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Exploring the Radical Nature of a Carbon Surface by Electron Paramagnetic Resonance and a Calibrated Gas Flow

While the first Electron Paramagnetic Resonance (EPR) studies regarding the effects of oxidation on the structure and stability of carbon radicals date ...

Bar Ilan University

The evolution of the solution microstructures during the formation of the hexagonal mesoporous material SBA-15 was studied by direct imaging and freeze-fracture replication cryo-TEM. A reaction mixture was sampled at different times after the addition of tetramethoxyorthosilane (TMOS) to an acidic solution of Pluronic P123 held at 50 degrees C. Solution microstructures were detected by direct imaging cryo-TEM in the time window of 6.5-40 min after the addition of the TMOS (t = 0). The micrographs revealed that the initial spheroidal micelles evolve into threadlike micelles, which become longer and straighter with time. Then bundles with the dimensions similar to those found in the final material appeared, although there was no sign of a hexagonal arrangement up to 40 min. Due to the appearance of a precipitate at 40 min the sample became too viscous, preventing clear observation of its content. To observe the structures present after 40 min, freeze-fracture replication was carried out as well. Such samples were collected also at 22 min and showed the presence of threadlike micelles in agreement with the direct imaging cryo-TEM micrographs. The 2 h samples showed some areas of hexagonal ordered structures, which become very clear at 2 h 50 min. The cryo-TEM measurements were carried out under the same reaction conditions used in earlier in situ EPR experiments, thus allowing us to correlate molecular level events with the microstructure shape evolutions. This showed that the elongation of the micelles is a consequence of a reduction of the polarity and the water content within the micelles due to silicate adsorption and polymerization. Similar experiments were carried out also on SBA-15 prepared with HCl and TMOS at 35 degrees C. The appearance of threadlike micelles, followed by clustering of the TLMs, was observed under these conditions as well, but the reaction rate was faster. This suggests that the observed mechanism for the formation of SBA-15 is general.

Resolving intermediate solution structures during the formation of mesoporous SBA-15.

Double electron electron resonance (DEER) is an experimental technique used to determine distance between electron spins. In this work, we show that it can be used to study the properties of micelles in solution, specifically their volume and the aggregation number. The feasibility of the method is tested on micelles of Pluronic block copolymers, PEO(x)-PPO(y)-PEO(x), built from chains of poly(ethylene oxide) (PEO), comprising the more hydrophilic corona, and a poly(propylene oxide) (PPO) block constituting the hydrophobic core. In this work, the dimensions of the hydrophobic core of micelles of Pluronic L64 (x = 13, y = 30), P123 (x = 20, y = 70), and F127 (x = 106, y = 70) and their aggregation number were studied. This was done using the spin-probe 4-hydroxy-tempo-benzoate (4HTB), which is hydrophobic and is localized in the hydrophobic core of the micelles and does not dissolve in aqueous solution. The measurements were carried out on frozen solutions, freeze quenched after equilibration at 50 degrees C. It was found that the hydrophobic core radii occupied by 4HTB in 7.5 wt % F127 and 6 wt % L64 are 4.0 +/- 0.05 and 3.8 +/- 0.1 nm, respectively, and the corresponding aggregation numbers are 57 +/- 2 and 206 +/- 14. The micelles of 6 wt % P123 were found to have a rod shape, and the addition of 4HTB at concentrations higher than 0.7 mM resulted in a phase transitioned to spherical micelles. Finally, this study also showed that the micelle structure is preserved upon rapid freezing.

Double electron electron resonance as a method for characterization of micelles.

