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The reaction between isatin and 2,5-dimethoxyaniline is described. The main product was identified as 3,3-bis(4-amino-2,5-dimethoxyphenyl)-1,3-dihydroindol-2-one. The antioxidant activity of the compounds isolated was evaluated with two methods. Three published antitumor E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-2-ones entered the same tests to search whether they are endowed with antioxidant activity too. 3,3-Bis(4-amino-2,5-dimethoxyphenyl)-1,3-dihydroindol-2-one and the three antitumor agents showed a good chemical antioxidant activity.

The cooperative effect of solvophobic interactions and hydrogen bonding has been exploited to self-assemble supramolecular helical architectures of 8-oxoguanosines in different environments. This self-assembly into helical structures is completely different from that of the parent guanosines which, in the same experimental conditions, form flat, ribbonlike structures. While optical microscopy and X-ray diffraction suggest a chiral columnar aggregate in the LC phase, NMR and Circular Dichroism reveal the presence of a helical structures in solution. Scanning Tunneling Microscopy made it possible to visualize hexagonally arranged G-quartets on graphite, which are sections of the helices packed with their long axis perpendicular to the basal plane of the substrate. Due to their rectifying electrical properties, such helices are interesting for fabricating (opto)electronic biodevices.

The anaerobic ribonucleotide reductase from Escherichia coli contains an iron-sulfur cluster which, in the reduced [4Fe-4S](+) form, serves to reduce S-adenosylmethionine and to generate a catalytically essential glycyl radical. The reaction of the reduced cluster with oxygen was studied by UV-visible, EPR, NMR, and Mössbauer spectroscopies. The [4Fe-4S](+) form is shown to be extremely sensitive to oxygen and converted to [4Fe-4S](2+), [3Fe-4S](+/0), and to the stable [2Fe-2S](2+) form. It is remarkable that the oxidized protein retains full activity. This is probably due to the fact that during reduction, required for activity, the iron atoms, from 2Fe and 3Fe clusters, readily reassemble to generate an active [4Fe-4S] center. This property is discussed as a possible protective mechanism of the enzyme during transient exposure to air. Furthermore, the [2Fe-2S] form of the protein can be converted into a [3Fe-4S] form during chromatography on dATP-Sepharose, explaining why previous preparations of the enzyme were shown to contain large amounts of such a 3Fe cluster. This is the first report of a 2Fe to 3Fe cluster conversion.

The program DYANA, for calculation of solution structures of biomolecules with an algorithm based on simulated annealing by torsion angle dynamics, has been supplemented with a new routine, PSEUDYANA, that enables efficient use of pseudocontact shifts as additional constraints in structure calculations of paramagnetic metalloproteins. PSEUDYANA can determine the location of the metal ion inside the protein frame and allows to define a single tensor of magnetic susceptibility from a family of conformers. As an illustration, a PSEUDYANA structure calculation is provided for a metal-undecapeptide complex, where simulated pseudocontact shifts but no NOE restraints are used as conformational constraints.

The pseudocontact shifts of NMR signals, which arise from the magnetic susceptibility anisotropy of paramagnetic molecules, have been used as structural constraints under the form of a pseudopotential in the SANDER module of the AMBER 4.1 molecular dynamics software package. With this procedure, restrained energy minimization (REM) and restrained molecular dynamics (RMD) calculations can be performed on structural models by using pseudocontact shifts. The structure of the cyanide adduct of the Met80Ala mutant of the yeast iso-1-cytochrome c has been used for successfully testing the calculations. For this protein, a family of structures is available, which was obtained by using NOE and pseudocontact shifts as constraints in a distance geometry program. The structures obtained by REM and RMD calculations with the inclusion of pseudocontact shifts are analyzed. Proteins 29:68–76, 1997. © 1997 Wiley-Liss, Inc.

