部分还原二氧化钛的NMR和EPR研究

Partially reduced TiO₂ nanomaterials have attracted significant interest because of their visible-light activity for catalysis and photodegradation. Herein, we prepared a partially reduced anatase TiO₂ (Re-A-TiO₂) nanoparticle material using a fast combustion method, demonstrating good activity toward decomposing methyl orange under visible light irradiation. The surface structure of the prepared material, after being surface-selectively ¹⁷O-labeled with H₂¹⁷O (¹⁷O-enriched water), was studied via ¹⁷O and ¹H solid-state magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy and electron paramagnetic resonance (EPR) spectroscopy, and the obtained results were compared to those of non-reduced anatase TiO₂ (A-TiO₂). The EPR results showed that the concentrations of paramagnetic species (i.e., oxygen vacancies (OV) and Ti³⁺) in Re-A-TiO₂ were much higher than that in A-TiO₂, while the former was associated with a higher OV/Ti³⁺ ratio. The intensities of the EPR signals were significantly affected by the adsorbed water, and this phenomenon was explored in combination with ¹H NMR spectroscopy. The ¹H species on Re-A-TiO₂ appeared at larger chemical shifts, denoting the increased acidity of the sample, and these ¹H species on Re-A-TiO₂ were more difficult to remove than those on A-TiO₂. On the other hand, different features were observed for the signals arising from the two-coordinated oxygen atoms (μ₂-O) in ¹⁷O NMR, suggesting a typical anatase TiO₂(101) surface on A-TiO₂, but a more complex surface environment for Re-A-TiO₂. Furthermore, a larger amount of hydroxyl groups (OH) were observed on Re-A-TiO₂ compared to that on A-TiO₂, indicating a larger proportion of exposed (001) facets on Re-A-TiO₂. However, the μ₂-O signals broadened and became similar when the drying temperature was increased to 100 ℃, indicating a non-faceted anatase TiO₂ surface in such conditions. Based on the EPR and NMR results, a significant fraction of the OH species is believed to be formed from the reaction of the paramagnetic centers and adsorbed water molecules. The ¹H→¹⁷O cross polarization (CP) MAS and two-dimensional heteronuclear correlation (2D HETCOR) NMR spectra were used to verify the spatial proximity of the hydrogen and oxygen species, confirming the spectral assignments of a strongly adsorbed water and one type of surface OH species. In particular, the ¹H NMR signals at approximately 11 ppm were ascribed to the hydrogen species in the intramolecular hydrogen bond. In summary, this study investigated the paramagnetic species and surface structure of anatase TiO₂ materials by combining EPR along with ¹H and ¹⁷O solid-state NMR spectroscopy. The differences in the surface structures of Re-A-TiO₂ and A-TiO₂ should be closely related to their different properties toward the photodegradation of methyl orange.     

Solid-state NMR and EPR study of fluorinated carbon nanofibers

Carbon nanofibers were fluorinated in two manners, in pure fluorine gas (direct fluorination) and with a fluorinating agent (TbF₄ during the so-called controlled fluorination). The resulting fluorinated nanofibers have been investigated by solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). This underlines that the fluorination mechanisms differ since a (CF)_n structural type is obtained, whatever the temperature, with the controlled reaction, whereas, during the direct process, a (C₂F)_n type is formed over a wide temperature range. Through a careful characterization of the products, i.e. density of dangling bonds (as internal paramagnetic centers), structural type (acting on molecular motion) and specific surface area (related to the amount of physisorbed O₂), the effect of atmospheric oxygen molecules on the spin-lattice nuclear relaxation has been underlined.     

Structural and dynamic characterization of copper(II) binding of the human prion protein outside the octarepeat region

Human prion protein (hPrP) fragments encompassing the 91-120 region, namely hPrP92-100 (SP1), hPrP106-113 (SP2), hPrP91-120 (LP1), and hPrP91-114 (LP2), were considered for delineation of the Cu(II)-binding site(s). NMR and EPR spectroscopy results obtained from LP1 or LP2 were compared with those obtained from SP1 and SP2. The coexistence of two binding sites, one centered at His96 and the other at His111, was evidenced and ratified by ESI mass spectrometry at low and high metal:peptide ratios. While room-temperature NMR spectroscopy data were consistent with the binding site centered on His111 being approximately fourfold stronger than that centered on His96, low-temperature EPR spectroscopy results yielded evidence for the opposite trend. This disagreement, which has also occurred in the literature, was clarified by temperature-dependent molecular dynamics runs that demonstrated Met112 approaching the metal at room temperature, a process that is expected to stabilize the His111-centered binding site through hydrophobic shielding of the metal coordination sphere.     

