Molecular organization of DHP membrane fragments

The phase transition of dihexadecyl phosphate (DHP) bilayered disks has been studied using EPR spectroscopy. In the acid form of DHP, a phase transition temperature exists, that we have monitored through the spin-spin interaction between the nitroxide molecules at high concentration (8%) in DHP bilayers. This spin-spin interaction is due to the gathering of solutes in a fluid defect of the membrane: the border. The fluorescence quenching of two probes by the nitroxide stearic acids in DHP bilayers has been studied by stationary and time-resolved fluorescence measurements. The quenching process is mainly static. Both magnetic and fluorescent probes are localized in the periphery of the bilayered disks. An erratum to this article is available at .     

Structural and atoms-in-molecules analysis of hydrogen-bond network around nitroxides in liquid water

In this study, we investigated the hydrogen-bond network patterns involving the NO moieties of five small nitroxides in liquid water by analyzing nanosecond scale molecular dynamics trajectories. To this end, we implemented two types of hydrogen-bond definitions, based on electronic structure, using Bader's atoms-in-molecules analysis and based on geometric criteria. In each definition framework, the nitroxide/water hydrogen-bond networks appear very variable from a nitroxide to another. Moreover, each definition clearly leads to a different picture of nitroxide hydration. For instance, the electronic structure-based definition predicts a number of hydrogen bonds around the nitroxide NO moiety usually larger than geometric structure-based ones. One particularly interesting result is that the strength of a nitroxide/water hydrogen bond does not depend on its linearity, leading us to question the relevance of geometric definition based on angular cutoffs to study this type of hydrogen bond. Moreover, none of the hydrogen-bond definitions we consider in the present study is able to quantitatively correlate the strength of nitroxide/water hydrogen-bond networks with the aqueous nitroxide spin properties. This clearly exhibits that the hydrogen-bonding concept is not reliable enough to draw quantitative conclusions concerning such properties.     

Orientational motion of nitroxides in molecular glasses: dependence on the chemical structure, on the molecular size of the probe, and on the type of the matrix

Echo detected electron paramagnetic resonance (EPR) study of orientational molecular motion of nitroxide spin probes in glassy solvents was performed by evaluating the anisotropic transverse relaxation rate 1/T(2) at different positions of the EPR spectrum. Experiments were done on nitroxides of different sizes and shapes, in different solvent glasses, with different deuteration degree, and at different temperatures. We found that the properties of the solvent glass have a much stronger impact on the relaxation rate than the size and shape of the nitroxide have. We concluded that the anisotropic relaxation is induced by reordering of the solvent cage and not by small angle fluctuations of the nitroxide in the cage or intramolecular motion of nitroxide.     

Electrostatic influence on rotational mobilities of sol-gel-encapsulated solutes by NMR and EPR spectroscopies

The rotational mobilities of small solute molecules encapsulated in tetramethyl orthosilicate (TMOS) sol-gels have been investigated by EPR spectroscopy of encapsulated nitroxide probes and by high-resolution NMR spectroscopic measurements of transferred NOE's (trNOE's), of T(1)'s, and of T(1)'s in the rotating frame (T(1)rho). The two spectroscopic methods are sensitive to motions on different time scales and hence, are nicely complementary. Suites of neutral, positively, and negatively charged nitroxide probes (EPR) and of simple diamagnetic small molecules (NMR) were selected to disclose influences of electrostatic interactions with the sol-gel walls and to probe the presence of multiple populations of molecules in distinct regions of the sol-gel pores. For neutral and negatively charged solute probes, both techniques disclose a single population with a significantly increased average rotational correlation time, which we interpret at least in part as resulting from exchange between free-volume and transiently immobilized surface populations. The electrostatic attraction between cationic probes and the negatively charged sol-gel walls causes the positively charged probes to be more effectively immobilized and/or causes a greater percentage of probes to undergo this transient immobilization. The EPR spectra directly disclose a population of cationic probes which are immobilized on the X-band EPR time scale: tau(c) greater than or approximately equal 10(-7) s. However, NMR measurements of trNOE's and of T(1)rho demonstrate that this population does exchange with the free-volume probes on the slower time scale of NMR. This approach is equally applicable to the study of solutes within other types of confined spaces, as well.     

