain DNP-enhanced 19 F and 19 F-13 C cross-polarization (CP) spectra for an active pharmaceutical ingredient and a fluorinated mesostructured hybrid material, using incipient wetness impregnation, with enhancements of approximately 25 and 10 in the bulk solid, respectively. This strategy is a general and straightforward method for obtaining enhanced 19 F MAS spectra from fluorinated materials.