The solution structure of the dicerium(III) complex of the N-terminal domain of calmodulin (Ce2-TR1C hereafter) has been solved employing paramagnetic T1 relaxation enhancements and pseudocontact shifts introduced by the Ce3+ ions, together with conventional NOE constraints. The use of pseudocontact shift constraints constitutes the first attempt to locate metal ions within a protein structure by NMR. Like calcium(II), paramagnetic cerium(III) has been found to bind to the two metal binding sites of the TR1C fragment of calmodulin in a cooperative manner. Due to the presence of pseudocontact interactions between the Ce3+ ions and protons of the 76-residue protein, the 1H NMR spectra of the complex show resonances shifted between +22 and -9 ppm. Eighty percent of its proton resonances could be assigned through a standard approach using TOCSY/COSY and NOESY spectra and through 1D NOE difference spectra for the broad resonances of protons close to the paramagnetic ions. A family of structures was calculated by means of the torsion angle dynamics program DYANA [Guntert, P., Mumenthaler, C., & Wuthrich, K. (1996) XVIIthInternational Conference on Magnetic Resonance inBiological Systems (Abstract)] using 1012 NOEs. Longitudinal proton relaxation times helped to roughly define the position of the metal ions within the protein. A total of 381 pseudocontact shift constraints, whose evaluation and use are critically discussed, have then been added to further refine the metal coordinates within the protein frame and to improve the structure resolution. A dramatic resolution improvement of the metal coordinates together with a sizable resolution improvement in the regions close to the paramagnetic centers, where the number of NOEs is low, is observed. The good quality of the solution structure permitted a meaningful comparison with the solid-state structure of calcium-loaded calmodulin at 1.7 A resolution [Chattopadhyaya, R., Meador, W. E., Means, A. R., & Quiocho, F. A. (1992) J. Mol. Biol. 228, 1177]. The Ce2-TR1C complex is overall more compact than the Ca form.

The availability of NOE constraints and of the relative solution structure of a paramagnetic protein permits the use of pseudocontact shifts as further structural constraints. We have developed a strategy based on: (1) determination of the χ tensor anisotropy parameters from the starting structure; (2) recalculation of a new structure by using NOE and pseudocontact shift constraints simultaneously; (3) redetermination of the χ tensor anisotropy parameters from the new structure, and so on until self-consistency. The system investigated is the cyanide derivative of a variant of the oxidized Saccharomyces cerevisiae iso-1-cytochrome c containing the Met80Ala mutation. The structure has been substantially refined. It is shown that the analysis of the deviation of the experimental pseudocontact shifts from those calculated using the starting structure may be unsound, as may the simple structure refinement based on the pseudocontact shift constraints only.

Oxidized and reduced forms of high-potential iron-sulfur protein (HiPIP) from the purple non-sulfur photosynthetic bacterium Rhodoferax fermentans have been characterized using 1H-NMR spectroscopy. Pairwise and sequence-specific assignments of hyperfine-shifted 1H-NMR signals to protons of cysteine residues bound to the [4Fe-4S]3+/2+ cluster have been performed using one-dimensional NOE and exchange spectroscopy experiments. 1H-NMR hyperfine shifts and relaxation rates of cluster-bound Cys beta-CH2 protons indicate that in the [4Fe-4S]3+ cluster one iron ion can be formally described as Fe(III), while electron density corresponding to one electron is unevenly delocalized onto the remaining three iron ions. This delocalization is effected by means of two different electronic distributions interconverting rapidly on the NMR time scale. The mechanism of paramagnetic proton relaxation, studied by analyzing longitudinal relaxation rates of Cys beta-CH2 protons in HiPIPs from six different sources as a function of the Fe-S-C beta-C alpha dihedral angle, indicate that the major contribution is due to a dipolar metal-centered mechanism, with a non-negligible contribution from a ligand-centered dipolar mechanism which involves the 3p orbital of the Cys sulfur atom. A semi-quantitative tool for extracting structural information from relaxation time measurements is proposed.

