EPR spectroscopic characterization of biological thiyl radicals as PBN spin-trap adducts

While the importance of thiols and their derivatives in biological processes is widely appreciated, the elucidation of the roles played by thiyl radicals in these processes — being hampered by the radical reactivity that makes their detection and characterization difficult — is lagging. The results of a spin-trap EPR study are reported which advance the capability for detecting and identifying thiyl radicals. Adducts with PBN (α-phenyl-N-_t-butylnitrone) of thiyl radicals derived from the biologically abundant low-molecular-weight thiols cysteine, homocysteine, and glutathione are examined. Significant differences in the β-proton hyperfine couplings of the various adducts are observed; both the EPR lineshapes and the radical adduct lifetimes show trends reflective of the molecular size of the trapped thiyl radical. These results indicate that EPR spectroscopy can be useful in identifying specific thiyl radicals that may be involved in the biochemical reactions of low-molecular-weight thiols, protein thiols, and their derivatives.     

Conformational analysis and vibrational spectroscopic studies on dapsone

In this study, the theoretical conformation analysis of free dapsone has been performed by single point energy calculations at both semi-empirical PM3 and DFT/B3LYP-3-21G theory levels and three stable conformers were determined. Both the IR and Raman spectra of the molecule in solid phase have been recorded. The IR intensities and harmonic vibrational wavenumbers of each conformer were calculated by DFT method at B3LYP/6-31++G(d,p) theory level. For the fundamental characterization, the total energy distribution (TED) calculations of the vibrational modes were done using parallel quantum mechanic solution program (SQM) and the fundamental modes were assigned. The theoretical results are in agreement with the experimental ones.     

μSR studies of various novel radicals

The aim of this work has been to study various muonium containing radicals not previously studied by the μSR technique with a view to determining the effect of a bound muonium atom on the radical structures as compared with their hydrogen analogues. In the first group, muonium addition to the aromatic rings of styrene, phenylacetylene and allylbenzene are compared with the more important addition to the substituent double or triple bonds. In the second group, β-muonium substituted cyclopentyl and cycloheptyl radicals are compared with their protiated versions. In the third group, radicals obtained following muonium addition to trimethylsilylethene, tri (methoxysilyl)-ethene, and 1-chloro-2, 2-dimethylethene are discussed with respect to the competition between the β-muonium atom, and β-silicon or β-chlorine substituents for control of the conformation of the radical.     

Relaxation of thiyl radicals as a measure of the probable timescale over which the missing fraction of muonium in water is lost

Muonium atoms produced in water react with thioacetamide (CH₃CSNH₂) to form a sulphur-centred (thiyl) free radical whose level-crossing-resonance from the CH₃-protons has been detected and analysed. Its amplitude increases with concentration below ∼10⁻² M but decreases at higher concentrations with a concommitant increase in linewidth. This is interpreted as indicating overlap with paramagnetic species from the muon track. It starts when the track is a few nanoseconds old.     

Kinetic studies of chlorobenzene reactions with hydrogen atoms and phenyl radicals and the thermochemistry of 1-chlorocyclohexadienyl radicals

Atomic H and Cl were monitored by time-resolved resonance spectroscopy in the vacuum ultraviolet, following 193 nm laser flash photolysis of C₆H₅Cl and mixtures with NH₃, over 300-1020 K and with Ar bath gas pressures from 30 to 440 mbar. Below 550 K simple exponential decays of [H] were observed, and attributed to addition to form chlorocyclohexadienyl radicals. This addition was reversible over 550-630 K and the equilibrium constant was determined by a third law approach. The addition rate constant can be summarized as (1.51 ± 0.11) × 10⁻¹¹exp((−1397 ± 29)/T) cm³ molecule⁻¹ s⁻¹ (300-630 K, 1σ uncertainties), and the C-H bond dissociation enthalpy in 1-chlorocyclohexadienyl was determined to be 108.1 ± 3.3 kJ mol⁻¹ at 298 K. At higher temperatures the photolysis of chlorobenzene yielded H atoms, which is attributed to the reaction of phenyl with chlorobenzene with a rate constant of (4.5 ± 1.8) × 10⁻¹⁰exp((−4694 ± 355)/T) cm³  molecule⁻¹ s⁻¹ over 810-1020 K. These and other reaction pathways are discussed in terms of information about the potential energy surface obtained via B3LYP/6-311G(2d,d,p) density functional theory.     

