Solid-Effect Dynamic Nuclear Polarization in Viscous Liquids at 9.4 T Using Narrow-Line Polarizing Agents.

Dynamic nuclear polarization (DNP) is a hyperpolarization method that is widely used for increasing the sensitivity of nuclear magnetic resonance (NMR) experiments. DNP is efficient in solid-state and liquid-state NMR, but its implementation in the intermediate state, namely, viscous media, is still less explored. Here, we show that a 1H DNP enhancement of over 50 can be obtained in viscous liquids at a magnetic field of 9.4 T and a temperature of 315 K. This was accomplished by using narrow-line polarizing agents in glycerol, both the water-soluble α,γ-bisdiphenylen-ß-phenylallyl (BDPA) and triarylmethyl radicals, and a microwave/RF double-resonance probehead. We observed DNP enhancements with a field profile indicative of the solid effect and investigated the influence of microwave power, temperature, and concentration on the 1H NMR results. To demonstrate potential applications of this new DNP approach for chemistry and biology, we show hyperpolarized 1H NMR spectra of tripeptides, triglycine, and glypromate, in glycerol-d8.

Implantable graded-index fibers for neural-dynamics-resolving brain imaging in awake mice on an air-lifted platform.

We demonstrate a versatile framework for cellular brain imaging in awake mice based on suitably tailored segments of graded-index (GRIN) fiber. Closed-form solutions to ray-path equations for graded-index waveguides are shown to offer important insights into image-transmission properties of GRIN fibers, suggesting useful recipes for optimized GRIN-fiber-based deep-brain imaging. We show that the lengths of GRIN imaging components intended for deep-brain studies in freely moving rodents need to be chosen as a tradeoff among the spatial resolution, the targeted imaging depth and the degree of fiber-probe invasiveness. In the experimental setting that we present in this paper, the head of an awake mouse with a GRIN-fiber implant is fixed under a microscope objective, but the mouse is free to move around an in-house-built flat-floored air-lifted platform, exploring a predesigned environment, configured as an arena for one of standard cognitive tests. We show that cellular-resolution deep-brain imaging can be integrated in this setting with robust cell-specific optical neural recording to enable in vivo studies with minimal physical restraints on animal models. The enhancement of the information capacity of the fluorescence signal, achieved via a suitable filtering of the GRIN-fiber readout, is shown to open routes toward practical imaging modalities whereby the deep-brain neuronal dynamics and axonal connections underpinning the integrative functions of essential brain structures can be studied in awake rodent models.

In Situ Labeling and Distance Measurements of Membrane Proteins in E. coli Using Finland and OX063 Trityl Labels.

In situ investigation of membrane proteins is a challenging task. Previously we demonstrated that nitroxide labels combined with pulsed ESR spectroscopy is a promising tool for this purpose. However, the nitroxide labels suffer from poor stability, high background labeling, and low sensitivity. Here we show that Finland (FTAM) and OX063 based labels enable labeling of the cobalamin transporter BtuB and BamA, the central component of the ß-barrel assembly machinery (BAM) complex, in E coli. Compared to the methanethiosulfonate spin label (MTSL), trityl labels eliminated the background signals and enabled specific in situ labeling of the proteins with high efficiency. The OX063 labels show a long phase memory time (TM ) of ≈5 µs. All the trityls enabled distance measurements between BtuB and an orthogonally labeled substrate with high selectivity and sensitivity down to a few µm concentration. Our data corroborate the BtuB and BamA conformations in the cellular environment of E. coli.

Multiquantum Counting of Trityl Radicals.

We demonstrate a series of multitrityl radical compounds where accurate spin-counting by pulsed electron paramagnetic resonance (EPR) can be achieved at X-band (9 GHz) frequencies, even for molecules with very short and flexible linkers. Multiquantum filter experiments, well-known from NMR, were used to count the number of coupled electron spins in these compounds. The six pulse double quantum filter sequence used in EPR for distance determinations in biradicals was used. Precise phase settings to separate higher quantum coherences were achieved by an arbitrary waveform generator. The trityl radicals have narrow spectral width so that homogeneous excitation of all spins by the pulses is possible. The transversal relaxation times of higher quantum coherences of trityl radicals are sufficiently long to allow their detection. Our results on model compounds show the potential of this approach to determine oligomeric states in protein complexes in their native environment using functionalized trityl spin labels.