Pulse double electron-electron spin resonance (DEER) measurements were applied to characterize the distribution and average number of guest-molecules (in the form of spin-probes) in Pluronic P123 micelles. Two types of spin-probes were used, one of which is a spin-labeled P123 (P123-NO), which is similar to the micelles constituent molecules, and the other is spin-labeled Brij56 (Brij56-NO) which is significantly different. Qualitative information regarding the relative location of the spin-labels within the micelles was obtained from the isotropic hyperfine coupling and the correlation times, determined from continuous wave EPR measurements. In addition, complementary small angle X-ray scattering (SAXS) measurements on the P123 micellar solutions, with and without the spin-probes, were carried out for an independent determination of the size of the core and corona of the micelles and to ensure that the spin-probes do not alter the size or shape of the micelles. Two approaches were used for the analysis of the DEER data. The first is model free, which is based on the determination of the leveling off value of the DEER kinetics. This provided good estimates of the number of radicals per micelle (low limit) which, together with the known concentration of the P123 molecules, gave the aggregation number of the P123 micelles. In addition, it provided an average distance between radicals which is within the range expected from the micelles' size determined by SAXS. The second approach was to analyze the full kinetic form which is model dependent. This analysis showed that both spin-labels are not homogeneously distributed in either a sphere or a spherical shell, and that large distances are preferred. This analysis yielded a slightly larger occupation volume within the micelle for P123-NO than for Brij56-NO, consistent with their chemical character.

Distribution of guest molecules in Pluronic micelles studied by double electron electron spin resonance and small angle X-ray scattering.

We studied the structural evolution during the formation of large-pore cubic Ia3d silica-based mesoporous materials, synthesized with Pluroinc P123 and butanol as structure directing agents. We used cryogenic high resolution scanning electron microscopy (cryo-HRSEM) and freeze-fracture-replication (FFR) transmission electron microscopy (TEM). Typically a silica precursor is added to an acid-catalyzed solution of Pluronic P123 and butanol. The latter serves as a cosolute, which can be added either at the beginning of the reaction, or after precipitation and the formation of a hexagonal phase. In this study we focused on the structural evolution from the hexagonal phase to the final cubic phase in the two different reactions. The same structural evolution with different kinetics was detected for both reactions. Cryo-HRSEM and FFR-TEM images revealed that from the hexagonal phase a perforated layer (PL) phase is formed, which later evolves into a bicontinuous structure. The final cubic phase forms within the layers, maintaining their orientation. We suggest a formation mechanism involving cylinder merging for the hexagonal to PL transition. Upon additional polymerization of the silica, the PL phase relaxes into the stable Ia3d cubic phase. Another minor mechanism detected involves the direct transition between the hexagonal to the final cubic phase through cylinder branching.

High-resolution cryogenic-electron microscopy reveals details of a hexagonal-to-bicontinuous cubic phase transition in mesoporous silica synthesis.

Characterizing the structure and dynamic properties of a single monolayer is a challenge due to the minute amount of material that is probed. Here, EPR spectroscopy is used for investigating the spatial and temporal organization of self-assembled monolayers of 5- and 16-doxyl stearic acid (5 DSA and 16 DSA, respectively) adsorbed on a GaAs substrate. The results are complemented with FTIR and ellipsometery measurements, which provide the evidence for the formation of monolayers. Moreover, a comparison with the FTIR spectrum of a monolayer of stearic acid shows that the monolayers of the spin labeled molecules are less packed due to the hindrance introduced by the labeling group. The EPR spectra provide a new insight on the ordering in the layer and more interestingly, it reveals the time dependence of the organization. For 5DSA, with the spin-label group situated close to the substrate, the EPR spectrum immediately after adsorption is poorly resolved and dominated by the spin-exchange interaction between neighboring molecules. As time increases (up to 1 week) the resolution of the 14N hyperfine coupling increases, revealing a better organized monolayer where the molecules are more homogenously spaced. Moreover, the spectrum of the layer, after reaching equilibrium, shows that there is no motional freedom near the GaAs surface. Orientation dependence measurements on the equilibrated sample show the presence of a preferred orientation of the molecules, although with a wide distribution. The spectrum of the 16DSA monolayer, where the nitroxide spin label is situated at the end of the chain, far from the surface, also showed a poorly resolved spectrum at short times, but unlike 5DSA, it did not exhibit any time dependence. Through EPR line-shape simulations and by comparison with FTIR results, the differences between 5DSA and 16DSA were attributed to difference in coverage caused by the bulky spin label near the surface in the case of 5DSA.

