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.

Redox-crippled MitoQ potently inhibits breast cancer and glioma cell proliferation: A negative control for verifying the antioxidant mechanism of MitoQ in cancer and other oxidative pathologies.

Mitochondria-targeted coenzyme Q10 (Mito-ubiquinone, Mito-quinone mesylate, or MitoQ) was shown to be an effective antimetastatic drug in patients with triple-negative breast cancer. MitoQ, sold as a nutritional supplement, prevents breast cancer recurrence. It potently inhibited tumor growth and tumor cell proliferation in preclinical xenograft models and in vitro breast cancer cells. The proposed mechanism of action involves the inhibition of reactive oxygen species by MitoQ via a redox-cycling mechanism between the oxidized form, MitoQ, and the fully reduced form, MitoQH2 (also called Mito-ubiquinol). To fully corroborate this antioxidant mechanism, we substituted the hydroquinone group (-OH) with the methoxy group (-OCH3). Unlike MitoQ, the modified form, dimethoxy MitoQ (DM-MitoQ), lacks redox-cycling between the quinone and hydroquinone forms. DM-MitoQ was not converted to MitoQ in MDA-MB-231 cells. We tested the antiproliferative effects of both MitoQ and DM-MitoQ in human breast cancer (MDA-MB-231), brain-homing cancer (MDA-MB-231BR), and glioma (U87MG) cells. Surprisingly, DM-MitoQ was slightly more potent than MitoQ (IC50 = 0.26 µM versus 0.38 µM) at inhibiting proliferation of these cells. Both MitoQ and DM-MitoQ potently inhibited mitochondrial complex I-dependent oxygen consumption (IC50 = 0.52 µM and 0.17 µM, respectively). This study also suggests that DM-MitoQ, which is a more hydrophobic analog of MitoQ (logP: 10.1 and 8.7) devoid of antioxidant function and reactive oxygen species scavenging ability, can inhibit cancer cell proliferation. We conclude that inhibition of mitochondrial oxidative phosphorylation by MitoQ is responsible for inhibition of breast cancer and glioma proliferation and metastasis. Blunting the antioxidant effect using the redox-crippled DM-MitoQ can serve as a useful negative control in corroborating the involvement of free radical-mediated processes (e.g., ferroptosis, protein oxidation/nitration) using MitoQ in other oxidative pathologies.

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.

Polyphenolic Boronates Inhibit Tumor Cell Proliferation: Potential Mitigators of Oxidants in the Tumor Microenvironment.

Boronate-based compounds have been used in brain cancer therapy, either as prodrugs or in combination with other modalities. Boronates containing pro-luminescent and fluorescent probes have been used in mouse models of cancer. In this study, we synthesized and developed polyphenolic boronates and mitochondria-targeted polyphenolic phytochemicals (e.g., magnolol [MGN] and honokiol [HNK]) and tested their antiproliferative effects in brain cancer cells. Results show that mitochondria-targeted (Mito) polyphenolic boronates (Mito-MGN-B and Mito-HNK-B) were slightly more potent than Mito-MGN and Mito-HNK in inhibiting proliferation of the U87MG cell line. Similar proliferation results also were observed in other cancer cell lines, such as MiaPaCa-2, A549 and UACC-62. Independent in vitro experiments indicated that reactive nitrogen species (e.g., peroxynitrite) and reactive oxygen species (e.g., hydrogen peroxide) stoichiometrically react with polyphenolic boronates and Mito-polphenolic boronates, forming polyphenols and Mito-polyphenols as major products. Previous reports suggest that both Mito-MGN and Mito-HNK activate cytotoxic T cells and inhibit immunosuppressive immune cells. We propose that Mito-polyphenolic boronate-based prodrugs may be used to inhibit tumor proliferation and mitigate oxidant formation in the tumor microenvironment, thereby generating Mito-polyphenols in situ, as well as showing activity in the tumor microenvironment.