Photochemistry of tris(2,3,5,6-tetrathiaaryl)methyl radicals in various solutions.

During the last decades, persistent tris(2,3,5,6-tetrathiaaryl)methyl radicals (TAMs) have attracted much attention due to their applications in oximetry, EPR tomography, and as spin labels in pulsed dipolar EPR spectroscopy. Recently, researchers proposed to use TAM radicals as spin labels and/or a partner for photoinduced spin labels. Thus, the questions of their photochemical stability and mechanism of degradation under UV irradiation have become relevant and important. In this study, steady-state photolysis and flash photolysis of TAM radicals were investigated. A detailed mechanism of TAM phototransformations was proposed and confirmed by NMR, gel permeation chromatography, and mass-spectrometric analyses of the products.

Methanethiosulfonate Derivative of OX063 Trityl: A Promising and Efficient Reagent for Side-Directed Spin Labeling of Proteins.

Trityl radicals (TAMs) have recently appeared as an alternative source of spin labels for measuring long distances in biological systems. Finland trityl radical (FTAM) served as the basis for this new generation of spin labels, but FTAM is rather lipophilic and susceptible to self-aggregation, noncovalent binding with lipophilic sites of proteins, and noncovalent docking at the termini of duplex DNA. In this paper the very hydrophilic OX063 TAM with very low toxicity and little tendency for aggregation is used as the basis for a spin label. Human serum albumin (HSA) labeled with OX063 has an intense narrow line typical of TAM radicals in solution, whereas HSA labeled with FTAM shows broad lines and extensive aggregation. In pulse EPR measurements, the measured phase memory time TM for HSA labeled with OX063 is 6.3 µs at 50 K, the longest yet obtained with a TAM-based spin label. The lowered lipophilicity also decreases side products in the labeling reaction.

Trityl-Aryl-Nitroxide-Based Genuinely g-Engineered Biradicals, As Studied by Dynamic Nuclear Polarization, Multifrequency ESR/ENDOR, Arbitrary Wave Generator Pulse Microwave Waveform Spectroscopy, and Quantum Chemical Calculations.

Trityl and nitroxide radicals are connected by π-topologically controlled aryl linkers, generating genuinely g-engineered biradicals. They serve as a typical model for biradicals in which the exchange (J) and hyperfine interactions compete with the g-difference electronic Zeeman interactions. The magnetic properties underlying the biradical spin Hamiltonian for solution, including J's, have been determined by multifrequency CW-ESR and 1H ENDOR spectroscopy and compared with those obtained by quantum chemical calculations. The experimental J values were in good agreement with the quantum chemical calculations. The g-engineered biradicals have been tested as a prototype for AWG (Arbitrary Wave Generator)-based spin manipulation techniques, which enable GRAPE (GRAdient Pulse Engineering) microwave control of spins in molecular magnetic resonance spectroscopy for use in molecular spin quantum computers, demonstrating efficient signal enhancement of specific weakened hyperfine signals. Dynamic nuclear polarization (DNP) effects of the biradicals for 400 MHz nuclear magnetic resonance signal enhancement have been examined, giving efficiency factors of 30 for 1H and 27.8 for 13C nuclei. The marked DNP results show the feasibility of these biradicals for hyperpolarization.

Triplet Fullerenes as Prospective Spin Labels for Nanoscale Distance Measurements by Pulsed Dipolar EPR Spectroscopy.

