Presentations with abstracts

Advances in NMR Imaging and Microscopy

Contribution to the conference:
Diffusion, dispersion and shear-delights.Pulsed Gradient Spin Echo NMR and Complex Fluids
Paul T. Callaghan
The range of experiments which can be performed using pulsed gradient spin echo NMR is now very large. This lecture will review some of the tricks of the trade and demonstrate some diverse applications. In particular the lecture will show how Fourier methods may be used to help understand experiments used to study translational motion of spin-bearing particles, with both spatial and temporal Fourier domains being explored. We will start by looking at the various applications of PGSE NMR to studies of diffusion and flow in porous media, covering the phenomena of diffusive and dispersive diffraction. This idea will be extended to consider frequency domain methods, including an examination of which type of gradient modulation scheme is most appropriate. A new theoretical method of handling the description of these generalised experiments will also be mentioned. The use of Pulsed Gradient Spin Echo methods in association with spatial encoding, enables one to image velocity and diffusion with a high degree of accuracy and precision. This lecture will summarize some unusual applications of NMR imaging concerning flow in different geometries, including capillary flow, cylindrical Couette flow, cone and plate flow and both twin-jet and four-roll-mill extensional flow. The study of fluids under shear using NMR imaging and NMR spectroscopy has led to the possibility of investigating the molecular origins of non-Newtonian rheological properties, thus leading to the new field of "Rheo-NMR". A range of examples will be presented in which the phenomena of shear-thinning, slip, spurt and shear banding will be demonstrated. 1. P.T. Callaghan and J. Stepisnik, "Generalised Analysis of Motion using Magnetic Field Gradients", Advances in Magnetic and Optical Resonance, 19, 325-388 (1996) 2. J.D. Seymour and P.T. Callaghan, "'Flow-diffraction' stuctural characterisation and measurement of hydrodynamic dispersion in porous media by PGSE NMR",Journal of Magnetic Resonance A 122, 90-93 (1996) 3. M.M. Britton and P.T.Callaghan, "Two-phase shear band structures at uniform stress " Phys. Rev. Lett ., 78 4930-4933 (1997) 4. P.T. Callaghan, "A simple matrix formalism for the spin echo analysis of restricted diffusion under generalised gradient waveforms", J. Magn. Reson.129, 74-84 (1997)

Contribution to the conference:
Paramagnetic complexes for the recognition of biological macromolecules
Mauro Fasano, Silvio Aime and Paolo Ascenzi
Paramagnetic complexes are widely used for the enhancement of the physiological contrast of MRI images. Their mechanism of action is based on the reduction of NMR relaxation times of water protons in the tissues where they distribute. In order to have the highest effect in the region of interest, these diagnostics should not be diluted in the whole body, rather they must concentrate in an area characterised by a given metabolic state or towards a given organ or tissue. The relaxation enhancement promoted by a contrast agent is usually increased through the formation of non-covalent adducts between the complex and a macromolecular substrate (such as serum albumin) as a consequence of the lengthening of the molecular reorientational correlation time. The targeting of a paramagnetic complex to a given tissue or organ may be pursued by introducing on the surface of a suitable GdIII or MnII complex a functionality able to recognise a protein whose concentration changes drastically during the course of a disease. In this paper we address the problem of targeting paramagnetic complexes to human haemoglobin derivatives and to serine proteases. We show that two GdIII chelates, rather different for structural and electronic characteristics, bind quite strongly to human adult haemoglobin and, more interestingly, they are able to distinguish between conformational states of the oxygen carrier protein. Thus, they may act as reporters of the tissue oxygen pressure. In the second application, a derivatised contrast agent has been developed to interact selectively with proteinases of the trypsin superfamily. The binding interaction is strong enough to form a macromolecular adduct, but in the same time weak enough to avoid activity inhibition.

Contribution to the conference:
Application of ultralow field NMR imaging to study of natural porous media
V.V. Frolov and K.V. Tyutyukin
Nature porous materials are caracterized by a large inhomogeneity of magnetic susceptibility due to various magnetic inclusions and the susceptibility differences between the rock material and the contained fluids. In general, the field nonuniformities cause disturbances in NMR images of liquids filling natural porous media, the more strong magnetic field the more important. Another effect that has an influence on NMR images gotten at various levels of magnetic field is the dispersion of relaxation rates, the must manifesting at weak field. Therefore images at weak field are little influenced of local magnetic inhomogeneities. A serie of samples of rock was studied using NMR imaging at 7 mT on the home- built imager [1]. [1] 5. V. Frolov. NMR imaging at low frequency Magnetic Resonance and Related Phenomena. Aug. 21-27, 1994. Extended Abstracts of the XXVII Congress AMPERE (ed. K. Salikhov), v. 2, p. 705-706.

Contribution to the conference:
The determination of domains of stagnation in hydrodynamic flow by NMR imaging at very weak magnetic field
V.V. Frolov, A.L. Fiodorov, S.V. Binevskaya, Yu.V. Osinzev and K.V. Tyutyukin
The NMR imaging at 7 mT [1] is applied to discovery domains of stagnation in liquid flow through models of elements of hydrodynamic system with obstacles to a flow. Using of NMR imaging with week field do the hydrodynamics study more accessible and economical. [1] V. V. Frolov. NMR imaging at low frequency Magnetic Resonance and Related Phenomena. Aug. 21-27, 1994. Extended Abstracts of the XXVII Congress AMPERE (ed. K. Salikhov), v. 2, p. 705-706.

Contribution to the conference:
NMR STUDIES OF POROUS CATALYST SUPPORT PELLETS
Sally Harding and Dr Lynn Gladden
NMR STUDIES OF POROUS CATALYST SUPPORT PELLETS Sally Harding* and Dr Lynn Gladden** *Department of Chemistry, Swedish University of Agricultural Sciences, PO Box 7015, Uppsala, SWEDEN. **Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, U.K. An understanding of the internal structure, pore-size distribution and macroscopic heterogeneities within porous catalyst support materials play a crucial role in determining their transport properties. The transport characteristics of the support material are intern important in determining the catalyst performance. We have employed a range of NMR experiments to study the pore size distribution and extent of the structural heterogeneities found within commercially available porous catalyst support pellets. Previous studies 1,2 have visualised the internal structure within porous catalyst support pellets by 2D NMR imaging. The porous structure however is a three dimensional network and the work reported here extends these preliminary studies to 3D. 3D spin density-, relaxation- and diffusion- mapping has shown that the porous pellets contain significant structural heterogeneity over length scales of 0.1-1 mm, much larger than the pore-size. The structural heterogeneity observed have been quantified using the concept of a 3D fractal dimension. It was found that pellets taken from the same batch were characterised by a single 3D spin density fractal dimension, whereas pellets manufactured by differing processes, had significantly different fractal dimensions. The 3D spin density fractal dimension is therefore considered to be a useful parameter to quantify heterogeneities in the voidage of the material. 3D T1 weighted images have also been used to study the effect of varying the pixel size in determining the distribution of T1 values. As the slice thickness is increased the distribution of T1 values determined from a 2D slice narrows and the high T1 tail representing higher pore-sizes is lost. This shows the importance of spatial averaging of the signal intensity in determining T1 distributions from spatially resolved maps. The spatial averaging of the signal used to determine the T1 relaxation also accounts for the narrower pore-size distribution determined by NMR relaxometry compared to that determined from nitrogen adsorption and NMR cryoporometry. NMR cryoporometry measures the depression in the melting point of a liquid confined within a porous media to determined the pore size and pore size distribution3. This technique has been used here to study the pore size distribution in a range of catalyst support materials using cyclohexane as the probe molecule. The results have shown good agreement with nitrogen adsorption data and NMR relaxation time analysis. A significant advantage of NMR cryoporometry is that it can be combined with NMR imaging to give a spatially resolvedpore- size distribution, thus enabling the distribution of pore-sizes within each pixel of an image to be separately determined4. Using this technique spatial variations of the pore-size distribution have been detected and these are in good agreement with heterogeneities detected from spin density-, relaxation- and diffusion-weighted images. 1 Gladden, L. F., Hollewand, M. P. and Alexander, P., 1995, AIChE, 41, 894. 2 Hollewand, M. P. and Gladden, L. F., 1995, Chem. Eng. Sci., 50, 327. 3 Strange, J. H., Rahman, M. and Smith, E. G., 1993, Phys. Rev. Lets., 71, 3589. 4 Strange, J. H and Webber J. B. W.,1997 Meas. Sci. Technol., 8, 555.

Contribution to the conference:
Investigation of carbohydrate transport and metabolism in plants by indirect detected 13C imaging and spectroscopy
M. Heidenreich, W. Koeckenberger, N. Chandrakumar, R. Bowtell, R. Kimmich
In many plants photoassimilates are transported within the phloem of the vascular bundles in the form of the disaccharide sucrose. This process plays a key role in providing energy rich compounds to non-photosynthetic plant tissue and therefore it is essential for the maintenance of growth in general and the development of crops and fruits or tubers in particular. The regulation and the mechanism of this transport over long distances is not well understood. Indirect detected 13C NMR by cyclic cross-polarization (CYCLCROP) offers the oppertunity to investigate metabolic and transport activities in an unperturbed state of the intact plant. The high NMR sensitivity of indirect 13C detection allows to perform 13C NMR imaging studies to visualize the synthesis, transport and unloading of 13C labelled sucrose in the hypocotyl of castor bean seedlings in a time resolved manner.

Contribution to the conference:
Application of mutipol moments in NMR
I.V.Lunegov

Contribution to the conference:
MRI Velocimetry Studies of Velocity Profiles Through and Over Arrays of Impermeable Cylinders
M.D. Mantle(1), B. Bijeljic(2), A.J. Sederman(1), L.F. Gladden(1) and T.D. Papathanasiou(3)
Affiliations: (1) Department of Chemical Engineering, University of Cambridge, Pembroke Street, CB2 3RA, England, UK. (2) Department of Chemical Engineering, Imperial College of Science and Technology, Univrsity of London, London, SW7, UK. (3) Department of Chemical Engineering, Swearingen Engineering Centre, University of South Carolina, Colombia, SC 29208, USA.
ABSTRACT
The ability of magnetic resonance imaging (MRI) velocimetry to examine flow phenomena non-invasively is becoming increasingly popular in process engineering applications such as resin transfer moulding (RTM) and structural reaction injection moulding (SRIM). Here we report both experimental and computational (using the Boundary Element Method, BEM) results concerning the distribution of fluid velocities across geometrical arrays of uniform cylinders (fiber tows) of varying porosity, e(epsilon), and radius r where appropriate, placed inside a Hele-Shaw cell. Specific cylinder arrays studied are: Rectangular: e=0.54 and e=0.75. Hexagonal: e=0.48 Semi-circular: r=11.4 mm, e=0.48 Semi-circular: r=12.1mm, e=0.72 etc Semi-circular: r=12.1mm, e=0.48 (137 cylinders) Semi-circular: r=11.4mm, e=0.72 (79 cylinders) Semi-circular in hexagonal arrangement:r=14.5mm, e=0.48 The model assumes absence of body forces and flow is also assumed to be linear, incompressible and inertialess, i.e. creeping flow regime with Reynolds number (Re) approaching zero. In particular, two components, (Vx, Vz) of gap-averaged velocities in the inter and intra-tow regions have been measured using the stimulated-echo phase difference method. The results presented here show the distribution of flow between the two regions, the open channel and the cylinder array. Good agreement is found between experimental and computational results.

Contribution to the conference:
NONSTATIONARY CONVECTION FLOW BY NMR IMAGING
A. Mohoric, J. Stepisnik, G. Planinsic and M. Kos

Contribution to the conference:
Double Quantum Filtered NMR as a New Modality for Imaging and Diffusion Measurements in Biological Tissues
G. Navon, Y. Sharf, H. Shinar, L. Tsoref, U. Eliav and Y. Seo.
Double quantum filtered (DQF) NMR of water protons and deuterons has the important property, that a signal is obtained only in anisotropic systems. This is a result of a non-zero residual dipolar or quadrupolar interaction for protons and deuterons respectively. As many biological tissues exhibit some degree of order, DQF NMR can be applied as a new modality for imaging, spectroscopy and diffusion measurements in these tissues. Examples will be given of (a) microscopic imaging of blood vessel walls showing contrast between their different layers, and reflecting the strain exerted on them; (b) microscopic imaging of tendon, where, unlike in conventional MRI, the image of collagen fibers is specifically enhanced and (c) discrimination between the different compartments in isolated nerves, the measurement of anisotropic diffusion of water molecules in each of the compartments, and the heavily restricted diffusion of the water molecules inside the axons.%EOR%%BOR%Dr.

Contribution to the conference:
Pore distribution in mortar
R.Valckenborg, B.Hartgers, L.Pel, A.Duif, K.Kopinga
Moisture in porous building materials plays a dominant role in various deterioration processes, like frost, salt crystallisation, and mould growth. A detailed knowledge of the moisture transport is needed to understand these processes. Nuclear magnetic resonance (NMR) offers the possibility to determine moisture profiles. Moreover, the relaxation of the NMR signal can provide additional information on the water distribution in the microstructure. For mortar it is shown that the transverse relaxation can provide information on the distribution of water in the gel and capillary pores. To get quantitative information, the surface relaxation was determined from separate NMR experiments, in which only a surface layer of water was present in the pores. Using inversion process the pore distribution was calculated. In addition the pore distribution was also determined using cryoporometry. This technique makes use of the fact the melting/freezing point of the fluid decreases in smaller pores. Using NMR the liquid water content was determined a function of temperature. Some preliminary results will be presented.

Contribution to the conference:
Moisture and ion transport in porous building materials
B.Hartgers, R.Valckenborg, L.Pel, A.Duif, K.Kopinga
The transport of moisture and salt is an important damaging mechanism in porous building materials such as masonry and concrete. In masonry, salt crystallization during drying causes defacing of the surface, surface chipping, and disintegration just below the surface. Chloride ion penetration in concrete accelerates the corrosion process in reinforced structures. It is a very complex phenomenon. It is clear that detailed information about the transport of salt and moisture is necessary for understanding the durability of these porous building materials. By using Nuclear Magnetic Resonance the moisture and ion-concentration profiles can be determined non-destructively. However, a complicating factor for the use of NMR with building materials is the presence of large amounts of paramagnetic ions (Fe) in these types of materials. This results in a reduction of the transverse relaxation times (typically less than 1 ms in fired-clay brick for 1H). In addition, the variations in the local magnetic susceptibility cause broadening of the resonance line (typically ~ 10 kHz), and thus limit the spatial resolution. Because of our interest in the moisture profile as well as the ion distribution, the setup has to be able to detect both H and Na at the same time. An especially designed home-built NMR apparatus is used with which it is possible to measure the hydrogen and sodium concentrations quasi simultaneously, as a function of both time and (one-dimensional) spatial position. Some preliminary results will be presented.

Contribution to the conference:
Short TE projection-reconstruction NMR microscopy of articular cartilage
P. Szomolanyi, V. Jellus, P. Latta, A. Piras, M. Bliznakoff, F. Vittur, O. Jarh and R. Toffanin
1Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, I-34127 Trieste, Italy 2Institute of Measurement Science, Slovak Academy of Sciences, Sk-842 19 Bratislva, Slovakia 3"Jozef Stefan" Institute, 1000 Ljubljana, Slovenia 4POLY-biós Research Centre, Area Science Park, I-34012 Trieste, Italy Magnetic resonance imaging is the most accurate non-invasive method to evaluate the articular cartilage. Conventional MR imaging sequences, however, offer only limited image contrast between cartilage and the surrounding structures and consequently on MR images cartilage thickness is measured at less than its true value. This fact has been attributed to a variety of causes which include susceptibility, partial volume or shortened T2 in the deep layers of cartilage. In this ex vivo study we describe the use of a short-TE 2D NMR microscopy method based on projection-reconstruction for accurately measuring cartilage thickness. The advantage of this method is that short echo times can be used to obtain MR signal from structures with short T2 relaxation times. In cartilage for example the signal from organised collagen structures is strongly influenced by the dependence of T2 relaxation on the relative orientation of the collagen fibers. Moreover, as a highly organised structure, articular cartilage is subject to rapid decay due to very short T2 relaxation times. Therefore, on high resolution short TE images the cartilage signal intensity is increased, thereby accurately delineating cartilage from the region of tide-mark/cortical bone. This means that by the proposed method it is possible to accurately determine the cartilage thickness. This finding is very important for future examination of joint cartilage thickness in clinical MR systems for patients suffering from osteoarthritis or similar pathologies.

Contribution to the conference:
NMR Imaging of Network Heterogeneity in Cured Isobutylene-Based Elastomers
A. Pollaris, P. Adriaensens, J.L. White, M. Kelchtermans, D. Vanderzande, J. Gelan
Institute for Materials Research - Limburg University - building D - SBG B-3590 Diepenbeek, Belgium. astro bin cdrom client dev disk2 etc home lib misc mnt net opt paging root sbin stand sw tcb tmp tmp_mnt users usr var Esso Chemical, Baytown Polymers Center, P. O. Box 5200, Baytown, Texas 77522. astro bin cdrom client dev disk2 etc home lib misc mnt net opt paging root sbin stand sw tcb tmp tmp_mnt users usr var Esso Chemical Europe Inc., Hermes Laan 2, B-1831 Machelen, Belgium. Proton nuclear magnetic resonance imaging (1H-MRI) was used to study the two- and three-dimensional network heterogeneity and cross-link density. Network homogeneity was investigated for isobutylene-p-methylstyrene-p-bromomethylstyrene terpolymers cured with inorganic versus organic curatives, and those unfilled versus filled with carbon black. Results from these NMR imaging experiments show that not only the cross-link density but mainly the voids and void distributions, are very important parameters to make a correlation with the mechanical tests data. MRI has been established as a method to provide imaging information over large sample areas (mm2) in a non-destructive manner. Because of their slow reorientational chain dynamics at room temperature, the rubbers had to be swollen with different solvents to decrease the correlation time for the chain motions. Using non-protonated solvents makes it possible to obtain images of the polymers, while protonated solvents may be used to image the swelling solvent. Solvent images of rubbers swollen in cyclohexane or benzene seemed to be superior towards the visualisation of voids. These are believed to be air bubbles occluded in the polymer matrix. CCl4 and benzene swollen rubbers are used to make rubber images (for benzene by chemical selective excitation of the rubber signal) which are superior towards the visualisation of the cross-link density. Swelling in CCl4 seems to be interesting for samples containing a significant olefinic resonance. Both an aliphatic and olefinic image can be made by means of chemical shift selective imaging. This is not possible for benzene swollen samples due to severe signal overlap.

Contribution to the conference:
NMR Study of Water Ingress into Nylon 4.6 plaques.
A. Pollaris, P. Adriaensens, V. Litvinov, J. Tijssen, D. Vanderzande, J. Gelan
NMR study of Water Ingress into Nylon 4.6 plaques. A. Pollaris, P. Adriaensens V. Litvinov,* J. Tijssen*, D. Vanderzande and J. Gelan Institute for Materials Research - Limburg University - building D - department SBG B-3590 Diepenbeek, Belgium. counter csz csz.cgi eenc98-list eenc98-register eenc98-shortlist fancycount fancyselfcount finger.cgi gaco guestboo.pl htimage nph-count realteam.pl test-cgi test2.cgi testscript.pl usluzbenci_eng.cgi usluzbenci_slo.cgi DSM Research, Geleen, The Netherlands. The uptake of water into Nylon 4.6 plaques was investigated by Magnetic Resonance Imaging (MRI). Due to the small amount of water, ingressed into the plaque at equilibrium, images were acquired with the inversion recovery technique and at 45°C to optimally suppress the surrounding water. In the MRI images, the intensity of the ingressed water into the native plaque and some pre-treated plaques (annealing, scraped edges, ...), was calibrated towards the image intensity of a reference capillary filled with doped water. These experiments clearly show that pre-treating the plaques has an effect on the molecular mobility of the ingressed water. From the MRI measurements of the water uptake into the plaques as a function of time, it appears that the diffusion is Fickian but one can clearly see that the edges of the Nylon 4.6 plaques take up more water. This is explained by a gradient in the amorphous density over the plaque due to the high crystallization rate of Nylon 4.6 and the thermal gradient present during cooling after injection moulding. This gradient is confirmed by the kinetics of the water ingress into pre-treated plaques compared to the native ones. The differences in amorphous phase density between the core and edges of a Nylon 4.6 plaque was further confirmed by 1H-wideline solid echo experiments.

Contribution to the conference:
Advances in Stray Field Techniques
E. W. Randall
To be supplied. It will be the same as the talk I have been invited to give at the ENC in March at Asilomar

Automated Spectral Assignment

Contribution to the conference:
New approaches to automated resonance assignments
Billeter et al.
The conventional sequence-specific assignment of resonances inspectra of biological macromolecules consists of several steps, and so does an automated approach to this problem. At the outset, a reliable peak picker is needed that must be able to correctly identify a sufficient number of individual cross peaks that allows their sequence-specific assignments and eventually complete structure calculations. An automatic peak picker will be presented that, when combined with a recently published automated tool for simultaneous NOESY assignments and structure calculations, yields structures of comparable quality than those from a cnmventional approach. Another application for this fully automatic peak picker is its combination with a program for automated sequence-specific assignments based on the matching of observed (and picked) peaks in various spectra with a list of atom-atom interactions that are derived only from the protein sequence and that are expected to give rise to signals in the NMR spectra.

Contribution to the conference:
Rapid removal of residual 1H NMR water signal in a PC
S. Nyman, J. Järvi and M. Komu
Proton (1H) magnetic resonance spectroscopy (MRS) has been valuablein detecting metabolites of dignostic interest. In all biological samples water is precent. The large water signal affects the quantiation and detection of metabolites in low concentration. Modeling the base line without removing the dominating water is very complicated and inaccurate. The removal of water signal in time domain will not affect the signals of interest. Singular value decomposition (SVD) methods are linear prediction that have gained popularity as a means of spectral analysis in MRS. This poster describes the implementation of Hankel SVD (HSVD) algorithm for rapid removal of water signal in a PC under Windows.

Biomedical MR Imaging

Contribution to the conference:
Radio-Frequency Current Density Imaging of Evoked Depolarization
K. Beravs, R. Frangež, U. Mikac, I. Serša and F. Demsar
Glutamic acid receptors are one of the most atractive targets for developing relevant neuroactive agents. As in vivo quantitative studies of drug-receptor interactions are essential for the development of novel neuroactive agents, the purpose of this study was to examine whether depolarization evoked by excitatory amino acids can be imaged quantitatively using Radio-Frequency Current Density Imaging (RF-CDI). The technique uses MR imager to measure magnetic field produced by synhronous RF current and theoretically offers much higher temporal resolution than blood oxygenation or flow dependant functional MRI. In this study, kainate as a glutamate receptor agonist was used to evoke depolarization in the rat’s brain and the fenomena was imaged using RF-CDI.

Contribution to the conference:
BROAD-LINE HIGH RESOLUTION IMAGING SEQUENCES APPLIED TO HUMAN EXTREMITIES AND CARTILAGE -IN VITRO AND IN VIVO INVESTIGATIONS
A. Berg and E. Moser
BROAD-LINE HIGH RESOLUTION IMAGING SEQUENCES APPLIED TO HUMAN EXTREMITIES AND CARTILAGE - -IN VITRO AND IN VIVO INVESTIGATIONS A. Berg and E. Moser Institut fuer Medizinische Physik, Universitaet Wien, Waehringerstr. 13, A-1090 Wien Introduction Semisolid organic tissue as, for instance hypodermic tissue and cartilage, containing a large amount of collagen fibres, exhibits short transversal relaxation times and relatively broad line shapes in spectroscopy. This is due to the high amount of interconnected longchained molecules and the sensitivity of MR-methods to the dynamics of molecular motions via the respective correlation time1. In standard 2DFT-imaging the encoding of the spatial position is achieved by phase encoding gradients, which are switched within a spin echo experiment between the rf-pulses. This type of encoding leads to an increase of the minimum achievable spin echo time. In case of tissue, containing mainly large molecules, the minimum achievable spin echo time will be significantly higher than the T2 values of the corresponding fibres resulting in a strong loss of signal5. The corresponding areas appear dark, i.e. without any contrast. Broadline imaging sequences, based on Backprojection2 and SPI4, offer very short detection times avoiding this disadvantage. It is the aim of this contribution to document the results of the application of these sequences to cartilage tissue, dissected human finger and toe, as well as an in vivo application to the human finger. Methods The MR-Backprojection technique avoids phase encoding and thus enable short spin echo times and strong signal3 especially in semirigid materials. The pulse sequence is based on a single echo detection with read gradient. Slice selection is achieved by subtraction. During the first scan an inversion adiabatic pulse inverts the magnetization of the slice leaving the rest unperturbed for read detection. The next scan gives an echo and projection of the whole sample. Subtraction offers a profile within the excited slice. Images with spin echo times of 3 ms have been obtained in vitro and in vivo. A bandwidth of 125 kHz was used in combination with a strong adapted gradient system offering 200 mT/m. The voxel size in case of the in vivo application is chosen to be 203 µm x 203 µm x 1000 µm. Single point imaging („SPI“)4 sequences avoid the detection of spin echo signals. One point out of a FID is phase encoded by gradients which are stepped in cycles for all three spatial directions. By this method, a detection time of 260 µs had been achieved for a 3D image of a dissected human toe. (TR = 1 ms, band width 125 kHz, Voxel size: 390 µm x 312 µm x 625 µm). Results The imaging results show that Broad-Line imaging sequences are capable of offering information that cannot be spatially detected by standard 2DFT- sequences, especially in tissues and materials in the human body containing large immobile molecules. The measurement time and SAR are tolerable. References: [1] Berg,A., Proc. EENC, Paris, 425 (1996) [2] Lauterbur, P.C., Nature 242, 190-191 (1973) [3] W. Kuhn,et. al, Macromolecules 27, 5773 (1994) [4] S. Emid, J.H.N. Creyghton, Physica 125B, 81 (1990) [5] H. K. Song, F. W. Wehrli, J. Ma, Magn. Res. Med. 37, 185-191 (1997)

Contribution to the conference:
Atherosclerosis in a transgenic mouse model
P.D.Hockings, T.J.Roberts,D.G.Reid, M.Benson
Atherosclerosis research has been hampered by the variability of disease progression in individual animals. We have followed disease progression in two transgenic mouse models (ApoE(-/-) and LDLR(-/-))using a 3D RARE sequence to directly image plaque in the aortic arch and carotid arteries with an isotropic pixel resolution of 140 microns. Combined respiratory and cardiac gating was used.

Contribution to the conference:
NMR relaxometry in study of x-ray contrast media interaction withblood plasma proteins.
V.Panov, I.Shabunin, O.Vetrov, N.Shimanovsky, L.Sibeldina, P.Sergeev
Roentgen contrast media (RCM) have different side effects. Most partof their influence on human organism is related with RCM osmoticactivity. The aim of this work was to investigate RCM influence onwater molecular motion in water and blood plasma proteins solutionsby water 1H T1 and Ds measurings which were obtained on proton MRrelaxo- meter IVR3 (Russia) with pulsed gradient of magnetic field. 6ionic and 3 nonionic RCM were examined in water solutions and insolutions of human serum albumin and gamma-globulin. RCM osmoticactivity, acute toxicity and lipophility were correlated with RCMinfluence on water motions. RCM effected to protein dehydrationprocess.

Biomolecular NMR/Proteins/Nucleic Acids

Contribution to the conference:
Solid-state NMR spectroscopy on polypeptides in oriented membranes
Bechinger, B.
Whereas multidimensional high-resolution NMR spectroscopy allows forthe characterization of the secondary structure of hydrophobic polypeptides in the presence of detergent micelles, solid-state NMR spectroscopy is used for structural investigations of peptide-phospholipid bilayers. Proton decoupled 15N solid-state NMR spectroscopy of uniaxially oriented peptide membranes has proven to be particularly usefull as it provides information to calculate the orientation and topology of bilayer-associated polypeptides. This technique also allows one to determine the secondary structure of membrane polypeptides. Examples of proteins, as well as antibiotic, channel forming and model peptides will be shown and the functional implications of the structural findings discussed.

Contribution to the conference:
Structure and Dynamics of Spinach Plastocyanin
A.Bergkvist, S.Young, C.Johansson, B.G.Karlsson
Plastocyanin is a small soluble protein from the thylakoid lumen in the chloroplasts. The function of the protein is to transport electrons from cytochrome b6f to photosystem I in the photosynthetic electron transport chain. Previous structures of plastocyanin include crystal structures of poplar and C.reinhardtii and NMR structures of french bean, parsley and A.variabilis. The current work has achieved a high-resolution structure, as well as mapping the protein backbone dynamics by NMR, using unlabelled and 15N labelled samples of spinach plastocyanin. The resulting structure is similar to previous structures of plastocyanin. The dynamics studies indicate a very rigid structure, which is expected from a beta-sheet protein. Small but significant increase in mobility is found at the 'acidic patches', which are believed to be part of the docking surface to cytochrome b6f.

Contribution to the conference:
Strategies for the NMR Resonance Assignment of (U C-13, N-15) labeled Peptides and Proteins in the Solid State
Anja Böckmann #, Tatyana E. Polenova ý, Gaetano T. Montelione § and Ann E. McDermott ý
ý Chemistry Department, Columbia University, New York, NY 10027, USA # present address: IBCP-CNRS, 7, passage du Vercors, 69367 Lyon, France § Center for Advanced Biotechnology and Medcine , Rutgers University, Piscataway, NJ 08854-5638, USA Some important classes of proteins, such as membrane proteins and insoluble proteins, are significantly under-represented in the database of protein structures. A protocol for structure determination in solid state proteins using high resolution solid state NMR methods can be envisioned using uniform C-13, N-15 labeling of solid biopolymers, resonance assignment, and structural characterization. Such a protocol would be a very important addition to structural biology methods. We have concentrated initially on the assignment strategies of the resonances. Simulation of protein spectra using chemical shift information from the BMRB (1), and line widths observed on model molecules, allowed us to evaluate the limits and the potential of bi- and tridimensional spectra showing intra- and inter-residue connectivities. We optimized homo- and heteronuclear chemical shift correlation experiments (2-5) on model amino acids, in order to maximize for the correlation of amide N shifts to CO, C-alpha shifts and of C-alpha shifts to C-beta, and to obtain spectra of sufficient resolution for assignment. Using preliminary multidimensional NMR spectra of uniformly C-13, N-15 labeled BPTI, we illustrate the issues affecting protein resonance assignments in the solid state. (1) Seavey, B. R., Farr, E. A., Westler, W. M., and Markley, J. L. J. Biomol. NMR 1991, 1 , 217-236. (2) Bennett, A. E.; Ok, J. H.; Griffin, R. G.; Vega, S. J. Chem. Phys. 1992, 96 , 8624-8627. (3) Sun, B. Q., Costa, P. R., Kocisko, D., Lansbury, P. T. and Griffin, R. G. J. Chem. Phys. 1995, 102 , 702-707. (4) Baldus, M., Geurts, D. G., Hediger, S. and Meier, B. H. J. Magn. Reson. A 1996, 118 , 140-144. (5) Sun, B. Q., Costa, P. R., and Griffin, R.G. J. Magn. Reson. A 1995, 112 , 191-198.

