Tetrathiatriarylmethyl Radicals Research Articles

Magneto-Structural Correlations in a Mixed Porphyrin(Cu2+ )/Trityl Spin System: Magnitude, Sign, and Distribution of the Exchange Coupling Constant.

Tetrathiatriarylmethyl radicals (TAM or trityl) are receiving increasing attention in various fields of magnetic resonance such as imaging, dynamic nuclear polarization, spin labeling, and, more recently, molecular magnetism and quantum information technology. Here, a trityl radical attached via a phenyl bridge to a copper(II)tetraphenylporphyrin was synthesized, and its magnetic properties studied by multi-frequency continuous-wave electron paramagnetic resonance (EPR) spectroscopy and magnetic measurements. EPR revealed that the electron spin-spin coupling constant J between the trityl and Cu2+ spin centers is ferromagnetic with a magnitude of -2.3 GHz (-0.077 cm-1 , convention) and a distribution width of 1.2 GHz (0.040 cm-1 ). With the help of density functional theory (DFT) calculations, the obtained ferromagnetic exchange coupling, which is unusual for para-substituted phenyl-bridged biradicals, could be related to the almost perpendicular orientation of the phenyl linker with respect to the porphyrin and trityl ring planes in the energy minimum, while the J distribution was rationalized by the temperature weighted rotation of the phenyl bridge about the molecular axis connecting both spin centers. This study exemplifies the importance of molecular dynamics for the homogeneity (or heterogeneity) of the magnetic properties of trityl-based systems.

Synthesis and Redox Properties of Water-Soluble Asymmetric Trityl Radicals.

Tetrathiatriarylmethyl (trityl) radicals have been recently shown to react with biological oxidoreductants including glutathione (GSH), ascorbic acid (Asc), and superoxide anion radical (O2•-). However, how the substituents affect the reactivity of trityl radicals is still unknown. In this work, five asymmetric trityl radicals were synthesized and their reactivities with GSH, Asc, and O2•- investigated. Under aerobic conditions, GSH induces fast decays for the thioether- (TSA) and N-methyleneglycine-substituted (TGA) derivatives and slow decay for the 4-carboxyphenyl-containing one (TPA). Under anaerobic conditions, the direct reduction of these radicals by GSH also occurs with rate constants (kGSH) from 1.8 × 10-4 M-1 s-1 for TPA to 1.0 × 10-2 M-1 s-1 for TGA. Moreover, these radicals can also react with O2•- with rate constants (kSO) from 1.2 × 103 M-1 s-1 for ET-01 to 1.6 × 104 M-1 s-1 for TGA. Surprisingly, these radicals are completely inert to Asc in both aerobic and anaerobic conditions. Additionally, the substituents exert an important effect on redox potentials of these trityl radicals. This work demonstrates that the redox properties of the trityl radicals strongly depend on their substituents, and TPA with high stability toward GSH shows great potential for intracellular applications.

Novel Acetylene Derivatives of Stable Tetrathiatriarylmethyl Radicals

Novel Acetylene Derivatives of Stable Tetrathiatriarylmethyl Radicals

Synthesis, Characterization, and Application of a Highly Hydrophilic Triarylmethyl Radical for Biomedical EPR.

Stable tetrathiatriarylmethyl radicals have significantly contributed to the recent progress in biomedical electron paramagnetic resonance (EPR) due to their unmatched stability in biological media and long relaxation times. However, the lipophilic core of the most commonly used structure (Finland trityl) is responsible for its interaction with plasma biomacromolecules, such as albumin, and self-aggregation at high concentrations and/or low pH. While Finland trityl is generally considered inert toward many reactive radical species, we report that sulfite anion radical efficiently substitutes the three carboxyl moieties of Finland trityl with a high rate constant of 3.53 × 108 M-1 s-1, leading to a trisulfonated Finland trityl radical. This newly synthesized highly hydrophilic trityl radical shows an ultranarrow linewidth (ΔBpp = 24 mG), a lower affinity for albumin than Finland trityl, and a high aqueous solubility even at acidic pH. Therefore, this new tetrathiatriarylmethyl radical can be considered as a superior spin probe in comparison to the widely used Finland trityl. One of its potential applications was demonstrated by in vivo mapping oxygen in a mouse model of breast cancer. Moreover, we showed that one of the three sulfo groups can be easily substituted with S-, N-, and P-nucleophiles, opening access to various monofunctionalized sulfonated trityl radicals.