Triple Stack of a Viologen Derivative in a CB[10] Pair.

A viologen-phenylene-imidazole (VPI) conjugate, previously shown to be singly complexed by CB[7] and doubly bound by CB[8], is herein shown to form antiparallel triple stacks in water with cucurbit[10]uril (CB[10]), pairwise complexing the guest trimer. The quinary host:guest 2:3 complex showed features assignable to charge-transfer interactions. Under reductive conditions, CB[10] could solubilize a VPI radical, even though CB[10] and reduced VPI are almost insoluble, thereby illustrating a possible new application for CB[10].

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.

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.

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.

TinyPols: a family of water-soluble binitroxides tailored for dynamic nuclear polarization enhanced NMR spectroscopy at 18.8 and 21.1 T.

Dynamic Nuclear Polarization (DNP) has recently emerged as a key method to increase the sensitivity of solid-state NMR spectroscopy under Magic Angle Spinning (MAS). While efficient binitroxide polarizing agents such as AMUPol have been developed for MAS DNP NMR at magnetic fields up to 9.4 T, their performance drops rapidly at higher fields due to the unfavorable field dependence of the cross-effect (CE) mechanism and AMUPol-like radicals were so far disregarded in the context of the development of polarizing agents for very high-field DNP. Here, we introduce a new family of water-soluble binitroxides, dubbed TinyPols, which have a three-bond non-conjugated flexible amine linker allowing sizable couplings between the two unpaired electrons. We show that this adjustment of the linker is crucial and leads to unexpectedly high DNP enhancement factors at 18.8 T and 21.1 T: an improvement of about a factor 2 compared to AMUPol is reported for spinning frequencies ranging from 5 to 40 kHz, with ε H of up to 90 at 18.8 T and 38 at 21.1 T for the best radical in this series, which are the highest MAS DNP enhancements measured so far in aqueous solutions at these magnetic fields. This work not only breathes a new momentum into the design of binitroxides tailored towards high magnetic fields, but also is expected to push the application frontiers of high-resolution DNP MAS NMR, as demonstrated here on a hybrid mesostructured silica material.

A single-crystal-to-single-crystal transformation affording photochromic 3D MORF crystals.

Metal-organic framework (MOF) type crystals in which rigid, viologen-based pillars are surrounded by cucurbit[7]uril (CB[7]) macrocycles are described. These metal-organic rotaxane frameworks (MORF) are obtained by desolvation of a crystal precursor featuring a network already near-optimal toward 3D metal-ligand reticulation and show reversible photochromism.

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.

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.

BDPA-Nitroxide Biradicals Tailored for Efficient Dynamic Nuclear Polarization Enhanced Solid-State NMR at Magnetic Fields up to 21.1 T.

Dynamic nuclear polarization (DNP) solid-state nuclear magnetic resonance (NMR) has developed into an invaluable tool for the investigation of a wide range of materials. However, the sensitivity gain achieved with many polarizing agents suffers from an unfavorable field and magic angle spinning (MAS) frequency dependence. We present a series of new hybrid biradicals, soluble in organic solvents, that consist of an isotropic narrow electron paramagnetic resonance line radical, α,γ-bisdiphenylene-ß-phenylallyl (BDPA), tethered to a broad line nitroxide. By tuning the distance between the two electrons and the substituents at the nitroxide moiety, correlations between the electron-electron interactions and the electron spin relaxation times on one hand and the DNP enhancement factors on the other hand are established. The best radical in this series has a short methylene linker and bears bulky phenyl spirocyclohexyl ligands. In a 1.3 mm prototype DNP probe, it yields enhancements of up to 185 at 18.8 T (800 MHz 1H resonance frequency) and 40 kHz MAS. We show that this radical gives enhancement factors of over 60 in 3.2 mm sapphire rotors at both 18.8 and 21.1 T (900 MHz 1H resonance frequency), the highest magnetic field available today for DNP. The effect of the rotor size and of the microwave irradiation inside the MAS rotor is discussed. Finally, we demonstrate the potential of this new series of polarizing agents by recording high field 27Al and 29Si DNP surface enhanced NMR spectra of amorphous aluminosilicates and 17O NMR on silica nanoparticles.