Precise nanoscale distance measurements by pulsed electron paramagnetic resonance (EPR) spectroscopy play a crucial role in structural studies of biomolecules. The properties of the spin labels used in this approach determine the sensitivity limits, attainable distances, and proximity to biological conditions. Herein, we propose and validate the use of photoexcited fullerenes as spin labels for pulsed dipolar (PD) EPR distance measurements. Hyperpolarization and the narrower spectrum of fullerenes compared to other triplets (e.g., porphyrins) boost the sensitivity, and superior relaxation properties allow PD EPR measurements up to a near-room temperature. This approach is demonstrated using fullerene-nitroxide and fullerene-triarylmethyl pairs, as well as a supramolecular complex of fullerene with nitroxide-labeled protein. Photoexcited triplet fullerenes can be considered as new spin labels with outstanding spectroscopic properties for future structural studies of biomolecules.

A radical containing injectable in-situ-oleogel and emulgel for prolonged in-vivo oxygen measurements with CW EPR.

Molecular oxygen, reactive oxygen species and free radicals derived from oxygen play important roles in a broad spectrum of physiological and pathological processes. The quantitative measurement of molecular oxygen in tissues by electron paramagnetic resonance (EPR) has great potential for understanding and diagnosing a number of diseases, and for developing and guiding therapies. This requires improvements in the free radical probe systems that sense and report molecular oxygen levels in vivo. We report on the encapsulation of existing free radical probes in lipophilic gel implants: an in-situ-oleogel and an emulgel, based only on well-known, safe excipients for the incorporation of lipophilic and hydrophilic radicals, respectively. The EPR signals of encapsulated radicals were not altered compared to dissolved radicals. The high solubility of oxygen in lipophilic solvents enhanced oxygen sensitivity. The gels extended the lifetime of the radicals in tissues from tens of minutes to many days, simplifying studies with extended series of measurements. The encapsulated radicals showed a good in vivo response to changes in oxygen supply and seem to circumvent concerns from toxicity of the radical probes. These gels simplify the development of new oxygen-sensitive free radical probes for EPR oximetry by making their in vivo stability, persistence and toxicity a function of the encapsulating gel and not a set of additional requirements for the free radical probe.

Triarylmethyl Radicals: EPR Study of 13C Hyperfine Coupling Constants.

Triarylmethyl (TAM) radicals are widely used in Electron Paramagnetic Resonance (EPR) spectroscopy as spin labels and in EPR imaging as spin probes for in vivo oxymetry. One of the key advantages of TAMs is extremely narrow EPR line, especially in case of deuterated analogues (~5 µT). Another advantage is their slow spin relaxation even at physiological temperatures allowing, in particular, application of pulsed dipolar EPR methods for distance measurements in biomolecules. In this paper a large series of TAM radicals and their deuterated analogues is synthesized, and corresponding spectroscopic parameters including 13C hyperfine constants are obtained for the first time. The negligible dependence of 13C hyperfine constants on solvent, as well as on structure and number of substituents at para-C atoms of aromatic rings, has been found. In addition, we have demonstrated that 13C signals at natural abundance can be employed for successful room-temperature distance measurements using Pulsed Electron Double Resonance (PELDOR or DEER).

Electron spin dynamics and spin-lattice relaxation of trityl radicals in frozen solutions.

Electron spin-lattice relaxation of two trityl radicals, d24-OX063 and Finland trityl, were studied under conditions relevant to their use in dissolution dynamic nuclear polarization (DNP). The dependence of relaxation kinetics on temperature up to 100 K and on concentration up to 60 mM was obtained at X- and W-bands (0.35 and 3.5 Tesla, respectively). The relaxation is quite similar at both bands and for both trityl radicals. At concentrations typical for DNP, relaxation is mediated by excitation transfer and spin-diffusion to fast-relaxing centers identified as triads of trityl radicals that spontaneously form in the frozen samples. These centers relax by an Orbach-Aminov mechanism and determine the relaxation, saturation and electron spin dynamics during DNP.

Selective High-Resolution Detection of Membrane Protein-Ligand Interaction in Native Membranes Using Trityl-Nitroxide PELDOR.

The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero-oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro-electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter-subunit interaction and the intra-subunit conformational changes in hetero-oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein-ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.

A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T1 relaxation enhancement.