Nitrogen nuclear spin flips in nitroxide spin probes of different sizes in glassy o-terphenyl: possible relation with α- and β-relaxations

The pulsed electron-electron double resonance (ELDOR) technique was employed to study nitroxide spin probes of three different sizes dissolved in glassy o-terphenyl. A microwave pulse applied to the central hyperfine structure (hfs) component of the nitroxide electron paramagnetic resonance spectrum was followed by two echo-detecting pulses of different microwave frequency to probe the magnetization transfer (MT) to the low-field hfs component. The MT between hfs components is readily related to flips in the nitrogen nuclear spin, which in turn are induced by molecular motion. The MT on the time scale of tens of microseconds was observed over a wide temperature range, including temperatures near and well below the glass transition. For a bulky nitroxide, it was found that MT rates approach dielectric α (primary) relaxation frequencies reported for o-terphenyl in the literature. For small nitroxides, MT rates were found to match the frequencies of dielectric β (secondary) Johari-Goldstein relaxation. The most probable motional mechanism inducing the nitrogen nuclear spin flips is large-angle angular jumps, between some orientations of unequal occupation probabilities. The pulsed ELDOR of nitroxide spin probes may provide additional insight into the nature of Johari-Goldstein relaxation in glassy media and may serve as a tool for studying this relaxation in substances consisting of non-rigid molecules (such as branched polymers) and in heterogeneous and non-polar systems (such as a core of biological membranes).     

Separation of polyelectrolyte chain dynamics and dynamics of counterion attachment by EPR spectroscopy

Rotational dynamics and local enrichment of counterions close to polyelectrolyte chains were studied by EPR spectroscopy in solvents of different viscosity. The results confirm previous findings (D. Hinderberger, G. Jeschke, and H. W. Spiess, Macromolecules 2002, 35, 9698) that electrostatic attachment of counterions to the chains is dynamic with lifetimes of contact ion pairs shorter than 1 ns. While in low-viscosity solvents linewidths for a dianionic nitroxide probe and their dependence on polyelectrolyte concentration are dominated by the gradient of local concentration in the vicinity of the chain, they are more strongly influenced by changes in rotational dynamics in a glycerol/water mixture. The slowdown of dynamics at higher viscosity strongly depends on polyelectrolyte concentration, suggesting that the lifetime of the attached state increases. The linewidths of trianionic triarylmethyl probes and of the center line of the nitroxide probes are dominated by local counterion enrichment both at low and high viscosity. Comparison of these linewidths and of the extent to which the lineshapes are non-Lorentzian indicates build-up of larger concentration gradients at higher viscosity.     

Polyaromatic Profluorescent Nitroxide Probes with Enhanced Photostability

Novel profluorescent mono‐ and bis‐isoindoline nitroxides linked to napthalimide and perylene diimide structural cores are described. These nitroxide‐fluorophore probes display strongly suppressed fluorescence in comparison to their corresponding non‐radical diamagnetic methoxyamine derivatives. The perylene‐based probe possessing two isoindoline systems tethered through ethynyl linkages was shown to be the most photostable in solution, demonstrating significantly enhanced longevity over the 9,10‐bis(phenylethynyl)anthracene fluorophore used in previous profluorescent nitroxide probes.     

Photophysical properties of the prefluorescent nitroxide probes QT and C343T

The photophysical properties of the nitroxide prefluorescent probes 4-(3-hydroxy-2-methyl-4-quinolinoyloxy)-2,2,6,6-tetramethyl-piperidin-4-yl) ester free radical (QT) and 2,3,4,6,7,8-hexahydro-quinolizino [1,10,9-gh] coumarin-3-carboxylic acid (1-oxyl-2,2,6,6-tetramethyl-piperidin-4-yl) ester free radical (C343T) were evaluated as a function of pH and solvent properties. The absorbance of QT showed high pH sensitivity. The pKa values for the different ionization forms involved in the acid-base equilibrium of the quinoline chromophore were determined in the ground and excited states. The fluorescence lifetimes of QT, and N-hydroxylamine (QTH) and quinoline methyl ester (QMe) derivatives, showed that the intramolecular quenching efficiency by the nitroxide moiety is independent of the quinoline ionization form. The fluorescence and absorbance of C343T were highly sensitive to solvent polarity in agreement with a charged transfer excited state of the chromophore. However, we noted a decrease in the intramolecular fluorescence quenching efficiency by the nitroxide moiety when changing the polarity of the solvent from hexane to water. This behavior has been attributed to a suppression of an energy transfer mechanism in the nitroxide quenching process in very polar solvents. The results obtained in micelles allow us to propose QT and C343T as sensors for pH and micropolarity, respectively, in addition to their role as monitors for free radicals or hydrogen transfer from phenols.     

