Comparative analysis of pulsed electron spin resonance spectrometers at X-band and S-band

Comparative analysis of pulsed electron spin resonance spectroscopy at X-band and at S-band indicates that despite the lower sensitivity at the lower frequency, electron spin echo spectroscopy at S-band provides valuable information on the electron-nuclear interactions in systems where the electron spin echo modulation is too small to study well at X-band. It is shown that independent experimental data on electron spin echo modulation and decay at both X-band and S-band put additional constraints on the structural parameters obtained by comparison of experimental and simulated nuclear modulation patterns, and can also help to elucidate the electron spin relaxation mechanism.     

Electron spin-echo envelope modulation at S-band. 1: Analysis of hyperfine interaction effects

Electron spin-echo techniques have been successfully applied to the study of disordered systems mainly due to the nuclear modulation of the echo signal, the analysis of which gives information on the structural arrangement around the paramagnetic center. Most applications to date have been carried out at X-band (≈9 GHz). Here we report on the analysis of two and three pulse modulation patterns simulated at S-band (≈3 GHz) for which the modulation depth is about tenfold deeper and more quantitative analysis ofI=1/2 nuclei becomes possible. The investigation is applied to two different nuclei of experimental interest at different electron-nucleus distances, and comparison is done with the corresponding X-band patterns.¹³C and¹⁵N nuclei, both with nuclear spin quantum valueI=1/2, are chosen due to their different nuclearg-factors that produce quite different modulation periods. Modulation features due to isotropic hyperfine couplings are also analyzed. It is shown that in several cases complicated patterns arise, that are successfully interpreted by Fourier transform in the frequency domain. Some conclusions are drawn concerning the experimental conditions under which the use of S-band is really of significant advantage with respect to X-band.     

Spin dynamics in continuous wave and pulsed ENDOR spectroscopy of coals

ENDOR experiments on coals recorded using continuous wave (CW) and pulsed techniques appear to give qualitatively different spectra. A matrix proton signal dominates the ENDOR spectrum of coals recorded in the CW ENDOR experiment while both a matrix and local proton ENDOR signals with huperfine couplings of up to 20 MHz are observed in spectra recorded using pulsed excitation techniques. Analysis of these spectra lead to different implications for the structure of the molecules that host the unpaired electron. Using a combination of pulsed EPR (Electron Spin Echo, FID detected hole burning) and pulsed Electron Nuclear Multiple Resonance (Sub-level relaxation, hyperfine selective ENDOR, EPR sub-spectra) experiments, we investigate the electron and nuclear spin dynamics in order to reconcile the different signal amplitudes observed in the CW and pulsed ENDOR spectra. In the CW ENDOR experiment, the results of the FID detected hole burning experiments prove that the low ENDOR signal intensity can not be attributed to spectral diffusion mechanisms competing with ENDOR mechanisms. Instead, we find that an unfavorable ratio of the electron and nuclear spin relaxation rates results in small local ENDOR signals. The matrix line dominates the spectrum because of the large number of matrix protons. In the pulsed ENDOR experiment, the hyperfine contrast selectivity mechanism suppresses the intensity of the matrix ENDOR signal and enhances the amplitudes of the local ENDOR signals. In addition, the ENDOR signal is not a function of the ratio of the electron and nuclear relaxation rates.     

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.     

Determination of the deuterium content of deuterated betaine arsenate by electron spin echo envelope modulation

An alternative method for the determination of the deuterium content in the hydrogen bonds of ferroelectric/antiferroelectric deuterated betaine arsenate is presented. Carbon radicals formed by gamma irradiation of the betaine arsenate have been used as paramagnetic probes. The deuterium content in the hydrogen bonds has been determined by analyzing the modulation in the electron spin echo spectra of the carbon radical (CH₃)₃N+CHCOO^? which arises because of the dipolar interactions between the paramagnetic radical and the deuterium nuclei.     

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.     

Electron spin-echo envelope modulation at S-band. 2: Hyperfine and weak nuclear quadrupole interaction effects forI>1/2

The analysis of the nuclear modulation often observed in the electron spin-echo signals gives information on the structural arrangement around the paramagnetic center and on the interactions undergone by the spin system. Most applications to date have been carried out at X-band (≈9 GHz). However, to run experiments at different operating frequencies yields more accurate results and increases the number of experimental situations that can be investigated. Here we analyze the two- and three-pulse modulation patterns simulated at S-band (≈3 GHz) where the modulation is due to two different nuclei of experimental interest at different electron-nucleus distances, and comparison is done with the corresponding X-band patterns.²H (I=1) and⁷Li (I=3/2) nuclei are chosen due to their low quadrupole moment, so that comparison can be done between the effects due to weak nuclear quadrupole interaction at the two bands. It is shown that at short distances the complicated patterns due to hyperfine couplings are successfully interpreted by Fourier transform in the frequency domain, and that the use of S-band is really of some advantage with respect to X-band in detecting the presence of a weak nuclear quadrupole interaction.     