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has become an important tool for measuring distances in proteins on the order of a few nm. For this purpose pairs of spin labels, most commonly nitroxides, are site-selectively introduced into the protein. Recent efforts to develop new spin labels are focused on tailoring the intrinsic properties of the label to either extend the upper limit of measurable distances at physiological temperature, or to provide a unique spectral lineshape so that selective pairwise distances can be measured in a protein or complex containing multiple spin label species. Triarylmethyl (TAM) radicals are the foundation for a new class of spin labels that promise to provide both capabilities. Here we report a new methanethiosulfonate derivative of a TAM radical that reacts rapidly and selectively with an engineered cysteine residue to generate a TAM containing side chain (TAM1) in high yield. With a TAM1 residue and Cu(2+) bound to an engineered Cu(2+) binding site, enhanced T1 relaxation of TAM should enable measurement of interspin distances up to 50Å at physiological temperature. To achieve favorable TAM1-labeled protein concentrations without aggregation, proteins are tethered to a solid support either site-selectively using an unnatural amino acid or via native lysine residues. The methodology is general and readily extendable to complex systems, including membrane proteins.

Facile and High-Yielding Synthesis of TAM Biradicals and Monofunctional TAM Radicals.

Facile and high-yielding procedures for synthesis of monocarboxylic acid derivatives of triarylmethyl radicals (TAMs) were developed. Reaction of methyl thioglycolate with tris(2,3,5,6-tetrathiaaryl)methyl cation smoothly afforded the monosubstituted TAM derivative, which was hydrolyzed to a monocarboxylic acid, with the TAM moiety attached to thioglycolic acid via the sulfur atom. Alternatively, the diamagnetic tricarboxylic acid precursor of Finland trityl was transformed to a trimethyl ester and partially hydrolyzed under controlled conditions. The diester product was isolated and the remaining fractions were converted back to the trimethyl ester for production of more diester. The first representatives of TAM biradicals with different TAM cores and interspin distances were obtained by reaction of these new TAM monocaboxylic acids with N,N'-dimethylethylenediamine.

Trityl-based alkoxyamines as NMP controllers and spin-labels.

Recently, new applications of trityl-nitroxide biradicals were proposed. In the present study, attachment of a trityl radical to alkoxyamines was performed for the first time. The rate constants kd of C-ON bond homolysis in these alkoxyamines were measured and found to be equal to those for alkoxyamines without trityl. The electron paramagnetic resonance (EPR) spectra of the products of alkoxyamine homolysis (trityl-TEMPO and trityl-SG1 biradicals) were recorded, and the corresponding exchange interactions were estimated. The decomposition of trityl-alkoxyamine showed more than an 80% yield of biradicals, meaning that the C-ON bond homolysis is the main reaction. The suitability of these labelled initiators/controllers for polymerisation was exemplified by means of successful nitroxide-mediated polymerisation (NMP) of styrene. Thus, this is the first report of a spin-labelled alkoxyamine suitable for NMP.

Triarylmethyl Labels: Toward Improving the Accuracy of EPR Nanoscale Distance Measurements in DNAs.

Triarylmethyl (trityl, TAM) based spin labels represent a promising alternative to nitroxides for EPR distance measurements in biomolecules. Herewith, we report synthesis and comparative study of series of model DNA duplexes, 5'-spin-labeled with TAMs and nitroxides. We have found that the accuracy (width) of distance distributions obtained by double electron-electron resonance (DEER/PELDOR) strongly depends on the type of radical. Replacement of both nitroxides by TAMs in the same spin-labeled duplex allows narrowing of the distance distributions by a factor of 3. Replacement of one nitroxide by TAM (orthogonal labeling) leads to a less pronounced narrowing but at the same time gains sensitivity in DEER experiment due to efficient pumping on the narrow EPR line of TAM. Distance distributions in nitroxide/nitroxide pairs are influenced by the structure of the linker: the use of a short amine-based linker improves the accuracy by a factor of 2. At the same time, a negligible dependence on the linker length is found for the distribution width in TAM/TAM pairs. Molecular dynamics calculations indicate greater conformational disorder of nitroxide labels compared to TAM ones, thus rationalizing the experimentally observed trends. Thereby, we conclude that double spin-labeling using TAMs allows obtaining narrower spin-spin distance distributions and potentially more precise distances between labeling sites compared to traditional nitroxides.