NMR and EPR studies of the reaction of nucleophilic addition of (bi)sulfite to the nitrone spin trap DMPO

The reaction of the nitrone spin trap 5,5‐dimethylpyrroline‐N‐oxide (DMPO) with sodium (bi)sulfite in aqueous solutions was investigated using NMR and EPR techniques. Reversible nucleophilic addition of (bi)sulfite anions to the double bond of DMPO was observed, resulting in the formation of the hydroxylamine derivative 1‐hydroxy‐5,5‐dimethylpyrrolidine‐2‐sulfonic acid, with characteristic ¹H and ¹³C NMR spectra. The reaction mechanism was suggested and corresponding equilibrium constants determined. The mild oxidation of the hydroxylamine results in the formation of an EPR‐detected spectrum identical with that for the DMPO adduct with sulfur trioxide anion radical. The latter demonstrates that a non‐radical addition reaction of (bi)sulfite with DMPO may contribute to the EPR detection of SO₃^?? radical. This possibility must be taken into account in spin trapping analysis of sulfite radical. Copyright © 2003 John Wiley & Sons, Ltd.     

Evolution of ordered films of copper phthalocyanine according to EPR data

The procedure for calculating the orientation distribution of molecules using the angular dependence of EPR spectra was employed to study copper(II) phthalocyanine (CuPc) films varying in thickness and obtained by depositing the molecular complex on flat quartz plates. At the first stage of deposition, a layer of the α-CuPc phase with preferable orientation of molecular stacks along the plate surface is formed. At the second stage, a layer with an orthogonal arrangement of molecular stacks is condensed over the first layer. The interaction with NO₂ forms CuPc binuclear associates. Analysis of the EPR spectra made it possible to determine the symmetry of the structure and the distance between the paramagnetic Cu²⁺ ions; the structure of the associates has been proposed. The orientation distribution of CuPc dimers in the film depends both on the initial ordering in the film and on processing conditions. Strong disordering of molecular stacks in ordered films during the α-CuPc to α-CuPc phase transition has been found.     

A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR,EPR and Time-Resolved Luminescence Studies

A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral Ln^III tag, referred to as C12 , is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with Tb^III or Tm^III ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron–electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the Tb^III complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.     

Copper(II) interaction with prion peptide fragments encompassing histidine residues within and outside the octarepeat domain: speciation, stability constants and binding details

A 31-mer polypeptide, which encompasses residues 84-114 of human prion protein HuPrP(84-114) and contains three histidyl residues, namely one from the octarepeat (His85) and two histidyl residues from outside the octarepeat region (His96 and His111), and its mutants with two histidyl residues HuPrP(84-114)His85Ala, HuPrP(84-114) His96Ala, HuPrP(84-114)His111Ala and HuPrP(91-115) have been synthesised and their Cu2+ complexes studied by potentiometric and spectroscopic (UV/Vis, CD, EPR, ESI-MS) techniques. The results revealed a high Cu2+-binding affinity of all peptides, and the spectroscopic studies made it possible to clarify the coordination mode of the peptides in the different complex species. The imidazole nitrogen donor atoms of histidyl residues are the exclusive metal-binding sites below pH 5.5, and they have a preference for macrochelate structure formation. The deprotonation and metal-ion coordination of amide functions take place by increasing the pH; all of the histidines can be considered to be independent metal-binding sites in these species. As a consequence, di- and trinuclear complexes can be present even in equimolar samples of the metal ion and peptides, but the ratios of polynuclear species do not exceed the statistically expected ones; this excludes the possibility of cooperative Cu2+ binding. The species with a (N(im),N,N)-binding mode are favoured around pH 7, and their stability is enhanced by the macrochelation from another histidyl residue in the mononuclear complexes. The independence of the histidyl sites results in the existence of coordination isomers and the preference for metal binding follows the order of: His111>His96>His85. Deprotonation and metal-ion coordination of the third amide functions were detected in slightly alkaline solutions at each of the metal-binding sites; all had a (N(im),N,N,N)-coordination mode. Spectroscopic measurements also made it clear that the four lysyl amino groups of the peptides are not metal-binding sites in any cases.     