Contribution to the conference:
Conformational variation of the central CG site in the (purine-ACGT-pyrimidine) step
CORDIER C.
Impact of the base sequence on the conformation of the central CG site within the two oligomers d(ATGACGTCAT)2 and d(GAAAACGTTTTC)2 has been examined by molecular modelling completed with 3D-similarity calculations. A full NMR study of each DNA fragment has been achieved using the standard 2D experiments: NOESY TOCSY and DQF-COSY. Experimental data have been used as constraints to refine the molecular models generated by energy minimisation with help of Jumna. A 3D-similarity procedure has allowed to compare the common central CG part and to quantify conformational 3D-homology of the two nucleotides.

Contribution to the conference:
NMR Investigations on a Novel,Synthetic Tumor -Associated Glycoconjugate.
C. Baby, S. Selvaraj ,and S.Subramanian
The Thomsen- Friedenreich (TF) and Tn carbohydrate antigens are expressed on a vast majority of human carcinomas and are associated with the aggressive behaviour of certain tumors. Eventhough the precise nature of the carbohydrate groups on carcinomas is yet to be defined, unmasked TF and Tn epitopes are found to be unique carcinoma markers. The use of TF like- and Tn like- antigens to produce MoAbs are well reported. Here, we report the solution structure of a synthetic tumor associated glycoconjugate (S-TAG) namely Gal-b(1-3)Gal NAc b-O-Ser(NHAc)GlyCOOMe. A combination of homo and hetero nuclear two dimensional techniques such as DQF COSY, TOCSY, ROESY and HMQC are applied to unravel the anomeric configuration, position of glycosidic linkages etc. 1D TOCSY (HOHAHA) combined with Z filtration technique has been done to generate pure phase subspectra and consequently the complete assignment of all the multiplet pattern in the 1H NMR spectrum.

Contribution to the conference:
High resolution three dimensional structures and dynamics of conotoxins determined by NMR spectrosopy
DJ Craik, PF Alewood, J Gehrmann, J Hill, N Kurniawan, R Lewis and K Nielsen
HIGH RESOLUTION THREE DIMENSIONAL STRUCTURES AND DYNAMICS OF CONOTOXINS DETERMINED BY NMR SPECTROSCOPY David J. Craik, Paul F. Alewood, John Gehrmann, Justine Hill, Nyoman Kurniawan, Richard Lewis and Katherine J. Nielsen Centre for Drug Design and Development, University of Queensland, Brisbane, QLD, 4072, Australia Marine snails of the genus Conus have evolved a prey hunting strategy that involves the injection of a venom which is made up of a cocktail of peptide toxins. The venom is injected via a hollow radula tooth and results in immediate immobilisation of the prey. The peptide components of the venom, referred to as conotoxins, are typically small (approximately 13-30 amino acids) but are rich in cysteine residues and are multiply disulfide bonded. These peptides often have very well defined three dimensional structures despite their small number of amino acid residues. The conotoxins are divided in several classes depending on their biological activities and framework of cysteine residues. They are widely used as probe ligands in neurophysiological studies and have great potential as leads in drug design applications. In this presentation the structures and activities of representative examples from the alpha, mu and omega classes of conotoxins are described. These target the nicotinic acetylcholine receptor, muscle-type sodium channels and calcium channels respectively. Applications in drug design are specifically illustrated using the omega conotoxins. Voltage-sensitive calcium channels (VSCC) are implicated in the control of a multitude of biochemical processes. The omega-conotoxins are of fundamental interest as they represent a group of related peptides with a wide range of specificities for the VSCCs. In this presentation the structures of naturally occurring omega-conotoxins with differing VSCC subtype specificities and several chimeric peptides are described. The chimeras were synthesised to probe the role of various regions of the molecules on VSCC specificity. The results indicate that subtle differences in the nature and positioning of amino acid sidechains significantly affect specificity. NMR relaxation measurements have been used to probe the dynamics of a series of conotoxins to assess whether their solution conformations are likely to provide a good model for receptor bound conformations. These measurements are illustrated with examples from the mu and omega conotoxin families. Finally, the role of disulfide bonds in determining the structures of alpha conotoxins is probed using synthetic peptides in which non-native disulfide bonds were engineered. The results indicate that although there may only be small differences in the thermodynamic stability of various disulfide bond isomers the native disulfide connections have a much greater conformational homogeneity than non-native forms.

Contribution to the conference:
The use of selective excitation for structure analysis andenhancement of complex carbohydrates
A. De Bruyn, J. Schraml, P. Herderuyn

Contribution to the conference:
43-Calcium NMR on Biomolecules
Torbjörn Drakenberg, Maria U. Johansson and Anders Malmendal
The calcium ion is considered as a generally very important metal ion that take part in the regulation of a multitude of biological processes. The calcium ion is difficult to study directly since it does have very few useful spectroscopic properties. However, 43-calcium has a non-zero spin quantum number (I=7/2) making it NMR active. It is also a quadrupolar nucleus with fast and strongly correlation time dependent relaxation. The free solvated ion has a relaxation time of a few seconds, resulting in a sharp NMR signal. When bound to a small protein e.g. calmodulin the 43-calcium resonance is ca. 1000 Hz broad and we have previously shown that the temperature dependence in the 43-calcium NMR spectrum in the presence of a calcium-binding protein can be used to obtain information regarding both the kinetics and thermodynamics of the calcium binding (1). Blood serum contains ca. 2 mM calcium and about half of this is known to be bound to proteins, mostly serum albumin. The calcium binding to serum albumin is, however, not very well defined, but there seems to be a general agreement that there are several week sites. The strongest site appear to have a dissosiation constant of ca. 1 mM. The binding has also been shown to be pH dependent in the pH range 7 to 9 and this has been interpreted as a binding site involving the N-terminus. We have now performed a 43-calcium NMR study of the calcium binding to human serum albumin (HSA) with some quite surprising results. In the titration experiment with increasing HSA concentrations at conctant calcium we observed to our surprise essentially no broadening of the calcium resonance. In a temperature dependence study on the other hand we found a dependence typical for a case with calcium ion in slow exchange at low temperature and fast exchange at high temperature. This is a most surprising result since it shows that the on-rate of calcium is about 5 orders of magnitude slower than the diffusion limit, indicating that the site is burried deep inside the protein and no such site has so far been found in the crystal structure of HSA. This together with some other findings using 43-calcium NMR will be discussed in some detail. (1) Vogel, H.J., Drakenberg, T. and Forsén, S. in "NMR of Newly Accessible Nuclei. Vol. 1", Ed. P.Laszlo, Academic Press, New York, 1983.

Contribution to the conference:
1H NMR INVESTIGATION OF THE SEQUENCE-SPECIFIC BINDING OF THE ANTIBIOTIC DAUNOMYCIN WITH DEOXYTETRANOCLEOTIDES IN AQUEOUS SOLUTION
R.Eaton, D.Davies, S.Osetrov, S.Baranovsky, V.Pahomov and A.Veselkov
The anthracycline antibiotics are widely used in chemotherapy of certain cancers. Complexation of the most prominent member of this family daunomycin (DN), with deoxytetranucleotides of different base sequence, 5'd(GpCpGpC), 5'-d(CpGpCpG), 5'd(TpGpCpA), 5'd(ApGpCpT) and 5'(ApCpGpT), in aqueous salt solution has been studied by one- and two-dimensional (2-D TOCSY and 2-D NOESY) 500MHz 1H NMR spectroscopy. Concentration and temperature dependences of proton chemical shifts have been measured and analysed in terms of the equilibrium reaction constants, the relative content of different types of complexes as a function of concentration and temperature, the limiting proton chemical shift of DN in different complexes and the thermodynamic parameters dH and dS of molecules complexation. The result show that preferred binding sites of DN are triplet nucleotide sequences containing three adjacent G-C base pairs in the tetranucleotide duplex. DN does not show any sequence specificity in binding to single-stranded deoxytetranucleotide sequences. The most favourable spatial structures of the 1:2 complexes between DN and the deoxytetranucleotides have been derived using calculated values of the induced chemical shifts for the DN protons. The results are in good agreement with the structure of the DN-deoxyoligonucleotide complex derived from X-ray crystallographic measurements. Distinctive features of the intercalated complexes of DN with deoxytetranucleotides of different base sequence in aqueous salt solution have been revealed. It is shown that binding of the second drug molecule with both single- and double-stranded tetramer is highly anti-cooperative. This can be explained by the existence in DN of aminosugar residue which causes steric barriers to the binding of the second molecule of antibiotic with relatively short tetranucleotide sequence. A comparison has been made of the characteristics of complexation of DN with previous results(1,2) for the typical intercalators, proflavine and ethidium bromide with the same deoxytetranucleotides at the same experimental conditions. (1). Davies D.B., Djimant L.N., Veselkov A.N. Nucleos.& Nucleot., 1994, 13, 637-655. (2). Davies D.B., Karawajew L., Veselkov A.N. Biopolymers, 1996, 38, 745-757.

Contribution to the conference:
Highly Sensitive HCN Experiments for Applications to Larger RNA Oligonucleotides
Radovan Fiala & Vladimir Sklenar
for an abstract see the entry of Prof. Vladimir Sklenar

Contribution to the conference:
The hydration of lichens as observed by proton magnetic relaxation.
S. Gazdzinski, H. Haranczyk, M. Olech
The hydration of lichens as observed by proton magnetic relaxation. S.GaŸdziñski*, H.Harañczyk* and M.Olech** *Institute of Physics and **Institute of Botany, Jagiellonian University, Cracow, Poland Lichens are living organisms capable of surviving extreme conditions of drastic desiccation and low temperature. Lichen resistance to low temperature is accompanied by the resistance to acute water stress. They can passively absorb water from gasous phase. These properties make lichens a convenient system to study the ability of living entities to survive acute water stress and to study the effect of dehydration on the structure and function of the system. The hydration of selected lichens (Cladonia mitis, Cladonia bellidiflora, Certaria islandica, Parmelia saxatilis and Xanthoria parietina) was investigated using gravimetry and proton magnetic relaxation (FIDs and T*2 hydration courses). The hydration from gaseous phase shows the first order kinetics. The proton free induction signal from lichen thallus has three components: (i) the component coming from the solid matrix of thallus structure, and two liquid signals coming from (ii) water tightly bound to the surface of thallus solid matrix and from (iii) loosely bound water. Dehydration (to gaseous phase) can completely remove the loosely bound water fraction, leaving the system below water percolation treshold and (probably) below water clustering point. It was shown that the proton spin-lattice magnetisation function versus hydration of Cladonia mitis has two or three T1 components. For each hydration course a minimum in T1 has been found due to the hydration caused changes in bound water corelation times.

Contribution to the conference:
Sensitivity gain in multi-quantum NMR techniques applied to 75% deuterated protein samples
Gerd Gemmecker, Ruth Gschwind
SENSITIVITY GAIN IN MULTI-QUANTUM NMR TECHNIQUES APPLIED TO 75% DEUTERATED PROTEIN SAMPLES Gerd Gemmecker, Ruth Gschwind Institute for Organic Chemistry and Biochemistry, TU Munich Lichtenbergstr. 4, D-85747 Garching (Germany) Partially deuterated protein samples are now routinely used for NMR studies of large proteins, since they offer dramatic improvements in terms of relaxation and signal linewidth. However, the carbon bound protons remaining in a partially deuterated sample, as well as the 13C nuclei directly bound to them, still undergo fast relaxation caused by the remaining strong dipolar coupling across the 1H-13C bond. In highly deuterated protein samples, these 1H-13C pairs can be described as isolated two- spin pairs in good approximation. It is long known that in this case, the introduction of 1H-13C multi-quantum coherences can greatly reduce relaxation and hence improve sensitivity and resolution in NMR experiments. We could show in a comparison of HMQC vs. HSQC experiments that this effect can indeed be observed in 75% deuterated protein samples, with significant intensity gains of ca. 20% in the HMQC version for the 31 kDa IIAMan domain (1). Such intensity gains can also be realized in more complex NMR pulse sequences. In the HBHA(CBCACO)NH experiment, the substitution of the single-quantum 1Ha / 1Hb evolution time by appropriate multi-quantum evolution times led to intensity gains of more than 40% for IIAMan (1). The improved relaxational behaviour of 1H-13C multi-quantum coherences also make it possible to use new editing techniques for the side-chain protons in the ed-H(CCCO)NH- TOCSY experiment. A recently proposed technique produces different signs for signals with different 13C-13C multiplicity (2), but could so far only be applied to rather small proteins (< 12 kDa) due to the required long 13C-13C coupling evolution delay of 28 ms. With the introduction of multi-quantum periods into the pulse sequence, it is now possible to apply this technique to large proteins like IIAMan with neglectable relaxation (3). References (1) R. Gschwind, G. Gemmecker, H. Kessler (1997) J. Biomol. NMR, in press. (2) M. Tashiro, C. B. Rios, G. T. Montelione (1995) J. Biomol. NMR 6, 211-216. (3) R. Gschwind, G. Gemmecker, H. Kessler (1998) J. Am. Chem. Soc., submitted.

Contribution to the conference:
New Methods for Structure Determination of Isotopically Labelled RNA: Direct Determination of Angles alpa and zeta by NMR.
Christian Richter, Bernd Reif, Karlheinz Wörner, Joachim Engels, Harald Schwalbe und Christian Griesinger*
13C-labelled [1] hairpin 5'-CGCUUUUGCG-3' (U = ribosyl 13C-labelled labelled nucleotide) has been studied by NMR. The conformation of the sugar pucker is obtained from 3J(H,H) [2] and 2J(C,H) and 3J(C,H) coupling constants which are obtained in a new method relying on alpha/beta selective HCCH-TOCSY experiments [3]. A new method for the measurement of 3J(C,P) coupling constants along the phosphodiester backbone including 3J(C4'i,Pi) and 3J(C4'i,Pi+1) based on a quantitative [4] HCP [5] experiment is presented and coupling constants obtained with this new method are compared with data from the P-FIDS-experiment [6]. Together with 3J(H,P) from the P-FIDS-experiment, these couplings yield the backbone angles beta and epsilon. A new method for the determination of alpha and zeta is presented. Torsion angle restraints are derived from quantification of the angular dependence of the dipole-CSA cross correlated relaxation on the angles alpha and beta as well as epsilon and zeta [7,8]. [1] S. Quant, R.W. Wechselberger, M. A. Wolter , K-H. Wörner, J.W. Engels, C. Griesinger and H. Schwalbe;THL (1994) 35 6649. [2] H. Schwalbe, J.P. Marino, S.J. Glaser, and C. Griesinger; JACS (1995 117 7251. [3] C. Richter, B. Reif, K.-H. Wörner, S. Quant, S. Glaser, J. W. Engels, C. Griesinger and H. Schwalbe; submitted. [4] G. W. Vuister, A. C. Wang and A. Bax; JACS (1993) 115 5334. [5] H. Schwalbe, W. Samstag, J.W. Engels, W. Bermel, and C. Griesinger; J.Biomol. NMR (1993) 2 479. [6] C. Richter, B. Reif, K. Wörner, S. Quant, J.P. Marino, J. W. Engels, C. Griesinger and H. Schwalbe; J.Biomol. NMR (1998) in press. [7] C. Richter, B. Reif, C. Griesinger, and H. Schwalbe; submitted. [8] B. Reif, M. Hennig, and C. Griesinger; Science (1997) 278 119.

Contribution to the conference:
Structure Determination of Calmodulin in Complex with C20W and C21W, Cognate Peptides of the Plasma Membrane Calcium Pump.
Elshorst,B.; Försterling,F.H.; Hennig,M.; Diener,A.; Schwalbe, Griesinger,C.; Nilges,M.; Krebs,J.
Calmodulin (CaM) is an ubiquitous Ca2+ binding protein which plays a pivotal role in the signal transduction chain at the cell membrane in eucariotic cells. Free CaM has a dumbbell type structure with two globular domains connected by a flexible linker. Binding of CaM to target proteins induces a dramatical conformational change in which the dumbbell like structure of free CaM collapses to a globular structure. In this study we used NMR spectroscopy to determine the difference in binding of CaM with two peptides (C20W and C21W) resembling overlapping regions of the binding domain of the plasma membrane Ca2+ pump. The complex was prepared using CaM enriched in C-13 and N-15 obtained from expression in E. coli, and unlabled peptides obtained by the procedure of Vorherr. Complete Sequence assignments of the complexes were obtained by a number of 3-D experiments (HNCACB, CBCA(CO)NH, HBHA(CO)NH, HCCH-TOCSY). Structural information was obtained by measurement of NOEs within CaM (NH-NOESY-HSQC and CH-NOESY-HSQC), within the peptide and between the two (filtered NOESY). Information about backbone dynamics was obtained using N-15 relaxation and hetero-NOE measurements. Our results indicate that C20W binds only to the C-terminal domain of CaM, leaving the general structure of CaM mainly intact. C21W on the other hand appears to interact with both domains of CaM. The resulting differences in structure and dynamics between the two complexes will be discussed.

Contribution to the conference:
Novel Solid State NMR Methods for Structural Studies on MembraneProteins and Peptides
G. Groebner, G. Choi, I. Burnett, C. Glaubitz, A. Watts

Contribution to the conference:
New NMR Structures of Protein/DNA complexes
Angela M. Gronenborn
A recurring theme of events that controls transcription, recombination and replication is the involvement of multiprotein-DNA complexes. The intricate architecture of these complexes relies not only on sequence specific protein-DNA interactions, but also on the interaction of multiple proteins whose DNA binding sites may be quite distant from one another. Thus the formation of such higher order structures usually requires that the conformation of the DNA template be bent or distorted in order to bring the requisite proteins into close proximity. In large part, this task is accomplished by architectural proteins which typically lack the potential to activate transcription or to carry out recombination on their own, but instead induce conformational changes in DNA, thereby facilitating the assembly and enhancing the overall stability and activity of multiprotein-DNA complexes. We solved high resolution three-dimensional structures for several proteins complexed to their cognate DNA sites by NMR, involving both major groove and minor groove recognition. Specific structural features and their relationship to functions of these major and minor groove transcription factors will be discussed. In addition, several new NMR experiments designed to elucidate structural features of protein/DNA complexes will be presented.

Contribution to the conference:
A ROESY approach to determine solution structures of DNA oligomers
Monika Ivancic, Victor L. Hsu
For DNA resonance assignments and structure determination, certain characteristics of the ROESY experiment were found to be advantageous. Although for macromolecules the NOESY experiment is more sensitive than the ROESY experiment, more accurate measurements of cross-relaxation rates, and thus better structure determination, can often be achieved with the ROESY experiment. Spin diffusion plays a different role in the two experiments; in the rotating frame dipolar relaxation affects cross-relaxation by attenuating spin diffusion. In a ROESY spectrum a spin diffusion peak will exhibit an opposite sign intensity from that of a direct cross-relaxation peak. It is this phenomenon that produces a typical cross-peak pattern for DNA oligos in the ROESY spectrum. The two most interesting regions are the H1' to H2', H2" cross-peaks and the H2', H2" to H6, H8 cross-peaks. In these regions most peaks exhibit direct cross-relaxation and have opposite sign intensity of the diagonal, however, several peaks exhibit relayed cross-relaxation and have the same sign intensity as the diagonal. This phenomenon makes unambiguous assignments of the H2' and H2" resonances possible. The characteristic pattern of peak sign intensities is due to the different interproton distances within the DNA oligo. These distances are dependent on the pseudorotation angle of the DNA backbone and are significantly different in the 3' endo and 2' endo puckers. As a result, the ROESY experiment can be used to directly differentiate particular DNA conformations, especially A-form from B-form DNAs. A DNA hairpin molecule was used to conduct a series of ROESY experiments to demonstrate these points, varying the spin-lock mixing time as well as the spin-lock power level.

Contribution to the conference:
Protein Dynamics by NMR
Oleg Jardetzky
Over the past decade the usefulness of NMR for determining the solution structure initially of small proteins and more recently of larger biological structures up to 37 kD has been demonstrated. The structure determination is predicated on a complete resolution and assignment of protein NMR spectra, which is achieved by resort to high frequency spectrometers (500-750 MHz), the use of multidimensional (2D homo- or heteronuclear, 3D and 4D) spectroscopy and isotopic spectral editing, especially for structures of MW above 15 kD. Several sequential assignments techniques have been developed - for small molecules the Gibbons paradigm, based on the use of coupling constants and NOEs along the peptide backbone, for larger molecules paradigms based on NOEs alone or on heteronuclear coupling constants. Simultaneously with the assignment one usually obtains the secondary structure pattern, and a 3D model of the protein can be constructed from long-range NOE distance constraints. Methods available for model building range from manual computer graphics, to distance geometry, to restrained molecular dynamics, to optimal filtering, with several specific algorithms available in each category. All of these methods reproduce te general topology of the molecules equally well, but energy minimization or molecular dynamics are necessary for structure refinement. The accuracy of NMR structures has been shown to be of the order of 1-2 Angstroms (RMSD). Today more sophisticated approaches are being developed to integrate dynamic aspects into the detailed structural description of proteins. Heteronuclear NMR studies of the relaxation behavior of the backbone and side chains of proteins and the extension of studies of protein dynamics to partly folded non- native states including folding intermediates allow an examination of protein structure across a broad timescale. The dynamic and structural changes that can thus be examined allow us to determine the function of different regions of the protein and how changes in protein structure are necessary to its function.

Contribution to the conference:
The signal transducer gp130: solution structure of the carboxy-terminal domain of the cytokine-binding module
Thomas Kernebeck, Stefan Pflanz, Stephan Grzesiek, Guenther Kurapkat, Gerhard Mueller-Neven, Peter C. Heinrich, Axel Wollmer and Joachim Groetzinger
GP130 is the signal transducing receptor subunit of the so-called interleukin-6- type cytokines. This transmembrane protein is a member of the cytokine-receptor superfamily predicted to consist of six domains in its extracellular part. The N- terminal domain 1 is believed to have an Ig-like fold. Domains 2 and 3 constitute the so-called cytokine-binding module. Domain 2 is characterized by a set of four conserved cysteine residues, domain 3 by a conserved WSXWS motif. Domains 4-6 are homologous to the so-called fibronectin-type-III-like domain. Domain 3, the carboxy-terminal domain of the cytokine-binding module, is involved in binding to the cytokine/cytokine-receptor complex. Here we report the solution structure of this domain determined by heteronuclear multidimensional NMR spectroscopy. The structure consists of seven beta-strands forming two antiparallel beta- sheets seen in several cytokine receptors.

Contribution to the conference:
Hydrophobicity of Amino Acids in biological Membranes; A new solid state NMR approach
Rudolf Kinder, Burkhard Bechinger
Tables for the prediction of membrane protein topology are often based on the partitions of amino acids between water and organic solvents (oil). This can only provide a crude estimate of the values relevant in biological membranes or interfaces. We therefore developed a new method to investigate the hydrophobicity in model membranes. 15N backbone labelled peptides are incorporated into oriented lipid multilayers. The chemical shift exhibits a direct correlation with the orientation of the helical peptide in the membrane. PH-dependent deprotonation of histidin in the artificial peptides causes a reorientation of the peptide from an „in-plane" to a transmembrane orientation in the bilayer. The pH value at which the peptides flip their orientation in the bilayer is dependent on the hydrophobicity of the transferred amino acids. Careful design of the peptides allows deducing a relative value of the hydrophobicity of specific amino acids in lipid environments.

Contribution to the conference:
Solution structures of the inactivation gate peptide of sodium channel and interaction between the peptide and local anesthetic dibucaine
Yoshihiro Kuroda, Masaru Matsumoto, Rie Morita, Hirosato Nasu, Nobutaka Fujii, and Terumichi Nakagawa
SOLUTION STRUCTURES OF THE INACTIVATION GATE PEPTIDE OF SODIUM CHANNEL AND INTERACTION BETWEEN THE PEPTIDE AND LOCAL ANESTHETIC DIBUCAINE Yoshihiro Kuroda, Masaru Matsumoto, Rie Morita, Hirosato Nasu, Nobutaka Fujii, and Terumichi Nakagawa Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan Sodium ion channels, which initiate the action potentials in electrically excitable cells, are considered to be the molecular targets of local anesthetic drugs. Inactivation of sodium channels terminates the sodium channel current. The local anesthetic drugs stabilize the inactivated state and block the inward sodium current. It has been reported that three clustered hydrophobic amino acids, IFM1488-90, located in the linker between domains III and IV of the rat brain type IIA sodium channel, is required for fast inactivation (West, J. W. et al. 1992, Proc. Natl. Acad. Sci. USA. 89:10910-10914). The fast inactivation is supposed to occur utilizing glycine (GG1484-85) and/or proline (P1512, P1514, P1516) residues on either side of the IFM domain as hinge points (Kellenberger, S. et al. 1997, J. Gen. Physiol. 109:607-617) and the IFM as a hydrophobic latch against the putative receptor sites which are composed of domains IIIS4-S5 linker (Smith, M. R. and A. L. Goldin 1997, Biophys. J. 73:1885-1895) and IVS4-S5 linker (Tang, L. et al. 1996, J. Gen. Physiol. 108:89-104). In our previous paper (Kuroda, Y. et al. 1996, Biophys. J. 71:1191-1207), we studied the interaction between local anesthetic dibucaine and a fragment peptide which includes the IFM residues (GGQDIFMTEEQK; G1484-K1495, MP-1A) by H-NMR spectroscopy and found the importance of pi-pi stacking interactions between the aromatic ring of the local anesthetic molecule and the phenyl ring of F1489. In the present study, in order to investigate the solution structures of the inactivation gate peptide, we synthesized peptides consisting of 17 amino acid residues (KKKFGGQDIFMTEEQKK; K1480-K1496, MP-3A) and the one in which F1489 has been substituted by Gln (MP-4A). We observed H-NMR (COSY, TOCSY, and NOESY) and CD spectra of the peptides in phosphate buffer, TFE-d2, and SDS-d25 micelle solutions. Interactions between dibucaine and the peptides have also been investigated by noting changes in chemical shifts of dibucaine caused by the peptides. Both MP-3A and MP-4A were found to take random conformations in phosphate buffer and alpha-helix structures in TFE-d2 solutions. In a micelle solution, however, the peptides, especially MP-3A takes a beta-sheet structure at around K1480-D1487 and an alpha-helix structure at around D1487-K1496. These results mean that GG1484-85 can act as a hinge point only in an amphiphilic environment.

Contribution to the conference:
Effects of CpG cytosine methylation in (A)nCG(T)n sequence
MENAGER L.
Impact of the methylation at position 5 of the cytosine residue in the CpG step flanked by 5'adenine and 3'thymine tracts has been studied by NMR and molecular modelling. 1H-NMR and 31P-NMR 2D-experiments have been achieved. Experimental results (NOE derived distance constraints, sugar phase calculated from simulation of DQF-COSY cross peaks patterns) were used in restained molecular modelling to generate conformers. Significant conformational variations induced by methylation of the cytosine have been observed for this privileged sequence.

Contribution to the conference:
Some NMR experiments and structure calculation using a 100%deuterated protein.
T.M.Mal, Helena Kovacs, I. D. Campbell and J. Boyd
Results from studies of an aqueous sample of 100% {15N, 2H} enrichedprotein, the SH3 domain from Fyn, will be presented. Experimental longitudinal and transverse 1H relaxation data, as well as quantitative measurement of the interaction between H2O solvent and some exchangeable protons will described. A method for decreasing the effective R1 of solvent exchangeable proton resonances will be illlustrated. Analysis of a series of 2D NOESY datasets, recorded as function of mixing time, indicates that spin diffusion processes are weak in comparison to that typically observed for a fully protonated sample. This allows the use of relatively long mixing times. Finally, structure calculation using only interamide NOEs will be presented.

Contribution to the conference:
Solution structures of thanatin and androctonin, two animal defence peptides with antibiotic properties, determined from NMR data
Mandard N., Sodano P., Labbe H., Hetru C., Bonmatin J.M., Ptak M. & Vovelle F.
As part of structure determinations of defence peptides, we studied thanatin and androctonin, two recently isolated molecules with broad spectrum of antibiotic activities. Solution structures of these two peptides have been solved by conventional 2D-NMR techniques followed by molecular modelling using distance geometry and molecular dynamics methods. Thanatin is a 21-residue inducible peptide from the hemipteran insect podisus maculiventris and shows activities against Gram+ and Gram- bacteria as well as fungi at physiological concentrations. Its structure mainly consists of a well-structured rigid antiparallel B-sheet formed by the strands Ile8-Asp12 and Lys17-Met21, and a well-defined cationic loop delineated by the disulfide bridge binding Cys11 and Cys18. Conversely, the N-terminal part (Gly1 to Val6) shows a large structural variability. Androctonin, a 25-residue constitutive peptide from the scorpion androctonus, acts as antibacterial and antifungal agent too. A twisted antiparallel B-sheet is localized between the two disulfide bridge Cys4-Cys20 and Cys10-Cys16. The 5-residue loop (Arg11-Gly15) is highly positively charged and presents some variable conformations. Androctonin structure is close to the tachyplesin one, an other antibacterial peptid from crab with which it displays sequence similarities. Acticity test experiments were carried out on native, truncated and mutated thanatin and androctonin peptides. Combined with our structural results, it was possible to establish structure-function relationships in order to understand their modes of antimicrobial actions.