Effects of cucurbit[n]uril (n = 7, 8, 10) hosts on the formation and stabilization of a naphthalenediimide (NDI) radical anion.

Most 1,4,5,8-naphthalenediimide (NDI) derivatives, especially those with mild π-acidity cannot achieve photoinduced electron-transfer (PET). Here we report our investigations on the binding interactions of a NDI diammonium dichloride salt (NDI·Cl2) with cucurbit[n]uril (CB[n], n = 8, 10) and the formation process of a NDI radical anion upon photoexcitation of the NDI derivative in the presence of CB[n] (n = 8, 10). As a comparison, the influence of CB[7] on the PET process was also evaluated. The results show that the NDI core can be partially or fully encapsulated in CB[8] or CB[10] to form inclusion complexes at molar ratios of 1 : 1 (CB[8]·NDI2+) or 1 : 2 (CB[10]·2NDI2+). In the presence of the host, quick formation of the NDI radical anion was observed with respect to no radical anion formation without the host. According to the spectral results, interesting CB[8]-promoted charge-transfer interactions between the NDI radical anion and NDI2+ are assumed during UV irradiation. Moreover, from the UV/Vis and EPR spectra, the observation of intense signals of the NDI radical anion in the presence of CB[10], could presumably be related to a much better stabilized NDI radical anion encapsulated in CB[10].

Detection and Characterization of Reactive Oxygen and Nitrogen Species in Biological Systems by Monitoring Species-Specific Products.

SIGNIFICANCE: Since the discovery of the superoxide dismutase enzyme, the generation and fate of short-lived oxidizing, nitrosating, nitrating, and halogenating species in biological systems has been of great interest. Despite the significance of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in numerous diseases and intracellular signaling, the rigorous detection of ROS and RNS has remained a challenge. Recent Advances: Chemical characterization of the reactions of selected ROS and RNS with electron paramagnetic resonance (EPR) spin traps and fluorescent probes led to the establishment of species-specific products, which can be used for specific detection of several forms of ROS and RNS in cell-free systems and in cultured cells in vitro and in animals in vivo. Profiling oxidation products from the ROS and RNS probes provides a rigorous method for detection of those species in biological systems. CRITICAL ISSUES: Formation and detection of species-specific products from the probes enables accurate characterization of the oxidative environment in cells. Measurement of the total signal (fluorescence, chemiluminescence, etc.) intensity does not allow for identification of the ROS/RNS formed. It is critical to identify the products formed by using chromatographic or other rigorous techniques. Product analyses should be accompanied by monitoring of the intracellular probe level, another factor controlling the yield of the product(s) formed. FUTURE DIRECTIONS: More work is required to characterize the chemical reactivity of the ROS/RNS probes, and to develop new probes/detection approaches enabling real-time, selective monitoring of the specific products formed from the probes. Antioxid. Redox Signal. 28, 1416-1432.

Synthesis and properties of a series of ß-cyclodextrin/nitrone spin traps for improved superoxide detection.

Three new DEPMPO-based spin traps have been designed and synthesized for improved superoxide detection, each carrying a cyclodextrin (CD) moiety but with a different alkyl chain on the phosphorus atom or with a long spacer arm. EPR spectroscopy allowed us to estimate the half-life of the superoxide spin adducts which is close to the value previously reported for CD-DEPMPO (t1/2 ≈ 50-55 min under the conditions investigated). The spectra are typical of superoxide adducts (almost no features of the HO˙ adduct that usually forms with time for other nitrone spin traps such as DMPO) and we show that at 250 µM, the new spin trap enables the reliable detection of superoxide by 1 scan at the position opposite to the corresponding spin trap without the CD moiety. The resistance of the spin adducts to a reduction process has been evaluated, and the superoxide spin adducts are sensitive to ascorbate and glutathione (GSH), but not to glutathione peroxidase/GSH, reflecting the exposed nature of the nitroxide moiety to the bulk solvent. To understand these results, 2D-ROESY NMR studies and molecular dynamics pointed to a shallow or surface self-inclusion of the nitrone spin traps and of nitroxide spin adducts presumably due to the high flexibility of the permethyl-ß-CD rim.