Understanding the g-tensors of perchlorotriphenylmethyl and Finland-type trityl radicals.

The 285 GHz EPR spectra of perchlorotriphenylmethyl and tetrathiatriarylmethyl radicals in frozen solution have been accurately measured. The relationship between their molecular structures and their g-tensors has been investigated with the aid of DFT calculations, revealing that the degree of spin density delocalization away from the central methylene carbon is an important determining factor of the g-anisotropy. In particular, the small amount of spin densities on the Cl or S heteroatoms at the 2 and 6 positions with respect to the central carbon have the strongest influence. Furthermore, the amount of spin densities on these heteroatoms and thus the anisotropy can be modulated by the protonation (esterification) state of the carboxylate groups at the 4 position. These results provide unique insights into the g-anisotropy of persistent trityl radicals and how it can be tuned.

Synthesis of Central Chirality-Containing Triarylmethanols and Triarylmethyl Radicals with Extraordinarily Stable Configurations.

Triarylmethanol adopts a propeller-shaped conformation with either right-handed (P) or left-handed (M) configuration. Herein, new triarylmethanols with two chiral centers were obtained via introduction of two cis-hydroxyl groups on the side chains, affording four stereoisomers. These four stereoisomers were easily separated by silica gel column chromatography into two pairs of propeller-shaped enantiomers, as shown by NMR and X-ray crystallographic studies. High-performance liquid chromatography (HPLC) studies showed that the configurations of the hydroxyl-bearing triarylmethanols are much more stable than those of the bulky tert-butyldimethylsilyl-protected precursors, inconsistent with the general strategy in which the steric repulsion is largely responsible for the configurational stability. Similarly, two hydroxyl-bearing tetrathiatriarylmethyl (TAM) radicals also exhibit excellent configurational stability and are thus separable by CS-HPLC into four stereoisomers. Interestingly, both helical chirality from triaryl group (M or P) and central chirality (R and S) on the side chain have little effect on their electron paramagnetic resonance properties. Our present study provides a new strategy to construct configurationally stable triaryl compounds and demonstrates that the side chain on TAM radicals is a new site for their structural modifications.

Synthesis and Characterization of Hydrophilic Trityl Radical TFO for Biomedical and Biophysical Applications.

Tetrathiatriarylmethyl (TAM, trityl) radicals have found wide applications as spin probes/labels for EPR spectroscopy and imaging, and as polarizing agents for dynamic nuclear polarization. The high hydrophilicity of TAM radicals is essential for their biomedical applications. However, the synthesis of hydrophilic TAM radicals (e.g., OX063) is extremely challenging and has only been reported in the patent literature, to date. Herein, an efficient synthesis of a highly water-soluble TAM radical bis(8-carboxyl-2,2,6,6-tetramethylbenzo[1,2-d:4,5-d']bis([1,3]dithiol-4-yl)-mono-(8-carboxyl-2,2,6,6-tetrakis(2-hydroxyethyl)benzo[1,2-d:4,5-d']bis([1,3]dithiol-4-yl)methyl (TFO), which contains four additional hydroxylethyl groups, relative to the Finland trityl radical CT-03, is reported. Similar to OX063, TFO exhibits excellent properties, including high water solubility in phosphate buffer, low log P, low pKa , long relaxation times, and negligible binding with bovine serum albumin. On the other hand, TFO has a sharper EPR line and higher O2 sensitivity than those of OX063. Therefore, in combination with its facile synthesis, TFO should find wide applications in magnetic resonance related fields and this synthetic approach would shed new light on the synthesis of other hydrophilic TAM radicals.