Contribution to the conference:
Protein dynamics by NMR relaxation
F. Cordier, M.J. Blackledge, B. Brutscher, J.-P. Simorre and D.Marion
Insight in protein dynamics can be obtained by NMR relaxation datacombined to molecular dynamics simulations. Several relaxation probes available on doubly labelled proteins are used in order to evidence concerted motion in protein backbone. Special emphasis is made on the data processing, which permits to derive overall and internal motional parameters and their confidence intervals without overinterpretating the experimental data. Several proteins are studied by these methods: in a first case, a covalently linked cofactor seems to modulate the internal dynamics; in another one, the dynamics of two adjacent modules in a modular protein is investigated. Finally, the implication of the solvent molecules in the dynamics of protein will be discussed on the case of two homologuous proteins with very distinct behaviors.

Contribution to the conference:
Mechanisms of Glutathione Reductase Inhibition by Cd ions
George L. Mendz
In situ inhibition of Campylobacter spp. glutathione reductase (EC 1.6.4.2) by Cd2+ and other cations was investigated employing 1H-, 13C- and 113Cd-NMR spectroscopy. Enzyme activity in bacterial lysates was measured from 1H-NMR time courses. Progress curves of the reaction in the presence of Cd2+ ions were biphasic, and characterised by an early phase of low enzymatic activity followed by a late phase with a faster rate of reaction; the duration of the early phase depended on Cd2+ concentration. To understand this biphasic behaviour the binding of Cd2+ to glutathione reductase, and to oxidised and reduced glutathione was measured employing 1H- and 113Cd-NMR spectroscopy. The results showed that Cd2+ ions bind to glutathione reductase and reduced glutathione, a product of the enzyme reaction, but not to oxidised gluthatione, a substrate of the reaction. It was concluded that tight binding of Cd2+ by reduced glutathione removed available cations from the medium, and thus decreased enzyme inhibition.

Contribution to the conference:
Selective relaxation rate measurements of backbone protons in proteins
O. Millet, E. Chiarparin, P. Pelupessy, M. Pons, G. Bodenhausen
A method based on selective cross polarization (CP) is presented for the selective measurements of proton relaxation rates in labeled macromolecules. In 15N labeled proteins and polipeptides the method allows the measurement of amide proton and alpha-proton spin-lattice and spin-spin relaxation rates. In this work we present T1 and T2 measurements in 15N ubiquitin and a partially labeled octapeptide (GFL). Selective two-way cross polarization is used in combination with classical schemes for relaxation measurements to focus on signals of interest. Particular emphasis is put on longitudinal relaxation rates of chosen amide protons,which have been measured under different conditions: 1) non-selective inversion of all the proton resonances, 2) semi-selective inversion of the amide region only and 3) selective inversion of the amide proton of interest. The selective inversion of a proton resonance is achieved with a BIRD like sequence, in which selectivity is obtained by selective cross-polarization transfer.

Contribution to the conference:
Multidimensional HoMQC applications for complex biomolecules
I. Pelczer

Contribution to the conference:
Energetic Evaluation of Steric Effects of Purine Nucleobases
M. Polak and J. Plavec

Contribution to the conference:
Energetic Evaluation of Steric Effects of Purine Nucleobases
M. Polak and J. Plavec

Contribution to the conference:
13C solid-state NMR of gramicidin D in a membrane
P-O Quist
The natural abundance 13C NMR spectrum of gramicidin A in a synthetic membrane was acquired under magic angle spinning conditions. With fast sample spinning (15 kHz) the resonances from several of the aliphatic, alfa-, beta-, aromatic, and carbonyl carbons in the peptide could be resolved. The resolution in the 13C spectrum was superior that observed with 1H NMR under similar conditions. The 13C linewidths was in the range 30 - 100 Hz, except for the alfa- and beta-carbons where the linewidth was about 350 Hz. Under slow sample spinning (500 Hz), the intensity of the spinning sidebands from 13C in the backbone carbonyls was used to determine the residual shift tensor. As expected, the elements of the residual shift tensor were consistent with the single-stranded, right-handed beta-6.3 helix structure proposed for gramicidin A in membranes.

Contribution to the conference:
The Structure of the HIV-1 Tat Protein
Markus Boehm, Heinrich Sticht, Paul Rosch
Lentiviral transactivator proteins (Tat) control the expression of early genes of this class of viruses, including the human immunodeficiency virus type 1 (HIV-1). Tat exerts its effects by binding to the Tat-responsive region (TAR), an RNA stem-loop structure located at the 5'-end of all lentiviral RNAs. The conformation of TAR in solution is known in its ligand bound as well as in its free form. Structure determination of the 86 amino acid Tat of HIV-1 was highly aggravated by the fact that Tat contains a cysteine rich region and the necessary concentration could not be obtained with Tat. We circumvented the problem by a novel approach, determining the structure of a Cys- HIV-1 Tat mutant on the basis of nearly 2000 restraints and putting the Cys-residues back into the protein by a molecular dynamics approach. This procedure thus resulted in the reduced form of the wt protein. The protein exhibits a mostly rigid structure stabilized by turns, and a less stable structure in the the cysteine rich and the TAR binding basic domain. The NH2-terminus is positioned in the center of the molecule interacting with the core, the basic, and the glutamine rich domain, and the COOH-terminal RGD (Arg-Gly-Asp) loop is solvent exposed. Known structural rearrangements upon RNA binding of this protein could be explained on the basis of the current experimental conformation, and a model structure of the Tat-TAR complex could be calculated. References Aboul -ela, F., Karn, J., & Varani, G., Nucleic Acids Res. 24, 3974 (1996) Bayer, P., Kraft, M., Ejchart, A., Westendorp, M., Frank, R.W., & Rösch, P., J. Mol. Biol. 247, 529 (1995) Churcher, M.J., Lamont, C., Hamy, F., Dingwall, C., Green, S.M., Lowe, A.D., Butler, P.J.G., Gait, M.J., Karn, J., J. Mol. Biol. 230, 90 (1993) Metzger, A.U., Schindler, T., Willbold, D., Kraft, M., Steegborn, C., Volkmann, A., Frank, R.W., & Rösch, P., FEBS Lett. 384, 255 (1996)

Contribution to the conference:
Influence of the mode of insertion of SIV peptides in membranes on the structure of model membrane
A. Schanck(1), R. Brasseur(2) and J. Peuvot(3) ; (1) CPMC, UCL, Louvain-la Neuve, (2) CBMN, Gembloux, (3) MDI, UCB S.A., Braine l'Alleud, Belgium.
The calculated mode of insertion of SIV peptides in the membrane is correlated to their fusogenic character. Only in the case of tilted insertion, these peptides are able to induce fusion. We have examined by P-31 NMR the influence, on model membrane organization, of a tilted fusion peptide of SIV and of a non fusogenic mutant of this peptide showing by molecular modeling a more vertical insertion in the membrane. Observations were made on large multilamellar vesicles. In the presence of the SIV fusion peptide, a dose dependent destabilization of the bilayer structure occurs: induced isotropic type structures are evidenced by the development of a narrow signal superimposed to the broad bilayer P-31 NMR signal. On the contrary, even at high concentration, the "vertical" mutant is unable to induce non lamellar structures. This study reinforces the correlation between the fusion phenomenon and the formation of small structures not organized in extended bilayers.

Contribution to the conference:
1H NMR Self-Diffusion in Compartmented Systems Containing Enzyme
Yury E.Shapiro
The multicomponent self-diffusion in nanocapsules and cryogel biocatalytic sistems containing chymotrypsin was studied with aid of the FT-PGSE NMR technique at various temperatures and compared to the diffusion of such systems without enzyme. Unilamellar vesicles were formed in water after 'coating' with Brij-97 of the poly-(N,N- diallyl-N,N-didodecylammonium bromide), poly-DDAB, nanocapsules. Cryogels was made from poly-(vinyl alcohol), PVA, aqueous solutions after freezing-thawing cyclic process. Both compartmented systems were used as vehicles of enzyme entrapped into inner aqueous cavities. Compartmented systems under study have outwardly the similar features by heating. Indeed, a comparison of the clouding for the 'coated' vesicles and syneresis of cryogels allows to conclude that both processes of the stability destruction have the similar morphological and structural properties. However, they have the only principal difference: clouding is a reversible process, but syneresis is irreversible one. This difference is due to distinction of the stabilization mechanisms for these colloid systems. The nanocapsules 'coated' by Brij-97 are enthalpic stabilized, but the PVA cryogels have the combined enthalpic-entropic type of stabilization. Encapsulation of chymotrypsin into the poly-DDAB/Brij-97 vesicles and the PVA cryogel diminishes the Ds values for all molecules, and shifts the cloud point to lower temperature. On the contrary, the syneresis point for the PVA cryogel is shifted to higher temperature by entrapment of enzyme. Besides entrapment of chymotrypsin into the cryogel promotes increase of the activation energies for the PVA diffusion on 2 kJ/mol. Such a difference indicates sensitivity of the H-bond system of PVA hydroxyl groups and water molecules to interference of a protein globule. Self-diffusion of the substrate molecules was measured also after addition of ATEE to the vesicular system containing chymotrypsin. For the substrate self-diffusion a two-state mobility model is applicable. Estimation of the diffusion restricted kinetics parameters using a small diffusion increment to Ds corresponding to movement through the bilayer from poly-DDAB/Brij-97, as well as values of the Michaelis constant and the maximal velocity allows to conclude that ATEE conversion is diffusion limited. The efficiency factor of process equals to 0.9.

Contribution to the conference:
Highly Sensitive HCN Experiments for Applications to Larger RNA Oligonucleotides
R. Fiala and V. Sklenar
Triple resonance NMR experiments utilizing H-1, C-13 and N-15 nuclei provide valuable information for structural studies of C-13, N-15 labeled RNA fragments. Optimized experimental protocols will be presented which allow simultaneous sugar to base correlations in a single experiment for all purine and pyrimidine nucleotides. A reduced-dimensionality version supplies chemical shifts for all H1',C1', N9/N1, C8/C6, and H8/H6 nuclei along and adjacent to the glycosidic bond in one two-dimensional spectrum. Additional sensitivity enhancement is obtained using a multiple-quantum evolution of CH coherences. Comparison of various 2D and 3D NMR experiments as applied to larger RNA fragments of 24, 36 and 40 nucleotides will be presented.

Contribution to the conference:
1H and 15N Assignments, Structure and Dynamics of Human Pancreatic Ribonuclease
El-Joubari, A., Bruix, M., Santoro, J., Rico, M., Kover, K.E., Batta, Gy., Szilagyi, L.
Human pancreatic ribonuclease is a 128-residue monomeric protein with potential antitumor activity. The study of its solution structure and dynamics may provide insights into the molecular basis of its biological activity. To that end, an NMR study was initiated. Assignments of many backbone and sidechain 1H resonances were accomplished on the basis of 2D TOCSY/NOESY spectra via the sequential approach (Wuthrich, 1986). 1H assignments were confirmed and extended and 15N resonances assigned by way of 3D TOCSY-HSQC/NOESY-HSQC measurements on a uniformly 15N labeled sample. Chemical shift prediction based on homologous proteins (Gronwald et al.,1997) proved to be of value as an adjunct tool in these processes. On the basis of a limited set of backbone NOEs a low-resolution solution structure could be calculated. As expected, the 3D structures of bovine and human RNAses look very similar, although some changes are observed mainly in the loop regions. A preliminary investigation of the backbone dynamics was made on the basis of 15N relaxation (T1, T2 and NOE) data. All relaxation data (15N T1, T2, NOE as well as 1H and 15N CSA/DD cross-correlation rates, (Tessari et al., 1997)) could be determined for all 15N/1H resonances resolved in the 2D HSQC spectrum. The 15N T1 relaxation times display a rather uniform distribution all over the structure, the T2 values show larger variations and so do the 15N NOE data too. 15N CSA/DD cross-correlation rates vary between 5 to 10 Hz whereas the respective 1H values display larger variations (2 to 10 Hz). From these rates, chemical shift anisotropy (CSA) values were calculated in view of the great interest of their correlation with H-bonding and secondary structure. This research was supported by an EC grant no. IC15CT96-0903 (Inco-Copernicus). References Gronwald,W., Boyko,R.F., Sonnichsen,F.D., Wishart,D.S., Sykes,B.D., J.Biomol.NMR, 10, 165 (1997). Tessari, M., Mulder,F.A.A., Boelens, R., Vuister, G.W., J. Magn. Res. 127, 128 (1997). Wüthrich, K., NMR of Proteins and Nuclei Acids, Wiley, 1986.

Contribution to the conference:
1H NMR STRUCTURAL AND THERMODYNAMICAL ANALYSIS OF THE COMPLEXATION OF ETHIDIUM BROMIDE TO A DNA OCTAMER, 5'-d(GpApCpApTpGpTpC), IN AQUEOUS SOLUTION
A.N.Veselkov, V.I.Pahomov, L.N.Djimant and D.B.Davies
The self-association of the self-complementary deoxyoctanucleotide, 5'd(GpApCpApTpGpTpC), and its complexation with phenanthridinium drug , ethidium bromide (EB) has been studied by one- and two-dimensional 500/600 MHz 1H NMR spectroscopy. Two-dimensional PMR spectra (2-D TOCSY and 2-D NOESY) was used to complete assignment of the non-exchangeable protons of the molecules and for a qualitative determination of the preferred binding site(s) of the ligand with the oligonucleotide chain. Quantitative determination of the equilibrium constants and thermodynamic parameters (free energy, enthalpy and entropy) of duplex formation of the octamer and its complexation with EB is based on investigation of the dependence of the proton chemical shifts of the molecules on temperature and on concentration (at three different temperatures, 298, 308 and 318 K)[1]. The experimental results were interpreted in terms of different types of drug-octamer complexes (1:2, 2:2, 3:2 and 4:2 complexes) containing different numbers of drug molecules in the octamer duplex. The most favourable structures of 1:2 and 2:2 drug-octamer complexes, corresponding to intercalation of EB into pyrimidine-purine sites of the deoxyoligonucleotide duplex have been constructed using calculated values of induced chemical shifts of EB protons. It is shown that (i) successive binding of drug molecules to octamer duplex is anti-cooperative and (II) the relative amount of each molecular complex depends on the ratio of the initial concentrations of the octamer and drug, and on the temperature of solution. It is concluded that hydrophobic interactions and conformational properties of the octamer duplex play major roles in the enthalpy changes on intercalative binding of the drug in solutions of relatively high ionic strength (0.1 mol/l Na+). [1]. Davies D.B., Veselkov A.N. J.Chem.Soc. Faraday Trans, 1996, 92, 3545-3557.

Contribution to the conference:
1H NMR STRUCTURAL AND THERMODYNAMICAL ANALYSIS OF THE COMPLEXATION OF ETHIDIUM BROMIDE TO A DNA OCTAMER, 5'-d(GpApCpApTpGpTpC), IN AQUEOUS SOLUTION
A.N.Veselkov, V.I.Pahomov, L.N.Djimant and D.B.Davies
The self-association of the self-complementary deoxyoctanucleotide, 5'd(GpApCpApTpGpTpC), and its complexation with phenanthridinium drug , ethidium bromide (EB) has been studied by one- and two-dimensional 500/600 MHz 1H NMR spectroscopy. Two-dimensional PMR spectra (2-D TOCSY and 2-D NOESY) was used to complete assignment of the non-exchangeable protons of the molecules and for a qualitative determination of the preferred binding site(s) of the ligand with the oligonucleotide chain. Quantitative determination of the equilibrium constants and thermodynamic parameters (free energy, enthalpy and entropy) of duplex formation of the octamer and its complexation with EB is based on investigation of the dependence of the proton chemical shifts of the molecules on temperature and on concentration (at three different temperatures, 298, 308 and 318 K)[1]. The experimental results were interpreted in terms of different types of drug-octamer complexes (1:2, 2:2, 3:2 and 4:2 complexes) containing different numbers of drug molecules in the octamer duplex. The most favourable structures of 1:2 and 2:2 drug-octamer complexes, corresponding to intercalation of EB into pyrimidine-purine sites of the deoxyoligonucleotide duplex have been constructed using calculated values of induced chemical shifts of EB protons. It is shown that (i) successive binding of drug molecules to octamer duplex is anti-cooperative and (II) the relative amount of each molecular complex depends on the ratio of the initial concentrations of the octamer and drug, and on the temperature of solution. It is concluded that hydrophobic interactions and conformational properties of the octamer duplex play major roles in the enthalpy changes on intercalative binding of the drug in solutions of relatively high ionic strength (0.1 mol/l Na+). [1]. Davies D.B., Veselkov A.N. J.Chem.Soc. Faraday Trans, 1996, 92, 3545-3557.

Contribution to the conference:
1H NMR STUDY OF HETERO-ASSOCIATION OF AROMATIC DRUG MOLECULES IN AQUEOUS SOLUTION
D. Veselkov, D. Davies and A. Veselkov
An NMR analysis has been developed for determining the structural and thermodynamical parameters of the hetero-association of aromatic molecules in aqueous solution. A statistical-thermodynamical model of the hetero-association of aromatic drug molecules forming indefinite aggregates for both self- association and hetero-association has been proposed and provides suitable analytical expressions for the analysis of NMR parameters. The analysis is based on measurements of the chemical shifts of the drug protons as a function of concentration and temperature, enabling determination of both thermodynamics and structures of hetero-associates in solution. The proposed NMR analysis has been applied to the hetero-association of phenanthridinium drug molecules, ethidium bromide (EB) and propidium iodide (PI), in aqueous salt solution and has been confirmed by the same results being given for two independent sets of experiments having either PI or EB constant and the other varied. Concentration and temperature dependences of proton chemical shifts (500MHz 1H NMR spectroscopy)of the drugs have been measured. Equilibrium reaction constants, parameters of cooperativity (1), the thermodynamic parameters (enthalpy and entropy) of the self-association reactions for EB and PI as well as the equilibrium constants of their hetero-association in aqueous salt solution have been determined, together with the limiting chemical shifts in the complexes. Analysis of the relative content of the different molecular associations has been made as a function of the drugs concentration and the temperature of solution. The most favourable spatial structures of the dimers of PI and EB and their 1:1 hetero-association complex have been derived using calculated values of the induced chemical shifts of the drug protons. The result show that the types of side chains and groups in the aromatic molecules influence both the thermodynamics and structures of the molecular associates being formed in aqueous solution; the hetero-association parameters for phenanthridinium drugs, PI and EB are intermediate between those observe for the self-association of these drugs. (1). Davies D.B., Djimant L.N., Veselkov A.N. J.Chem.Soc.Faraday Trans., 1996, 92, 383-390.

Contribution to the conference:
1H NMR STUDY OF HETERO-ASSOCIATION OF AROMATIC DRUG MOLECULES IN AQUEOUS SOLUTION
D. Veselkov, D. Davies and A. Veselkov
An NMR analysis has been developed for determining the structural and thermodynamical parameters of the hetero-association of aromatic molecules in aqueous solution. A statistical-thermodynamical model of the hetero-association of aromatic drug molecules forming indefinite aggregates for both self- association and hetero-association has been proposed and provides suitable analytical expressions for the analysis of NMR parameters. The analysis is based on measurements of the chemical shifts of the drug protons as a function of concentration and temperature, enabling determination of both thermodynamics and structures of hetero-associates in solution. The NMR analysis has been applied to the hetero-association of phenanthridinium drug molecules, ethidium bromide (EB) and propidium iodide (PI), in aqueous salt solution and has been confirmed by the same results being given for two independent sets of experiments having either PI or EB constant and the other varied. Concentration and temperature dependences of proton chemical shifts (500MHz 1H NMR spectroscopy)of the drugs have been measured. Equilibrium reaction constants, parameters of cooperativity (1), the thermodynamic parameters (enthalpy and entropy) of the self-association reactions for EB and PI as well as the equilibrium constants of their hetero-association in aqueous salt solution have been determined, together with the limiting chemical shifts in the complexes. Analysis of the relative content of the different molecular associations has been made as a function of the drugs concentration and the temperature of solution. The most favourable spatial structures of the dimers of PI and EB and their 1:1 hetero-association complex have been derived using calculated values of the induced chemical shifts of the drug protons. The result show that the types of side chains and groups in the aromatic molecules influence both the thermodynamics and structures of the molecular associates being formed in aqueous solution; the hetero-association parameters for phenanthridinium drugs, PI and EB are intermediate between those observe for the self-association of these drugs. (1). Davies D.B., Djimant L.N., Veselkov A.N. J.Chem.Soc.Faraday Trans., 1996, 92, 383-390.

Contribution to the conference:
Probing the structure of kinetic protein folding intermediates by NMR
Jonathan Waltho, Jeremy Craven, Laszlo Hosszu, Martin Parker, Mark Lorch and Anthony Clarke
Advances in isotopic labelling and resonance assignment methods have allowed the refolding of larger proteins to be investigated with per-residue resolution using amide exchange methods. As part of a study of the refolding of the 45 kDa two-domain protein 3-phosphoglycerate kinase from Bacillus stearothermophilus we have prepared the isolated N-terminal domain. Using triple resonance NMR methods we have shown that despite the removal of interdomain contacts, including the C-terminal tail which packs against the N-terminus, the isolated N-domain (N-PGK) remains folded essentially as when part of the intact protein. A combination of equilibrium amide exchange and kinetic folding data show that the essential features of the complex topology of N-PGK are established during refolding from a denatured state on a millisecond or faster timescale and before the rate-limiting step in the folding pathway commences. The high stability of the kinetic protein folding intermediate with native-like topology cannot be established using traditional amide trapping experiments, and thus the organisational events of the early stages of protein refolding may have been underestimated.

Contribution to the conference:
Preventing Artifacts in NMR Hydration Studies Makes Possible the Observation of the Complete Surface Hydration Shell of a Protein.
G. Wider, A. Sobol, H. Iwai and K. Wuthrich
The water-protein interactions are of special interest to understanding protein folding, surface structure of proteins, or enzyme reactions. Special NMR techniques allow to identify individual hydration water molecules in surface sites. Such NMR experiments must be laid out for the detection of weak signals in the presence of very intense solvent lines and are therefore particularly prone to deterioration by artifacts originating from specific NMR properties of the solvent, such as radi- ation damping and the nuclear demagnetizing field. During a typical NMR pulse sequence the magnetic fields caused by these effects be- come time-dependent, which makes the system non-linear and may, for example, measurably influence the precession frequencies of all nuclei in the sample. This presentation demonstrates the presence of such artifacts in experiments at 750 MHz. Strategies will be discussed for the elimination of these unwanted signals. An additional complication arises from the hydroxyl groups of amino acid sidechains which interfere with the observation of water-protein NOEs because rapid exchange between bulk water and hydroxyl pro- tons may give strong intramolecular hydroxyl proton-protein NOEs at the same chemical shift. To overcome this difficulty we redesigned a helical protein, phage 434 repressor, for detailed studies of the surface water molecules. All hydroxyl protons are eliminated by means of site- directed mutagenesis. With this redesigned protein and the careful consideration of effects from radiation damping and from the demag- netizing field we achieved a rather complete characterization of the "first hydration shell".

Contribution to the conference:
Analysis of unsaturated fatty acid resonances in blood plasma lipids of patients with hepatic encephalopathy
Wieland Willker and Dieter Leibfritz
Subacute and chronic liver disease result in neuropathological damage (hepatic or portocaval encephalopathy, HE). NMR spectra of blood plasma lipids from patients with hepatic encephalopathy show characteristic differences compared to normal blood. The main differences are the degree of unsaturation and the composition of the unsaturated fatty acids. Several new signals arise from fatty acids with conjugated double bonds, for example 13-keto or 13-hydroxy-9,11-octadecadienoic acid. The increase of fatty acids with conjugated double bonds indicates a reduced detoxification of the liver. The amount of conjugated double bonds is a sensitive indicator for the therapy response. Whereas the PUFA content remains almost unchanged after 5 weeks the conjugated double bond signals are noticeably reduced.

Exotica and Alternative Ways of Doing Magnetic Resonance

Contribution to the conference:
Dipolar Field Effects in Liquid State NMR
R. Bowtell and P. Robyr
The effect of the magnetic field generated by the nuclei themselves is normally neglected in NMR experiments because of its small magnitude. In highly polarised samples it can however give rise to significant effects including the production of multiple spin echoes and unexpected multiple quantum coherences. Here we describe how dipolar field effects can be used to make structural measurements at the microscopic scale and to allow indirect detection of low gyromagnetic species and those with short relaxation times. This work has been facilitated by the design and construction of a very strong gradient coil, capable of generating gradients of up to 50 T/m.

Contribution to the conference:
NMR of laer polarized noble gases in porous media
R Mair, R Walsworth et.al.
To be determined at a later date

Contribution to the conference:
The nuclear magnetic resonance ring - original arrangement for model hydrodynamic measurements in Earth's magnetic field.
Mel'nikov A.V., Borodin P.M., Vecherukhin N.M.
The arrangement and experimental results of hydrodynamics effects study in a moving liquid by NMR in the geomagnetic field has been described. The arrangement consist of electronic device and the "NMR hydrodynamic ring". The ring represent two cylindrical vessels, one rotating and another statical, surrounded by the system of coils, that provide for the nuclear prepolarization and receiving of the signal. The coil system are providing the periodically symmetrical picture of prepolarization field, which serve as main factor for functioning of ring (1). The behavior of nuclear magnetization in this case is depicted by equations system of Bloch - Stejkal. By means of this device the liquid velosity and level of flow turbulence were monitored without the loss of magnetization due to liquid leakage. (1) P.M. Borodin, N.M. Vecherukhin, A.V. Mel'nikov and A.A.Morozov. Tech.Phys. 41 (3), 245 (1996).

Contribution to the conference:
The new NMR phenomenon - spin-echo by turbulent flow of liquid in uniform geomagnetic field.
Mel'nikov A.V., Vecherukhin N.M. and Borodin P.M.
The pulse sequence 90-t-180 for the observation of turbulent spin-echo was been used. It is necessary, to made prepolarization of nuclear system by periodical symmetrical magnetic field, that is provided the distribution of unitial phases of nuclear precessions according to the picture of the magnetic field lines (1). Laminar flow don't dislocate this distribution of initial phases, therefore don't break the coherency of the spin system. Result of turbulent mixing of moving liquid is the loss of coherency, that correspond to the nonuniform broadening of spectrum line in uniform geomagnetic field. Processes rephasing of the spin system and observation of signal NMR are analogical for a classic spin-echo. (1). P.M. Borodin, N.M. Vecherukhin, A.V. Mel'nikov and A.A.Morozov Tech.Phys. 41 (3), 245 (1996).

Contribution to the conference:
Spin-echo in the strong gradient field
G.Planinsic, M.Symms
Gorazd Planin¹iè1, Mark Symms2
1-Oddelek za fiziko, FMF, Jadranska 19, 1000 Ljubljana, Slovenia
2-Intitute of Neurology, London, WC1N 3BG, UK
Concomitant gradients are a known source of image distortion (1) in regimes where large gradients are used in conjunction with low static fields (on example DNP imaging (2,3) and low field MRI (4)). We are working for several years on MRI in the Earth’s magnetic field. Weak field NMR methods provide a special conditions that can not be achieved in strong magnetic fields, such as strong gradient field on the size of the sample compared to the static magnetic field, and very fast gradient rise times compared to Larmour time. Here we show how the dephasing due to concomitant gradient fields can be rephased by a spin-echo sequence.
(1)D.G.Norris, J.M.S. Hutchison. Magn.Reson.Imag. 8, 33 (1993). (2) D.J.Lurie et al, J.Magn.Reson. 84, 431 (1989). (3) D.Grucker et al, Mag.Res.Med. 11, 691 (1993). (4)J.Stepi¹nik et al, Mag.Res.Med. 15, 386 (1990).

Macromolecules/Polymers/Liquid Crystals

Contribution to the conference:
Surface analysis by NMR
M.I. Burgar

Contribution to the conference:
Analogy of the Behaviour of Liquid Crystals in Rotating MagneticFields to the Free Induct
with Ulf Schmeisser

Contribution to the conference:
Physical properties of a ferroelectric liquid crystal as studied bymeans of noble gas NMR
P. Ingman, J. Jokisaari and M. Ylihautala
Physical properties of a ferroelectric liquid crystal (FELIX-R&D)were studied by means of the 21Ne, 129Xe and 131Xe NMR spectra of the corresponding gases dissolved in the LC. The spectra were recorded over the temperature range covering the isotropic, nematic, smectic A and smectic C* phases. The analysis of the data is based on the application of the theoretical model (J. Chem. Phys. 97, 8977 (1992) and the McMillan molecular model for the smectic A phase of liquid crystals Phys. Rev. A. 4, 1238 (1971)) LC order parameters, tilt angle of the smectic C phase, and external electric field gradient will be reported and discussed.

Macromolecules/Polymers/Liquid crystals

Contribution to the conference:
NMR Studies of Artificial Proteins
J. BORBELY, Gy. BATTA, K.E. KOVER and A. KRECZ
NMR STUDIES OF ARTIFICIAL PROTEINS (1) Janos BORBELY*, Gyula BATTA**, Katalin E. KOVER*** and Agnes KRECZ* *Colloid and ***Organic Chemistry Departments, Kossuth L. University **Research Group for Antibiotics of the Hungarian Academy of Sciences H-4010 Debrecen, Hungary Poly(AG)4EG and Poly(AG)4PEG artificial proteins with repeating units have been expressed in cultures of recombinant Escherichia Coli strains (2). MNR studies were performed to determine the primary and secondary structure of polypeptides -[(AG)4EG]-28 and -[(AG)4PEG]-16. The primary structures were assigned by HSQC, HSQC TOCSY and D-HMBC NMR experiments. The AG diads adopt beta-sheets and the EG and PEG units form turns. Secodary structure was calculated with Macromodel program, and the type of turns (beta and gamma turns) was estimated on the basis of NOESY NMR experiments. (1) This work was supported by grant from Ministry of Culture (Project Number: MKM FKFP 0444/1997) and by US-Hungarian Joint Scientific Fund (JFn: 355). (2) Strains were taken from the University of Massachusetts, Polymer Sceience Department, Amherst, MA, USA

Contribution to the conference:
INVESTIGATION OF STRUCTURE ELEMENTS MOTION IN LYOTROPIC LIQUID CRYSTALS
V.M. Chernov, R.I. Valeev, V.D. Buchelnikov
Water solution of caesium perfluorononanoate in isotropic liquid and nematic phases was studied by 19F NMR pulse method. Relaxation times T1, T2 and T2ef were determined at different temperatures and sample orientation in magnetic field. On the basis of the experimental data and model in which the micelle motion is considered as a whole the angular amplitudes and correlation time of the micelle swing motion in the nematic phase as well as the correlation time of the micelle rotation in the isotropic-liquid phase were computed.