A nuclear magnetic resonance (NMR) method was implemented to assess in vivo oxygenation levels by a quantitative determination of the 1H NMR myoglobin (Mb) resonances. The proximal His-F8 NδH at 70–90 ppm and Val-E11 γCH3 resonance at –2.8 ppm, reflecting deoxygenated (deoxy-Mb) and oxygenated (met-Mb) states, were alternately recorded. The method was developed in vitro choosing a couple of NMR sequences that could each maximize the signal-to-noise ratio (SNR) while avoiding baseline rolling and suppressing the water signal. Two quantitative calibration methods were implemented for deoxy- and met-Mb samples (0.1–1 mM), respectively. The respective limit of detection (LOD) and limit of quantification (LOQ) were 0.015 and 0.05 mM for met-Mb and 0.013 and 0.042 mM for deoxy-Mb. Sequences and calibration curves were employed in vivo in Arenicola marina to obtain, for the first time, an accurate measurement of oxy- and deoxy-Mb actual concentrations. In Arenicola, the peaks at approximately 87 and –2.7 ppm, reflecting the deoxy- and oxy-Mb states, respectively, were alternately recorded during increasing hypoxia. The deoxy-Mb concentrations were obtained from the calibration curve. The oxy-Mb concentrations were calculated from the calibration of met-Mb because it was proved that oxy- and met-Mb gave the same NMR molar response. From oxy- and deoxy-Mb concentrations, the intracellular oxygen partial pressure (PiO2) trend was determined.

The effect of exocyclic conjugation on the inversion of a saturated six-membered ring is shown in a dynamic nmr study of a series of N-substituted morpholines where the substituents have similar shapes but different conjugating ability. As conjugation increases the ring inversion barrier decreases in contrast to previous observations of an unsaturated ring with conjugating substituents.

The most effective remedy for iron deficiency is the use of synthetic iron chelates, specifically chelates derived from polyamine-carboxylic acids as EDDHSA (N,N'-ethylenediamine-di-(2-hidroxy-5-sulfophenylacetic) acid). A gradient ion-pair chromatographic method was developed to quantify the total amount of chelated iron in EDDHSA/Fe-3divided by fertilizers. Two mobile phases were used containing. respectively, 35 and 75% acetonitrile in a 0.005 M tetrabutylammonium hydroxide aqueous solution at pH 6.0. The stationary phase was a reverse phase C-18 column (150 mm x 3.9 mm i.d., dp = 5 mum). Two chromatographic peaks appeared and were identified by Electrospray Mass Spectrometry. The first peak corresponds to the monomer of EDDHSA/Fe-3divided by and the second peak has been assigned to condensation molecules. Quality parameters indicate that the method is suitable for the quantification of iron chelate by EDDHSA fertilizers.

In this paper chemometrics (ANOVA and PCR) is used to measure unbiased correlations between NMR spin-echo decays of pork M. Longissimus dorsi obtained through Carr–Purcell–Meiboom–Gill (CPMG) experiments at low frequency (20 MHz) and the values of 14 technological parameters commonly used to assess pork meat quality. On the basis of the ANOVA results, it is also found that the CPMG decays of meat cannot be best interpreted with a “discrete” model (i.e., by expanding the decays in a series of a discrete number of exponential components, each with a different transverse relaxation time), but rather with a “continuous” model, by which a continuous distribution of T 2 's is allowed. The latter model also agrees with literature histological results.

An inexpensive external unit that allows the use of a commercial high-resolution NMR spectrometer as a very low frequency instrument is described. The external unit is phase coherent, the pulse timing being given by the parent spectrometer. With the exception of the probe, the external unit does not contain any tuned elements. This permits easy change of frequency in the range 100 kHz–1 MHz. The external unit may be appropriately employed in food science where, in several cases, low frequency is desirable. An application to hen shell eggs at the frequency of 700 kHz is described.