Dextran-conjugated tetrathiatriarylmethyl radicals as biocompatible spin probes for EPR spectroscopy and imaging.

Tetrathiatriarylmethyl (TAM) radicals represent soluble paramagnetic probes for biomedical electron paramagnetic resonance (EPR)-based spectroscopy and imaging. There is an increasing demand in the development of multifunctional, biocompatible and targeted trityl probes hampered by the difficulties in derivatization of the TAM structure. We proposed a new straightforward synthetic strategy using click chemistry for the covalent conjugation of the TAM radical with a water-soluble biocompatible carrier exemplified here by dextran. A set of dextran-grafted probes varied in the degrees of Finland trityl radical loading and dextran modification by polyethelene glycol has been synthesized. The EPR spectrum of the optimized macromolecular probe exhibits a single narrow line with high sensitivity to oxygen and has advantages over the unbound Finland trityl of being insensitive to interactions with albumin. In vivo EPR imaging of tissue oxygenation performed in breast tumor-bearing mouse using dextran-grafted probe demonstrates its utility for preclinical oximetric applications.

New Spin Probes: Tri- and Hexacationic Derivatives of Stable Tetrathiatriarylmethyl Radicals

The reaction of Finland trityl with tertiary aliphatic alcohols containing a terminal tertiary amino group, followed by quaternization of the amino groups, afforded new highly polar tri- and hexacationic derivatives. The obtained compounds are characterized by a sharp singlet signal in the EPR spectrum and are soluble in water over a wide pH range.

C-C Cross-Coupling Reactions of Trityl Radicals: Spin Density Delocalization, Exchange Coupling, and a Spin Label.

Organic radicals are usually highly reactive and short-lived species. In contrast, tetrathiatriarylmethyl radicals, the so-called trityl- or TAM-radicals, are stable and do survive over longer times even under in-cell conditions. In addition, they show strong EPR signals, have long phase memory times at room temperature, and are reporters on local oxygen and proton concentrations. These properties facilitated their use for magnetic resonance imaging, dynamic nuclear polarization, and spin-labeling EPR under in-cell conditions. Thus, synthetic approaches are required for functionalization of TAM radicals tailored to the desired application. However, most TAM derivatives reported in the literature are based on esterification of the Finland trityl, which is prone to hydrolysis. Here, we report on an approach in which TAM is site-selective iodinated and subsequently C-C cross-coupled to various building blocks in a modular approach. This yields conjugated trityl compounds such as a trityl attached to a porphyrin, an alkinyl functionalized trityl radical, and a strongly exchange-coupled trityl biradical. This synthesis approach thus has implications not only for magnetic resonance spectroscopy but also for the design of molecular magnets or quantum computing devices.

Synthesis of hydroxyethyl tetrathiatriarylmethyl radicals OX063 and OX071.

We report the synthesis of hydroxyethyl tetrathiatriarylmethyl radical OX063 and its deuterated analogue OX071 for biomedical EPR applications.

Preparation and evaluation of trityl-loaded lipid nanocapsules as oxygen sensors for electron paramagnetic resonance oximetry

Oxygen is essential in physiology and pathophysiology. Electron paramagnetic resonance (EPR) oximetry, using oxygen sensitive paramagnetic materials, could be attractive for measuring oxygen in tissues. The aim of the present study was to assess the properties of lipid nanocapsules (LNCs) loaded with the nitroxide tempo-benzoate (TB) or tetrathiatriarylmethyl (TAM) radicals. LNCs loaded with the EPR probes were successfully prepared by the phase inversion process leading to nanocapsules of about 60 nm. LNCs protected the TB radical against reduction in vitro. The calibration of the EPR line width (LW) as a function of the pO2 showed a two-fold increase in sensitivity with TAM-LNC compared to hydrophilic trityl radical. The TAM-LNCs were evaluated in vivo. Contrarily to unencapsulated TAM, for which a rapid decrease in EPR signal was observed, the half-life of TAM-LNCs administered in muscles or in tumours exceeded an hour. Carbogen-challenges in mice demonstrated that the TAM-LNCs responded well to changes in oxygen environment. However, the apparent pO2 values acquired were higher than the expected physiological values. These results warrant further investigation in the formulation of stable nano-objects encapsulating EPR oxygen sensitive probes.