Contribution to the conference:
Structure-Dynamics Relations in Lipid-Water-Mesostructures - an NMR Study
T.Feiweier, E.-M. Pospiech, B.Geil, J.Eisenblaetter, F.Fujara and R.Winter
Amphiphile molecules like lipids show the interesting property of self-organization when mixed with water [1]. Here we present a study of the monoolein/water-system, monoolein beeing a lipid involved in fat digestion. This system organizes in a wide variety of structures on a mesoscopic length scale (50 - 150 angstroms). While the geometry of those phases - inverse micellar, lamellar, inverse hexagonal and cubic - is well known today [2], there are still many open questions concerning the dynamics. In our work we try to understand some aspects of the water and lipid dynamics within this system and the correlation to the confined geometries. The methods employed - field-gradient-NMR, deuteron-NMR-spectroscopy, 2-dimensional exchange spectroscopy and spin-lattice-relaxation - provide information about the translational and rotational processes taking place. Our results show clearly the expected interrelation of dynamics and structure. At the same time new questions arise about the mechanism of 'trapped' water molecular motion, which strongly deviates from that of bulk water. [1] G.Lindblom and L.Rilfors, Biochim.et Biophys.Acta 988 p.221 (1989) [2] S.T.Hyde et.al., Zeitschrift fuer Kristallographie 168 p.213 (1984)

Contribution to the conference:
13C Spin lattice relaxation study on cross-linked polybutadiene
A.Ferrando, D.Tosetto, C.Maestrini, G.P.Ravanetti
The control of the cross-linking degree (CLD) in polybutadiene (PB) is a key factor in order to assess its properties and final performance. The traditional way of CLD evaluation is based on swelling phenomena taking place in contact with solvents. The first method was developed by Flory in 1943, who associated the swelling to the network chain density, considering the molar volume and the polymer-solvent interaction parameter as constants. However the original Flory approach is time consuming and somehow controversial in two phases materials, like polystyrene-polybutadiene (PS-PB) copolymers. Purpose of this work is then to suggest a new NMR-based method, able to overcome the mentioned problems and aimed mainly to PS-PB copolymers, whose performance is known to be strongly affected by the rubber CLD. 13C spin lattice relaxation time (T1) measurements were considered, following the standard inversion recovery sequence under magic angle spinning rotation (5KHz), but without cross polarisation, because of the liquid-like nature of PB. Several differently cross-linked samples characterised according to Flory were tested at 3 different temperatures: 300K, 320K and 350K. Using an empirical equation a good correlation is found between T1 and the CLD according to Flory, specially in measurements performed at 320K on poorly and medium cross-linked samples. The fit problems depend on the fact that the Flory method is scarcely sensitive when applied to highly cross linked samples, for which also the NMR results are impaired by the low signal/noise ratio and the pseudo solid like behaviour. Furthermore measurements done at 300K are too close to the narrow limit, while the highest temperature (350K), besides the natural signal reduction, can induce some damage on the samples (oxidation and/or uncontrolled cross-linking). The considered relationship assumes that the PB correlation times distribution only depends on the CLD in value, and it relates the measured T1 to the T1 of uncross linked PB by means of a factor containing a single adjustable parameter that, after the experimental fit, results close to the monomer molecular weight. Some two phases PS-PB copolymers were also tested with promising results.

Contribution to the conference:
NMR Relaxation and Diffusion Studies of Surfactant Systems
I. Furó,
Micelles, that form when surfactants are dissolved in water, present some interesting structural and dynamical problems which are well suited to be answered by NMR. The structure of the constituting surfactant molecules is described by the variation of the bond order parameters along the chain which can be accessed by field-dependent NMR relaxation studies. The same experiment also provides the surface diffusion coefficient of the molecules and, when measured in a broad enough frequency range, the axial ratio of the micelles as well. This latter parameter is also available via the translational diffusion coefficient of the micelles. Examples that will illustrate these points include field-dependent 81Br and 2H NMR relaxation and accurate 1H PGSE NMR measurements on the aqueous solution of cetyltrimethylammonium bromide (CTAB).

Contribution to the conference:
Low- and Stray-Field MRI Studies of the Sodium Polyacrylate Water Uptake
Genevieve Guillot, Teresa G. Nunes and J. Moura Bordado

Contribution to the conference:
Competition Between Na+ and Li+ for Unsealed and Cytoskeleton-Depleted Human RBC Membrane: A 23Na Multiple Quantum Filtered NMR Study
Carlos F.G.C. Geraldes, Chandra Srinivasan, Duarte Mota de Freitas and Daniel Graham
The MQF behaviour of 23Na NMR signals of Na+ present in human RBC membrane suspensions in the presence of increasing concentrations of Li+ was investigated providing evidence for Li+/ Na+ competition for membrane binding sites. Evidence for competition between Li+ and Na+ for unsealed and cytoskeleton-depleted human RBC membranes was obtained from the observed effect of Li+ addition upon the characteristics of 23Na DQF and TQF NMR signals. The observed TQC provided evidence for Li+/Na+ competition for isotropic binding sites only, while the observed DQC, which allows probing of both isotropic and anisotropic Na+ motion, provided evidence for Li+/Na+ competition for both types of sites at the membrane. Thus, a detailed picture of the membrane molecular sites of Li+/Na+ competition and of the role played by the cytoskeleton on the competitive binding of those ions was obtained in this work.

Contribution to the conference:
Novel materials for MRI radiation dosimetry
M.Bero P.Glover S.Doran & J.Keddie
Both Fricke Gels and polymers can be used for spatial readout, using MRI, of the three dimensional radiation dose map generated by a radiotherapy linac. Both these materials have their own particular disadvantages. Two new approaches to these problems will be presented.

Contribution to the conference:
Interaction of Oligomers and Polymers with Clays Suspended in Aqueous Solutions
J. Grandjean
Adsorption of surfactants and polymers onto clays is of interest for environmental issues and industrial applications. Smectite clays suspended in aqueous solutions of polyethyleneglycol monoalkylethers or of polyethyleneoxides have been studied by NMR methods. Analysis of water deuteron quadrupolar splittings, sodium-23 or lithium-7 relaxation rates of counterions, carbon-13 relaxation rates and proton self-diffusion coefficients of oligo(poly)mers provides a convergent picture on the organic molecule interaction with clays. Such studies have been performed as a function of the clay charge and clay structure.

Contribution to the conference:
Chain Motion Polyethylene Oxide-Urea Complex
Kunio Hikichi and Daisuke Oka
Polyethylene oxide(PEO) is in a 7-residue 2-turn helix in the crystalline state. We reported that the helix in the crystal starts to undergo an oscillational motion about the helical axis above room temperature. It is well known that PEO forms a complex with urea; the PEO chain is accomodated in the channel formed urea.. It is of interest to examine how molecular motion of PEO is modified by complexing. We observed 13C CP/MAS NMR spectra in a temperature range where pure PEO starts to undergo motion. The results show that the chain motion of PEO in the urea complex is greatly modified by complexing.

Contribution to the conference:
The determination of domain morphology in segmented polyurethanes by NMR
Idiyatullin D.Sh.
The development of Goldman-Shen method based on the study of recovery factor of spin systems after selected distorted magnetization is proposed. The method provides information about the domain morphology in segmented polyurethanes. Moreover the method proposed allows to obtain the dependence of molecular mobility in soft domains on the distances to their boundaries. The experimental data together with computer simulation are discussed. The possibilities of the determination of spin diffusion coefficient from the rates of dipole echo decay are considered.

Contribution to the conference:
NMR Chiral Recognition through NMR in Cholesteric Liquid Crystals : an Order Affair

The various NMR techniques used to analyse chiral molecules in isotropic solvents are often deceptive. On the other hand, the chiral discriminating power of biopolymers such as DNA, polysaccharides or polypeptides is often amazing. One may then note that these naturally occurring biologic media often share two important properties : they possess a helical superstructure and they are ordered. It will be shown that NMR performed in a medium which does possess those two properties, that is using a cholesteric liquid crystal as the solvent, furnishes a very powerful tool to measure enantiomeric excess. It will be demonstrated that the chiral discrimination originates from a difference in the ordering parameters of the molecules dissolved into the chiral mesophase. This differential ordering of enantiomers affects all the order dependant NMR interactions, namely the chemical shift anisotropy, the dipolar couplings and the quadrupolar splittings for nuclei larger than 1/2.         The power and the generality of this new technique will be illustrated through numerous organic and inorganic examples. A particular attention will be devoted to small chirality cases including isotopic chirality. As a conclusion it will be shown how this technique evolves toward a true shape recognition NMR method via the molecular ordering parameters. Bibliography. 1 - Visualisation of Enantiomers in Cholesteric Solvents through Deuterium NMR.        J. Courtieu, E. Lafontaine, J.M. Pechine et C.L. Mayne. Liquid Crystals, 7, 293 (1990). 2- Observation of Enantiomers, Chiral by Virtue of Isotopic Substitution, through Deuterium NMR in a Polypeptide liquid crystal. A. Meddour, I. Canet, A. Loewenstein et J. Courtieu. J. Amer. Chem. Soc. , 116, 9652 (1994). 3 - Enantiomeric Analysis in a Polypeptide Lyotropic Liquid Crystal by Deuterium NMR. A. Meddour, I. Canet, A. Loewenstein, J.M. Pechine et J Courtieu. J. Amer. Chem. Soc. , 117, 6520 (1995). 4 - Discrimination of the NMR spectra of enantiomers dissolved in chiral liquid crystal solvents through 2D correlation experiments. P. Lesot, D. Merlet, J. Courtieu et J.W. Emsley. Liquid Crystals;  21, 427, (1996). 5-  An achiral deuterated derivatizing agents for enantiomeric analysis through NMR in a liquid crystal solvent. A. Meddour, A. Loewenstein, J.M. Pechine et J. Courtieu. Tetrahedron Asym., 8, 485 (1997). 6 - Proton-decoupled carbon-13 NMR spectroscopy in the PBLG/Organic solvent system as an analytical method for the study of chirality. A. Meddour, P. Berdague, A. Hedli, J. Courtieu et P. Lesot. J. Am. Chem. Soc.,119, 4502 (1997)

Contribution to the conference:
Experimental Studies of Cross-correlations in Oriented Systems
Anil Kumar, P.Kumar and Kavita Dorai
The study of cross-correlations has become significant and highly relevant with the availability of high field spectrometers in recent years. We have observed significant CSA/Dipolar cross correlation in the fluorine relaxation of an oriented fluorobenzene and in the carbon relaxation of several liquid crystal molecules just above the phase transition into the isotropic phase. These results will be presented.

Contribution to the conference:
Combined use of NMR and other techniquesto investigate the motional cooperativity in polymers
Françoise Lauprêtre
Combined use of NMR and other techniques to investigate the motional cooperativity in polymers Françoise Lauprêtre Laboratoire de Physico-Chimie Structurale et Macromoléculaire associé au C.N.R.S., E.S.P.C.I., 10 rue Vauquelin, 75 231 Paris Cedex 05, FRANCE. The local dynamics play an important role in some properties of polymers. For example, at high temperature, the glass transition phenomenon is due to cooperative motions of the main chain. Secondary relaxations, which occur at temperatures below the glass transition temperature, arise from local motions involving a small number of main-chain or side-chain atoms. When investigating local motions, the main questions to be addressed are the following: i/ what are the moving sites in the polymer considered? ii/ what is the size of the moving unit or the cooperativity of the motion? iii/ what is the geometry of the motion? The answer to the first question is classically obtained by high-resolution 13C NMR, either in the solution, melt or solid state, or by 2H NMR using selectively deuterated samples. In principle, information on the size of the moving unit or the cooperativity of slow motions can be obtained by using multidimensional solid-state NMR (1). However, in ordinary polymers, which are not 13C-enriched, these techniques are usually too time-consuming to be of practical use. Another method consists in following the changes in motional behavior when progressive changes are introduced in the chemical structure of the polymer or miscible additives such as antiplasticizers are added. Finally, only computer simulations of the local dynamics can provide the details of the geometry of the motion. The examples of polybutadienes with different microstructures (2), and epoxy resins based on diglycidylether of bisphenol-A and hexamethylenediamine (3,4) will illustrate the potential of the combined use of NMR and other techniques for characterizing the motional cooperativity in polymers, either above or below their glass transition temperature. References (1) K.Schmidt-Rohr and H.W.Spiess, «Multidimensional solid-state NMR and polymers» Academic Press, London (1994) (2) M.A.Krajewski-Bertrand and F.Lauprêtre, Macromolecules 29, 7616 (1996) (3) L.Heux, J.L.Halary, F.Lauprêtre and L.Monnerie, Polymer 38, 1767 (1997) (4) L.Heux, F.Lauprêtre, J.L.Halary and L.Monnerie, Polymer 39, 1269 (1998)

Contribution to the conference:
NMR study of topological constraints in polydimethylsiloxanes
T. ANDROV, F. LAUPRETRE, L. MONNERIE
NMR STUDY OF TOPOLOGICAL CONSTRAINTS IN POLYDIMETHYSILOXANES T. Androv, F. Lauprêtre, L. Monnerie Laboratoire de Physico-Chimie Structurale et Macromoléculaire -ESPCI, 10 rue Vauquelin 75231 Paris cedex 05. In entangled or crosslinked melts, the topological constraints induce a slowing down of the frequency and amplitude of local motions. Therefore, the 1H-1H dipole-dipole interactions are not entirely averaged out by the motions, which results in a pseudo-solid NMR behavior(1). In the present work, the residual dipole-dipole interaction was used as a tool for characterizing the influence of both entanglements and crosslinks. It was determined by studying the 1H spin-spin relaxation using both the Hahn echo (2) and the stimulated echo pulse (3) sequences. The samples under study were linear polydimethylsiloxanes (PDMS) with low and high molecular weights, and UV-crosslinked PDMS with various molecular weights between crosslinks. The 1H NMR experiments were carried out on a 100 MHz Bruker spectrometer as a function of temperature. Hahn echo data were interpreted following Sotta et al.(4) and Cohen-Addad et al.(5). In the stimulated echo experiments the residual dipole-dipole interaction was quantitatively determined from the ratio Qdc of the intensity of the primary echo Ep over the intensity of the stimulated echo Est. Both NMR experiments underline the reduction in mobility in linear and crosslinked samples when the density of topological constraints increases. - In linear PDMS with a high molecular weight, the residual dipolar interaction reflects the presence of entanglements. - Results obtained in crosslinked PDMS depend on the density of crosslinks and the nature (unimodal or bimodal) of the network. - When the temperature increases, the residual dipolar interaction decreases for samples that exhibit temporary constraints i.e entanglements, only highly crosslinked samples show no temperature dependence of the residual 1H-1H dipole-dipole interaction in the temperature range investigated. (1) Cohen Addad, J.P., In Progress in Nuclear Magnetic Spectroscopy; NMR and Fractal Properties of Polymeric Liquids and Gels, Pergamon Press: Oxford, England, 1993. (2) Hahn,E.L. , Physical Review 80, 580 (1950) (3) Kimmich,R. Fischer,E. Callaghan,P. and Fatkullin,N., J. Magn. Reson., A, 117, 53 (1995) (4) Sotta,P. Fülber,C. Demco,D.E. Blümich,B. and Spiess,H.W., Macromolecules 29, 6222 (1996) (5) Cohen-Addad,J-P., Soyez,E., Viallat,A., Queslel,J.P., Macromolecules 25, 1259 (1992)

Contribution to the conference:
Application of Z-COSY for the analysis of the spectra of oriented molecules
R. Christy Rani Grace and N. Suryaprakash
NMR spectroscopy of molecules oriented in liquid crystalline matrices provides information on the molecular structure and order parameters. In general, the spectra of these oriented molecules are strongly coupled dominated by the intramolecular dipolar couplings. The complexity of the spectra increases with the increase in the number of interacting spins and decreasing symmetry of the molecules. Thus the analyses of such spectra may be non-trivial even for molecules with three interacting spins and lower symmetries. Any technique which providces some information on the energy levels aids the analyses of such spectra. Two-dimensional Z-COSY experiment is one such technique which provides information on the connectivity of transitions. The use of the Z-COSY technique is demonstrated for teh analysis of the proton NMR spectra of meta-fluoroiodobenzene, a system of five interacting spins and planar symmetry. The carbon-proton dipolar couplings are also derived with the sub-spectral analysis of proton coupled Carbon-13 spectra. The advantage of the Z-COSY technique and the structural and orientational information derived will be discussed.

Contribution to the conference:
An PFG NMR Investigation of Probe Diffusion in Ethyl(hydroxyethyl)cellulose Matrix.
Magnus Nydén and Olle Söderman
The echo-decays for different molar masses of polyethyleneoxide are measured by the PFG NMR technique in solution and in gel matrixes formed in Ehtyl(hydroxyethyl)cellulose/water. From accurate experiments, covering a large range of field gradient strengths, it is found that for small molar masses the echo-decays show gaussian diffusion behaviour. For high molar masses, a more complex behaviour is noted as revealed by an observed curvature in the echo-decay, when plotted in a Stejskal-Tanner plot. For high concentration of matrix (6%), the molar mass scaling of the mean square displacement reveals interesting behaviour concerning the molar mass scaling of the diffusion coefficient. The results are interpreted according to diffusion in heterogeneous media.

Contribution to the conference:
Pfg-NMR measurements of lateral diffusion in lamellar liquid crystals
G. Orädd

Contribution to the conference:
CHAIN DYNAMICS IN THE ORTHORHOMBIC AND HEXAGONAL CRYSTALLINE PHASES OF POLYETHYLENE,- A HIGH-PRESSURE NMR STUDY
M. de Langen and K.O. Prins
A crystalline phase of polyethylene with a hexagonal structure has been shown to exist at rather extreme conditions, namely in a region of high pressure (more than 3200 bar) and high temperature (more than 493 K) [1]. At room temperature and ambient pressure the crystal structure of PE is orthorhom- bic. High-pressure crystallization from the melt through the hexagonal phase results in so-called chain-extended crystals in the orthorhombic phase. We present the first NMR investigation of the chain dynamics of polyethylene in its hexagonal crystal structure. For this project we developed a titanium high-pressure (10 kbar), high-temperature NMR probe [2]. We will discuss the behaviour of the proton and deuteron NMR line shapes and spin-lattice relaxation rates. From these properties, the transition from the strongly ordered, rather immobile orthorhombic struc- ture to the highly mobile hexagonal phase can be very clearly observed. Deuteron quadrupole echo spectra have been used in the hexagonal phase to determine the character and rate of local reorientations of the molecular chains. This motion appears to be similar to the one observed in the rotator phases occurring in the long normal alkanes. From proton rotating frame relaxation data we derived the rate of translational diffusion in the chain direction. These observations contribute to the understanding of the important role [3] of the hexagonal phase in the growth of chain-extended polyethylene crystals. References: [1] D.C. Bassett and B. Turner, Phil.Mag.29,285,925(1974); [2] M. de Langen and K.O. Prins, Rev.Sci.Instrum.66, 5218(1995); [3] A. Keller, M. Hikosaka, S. Rastogi, A. Toda, P.J. Barham and G.Goldbeck-Wood, J.Mater.Sci.29,2579(1994).

Contribution to the conference:
NMR 1H AND 13C STUDY OF NICKEL(II) COMPLEXES WITH SHIFF BASE IN SOLUTION AND SOLID STATE
A.I. Rakhmatullin, A.V. Aganov
The structure of nickel (II+) paramagnetic complexes with C6H4(OH)CH=NCH(CH3)2, CH3OC6H3(OH)CH=NC4H9, C12H25OC6H4C(O)OC6H3(OH)CH=NCH2CH(CH3)2 ligands in solution, isotopie liquid, mesophase and solid state is studied by NMR methods 1H and 13C. We defined the existance in solution as an equilibrium of assos.(param.), planes (diam.), tetrahedral (param.), the position of which depends on the temperature, solvent and concentration of solution. We also defined absence of spectrum NMR 13C CP/MAS dependence on first-order-transition (mesophase and solid state) and second-order-transition (isotopie liquid and mesophase). The relationship between the length of radical and the construction of chelate node was traced.

Contribution to the conference:
Deuteron relaxation dispersion in aqueous colloidal silica
P. Roose, J. Van Craen, H. Eisendrath
We report on an extensive study of the deuteron relaxation in aqueouscolloidal silica systems. The relaxation dispersions were measured asa function of pD and temperature. The experimental data show that themain relaxation contribution is due to exchanging silanol deuterons,especially at alkaline pH values.

Contribution to the conference:
AN ISOTOPE FRACTIONATION MODEL FOR THE PROTON SPIN-LATTICE RELAXATION OF AMORPHOUS COLLOIDAL SILICA SUSPENSIONS IN H2O/D2O MIXTURES
J. Van Craen, P. Roose, H. Eisendrath
The proton spin-lattice relaxation rates of amorphous colloidal silica suspensions are reported over a wide Larmor frequency range for varying degrees of isotopic dilution. At low magnetic fields, the amplitude of the relaxation dispersion reveals a non-linear dependence upon deuteration. This behaviour is primarily attributed to isotopic fractionation effects of slow exchanging hydrogens situated at solvent-shielded sites on the silica surface, but which are involved in dipolar interactions with readily solvent-accessible proton species. A theoretical spin relaxation model is proposed which accounts for the isotope dilution effects in a multiple-phase system composed of the abundant slow-relaxing bulk spins and spin environments in mutual dipolar interaction at the oxide interface. The broadness of the observed relaxation dispersions is treated as a superposition of a discrete number of lorentzian-like power spectra. At least two relaxation components are resolved from the present dispersion data which obviously reflects the heterogeneity of the proton sites at the silica surface. Each component seems to consist of a water and a pH dependent silanol contribution. The exchange of the observed proton classes has been estimated to occur on the µs time scale. An overall quantitative analysis of our relaxation dispersion data at different H/D ratios reveals considerable isotopic fractionation between some slow exchanging surface hydrogens and the water solvent. Fractionation factor values lower than 0.5 were obtained.

Contribution to the conference:
Deuteron NMR Study of the Microconfined Smectogenic Liquid Crystal 8CB
N. Vrbanèiè-Kopaè, M. Vilfan, S. Žumer, R. Blinc, G. P. Crawford, and R. Ondris-Crawford

Contribution to the conference:
NMR Evidence for Magnetic Field Reorientation-Induced Modulation of Soliton Density in Ferroelectric Liquid Crystal
B. Zalar, A. Gregoroviè, M. Simsiè, A. Zidanšek, and R. Blinc
Deuteron NMR was used for the detection of the magnetic field reorientation-induced changes in the spatial modulation of the incommensurate molecular tilt wave in the SmC* phase of a bulk ferroelectric liquid crystal CE8. A step motor-driven sample tilting mechanism, synchronized with the NMR pulse sequence, was applied in order to investigate the quasi-equilibrium state with smectic layers being non-parallel to the external magnetic field. The system was found to exhibit a reentrant SmC*-SmC-SmC* behaviour. At intermediate orientations, the critical field and the soliton density are much lower than the ones found when the magnetic field is parallel to smectic layers. Experimental observations agree very well with the predictions of the Landau theory with the inclusion of the non-parallel field magnetic energy term in the thermodynamic potential.

Contribution to the conference:
Chain dynamics in the orthorhombic and hexagonal crystalline phases of polyethylene, a high pressure NMR study.
M. de Langen and K.O. Prins
A crystalline phase of polyethylene with a hexagonal structure has been shown to exist at rather extreme conditions, namely in a region of high pressure (more than 3200 bar) and high temperature (more than 493 K) [1]. At room temperature and ambient pressure the crystal structure of PE is orthorhom- bic. High-pressure crystallization from the melt through the hexagonal phase results in so-called chain-extended crystals in the orthorhombic phase. We present the first NMR investigation of the chain dynamics of polyethylene in its hexagonal crystal structure. For this project we developed a titanium high-pressure (10 kbar), high-temperature NMR probe [2]. We will discuss the behaviour of the proton and deuteron NMR line shapes and spin-lattice relaxation rates. From these properties, the transition from the strongly ordered, rather immobile orthorhombic struc- ture to the highly mobile hexagonal phase can be very clearly observed. Deuteron quadrupole echo spectra have been used in the hexagonal phase to determine the character and rate of local reorientations of the molecular chains. This motion appears to be similar to the one observed in the rotator phases occurring in the long normal alkanes. From proton rotating frame relaxation data we derived the rate of translational diffusion in the chain direction. These observations contribute to the understanding of the important role [3] of the hexagonal phase in the growth of chain-extended polyethylene crystals. References: [1] D.C. Bassett and B. Turner, Phil.Mag.29,285,925(1974); [2] M. de Langen and K.O. Prins, Rev.Sci.Instrum.66, 5218(1995); [3] A. Keller, M. Hikosaka, S. Rastogi, A. Toda, P.J. Barham and G.Goldbeck-Wood, J.Mater.Sci.29,2579(1994).

Contribution to the conference:
Microstructural investigations of monoglyceride gels by PFG NMR
J. van Duynhoven, R. Raynal, A. Sein
Microstructural investigations of monoglyceride gels by PFG NMR J.P.M. van Duynhoven, R. Raynal, A. Sein Monoglycerides can act as surfactants and when mixed with water they can occur in several liquid crystal forms. Because of their ability to bind large volumes of water these molecules can be employed as fat-replacers in food technology. The macroscopic properties of these materials are governed by the molecular and microstructural organisation of the monoglyceride surfactants. Expanding our knowledge in structural-property relations at the molecular and microstructural level will permit us to have better understanding in these water-structured products. NMR PFG water self-diffusion measurements were used for the microstructural investigation of these materials and it has been demonstrated that this provides a method for the determination of two important microstructural parameters, i.e. network tortuosity and surface to volume ratio. Below a threshold value, both tortuosity and surface to volume ratio vary proportional with concentration. This is due to the formation of more crystal plates in the network when more monoglyceride is added. At higher monoglyceride concentration, the diffusional properties of water become invariant: no additional crystal-plates are formed. The microstructural parameters of monoglyceride gels, as assesed with PFG NMR, also vary with monoglyceride type and processing conditions. The conclusions drawn from the PFG NMR results are in accordance with electron and light microscopy observations.

Molecular Material Science

Contribution to the conference:
Ferromagnetic Resonance
D. Arèon, P. Cevc, A. Omerzu, and R. Blinc
Until recently, the occurence of ferromagnetism in purely organic compounds has been restricted to temperatures below approximately 1 K. However, this has changed significantly by the report [1] of a soft ferromagnetic state below 16.1 K in the fullerene compound TDAE-C60. Here we report on the first observation of a non-linear variation of the electron resonance frequency with resonance field characteristic of ferromagnetic resonance in a TDAE-C60 in the radion-frequency region. The results not only definitely prove the existence of long range order below Tc, but also show that well annealed TDAE-C60 behaves as an easy axis three dimensional Heisenberg ferromagnet with an exceptionally small anisotropy field Ha=29 Gauss. The easy axis coincides with the crystal c-direction which is the direction of closest approach of the C60 molecules. [1] P. -M. Allemand et al., Science 253, 301 (1991).

Contribution to the conference:
The study of phase transitions in polymers confined in porous media to characterize the media geometry
A. Filippov, R. Sh. Vartapetyan, M.M. Doroginizkij

Contribution to the conference:
Molecular dynamics of benzene in the zeolite NaY investigated with deuteron NMR
O.Isfort, B.Boddenberg, F.Fujara, R.Grosse
Zeolites are nanoporous crystalline materials with large internal surface area which are commercially used as molecular sieves and catalysts. We present the investigation of the molecular transport mechanisms of benzene in the faujasite type zeolite NaY with NMR methods. A main objective is the elucidation of the relation between microscopic dynamic processes and long range transport behavior. Our methods are multidimensional deuteron NMR, NMR relaxation and static field gradient NMR. The experiments show that the benzene molecules perform tetraedral reorientations between adjacent adsorption sites in the large voids (supercages) of the zeolite. Additionally we obtain evidence for small angle reorientations due to intermolecular interactions between neighbouring molecules. We obtain quantitative information on reorientation angles, time scales, and activation energies of the investigated molecular dynamics.

