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Being a low-toxic and hydrophilic representative of TAM, OX063 has shown its suitability for in-vivo and in-cell EPR experiments and design of spin labels. Using 13 C labeling, we investigated the course of oxidative degradation of OX063 into quinone-methide (QM) under the influence of superoxide as well as further thiol-promoted reduction of QM into TAM radical, which formally corresponds to substitution of a carboxyl function by a hydroxyl group. We found these transformations being quantitative in model reactions mimicking specific features of biological media and confirmed the presence of these reactions in the blood and liver homogenate of mice in vitro. The emergence of the trityl with the hydroxyl group can be masked by an initial TAM in EPR spectra and may introduce distortions into EPR-derived oximetry data if they have been obtained for objects under hypoxia. 13 C labeling allows one to detect its presence, considering its different hyperfine splitting constant on 13 C 1 (2.04 mT) as compared to OX063 (2.30 mT). The potential involvement of these reactions should be considered when using TAM in spin-labeling of biopolymers intended for subsequent EPR experiments, as well as in the successful application of TAM in experiments in vivo and in cell., (© 2024 Wiley-VCH GmbH.)

The combination of fluorine labeling and pulsed electron-nuclear double resonance (ENDOR) is emerging as a powerful technique for obtaining structural information about proteins and nucleic acids. In this work, we explored the capability of Mims 19 F ENDOR experiments on reporting intermolecular distances in trityl- and 19 F-labeled DNA duplexes at three electron paramagnetic resonance (EPR) frequencies (34, 94, and 263 GHz). For spin labeling, we used the hydrophobic Finland trityl radical and hydrophilic OX063 trityl radical. Fluorine labels were introduced into two positions of a DNA oligonucleotide. The results indicated that hyperfine splittings visible in the ENDOR spectra are consistent with the most populated interspin distances between 19 F and the trityl radical predicted from molecular dynamic (MD) simulations. Moreover, for some cases, ENDOR spectral simulations based on MD results were able to reproduce the conformational distribution reflected in the experimental ENDOR line broadening. Additionally, MD simulations provided more detailed information about the melting of terminal base pairs of the oligonucleotides and about the configuration of the trityls relative to a DNA end.

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 1 H 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-β-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 1 H NMR results. To demonstrate potential applications of this new DNP approach for chemistry and biology, we show hyperpolarized 1 H NMR spectra of tripeptides, triglycine, and glypromate, in glycerol- d 8 .

Recently, Marina Bennati and coworkers (M. Bennati et al. , Angew. Chem., Int. Ed. , 2020, 59 , 373-379., M. Bennati et al. , J. Magn. Reson. , 2021, 333 , 107091) proposed to use electron nuclear double resonance (ENDOR) spectroscopy in the W-band for a pair of labels, nitroxide and 19 F, for measurements of short (0.5-1.0 nm) distances in biomolecules. In our paper, we investigated the suitability of high-field ENDOR spectroscopy in the W-band for pairs of triarylmethyl and fluorine labels using five newly synthesized model compounds. It is shown that the application of strong magnetic fields allows distinguishing nuclear frequencies of 19 F and protons with sufficient resolution. On the one hand, in contrast to nitroxides, for triarylmethyl radicals, it is not necessary to obtain spectra in different orientations owing to low g -factor anisotropic and long electron spin relaxation times of triarylmethyls. On the other hand, the size of the triarylmethyl radical is substantially larger than that of nitroxide and comparable with measured distances. We theoretically analyzed the suitability of the dipole-dipole approach for triarylmethyl to be used in a 19 F ENDOR experiment and determined limitations of this approach. Finally, for comparison, we performed paramagnetic relaxation enhancement (PRE) NMR on the same compounds. In addition, we applied this approach to study the process of a thiol exchange between molecules of triarylmethyl-labeled and 19 F-labeled human serum albumin (HSA).

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.

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 T M 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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 13 C hyperfine constants are obtained for the first time. The negligible dependence of 13 C 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 13 C signals at natural abundance can be employed for successful room-temperature distance measurements using Pulsed Electron Double Resonance (PELDOR or DEER)., Competing Interests: NOTES The authors declare no competing financial interests.

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 k d 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., Competing Interests: Notes The authors declare no competing financial interest. The authors were ordered alphabetically.

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.

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

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.

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.

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).

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.

The electron nuclear dipolar interactions responsible for some dynamic nuclear polarization (DNP) mechanisms also are responsible for the presence formally in CW EPR spectra of forbidden satellite lines in which both the electron spin and a nuclear spin flip. Such lines arising from (1)H nuclei are easily resolved in CW EPR measurements of trityl radicals, a popular family of DNP reagents. The satellite lines overlap some of the hyperfine features from (13)C in natural abundance in the trityl radical, but their intensity can be easily determined by simple simulations of the EPR spectra using the hyperfine parameters of the trityl radical. Isotopic substitution of (2)H for (1)H among the hydrogens of the trityl radical and/or the solvent allows the dipolar interactions from the (1)H on the trityl radical and from the solvent to be determined. The intensity of the dipolar interactions, integrated over all the (1)H in the system, is characterized by the traditional parameter called reff. For the so-called Finland trityl in methanol, the reff values indicate that collectively the (1)H in the unlabeled solvent have a stronger integrated dipolar interaction with the unpaired electron spin of the Finland trityl than do the (1)H in the radical and consequently will be a more important DNP route. Although reff has the dimensions of distance, it does not correspond to any simple physical dimension in the trityl radical because the details of the unpaired electron spin distribution and the hydrogen distribution are important in the case of trityls., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

Triarylmethanols - the direct precursors of persistent trityl radicals - are racemic mixtures of chiral three-bladed molecular propellers. Depending on bulkiness of aryl groups they exhibit various liabilities to interconversion, the half- life time of room temperature racemization varying in a range between 8.4 hours and 1.32 years. NOESY/EXSY experiment performed on two representative models strongly supports the two-ring flip mechanism for the configurational interchange.

Diffusion in porous media is a general subject that involves many fields of research, such as chemistry (e.g. porous catalytic pallets), biology (e.g. porous cellular organelles), and materials science (e.g. porous polymer matrixes for controlled-release and gas-storage materials). Pulsed-gradient spin-echo nuclear magnetic resonance (PGSE NMR) is a powerful technique that is often employed to characterize complex diffusion patterns inside porous media. Typically it measures the motion of at least approximately 10(15) molecules occurring in the milliseconds-to-seconds time scale, which can be used to characterize diffusion in porous media with features of approximately 2-3 mum and above (in common aqueous environments). Electron Spin Resonance (ESR), which operates in the nanoseconds-to-microseconds time scale with much better spin sensitivity, can in principle be employed to measure complex diffusion patterns in porous media with much finer features (down to approximately 10 nm). However, up to now, severe technical constraints precluded the adaptation of PGSE ESR to porous media research. In this work we demonstrate for the first time the use of PGSE ESR in the characterization of molecular restricted diffusion in common liquid solutions embedded in a model system for porous media made of sub-micron glass spheres. A unique ESR resonator, efficient gradient coils and fast gradient current drivers enable these measurements. This work can be further extended in the future to many applications that involve dynamical processes occurring in porous media with features in the deep sub-micron range down to true nanometric length scales.