Yangping Liu

Pulsed ESR dipolar spectroscopy for distance measurements in immobilized spin labeled proteins in liquid solution.

Pulsed electron spin resonance (ESR) dipolar spectroscopy (PDS) in combination with site-directed spin labeling is unique in providing nanometer-range distances and distributions in biological systems. To date, most of the pulsed ESR techniques require frozen solutions at cryogenic temperatures to reduce the rapid electron spin relaxation rate and to prevent averaging of electron-electron dipolar interaction due to the rapid molecular tumbling. To enable measurements in liquid solution, we are exploring a triarylmethyl (TAM)-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. In this preliminary study, TAM radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or 65 and 76 in T4 lysozyme immobilized on Sepharose. Interspin distances determined using double quantum coherence (DQC) in solution are close to those expected from models, and the narrow distance distribution in each case indicates that the TAM-based spin label is relatively localized.

Efficient Dynamic Nuclear Polarization at 800 MHz/527 GHz with Trityl-Nitroxide Biradicals

Cross-effect (CE) dynamic nuclear polarization (DNP) is a rapidly developing technique that enhances the signal intensities in magic-angle spinning (MAS) NMR spectra. We report CE DNP experiments at 211, 600, and 800 MHz using a new series of biradical polarizing agents referred to as TEMTriPols, in which a nitroxide (TEMPO) and a trityl radical are chemically tethered. The TEMTriPol molecule with the optimal performance yields a record (1) H NMR signal enhancement of 65 at 800 MHz at a concentration of 10 mM in a glycerol/water solvent matrix. The CE DNP enhancement for the TEMTriPol biradicals does not decrease as the magnetic field is increased in the manner usually observed for bis-nitroxides. Instead, the relatively strong exchange interaction between the trityl and nitroxide moieties determines the magnetic field at which the optimum enhancement is observed.

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.

Esterified Trityl Radicals as Intracellular Oxygen Probes

Triarylmethyl (trityl) radicals exhibit high stability and narrow linewidth under physiological conditions which provide high sensitivity and resolution for the measurement of O2 concentrations, making them attractive as EPR oximetry probes. However, the application of previously available compounds has been limited by their poor intracellular permeability. We recently reported the synthesis and characterization of esterified trityl radicals as potential intracellular EPR probes and their oxygen sensitivity, redox properties, and enzyme-mediated hydrolysis were investigated. In this paper, we report the cellular permeability and stability of these trityls in the presence of bovine aortic endothelial cells. Results show that the acetoxymethoxycarbonyl-containing trityl AMT-02 exhibits high stability in the presence of cells and can be effectively internalized. The intracellular hydrolysis of AMT-02 to the carboxylate form of the trityl (CT-03) was also observed. In addition, this internalized trityl probe was applied to measure intracellular O2 concentrations and the effects of menadione and KCN on the rates of O2 consumption in endothelial cells. This study demonstrates that these esterified trityl radicals can function as effective EPR oximetry probes measuring intracellular O2 concentration and consumption.

Fast Reactivity of a Cyclic Nitrone−Calix[4]pyrrole Conjugate with Superoxide Radical Anion: Theoretical and Experimental Studies†

Nitrone spin traps have been employed as probes for the identification of transient radical species in chemical and biological systems using electron paramagnetic resonance (EPR) spectroscopy and have exhibited pharmacological activity against oxidative-stress-mediated diseases. Since superoxide radical anion (O2(•-)) is a major precursor to most reactive oxygen species and calix[4]pyrroles have been shown to exhibit high affinity to anions, a cyclic nitrone conjugate of calix[4]pyrrole (CalixMPO) was designed, synthesized, and characterized. Computational studies at the PCM/B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) level suggest a pendant-type linkage between the calix[4]pyrrole and the nitrone to be the most efficient design for spin trapping of O2(•-), giving exoergic reaction enthalpies (ΔH(298K,aq)) and free energies (ΔG(298K,aq)) of -16.9 and -2.1 kcal/mol, respectively. (1)H NMR study revealed solvent-dependent conformational changes in CalixMPO leading to changes in the electronic properties of the nitronyl group upon H-bonding with the pyrrole groups as also confirmed by calculations. CalixMPO spin trapping of O2(•-) exhibited robust EPR spectra. Kinetic analysis of O2(•-) adduct formation and decay in polar aprotic solvents using UV-vis stopped-flow and EPR methods gave a larger trapping rate constant for CalixMPO and a longer half-life for its O2(•-) adduct compared to the commonly used nitrones. The unusually high reactivity of CalixMPO with O2(•-) was rationalized to be due to the synergy between the α-effect and electrostatic effect by the calix[4]pyrrole moiety on O2(•-) and the nitrone, respectively. This work demonstrates for the first time the application of an anion receptor for the detection of one of the most important radical intermediates in biological and chemical systems (i.e., O2(•-)).

Synthesis of 14N- and 15N-labeled Trityl-nitroxide Biradicals with Strong Spin−Spin Interaction and Improved Sensitivity to Redox Status and Oxygen

Simultaneous evaluation of redox status and oxygenation in biological systems is of great importance for the understanding of biological functions. Electron paramagnetic resonance (EPR) spectroscopy coupled with the use of the nitroxide radicals have been an indispensable technique for this application but are still limited by low oxygen sensitivity and low EPR resolution in part due to the moderately broad EPR triplet and spin quenching through bioreduction. In this study, we showed that these drawbacks can be overcome through the use of trityl-nitroxide biradicals allowing for the simultaneous measurement of redox status and oxygenation. A new trityl-nitroxide biradical TNN14 composed of a pyrrolidinyl-nitroxide and a trityl and its isotopically labeled (15)N analogue TNN15 were synthesized and characterized. Both biradicals exhibited much stronger spin-spin interaction with J > 400 G compared with that of the previous synthesized trityl-nitroxide biradicals TN1 (∼160 G) and TN2 (∼52 G) with longer linker chain length. The enhanced stability of TNN14 was evaluated using ascorbate as reductant, and the effect of different types of cyclodextrins on its stability in the presence of ascorbate was also investigated. Both biradicals are sensitive to redox status, and their corresponding trityl-hydroxylamines resulting from the reduction of the biradicals by ascorbate share the same oxygen sensitivity. Of note is that the (15)N-labeled TNN15-H with an EPR doublet exhibits improved EPR signal amplitude as compared with that of TNN14-H with an EPR triplet. In addition, cyclic voltammetric studies verify the characteristic electrochemical behaviors of the trityl-nitroxide biradicals.