Contribution to the conference:
NMR -apparature for on-line control of Phys.Chem.parameters inoil-water emulsion
R. S. Kashaev

Contribution to the conference:
Physicochemical Characterization of Emulsions by Diffusion NMR
W. Kuhn

Contribution to the conference:
C60 containing molecular crystals
Elizabeth Shabanova, Kjeld Schaumburg

Contribution to the conference:
LIQUID SELF-DIFFUSION IN POROUS GLASS "VYCOR" : NMR DIFFUSOMETRY STUDY.
M.V. Skirda, V.D. Skirda, V.A. Sevrjugin
The self-diffusion of the alkanes molecules with molecular weights from 86 to 212 and the water molecules, confined in the controlled pore glass (CPG) "Vycor" with average pore diameter 40 A are measured within 3 decimal orders of diffusion time t interval from 3 ms to 1000 ms by NMR pulsed field gradient ( NMR PFG) method at 303 K. The self-diffusion echo attenuations (SDEA) are ploted concretely the gradient magnitude. The duration of gradient is lesser 0.1 ms and the maximum gradient amplitude is 200 T/m. SDEA are exponentional in all cases. Previously the water time-dependent self-diffusion coefficient D(t) was measured by field static gradient NMR [1]. It was shown that the dependence D(t) follows the power law D(t)~t^-0.11. This behavior of D(t) can be described in terms of fractal CPG "Vycor" structure. We reliably observe the dependence D(t)~t^0 , i.e. SDC is not dependent on the self-diffusion time. It is found for all the studied liquids. This results allows us to suggest that the fractal structure of CPG "Vycor" with average pore diameter 50 A is not displayed in the spatial scales from 10^-7 m to 240*10^-7 m. The second important result is the observation of power law dependence of D on the liquid molecule size. It is shown, that the accounting of this result is necessary for the estimation of tortuosity as well as the form-factor F of the porous media according to the self-diffusion data. For the system "Vycor"-water we observe the peculiarities wich can be explained only by structure ordering caused by the strong hydrogen bonds. We suggest that our results can be used to study the association phenomenon of low molecular weight liquids by NMR PFG method. REFERENCES Chang I, Fujara F, Geil B, Hinze G, Sillesku H, Tolle A, J Non-Cryst Sol, 172-174, 674 (1994)

NMR Computations/Simulations

Contribution to the conference:
Lattice elctric field gradient tensors calculation in K_2ZnCl_4
M.N. GRECU and S. CONSTANTINESCU
LATTICE ELECTRIC FIELD GRADIENT TENSORS CALCULATION IN K2ZnCl4 M.N. Grecu and S. Constantinescu National Institute for Materials Physics, 76900 Bucharest, Romania IN THIS Communication we report on results for K2ZnCl4 in all phases, as derived from a numerical computation of the lattice electric field gradient tensor Va b , local electric potential V and electric field intensityï Eï at K and Zn sites, in an ionic point charge and rigid lattice approximations. A special computing program QSCOPM [1] was used. The lattice of rectangular unit cell was build by the subroutine LATTICE with the origin of coordinates in the crystallographic positions 4c, 8d and 4a for normal, and ferroelectric phase, respectively. Two different values for the atomic fractional coordinates were taken into account for high and low part of the incommensurate phase, as derived from structural neutron analysis [2]. The principal components of the electric field gradient tensor were provided by subroutine GRADIENT, taking into account the LATTICE output data for N=756 ions (Pnam normal phase) and N=2268 ions (Pna21 superstructure ferroelectric phase) situated at their correct position, i.e. contained within a radius of 26 unit cells. The numerical data obtained at each K and Zn positions are correlated with the corresponding experimental 39K NMR, Cu2+ EPR and 57Fe Mössbauer results. Some differences were observed in Mössbauer experiments [3] in rigid lattice approximation. To improve them, this hypothesis was relaxed. The probe-ion site was gradually displaced from the nominal values along the crystallographic axes and the electric field gradient tensor components were calculated. A good agreement with experimental values was obtained for a probe-ion in certain shifted positions. [1] S. Constantinescu and S. Calogero, QCMP 172, QCPE Bulletin 17 (1997) 1 [2] M. Quilichini, P. Bernede, J. Lefebvre and P. Schweiss, J. Phys.: Condens. Matter 2 (1990) 4543 [3] S.Constantinescu and M.N. Grecu, Rom. Rept. Phys. Suppl., 50 (1998) in press

Contribution to the conference:
On the prediction of stereochemically different carbons (tentative)

Contribution to the conference:
Efficient simulation of phase cycling and sequences of pulsed field gradients
Alexej Jerschow and Norbert Müller
The success of new and useful NMR pulse sequences is to a major part due to the capabilities of coherence pathway selection offered by phase cycling and pulsed field gradients. Radio frequency pulses are relatively non-selective with respect to coherence orders and their interconversion. Although it is straightforward to design a pulse sequence with a phase cycle and gradient sequence to follow an intended coherence transfer pathway two major problems remain: 1.) Estimating the ``leakage'' of unwanted pathways due to imperfections 2.) Reducing the number of phase cycling steps without degradation of the performance. We present an efficient and general algorithm to simulate the effects of phase cycles and sequences of pulsed field gradients, including the effects of diffusion and flow. It is a convenient method to check and develop optimum schemes for coherence pathway selection. Since it does not a priori include parameters associated with spin system specifics it is applicable to both liquid and solid state NMR, as well as to EPR. The program (called CCCP - Complete Calculation of Coherence Pathways) has been implemented in MATLAB and will be made available to the NMR community.

Contribution to the conference:
Component-Resolved Spectroscopy (CORE)
Peter Stilb
For complex multi-component systems, the application of traditional FT-PGSE techniques for measuring self-diffusion may be difficult, and the data evaluation uncertain. Underlying reasons may be extensive signal overlap and poor signal/noise in the experiment. Prompted by such problems in NMR-based FT-PGSE self-diffusion studies^1 of polymer-surfactant systems^2 a new data processing approach named CORE (COmponent-REsolved) spectroscopy was developed^3. This approach accounts for all of the available information in the data set, including prior knowledge that the whole bandshape of a given component stays constant at all gradient settings - only its amplitude varies. Component bandshapes are therefore directly extracted by the CORE processing. In addition the effective signal/noise of the experiment is increased by more han an order of magnitude in comparison with traditional peak height fitting procedures. Through CORE processing of the acquired data FT-PGSE can confidently and routinely be applied to complex multicomponent systems. The generic CORE-approach has been shown to be of great potential value also for the analysis of multi- wavelength kinetic or time-resolved fluorescence data^4 and can also be applied to some types of 2D NMR data, and sets of images. 1) P. Stilbs, Prog. Nucl. Magn. Reson. Spectrosc. 19, 1 (1987). 2) O. Soderman, P. Stilbs, Prog. Nucl. Magn. Reson. Spectrosc. 26, 445 (1994). 3) P. Stilbs, K. Paulsen, P. C. Griffiths, J. Phys. Chem. 100, 8180 (1996). 4) P. Stilbs, K. Paulsen, Rev. Sci. Instrum. 67, 4380 (1996).

Contribution to the conference:
Spectral Analysis of 8-Azabicyclo[3.2.1]octane Derivatives
Anu Airaksinen, Jouko Vepsäläinen, Matthias Niemitz and Reino Laatikainen
The knowledge about long-range couplings in bicyclic structures is still scarce. These couplings usually form a nuisance for spectral analysis, leading often to broad, poorly resolved multiplets. In this work we have analysed high-resolution 1H NMR spectra of tropinone and some 8-aza-bicyclo[3.2.1]octane derivatives (e.g. cocaine) using a procedure based on the use of integral transforms (1). The analysis based on very strongly resolution enhanced spectra, with line-widths of typically less than 0.1 Hz, yielded the values and in many cases also the signs of the coupling constants. Conformational dependencies of the coupling constants are discussed in the poster. It will be also shown that the integral transform procedure provides good estimates of conformationally important couplings without complete analysis of the small long-range couplings.

Contribution to the conference:
Three parameter model fit .. plant MRI data
P.A. de Jager, F.J. Vergeldt and H. Van As
MRI relaxation data of plants can be fitted with the sum of a discrete number of exponentials. Better images are obtained by fitting with a simple three parameter model.

NMR in Liquids

Contribution to the conference:
Combined Solvent Hetero-NOE and Oversampling - Quaternary CarbonsEnhanced
Walter Bauer
The 13C-NMR-signals of fullerenes and related compounds lack the intensity gain from intramolecular NOE due to the absence of protons. However, the usage of protonated instead of deuterated solvents provides intermolecular NOE from the bulk solvent protons. A draw- back of using protonated solvents is the strong solvent 13C-NMR- signal that is likewise present in the spectrum. Additional oversampling compensates for dynamic range problems in dilute solutions. The method is demonstrated for appropriate samples. The signal-to-noise ratio may be increased up to 87%, equivalent to a 3.5 time savings factor.

Contribution to the conference:
New NMR and MNDO/PM3 Insights into the Mechanism of the ortho-Lithiation
Juergen Betz and Walter Bauer
Depending on the reaction conditions, nBuLi reacts with 1-methoxynaphthalene in the 2- and/or 8-position. Using tBuLi, lithiation exclusively in the 8-position is found. We have studied the reaction mechanism by using 6-Li labeled material and the HOESY pulse sequence. Whereas nBuLi forms a tight precursor complex, no such complex is found when using tBuLi. Semiempirical calculations indicate that the nBuLi reaction proceeds under kinetic control whereas in the tBuLi case thermodynamic control is indicated.

Contribution to the conference:
FIRST DIRECT NMR OBSERVATION OF A LANTHANIDE COORDINATED WATER MOLECULE WHOSE EXCHANGE RATE IS MARKEDLY STEREOCHEMICALLY DEPENDENT
Mauro Botta, Silvio Aime, Alessandro Barge, Alvaro S. De Sousa and David Parker
Mauro Botta, Silvio Aime, Alessandro Barge, Alvaro S. De Sousa° and David Parker° Dipartimento di Chimica I.F.M., Università di Torino, Via P. Giuria 7, 10125 Torino (Italy) ° Department of Chemistry, Durham University, South Road, DH1 3LE Durham (UK)
The issue of the water exchange rate in lanthanide(III) complexes is of paramount importance in the development of novel contrast agents (CA) for MRI. In fact, the residence lifetime of the coordinated water molecule(s) to a Gd(III) complex plays a particularly important role since it contributes directly to the modulation of the electron-nucleus dipolar interaction and it controls the efficiency of the transfer of the paramagnetic effect to the bulk water.[1] In lanthanide(III) complexes, water is considered a very labile ligand and then in fast exchange, on the NMR time-scale, with the bulk. Recently, we reported that a Gd(III) complex with a tetraazamacrocyclic ligand containing four N-methylcarboxamide groups in place of four carboxylates displays a large increase of the residence lifetime of the coordinated water.[2] The slow exchange rate is a consequence of the stronger Ln-OH2 interaction and a stabilizing H-bonding interaction to the proximate anions. We thought that the slow exchange of the coordinated water in this type of complex may allow further insight into the relationship between the solution structure of the lanthanide (III) chelate and the water exchange rate to be gained. Herein we report that, in the case of the Eu(III) complex with a tetraazamacrocyclic ligand bearing four carbamoylmethyl groups (DOTAM), the exchange may be frozen out at low temperature in acetonitrile solution. This allows for the first time the direct observation of the NMR signal of the coordinated water in a paramagnetic lanthanide(III) complex. Moreover, EuDOTAM is found to exist in solution as a mixture of two diasereoisomers (M and m) and the band-shape analysis of the VT-1H NMR spectra evidences a remarkable difference in their water exchange rate (M isomer, DH = 79.0 (± 1.9) KJ/mol, DS = 65.7 (± 6.5) J/(mol K); m isomer, DH = 79.0 (± 2.5) KJ/mol, DS = 110.8 (± 10.4) J/(mol K)). This marked stereochemical dependence of the bound water residence lifetime is of considerable importance for the optimum design of more efficent contrast agents for magnetic resonance imaging application.
References: [1] a) R. B. Lauffer, Chem Rev. 1987, 87, 901-927; b) J. A. Peters, J. Huskens, D. J. Raber, Prog. NMR Spectrosc. 1996, 28, 283-350; c) S. Aime, M. Botta, M. Fasano, E. Terreno, Chem. Soc. Rev. 1997, 26, 1-11. [2] S. Aime, A. Barge, M. Botta, A. De Sousa, D. Parker, J. Am. Chem. Soc. 1997, 119, 4767-4768.

Contribution to the conference:
NMR of confined metals near the melting and freezing phasetransitions
E.V.Charnaya, W.-D.Hoffmann, D.Michel, I.Sorina, and Yu.A.Kumzerov
NMR of gallium and mercury confined within porous glasses wasobserved in temperature ranges covering the melting and freezing phase transitions for embedded metals. Peculiarities of the freezing and melting processes in confined geometries were studied for various pore sizes and compared with predictions of different theoretical models. Temperature dependences of the Knight shift and of the line shape are discussed for confined liquid metals. NMR results for melting and freezing are compared with those obtained for the same samples using other experimental techniques.

Contribution to the conference:
Selective Cross-Polarization in Solution-State NMR
Brian Cutting, Philippe Pelupessy, Elisabetta Chiarparin and Geoffrey Bodenhausen
Cross-Polarization has long been recognised as a useful method in isotropic liquids to transfer coherence between scalar-coupled nuclei such as protons and carbon-13. Unfortunately, the transfer efficiency is strongly attenuated through RF inhomogeneity. This presentation demonstrates that, provided the applied RF amplitudes are comparable to the heteronuclear coupling constants, difficulties attributed to RF inhomogeneity are greatly reduced. Comparisons to selective INEPT illustrate that selective cross-polarization is more efficient, is insensitive to experimental instabilities, and may be easily implemented without careful calibrations.

Contribution to the conference:
ORSAT for quick Assignment to C, CH, CH2 and CH3-Groups
U.Beckmann, W.Dietrich, R.Radeglia
Generally three spectral traces for the assignment of C, CH, CH2 and CH3 to a carbon spectrum are necessary, a broadband decoupled one and 2 DEPT spectra. It can be shown that besides the pulse sequence "ORSAT" (Off Resonance Syncronized Attachment Test) only DEPT 135 is needed for the complete assignment of all carbons. ORSAT is optimized in time for the quaternary and optimized in residual splittings for the protonized carbons.

Contribution to the conference:
ONE-DIMENSIONAL SELECTIVE NOE MEASUREMENTS IN LABELED MACROMOLECULES USING 1H-15N-1H CROSS-POLARIZATION TRANSFER
THOMAS EYKYN; ELISABETTA CHIARPARIN; BRIAN CUTTING; DAMIEN JEANNERAT; PHILIPPE PELUPESSY; GEOFFREY BODENHAUSEN
A novel selective 1D NOE experiment is presented. In 15N or 13C labeled macromolecules it is possible to selectively excite signals in crowded proton spectra via cross-polarization using low amplitude continuous-wave radio-frequency fields, provided the protons to be excited are bound to heteronuclei with differing resonance frequencies. After selective excitation a mixing period tm is inserted to allow cross-relaxation with dipolar-coupled spins in the vicinity of the source spin. The resulting 1D spectrum corresponds to a slice taken through a 3D HSQC-NOESY spectrum at the w1 and w2 frequencies corresponding to the chemical shifts of the heteronucleus and source proton. The utility of this method is demonstrated for a sample of 15N labelled human ubiquitine. Inter-residue and intra-residue NOE’s from a selected amide proton to protons on non-contiguous amino acids are clearly observed and build-up curves measured.

Contribution to the conference:
1H and 13C NMR Studies of Some Steroidal Neuromuscular Blocking Drugs: Solution Conformation and Dynamics
L. Fielding
The 1H and 13C NMR spectra of the steroidal neuromuscular blocking drugs, pancuronium bromide, vecuronium bromide, rocuronium bromide and Org 9487 are presented. The 13C NMR spectra are fully assigned. NOE data and variable temperature studies show that there is considerable conformational freedom in both the ring A substituents and the sterically more crowded ring D substituents. The quaternised ring D piperidine (or pyrollidine) rings are free to rotate with respect to the steroid.

Contribution to the conference:
Minimising diffusion losses in GROESY and GOESY experiments
M.A. Delsuc, C. Roumestand, J.A. Gavín and T. Parella
A serious drawback of the GOESY and GROESY experiments are the inherent sensi- tivity losses due to diffusion effects. In this work we show that improved sensi- tivity can be achieved by simple sample rotation. The maximum NOE (or ROE) enhan- cement obtained from the GOESY and GROESY experiments is achieved around 400 ms. in a non-rotation sample. In addition, the sensitivity of these experiments lar- gely depends on the intensity and/or duration of the gradients and, therefore, careful calibration must be taken into account when performing such experiments. We have observed that the sensitivity of these experiments dramatically changed when the data were recorded when the sample was spinning, whereas the maximum signal was obtained around 800 ms., which value was very similar to that obtained with the non-refocused version.

Contribution to the conference:
NMR Measurements of Residual Dipolar 1H-13C Couplings in a 19mer RNA Hairpin
Geierstanger, Bernhard H., Marino, John P., Schwalbe, Harald and Griesinger, Christian
At high magnetic fields proteins and nucleic acids slightly align relative to the magnetic field because of their anisotropic magnetic susceptibility tensors (Tolman et al., 1996; Kung et al., 1995). This alignment gives rise to residual dipolar coupling constants that are observed on top of the scalar couplings in high-resolution spectra. The residual dipolar contributions to the 1H-13C couplings in a uniformely labeled RNA 19mer (Eguchi & Tomizawa, 1991; Marino, 1995; Marino et al., 1995) were determined from 1JCH-modulated CT-HSQC (Tjandra & Bax, 1997) experiments at 14.1 and 18.8 Tesla. These measurements can provide information about the relative orientation of bond vectors in large biomolecules (Bax & Tjandra, 1997; Tolman et al.,1997) and yield additional constraints that can improve the accuracy of NMR structures particularly of nucleic acids and their complexes (Tjandra et al., 1997). Here we report on our progress to apply this methodology to a 19mer RNA hairpin and discuss the implications of using this new approach on the RNA hairpin structure. References: Bax & Tjandra (1997) Nat. Struct. Biol. 4, 254-256 Eguchi & Tomizawa (1991) J. Mol. Biol. 220, 831-842. Marino (1995) Ph.D. dissertation, Yale University. Marino, Gregorian, Csankovszki & Crothers (1995) Science 268, 1448-1454 Kung, Wang, Goljer & Bolton (1995) J. Magn. Res. Series B 109, 323-325. Tjandra & Bax (1997) J. Magn. Res. 124, 512-515 Tjandra, Omichinski, Gronenborn, Clore & Bax (1997) Nat. Struct. Biol. 4, 732-738 Tolman, Flanagan, Kennedy & Prestegard (1997) Nat. Struct. Biol. 4, 292-297 Tolman, Flanagan, Kennedy & Prestegard (1996) Proc. Natl. Acad. Sci. USA, 9279-9283

Contribution to the conference:
Projection Restraints as Tool for Structure Elucidation
B. Reif, T. Carlomagno, M. Maurer, M. Hennig, H. Steinhagen, A. Diener, B. Elshorst, A. Tüchelmann, M. Reggelin, H. Schwalbe, C. Griesinger
Projection restraints as a Tool for Structure Elucidation B. Reif, T. Carlomagno, M. Maurer, M. Hennig, H. Steinhagen, A. Diener, B. Elshorst, A. Tüchelmann, M. Reggelin, H. Schwalbe, C. Griesinger * Institut für Organische Chemie, Johann Wolfgang Goethe-Universität Frankfurt/Main, Marie-Curie-Str. 11, D-60439 Frankfurt. Cross correlated relaxation of two dipoles as well as CSA and one dipole is measured for isotopically labelled proteins [1]. Structural information as well as dynamical information can be derived from these measurements. The impact of this novel structural restraint will be demonstrated. The potential of the method with respect to precision and long range information will be demonstrated on a metalloorganic catalytical intermediate that has been partly 13C labelled. The precision obtained is less than ±5° for the projection angles. The projection restraint has been incorporated into DG calculations. Finally, the impact of the measurement of cross correlated relaxation about the peptide bond for the elucidation of dynamics will be presented. [1] B. Reif, M. Hennig, and C. Griesinger, Science, 276, 1230-33 (1997). [2] B. Reif, H. Steinhagen, B. Junker, M. Reggelin, and C. Griesinger, Angew. Chem. in press

Contribution to the conference:
Magnetically Anisotropic Tags for Biomolecules
Ariane Tüchelmann, Harald Schwalbe, Mauro Cremonini, Claudio Luchinat, Christian Griesinger
The partial alignment of biomolecules [1] in the magnetic field caused by paramagnetically anisotropic molecules is of great interest to obtain additional and long range structural restraints from the evaluation of dipolar shifts and field dependent residual dipolar couplings. The poster presents the synthesis of magnetically orientating tags and the NMR-spectroscopic characterization of the observed paramagnetic effects. Three approaches are discussed: 1.) Covalent ligation to metallated porphyrines (Co-porphyrines), the synthesis of which is also compatible with the solid phase synthesis of peptides so that porphyrinopeptides with the porphyrine at different positions in the peptide can easily be synthesized. Measured dipolar shifts in a rigid molecule attached to the orienting tag were incorporated in a structural calculation revealing long range order. 2.) Complexation of Lanthanide ions such as Dy, Pr, Yb which exhibit larger paramagnetic effects due to large spin-orbit-coupling with DTPA (Diethylentriaminpentaaceticacid). DTPA was attached to dipeptides and amino acids on the solid phase. 3.) Complexation of Lanthanide ions such as Dy, Pr, Yb to His-tags and Asp-tags. This approach offers the possibility to obtain large peptides with the chelating sequence of amino acids by biological methods with no further chemical modification. [1] J.R. Tolman, J.M. Flanagan, M.A. Kennedy, J.H. Prestegard Proc. Natl. Acad. Sci. USA (1995) 92 9279; H.C. Kung, K.Y. Wang, I. Goljer, P.H. Bolton J. Magn. Reson. (1995) 109 323; N. Tjandra, A. Bax Science (1997) 278 1111.

Contribution to the conference:
Relaxation NMR study on Hg(CN)2
A. Gryff-Keller, S. Molczanow

Contribution to the conference:
Trinuclear Lanthanide(III) Complexes Studied by 17O NMR, 1H NMRD and EPR
Éva Toth, Lothar Helm, André Merbach, András Jánossy, Roman Hediger, Kaspar Hegetschweiler
The trinuclear [Gd3(H-3taci)2(H2O)6]3+ complex has been characterized in aqueous solution from the point of view of magnetic resonance imaging (MRI): The parameters that affect proton relaxivity have been determined in a combined variable temperature, pressure and multiple field 17O, NMR, EPR and 1H NMRD study. The solution structure of the complex shows that the total coordination number of each lanthanide(III) ion is eight with two inner sphere water molecules. Variable field EPR measurements (up to 8.09 T) show the presence of a strong intramolecular dipole-dipole interaction between Gadolinium(III) electron spins. Its contribution to the overall electron spin relaxation is important at high magnetic fields (B > 5T) but only minor at fields used by MRI. [Gd3(H-3taci)2(H2O)6]3+ undergoes a relatively slow water exchange (kex = 1.1 107 s-1) compared to the Gd(III) aqua ion (kex = 8.04 108 s-1), while the mechanism is much more associatively activated as shown by the large negative activation volume V# = -12.7 cm3 mol-1 obtained from variable pressure 17O NMR. The slow exchange rate can be explained with the rigidity of the [Gd3(H-3taci)2(H2O)6]3+ complex which considerably slows down the transition from the eight-coordinate reactant to the nine-coordinate transition state. Information on the rotational correlation time, c, of the complex can be obtained from 17O T1 relaxation rates and from 1H nuclear magnetic relaxation dispersion (NMRD).

Contribution to the conference:
NMR Spectroscopy in Wine Analysis
I. Kosir, M. Kocjancic and J. Kidric

Contribution to the conference:
Transverse relaxation optimized spectroscopy (TROSY) opens an avenue to NMR structures of very large proteins in solution
Konstantin Pervouchine, Roland Riek, Gerhard Wider and Kurt Wüthrich*
Fast transverse relaxation of 1H, 15N and 13C by dipole-dipole coupling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacromolecular structures that can be studied by NMR spectroscopy in solution. TROSY (Transverse Relaxation- Optimized SpectroscopY) [1] is a new approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA. For example, a TROSY-type [1H,15N]-correlation experiment with a 15N-labeled, protonated 110 kDa homooctameric S. aureus dihydroneopterin aldolase showed that 15N relaxation during 15N chemical shift evolution and 1HN relaxation during signal acquisition are both many-fold reduced resulting in a well resolved 2D NMR spectrum which is otherwise impossible to obtain. TROSY significantly improves the sensitivity of NMR experiments for aromatic spin systems in proteins. In many 1H-13C, 13C-13C correlation experiments up to ten-fold increase in the signal-to-noise ratio is achieved for the uniformly 13C-labeled, fully protonated 18 kDa protein cyclophilin A at 10° C when compared to the corresponding conventional NMR experiments. A number of triple resonance experiments for resonance assignment and collection of structural constraints are constructed based on the TROSY method, which constitute a basis for the NMR structural studies of large proteins in solution.

Contribution to the conference:
NMR Spectroscopy in Wine Analysis
I. Kosir, M. Kocjancic and J. Kidric

Contribution to the conference:
Anysotropic Effects of Carbonyl and Thione Groups
Sh. Latypov, V.Kataev, E.Qunioa, R.Riguera
Anisotropic effects of carbonyl and thione groups are useful instrument in fine structure determination of organic molecules by NMR. It was established for aldehydes that carbonyl bond produces shielding effect for a nuclear near to the bond. However, inverse model was derived for thione bond in thioesters. According to our experimental (DNMR) and theoretical (MM, AM1 and PM3) data anisotropic effects of carbonyl and thione bonds in carbo- and thio-esters should be essntially the same as in aldehydes and there no need to modify the model if to carry out conformational analysis correctly.

Contribution to the conference:
NMR spectroscopy of hydroxyl protons; A probe for conformational analysis
B.R. Leeflang, C. Sandstrom, L. Kenne and J.F.G. Vliegenthart
Hydrogen bonding plays an important role in molecular structure of carbohydrates. This study focusses on the problems and possibilities of using NMR spectroscopy of hydroxyl protons.

Contribution to the conference:
Facial Discrimination in Molecules Containing a Prochiral Group by NMR in PBLG Liquid Crystalline System
P.Lesot, D.Merlet, A.Loewenstein and J.Courtieu
The quantitative description of facial discrimination in molecules containing a prochiral group oriented in a polypeptyde liquid crystalline system is reported. Analysis of proton, carbon-13 and deuterium NMR of ethanol dissolved in an organic solution of PBLG yields the full molecular ordering matrix and the principal axis system. It is shown that five non-zero independent order parameters are needed to describe correctly the molecular ordering of ethanol in this media. The nonequivalence, in terms of the dipolar couplings of the two enantiotopic protons, enables us to determine the magnitude and sign of the geminal coupling between them. Natural abundance deuterium NMR in this and other molecules is shown to be a useful tool for enantiomeric analysis.

Contribution to the conference:
Particle dynamics in LiNO3-Ca(NO3)2-H2O solution as studied by multinuclear NMR
V.V.Matveev
The relaxation rates have been studied for all nuclei in the metastable ternary system 13mLiNO3-6.5mCa(NO3)2-H2O in the temperature range 0-25 C. Experimental temperature dependencies indicate the same activation energy for water, both cations and nitrate anion. It means that the dynamics of all particles is correlated closely one to another, i.e. a complete ionic association exists in the system ( at least from the dynamic point of view ). The comparison of 14N and 17O experimental data indicates that the correlation times of a nitrate anion reorientation around two different axis are equal. That means that the anion reorientates as a part of some pseudo-spherical complex and gives another independent confirmation of an essential ionic association in the system investigated. Possible composition and structure of the associates is discussed on the basis of ions and solvent relaxivities in the system as compared to the proper dilute solutions.

Contribution to the conference:
THE MOLECULAR BASIS OF HYPERTENSION
T. MAVROMOUSTAKOS, A. KOLOCOURIS, M. ZERVOU, I. MATSOUKAS
National Hellenic Research Foundation, Institute of Organic and Pharmaceutical Chemistry, Vas. Constantinou 48, 11635, Athens, Greece. University of Patras, Chemistry Department, Patras 26000, Greece. Ang II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) is a peptide hormone that is implicated in the cause of hypertension, a recently wide spread disease due to the stressfull life style of our age in modern civilized societies. For this reason, the design and synthesis of a novel drug that blocks the undesirable biological effect of Ang II would be of great beneficial to humanity. Hitherto, the best developed drugs are the non peptide AT1 receptor antagonists. The efforts were rewarded by the synthesis of Losartan, a drug already in the market, that exerts its biological action as a metabolite known EXP-3174. In the last two decades our laboratory has been involved in the synthesis and the study of conformational analysis of Ang II and various analogs, peptide or not in nature, that possess either agonist or antagonist activity. The design of synthetic molecules has been based upon the conformational analysis obtained by modern 1D and 2D NMR spectroscopy coupled with theoretical calculations and molecular modelling. The obtained results showed that the three key aminoacids Tyr4, His6 and Phe8 present in Ang II form a charge relay system. The relay system results from the clustering of the triad of aminoacids and it is observed only on the peptide analogs that possess agonist activity. The picture is extended to molecules which are not peptide in nature but mimick the C-terminal part of Ang II. Thus, Losartan and its active metabolite EXP-3174 lacks a superimposition with Ang II in which to favour transfer of the relay system. However, this is possible with the only known synthetic non peptide AT1 agonist, L-163,491. The data build a model in which the molecular basis of hypertension can be explained. Agonist activity is exerted only by molecules that possess stereoelectronic features able to mimick the template of the three important aminoacids Tyr4, His6 and Phe8 present in Ang II. It remains to fully elucidate the stereochemical requirements for antagonist activity. The basis has been already founded. Molecules which possess antagonist activity must closely resemble the stereoelectronic characteristics of the triad of aminoacids His-Tyr-Phe but must lack of the ability to form a relay system. The better understanding of molecular features that lead to antagonist activity can drive synthetic chemists to design drugs with better biological profile.

Contribution to the conference:
Gradient Accordion Used for DIffusion (GAUDI): a new method for measuring diffusion coefficients by 2D NMR
Oscar Millet, Miquel Pons
A new method for the determination of translational diffusion coefficients by 2D NMR is presented. When evolution time and gradient strength are incremented in a concerted way, traslational diffusion introduces a gaussian attenuation of the signal in t1. This is analogous to what is found in accordion spectroscopy. After Fourier transformation the information is given as an additional gaussian broadening in f1. GAUDI strategy can be applied to different homo- and heteronuclear experiments and gives diffusion coefficients in good agreement with those determined by standard methods.

Contribution to the conference:
MULTINUCLEAR (1H, 31P, 183W) MAGNETIC RESONANCE STUDIES OF THE REACTIONS BETWEEN A MIXED LIGAND BIS(DITHIOLATE) TUNGSTEN DICARBONYL AND A SERIES OF PH
Dimitris Argyropoulos, Christiana A. Mitsopoulou
The reactions of a new asymmetric ligand tungsten bis-(dithiolate) complex {W(S2C2HPh)2(CO)2-} with a series of phosphine ligands have been investigated by multinuclear magnetic resonance spectroscopy. As it has been shown1,2 homoleptic as well as mixed ligand tungsten tris dithiolenes have a trigonal prismatic geometry in solution. They are also stereochemically non rigid at room temperatures. In order to investigate further the trigonal prismatic geometry, we react the W(S2C2HPh)2(CO)2- complex with a series of phosphines in acetone. The corresponding phosphine complexes are readily formed. The 1H-{183W}-HMQC spectra shows the existence of the W(II), according to the appearence the 183W signal below 1000 ppm. The 31P NMR spectra of the bis(dithiolate) tungsten complexes at low temperature are consistent with a cis stereochemistry of the phosphine ligands and isomerism with respect to inversion at sulfur and relative positioning of the hydrogen groups on the chelating 1,2- S2C2HPh ligands. The singlet in the 31P NMR spectrum at room temperature arises from rapid interconversion of these isomers. In addition,the variable temperature 1H NMR spectra show that some of the phoshine bis(dithiolate) tungsten complexes are trigonal prismatic in solution and stereochemically non rigid at room temperatures, with varying rates of isomerisation. 1.Argyropoulos, D.; Mitsopoulou,C.A.; Katakis, D.; Inorg. Chem., 1996, 35, 5549. 2. Argyropoulos, D.; Lyris, E.; Mitsopoulou,C.A.; Katakis, D.; J. Chem. Soc., Dalton Trans., 1997, 615

Contribution to the conference:
ADDITIVITY OF DEUTERIUM-INDUCED ISOTOPE EFFECTS IN 13C NMR SPECTRA OF BENZOCYCLOBUTENE ISOTOPOMERS
Predrag Novak, Zoran Glasovac, Mirjana Eckert-Maksiæ and Zlatko Meiæ
One of the main characteristics of isotope effects on NMR parameters, i.e. chemical shifts and spin-spin coupling constants, is their additivity. Linear dependence of isotope effects upon the number of isotopic nuclei has been observed in many cases. Nevertheless, we have recently demonstrated that a significant non-additivity in molecules of a medium size containing one or two phenyl rings (benzoic acid, benzophenone, cis- and trans-stilbene, trans-N-benzylideneaniline, trans-azobenzene etc.), could indicate changes in equilibrium upon isotopic substitution. Polydeuteriation was supposed to change rotamer population distribution which affected chemical shifts. Benzocyclobutene, unlike the above mentioned benzene derivatives, is a rigid system which possesses a fixed geometry. Therefore, it is of interest to check whether the additivity rule is obeyed in a series of benzocyclobutene isotopomers. In order to compare experimental observations and theory, quantum chemical calculations have been performed. Geometries of all the studied isotopomers were optimised at DFT level of theory by using B3LYP hybrid functional. Shielding constants were calculated employing GIAO approach.

