Discrimination of radiation quality through second harmonic out-of-phase cw-ESR detection.

The ability to discriminate the quality of ionizing radiation is important because the biological effects produced in tissue strongly depends on both absorbed dose and linear energy transfer (LET) of ionizing particles. Here we present an experimental electron spin resonance (ESR) analysis aimed at discriminating the effective LETs of various radiation beams (e.g., 19.3 MeV protons, (60)Co photons and thermal neutrons). The measurement of the intensities of the continuous wave spectrometer signal channel first harmonic in-phase and the second harmonic out-of-phase components are used to distinguish the radiation quality. A computational analysis, was carried out to evaluate the dependence of the first harmonic in-phase and second harmonic out-of-phase components on microwave power, modulation amplitude and relaxation times, and highlights that these components could be used to point out differences in the relaxation times. On the basis of this numerical analysis the experimental results are discussed. The methodology described in this study has the potential to provide information on radiation quality.

Degradation products from naturally aged paper leaves of a 16th-century-printed book: a spectrochemical study.

In this work, we present a wide-range spectrochemical analysis of the degradation products from naturally aged paper. The samples obtained from wash waters used during the de-acidification treatment of leaves from a 16th-century-printed book were analysed through NMR, IR, Raman UV/Vis, EPR and X-ray fluorescence (XRF) spectroscopy and HPLC-MS and inductively coupled plasma (ICP) analysis. By these methods we also studied some of the previous samples treated by acidification (sample AP) and catalytic hydrogenation (sample HP). Crossing all the data, we obtained precise indications about the main functional groups occurring on the degraded, water-soluble cellulose oligomers. These results point out that the chromophores responsible for browning are conjugated carbonyl and carboxyl compounds. As a whole, we show that the analysis of wash waters, used in the usual conservation treatments of paper de-acidification, gives much valuable information about both the conservation state of the book and the degradation reactions occurring on the leaves, due to the huge amount of cellulose by-products contained in the samples. We propose therefore this procedure as a new very convenient general method to obtain precious and normally unavailable information on the cellulose degradation by-products from naturally aged paper.

Interpretation of cw-ESR spectra of p-methyl-thio-phenyl-nitronyl nitroxide in a nematic liquid crystalline phase.

In this paper we report on the characterization by continuous wave electron spin resonance spectroscopy (cw-ESR) of a nitronyl nitroxide radical in a nematic phase. A detailed analysis is performed by exploiting an innovative modeling strategy alternative to the usual spectral simulation approach: most of the molecular parameters needed to calculate the spectrum are evaluated a priori and the ESR spectrum is obtained by direct application of the stochastic Liouville equation. Allowing a limited set of fitting parameters it is possible to reproduce satisfactorily ESR spectra in the temperature range 260 K-340 K including the nematic-to-isotropic phase transition (325.1 K). Our results open the way to a more quantitative understanding of the ordering and mobility of nitronyl nitroxide radicals in nanostructured environments.

Triplet states of the nonlinear optical chromophore DCM in single crystals of potassium hydrogen phthalate and their relationship to single-molecule dark states.

Single-molecule dark states are often attributed to photoexcited triplets with scant evidence of the participation of paramagnetic molecules. The photodynamics of blinking single molecules of 4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM) in crystals of potassium hydrogen phthalate (KAP) were compared with the lifetimes of DCM triplet states, likewise in KAP, whose zero-field splitting (ZFS) tensors were fully characterized by time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy. Luminescent mixed crystals of KAP were grown from solutions containing 10(-4) -10(-9) M DCM, a model optically nonlinear chromophore. The luminescent dye was localized in the {111} crystalline growth sectors. The photoexcited triplets states of DCM in the heavily dyed (10(-4) M) crystals were analyzed by TR-EPR spectroscopy. The photoexcited singlet states of DCM in lightly dyed crystals (10(-9) M) were analyzed by single-molecule microscopy. Large blue shifts in the absorption and emission spectra of DCM in KAP were interpreted as a consequence of protonation at the dimethylamino nitrogen atom, an assignment supported by calculations of the zero-field splitting (ZFS) tensors of molecules in their triplet states. Experimental ZFS tensors with eigenvalues comparable to those of the computed tensors were determined from the angular dependence of the EPR spectra of DCMH(+) triplets within KAP single crystals with respect to the applied magnetic field. Data from individual growth sectors failed to show magnetically equivalent site occupancies, evidence of the kinetic ordering during growth. The intermittent fluorescence of individual chromophores was analyzed. The distributions of on(off) times were characterized by distributed rates fit to power laws. The lifetime of the triplet states was analyzed from the time decay of the EPR signals between 100 and 165 K. The data were well fit with a single time constant for the signal decay, a result wholly inconsistent with the blinking of single molecules with off times commonly of tens of seconds. Triplet decay was extrapolated to approximately 25 micros at room temperature. Therefore, the assumption that single-molecule dark states originate with triplet excited states is not sustainable for single DCM molecules in KAP.