Evaluation of Conformational dynamics properties of spin-labelled proteins at different degrees of nitroxide radicals immobilization

Spin-labelling has found wide applications in elucidation of the dynamic behaviour of biological macromolecules in aqueous media and biomembranes. Most of the proposed methods aimed at estimation of macromolecular correlation times (τ_c) assume, however, spin label molecules rigidly bound within the protein matrix. To avoid this limitation theoretical models which involve additional dynamic parameters to characterize the spin label motion should be considered. We have used ESR spectra analysis technique which permits quantitative separation of slow macromolecular rotation (described by the rotational correlation time, τ_c) and fast anisotropic relative to protein nitroxyl radical motion (described by the “order parameter”,S). This method was applied to study: i) conformational dynamics of covalently and non-covalently spin-labelled human serum albumin (HSA) in solution; ii) protein-protein (antigen-antibody) interactions in a model system containing spin-labelled bovine serum albumin (BSA) and anti-BSA immunoglobulin (IgG) in solution; and iii) dynamic properties of membrane-bound proteins: H⁺-ATPase (CF₁-CF₀ coupling factor of photophosphorylation) and Photosystem I pigment-protein reaction centre complex (PSI RC) isolated from spinach chloroplasts and reconstituted in proteoliposomes.     

Conformational studies of ethylene glycol and its two methyl ether derivatives

As a theoretical analysis of the conformational equilibria of ethylene glycol, methoxyethanol and dimethoxyethane, the energy of each stable conformational isomer (rotamer) of these molecules was calculated for various temperatures and solvent dielectric constants. Classical semi-empirical potential functions were used. Besides intrinsic potentials for rotation about single bonds, intramolecular dispersion and repulsive interaction, dipole-dipole interaction and hydrogen bonding energies were included. Interaction with the solvent was considered only in terms of a continuous dielectric medium interacting with the local dipoles and quadrupoles of the molecule. For each rotamer, the dihedral angles giving the lowest energy were determined. From the energies of each rotamer, Boltzmann distributions of populations were obtained, and total concentrations were calculated in various physically distinguishable states, e.g. those with and without internal hydrogen bonds, or those in which the central C-C bond takes a trans or a gauche conformation. It is shown that the equilibrium constants, K _HB and K _TG, for these two cases are not identical. While changes in the dielectric constant may alter strongly the geometries and energies of individual rotamers, their effect on the average geometries and on the two equilibrium constants is small. The same is true of temperature changes, and is due to the presence of several rotamers in each of the physical states considered. Thus the small temperature dependence of some observed physical properties is shown to be consistent with the distribution of molecules over several conformational states. In solution, the fraction of ethylene glycol molecules with two free OH groups (i.e. without an intramolecular hydrogen bond) is predicted to be at least 20 per cent. This shows that the presence of three-dimensional hydrogen-bonded structures in the liquid, which we propose, is possible in principle.     

High-frequency EPR studies on cofactor radicals in photosystem I

Electron paramagnetic resonance (EPR) spectroscopy at W-band (94 GHz) is used to resolve theg-tensors of the radical ions of the primary chlorophyll donor P ₇₀₀ ^+? and the quinone acceptor A ₁ ^?? in photosystem I. The obtainedg-tensor principal values are compared with those of the isolated pigment radicals in organic solvents and the shifts are related to an impact of the protein environment. P ₇₀₀ ^+? has been investigated in photosystem I single crystals at 94 GHz. W-band EPR applied to the photoinduced radical pair P ₇₀₀ ^+? A ₁ ^?? is used to correctly assign theg-tensor axes of P ₇₀₀ ^+? to the molecular structure of the primary donor. Density functional theory calculations on a model of A ₁ ^?? in its binding pocket derived from the recent crystal structure of photosystem I were utilized to correlate experimental magnetic resonance parameters with structural elements of the protein.     