A Double‐Armed,Hydrophilic Transition Metal Complex as a Paramagnetic NMR Probe

Synthetic metal complexes can be used as paramagnetic probes for the study of proteins and protein complexes. Herein, two transition metal NMR probes (TraNPs) are reported. TraNPs are attached through two arms to a protein to generate a pseudocontact shift (PCS) using cobalt(II), or paramagnetic relaxation enhancement (PRE) with manganese(II). The PCS analysis of TraNPs attached to three different proteins shows that the size of the anisotropic component of the magnetic susceptibility depends on the probe surroundings at the surface of the protein, contrary to what is observed for lanthanoid‐based probes. The observed PCS are relatively small, making cobalt‐based probes suitable for localized studies, such as of an active site. The obtained PREs are stronger than those obtained with nitroxide spin labels and the possibility to generate both PCS and PRE offers advantages. The properties of TraNPs in comparison with other cobalt‐based probes are discussed.     

A Double‐Armed,Hydrophilic Transition Metal Complex as a Paramagnetic NMR Probe

Synthetic metal complexes can be used as paramagnetic probes for the study of proteins and protein complexes. Herein, two transition metal NMR probes (TraNPs) are reported. TraNPs are attached through two arms to a protein to generate a pseudocontact shift (PCS) using cobalt(II), or paramagnetic relaxation enhancement (PRE) with manganese(II). The PCS analysis of TraNPs attached to three different proteins shows that the size of the anisotropic component of the magnetic susceptibility depends on the probe surroundings at the surface of the protein, contrary to what is observed for lanthanoid‐based probes. The observed PCS are relatively small, making cobalt‐based probes suitable for localized studies, such as of an active site. The obtained PREs are stronger than those obtained with nitroxide spin labels and the possibility to generate both PCS and PRE offers advantages. The properties of TraNPs in comparison with other cobalt‐based probes are discussed.     

Self-assembly of beta-cyclodextrin in water. 2. Electron spin resonance

The interaction of amphipilic spin labels with beta-cyclodextrin was investigated using conventional electron spin resonance (ESR) spectroscopy to explore the aggregation of cyclodextrins in water. Methyl 5-doxylstearate (5-DMS) and stearic acid spin probes (n-DSA), which contain a cyclic nitroxide moiety with unpaired electrons covalently linked to the aliphatic chain carbon in positions 5, 7, 12, and 16, show that different dynamic domains coexist in beta-CD water solutions above 3 mM. The results are consistent with the formation of beta-CD self-assembled structures in water above a critical aggregation concentration and confirm the previous findings that were reported in the part 1 article of this series.     

Chemical surface modification of self-assembled monolayers by radical nitroxide exchange reactions

This article describes the application of nitroxide exchange reactions of surface-bound alkoxyamines as a tool for reversible chemical modification of self-assembled monolayers (SAMs). This approach is based on radical chemistry, which allows for introduction of various functional groups and can be used to reversibly introduce functionalities at surfaces. To investigate the scope of this surface chemistry, alkoxyamines with different functionalities were synthesized and were then applied to the immobilization of, for example, dyes, sugars, or biotin. Surface analysis was carried out by contact angle, X-ray photoelectron spectroscopy, and fluorescence microscopy measurements. The results show that this reaction is highly efficient, reversible, and mild and allows for immobilization of various sensitive functional groups. In addition, Langmuir-Blodgett lithography was used to generate structured SAMs. Site-selective immobilization of a fluorescent dye could be achieved by nitroxide exchange reactions.     

Distinctive pressure effects on partitioning and rotational motions of nitroxide spin probes in dispersions of a triglyceride membrane

The effects of pressure were examined for the partitioning and rotational correlation time of nitroxide probes, TEMPO and DTBN, in dispersions of a triglyceride membrane. As external pressures increased, nitroxide probes shifted from the vesicle phase to the aqueous phase. Rotational correlation times of TEMPO and DTBN in the vesicle phase, determined from ESR line broadening, increased remarkably with increasing pressure, dependent of the molecular size of the nitroxide probes: ΔV^‡ = 65.6 cm³ mol⁻¹ for TEMPO and 24.4 cm³ mol⁻¹ for DTBN. Based on the results, the microscopic properties inside the triglyceride membrane are discussed.     