EPR studies on the organization of self-assembled spin-labeled organic monolayers adsorbed on GaAs.

Herein, we identify the coordination environment of Cu²(+) in the human α1-glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel superfamily (pLGIC) that mediate fast signaling at synapses. Metal ions like Zn²(+) and Cu²(+) significantly modulate the activity of pLGICs, and metal ion coordination is essential for proper physiological postsynaptic inhibition by GlyR in vivo. Zn²(+) can either potentiate or inhibit GlyR activity depending on its concentration, while Cu²(+) is inhibitory. To better understand the molecular basis of the inhibitory effect we have used electron spin resonance to directly examine Cu²(+) coordination and stoichiometry. We show that Cu²(+) has one binding site per α1 subunit, and that five Cu²(+) can be coordinated per GlyR. Cu²(+) binds to E192 and H215 in each subunit of GlyR with a 40 μM apparent dissociation constant, consistent with earlier functional measurements. However, the coordination site does not include several residues of the agonist/antagonist binding site that were previously suggested to have roles in Cu²(+) coordination by functional measurements. Intriguingly, the E192/H215 site has been proposed as the potentiating Zn²(+) site. The opposing modulatory actions of these cations at a shared binding site highlight the sensitive allosteric nature of GlyR.

Pulsed electron spin resonance resolves the coordination site of Cu²(+) ions in α1-glycine receptor.

Once coal is excavated it comes into contact with atmospheric oxygen and begins to undergo low temperature oxidation. The mechanism by which the molecular oxygen interacts with the coal macromolecule is suggested to occur in several steps. These steps primarily involve O(2) diffusion to the surface where physical adsorption followed by chemical adsorption takes place. The chemical adsorption forms several types of oxides that can subsequently react to form several products, primarily CO(2). It has also been suggested that some of these oxidation mechanisms might involve radical reactions. As the previous studies were conducted under conditions where significant structural changes occur it is possible that in the low temperature range (T < 100 °C) the oxidation mechanism is different. Several different rank (lignite-subbituminous-bituminous) coals were isothermally heated at 95 °C in an air atmosphere for a period of up to 6 months and samples were collected at two week intervals. The radical concentration of each sample was measured by Continuous Wave Electron Paramagnetic Resonance (CW-EPR). It is apparent that there are distinct differences between the lower rank (lignite) and the higher rank (subbituminous, bituminous) coals. The lower rank coals exhibited only carbon centered radicals with an adjacent oxygen atom and the higher rank coals exhibited only carbon centered radicals. Interestingly, the lower rank coals exhibited no change in radical concentration due to the long term oxidation treatment while the higher rank coals showed a distinct increase in the radical concentration. These findings shed new light on the complex heterogeneous low temperature oxidation reactions occurring at the coal surface.

Stable radicals formation in coals undergoing weathering: effect of coal rank.

We report the discovery that a flow of CO2, N2 or He can sufficiently reduce the spin-spin interactions of specific stable carbon centered radicals by displacing the molecular oxygen in the atmosphere enabling their detection via electron paramagnetic resonance (EPR). This finding unlike other reported effects on carbon radicals occurs under STP conditions and is reversible.

Reducing the spin-spin interaction of stable carbon radicals.

Double quantum coherence (DQC) ESR spectroscopy is applied to measure the Cu(2+)-Cu(2+) distance in the EcoRI-DNA complex. A simple method is proposed to reduce the contribution of nuclear hyperfine and quadrupole interactions to such data. The effects of such interactions between the electron spin of Cu(2+) and neighboring nuclei on the DQC data make it difficult to measure the nanometer range interspin distance. The DQC data is in good agreement with results obtained by double electron electron resonance (DEER) spectroscopy. At the same time, the signal-to-noise ratio per shot in DQC is high. Taken together, these results provide impetus for further development of paramagnetic metal ion-based DQC techniques.