Structure and dynamics of radicals in zeolite matrices: ESR and theoretical studies

Amine radical cations of the type R₃N^·+ and [R₃NCH₂]^·+, R=CH₃, C₃H₇, and nitric oxide, NO, have been used to probe the bonding to the surface and the dynamics of the radicals trapped in the confined space of cages or channels in the zeolite. Regular continuous-wave electron spin resonance (ESR) was employed to study the internal motion of the cation radicals formed by γ-irradiation of amines and related ammonium ions, introduced during the synthesis of the zeolites Al-offretite, SAPO-37, SAPO-42 and AlPO₄-5. The ESR spectra of [(CH₃)₃NCH₂]^·+ radical cation in several studied systems changed reversibly with temperature, indicating dynamical effects. Free rotation about the >N?CH₂ bond of the [(CH₃)₃NCH₂]^·+ species was found to occur in the temperature range of 110 to 300 K, while the rotation about the >N?CH₃ bonds was hindered. The observations confirm the theoretical prediction on the basis of density functional theory calculations, which indicate that the corresponding barriers are of the order of 0.3 and 7 kJ/mol, respectively. The radical cations of the type R₃N^·+ with R=C₂H₅, C₃H₇ were found to undergo a different type of dynamics, involving a two-jump process of the methylene hydrogens next to the nitrogen. A cage or channel size effect on the stability and molecular dynamics was inferred in some cases. Pulsed ESR was employed to study the (NO)₂ triplet-state dimers in Na-A type zeolite, with the purpose to resolve the interaction with surface groups, and to elucidate the role of the zeolite on stabilizing the triplet rather than the usual singlet state. Measurements performed at 5 K gave rise to Fourier transform spectra that were assigned to the dimer species interacting with one or more²³Na nuclei, with approximative parameters A(²³Na)=(4.6, 4.6, 8.2) MHz and Q(²³Na)=(?0.3, ?0.3, 0.6) MHz for the hyperfine and nuclear quadrupole coupling tensors, respectively. The values are of similar magnitude as those determined for the NO?Na⁺ complex. The stability of the triplet-state structure was attributed to unusual geometric structure imposed by the zeolite matrix, with the N?O bonds along a line as in [O?N?Na⁺?N?O], which according to UHF ab initio calculations has a triplet ground     

Conformational studies of poly(methacrylic acid). I. Conformational energy calculations on lsotactic and syndiotactic poly(methacrylic acid)

The conformational energies of isotactic and syndiotactic poly (methacrytic acid) were calculated for isolated chains in order to determine the most probable helical conformations. It was concluded that the most probable backbone conformation of the isotactic polymer is either a 5₂ or 8₃ helix, where the internal rotations of the two helices are nearly equal. The left-handed and right-handed conformations were also essentially equienergy conformations. Two lowenergy helical conformations were found for the syndiotactic polymer. One of these conformations had backbone internal rotations nearly identical to those of the 5₂ and 8₃ helices of isotactic poly(methacry1ic acid). The second conformation was a nearly planar structure containing two monomer units in the axial repeat unit, with backbone rotation angles nearly identical to those of the 5₂ and 8₃ helices or their negatives.

Deformation of the backbone C─C─C bond angles was considered in the conformational energy calculations. The optimum bond angles for both isotactic and syndiotactic polymer chains were considerably larger than the normal tetrahedral bond angle. The relative energies calculated for helical conformations were influenced by the backbone bond angles. It was demonstrated that the failure to consider backbone bond-angle deformation may lead to erroneous conformational assignments in disubstituted vinyl polymers.     