Contribution to the conference:
The Hetero nOe, a tool for structure determination of fluorinated compounds
M.Ourevitch
The fluorine nucleus acts as a "natural marker" by the couplings it shares with the proximate nuclei; 19F is also involved in long range couplings which are either transmitted through bonds or through space. Hetero nuclear Overhauser effect is the only technique that enables to distinguish between the two ways of transmission. Examples of configuration assignments of conformational study are given in fluorinated analogs of natural bioactiv compounds.and in taxoid fluorinated analogs

Contribution to the conference:
The study of the nuclear magnetic relaxation of some alkane liquids in zeolite ZSM-5.
G.G. Pimenov, O.P. Opanasyuk, V.D. Skirda
The molecular mobility of n-hexane, n-octane, n-decane and cyclohexane in zeolite ZSM-5, was sdudied by NMR. The concentration and temperature dependencies of a free induction decay (FID), longitudinal and transverse relaxation times were obtained. The free induction decay of investigated liquids have the obvious nonexponential shape and can be decomposed on two components. The first "liquid-like" FID component has the exponential shape and transverse relaxation timeas in range from 10 ^ -1 up to 10 ^ -3 seconds. The fraction of this component in the FID signal decreases with decreasing of concentration of fluid in zeolite. We have related this component to molecules of liquid located outside of a zeolite crystalline structure. The second FID component has "solid-like" ( for example, for it the dipolar echo have been observed which is typical for solids) nonexponential shape which depends on a type of entered liquid. This FID component was related by us to molecules of liquid located inside zeolite channels. The feature of this component is it can not be described neither by single Gaussian line nor by superposition of such lines. On our sight, the complex shape of this FID part is a corollary of restrictions of molecular mobility in zeolite pores, causing an anisotropy of molecular rotational mobility. Nevertheless, on the present moment a clear correlation of the FID shape with the sizes or structure of molecules of entered liquid have not bee detected. Using techniques of selective separation of a NMR signal it was found, that the spin-latice relaxation for molecules inside and outside of a zeolite crystalline structure differ. This difference decreases with decreasing of liquid concentration. Such behaviour can be explained by availability of molecular exchange between inside and outside of zeolite crystals. The existence of exchange is confirmed by application of special Goldman-Shen type pulse sequences for n-hecsane/zeolite, n-octane/zeolite systems. At the same time for a decane/zeolite system indications of molecular exchange does not find out and, besides, the inversion of T1 relaxation times is observed with decreasing of liquid concentration: the T1 time of molecules outside of crystals becomes shorter T1 time of molecules located in zeolite channels while concentration of fluid decreases lower 10% . The obtained results are considered in the supposition of difference in degrees of an anisotropy of rotational molecular mobility inside and outside of zeolite channels. The translational molecular mobility data measured by NMR PFG method are used for analisys . This work was supported by the INTAS grant, project N 96-1177.

Contribution to the conference:
Improved sensitivity in Heteronuclear Gradient-based 1D experiments
T. Parella, J. Belloc, A. Virgili, and F. Sanchez-Ferrando
The incorporation of coherence selection by pulsed field gradients in 1D inverse experiments usually suffers of sensitivity losses by a factor of 2. Such defficiencies can be overcomed incorporating the so-called PEP methodology in such experiments. Thus, the same signal-to-noise ratios are obtained as the phase-cycled counterparts. In addition, the improved spectral quality obtained and the better performance are two important factors to be considered when using these experiments. Examples will be given for selective and non-selective HMQC, HSQC and INEPT experiments

Contribution to the conference:
Selective Gradient-based 1D experiments using Heteronuclear Cross-polarization
T. Parella, J. Belloc, A. Virgili, and F. Sanchez-Ferrando
New pulse sequences and practical details on new selective 1D experiments based on heteronuclear cross-polarixation will be given. The use of such approaches to measure accurate long-range proton-carbon coupling constants from highly desired in-phase multiplets will be illustrated.

Contribution to the conference:
Unambiguos unfolding of 2D-INADEQUATE-type spectra with deliberate folding in F1.
Henrik Pedersen
Deliberate folding to half size in F1 is a well recognized technique for improving S/N in the very insensitive, but highly informative carbon-13 detected 2D-INADEQUATE experiment. However the folded matrix gives less easy interpretation of the spectral data. Based on the geometry of the cross peak distribution, a method for unambiguous unfolding of 2D-INADEQUATE-type spectra is described. The unfolding may be executed within a proper NMR software package, and this is examplified by an AU program for execution within Brukers XWINNMR software. Also 2D proton-proton double quantum spectra can be unfolded by this procedure. For the 2D-INADEQUATE experiment the method is illustrated by 2D spectra of menthol and the selective seretonin reuptake inhibitor citalopram, and for the 2D proton-proton double quantum experiment the method is illustrated by 2D spectra of 2 mM sucrose in H2O/D2O.

Contribution to the conference:
Dynamics of Water in Glass Pores at Sub-zero Temperatures
Changho Choi, James Benson, J. A. Chris Delaney, and M. M. Pintar
In the simplest model of pore water a thin layer of water is thought to be attached to the glass surface while the pore core water is bulk-like. These two states of water are shared by all water molecules due to fast molecular exchange between core and surface states. For this reason the pore water relaxes with one (average) NMR relaxation time. On the surface, the water reorients around its surface bond while in the pore core the water undergoes fast rotational diffusion. With much slower exchange jumps, water changes its surface state to bulk state. This model, which has been tested many times, agrees with the experiments qualitatively. We have extended the room temperature NMR research employing the above model to the temperature range below the freezing point of water and will report on hydration/dehydration hysteresis of water configuration in model pores and on water dynamics in the so called "non-freezable state". References: R. Holly, H. Peemoeller, C. Choi, and M. M. Pintar, J. Chem. Phys., 108(10), March 8 issue, 1998. J. Dolinsek, J. Bharatam, M. B. Dusseault, and M. M. Pintar, to appear in Phys. Rev. B.

Contribution to the conference:
Multinuclear Magnetic Resonance Study of the Coordination of Aluminium (III) with Citric Acid in Aqueous Solution
Ana Isabel Rodrigues, Teresa Nunes and Geoffrey Hawkes
(+)-Tartaric acid is usually added, in small amounts, to improve the manipulation of glass-ionomer dental cement pastes by modifying the setting rate. The role of this acid was shown to suppress the early formation of calcium polyacrylate and to enhance the aluminium polyacrylate formation (1). More recently, the study of the coordination of Al(III) with (+)-tartaric acid contributed for the understanding of these systems (2). Aiming to investigate the potencial replacement of (+)-tartaric acid by citric acid in dental cement pastes, the coordination of Al(III)with citric acid in aqueous solutions at various pH values, was studied using 1-H, 13-C and 27-Al n.m.r. techniques. In particular, high field 27-Al n.m.r. allowed the identification of 1:1, 1:2 and 1:3 complexes of Al(III) with citric acid. The binding of citrate to aluminium takes place through the hydroxyl and carbonyl groups, resulting in the formation of six- and seven-membered chelates rings. References (1) S.Crisp, B.G.Lewis and A.D.Wilson, J.of Dental Res., 55, 1032 (1976) (2) F.R.Venema, J.A.Peters and H. van Bekkum, Inorg. Chem. Acta, 191, 261 (1992) Acknowledgements The authors acknowledge a collaborative research grant under the NATO Scientific Exchange Programmes (CRG970270).

Contribution to the conference:
29-Si NMR Tagging with TMS or TBDMS group
Schraml J., Blechta V., Kvicalova M.
Potentals of 29-Si NMR tagging based on TMS (trimethylsilyl) and TBDMS (tertbutydimethylsilyl) groups will be compared and examples presented.

Contribution to the conference:
H-1 and C-13 NMR Spectroscopic Evidence of the Protonation of Imino and Azo Group in Aromatic Compounds
Vilko Smrecki, Zlatko Meic
In this study trans-N-benzylideneaniline, trans-N-salicylideneaniline and trans-azobenzene were chosen as model molecules for aromatic compounds containing the imino and azo group, respectively. Protonation of studied compounds was made using trifluoroacetic acid (CF3COOH), which was a proper choice, because it could also serve as a solvent. Deuteration using CF3COOD was made to confirm the assignment. Spectroscopic evidence from H-1 and C-13 NMR spectra for the nitrogen protonation in the imino and azo group of model molecules will be shown. Conformational changes due to protonation will be discussed. Differencies between hydrogen-bonded and non-hydrogen-bonded systems will be considered.

Contribution to the conference:
Solution State Multinuclear Magnetic Resonance of Cobalt Complexes and Clusters. Study of Relaxation Mechanisms Leading to „"Self-Decoupling”" of 31P Nucleus from the Carbon Atoms in PPh1-3 Ligands.
G.Szalontai, S.Vastag and L.Markó
During the past years we have studied numerous organometallic compounds containing tri- or diphenylphosphines as ligands. In many of them the P atoms are chemically equivalents, therefore the valuable information offered by the relevant two- and three-bond P... P scalar couplings is not readily available. Furthermore occasionally we observed complete or partial "washing out" of the P...C scalar couplings from the 13C spectra of these compounds. Since breaking of the relevant P-C bonds was out of question, other possibilities, such as the „self-decoupling” phenomena, had to be looked for. If observed, P ... C scalar couplings to the carbon atoms of the phenyl group are sensitive reporters of the ligand co-ordination to metals, the numbers of phosphine ligands and also of the relaxation rate of the P atoms. We report here on the 13C and 31P spectra of mononuclear Co(I) and Cu(I) complexes and trinuclear Co carbonyl clusters (some of them of known geometry) in order to draw attention to the information available under certain conditions from these spectra. While in solutions of planar or tetrahedral mononuclear complexes the P...C and P...P scalar couplings are often lost due to the fast T2 relaxation of the phosphorus atoms, in tetrahedral clusters sometimes this information is available from the simulation of the resulting AXX’ spin systems. Examples will be given for both situations. The dependence of P...P couplings on molecular structure will also be demonstrated. In case of a large one-bond metal-phosphorus coupling constant (such as 1J59Co-31P which are in the range of 450-600 Hz as obtained from solid state spectra) contribution of the scalar coupling relaxation mechanism of the second kind can be significant. Depending, however, on the ligand-complex equilibria of these molecules in solution the paramagnetic contribution of the Co(II) or Cu(II) ions, almost ever present as impurities, often prevails. Effects of such paramagnetic species were thoroughly studied. T1 and T2 measurement of the P nuclei in these molecules confirmed significant contribution of the scalar coupling mechanism to the line widths, at the same time no evidence for the effect of the quadrupolar-CSA interference term has been observed.

Contribution to the conference:
NMR study of "through space" 19F-13C couplings
P. Szczecinski

Contribution to the conference:
Studies by 1^H NMR and fluorescence of a complex of wheat non-specific lipid transfer protein with its natural ligand
Tassin S., Marion D., Vovelle F. and Sodano P.

Contribution to the conference:
NMR studies of self-diffusion in zeolite NaX
Uryadov A.V. Skirda V.D.
NMR studies of self-diffusion in zeolite NaX The self-diffusion of decane in zeolite NaX was measured by pulsed magnetic field gradient NMR method. It was carried out using homebuild NMR spectrometer at proton resonance frequency 60MHz. The maximum magnitude of the gradient pulse was more than 100T/m. The quantitative characteristics of self-diffusion was the effective self-diffusion coefficient (D) which was extracted from the initial slope of spin echo attenuation. The diffusion observation time was varied in the range 2-300 ms. We obtained the anomalous time dependencies of effective self-diffusion coefficient for the range of temperatures ( 303-393K ). For temperature 333K the time dependence of effective self-diffusion coefficient can be determined by the following equation: D ~ t^-n , where t is the observation time of diffusion, n=0.5...0.7. Such diffusional behavior of decane is similar to the single-file diffusion [1] . However we would like to note that the single-file diffusion was observed in zeolites with one dimensional channels but the NaX is not the sample of such type zeolite. In the report we present the detail discussion of these results. REFERENCES 1. K.Hahn, J.Kaerger, and V.Kukla, Phisycal Review. Letters, 2762, 76 (1996). This work was supported by INTAS N 96-1177

Contribution to the conference:
THE REGISTRATION OF MOLECULAR EXCHANGE PROCESSES FOR LIQUID SELF-DIFFUSION IN TRACK MEMBRANES
E.N. Vasina, V.D. Skirda
The translational diffusion of water molecules in the system of water and track membrane, was investigated with using NMR PFG. The system was prepared as a package of the parallel set membrane films, thus the periodic structure was formed. The investigations were performed on the H1 64 MHz resonance frequency with using the stimulated spin echo pulse sequence. The maximum of the pulse field gradient amplitude was about 200 T/m. At large diffusion times, when the gradient vector was parallel to the direction of the system periodicity, the «diffraction» peaks on diffusion attenuations were observed. The magnitude of the pore spacing (the period of the system) corresponded to the sum of membrane film thickness and the average distance between neighbour membrane films, that was evaluated from the small (relatively) diffusion time data. The passage time of the pore spacing was formed as a time for the passage through single pore and the diffusion time for the passage between neigbour membrane films (from the pore of one membrane film to the pore of the neighbour membrane film). From experimental data at small diffusion times, the average lifetime in pores and the average lifetime between membrane films were estimated. From the data of the molecular exchange (lifetime data) the time of appearance of the «diffraction» peaks was evaluated.

Contribution to the conference:
Application of LC-NMR to Natural Products

Contribution to the conference:
NMR AND MOLECULAR DYNAMICS STUDIES OF THE BINDING AND STRUCTURE OF ADRENOCORTICOTROPIN AND SUBSTANCE P PEPTIDES IN MEMBRANE MIMICS
TUCK C. WONG, Troy Wymore and Xinfeng Gao
NMR AND MOLECULAR DYNAMICS STUDIES OF THE BINDING AND STRUCTURE OF ADRENOCORTICOTROPIN AND SUBSTANCE P PEPTIDES IN MEMBRANE MIMICS. Tuck C. Wong, Troy Wymore and Xinfeng Gao, Department of Chemistry, University of Missouri, Columbia, Missouri 65211 Two series of biologically active peptides, adrenocorticotropin hormones (ACTH) and Substance P (SP) peptides, in sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles have been studied by two-dimensional NMR, pulsed-field gradient diffusion techniques, and by molecular dynamics (MD) simulations involving explicit micelle-peptide complexes. The partition coefficients of these peptides in the micelles (as determined from diffusion measurements) and the secondary structures of the peptides upon binding to the micelles have been determined and compared with the results when these peptides are partitioned in lipid bilayers. Major differences between the binding patterns of the peptides with micelles and with bilayers were found. PFG diffusion measurements were also made on the same peptides in POPC vesicles. The mode of binding of these peptides to the micelles was studied by transverse relaxation rates of the protons in individual segments, by intermolecular NOE between the peptides and the micelles, and by MD simulations. In addition, the thermodynamic functions (enthalpy and entropy) of partitioning of these peptides in DPC micelles were determined by temperature dependent measurement of the partition.

New Methods/Instrumentation/Hardware

Contribution to the conference:
The effect of rf power and spinning speed on MQMAS NMR
J.P.Amoureux ; M.Pruski ; D.Lang ; C.Fernandez
It is known that the patterns of spinning sidebands observed in the multiple-quantum dimension of MQMAS spectra are often significantly wider than expected from the anisotropies of relevant interactions. It has been recently shown by others that these sidebands are generated due to the rotor-driven reorientations that the quadrupole tensors of the crystallites undergo during the evolution period between the multiple- and single-quantum conversion processes. We present an experimental and theoretical study of the effects of the spinning speed and rf field strength on the development of these sideband patterns. The theoretical analysis relies upon numerical simulations and includes propagation of the density matrix during the entire MQMAS experiment. The possibility of additional rotational encoding during the rf pulses is discussed. Both the theoretical and experimental results show the benefits of using the highest available spinning speed and rf field.

Contribution to the conference:
Local-order and connectivities analyzed by MQMAS methods
Amoureux,J-P ; Fernandez,C ; Pruski,M ; Delevoye,L
MQMAS is a recent very powerful two-dimensional NMR method for high-resolution analyses of quadrupolar nuclei in solids. Up to now,its developments have been aimed increasing its efficiency and resolution by introducing : the z-filtering 3-pulse sequence, the t1 rotor synchronization, the multiple-quantum decoupling... Three years after its discovery, MQMAS can presently be regarded as a "routine" experimental method for characterization of semi-integer quadrupolar nuclei, as long as the single-nucleus aspect is concerned. One can easily obtain for these nuclei: the number of different species, their relative concentrations, their actual chemical shifts and their quadrupole interactions. Moreover, when samples are not correctly crystallized, their surrounding distributions can be quantified. However, connectivities with other nuclei are not directly accessed with MQMAS. The first attempts to analyze the inter-nuclei distances between spins 1/2 and quadrupolar spins n/2, use the dipolar interaction as local "tool". This interaction can be introduced qualitatively through cross-polarization(CP-MQMAS or HETCOR-MQMAS) or quantitatively through methods such as REDOR-MQMAS or REAPDOR-MQMAS.

Contribution to the conference:
Suppression of Strong Coupling Artifacts in Hetero-Correlated Spectra
Katalin E. Kövér and Gyula Batta
Misinterpretation of strong-coupling effects (e.g. as intermolecular dipolar interaction) is of potential danger, and can lead to false structural conclusion. Therefore a general strategy is presented for suppression of strong-coupling artifacts (1-4) in heteronuclear correlation experiments. Properly designed X/1H-filters inserted in the preparation period of the pulse sequence (X-detection) or prior to aquisition (1H-detection) eliminate the undesired signals of strong-coupling origin. The proposed method is applicable both for scalar and dipolar heteronuclear correlated experiments. In HOESY, efficient suppression is achieved irrespective of the motional properties of the molecule in study, and both direct and long-range HOE-s pass the filter. This research was supported by Hungarian National Funds from OTKA T-014982 and OTKA D-23749. (1) P. H. Bolton, J. Magn. Reson. 51, 134 (1983). (2) G. A. Morris and K. J. Smith, J. Magn. Reson. 65, 506 (1985). (3) K. E. Kövér and Gy. Batta, J. Magn. Reson. 74, 397 (1987). (4) G. Esposito, F. Fogolari, H. Molinari, M. Pegna and L. Zetta, J. Magn. Reson. Ser. B101, 240 (1993).

Contribution to the conference:
DEPTQ: Distorsionless Enhancement by Polarization Transfer including the detection of Quaternary nuclei
R. Burger and P. Bigler
Multiplicity dependent 1D 13C-experiments such as DEPT, INEPT or APT belong nowadays to the most common and popular tools for the elucidation and characterization of moclecular structures on a routine level. In contrast to the less sensitive APT sequence DEPT and INEPT yield exclusively the signals of proton bearing carbons CHn, whereas the signals of quaternery carbons Cq are suppressed. As a consequence and to complete 13C data the latter two pulse sequences are usually combined with a simple one pulse 13C experiment. To avoid this additional experiment and to save spectrometer time the PENDANT pulse sequence has recently been proposed. In contrast to DEPT or INEPT the signals of all carbon types - including Cq - may be obtained together with the corresponding multiplicity information in one single experiment. Since it is based on the INEPT experiment the PENDANT pulse sequence is highly sensitive to deviations of pulse angles and delays from optimal values and may cause sporadic but severe problems as with olefinic methylene groups. Furthermore severe line distorsions are obtained for 1H-coupled PENDANT spectra. Most important however, this pronounced dependence on experimental parameters makes PENDANT especially less suitable for the generation of multiplicity selective subspectra (spectral editing), a procedure most successfully used in automatic spectral analysis. The aim of our efforts was to develop a pulse sequence based on DEPT yielding the signals of all carbon multiplicities including the signals of quaternary carbons and encompassing all of its advantages such as insensitiveness to experimental parameters and suitabilty for spectral editing. The correspondingly modified pulse sequence (DEPTQ) should be compared critically with PENDANT and the basic DEPT sequence.

Contribution to the conference:
The NMR-MOUSE: Pulse Sequences and Applications
B. Blümich, G. Zimmer, A. Guthausen, P. Blümler
The NMR-MOUSE is a realization of single-sided NMR, where the polarizing magnetic field B0 and the rf magnetic field B1 are applied from one side to the object. Such a device can be constructed from permanent magnets and a surface coil. The MOUSE can be used as a probe to any pulsed NMR spectrometer, which is capable to operate in the frequency region of about 20 MHz. If connectd to a PC-based spectrometers the equipment becomes mobile and can be transported to arbitrarily large objects. Because the probe resembles a computer mouse in size and function, and because surface-near volume elements are accessible by this technique, the acronym MOUSE is interpreted as MObile Universal Surface Explorer. Essentially most NMR techniques can be adapted for operation with the MOUSE, but subtle pecularities arise, because both, B0 and B1 are inhomogeneous. The flip angles are illdefined. But in the strong B0 gradient of about 10 T/m the sensitive volume is so small, that B1 is approximately homogeneous and the amplitude dependence of the flip angle is a sinusoidal function in good approximation. The sensitive volume is defined by the B0 and B1 gradients and the mean values of B0 and B1. By changing the rf frequency the sensitive volume is displaced from the surface of the MOUSE. The signal strength depends on the size of the sensitive volume as well as on the orientation of the laboratory frame in each voxel. Different types of relaxation measurements can readily be performed including relaxation measurements under average Hamiltonians invoked by multi-pulse excitation. But spin-lock and turn-on effects effects can arise, which are particular to inhomogeneous fields. Although the B0 field gradient varies within the sensitive volume, constants of translational selfdiffusion can be measured with good accuracy. Because B0 is already inhomogeneous, measurement can be performed in the presence of ferromagnetic components. For example, polymer coatings can be investigated on iron sheets. For optimum signal-to-noise ratios measurements can be performed in dynamic equilibrium with the excitation, similar to NMR by steady-state free precession. Several NMR parameters have been explored for measurements by the NMR-MOUSE, and their correlations with material properties have been investigated. In soft matter like rubber and tendon, temperature, macroscopic molecular orientation, stress and strain, and cross-link density can be probed. Medical applications are envisioned in the characterization of malignant skin, and the assessment of muscle and tendon by measuring the relaxation anisotropy, which is inaccessible in conventional medical imagers. References: 1. G. A. Matzkanin, A Review of Nondestructive Characterization of Composites Using NMR in: P, Höller, V. Hauck, C. O. Rund, R. E. Green (eds.) Nondestructive Characterization of Materials, p. 655, Springer, Berlin, 1989. 2. R. L. Kleinberg, NMR Well Logging in: D. M. Grant, R. K. Harris (eds.) Encyclopedia of NMR, 8, p. 4960, Wiley, New York, 1996. 3. G. Eidmann, R. Savelsberg, P. Blümler, B. Blümich, The NMR MOUSE, a Mobile Universal Surface Explorer, J. Magn. Reson. A 122 (1996) 104. 4. G. Zimmer, A. Guthausen, U. Schmitz, K. Saito, Weathering Investigation of PVC Coatings on Iron Sheets by the NMR MOUSE, Adv. Materials 12 (1997) 987. 5. A. Guthausen, G. Simmer, P. Blümler, B. Blümich, Analysis of Polymer Materials by Surface NMR via the NMR MOUSE, J. Magn. Reson. 130 (1998) 1.

Contribution to the conference:
Phase-Independent Spin-State Orthogonal Excitation. An Alternative to S3E
J.Santoro, M.Bruix and M. Rico
Phase-Independent Spin-State Ortogonal Excitation (PSOE) An Alternative to S3E J.Santoro, M.Bruix and M. Rico Instituto de Estructura de la Materia, CSIC Serrano 119, 28006 Madrid, Spain Coupling constant measurements should play an important role in the determination of protein structures (Bax et al. 1994). Despite the arsenal of experimental techniques available, it is still rare to see protein coupling constants reported in the literature, indicating the existance of experimental difficulties in their determination. Recently, a very promising new technique has been introduced (Meissner et al. 1997). This technique imposes a restriction in the preparation sequence to allow editing in two seperate subspectra, one corresponding to the spin of the attached nucleus being in the a state and one in the b state. In our hands, however, this new pulse sequence element, dubbed spin-state-selective excitation (S3E) by the authors, does not produce the expected results. An investigation of this misfunction revealed that the offset dependence of the phase of the magnetization after the first ex- citation pulse, that should be exactly pi/4 for the proper functioning of S3E, was the origin of our problem. To overcome this difficulty, we have designed an alterative to S3E that achieves the same end independently of the excitation phase. This derived sequence, Phase-Independent Spin-State Ortogonal Excitation (PSOE), should allow a more flexible use of the selective excitation module to measure coupling constants. References -A. Bax, G.W. Vuister, S. Grzesiek, F. Delaglio, A. C. Wang, R. Tschudin and G. Zhu, Methods Enzymol. 239, 79-105 (1994) -A. Meissner, J. O. Duus and O. W. Sorensen, J. Magn. Reson. 128, 92-97 (1997).

Contribution to the conference:
Litz RF Coils - User-Friendly Double-Resonance Coils for MRI
F. David Doty, George Entzminger Jr., Y. Andy Yang
A new class of NMR rf volume coils has been developed that permits improved B1 homogeneity, tuning range, tuning stability, and sensitivity compared to birdcages under a wide range of practical conditions, especially for microscopy and wrap-around flexible applications. They are denoted litz coils, as their flux transparency and current distribution is obtained from woven foil patterns with insulated crossovers. Contrary to design criteria in phased arrays, the parallel routes in litz coils require high coupling coefficients to achieve an optimal current distribution that is independent to first order of tuning, balancing, and matching adjustments and is compatible with multiple capacitive segmentation. The motivation for exploring novel current topologies arose primarily from work in vertical bore magnets (7 T to 14 T) where tight space constraints make it extremely difficult to obtain high B1 homogeneity for asymmetric loads in birdcages, as the high-order rf homogeneity errors can only be addressed by a complex tuning procedure and the parasitics cannot be precisely controlled. Numerical modeling and experimental comparisons with balanced-high-pass birdcages are presented for a variety of birdcage and litz coils with frequency-diameter products from 5 to 30 MHz-m for frequencies at least to 600 MHz. Both linear and quadrature versions are presented for both single and double-resonance.

Contribution to the conference:
Unification of Mossbauer and NMR Spectroscopies
A.Ya. Dzyublik, R.A.Manapov, V.Yu.Spivak, F.G.Vagizov
The Double \gamma-Nuclear Magnetic Resonance is investigated both theoretically and experimentally. General equations for absorption and scattering cross sections of Mossbauer radiation are derived when the NMR transition are generated between sublevels of the Mossbauer nucleus both in the ground and excited states. We observed splitting of absorption lines in hard ferromagnet subject to radio-frequency (rf) magnetic field. Besides, we investigated soft ferromagnets, whose magnetization reverses under the influence of an rf magnetic field. The collapse of the hyperfine structure of the scattering Mossbauer spectrum is predicted analogous to familiar phenomenon in absorption geometry. Furthermore, the splitting of Mossbauer sidebands and central peak is predicted, when both radio-frequency and static magnetic field act on the crystal. In such case there are asymmetric reversals of the magnetization and magnetic field at the nucleus. We observed this effect experimentally as well. The Pfeiffer's spectra in Permalloy subject to rf field were explained, when we took into account two mechanisms of sidebands formation: due to exchange by rf photons and due to magnetistrictive vibrations. We studied also transient phenomena following abrupt reversal of the magnetic field. Moreover, interesting interference effects were found, when absorber placed in rf field vibrates as a whole.

Contribution to the conference:
Suppression of Strong Coupling Artifacts in Hetero-Correlated Spectra
Katalin E. Kövér and Gyula Batta
Misinterpretation of strong-coupling effects (e.g. as intermolecular dipolar interaction) is of potential danger, and can lead to false structural conclusions. Therefore a general strategy is presented for suppression of strong-coupling artifacts (1-4) in heteronuclear correlation experiments. Properly designed X/1H-filters inserted in the preparation period of the pulse sequence (X-detection) or prior to aquisition (1H-detection) eliminate the undesired signals of strong-coupling origin. The proposed method is applicable both for scalar and dipolar heteronuclear correlated experiments. In HOESY, efficient suppression is achieved irrespective of the motional properties of the molecule in study, and both direct and long-range HOE-s pass the filter. This research was supported by Hungarian National Funds from OTKA T-014982 and OTKA D-23749. (1) P. H. Bolton, J. Magn. Reson. 51, 134 (1983). (2) G. A. Morris and K. J. Smith, J. Magn. Reson. 65, 506 (1985). (3) K. E. Kövér and Gy. Batta, J. Magn. Reson. 74, 397 (1987). (4) G. Esposito, F. Fogolari, H. Molinari, M. Pegna and L. Zetta, J. Magn. Reson. Ser. B 101, 240 (1993).