Sensitive Cu2+-Cu2+ distance measurements in a protein-DNA complex by double-quantum coherence ESR.

The Arabidopsis cysteine-rich GASA5 is a redox-active metalloprotein that suppresses gibberellin responses.

The use of pulsed electron spin resonance (ESR) to measure interspin distance distributions has advanced biophysical research. The three major techniques that use pulsed ESR are relaxation rate based distance measurements, double quantum coherence (DQC), and double electron electron resonance (DEER). Among these methods, the DEER technique has become particularly popular largely because it is easy to implement on commercial instruments and because programs are available to analyze experimental data. Researchers have widely used DEER to measure the structure and conformational dynamics of molecules labeled with the methanethiosulfonate spin label (MTSSL). Recently, researchers have exploited endogenously bound paramagnetic metal ions as spin probes as a way to determine structural constraints in metalloproteins. In this context Cu(2+) has served as a useful paramagnetic metal probe at X-band for DEER based distance measurements. Sample preparation is simple, and a coordinated-Cu(2+) ion offers limited spatial flexibility, making it an attractive probe for DEER experiments. On the other hand, Cu(2+) has a broad absorption ESR spectrum at low temperature, which leads to two potential complications. First, the Cu(2+)-based DEER time domain data has lower signal to noise ratio compared with MTSSL. Second, accurate distance distribution analysis often requires high-quality experimental data at different external magnetic fields or with different frequency offsets. In this Account, we summarize characteristics of Cu(2+)-based DEER distance distribution measurements and data analysis methods. We highlight a novel application of such measurements in a protein-DNA complex to identify the metal ion binding site and to elucidate its chemical mechanism of function. We also survey the progress of research on other metal ions in high frequency DEER experiments.

Paramagnetic metal ions in pulsed ESR distance distribution measurements.

Recently, the nature of the carbon radicals stabilized in various coals was characterized using Electron Paramagnetic Resonance (EPR) spectroscopy. It was demonstrated that introducing diamagnetic gases, such as He, CO2, or N2, under STP conditions to the coal surface induces the appearance of a new type of carbon surface radical. This interesting phenomenon was not observed for all coal types, which suggests that the use of EPR measurements can provide information on functional groups that exist on the carbon surface. In the current study coupling Nuclear Magnetic Resonance (NMR) with gas flow in situ EPR measurements significantly enhances the ability to characterize the nature of these radicals and the surface functional groups of coal samples. It was observed that the oxidative reaction with aliphatic groups leads to the increase in stable carbon centered radicals. In addition, there are some species of carbon centered radicals that show reversible binding to O2. This phenomena, however, is dependent on the coal rank, sample porosity and the degree of the coal sample to undergo structural changes under the LTO process. These findings shed new light onto the complex heterogeneous low temperature oxidation reactions occurring at the coal surface.

Elucidating the role of stable carbon radicals in the low temperature oxidation of coals by coupled EPR-NMR spectroscopy - a method to characterize surfaces of porous carbon materials.

Sharon Ruthstein

Chemistry Department,
Faculty of Exact Science,
Chemistry Department, Faculty of Exact Science

Bar Ilan University

Exploring the Radical Nature of a Carbon Surface by Electron Paramagnetic Resonance and a Calibrated Gas Flow

Sharon Ruthstein

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Chemistry Department,Faculty of Exact Science,Chemistry Department, Faculty of Exact Science

Bar Ilan University

Exploring the Radical Nature of a Carbon Surface by Electron Paramagnetic Resonance and a Calibrated Gas Flow

Chemistry Department,
Faculty of Exact Science,
Chemistry Department, Faculty of Exact Science