Synthesis and Characterization of the Perthiatriarylmethyl Radical and Its Dendritic Derivatives with High Sensitivity and Selectivity to Superoxide Radical.

EPR spectroscopy, coupled with the use of tetrathiatriarylmethyl (TAM) radicals, has been a reliable method to detect the superoxide radical (O2.- ). However, the specificity and biocompatibility of TAM radicals need to be further improved. Although derivatization may overcome the drawbacks of current TAM radicals, esterification or amidation through the carboxylic groups greatly changes their redox properties and makes them inert to O2.- . Herein, the synthesis of a perthiatriarylmethyl (PST) radical and its dendritic derivatives, PST-TA and PST-NA, in which PST is covalently linked with dendrons containing three (TA) and nine (NA) carboxylic acids, respectively. The results show that PST rapidly reacts with O2.- to yield a unique quinone methide product. Dendritic modification of PST slightly decreases the reactivities of PST-TA and PST-NA, but notably increases their biostability toward various oxidoreductants. The detection limit of PST-NA to O2.- was estimated to be 2.1 nm min-1 over 60 min of detection. Importantly, PST-NA shows threefold higher sensitivity to O2.- in the presence and absence of ascorbic acid than that of the classic spin-trapping technique. In addition, the application of PST-NA to detect extracellular O2.- generation in stimulated RAW 264.7 macrophages was also explored. This study demonstrates that PST-NA has great potential for specific detection and quantitation of O2.- in extracellular sites.

Synthesis and Characterization of PEGylated Trityl Radicals: Effect of PEGylation on Physicochemical Properties.

Tetrathiatriarylmethyl (TAM, trityl) radicals have attracted considerable attention as spin probes for biological electron paramagnetic resonance (EPR) spectroscopy and imaging owing to their sharp EPR singlet signals and high biostability. However, their in vivo applications were limited by the short blood circulation lifetimes and strong binding with albumins. Our previous results showed that PEGylation is a feasible method to overcome the issues facing in vivo applications of TAM radicals. In the present study, we synthesized a series of new PEGylated TAM radicals (TTP1, TPP2, TNP1, TNP2, d-TNP1, and d-TNP3) containing various lengths and numbers of mPEG chains. Our results found that the pattern of PEGylation exerts an important effect on physicochemical properties of the resulting TAM radicals. Dendritic PEGylated TAM radicals, TNP1 and TNP2, have higher water solubility and lower susceptibility for self-aggregation than their linear analogues TPP1 and TPP2. Furthermore, dendritic PEGylated TAM radicals exhibit extremely high stability toward various biological oxidoreductants as well as in rat whole blood, liver homogenate, and following in vivo intravenous administration in mice. Importantly, the deuterated derivatives, especially d-TNP3, exhibit excellent properties including the sharp and O2-sensitive EPR singlet signal, good biocompatibility, and prolonged kinetics with half-life time of ≥10 h in mice. These PEGylated TAM radicals should be suitable for a wide range of applications in in vivo EPR spectroscopy and imaging.

Nitro-Triarylmethyl Radical as Dual Oxygen and Superoxide Probe.

Superoxide radical is involved in numerous physiological and pathophysiological processes. Tetrathiatriarylmethyl (TAM) radicals are known to react with superoxide allowing measurement of superoxide production in biological media. We report the synthesis of a Nitro conjugated TAM radical showing a rate constant of 7×105M-1s-1 which is two order of magnitude higher than other TAMs, allowing high sensitivity measurement of superoxide.