In‐Cell NMR and EPR Spectroscopy of Biomacromolecules

The dream of cell biologists is to be able to watch biological macromolecules perform their duties in the intracellular environment of live cells. Ideally, the observation of both the location and the conformation of these macromolecules with biophysical techniques is desired. The development of many fluorescence techniques, including superresolution fluorescence microscopy, has significantly enhanced our ability to spot proteins and other molecules in the crowded cellular environment. However, the observation of their structure and conformational changes while they attend their business is still very challenging. In principle, NMR and EPR spectroscopy can be used to investigate the conformation and dynamics of biological macromolecules in living cells. The development of in‐cell magnetic resonance techniques has demonstrated the feasibility of this approach. Herein we review the different techniques with a focus on liquid‐state in‐cell NMR spectroscopy, provide an overview of applications, and discuss the challenges that lie ahead.     

Use of 19F NMR spectroscopy to probe enzymatic oxidation of fluorine‐tagged sulfides

A novel ¹⁹F NMR‐based method for monitoring the enzymatic oxidation of thia fatty acid analogues is presented. Our approach is based on the observation that methyl ω‐monofluorinated 9‐thia‐ and 10‐thiaoctadecanoates and their S‐oxide and S‐dioxide derivatives are easily distinguishable via their ¹H‐decoupled ¹⁹F spectra. These long‐range substituent effects were used to probe the regio‐ and chemoselectivity of stearoyl ACP (acyl carrier protein) Δ⁹ desaturase‐mediated sulfoxidation. The results clearly demonstrate that mono‐oxygenation of a 10‐thia analogue ACP Δ⁹ desaturase was more efficient than that of a 9‐thia substrate. A product previously undetected by TLC was observed for the first time in the product mixture obtained from 18‐fluoro‐9‐thiaoctadecanoyl‐ACP. Copyright © 2002 John Wiley & Sons, Ltd.     

Characterisation of microstructure and evaluation of optical and EPR properties of superhydrophobic copper dodecanoate films

Copper dodecanoate films prepared by emulsion method exhibit superhydrophobic property with water contact angle of 155° and sliding angle of <2°. The films have been characterised by using X‐ray diffraction, field emission scanning electron microscopy and Fourier transform infrared spectroscopy techniques. Surface microstructure of copper dodecanoate consists of numerous microscale papillas of about 6–12 µm in length with a diameter in the range of 360–700 nm. The superhydrophobicity of the films is due to their dual micronano surface morphology. The wetting behaviour of the film surface was studied by a simple water immersion test. The results show that copper dodecanoate film retained superhydrophobic property even after immersing in water for about 140 h. The optical absorption spectrum exhibits two broadbands centred at 388 and 630 nm that have been assigned to ²B_1g → ²E_g and ²B_1g → ²B_2g transitions of Cu²⁺ ions, respectively. The electron paramagnetic resonance spectrum exhibits two resonance signals with effective g values at g_|| ≈ 2.308 and g_⊥ ≈ 2.071, which suggests that the unpaired electron occupies d_x2–y2 orbital in the ground state. Copyright © 2011 John Wiley & Sons, Ltd.     

Evidence for alkene cis-aminocupration, an aminooxygenation case study: kinetics, EPR spectroscopy, and DFT calculations

Alkene difunctionalization reactions are important in organic synthesis. We have recently shown that copper(II) complexes can promote and catalyze intramolecular alkene aminooxygenation, carboamination, and diamination reactions. In this contribution, we report a combined experimental and theoretical examination of the mechanism of the copper(II)-promoted olefin aminooxygenation reaction. Kinetics experiments revealed a mechanistic pathway involving an equilibrium reaction between a copper(II) carboxylate complex and the γ-alkenyl sulfonamide substrate and a rate-limiting intramolecular cis-addition of N-Cu across the olefin. Kinetic isotope effect studies support that the cis-aminocupration is the rate-determining step. UV/Vis spectra support a role for the base in the break-up of copper(II) carboxylate dimer to monomeric species. Electron paramagnetic resonance (EPR) spectra provide evidence for a kinetically competent N-Cu intermediate with a Cu(II) oxidation state. Due to the highly similar stereochemical and reactivity trends among the Cu(II)-promoted and catalyzed alkene difunctionalization reactions we have developed, the cis-aminocupration mechanism can reasonably be generalized across the reaction class. The methods and findings disclosed in this report should also prove valuable to the mechanism analysis and optimization of other copper(II) carboxylate promoted reactions, especially those that take place in aprotic organic solvents.     

