Very-high-field EPR and its applications

A kind of review of recent developments of the very-high-field EPR technique and applications is presented, the largest part of attention being attracted to studies of molecular structures and molecular and chemical dynamics of free radicals, biradicals and some paramagnetic complexes. The very recent results on thermal spin polarization effects on high-field/low-temperature EPR lineshape are overviewed. These effects are proved to provide a novel approach to study systems withS>1/2, particularly radical pairs, radical clusters and biradicals.     

A program for simulation of powder-type EPR spectra

An algorithm of a computer program for simulation of powder type EPR spectra (S=1/2,I≠0) is described. For determination of the energy levels, the perturbation approach is used and corrections up to second order due to hyperfine, quadrupole, and nuclear Zeeman coupling are taken into account. No restrictions on the mutual orientations of the eigenvectors ofg, hyperfine, and quadrupole tensors are imposed. Simulated spectra for a model paramagnetic system (S=1/2,I=3/2) with axial symmetry and noncoinciding directions ofg _‖ andA _‖ are presented.     

Inosine — Adenosine base pairing in d-GGTACIAGTACC: Duplex and hairpin with a two-base loop

An oligonucleotide dodecamer d-GGTACIAGTACC containing two inosine-adenosine mismatched base-pairs has been studied by¹H and³¹P NMR spectroscopy. Unique assignments of¹H and³¹P spins have been achieved by using a recently proposed two-dimensionalheteronuclearlong rangecorrelation (2D-HELCO) technique. The 2D nuclear Overhauser enhancement spectroscopy (NOESY) spectrum recorded in a mixed solvent of 90% H₂O+10%²H₂O has been used to assign all the exchangeable imino and amino proton resonances. NMR results indicate that at concentrations above 5 mM, the molecule adopts a duplex structure where the inosine imino protons are hydrogen bonded. Cross-strand NOEs between the imino proton of I6 and H2 proton of A7 and that between H2 of I6 and H2 of A7 provide evidence for inosine-adenosine base-pairing, with both bases I6 and A7 inanti conformation. The observed NOE connectivitiesd1,d2 andd3 from almost the middle of the molecule to the two ends are consistent with a right handed B-DNA conformation. However, the molecule adopts a more extended conformation in the mismatch region to accomodate the bulky purine-purine (IA) base pairs. NMR experiments at lower concentrations indicate the coexistence of a monomeric hairpin with a two base loop, consisting of I6 and A7 units, in equilibrium with the duplex. 2D-ROESY experiment at such a concentration shows that the conformation of the stem of the hairpin does not deviate much from the corresponding region of the duplex and four nucleotides C5, I6, A7 and G8 undergo significant structural changes during the formation of hairpin loop.     

Low-frequency detected EPR: Principles and applications

The experimental works described are performed by the authors over last two decades by means of the LFD EPR technique. The essence of this method is low-frequency detection of the longitudinal spin magnetization while the magnetic resonance is excited by a strong microwave field. The first kind of LFD EPR is the enchanced longitudinal susceptibility effect (ELSE) which has been elaborated and applied to study spin thermodynamics in solids since 1972. Various applications of ELSE are described such as direct measuring of the spin-spin interaction temperatureT _ss in the course of resonance saturation, spin-lattice and cross relaxation, dynamic nuclear polarization etc. Another version of LFD EPR was employed to study electron spin-lattice relaxation of paramagnetic centers in high-temperature superconductors (HTSC). Recent experimental data are presented on the electron spin-lattice relaxation of Cu²⁺ ions in YBa₂Cu₃O_6+x at various temperatures andx values.     

Mono and bidimensional1H NMR and inverse detected13C NMR of the duplex d(CA)3·d(TG)3

The unambiguous assignment of the aromatic protons of the duplex d(CA)₃·d(TG)₃ was carried out with the aid of HMQC and HMBC inverse detected¹³C NMR experiments atT=278 K. The NOE connections of these protons with the imino protons of the complementary bases — established by 2D NOESY experiments — indicated that the duplex is characterized by a canonical Watson-Crick basepairing pattern. Monodimensional¹H NMR experiments, also carried out atT=278K, in the presence of varying amounts of NaCl and with water suppression by presaturation and by spin echo techniques, showed that the imino protons of the TA and CG pairs of the duplex exchange with water through a process of local base pair opening and with different exchange rates.     

New band system of YbI molecule

Thermal emission spectrum of YbI molecule has been photographed for the first time in the spectral regionλλ 6100–6400 Å using Saha’s high temperature furnace at a reciprocal linear dispersion of 7.3 Å/mm. A total of 52 single-headed and violet degraded bands have been recorded and are classified into a single system. Vibrational analysis has been carried out and it has been suggested that system arises from the ground state with the vibrational constantω″ _e=153.0 cm^?1.     