Analysis of the spatial distribution of free radicals in ammonium tartrate by pulse EPR techniques.

Using pulse electron paramagnetic resonance (EPR) on a series of l(+)-ammonium tartrate (AT) dosimeters exposed to radiations with different linear energy transfer (LET), we assessed the ability of pulse EPR spectroscopy to discriminate the quality of various radiation beams such as (60)Co gamma-ray photons, protons and thermal neutrons at various doses by analyzing the local radical distributions produced by the different beams. We performed two types of pulse EPR investigations: two-pulse electron spin echo decay obtained by varying the microwave power, and a double electron-electron resonance (DEER) study. Both methods provide information about the dipolar interactions among the free radicals and about their spatial distributions. The first method provided information on the instantaneous diffusion and hence the microscopic concentration of the radicals that is compared with the macroscopic one obtained by CW-EPR. The DEER spectra yielded the distributions of distances between pairs of radicals two to five crystal cells apart produced by the same radiation event, a result reported here for the first time. The inter-radical distributions given by the DEER results have been simulated by modeling the radical distributions according to the details of the matter-radiation interactions for the various beams. The results of both types of pulse experiments are strongly dependent on the radiation quality. This was also observed for samples giving indistinguishable CW-EPR spectral profiles. We conclude that the pulse EPR measurements can be valuable tools for distinguishing the LET of the radiation beams, an important parameter for radiobiological considerations.

Three different tyrosyl radicals identified in L-tyrosine HCl crystals upon gamma-irradiation: magnetic characterization and temporal evolution.

High-frequency electron paramagnetic resonance (EPR) and X-band electron-nuclear double resonance (ENDOR) spectroscopies were used to investigate the effect of gamma-irradiation on single crystals of L-tyrosine hydrochloride at room temperature. The oxidation product is the tyrosyl radical formed by hydrogen abstraction from the phenolic group; interestingly, on freshly irradiated crystals, two tyrosyl radicals were identified, characterized by slightly different magnetic parameters. In particular, one of the two radicals, with a gxx value of 2.00621, has its phenoxyl oxygen strongly hydrogen-bonded to one or more donors; to our knowledge, this is the lower gxx value reported for tyrosyl radicals. These two oxidation radicals are found to evolve very slowly to a third, single more stable radical conformation. To interpret the experimental data, a possible molecular scenario is presented, where the process of radical formation can be seen as a hydrogen atom transfer or a proton-coupled electron transfer. These processes seem to be controlled by the specific network of hydrogen-bond interactions present in the crystal. The results are discussed in relation to their relevance for the interpretation of EPR spectra of tyrosyl radicals in biological systems.

Conformational role of the divalent metal in bovine heart mitochondrial F1-ATPase: an electron spin echo envelope modulation study.

The catalytic sites of beef heart mitochondrial F1-ATPase were studied by electron spin echo envelope modulation (ESEEM) spectroscopy, using Mn(II) as a paramagnetic probe, which replaces the naturally occurring Mg(II), maintaining the enzyme catalytic activity. F1-ATPase was purified from beef heart mitochondria. A protein still containing three endogenous nucleotides, named MF1(1,2), is obtained under milder conditions, whereas a harsher treatment gives a fully depleted F1, named MF1(0,0). Several samples were prepared, loading MF1(0,0) or MF1(1,2) with Mn(II) or MnIIADP in both substoichiometric and excess amounts. When MF1(1,2) is loaded with Mn(II) in a 1:0.8 ratio, the FT-ESEEM spectrum shows evidence of a nitrogen interacting with the metal, while this interaction is not present in MF1(0,0) + Mn(II) in a 1:0.8 ratio. However, when MF1(0,0) is loaded with 2.4 Mn(II), the FT-ESEEM spectrum shows a metal-nitrogen interaction resembling that present in MF1(1,2) + Mn(II) in a 1:0.8 ratio. These results strongly support the role of the metal alone in shaping and structuring the catalytic sites of the enzyme. When substoichiometric ADP is added to MF1(1,2) preloaded with 0.8 equiv of Mn(II), the ESEEM spectra show evidence of a phosphorus nucleus coupled to the metal, indicating that the nucleotide phosphate binding to Mn(II) occurs in a catalytic site. Generally, 14N coordination to the metal is clearly identified in the ESEEM spectra of all the samples containing more than one metal equivalent. One point of note is that the relevant nitrogen-containing ligand(s), responsible for the signals in the ESEEM spectra, has not yet been identified in the available X-ray structures.