Room-Temperature Electron Spin Relaxation of Triarylmethyl Radicals at the X- and Q-Bands.

Triarylmethyl radicals (trityls, TAMs) represent a relatively new class of spin labels. The long relaxation of trityls at room temperature in liquid solutions makes them a promising alternative for traditional nitroxides. In this work we have synthesized a series of TAMs including perdeuterated Finland trityl (D36 form), mono-, di-, and triester derivatives of Finland-D36 trityl, the deuterated form of OX63, the dodeca-n-butyl homologue of Finland trityl, and triamide derivatives of Finland trityl with primary and secondary amines attached. We have studied room-temperature relaxation properties of these TAMs in liquids using pulsed electron paramagnetic resonance (EPR) at two microwave frequency bands. We have found the clear dependence of phase memory time (Tm ∼ T2) on the magnetic field: room-temperature Tm values are ∼1.5-2.5 times smaller at the Q-band (34 GHz, 1.2 T) than at the X-band (9 GHz, 0.3 T). This trend is ascribed to the contribution from g-anisotropy that is negligible at lower magnetic fields but comes into play at the Q-band. In agreement with this, the difference between T1 and Tm becomes more pronounced at the Q-band than at the X-band due to increased contributions from incomplete motional averaging of g-anisotropy. Linear dependence of (1/Tm - 1/T1) on viscosity implies that g-anisotropy is modulated by rotational motion of the trityl radical. On the basis of the analysis of previous data and results of the present work, we conclude that, in the general situation where the spin label is at least partly mobile, the X-band is most suitable for application of trityls for room-temperature pulsed EPR distance measurements.

Preparation of Diversely Substituted Triarylmethyl Radicals by the Quenching of Tris(2,3,5,6-tetrathiaaryl)methyl Cations with C-, N-, P-, and S-Nucleophiles.

C-, N-, P-, and S-nucleophiles reacted with symmetrical tris(2,3,5,6-tetrathiaaryl)methyl cations, generated from the corresponding triarylmethanols by strong acids, to give a variety of asymmetrical monosubstituted persistent triaryl-methyl (TAM) radicals as the major products. The only byproducts were symmetrical TAMs.

Physiological-temperature distance measurement in nucleic acid using triarylmethyl-based spin labels and pulsed dipolar EPR spectroscopy.

Resolving the nanometer-scale structure of biomolecules in natural conditions still remains a challenging task. We report the first distance measurement in nucleic acid at physiological temperature using electron paramagnetic resonance (EPR). The model 10-mer DNA duplex has been labeled with reactive forms of triarylmethyl radicals and then immobilized on a sorbent in water solution and investigated by double quantum coherence EPR. We succeeded in development of optimal triarylmethyl-based labels, approach for site-directed spin labeling and efficient immobilization procedure that, working together, allowed us to measure as long distances as ~4.6 nm with high accuracy at 310 K (37 °C).

Generation of Trityl Radicals by Nucleophilic Quenching of Tris(2,3,5,6-tetrathiaaryl)methyl Cations and Practical and Convenient Large-Scale Synthesis of Persistent Tris(4-carboxy-2,3,5,6-tetrathiaaryl)methyl Radical.

Tris(2,3,5,6-tetrathiaaryl)methyl cations, which were generated from the corresponding triarylmethanols in the presence of strong acids, underwent reaction with nucleophiles to give trityl radicals, as the product of a one-electron reduction of the carbocation. Depending on the nature of the nucleophile, the only byproducts were either diamagnetic quinone methides or asymmetrical monosubstituted trityl radicals. Herein, we report a protocol for the large-scale synthesis of the Finland trityl, which has the advantage of high overall yield and reproducibility.