Dipolar broadening of electron spin resonance lines of free radicals: Spin-trapping studies of free radicals in cigarette smoke

Electron spin resonance spectra of spin-trapped free radicals from tobacco smoke have been examined. The cigarettes studied were standard IRI research cigarettes supplied by the University of Kentucky Tobacco and Health Research Institute. Substantial changes in the spectra were observed upon aging of the samples. Aging for several days resulted in at least two remaining long-lived spin-adducts. For the experimental configuration employed the efficiency of the spin-trapping apparatus was determined to be about 47%. Utilizing measurement of the broadening of the magnetic resonance lines as function of the amount of smoke passed through the phenyl-t-butyl nitrone spin-trapping solution, a concentration of about 1 × 10¹⁸ free radicals per cigarette puff was deduced. The method employed in this determination was tested with a number of stable free radicals in several solvents.     

Gas,liquid and solid state studies of muon spin relaxation in organic radicals

A distinctive field dependence of longitudinal muon spin relaxation in acetone liquid and vapour suggests that modulation of the isotropic hyperfine coupling of the (CH₃)₂COMu radical is the dominant relaxation mechanism. The temperature dependent correlation time extracted from the data then corresponds to the lifetime of the states of internal libration of the molecule. The variation of relaxation rate may also be followed into the solid phase, peaking at the freezing transition.     

REMPI as a tool for studies of OH radicals in catalytic reactions

2 +? O₂→H₂O on polycrystalline Pt foils has been studied by detection of desorbing OH radicals using the Resonance Enhanced Multiphoton Ionization, REMPI, technique. The measurements were performed at catalyst temperatures of 1000–1400 K and a total pressure below 10^-4 mbar. The studies of OH desorption by REMPI were achieved using a two-photon excitation D²Σ^-–X²Π (1–0), followed by one-photon ionization. The ions were detected in a time-of-flight mass spectrometer, TOF-MS, in order to avoid interference from non-resonantly ionized molecules. By applying TOF-MS, a simultaneous non-resonant ionization and detection of H₂, O₂ and H₂O was achieved. Recorded REMPI spectra were compared with spectra simulated using known molecular constants. The kinetics of the reaction derived from the measurements were compared with what was obtained in earlier LIF detection of OH, performed at higher total reactant pressure using the A–X transition. REMPI TOF-MS is shown to be a complement to LIF for reaction studies below 5×10^-4 mbar total pressure, where LIF is too insensitive for quantification. The reaction kinetics was found to be in agreement with a theoretic model and previous LIF studies. Received: 8 March 1996/Revised version: 4 October 1996     

Liquid-phase EPR, ENDOR, and TRIPLE resonance studies on corrole and isocorrole cation radicals

The “Contracted Porphyrins” corrole and the recently synthesized isocorrole (as octaethyl derivatives) represent novel porphyrinoid macrocycles of biochemical interest. The radical cations of the free-bases of both corrole and its isomer are studied by EPR, ENDOR, and TRIPLE resonance in liquid solution to measure isotropic hyperfine couplings including signs. They yield detailed information of the electronic structure of the radical cations. The experimental findings are compared with results of all-valence-electrons self-consistent field molecular orbital calculations (RHF-INDO/SP). The comparison shows that the free-bases of corrole and isocorrole undergo protonation when oxidized to cation radicals in CH₂Cl₂ solution. Excellent agreement of the measured and calculated spin density distributions is achieved for the protonated dication radicals, which exhibit the C₂ symmetry observed for the hyperfine structure. The analysis of the hyperfine data discriminates against NH tautomerism as the sole source for an effective C₂ symmetry of the macrocycle.     

Density matrix renormalization group studies on one-dimensional -conjugated organic ferromagnet with side radicals

By using the density matrix renormalization group (DMRG) and the self-consistent numerical method, we obtain a high spin ground state with localized spin density describing spin localization and the soliton describing the distortion of the lattice configurations along the main chain. Different electron-phonon interactions result in different configurations of solitons. When the electron-phonon coupling along the main chain is larger than a critical value , a transition from a single soliton-like distortion to a pair of soliton-like distortions along the main chain takes place. Such critical value depends mainly on the intersite Coulomb interactions. The spin density wave along the main chain is always localized around the center of soliton-like distortions. Received 2 July 2001 and Received in final form 25 September 2001