Development and validation of an integrated computational approach for the modeling of cw-ESR spectra of free radicals in solution: p-(methylthio)phenyl nitronylnitroxide in toluene as a case study.

In this work we address the interpretation, via an ab initio integrated computational approach, of continuous wave electron spin resonance (cw-ESR) spectra of p-(methylthio)phenyl nitronylnitroxide (MTPNN) dissolved in toluene. Our approach is based on the determination of the spin Hamiltonian, averaged with respect to fast vibrational motions, with magnetic tensor parameters (Zeeman and hyperfine tensors) characterized by quantum mechanical density functional calculations. The system is then described by a stochastic Liouville equation, with inclusion of diffusive rotational dynamics. Parametrization of diffusion rotational tensor is provided by a hydrodynamic model. Cw-ESR spectra of MTPNN are simulated for a wide range of temperatures (155-292 K) with minimal resorting to fitting procedures, proving that the combination of sensitive ESR spectroscopy and sophisticated modeling can be highly helpful in providing structural and dynamic information on molecular systems.

Photoexcited triplets of dyes in zeolitic nanostructured channels. A time resolved EPR study.

Two dyes (4-nitrostilbene, NST and 4-N,N-dimethylamino-4'-nitrostilbene, DANS) included in zeolites with nanometric channels and different Si : Al ratios have been photoexcited and their triplet state studied by time resolved EPR (TR-EPR). This is the first time that a TR-EPR spectrum of photoexcited triplet states of dyes in zeolites has been observed. The zeolites used were ZSM-5 and mordenite, with either protons or lithium as charge compensating ions, and the aluminium-free porosil. The ZFS parameters and the polarized spin populations have been obtained, and compared with those obtained in glassy solutions and in the organic nanostructured matrix perhydrotriphenylene (PHTP). For (3)DANS in neutral solvents and in PHTP the dimethylamino group is pi conjugated, whereas only its acid form is detected in all the zeolites. In the latter the spectra of the radical cations formed by spontaneous oxidation have also been observed both by cw-EPR and TR-EPR, the last spectra being spin polarized in emission. The mobility of the triplets is discussed, taking into account the spin polarization of the radical cations indicating a strong radical-triplet interaction.

Time-resolved EPR spectra of the triplet excited states of diaminoacridine guests in polar potassium hydrogen phthalate single crystals.

Mixed crystals of potassium hydrogen phthalate containing 3,6-diaminoacridine were photoexcited with visible light and the resulting triplet excited states were analyzed by time resolved EPR spectroscopy. Spectra from discrete growth sectors were compared with powders and polycrystalline glasses prepared at various pHs. The data yield the predominant protonation state and orientation of the triplets in each of a pair of growth sectors bounding the positive and negative ends of the polar crystal.

Structure and dynamics of the triplet state of oligothiophenes in isotropic and partially oriented matrices.

The photoexcited triplet states of 4,4'-dipentoxy-2,2'-dithiophene (4-T2), 3,3'-dipentoxy-2,2'-dithiophene (3-T2), and 4,4'''-dipentoxy-2,2':5',2'':5'',2'''-tetrathiophene (4-T4) have been investigated by time-resolved electron paramagnetic resonance in glassy toluene and in a frozen oriented liquid crystal, which provides a partially ordered medium. The preferential orientation of the rod-like 4-T2 and 4-T4 is compared to that of the disk-like 3-T2. The use of an oriented matrix coupled to simple semiempirical calculations allowed us to determine the orientation of the principal axes of the fine interaction with respect to the molecular axes. The motional behavior of the molecules in the isotropic and anisotropic matrices has been studied by comparing the spectral profiles of the Echo detected EPR (Echo-EPR) spectra with those of the continuous wave time resolved EPR (TR-EPR). A model considering the modulation of the zero-field splitting (ZFS) by molecular libration accounts for the Echo-EPR lineshape, on the basis of motions around preferential axes depending on the embedding matrix. The differences in the ZFS parameters of the two isomers 4-T2 and 3-T2 are attributed to a mesomeric effect due to the substituents.