Tetrathiatriarylmethyl radical with a single aromatic hydrogen as a highly sensitive and specific superoxide probe

Superoxide (O(2)(•-)) plays crucial roles in normal physiology and disease; however, its measurement remains challenging because of the limited sensitivity and/or specificity of prior detection methods. We demonstrate that a tetrathiatriarylmethyl (TAM) radical with a single aromatic hydrogen (CT02-H) can serve as a highly sensitive and specific O(2)(•-) probe. CT02-H is an analogue of the fully substituted TAM radical CT-03 (Finland trityl) with an electron paramagnetic resonance (EPR) doublet signal due to its aromatic hydrogen. Owing to the neutral nature and negligible steric hindrance of the hydrogen, O(2)(•-) preferentially reacts with CT02-H at this site with production of the diamagnetic quinone methide via oxidative dehydrogenation. Upon reaction with O(2)(•-), CT02-H loses its EPR signal and this EPR signal decay can be used to quantitatively measure O(2)(•-). This is accompanied by a change in color from green to purple, with the quinone methide product exhibiting a unique UV-Vis absorbance (ε=15,900 M(-1) cm(-1)) at 540 nm, providing an additional O(2)(•-) detection method. More than five-fold higher reactivity of CT02-H for O(2)(•-) relative to CT-03 was demonstrated, with a second-order rate constant of 1.7×10(4) M(-1) s(-1) compared to 3.1×10(3) M(-1) s(-1) for CT-03. CT02-H exhibited high specificity for O(2)(•-) as evidenced by its inertness to other oxidoreductants. The O(2)(•-) generation rates detected by CT02-H from xanthine/xanthine oxidase were consistent with those measured by cytochrome c reduction but detection sensitivity was 10- to 100-fold higher. EPR detection of CT02-H enabled measurement of very low O(2)(•-) flux with a detection limit of 0.34 nM/min over 120 min. HPLC in tandem with electrochemical detection was used to quantitatively detect the stable quinone methide product and is a highly sensitive and specific method for measurement of O(2)(•-), with a sensitivity limit of ~2×10(-13) mol (10 nM with 20-μl injection volume). Based on the O(2)-dependent linewidth broadening of its EPR spectrum, CT02-H also enables simultaneous measurement of O(2) concentration and O(2)(•-) generation and was shown to provide sensitive detection of extracellular O(2)(•-) generation in endothelial cells stimulated either by menadione or with anoxia/reoxygenation. Thus, CT02-H is a unique probe that provides very high sensitivity and specificity for measurement of O(2)(•-) by either EPR or HPLC methods.

Lipophilic β-cyclodextrin cyclic-nitrone conjugate: Synthesis and spin trapping studies

Nitrone spin traps are commonly employed as probes for the identification of transient radicals in chemical and biological systems using electron paramagnetic resonance (EPR) spectroscopy. Nitrones have also found applications as therapeutic agent in the treatment of radical-mediated diseases. Therefore, a spin trap that incorporates high reactivity to superoxide radical anion (O2(*-)), more persistent superoxide adduct, enhanced bioavailability, and selective targeting in one molecular design is desirable. In this work, the synthesis of a nitrone spin trap, 4, that is tethered via amide bonds to a beta-cyclodextrin (beta-CD) and a dodecyl chain was achieved with the expectation that the beta-cyclodextrin would lead to increased reactivity to O2(*-) and persistent O2(*-) adduct while the lipophilic chain would impart membrane targeting property. The two constitutional racemic isomers, 4a and 4b, were separated using preparative HPLC, and structural analysis and self-aggregation properties were carried out using NMR, induced circular dichroism, dynamic light scattering, transmission electron microscopy, and computational approach. EPR spin trapping of O2(*-) by 4a and 4b was only successful in DMSO and not in an aqueous system, due most likely to the amphiphilic character of 4 that can favor conformations (or aggregation) hindering radical addition to nitrone. Kinetics of formation and decay of the 4a-O2H adduct in polar aprotic solvents show faster reactivity to O2(*-) and more persistent O2(*-) adduct compared to nitrones not conjugated to beta-CD. Computational analysis of 4a and 4b as well as 4a-OOH and 4b-OOH adducts were carried out, and results show that isomerism, both constitutional and stereochemical, affects the orientations of aminoxyl-NO and/or hydroperoxyl groups relative to the beta-CD annulus for optimal H-bond interaction and stability.

Trityl-nitroxide biradicals as unique molecular probes for the simultaneous measurement of redox status and oxygenation

Novel trityl-nitroxide biradicals were synthesized and exhibited enhanced sensitivity and stability for rapid and simultaneous measurement of redox status and oxygenation by electron paramagnetic resonance spectroscopy.

Highly stable dendritic trityl radicals as oxygen and pH probe

Novel dendritic trityl radicals (DTR1 and DTR2) with a TAM radical core, PAMAM branching and carboxylate exterior surface exhibit high stability towards oxidoreductants as evidenced by their electrochemical and EPR properties, offering potential application as dual oxygen and pH probe.