Valence state of paramagnetic centers in fullerenes

Charge distribution on individual carbon atoms of a fullerene molecule C_n depends on the number of atoms it comprises,n. Only C₆₀ has the same charge on all its carbon atoms while other molecules containing fromn=32 ton=84 molecules are characterized by a variety of charge distributions. A neutral C₆₀ molecule is diamagnetic and does not show EPR signal. Charged C₆₀ ^v molecules, wherev is the valence taking values fromv=-6 tov=+1, show EPR signals for oddv and forv=2 when a fullerene molecule is in the triple state. EPR lines of isolated fullerene ions are narrow in the whole temperature range whereas EPR lines of Me_xC₆₀ ^v in ionic compounds Me_xC₆₀ are wide and their width decreases with temperature. A model of paramagnetic centers and an empirical formula relatingg-factor to valence have been given.     

Very high frequency EPR — 94 GHz instrument and applications to primary reaction centers from photosynthetic red bacteria and to other disordered systems

There are many advantages to carrying out electron paramagnetic resonance experiments at very high frequencies, either as part of a multifrequency strategy for solving problems or for special characteristics of high frequencies. These special characteristics include the potential for high point sensitivity, enhanced resolution, separation of similar species, altered sensitivity to motion, suppression of motional effects, and many others. This paper describes a three-millimeter-wavelength (W-band, 94 GHz) EPR spectrometer built for a multi-user facility and illustrates with some examples, most of them being disordered systems. One significant example is the oxidized primary reaction center, P ₈₆₅ ⁺ , isolated from the red photosynthetic bacteriaRhodobacter Sphaeroides R-26. The W-band technique applied to both centers isolated from bacteria grown from either deuterated or ordinary growth media allows extraction of the fullg anisotropy in these centers and sets the stage for multifrequency EPR spectroscopy to yield a full analysis of the various contributions to linewidths in these systems.     

31P NMR studies on the interaction of morpholinyl anthracyclines and related compounds with d(CGTACG)2. Thermodynamic and kinetic parameters

³¹P NMR spectroscopy has been used to study the intercalation of the anthracyclines doxorubicin1, daunorubicin2, 4-demethoxydaunorubicin3, morpholinodoxorubicin4, methoxymorpholinodoxorubicin5 and 9-deoxydaunorubicin6 with the DNA fragment d(CGTACG)₂. The individual phosphate resonances of the oligonucleotide were assigned in the free as well as in the intercalated species. The³¹P chemical shift variations allowed us to identify the intercalation sites, which resulted to be the same for all compoundsi.e. between the terminal CG base-pairs of the helix (two molecules of drugper duplex). The binding constants, the dissociation rate constants and ΔG ^# values have been determined in different conditions of ionic strength and temperature. The kinetic constant (k _off) of the slow step of the anthracycline/duplex intercalation process has been directly measured by NOE exchange techniques. Binding constants depend on the ionic strength and on the self-association process so greatly, that their use to study by NMR anthracycline/DNA interactions is questionable. On the contrary, thek _off are not affected by these phenomena and present an interesting trend for1–6, thus showing that the average lifetime of the drug in the intercalation site appears to be important for determining the cytotoxicity and the antimitotic activity.     

The EPR dipolar broadening of irradiated solids as detected using FID and ESE following selective hole burning

A novel electron spin echo method employing detection of FID and transient echo shape following selective hole burning in the EPR spectrum has been applied to study γ-irradiated malonic acid. The dipolar lineshapes of stabilized free radicals are separated into bulk and pairwise contributions; both of them have proved to be Lorentzian.     

Photo-CIDNP study of the splitting of the dinucleotidecis,syn-thymine dimer with reduced flavin as a sensitizer: Evidence for a thymine radical anion intermediate

Photochemically induced dynamic nuclear polarization (photo-CIDNP) studies were performed on the splitting ofcis,syn 2′-deoxythymidylyl-(3′→5′)-2′-deoxythymidine cyclobutane dimer (cs-dTp dT), using reduced flavin as a sensitizer. This system serves as a model for the light-induced repair mechanism of thymine dimers in DNA by the enzyme photolyase. The CIDNP spectrum shows enhanced absorption of the two C6-H protons of the corresponding monomer dTpdT, which demonstrates that a thymine radical anion is involved in the splitting of the dimer. This is supported by a similar CIDNP spectrum that is obtained with the electron-donorN _α-acetyl tryptophan as a sensitizer. This result suggests that the light-induced splitting of thymine dimers in DNA by photolyase also proceedsvia the thymine radical anion. The small difference in intensity of the two CIDNP signals belonging to the C6-H protons shows that the unpaired electron in the monomer radical has a slight preference for the thymine moiety at the 5′ terminus.     