Dynamics of alkoxy-oligothiophene ground and excited states in nanochannels.

Two oligothiophenes, 4,4'-dipentoxy-2,2'-dithiophene and 4,4"-dipentoxy-2,2':5',2":5",2' ''-tetrathiophene, have been included in the nanochannels of the autoassembling host TPP (tris-o-phenylenedioxycyclotriphosphazene). The effect of the confinement on the structure and properties of the two dyes, as conformational arrangements, dynamics, and photophysical behavior, was addressed by the combination of high spinning speed solid-state NMR and time-resolved EPR spectroscopy. We compared the conformations of the dyes in their ground and photoexcited triplet states and described in detail the dynamics of the supramolecular adducts from 4 K to room temperature. Above 200 K surprisingly fast spinning rates of the dithiophene core were discovered, while the side chains show far slower reorientation motion, being in bulky gauche-rich conformations. These lateral plugs keep the planar core as appended in the space like a nanoscale gyroscope, allowing a reorientation in the motion regime of liquids and a long triplet lifetime at unusually high temperature. The nuclear magnetic properties of the guest dyes are also largely affected by the aromatic rings of the neighboring host, imparting an impressive magnetic susceptibility effect (2 ppm proton shift). The high mobility is due to the formation of a nanocage in a channel where aliphatic and aromatic functions isolate the thiophene moieties. Instead, two conformers of the tetrathiophene twisted on the central bond are stabilized by interaction with the host. They interconvert fast enough to be averaged in the NMR time scale.

High-affinity metal-binding site in beef heart mitochondrial F1ATPase: an EPR spectroscopy study.

The high-affinity metal-binding site of isolated F(1)-ATPase from beef heart mitochondria was studied by high-field (HF) continuous wave electron paramagnetic resonance (CW-EPR) and pulsed EPR spectroscopy, using Mn(II) as a paramagnetic probe. The protein F(1) was fully depleted of endogenous Mg(II) and nucleotides [stripped F(1) or MF1(0,0)] and loaded with stoichiometric Mn(II) and stoichiometric or excess amounts of ADP or adenosine 5'-(beta,gamma-imido)-triphosphate (AMPPNP). Mn(II) and nucleotides were added to MF1(0,0) either subsequently or together as preformed complexes. Metal-ADP inhibition kinetics analysis was performed showing that in all samples Mn(II) enters one catalytic site on a beta subunit. From the HF-EPR spectra, the zero-field splitting (ZFS) parameters of the various samples were obtained, showing that different metal-protein coordination symmetry is induced depending on the metal nucleotide addition order and the protein/metal/nucleotide molar ratios. The electron spin-echo envelope modulation (ESEEM) technique was used to obtain information on the interaction between Mn(II) and the (31)P nuclei of the metal-coordinated nucleotide. In the case of samples containing ADP, the measured (31)P hyperfine couplings clearly indicated coordination changes related to the metal nucleotide addition order and the protein/metal/nucleotide ratios. On the contrary, the samples with AMPPNP showed very similar ESEEM patterns, despite the remarkable differences present among their HF-EPR spectra. This fact has been attributed to changes in the metal-site coordination symmetry because of ligands not involving phosphate groups. The kinetic data showed that the divalent metal always induces in the catalytic site the high-affinity conformation, while EPR experiments in frozen solutions supported the occurrence of different precatalytic states when the metal and ADP are added to the protein sequentially or together as a preformed complex. The different states evolve to the same conformation, the metal(II)-ADP inhibited form, upon induction of the trisite catalytic activity. All our spectroscopic and kinetic data point to the active role of the divalent cation in creating a competent catalytic site upon binding to MF1, in accordance with previous evidence obtained for Escherichia coli and chloroplast F(1).