A NMR method to probe the nature of liquid clathrates

The liquid clathrate formed from [N(CH₃)₄][Al₂(CH₃)₆I] and benzene has been studied using 200 MHz¹H FT-NMR. Two resonances, corresponding to the free and guest benzene are observed. The¹H spin lattice relaxation times,T ₁, for these molecules were measured and found to be distinctly different. The guest benzene protons relax nearly four times faster than the free molecules which is consistent with a more ordered structure of the benzene molecules within the clathrate.     

The Transient Complex of Cytochrome c and Cytochrome c Peroxidase: Insights into the Encounter Complex from Multifrequency EPR and NMR Spectroscopy

We present a novel approach to study transient protein-protein complexes with standard, 9 GHz, and high-field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso-1-cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate] attached at position 81 of Cc (SL−Cc). A dissociation constant K_D of 20±4×10⁻⁶ M (EPR and NMR) and an equal amount of stereo-specific and encounter complex (NMR) are found. The EPR spectrum of the fully bound complex reveals that the encounter complex has a significant population (60 %) that shares important features, such as the Cc-interaction surface, with the stereo-specific complex.     

Solid‐state NMR and EPR Spectroscopy of Mn2+‐Substituted ATP‐Fueled Protein Engines

Paramagnetic metal ions deliver structural information both in EPR and solid‐state NMR experiments, offering a profitable synergetic approach to study bio‐macromolecules. We demonstrate the spectral consequences of Mg²⁺/ Mn²⁺ substitution and the resulting information contents for two different ATP:Mg²⁺‐fueled protein engines, a DnaB helicase from Helicobacter pylori active in the bacterial replisome, and the ABC transporter BmrA, a bacterial efflux pump. We show that, while EPR spectra report on metal binding and provide information on the geometry of the metal centers in the proteins, paramagnetic relaxation enhancements identified in the NMR spectra can be used to localize residues at the binding site. Protein engines are ubiquitous and the methods described herein should be applicable in a broad context.     

Paramagnetic properties of the halide-bound derivatives of oxidised tyrosinase investigated by 1H NMR spectroscopy

The (1)H NMR relaxation characteristics of the histidines in the oxidised type-3 copper site of tyrosinase (Ty(met)) from the bacterium Streptomyces antibioticus in the halide-bound forms (Ty(met)X with X = F(-), Cl(-), Br(-)) have been determined and analysed. The (1)H NMR spectra of the Ty(met)X species display remarkably sharp, well-resolved, paramagnetically shifted (1)H signals, which originate from the protons of the six His residues coordinated to the two Cu(II) ions in the type-3 centre. From the temperature-dependence of the (1)H paramagnetic shifts the following values for the exchange-coupling parameter -2J were determined: 260 (Ty(met)F), 200 (Ty(met)Cl) and 162 cm(-1) (Ty(met)Br). The (1)H T(1) relaxation is dipolar in origin and correlates with the Cu--H distances. Electronic relaxation times tau(S) derived from the (1)H T(1) data amount to about 10(-11) s and follow the order Ty(met)F>Ty(met)Cl>Ty(met)Br. They are two orders of magnitude shorter than the tau(S) values reported for mononuclear copper systems, in accordance with the sharpness of the (1)H signals. The results corroborate the Cu(2) bridging mode of the halide ions. On the basis of the measured hyperfine interaction constants for the ligand histidine nuclei, it is concluded that 70-80 % of the spin density in the excited triplet state resides on the two copper ions and the bridging atoms.     

Structural investigations of paramagnetic metalloproteins using high-resolution NMR spectroscopy: iron-sulfur and copper proteins

Current approaches to the structural investigations of paramagnetic metal-containing proteins in solution using NMR spectroscopy are surveyed taking iron-sulfur and copper-containing proteins as examples.