EPR spectroscopy of poorly characterized systems: A historical and current view

Modeling of the CW-EPR spectra due to transition metal ions in non-crystalline systems grew in sophistication from the early 1960’s until fairly recently. A number of important effects have been introduced into the simulation of CW spectra over the past decade or so. These include allowance for local strain effects, the so-called “g-strain” and correlated “g-A strain”, the latter especially important in multifrequency spectroscopy. Limits have been reached in what is reasonable by way of guesses regarding the underlying lineshape function and further information can only be sought via ENDOR or ESEEM experiments in favourable cases. A clear understanding of the simplest way to describe field swept EPR via a master equation derived in the frequency domain will be reviewed in which asymmetric field swept lines sometimes occur naturally as a consequence of the theory. The paper will provide a review of the history of computer simulations and various statistical approaches to correlation effects covering examples as diverse as proteins and glasses. It is hoped that sufficient progress will have been achieved with a new Bruker ESP 380 pulsed spectrometer that some new results can also be reported.     

ESR study of Cu(II)-indomethacin and its pyridine and DMF adducts

Powder ESR spectra of [Cu₂(Ind)₄](H₂O)₂ at room temperature show the presence of copper acetate like dimers characterized by a strong antiferromagnetic exchange interaction (J≈?338 cm^?1). Two different monomeric species were evidenced in 40%Py+60%DMF solution absorbed on NaY zeolite: the Cu(Ind)₂(DMF)₂ with an elongated tetragonal-octahedral symmetry and the Cu(Ind)₂(Py)₂ with a CuN₂O₂ chromophore in atrans square-planar arrangement. Only one monomeric species Cu(Ind)₂(Py)₂ was identified in 20%Py+80%DMF and 20%Py+80% Chloroform solutions. The Cu(Py)₄ ²⁺ species of CuN₄ chromophore occurs in pyridine Cu(II)-indomethacin solution suggesting a decomposition of the solvit compound.     

Structure of the product of intracomplex alkylation of target octanucleotide pd[TGTTTGGC] by means of 4-[N-methyl-N-(2-chloro-ethyl)-amino]benzyl-5′-phosphamido derivative of heptanucleotide pd[CCAAACA]: Two-dimensional1H-NMR spectroscopy and restrained molecular mechanics refinement

The spatial structure of a covalent adduct — the product of intracomplex alkylation at N-3-position of dC-8 nucleoside residue of target octanucleotide pd[TGTTTGGC] by means of 4-[N-methyl-N-(2-chloroethyl)amino]benzyl-5′-phosphamido derivative of heptanucleotide pd[CCAAACA] — has been investigated in aqueous solution by two-dimensional (2D)¹H-NMR spectroscopy and restrained molecular mechanics calculations. By using COSY, COSY-DQF and NOESY experiments the assignment of oligonucleotide protons as well as protons of modifying group was carried out. The correlation times of benzylamide fragment protons and those of neighbouring nucleotide residues dC-1 and dC-8 were shown to be equal and shorter thanτ _c of intraduplex nucleotides. The analysis of proton-proton coupling constants for H1′, H2′a, H2′b and H3′ protons showed all sugar residues to be in 2′-endo conformation. The distances between protons closed in the space were determined by means of a set of one-dimensional (1D) NOE experiments. The experimental distances were used as the constraints for energy minimization by molecular mechanics calculations. Eight conformations of benzylamide fragment of the covalent adduct differed with orientations of 4-N-methyl residue of the alkylating group and 1-methyleneamide linker function were constructed as starting structures in energy minimization procedure. Among of the resulting optimized structures only one S(Alk)8^* was found to satisfy both experimental data and energetic criteria. The benzylamide fragment in conformation S(Alk)8^* has been shown to localize in the region of lacked nucleoside residue of the duplex at the end of heptanucleotide chain less than 5 Å apart from the residues dC-1 and alkylated dC-8.     

Orientation of the imidazole type ligands in copper complexes studied by ESEEM

Effects of nitrogen quadrupole coupling tensors on ESEEM spectra are discussed. It is shown from theoretical considerations that the spectra, especially the double quantum transition line, are strongly affected by changes of tensor orientation in the magnetic field. Model calculations for angle-selected nitrogen ESEEM spectra of copper complexes in the disordered state show that the double quantum transition line changes its intensity with the resonance magnetic field set position in the EPR spectrum. These model calculations also show that estimation of the orientation of the imidazole type ligands with respect to theg principal axes may be easily made without any tedious processes of computer simulation of the whole ESEEM spectral pattern. The usefulness of the method is experimentally shown by applying it to the structural studies of the copper complexes of 2-(2′-pyridyl)imidazole and 2(2′-pyridyl) benzimidazole and their six coordinated complexes with nitrogeneous bases.     