Pulsed 95 GHz high-field EPR heterodyne spectrometer with high spectral and time resolution

Various time resolved EPR methods are applied to different test samples to demonstrate the abilities of pulsed high-field EPR spectroscopy. Two-pulse-echo field swept EPR spectroscopy on a nitroxide radical shows the increased spectral resolution by separating different spin systems by their relaxation properties. Additionally N¹⁴ electron-spin-echo-envelope-modulation (ESEEM) is observed for these systems at fields as high as 3.5 T. Thus, the N¹⁴ hyperfine interaction couplings can be probed by ESEEM and pulsed ENDOR (electron-nuclear-double-resonance) experiments. The sensitivity of pulsed ENDOR experiments is compared with cw-ENDOR. The different linewidths and amplitudes of the two methods are discussed. Transient nutation experiments on light induced triplet states demonstrate the high sensitivity and time resolution of high-field pulsed EPR. The sensitivity and time resolution of our 95 GHz spectrometer are determined and compared with pulsed X-band EPR spectrometer performances.     

An ENDOR study of radical spin adducts derived from novel 2-substituted-5,5-dimethyl-pyrroline-N-oxide spin traps

ENDOR spectra of a series of carbon- and oxygen-centered radical adducts of 2-substituted DMPO-type nitrones are reported. They include the novel cyclic nitrones, 2-phenyl-5,5-dimethyl-pyrroline-N-oxide (2-Ph-5,5-M₂PO), 2,5,5-trimethyl-l-pyrroline-N-oxide (2,5,5-M₃PO), and 2-phenyl-3,3,5,5,-tetramethyl-l-pyrroline-N-oxide (2-Ph-3,3,5,5-M₄PO). Electron paramagnetic resonance (EPR) was used to ascertain the nitrogen hyperfine splittings (hfs’s) while¹H ENDOR was employed to determine the long-range (γ) hydrogen hfs’s. The magnitude of the nitrogen hfs combined with the numbers and sizes of the long-range γ-H hfs’s of spin adducts of these new spin traps are shown to help disclose the identities of various added radicals (or radical addends). It should be noted that the three new spin traps presented here are keto-nitrones not aldo-nitrones. Thus, there is alkyl (e.g. CH₃) or aryl (e.g. C₆H₅) substitution at the 2-position of the pyrroline-N-oxide ring. This feature is part of our search for modified spin traps that yield spin adducts with greater stability.     

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.     

Nucleic acid spin labeling study of interaction between polyadenylic acid: Polyuridilic acid duplex and egg phosphatidylcholine vesicles. Involvement of terminal and internal nucleotides and sugar-phosphate backbone

Model DNA-membrane contacts were studied in the system containing polyadenylic acid (polyA): polyuridilic acid (polyU) duplex and egg phosphatidylcholine (PC) vesicles without or in the presence of MgCl₂. PolyU containing O-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carbonyl) spin label at 2′-OH moiety was used for spin labeling study of nucleic acid-phospholipid interactions. For polyA labeled with the same nitroxyl radical EPR spectra from spin labels attached at terminal and at internal nucleotide units contribute into the EPR spectrum of d.s. polynucleotide without or in the presence of egg PC vesicles and Mg ions. Internal spin labels are more sensitive to the interaction with PC-MLV than terminal ones. The complexation between s.s. polynucleotide and PC-MLV was shown. The values of spin labels’ melting temperature of polyA: SL-polyU duplex at 1 mM MgCl₂ reflect the changes in rotational mobility of terminal spin labels connected with duplex→triplex transition. Arrhenius plots of temperature dependence of EPR spectra parameters prove that ribose moieties of sugarphosphate backbone are involved into the interaction between polynucleotide duplex and phospholipid bilayer. Egg PC vesicles demonstrate stabilizing effect on polyA: polyU duplex at 1 mM of Mg²⁺ and destabilizing effect at 5 mM Mg²⁺.     

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.     

A new algorithm for analysis of ESEEM from distant matrix nuclei

A new algorithm is developed for obtaining information about the structure of the paramagnetic center’s surroundings by distant matrix deuterons, explicitly accounting for the weak quadrupole interaction of deuterium nuclei and for the multiplicativity of nuclear contributions into ESE modulation.     

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.     

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.     

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.     

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.     

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.     

Interactions between polyamines and nucleotides studied by31P and1H NMR

The formation of complexes between poly- and mono-amines and some polyphosphate molecules such as ATP, ADP and tripolyphosphate was studied by³¹P and¹H NMR. Evidence for formation of complexes was obtained by¹H NMR, while the values of dissociation constants of the complexes between nucleotides and spermine and spermidine, calculated from the shift of the³¹P NMR, were in mM range.