Contribution to the conference:
Measurement of Heteronuclear Dipolar Interactions Between Quadrupolar and Spin-1/2 Nuclei in Solids by Multiple-Quantum REDOR NMR
Fernandez,C ; Pruski,M ; Amoureux,J-P ; Lang,D
A new technique is presented that adds to the group of solid state NMR methods which utilize heteronuclear recoupling for measurements of the dipolar interactions in rotating solids. The experiment combines the capabilities of multiple-quantum magic angle spinning (MQMAS) with the rotational-echo double resonance (REDOR). We present a detailed description of the experiment and demonstrate its usefulness in a study of 19F-27Al spin pairs in chabazite-like AlPO4 aluminophosphate. The advantage of the MQ-REDOR technique is that it provides direct inference of the connectivities between spin-1/2 (e.g. 1H, 19F, 31P) and quadrupolar (e.g. 11B, 17O, 23Na, 27Al) nuclei in solids under high resolution conditions. In addition, we have also shown that analysis of the MQ-REDOR data yielded accurate rAl-F distances for the three distinct aluminum sites in fluorinated AlPO4.

Contribution to the conference:
Proton donator properties of zeolites studied by laser supported in situ 1H MAS NMR
D. Freude and T. Mildner
In situ 1H MAS NMR spectroscopy of the proton transfer between bridging hydroxyl groups and benzene molecules yields temperature dependent exchange rates over more than five orders of magnitude. H-D exchange experiments were performed by laser heating and conventional heating up to 600 K. The first 2D 1H NOESY MAS NMR spectrum, which has been measured at 520 K, shows the chemical exchange between benzene and bridging hydroxyl groups. The rates of the proton transfer in the temperature region of 350-600 K increase by a factor of ten going from zeolite HY-85 (Si/Al = 2.4) to HY-92 (Si/Al = 3.1), respectively. The weak dealumination, which causes only a small change of the chemical shift of the bridging hydroxyl groups, has a strong influence to the dynamic measure of Brønsted acidity, which is given by the rate of the proton transfer.

Contribution to the conference:
Structure Refinement of Rhodniin using Cross Correlated Relaxation Rates
Alexander Diener, Bernd Reif, Mirko Hennig, Marcus Maurer and Christian Griesinger
Information about the projection angle between two atom-atom vectors has so far been restricted to the measurement of torsional angles via scalar 3J couplings. They relate two vectors that are separated by a single bond. A new method relying on cross correlated relaxation makes it possible to measure projection angles between atom-atom vectors separated by arbitrarily many bonds [1]. The dipole(15N-1H) dipole(13C_alpha-1H_alpha) cross correlated relaxation rates are used to refine the backbone angle psi in the anticoagulant protein Rhodniin [2]. As the dependence of the relaxation rate on the psi-angle can be approximated by a Karplus-like function, the restraints for the relaxation rates could be implemented into X-PLOR [3] using the coupling energy term. In a 100 ps restrained MD protocol the energy constant for the relaxation rates is increased exponentially with time. For some psi-angles, convergence to multiple values is observed. Free MD simulations suggest that this could reflect an internal mobility of these regions. The complementary CSA(15N) dipole(13C_alpha-H_alpha) cross correlated relaxation rates could help to overcome the ambiguities in the 3cos2theta-1 dependence of the dipole dipole relaxation rates. Literature [1] B. Reif, M. Hennig, C. Griesinger, Science (1997) 276, 1230-1233. [2] T. Friedrich et al., J. Biol. Chem. (1993) 286, 16216-16222. [3] A.T. Bruenger, X-PLOR Version 3.1, Yale University Press, New Haven 1992.

Contribution to the conference:
NMR Measurements with DC SQUID Magnetometer
Z. Jaglicic, J. Pirnat, J. Luznik, V. Jazbinsek, Z. Trontelj
SQUID (Superconducting QUantum Interference Device) as an ultrasensitive magnetic flux detector offers a possibility to directly detect the longitudinal magnetization in nuclear magnetic resonance experiments. The sensitivity of the SQUID, up to the maximum frequency which can be even in MHz region, is frequency independent. A comparation of a signal to noise (S/N) ratio for standard NMR detection with the S/N ratio for SQUID NMR detection shows that the application of SQUID szstem for NMR detection is reasonable in the case of low Zeeman magnetic fields and broad resonance lines. A DC SQUID with first order gradiometer configuration of pick-up coil has been used to detect longitudinal magnetization of samples with long spin-lattice relaxation times. The sample is placed inside one of gradiometer coils of the flux transformer. Zeeman magnetic field is generated by permanent cobalt-samarium disc magnets (magnetic field up to 200 mT) or magnetic flux trapped inside a superconducting lead cylinder (magnetic field up to 15 mT). RF magnetic field is generated by a small copper coil wound tight around the sample. Pulse and CW measurements were done on proton and fluor nuclei. Comparison of line shapes reconstructed from both techniques shows that the pulse technique gives more reliable line shape while the line shape obtained from the CW experiment can be distorted due to slow spin-lattice relaxation.

Contribution to the conference:
Collective effects in high resolution liquid NMR. Radiation dampingand dipôlar field.
J. Jeener and P. Broekaert
A summary will be given about recent developments: control and use ofradiation damping as a tool, dipolar field effects in solvents withspectra consisting of many lines, ...

Contribution to the conference:
Matrix RT shim system for NMR and MRI
P. Konzbul and K. Sveda

Contribution to the conference:
Overhauser Techniques for Imaging Free Radicals
D.J. Lurie, M.A. Foster, J.M.S. Hutchison
There is increasing interest in the role of free radicals in human disease, and many groups are working on low-frequency EPR in order to study free radicals in small animals. Conventional EPR imaging can produce high-resolution images showing the distribution of narrow-line free radicals, but the spatial resolution is compromised by the broad lines typical of most nitroxides or spin-trapped radicals. EPR imaging has so-far been limited to samples of diamater ~5 cm (using RF EPR at ~300 MHz), because of RF penetration and gradient strength limitations. To counter these problems we have developed free radical imaging techniques based on the Overhauser effect. A conventional NMR image of the sample is obtained, and during the pulse sequence the free radical's EPR is irradiated. Under conditions of partial EPR saturation an enhancement of the NMR signal can occur, and regions of the sample containing free radical then appear with modified intensity. We call this method PEDRI, standing for Proton Electron Double Resonance Imaging. Since the spatial information is obtained by NMR, the EPR linewidth does not affect the resolution. Only modest, pulsed MRI gradients are necessary, potentially allowing large samples to be studied, however PEDRI is still restricted to small samples by limited RF penetration (~300 MHz at 10 mT). The situation can be improved by using Field-Cycled PEDRI (FC-PEDRI) in which the applied magnetic field B0 is switched during the pulse sequence. The EPR irradiation is carried out at low field (B0e, the "evolution" field), at low frequency, and the field is then increased to a higher value (B0d, the "detection" field), when the NMR detection and imaging gradients are applied. We have constructed a whole-body sized FC-PEDRI imager. It uses the field- compensation method of field cycling, with a permanent, 60 mT, vertical field primary magnet and a resistive secondary magnet. The field can be switched from zero to 60 mT (or vice versa) in 40 ms. We have used this instrument to image exogenous nitroxide free radicals in living, anaesthetised rabbits (~2 kg), using EPR irradiation at 51 MHz at B0e = 3 mT, and NMR detection at 2.5 MHz at B0d = 60 mT. We are working to improve the sensitivity of the instrument, and have had some success with detecting complexed nitric oxide in in-vitro studies.

Contribution to the conference:
Aspects of New Coil Designs for MRI
P. Mansfield
The principle of active magnetic screening of gradient coils introduced by Mansfield and Chapman in 1986 was rapidly assimilated by the major manufacturers of MRI equipment and is now standard on most MRI scanners. This principle allows rapid switching of field gradient within the close confines of a superconductive magnet without inducing extraneous, unwanted currents in the surrounding magnet structure. However, in this work and also in all gradient coil structure constructed to date, an important aspect of gradient design has been so far overlooked. This is the acoustic noise, which accompanies high rapidly switched gradient coils. In some experimental gradient structures we have measured noise output in excess of 110 dB. At this levels acoustic noise can become dangerous unless ear defenders and earplugs are used to protect patients and scanner personnel. However, there are classes of patient where it is difficult if not impossible to use ear defenders, for example very young children and babies, and especially foetuses in utero. Other classes of subject include animal studies. Some manufacturers have been concerned with noise levels and have attempted to quieten their products by including sound absorbing materials. Many of these approaches are really treating the symptoms rather than going to the root cause of the problem. At Nottingham over the last five years we have been attempting to tackle the acoustic problem in a more fundamental way. This talk will be review of an approach we tried, together with an update on the current theory of sound propagation in simplified model system. References 1. Active Magnetic Screening of Gradient Coils in NMR Imaging. P. Mansfield and B. Chapman, J. Mag. Res. 66, 573-576 (1986). 2. Sound generation in Gradient Coil Structures for MRI. P. Mansfield. P.M. Glover and J. Beaumont, Mag. Res. Med. 39 (1998), in press.

Contribution to the conference:
Spin-State-Selective NMR Techniques for Measurement of J Coupling Constants and Deuterium Isotope Effects in Proteins
Axel Meissner, Morten D. Sørensen, Thomas Schulte-Herbrüggen, Jacques Briand, and Ole W. Sørensen
We have recently introduced a number of Spin-State-Selective (S3) NMR techniques for measurement of J coupling constants in 13C- or 15N-labeled proteins. These techniques edit E.COSY-type multidimensional NMR spectra into two subspectra corresponding to the spin state of a passive spin relevant for measurement of J coupling constants in an E.COSY way. This editing effectively doubles the spectral resolution and also allows suppression of large one-bond couplings. This contribution reviews the S3 techniques and presents related novel multidimensional experiments for measurement of deuterium isotope effects on the chemical shifts of the nuclei in protein backbones. These ISP (Isotope Selective Polarization) experiments edit heteronuclear triple resonance spectra into two subspectra according to there being a deuterium or a proton attached to 15N for the pertinent correlations. References: J. Magn. Reson. 128, 92-97 (1997), J. Biomol. NMR 10, 89-94, 181-186 (1997).

Contribution to the conference:
Convection Compensation in Gradient Enhanced NMR Pulse Sequences
Norbert Müller and Alexej Jerschow
With the GOESY and GROESY pulse sequences1,2 the possibility of acquiring NOE and ROE difference spectra in a single scan thus entirely avoiding subtraction artifacts was introduced. However, a major disadvantage of these original pulse sequences is their sensitivity to translational motions in the sample (diffusion and convection). We show that convection in the sample caused by temperature gradients in the NMR probe assembly gives rise to sinc-function like modulations of NOE and ROE buildup curves. This not only reduces sensitivity but also prevents any reasonable quantification of NOE and ROE data acquired in this way. Computational correction for the effects of self diffusion is also not possible if convection affects the spectral amplitudes. Two approaches are possible to compensate for the effects of constant laminar convection flow. Their common principle was applied earlier in the field of NMR imaging and has been adapted recently for diffusion separated spectroscopy3: The zeroth and first moments of the effective gradients over the entire pulse sequence must vanish. This can be realized either by adjusting the amplitudes of the 3 gradient pulses in the original sequence to fulfill these conditions. This is the method of choice at short mixing times. Alternatively the mixing time can be split to provide for a double stimulated echo3, which yields better results at longer mixing times. The general principle of convection compensation is not restricted to the two applications presented here. It can readily be applied to most gradient enhanced pulse sequences suffering from convection artifacts.4 Naturally those sequences with long inter-gradient delays are the prime candidates for such an improvement.

Contribution to the conference:
Three Dimensional Excitation of Complex Shapes in NMR
Igor Sersa and Slobodan Macura
A goal of spectroscopic imaging techniques is to reveal spectral sample information as a function of spatial coordinates. Techniques that can enable this are usually very time consuming. For most diagnostic cases it is enough to access the spectrum from body part or organ under investigation. One way to do this is to excite nuclear magnetization only in selected region that matches investigated part of the object, then acquire the free induction decay signal and finally calculate corresponding spectrum. Here we propose an efficient way of spatially selective excitation in 3D which we named CARVE after Completely Arbitrary Regional Volume Excitation. CARVE excitation sequence is based on DANTE train of short RF pulses which are interleaved with gradient pulses. We showed theoretically and experimentally that for a given ideal excitation profile there exists a sequence of RF pulses with precisely determined amplitude and phase which provided that they are interleaved with gradient pulses with adequate magnitude and direction can excite the profile close to the ideal one.

Contribution to the conference:
SELECTIVE MULTIPLE-QUANTUM NMR SPECTROSCOPY: ZZ-MUSEX method inexchange NMR experiment
V.S. Tsygankov, Ju.E. Chernysh, B.S. Lukyanov
ZZ-exchange NMR spectroscopy in 1D and 2D dimensions has benn well -know as an up-to-date method for studying molecular dynamicprocesses. ZZ-exchange technique has recently found use in moleculardynamics as well to take opportunity of obtaining the specialinformation concerning coupled-spin system processes undergoing suchas chemical exchange and relaxation ones et cetera.

Contribution to the conference:
Generating and Exploiting Intermolecular Multiple-Quantum Coherences in Solution NMR and MRI
W. S. Warren
In the last few years we have discovered, much to our surprise, that in a wide variety of two-dimensional solution NMR experiments, samples with at least one concentrated component (such as proteins in water) can grossly violate the predictions of conventional NMR theory. Slightly modified conventional sequences, which should give absolutely no signal, instead give strong intermolecular cross-peaks and new transitions in the indirectly detected dimension. I will discuss the theoretical picture of these effects which has evolved over the last few years, which requires reexamination and rejection of the two most fundamental fifty-year-old assumptions of NMR theory, but now gives analytical solutions which even include the competition between intramolecular scalar and intermolecular dipolar couplings. I will also show experimentally that these effects lead to a new MRI method (with contrast enhancement based on exciting intermolecular zero-quantum coherences demonstrated in rat brains), and to a technique which should permit high resolution spectra at double today's highest resonance frequencies with existing magnets.

Solid State NMR

Contribution to the conference:
EXPERIMENTAL OPTIMIZATION OF QUADRUPOLAR ECHOES IN SOLID STATE NMR
Y.DUMAZY , J.HANNA , J.P.AMOUREUX , C.FERNANDEZ
In solid state NMR semi-integer spin nuclei (I > 1/2), which represent 70% of chemical elements, are subjected to the quadrupolar interaction. In the case of a strong quadrupolar interaction, only the central transition remains visible on a static powder spectrum. Even if this transition is broadened by the second order quadrupolar interaction, its lineshape exibits singularities which provide important information on the local surroundings of the studied nucleus. In such a case, the echo sequence is a first-class experiment to avoid the electronic dead-time of the probe and hence the resulting distortions. This poster deals with the optimization of the quadrupolar echo sequence and its experimental application on several compounds. The main purpose is to acquire the most efficient echo signal of the central transition with a minimum of distortions. Using an home-made powder spectrum simulation software package (PULSAR), taking into account all interactions and pulse effects during the entire sequence, we have determined the best experimental conditions for each semi-integer spin. The experimental static spectrum, calculated at the top of the echo is then compared to the simulated one obtained with a single perfect pulse. Finally, we show that optimal experimental conditions can be found, leading to a good determination of the characteristic interaction parameters for several compounds : 23Na, 87Rb, 63/65Cu, 59Co and 93Nb.

Contribution to the conference:
Deuterium-Carbon Correlation Experiments for the Assignment and the Measurement of Quadrupolar Couplings in Oriented Materials
Celine Auger, Anne Lesage, Stefano Caldarelli, Paul Hodgkinson, and Lyndon Emsley
The measurement of deuterium quadrupolar coupling is of particular interest to access local order parameters in liquid crystals, so as to permit the study of structure and dynamics. Although deuterium spectra are often relatively simple, consisting of a superposition of quadrupolar doublets, assignment in uniformly labelled materials is very difficult, due to the lack of chemical shift differences. To overcome this problem, we have developed a two-dimensional deuterium-carbon correlation experiment. The assignment of the deuterium spectrum is obtained from the known carbon spectrum. The experiment is based on polarization transfer from a deuterium atom to the attached carbon atom. The normal Hartmann-Hahn cross-polarization matching condition is dependent on the value of the quadrupolar coupling and is ineffective over the complete width of the deuterium spectrum. In order to render the process broadband we make use of variable amplitude cross-polarization, based on adiabatic sweeps of the RF developed for CP in spin-1/2 systems. We have extended the existing theory of spin-1 to spin-1/2 CP to the « intermediate » regime where the RF field amplitude is comparable to the quadrupolar coupling. DEuterium CORrelation (DECOR) spectra of a model nematic liquid crystal allow, for the first time, the resolution, assignment and measurement of each of the quadrupolar couplings in a uniformly deuterated molecule. Additionaly, the experiment allows the direct observation and measurement of deuterium-carbon dipolar couplings. If this information is not required, we show that refocusing the heteronuclear coupling leads to significant improvements in resolution and sensitivity. Short cross-polarization times are demonstrated to yield an exclusively one-bond CP, while longer cross-polarization times reveal longer range transfers which help to resolve remaining ambiguities, and can potentially provide additional information about molecular structure and dynamics.

Contribution to the conference:
31P CP/MAS SOLID STATE AND MULTINUCLEAR ISOTROPIC NMR SPECTRA OF [Co(III)(Hdmg)2XPPh3] COMPOUNDS
Fioretta Asaro$, Roberto Gobetto#, Giorgio Pellizer$ and Giorgio Rosso$-$Università di Trieste- #Università di Torino
The 31P CP/MAS spectra of the organotriphenylphoshinecobaloximes [Co(III)(Hdmg)2XPPh3] (X= nPr(1), iPr(2), CH2Ph(3), Ph(4), Hdmg= monoanion of dimethylglyoxime) will be discussed. The spectra of (1), (3) and (4) show eight-peak patterns (59Co I= 7/2), from which the value of 1J(31P, 59Co) is readily obtained, whilst, owing to the self decoupling of the fast relaxing cobalt nucleus, the solution 31P spectra allow, at best, an indirect estimation of this coupling constant. The J values show appreciable differences in the three compounds, in line with the good sensitivity of the metal-phosphorus coupling constant towards the trans-influence of the X ligand. Furthermore, they show the same trend observed for the 1J(31P, 103Rh) in the analogous rhodium compounds, which are isostructural. As expected, the reduced constant 1K(P, Co) is lower than 1K(P, Rh), the ratio being about 0.45 : 1. The 31P CP/MAS spectrum of [Co(Hdmg)2CH2Ph(PPh3)] (3) evidences the presence of two molecules that give distinct octets (0.6 ppm apart) with the same J. The spectrum of [Co(Hdmg)2iPrPPh3] (2) is remarkable because the multiplet has collapsed. This can be attributed to the fast relaxation of the quadrupolar nucleus even in the solid, much alike the [Co(Hdmg)2ClPBu3] [1]. It is mentionworthy that thermal disorder has been evidenced for the isopropyl ligand in the x ray structure of (2) at room temperature [2]. In organocobaloximes 31P CP/MAS spectra the multiplet distortions are small and comparable in magnitude. Instead, in the halidecobaloximes (X= Br (5), I (6)) 31P CP/MAS spectra the multiplet is severely distorted by the much stroger quadrupolar coupling and broadened, probably because of magnetic coupling between phosphorus and halogen atoms (79Br, 81Br I= 3/2; 127I I= 5/2). In agreement with this, the 59Co linewidth in solution is very large for compounds (5) and (6). References [1] R. W. Schurko, R. E. Wasylishen and J. H. Nelson, J. Chem. Phys., 100 (1996) 8057. [2] L. Randaccio, N. Bresciani-Pahor, P. J. Toscano and L. G. Marzilli, J. Am. Chem. Soc., 102 (1980) 7372.

Contribution to the conference:
Field dependence studies in 59Co solid state NMR and estimation of quadrupole and chemical shielding tensor parameters
Ganapathy Subramanian; Dumazy Yves; Fernandez Christian; Amoureux Jean-Paul; Bailly Alain
59Co NMR is useful as a sensitive method for understanding the influence of the immediate environment of the cobalt atom. Recent improvement in NMR pulse methodology and instrumentation make the study of solid state 59Co NMR more accessible, especially in the context of the measurement of chemical shift anisotropy and quadrupolar coupling. When more than one cobalt site/environment is present and more than one interaction contribute to the spectral frequency and intensity, measurements at different magnetic field strengths are required for a satisfactory characterization of the cobalt environments. In this presentation, a run down of the methodology to record distortionless 59Co line shapes and their analysis thereof, using the simulation program QUASAR, will be presented. 59Co NMR experiments conducted at different magnetic field strengths (2.35T, 7.05T, 9.4T and 14.09T) on the inorganic complex K3Co(CN)6 and the low dimensional oxide LiCoO2 and their spectral simulations are illustrative of this approach in the estimation of accurate values of the quadrupole and chemical shielding tensor parameters.

Contribution to the conference:
Multinuclear solid state NMR investigations of structure and dynamic behavior of inorganic-organic hybrid catalysts
A. Baumann, F. Hoehn, E. Lindner, H. A. Mayer
Inorganic-organic hybrid catalysts have recently attracted much interest, since these materials combine the advantages of homogeneous and heterogeneous cataylsis. The materials consist of an inorganic carrier matrix obtained by the sol-gel process of silyl functionalized complexes, a reactive center consisting of an organometallic transition metall complex, which is connected via a spacer unit to the matrix. The inorganic-organic hybrid materials are excellent candidates for multinuclear NMR investigation, since a variety of NMR active nuclei (e. g. 1H, 13C, 31P, 29Si) are available. By selecting the appropiate nucleus one is able to get insight into different parts of the complex material: The inert carrier matrix is well characterized with 29Si NMR. 13C solid state NMR yields information about spacer unit and the ligand backbone. 31P NMR characterizes the reactive center, allowing direct comparison with analogous homogeneous catalysts. Catalytic activity is directly dependend on the dynamic behavior of these materials. Dynamic NMR investigations are applied to determine mobility, which is not only observed by determination of the cross polarization parameters (e. g. T_1rho; T_XH etc.), but also by application of spin diffusion experiments. The application of the 2D WISE (wideline separation) experiment correlates the structure directly with mobility, thus allowing a site dependend investigation of the mobility.

Contribution to the conference:
NMR in Multiple-q Modulated Incommensurate Systems
R. Blinc
In incommensurate (I) systems the periodicity of at least one the modulation waves cannot be expressed as a rational fraction of the periodicity of the underlying lattice. As a result of that the translational lattice periodicity is lost in spite of the existence of perfest long range order. Phase transitions leading to such I phases are described by order parameters with a minimum dimensionally two (n = 2), four (n = 4) or six (n = 6) resulting in systems with one (1-q), two (2-q) and three (3-q) incommensurate modulation waves. Nuclear magnetic resonance and nuclear quadrupole resonance are ideally suited to determine: a) the number of independent modulation waves, b) the relative phases of the modulation waves if they are correlated, c) the plane wave respectively soliton character of the modulation wave. The use of this technique is shown on the example of the 1-q system Rb2ZnCl4, the 2-q system biphenyl, the planar 3-q system AlPO4 and the non-planar 3-q system Ag3AsS3. The effects of the phase fluctuation excitations on the spin-lattice relaxation behaviour of these systems is also discussed.

Contribution to the conference:
Determination of Torsional Angles using Solid State NMR
Peter V. Bower, Manish Mehta, Gary P. Drobny
We show that the Double Quantum spectrum of the carbonyls on two adjacent amino acids has a significant dependence on the torsinal angles in a peptide or protein. The effect of adjacent NMR active nuclei has been investigated experi- mentally and through simulations. Good DQ efficiencies have been obtained with the DRAWS pulse sequence and the investigation has been supported by NMR distance measurements.

Contribution to the conference:
MAT Techniques Using Naturally-abundant Nuclei
Brouwer, E. B.; Harris, R. K.

Contribution to the conference:
REDOR Spectroscopy of doubly and singly labeled Amides
Gerd Buntkowsky*+,Ingolf Sack+, A. Goldbourt#, S.Vega#, H.-H. Limbach+
REDOR Spectroscopy of doubly and singly labeled Amides Gerd Buntkowsky*+,Ingolf Sack+, A. Goldbourt#, S.Vega# and H.-H. Limbach+ Institut für Organische Chemie der Freien Universität Berlin+, Takustrasse 3, D-14195 Berlin, Germany and Weizmann Institute#, Rehovot,Israel 15N- 13C, 15N- 2H and 13C- 2H rotational echo double resonance (REDOR) experiments on doubly labeled acetanilide, gluconamide and glycine and singly 15N respectively 2H labeled natural abundance 13C gluconamide and acetanilide have been performed. While the 15N- 13C REDOR experiments were performed in the usual way using simple p-pulses, for the X- 2H REDOR composite pulse sequences had to be employed, due to the insufficient 2H-B1 field strength in our commercial triple resonance probe. From the 13C- 15N experiments magnetic dipolar couplings in the range from 45Hz to 1220Hz have been elucidated and the corresponding CN distances varying from 4.1Å down to 1.35 Å have been determined. These dipolar couplings have been employed to elucidate the secondary structure of a crystalline modification of the gluconamide for which no X-ray structure exist. The resulting structure has been compared to X-ray data of a second crystalline modification of gluconamide and NMR data of the gel modification of gluconamide. The main structural differences are the bond length of the amide bond in the crystalline modification is increased as compared to the gel modification and that the gluconamide molecule has a sickle shaped form in this modification. For the evaluation of the X- 2H REDOR a special program has been written, which takes into account the influence of the finite RF power under MAS conditions by numerical solution of the density matrix equation. Using this program and a careful calibration of all NMR parameters it was feasible to evaluate the X- 2H REDOR from first principles. As an experimental example the molecular geometry of acetanilide has been studied by X- 2H REDOR.

Contribution to the conference:
Characterization of extra framework cations in ETS-10 and ETAS-10 molecular sieves by MQ-MAS and molecular modeling techniques
Delevoye Laurent; Ganapathy Subramanian; Fernandez Christian; Amoureux Jean-Paul
Pure and highly crystalline ETS-10 and ETAS-10 titano-silicate molecular sieves, devoid of the impurity ETS-4 phase, were synthesised and fully studied by MQ-MAS NMR. Triple quantum 23Na MAS experiments point to the presence of three distinct cation environments in ETS-10. Similar experiments in ETAS-10 additionally confirm that sodium environments are in tact in the aluminium substituted material, indicating thereby an isomorphic aluminium substitution in the ETS-10 lattice. The MQ-MAS results are further rationalised in terms of sodium ion locations as determined by cation modeling of the two ordered polymorphs, which are known to exist for the structure of ETS-10.

Contribution to the conference:
Dynamical properties of quasi-one-dimensional hydrogen-bonded crystals
O. Derzhko, T. Krokhmalskii

Contribution to the conference:
Quasicrystals studied by 2D exchange NMR
J. Dolinšek
Quasicrystals are solid materials having a new type of a perfect long range order without translational periodicity. Their symmetries (icosaedral, dodecagonal, decagonal, octagonal, pentagonal) involve symmetry elements such as fivefold rotation axis which are incompatible with the periodicity of a Bravais lattice. A consequence of nonperiodicity is that phonons and electrons can not propagate through the lattice so that quasicrystals-being alloys of pure metallic elements-exhibit almost isolating properties. The lack of translational periodicity implies that NMR spectra of quasicrystals are extremely broad and the absorption lines in many cases extend over several Tesla. Special measuring techniques have thus to be applied such as magnetic field sweeping and recording of the spin echo intensity as a function of the field strength. A subject of high interest has recently become the dynamics of the quasicrystalline structure. It was predicted that the dynamics of quasicrystals is determined by the phason motions, where the atoms diffuse relatively to each other in the vicinity of an imperfection in the quasicrystalline lattice. Here we report the first observation of the phason motion in an Al-Pd-Mn quasicrystal using the technique of 27Al 2D exchange NMR. With this method the motional time constants for the reorientation of the basic building element of the Al-Pd-Mn quasicrystalline structure-the pseudo Mackay icosaedron-have been determined in a large temperature interval. The time constants were found to be str ongly temperature dependent, thus showing that the  phason motions in Al-Pd-Mn system are thermally activated.

Contribution to the conference:
Solid-State NMR of Paramagnetic Materials: Hyperfine Interactions in a Sodalite-Supported Cubic Lattice of Unpaired Electrons
G.Engelhardt, O.Groeger, D.Markgraber, V.Srdanov, E.Lippmaa, I.Heinmaa
Submitted seperately

Contribution to the conference:
17O multiple-quantum and 1H MAS NMR studies of the zeolite ZSM5
C.Fernandez, D.Freude,D.Michel,F.Bauer,H.Ernst,U.T.Pingel,J.P.Amoureux
Oxygen 17 multiple-quantum magic-angle spinning nuclear magnetic resonance studies were performed, for the first time,in the high field of 17.6 Teslas. A good resolution between two oxygen signals due to SiOSi and SiOAl fragments could be obtained. From the residual linewidth of the SiOSi signal, it is concluded that a change of the bridging SiOSi angle by 13.4° gives rise to a change in the isotropic chemical shift of less than 5.7ppm.

Contribution to the conference:
Redor/Reapdor experiments on zeolites using high speed CP-MAS probehead
Ganapathy Subramanian; Delevoye Laurent; Fernandez Christian; Amoureux Jean-Paul
In zeolites both 1H and 29Si nuclei offer good detection sensitivity. Besides, being spin 1/2 nuclei, their spin manipulations by hard r.f pulses can be readily accomplished in heteronuclear dipolar recoupling experiments. From the experimental point of view also, a classical CP-MAS probehead would suffice for an easy implementation of the desired experiment and the high speed sample spinning (ca. 15 kHz) allows one to measure the REDOR/REAPDOR behavior at short dephasing times. This latter feature allows us to estimate shorter internuclear distances. We have carried out 29Si-1H REDOR and 1H-27Al REAPDOR experiments in the zeolites H-Y, H-mordenite and H-ZSM-12. Highly resolved proton resonances due to the bridging hydroxyl groups of the zeolite framework (3.9 to 5.6 ppm), terminal SiOH groups at crystal surface and crystal defects (1.8 ppm) and residual NH4 ions (7.0-7.5 ppm) allow us to measure the individual REDOR/REAPDOR behavior. In each case, the observed dephasing curves could be calculated by considering either an isolated pairwise interaction (Si(OH)Al) or a sum over N equivalent interactions (for the ammonium ions), using PULSAR.