Determination of the asymmetry parameter of EFG tensor from moments of NQR nutation spectra in powders

A novel approach to determination of the asymmetry parameter of the EFG tensor from zero-field nutation NQR spectra of the spinI = 3/2 nuclei in powder samples is reported. The proposed theoretical treatment uses lineshape analysis of the nutation NQR spectra by the method of line moments. The analytical formulas for the lineshape of the powder nutation spectrum are given. It is shown that the asymmetry parameter can be determined from the second moment 〈ω²〉 and the frequency of only one singularity ω₂ of the nutation spectrum. It is also shown that the asymmetry parameter can be determined from the second and fourth spectrum moments alone. The method is successfully demonstrated for the simulated nutation NQR spectra of the spinI = 3/2 nuclei in powder samples.     

A solid-state (CP-MAS)13C nuclear magnetic resonance study of selected photo(thermo)chromic spiro-(indolinonaphthoxazine)s

Liquid and solid-state¹³C NMR data are reported for three photo(thermo)chromic spiro-(indolinonaphthoxazine)s,1–3. The isotropic¹³C chemical shifts are almost identical in the solid state and in solution. Therefore, the structures in the solid state are fully retained in solution. Quantitative analysis of the spinning side-band pattern of¹³C CP-MAS NMR spectra gave shielding anisotropies and asymmetry parameters different for1–3 and related to the substitution pattern. For3 the tensor componentσ ₃₃, assumed to be perpendicular to the aromatic ring, is more shielded than the corresponding components of1 and2 probably because of the near orthogonality between the substituent piperidinyl ring and the naphthoxazinic system. A correlation between shielding anisotropy and thermochromic activity is suggested.     

Observation of broad EPR spectra of transient free radicals by FT-EPR

Application of electron spin echo Fourier transform EPR (ESE-FT-EPR) to photo-induced chemical reactions is presented. Main purpose of this study is to observe broad EPR spectra of free radicals having very shortT ₂ ^* by means of the ESE-FT-EPR technique. Details of the experimental procedures are described. In ESE experiments design of the resonator is important to obtain sufficient spectral bandwidth because of use of multiple pulses which decrease the bandwidth. We designed and constructed Loop-Gap-Resonantors (LGR) for light irradiation experiments and their specifications were examined. The phase cycling method is essential to obtain pure ESE signals and proper time resolution by eliminating unwanted FID signals which result from imperfect pulse angles. We applied this technique to observe the photo-induced electron transfer reaction between tetraphenylporphinato zinc(II) (ZnTPP) and duroquinone (DQ) in an ethanol solution, and successfully observed the time resolved EPR spectra of the both Zn(TPP) cation and DQ anion radicals by ESE-FT-EPR of the Hahn echo. The half-height full-width of envelope of EPR spectrum of Zn(TPP)⁺, which is never observed in ordinary FT-EPR, is about 16 MHz. Specificity of spectra and the time resolution are compared among the ESE-FT-, FT- and cw-Time-Resolved-EPR (cw-TREPR) techniques.     

Multinuclear study of RF losses in NaCl/D2O solutions

The RF losses in NMR experiments on ionic solutions have been investigated by multi-NMR. The change of π/2 pulse length (PW)₉₀ for²³Na,³⁵Cl,²D,¹⁷O and¹H nuclei in NaCl/D₂O solutions has been systematically studied. Quantitative relation between (PW)₉₀ values and the sample specific conductivity σ has been established. The increase in (PW)₉₀ values has been experimentally correlated to the reduction of the signal-to-noise ratio through¹⁷O measurements.     

EPR study of carbonate derived and ozonide radicals in carbonated apatites synthesized from aqueous solutions

In this study, a series of Na⁺ and¹²CO ₃ ^2? containing apatites synthesized by a hydrolysis of octo-calciumphosphate (OCP) and dried at 25°C until constant weight, were examined with EPR after X-irradiation. A variety of different paramagnetic radicals was formed, giving rise to composite and hence complex EPR powder spectra. The spectra were successfully decomposed into their basic components using a multivariate statistical technique. The different components could be identified unambiguously. In this way, it was found that an O^?, an O ₃ ^? , a CO ₃ ^? , two types of CO ₂ ^? and two types of CO ₃ ^3? ions were formed upon X-irradiation. Also resonances from atomic hydrogen were observed. The most striking feature is the presence of the ozonide ion, which is only rarely observed in irradiated hydroxyapatites. A comparison is made between the EPR spectra of apatite samples prepared by hydrolysis of OCP on the one hand, and those of samples prepared by hydrolyzing monetite, and the tooth enamel spectrum on the other hand.