Dynamic Nuclear Polarization/Solid-State NMR Spectroscopy of Membrane Polypeptides: Free-Radical Optimization for Matrix-Free Lipid Bilayer Samples.

Dynamic nuclear polarization (DNP) boosts the sensitivity of NMR spectroscopy by orders of magnitude and makes investigations previously out of scope possible. For magic-angle-spinning (MAS) solid-state NMR spectroscopy studies, the samples are typically mixed with biradicals dissolved in a glass-forming solvent and are investigated at cryotemperatures. Herein, we present new biradical polarizing agents developed for matrix-free samples such as supported lipid bilayers, which are systems widely used for the investigation of membrane polypeptides of high biomedical importance. A series of 11 biradicals with different structures, geometries, and physicochemical properties were comprehensively tested for DNP performance in lipid bilayers, some of them developed specifically for DNP investigations of membranes. The membrane-anchored biradicals PyPol-C16, AMUPOL-cholesterol, and bTurea-C16 were found to exhibit improved g-tensor alignment, inter-radical distance, and dispersion. Consequently, these biradicals show the highest signal enhancement factors so far obtained for matrix-free membranes or other matrix-free samples and may potentially shorten NMR acquisition times by three orders of magnitude. Furthermore, the optimal biradical-to-lipid ratio, sample deuteration, and membrane lipid composition were determined under static and MAS conditions. To rationalize biradical performance better, DNP enhancement was measured by using the 13 C and 15 N signals of lipids and a peptide as a function of the biradical concentration, DNP build-up time, resonance line width, quenching effect, microwave power, and MAS frequency.

Tailoring of Polarizing Agents in the bTurea Series for Cross-Effect Dynamic Nuclear Polarization in Aqueous Media.

A series of 18 nitroxide biradicals derived from bTurea has been prepared, and their enhancement factors ɛ ((1)H) in cross-effect dynamic nuclear polarization (CE DNP) NMR experiments at 9.4 and 14.1 T and 100 K in a DNP-optimized glycerol/water matrix ("DNP juice") have been studied. We observe that ɛ ((1)H) is strongly correlated with the substituents on the polarizing agents, and its trend is discussed in terms of different molecular parameters: solubility, average e-e distance, relative orientation of the nitroxide moieties, and electron spin relaxation times. We show that too short an e-e distance or too long a T1e can dramatically limit ɛ ((1)H). Our study also shows that the molecular structure of AMUPol is not optimal and its ɛ ((1)H) could be further improved through stronger interaction with the glassy matrix and a better orientation of the TEMPO moieties. A new AMUPol derivative introduced here provides a better ɛ ((1)H) than AMUPol itself (by a factor of ca. 1.2).

EPR Studies of the Binding Properties, Guest Dynamics, and Inner-Space Dimensions of a Water-Soluble Resorcinarene Capsule.

Nitroxide free radicals have been used to study the inner space of one of Rebek's water-soluble capsules. EPR and (1) H NMR spectroscopy, ESI-MS, and DFT calculations showed a preference for the formation of 1:2 complexes. EPR titrations allowed us to determine binding constants (Ka ) in the order of 10(7) M(-2) . EPR spectral-shape analysis provided information on the guest rotational dynamics within the capsule. The interplay between optimum hydrogen bonding upon capsule formation and steric strain for guest accommodation highlights some degree of flexibility for guest inclusion, particularly at the center of the capsule where the hydrogen bond seam can be barely distorted or slightly disturbed.