Tetrathiatriarylmethyl Radicals Conjugated to an RGD‐Peptidomimetic

AbstractTetrathiatriarylmethyl (TAM) radicals are favourite spin labels for electron paramagnetic resonance imaging (EPRI). In this work, we report a straightforward synthesis of new TAM radicals bound to an Arg‐Gly‐Asp (RGD)‐peptidomimetic known to be a cell‐targeting agent. These new radicals are stable in solution, and sensitive to oxygen, and their experimental EPR data is consistent with density functional theory (DFT) calculations.

Synthesis of two persistent fluorinated tetrathiatriarylmethyl (TAM) radicals for biomedical EPR applications

Tetrathiatriarylmethyl radicals are attractive spin probes extensively used in biomedical magnetic resonance applications. We report a straightforward synthesis of two original tetrathiatriarylmethyl radicals incorporating, respectively, 15 and 45 fluorine atoms, and thus possessing a high affinity to fluorous media. F15T-03 and F45T-03 exhibit a single sharp EPR spectrum and their EPR line broadening is highly sensitive to molecular oxygen. These spin probes are specially designed for assessment of tumor oxygenation using perfluorocarbon formulations.

Synthesis and Characterization of Ester-Derivatized Tetrathiatriarylmethyl Radicals as Intracellular Oxygen Probes

Electron paramagnetic resonance (EPR) spectroscopy using paramagnetic probes has been employed as an important tool for the accurate determination of oxygen (O2) concentrations in biological systems. However, paramagnetic probes are still limited by their intracellular penetrability. Various esterified trityl derivatives were synthesized and characterized, and an X-ray structure of one of the triyl radicals was determined. The ester-derivatized trityls exhibited higher sensitivity to O2 concentration compared to the trityl tricarboxylate CT-03. Cyclic voltammetry was also carried out to assess the susceptibility of the trityl radicals to oxidation and reduction. Among all of the ester-derivatized trityls studied, facile hydrolysis of the acetoxymethoxy esters to the respective carboxylate was observed using porcine liver esterase. This study demonstrates that cellular permeability of the trityl radicals can be achieved by varying the type and number of ester groups. Therefore, ester-derivatized trityl radicals show great potential as intracellular EPR oximetry probes and imaging agents.

Large-scale synthesis of a persistent trityl radical for use in biomedical EPR applications and imaging

Tetrathiatriarylmethyl radicals are ideal spin probes for biological electron paramagnetic resonance (EPR) spectroscopy and imaging. The wide application of trityl radicals as biosensors of oxygen or other biological radicals was hampered by the lack of affordable large-scale syntheses. We report the large-scale synthesis of the Finland trityl radical using an improved addition protocol of the aryl lithium monomer to methylchloroformate. A new reaction for the formal one-electron reduction of trityl alcohols to trityl radicals using neat trifluoroacetic acid is reported as well. Initial applications show that the compound is very sensitive to molecular oxygen. It has already provided high-resolution EPR images on large aqueous samples and should be suitable for a broad range of in vivo applications.

Reactivity of Molecular Oxygen with Ethoxycarbonyl Derivatives of Tetrathiatriarylmethyl Radicals

Tetrathiatriarylmethyl (TAM) radicals are commonly used as oximetry probes for electron paramagnetic resonance imaging applications. In this study, the electronic properties and the thermodynamic preferences for O2 addition to various TAM-type triarylmethyl (trityl) radicals were theoretically investigated. The radicals' stability in the presence of O2 and biological milieu was also experimentally assessed using EPR spectroscopy. Results show that H substitution on the aromatic ring affects the trityl radical's stability (tricarboxylate salt 1-CO2Na > triester 1-CO2Et > diester 2-CO2Et > monoester 3-CO2Et) and may lead to substitution reactions in cellular systems. We propose that this degradation process involves an arylperoxyl radical that can further decompose to alcohol or quinone products. This study demonstrates how computational chemistry can be used as a tool to rationalize radical stability in the redox environment of biological systems and aid in the future design of more biostable trityl radicals.