O2 penetration and proton burial depth in proteins: applicability to fold family recognition

Paramagnetically induced relaxation effects of O2 and the nitroxide 4-hydroxy TEMPO were measured for the amide protons of perdeuterated rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium pasteurianum. For both O2 and the impermeant nitroxide, the induced relaxation at the static solvent inaccessible amide sites is dominated by long-range interactions with the paramagnetic species in the bulk aqueous phase. The upper bound of O2 solubility in the internal matrix of the rubredoxins is one-tenth that of the bulk aqueous phase. Furthermore, the difference between the oxygen solubilities inside the two rubredoxins is at most 1% that of bulk water O2 solubility, suggesting that there are only modest differences in this measure of fluidity for the mesophile vs hyperthermophile protein interiors. Calculations based on the assumption of a paramagnet uniformly distributed on the protein exterior yield accurate predictions at nearly all amide sites for the minimum relaxation value observed from either the O2 or nitroxide data. Model calculations indicate that the readily obtained paramagnetically induced relaxation effects should prove effective in recognition of structural homology for proteins that are too widely diverged for sequence-based recognition.     

Microwave-assisted synthesis of amphiphilic spin probes

New amphiphilic nitroxide spin probes have been synthesized. The key reaction is based on microwave-assisted epoxide ring opening with amines as nucleophiles using calcium trifluoromethanesulfonate as a catalyst. High yields, in short reaction times, were obtained without any detectable nitroxide decomposition.     

Development of nitroxide‐based theranostic compounds that act both as anti‐inflammatory drugs and brain redox imaging probes in MRI

Theranostic probes provide both therapeutic and diagnostic imaging capabilities in one molecule and show significant promise for use in magnetic resonance imaging (MRI) examinations. The present study describes for the first time the synthesis and utility of nitroxide‐based contrast agents exhibiting a nonsteroidal anti‐inflammatory drug effect. The target theranostic probes were prepared by connecting the carboxyl group of ibuprofen or ketoprofen to the hydroxyl group of 3‐hydroxymethyl‐2,2,5,5‐tetramethylprrolidine‐1‐oxyl by a condensation reaction in the presence of dicyclohexylcarbodiimide and 4‐dimethylaminopyridine in dichloromethane. MRI of mouse heads after administration of either synthesized theranostic probe indicated that the probes enter the brain by passing through the blood–brain barrier (BBB), resulting in T1 contrast enhancement in mouse brain. This enhancement persisted for the duration of the half‐life of about 40 min, which is longer than that obtained by most of pyrrolidine nitroxide molecules. The therapeutic capacities of these theranostic probes were examined using a lipopolysaccharide (LPS)‐induced brain inflammation model. The production of nitric oxide, an inflammation marker in septic mouse brain induced by LPS, was remarkably inhibited by the addition of either synthesized probe, indicating that they also act as anti‐inflammatory drugs. The present results indicate that nitroxide‐based theranostic probes act as both BBB‐permeable redox‐sensitive contrast agents and as an anti‐inflammatory drug in septic mouse brain. Copyright © 2016 John Wiley & Sons, Ltd.     

Electron spin exchange in isoviscous solvent mixtures

Electron spin exchange rate constants and hyperfine coupling constants have been measured for two nitroxide spin probes in a number of isoviscous mixed solvents. Collision rate constants, normalized to 1 cP, are lower for solvents in which the major component is water. Further anomalies in the coupling constant for the systemtert-butanol-water are explained in terms of the marked concentration fluctuations known to occur in this solvent mixture. Substitution of a hydrophobic spin probe by one containing an –OH group leads to lowering of the spin exchange rate, possibly due to solvation of the probe.     

Two-photon fluorescence microscopy imaging of cellular oxidative stress using profluorescent nitroxides

A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore's usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H(2)O(2)-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially "light up" in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration.     

gem-Diethyl Pyrroline Nitroxide Spin Labels: Synthesis,EPR Characterization,Rotamer Libraries and Biocompatibility

The availability of bioresistant spin labels is crucial for the optimization of site-directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins’ fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin-labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide- and maleimide-functionalized spin labels based on the gem-diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem-dimethyl analogs by site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the “true” biocompatibility of the gem-diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations.     

The determination of the oxygen consumption in autoxidation studies by means of EPR spectroscopy

Stable paramagnetic species give rise in the presence of oxygen to EPR spectra characterized by broad lines due to Heisenberg spin exchange. This property can be conveniently used to determine the amount of oxygen present in the system under investigation. In the present paper we describe the use for this purpose of two radical probes: one is the stable aliphatic nitroxide 2,2,6,6-tetramethyl-l-piperidinyloxyl (TEMPO) which is employed as a homogeneous probe and the other is fusinite, a derivative of coal used as a finely grinded black powder totally insoluble both in water and in organic solvents. Both have been employed to follow the autoxidation reaction of three representative oxidizable substrates, i.e. cumene, styrene and methyl linoleate, in solution of chlorobenzene or of tert-butyl alcohol. The advantages of this technique with respect to traditional methods are emphasized.