Contribution to the conference:
Chemical bonding of strongly polarized ions on the NMR data
Gabuda S.P., Kozlova S.G.
Electronic configuration (n-1)d10s2 of some ions (Tl+, Pb2+, Sn2+,Te4+, Xe6+, etc) is generally supposed to be stable and chemically inert. At the same time, there is a lot of qualitative evidence that such ions chemically interact with each other in definite compounds. Here the authours describe a procedure for study of strengths and directions of chemical bond in crystals, and present results of investigation of previously unnoted interactions of strongly polarized ions. The approach that we are attempting is based on a nuclear magnetic resonance spectroscopy of single crystals. As an illustration the 125Te NMR spectra of tellurium dioxide TeO2 are used. It is revealed that abnormal far-distant (~4A) Te-Te exchange interaction is associated with a strong polarization of Te4+ ion under the intramolecular electric field and with spike-like stretching of 5s2 electron density along the Te-Te bondings. This kind of chemical bonding may be general for other strongly polarized ions, and might have wider importance for the physical chemistry and biochemistry and for matterial science. In the report will be submitted and are considered the NMR data on such interactions in the substances SnO, TlF, TlZrF5 and other.

Contribution to the conference:
11B and 15N solid state NMR studies of reference compounds containing B-N bonds. Application to the characterization of a polyborazilene.
C. Gervais, F.Babonneau, C.Bonhomme, J.Maquet, M.Vaultier, E. Framery, C.Duriez, D.Massiot, P. Florian
The present study is part of a project whose main objective is the NMR characterization of the polymerization and pyrolysis processes that transform borazine into h-BN, via the formation of polyborazilene. Due to the lack of previous studies on compounds containing B-N bonds. 15N and 11B solid state NMR investigations have been performed first on some reference compounds, with representative N and B environments that could be found in the various intermediates formed during the polymerization of borazine and pyrolysis steps of polyborazilene. The study was then extended to the polyborazilene.

Contribution to the conference:
Stereochemistry of Drugs Using Solid-State NMR
Robert Glaser
The stereochemistry of a series of drugs was studied by the vacp/tppmtechnique [variable applitude cross polarization/two pulse phase modulation decoupling] together with x-ray crystallographic structure analysis. Some of the drugs exist in polymorphic or pseudopolymorphic forms having different conformations. A number of 13C chemical shifts were found to be diagnostic for the particular molecular conformation. These solid-state studies together with the time- averaged chemical shifts in solution can then be used to ascertain the conformation of the major solution-state contributor. A case of a pressure dependent [spin-rate] solid-state phase-phase transition involving a mixture of polymorphic drug forms will also be discussed.

Contribution to the conference:
A SENSITIVE, HIGH-RESOLUTION MAGIC-ANGLE-TURNING EXPERIMENT FOR MEASURING CHEMICAL-SHIFT PRINCIPAL VALUES
David M. Grant, Gary McGeorge, James K. Harper, D. W. Alderman, Ronald J. Pugmire
Chemical-shift principal values from powdered-solid samples have been measured from the breakpoints in powder patterns on stationary samples or from the amplitudes of sidebands from spinning samples. When a number of different tensors are present, the stationary powder patterns usually overlap, making the breakpoints difficult to identify, thus complicating the extraction of the principal values for more than 2 or 3 tensors. The spinning-sideband method can handle more tensors in those cases where the relatively-narrow sidebands can be resolved, but still it is usually impractical to treat more than 5 or 6 tensors with this technique. The preferable 2D spinning method, referred to as a magic angle turning (MAT) experiment, was first proposed by Gan [J. Am. Chem. Soc. 114, 8307 (1992)] and developments latter gave our 5-pi pulse experiment [J. Magn. Reson. 105, 82 (1993)]. These 2D methods separate the individual powder patterns on the basis of their isotropic shifts. Themethod average the shifts along one of the 2D dimensions into a set of isotropic values. Recently, a modified MAT experiment (PHORMAT), that employs slow sample rotation (30 Hz), was introduced [J. Magn. Reson. 113, 210 (1994)] from this laboratory. All of the MAT type of experiments are achieved with slow sample rotation by synchronizing pulses to 1/3 of the rotor period, since at these rotor positions separated by 120° the three instantaneous precession frequencies always average to the isotropic frequency. MAT experiments significantly increase the number of tensors that can be measured in a sample, and a case of more than 25 different 13C tensors has already been published. This talk describes a further development of the 5-pi experiment that overcomes its evolution-dimension resolution limitations at higher spinning speeds. This new 5-piXR experiment incorporates two additional features, a data rearrangement technique derived from Gan's method [J. Magn. Reson. 109, 253 (1994)] for spinning-sideband suppression using a pseudo-2D experiment, and TIGER processing [J. Magn. Reson. 129, 134 (1997)] which is an extension of work by Manassen, Navon and Moonen [J. Magn. Reson. 72, 551 (1987); J. Magn. Reson. 79, 291 (1988)]. These 2D spectra display spinning-sideband patterns separated by their isotropic shifts with virtually no loss of sensitivity compared to 1D spectra. The 5-piXR method requires a high degree of synchronization of the pulses and acquisition to the rotor position, achieved with a modified version of a previously described electronic circuit. Applications to natural products, including drugs and related organic molecules with MW in the range of 500-800 Daltons, will be used to illustrate the very high resolution that is now achievable with these new powerful solid state techniques.

Contribution to the conference:
14N quadrupole-perturbed NMR of deuteron glasess
A.Gregorovic, B.Zalar, and R.Blinc
14N spin-lattice relaxation time(T1) temperature dependence of Rb(1-x)(ND4)xD2PO4 solid mixtures was measured in order to investigate the role of ammonius ions in the freezing behaviour of acid deuterons. In contrast to the ND4 2H T1 data where only one T1 minimum at T~140K, related to the slowing down of the ND4 reorientational dynamics, was found, a second T1 minimum at T~65K is present in the 14N T1(T) dependence. This shows that 14N probes sensitively both the reorientations of the ND4 ions as well as the intra hydrogen-bond acid deuteron dynamics.

Contribution to the conference:
CRYSTAL STRUCTURE OF MICROPOROUS ULM COMPOUNDS, MQMAS AND DOR STUDIES
Clarisse HUGUENARD, Ago SAMOSON, Francis TAULELLE, Thierry LOISEAU, Gérard FEREY
Clarisse HUGUENARD,(1) Ago SAMOSON,(2) Francis TAULELLE,(1) Thierry LOISEAU,(3) Gérard FEREY(3) (1) RMN et Chimie du Solide, UMR 7510 CNRS, Université Louis Pasteur, 4 rue Blaise Pascal, 67070 Strasbourg Cedex, France. (2) Institute of Chemical Physics and Biophysics, AKAD TEE 23, Tallinn 0026, Estonia (3) Institut Lavoisier, IREM, UMR 173 CNRS, Université de Versailles St-Quentin, 45 avenue des Etats-Unis, 78035 Versailles Cedex, France. Microporous materials under study are fluorinated aluminophosphates of the ULM type, obtained by hydrothermal synthesis using a diamine as a template [1]. These materials display an interesting variety of coordinence states for the oxyfluorinated aluminum sites. Our solid NMR study takes part to a more general investigation concerning the mechanism of synthesis of these kind of materials where organic molecules are involved in the organization of the inorganic framework [2a,b]. Combining NMR with X-ray diffraction has appeared powerful to elucidate crystal structures. Joined to other nuclei studies, 27Al NMR experiments presented here provide a direct tool for crystallographic inequivalent sites visualization. Site counting and characterization is far from obvious on 27Al MAS spectra because of strong quadrupolar interactions which cause second order broadening and overlapping of signals. To achieve high resolution second order quadrupolar interaction has to be removed. We report here synchronized DOR [3] and Triple Quantum Magic Angle Spinning [4] experiments results. DOR measurements were performed with outer rotor spinning speed wouter=1600 Hz and winner/wouter=4. Using a 2.5 mm rotor probe with high spinning speed (30 kHz) allowed us to synchronize 3QMAS measurements so that 2D spectra are free from spinning sidebands [5]. A three-pulse sequence with z-filtering was implemented [6]. In these experimental conditions, DOR gives a better resolution than 3QMAS and allows direct quantification. From 3QMAS spectra, other useful information can be obtained: the absence of chemical shift distributions reveals the quality of the crystal and quadrupolar parameters, easily determined on sites separated in the isotropic dimension, give an insight into local environments of aluminum atoms. Thus, these two techniques contribute in a complementary way to our elucidation of crystal structures. [1] J. Renaudin, T. Loiseau, F. Taulelle, G. Férey, C. R. Acad. Sci. Paris, Série II b, 323, p 545-553, (1996) [2] (a) C. In-Gerardin, M. In, F. Taulelle, J. Chim. Phys., 92, p 1877-1880, (1995) (b) G. Ferey, J. Fluorine Chem., 72, p 187-193, (1995) [3] A. Samoson, Chem. Phys. Lett., 214, p 456-458, (1993) [4] L. Frydman, S. Harwood, J. Am. Chem. Soc., 117, p 5367-5368, (1995) [5] D. Massiot, Journ. Magn. Res., series A, 122 (2), p 240-244, (1996) [6] J. P. Amoureux, C. Fernandez, S. Steurnagel, Journ. Magn. Res., series A, 123, p 116-118, (1996)

Contribution to the conference:
Solid state NMR investigation of the proton conducting properties ofimidazole
B.S.Hickman, J.J.Titman

Contribution to the conference:
Field-Cycling Studies of Tunnelling in the Hydrogen Bond
A.J.Horsewill, D.F.Brougham, C.J.McGloin, R.I.Jenkinson
At low temperature the dynamics of proton transfer in the hydrogenbond is dominated by quantum tunnelling. Direct measurements of the spectral density functions associated with this motion have been made using magnetic field-cycling NMR relaxometry. To a first approximation the behaviour is characteristic of a particle in a double minimum potential which is coupled to a bath of quantum oscillators. A range of materials have been studied and tunnelling rates in the range 1x10^7 to 2x10^9 s^-1 have been measured. There is strong evidence of a correlation between the hydrogen bond length and the tunnelling rate. This has been further investigated by means of applying hydrostatic pressure. We have studied the dynamics to higher temperatures as the system smoothly evolves towards quasi-classical behaviour. The role of excited states has been revealed in these investigations of the quantum-to-classical transition.

Contribution to the conference:
Bond-Angle Determination by MAS TOBSY Exchange NMR
Robbie J. Iuliucci and Beat H. Meier
Through-Bond-Correlation Spectroscopy,TOBSY,(1) relying on the scalar coupling interaction as the driving force for polarization transfer, offers a) an indirect measure of the scalar-coupling constant, b) isotropic transfer leading to a high transfer efficiency, and c) through-bond transfer providing information about the chemically bonded network. This latter point has the advantage that polarization transfer can be restricted to within an intramolecular spin pair even in the presence of strong intermolecular dipolar couplings. Thus, the relative orientation of a selected scalar-coupled spin pair, coexisting in multi-spin environment of a disordered sample, can readily be obtained by correlating the corresponding chemical-shift tensors. No dilution of the spin system is warranted. The crosspeak intensity of the sideband manifold, observed from a rotor synchronized MAS 2D exchange experiment, encodes the orientational information. The MAS condition furnishes the needed spectral resolution and an increased signal-to-noise ratio. While the procedure is general and suitable for any scalar- coupled spin 1/2 system, the concept will be demonstrated with 31P NMR on a sample of cubic phase SiP2O7. Here, isolation of the pyrophosphate groups from non-bonded phosphorous nuclei within the unit cell would otherwise be impossible. The six pyrophosphate units of SiP2O7 will be identified and the relative orientation of their principal axis system obtained. Exploiting the knowledge about the orientation of the CSA tensor to the molecular frame, the P-O-P bond angles can be evaluated. For SiP2O7, two classes of P-O-P units were observed, linear with an angle larger than 162 degrees and bent with angles ~148 degrees. The NMR data, presented here, eliminate the explanation of linear P-O-P bonds in M4+(P2O7) compounds by static spacial disorder. (1) Baldus, M.; Meier, B.H. J.Magn.Reson. A 1996, 121, 65­69.

Contribution to the conference:
Cross polarization from half-integer quadrupole nuclei to spin 1/2nuclei under MAS conditions- Theory and Application to InorganicMaterials
G. Jeschke and M. Jansen
Long spin-lattice relaxation times and in many cases low naturalabundance make solid-state NMR experiments on spins 1/2 in inorganic materials cumbersome. In many cases, half-integer quadrupole nuclei with much shorter relaxation times and higher natural abundance like Li-7, B-11, Na-23, or Al-27 are present in such materials. To use them as a polarization source for a more sensitive detection of spin 1/2 nuclei, the problem of spin locking magnetization on the central transition under MAS conditions has to be solved. A theoretical description for spin locking under MAS conditions is derived for the case of I=3/2 by expressing the Hamiltonian and the density matrix in terms of abstract spin 1/2 operators and applying Floquet formalism to render the Hamiltonian time-independent. Abstract spin formalism is a factorization of Hilbert space that allows one to formulate spin 3/2 problems in terms of two coupled spins 1/2 and use product operator formalism in calculations. The results are applied to cross polarization from Na-23 to N-15 in the model compound sodium nitrite and to cross polarization from B-11 to N-15 in hexagonal boron nitride (natural abundance) and Si-B-N ceramics (partially enriched in N-15).

Contribution to the conference:
QUADRUPOLE SPLITTING OF 7Li NMR LINE IN LITHIUM NIOBATE CRYSTALS WITH VARIOUS STOICHIOMETRY
E.V.Charnaya, N.A.Grigor'eva, V.S.Kasperovich, and M.G.Shelyapina
Lithium niobate crystals have various technological applications due to their electro-optical, nonlinear-optical, acoustic, and photorefractive properties. Recently, it was found by us that the temperature coefficient of the quadrupole constant for 7Li nuclei in the range 290 to 600 K differs significantly for congruent and stoichiometric crystals (the ratio [Li]/[Nb] equal to 0.95 and 1, respectively) [1]. The purpose of the present work is to compute the quadrupole constant for the lithium niobate crystals at room and high temperatures and to examine the difference in values of it for stoichiometric and congruent crystals above room temperature within the framework of theoretical models developed. For this we used the model of point multipoles. Calculations of the field-gradients were made for the charge on the niobium ion ranging from 0 to +5 and for the oxygen polarizability ranging from 0 to 2.6. The hexagonal cell and positional parameters were taken from [2]. A good agreement between experimental and theoretical values of the quadrupole constant at room temperature was achieved for the niobium charge in the range +4.5 to +5 and the oxygen polarizability around 0.7 and 1.7 for the positive and negative sign of the gradient, respectively. Then the values obtained were used to compute the quadrupole constant at high temperatures. We found that the positive temperature coefficient for the 7Li quadrupole constant can be explained on the base of changes in positional parameters with increasing temperature. 1. E.V.Charnaya, V.T.Gabrielyan, V.S.Kasperovich, and S.Klimko, Ferroelectrics, 202, 115 (1997). 2. S.C.Abrahams and P.Marsh, Acta Cryst.B, 42, 61 (1986).

Contribution to the conference:
Structural transformations in nanosystems on the NMR data
Voitsehovskii M.B., Kozlova S.G., Gabuda S.P.
Theoretical researches of structure and properties of nanosystemsshow, that depending on character and the sizes of of interactions in these systems exit areas in phase space and in space of parameters, in which such phenomena as the formation of incommensurate structures, occurrence of a chaotic movement, abnormal increase of mobility and etc. can be observed. The natural zeolites are considered as real objects of the nanosystems. The molecules of water, being in cavities cooperate with a skeleton and among themselves. The character of interaction changes and various srtuctural transformations are probable at change of density of filling by water molecules of the zeolite cavities. In the given work the results of the NMR research are submitted. Were investigated the dependencies of parametrs of the avered local magnetic field tenzor of water molecules in the natural zeolites at change of molecule content. A chaotic condition for systems of laumontite-leongardite is found out; a structural transformations in habazite are found out; the mechanism of occurrence of abnormal mobility of water molecules in natrolite is revealed.

Contribution to the conference:
Solid state NMR relaxation and spectral densities of a complex molecular motion in the asymmetric local environment
L. Latanowicz, E.C. Reynhardt, and W. Medycki

Contribution to the conference:
Measuring Molecular Geometry in Solids by Multiple-Quantum Coherence
M. H. Levitt
In the solid state, molecular torsional angles may be estimateddirectly by using double-quantum coherence to probe the correlations between local fields at neighbouring spin sites. We have used this approach to measure molecular torsional angles in peptides, retinals, the membrane protein rhodopsin, and a photointermediate of rhodopsin involved in the visual process. Methodlogical advances and applications will be described.

Contribution to the conference:
Continuous-Wave NMR Imaging of Solids
G.R. Davies, S.J. McCallum, J.M.S. Hutchison, D.J. Lurie
There is increasing interest in the use of NMR to image solid materials, which are characterised by very short transverse relaxation times (T2 longer than 5µs and shorter than 1ms), making them unsuitable for imaging by conventional 2DFT methods. A number of research groups have developed methods to image solids, some of which use line-narrowing strategies to overcome the broad NMR lines, and some of which use very strong gradients to produce images. Probably the most successful in terms of applications is stray-field imaging (STRAFI) which uses the continuous, very strong gradient present in the stray field of a superconducting magnet. We have developed an alternative scheme for solid imaging, based on continuous-wave (CW) NMR in the presence of moderately-strong field gradients, and have succeeded in producing two-dimensional images of Perspex (Plexiglass) materials with T2~10µs. Our CW-NMR system uses an Oxford Instruments 7 Tesla magnet with 183mm horizontal bore. The 300MHz CW-NMR system uses a birdcage resonator (6cm diameter) connected to a conventional homodyne reflection bridge. Lock-in detection is used to improve the signal-to-noise ratio, a Stanford DSP lock-in amplifier providing a reference signal at ~1kHz which is used to modulate the magnetic field. The magnetic field is swept using a split-solenoid co-axial with the birdcage resonator. In our prototype apparatus a single transverse gradient is provided by a water-cooled Golay set, capable of generating continuous gradients up to 300mT/m. A projection of the object is obtained by recording the output of the lock-in amplifier while the magnetic field is swept in the presence of a gradient. Data for a two-dimensional image can be obtained from multiple projections and reconstructed using filtered back-projection. Since the prototype system only has one gradient, the multiple projections are achieved by stepwise rotation of the object under study. A number of different materials have been imaged successfully using our CW-NMR system, which can accommodate samples up to 5cm in diameter. Examples include ABS plastic (a "Duplo" brick, with T2~300µs), Perspex (T2~10µs) and concrete. The latter was especially interesting as two separate components could easily be distinguished in the CW-NMR spectra, belonging to "bound" and "free" water in the sample, with T2s of ~7µs and ~500µs respectively. We are currently improving our apparatus by adding a second gradient coil which will allow rotation of the gradient field. This will improve the accuracy of gradient generation, and will remove the problem of sample rotation affecting the birdcage’s resonance. The two-gradient system will also enable data to be averaged (at least with stable samples), improving the SNR. CW-NMR imaging is a promising addition to the armoury of solid imaging techniques.

Contribution to the conference:
PROTONIC MAGNETIC RESONANCE STUDIES OF PHASE TRANSITIONS IN NH4NO3
F. MARICA, V. V. GRECU, R. GROSESCU*
It has been measured, for first time, the dipolar relaxation time together with the Zeeman relaxation time on NH4NO3 in the temperature range 125K-390K, obtaining supplementary information of relaxation mechanisms from various phases of this crystal. From the experimental data and from symmetry considerations, we have obtained information about the molecular dynamics in all crystalline phases. The molecular group NH4+ have a libration movement around of S4 axis with a frequency depending by temperature, simultaneously with the rotation around C2 and C3 axes. In the phases II and V, the correlation time of the libration movements is very short (fast movement). Near to the transition points V-IV and II-IV, the libration movements experience a slowing down process, so that when the crystalline probe transforms in the phase IV, the correlation time becomes long (hindered vibration). The Landau theory led us to the conclusion that the phase transitions are not of the second order.

Contribution to the conference:
IMPURITY NUCLEAR SPIN-LATTICE RELAXATION SUPPRESSION AND CHARGE-EXCHANGE OF CHROMIUM IONS IN A g-IRRADIATED RUBY CRYSTAL
E.V.Charnaya, V.M.Mikushev, A.M.Ulyashev, and Ali Chandoul,
In real crystals, the spin-lattice relaxation time of quadrupole nuclei is known to be determined by contributions from two additive mechanisms: a lattice mechanism, which is responsible for the relaxation in perfect samples, and an impurity one. Recently, a new method of separating the lattice and impurity contributions under the condition of a steady additional saturation of NMR line has been proposed in [1,2]. The method is based on the following thermodynamic ideas. When the concentration of paramagnetic centers is small, the impurity relaxation occurs by means of spin diffusion. It is effective when the local inverse spin temperature near defects is closer to the equilibrium value equal to the inverse lattice temperature than the average value over the sample. A rather strong additional magnetic (as well as acoustic or electric) steady saturation produces local heating of the nuclear spin-system [3]. This means that the regions near impurities became warmer than the spin-system in the main crystal volume. In this case the impurity relaxation should be turned off and the restoration of the magnetization signal in the sample volume should be caused by the lattice mechanism only. In the present paper we report results of using this method to study the spin-lattice relaxation of 27Al nuclei in a g-irradiated ruby crystal. It will be shown that the contributions from trivalent and charge-exchanged chromium ions to the impurity relaxation of Al nuclei can be separated. This allowed us to find the number of charge-exchanged chromium ions and to estimate the spin relaxation time of Cr4+ centers. 1. I.Mavlonazarov, V.M.Mikushev, and E.V.Charnaya, JETP Lett. 56, 13 (1992). 2. P.Yu.Efitsenko, V.M.Mikushev, and E.V.Charnaya, JETP Lett. 54, 587 (1991). 3. E.V.Charnaya, I.Mavlonazarov, and V.M.Mikushev, J.Magn.Res. A 112, 96 (1995).

Contribution to the conference:
Novel Approaches to Efficient Re- and Decoupling in Solid-State NMR
N.C.Nielsen, M.Hohwy, M.Bak, F.H.Larsen, T.Vosegaard, P.D.Ellis,H.J.Jakobsen
Preliminary abstract: Novel approaches to efficient re- and decoupling of anisotropic interactions in solid-state NMR spectroscopy will be presented. The topics may include broadband recoupling of homonuclear dipolar couplings, simultaneous recoupling of anisotropic shielding and dipolar interactions, high-order elimination of homonuclear dipolar interactions, and novel approaches to high-sensitivity quadrupolar-echo and MQMAS NMR.

Contribution to the conference:
Solid State Multinuclear MAS, 1H CRAMPS and High Resolution 1H and 13C NMR Study of Geochemical Samples
Yuri Oprunenko, Alexander Rebrov, Andrej Sirin and Andrei Timerbaev
Modern NMR spectroscopy is a prospective nondestructive analytical tool for investigation the composition and isotope fractionation of natural samples. So far, this technique found no wide application in geochemistry, espesially, to dating the fossil samples of geochemical and geological origin. A number of such samples, powdered or sorbed on inorganic oxides, including peats, coals, mammoth and bison bones and meat of different age (from 10 to 40kY), were investigated without any pretreatment (with except of drying and grinding)by means of multinuclear (including 1H CRAMPS) solid state MAS NMR. Additionally, some samples were extracted by water or various samples and concentrated by evaporation or were chemically modified (fermentation or enzimatic degradation) and then measured by multinuclear (1H, 13C, 15N, 2D) and 2D high resolution NMR spectroscopy. A range of spectral characteristics, such as aromatic region integrals in 13C and 1H spectra and isotope fractionation (12C/13C, 1H/2H) for peats and extracts from peats, some cross-peak intensities in 2D spectra and an extent of aminoacids racemization in the presence of optical lanthanide shift reagents, were correllated with data on 14C labelling and spore-pollen diagrams. This work was supported by Russian Foundation for Basic Research under grant N 97-05-64321

Contribution to the conference:
A NON INVASIVE NMR TECHNIQUE FOR PROBING MICROPOROSITY AND PROTON TRANSPORT IN REACTIVE POWDER CONCRETE
S. PHILIPPOT, J. P. KORB, D. PETIT, H. ZANNI
A NON INVASIVE NMR TECHNIQUE FOR PROBING MICROPOROSITY AND PROTON TRANSPORT IN REACTIVE POWDER CONCRETE S. PHILIPPOT*, J. P. KORB**, D. PETIT**, H. ZANNI* *Laboratoire de Physique et Mécanique des Milieux Hétérogènes, URA CNRS 857, ESPCI, 10 rue Vauquelin, 75231 Paris Cedex 05, France. **Laboratoire de Physique de la Matière Condensée, URA CNRS 1254, Ecole Polytechnique, 91128 Palaiseau Cedex, France. Reactive Powder Concretes (RPC) exhibit very high mechanical and durability properties which could be useful for nuclear waste confinement. Interesting questions could be addressed about the proton transport properties, at various lenghtscales, in this highly disordered material. Here we propose a noninvasive technique, based on proton nuclear relaxation at various frequencies, to relate these properties to the microstructure of RPC. At each frequency, the observed non-exponential recovery is explicitely resolved in term of a discrete distribution of four spin-lattice relaxation times, T1i, by the inverse Laplace method. The typical frequency dependence of the four relaxation times is characteristic of a nuclear paramagnetic relaxation mechanism. These data have been interpreted in terms of a biphasic fast exchange model and proton nuclear relaxation of hydrated paramagnetic ions at the surface of four nanopores hierarchically distributed in sizes. Finally we show that our nuclear relaxation technique is useful to follow in situ the kinetics of hydration and setting of such a concrete.

Contribution to the conference:
Spin-Locking in One Pulse NMR Experiments.
G. B. Furman, A. M. Panich, and S. D.Goren
Spin-Locking in One Pulse NMR Experiments. G. B. Furman, A. M. Panich, and S. D.Goren Department of Physics, Ben-Gurion University of the Negev, P.O.Box 653, 84105 Be'er-Sheva, Israel The response of a spin system to a long (in comparison to spin-spin relaxation time T2) radiofrequency pulse has been studied. We observed that the magnetization after the long pulse does not fall to zero at time t>>T2 for both on-resonance and off-resonance conditions and exhibits spin-locking behavior. The dependencies of the magnetization on frequency offset, linewidth and radiofrequency power are investigated both theoretically and experimentally. The question of the effective field direction is also discussed.

Contribution to the conference:
In Situ Solid State NMR Mechanistic Study of Alkylation Reactions Over Zeolite Catalysts
Elena B. Pomakhina
It has been demonstrated recently that high resolution solid state NMR spectroscopy is a superior techniques for the in situ studies of the mechanisms of heterogeneous reactions. In this contribution, solid state NMR has been used for the investigation of alkanes transformations over microporous solid catalysts like zeolites. 13C MAS NMR study of the early stages of propane 2-13Ñ and isobutane 2-13C activation was performed over H-ZSM-5 catalysts with various content of protonic and aprotonic sites. The technique used to produce samples, sealed under controlled atmospheres in precision glass ampoules, suitable for MAS in the region 2 to 4 kHz is described. Solid state NMR spectroscopy such as CP-MAS, CRAMPS, 2D solid-state spin diffusion 13C NMR experiment in situ apply for the investigation of alkylation reaction of benzene with alkanes. This techniques can give a useful information on the mobility of the adsorbed molecules and on the distribution of reactants and products in microporous solids.

Contribution to the conference:
Solid State NMR Spectroscopy of Hydrides fullerenes of C60 and C70
A. I. Rebrov a*, A. S. Lobachb
The value of the high-resolution solid state technique 13C CP-MAS and CRAMPS for the observation of different type of fullerenes, fullerenes chemisorbed or physisorbed on silica and hydrides fullerenes of C60 and C70. The resolution sufficient to resolve resonance signals of the different molecular symmetry groups. Fullerene hydrides are prepared by transfer hydrogenation of fullerenes C60 by 9,10 - dihydroanthracene. The main product of C60 hydrogenation is C60H36, which is a sufficiently stable molecule.. The comparison of IR and solid state 1H and 13C NMR data for C60H36 with the theoretical ones allows the suggestion that fullerene hydride has a T symmetric structure and contains 4 isolated benzenoid rings located at tetrahedral position on the surface of a closed skeleton of molecule. In the frame of systematic search of fullerenes investigated high-pressure modified fullerenes by means of solid state NMR.

Contribution to the conference:
DOR NMR at very high magnetic fields
A. Samoson
The fast sample double rotation provides for maximum real-timeresolution of the spectra of quadrupole nuclei with a half-integer spin. Repeated measurements at different fields or application of multi-quantum evolution enable immediate determination of the isotropic resonance parameters. The resolution generally, but not always, improves with increase of the magnetic field. The conclusions are derived from, and illustrated by data of B-11, O-17, Na-23 and Al-27 at magnetic fields up to 17 T.

Contribution to the conference:
Oxygen-17 NQR in hydrogen bonded organic solids
Janez Seliger
Oxygen-17 nuclear quadrupole resonance data from various organic solids are analysed in a simple model, where the strength of the hydrogen bond is reflected on the principal value V33 of the electric field gradient (EFG) tensor along the principal direction pointing perpendicular to either the C-O-H or the C=O...H plane. A nearly linear correlation between V33 and the other two principal values of the EFG tensor is observed through the complete region of the C-O-H and C=O oxygens. A correlation between V33 at the C-O-H oxygen site and V33 at the corresponding C=O...H oxygen site is as well observed. Further we observe a correlation between V33 at either the C-O-H oxygen site or the C=O...H oxygen site and the O-O distance in a C-O-H...O=C hydrogen bond. The observed correlations are of particular value in interpreting the oxygen-17 NQR and quadrupole perturbed NMR spectra in hydrogen bonded organic solids.

Contribution to the conference:
The transient coherent formation under double inhomogenity
L.N. Shakhmuratova, D.M. Chaplin and D.K. Fowler

Contribution to the conference:
31P-Solid state NMR and electron microscopy studies of phospholipid vesicles. The effect of freeze-thawing cycles
TRAIKIA Mounir a, Michel RECOUVREUR b, Jean CARTAUD b & Philippe F. DEVAUX a
a) Laboratoire de Biophysique Membranaire UPR 9052, IBPC, 13 rue Pierre et Marie Curie 75005 Paris, France b) Laboratoire de Biologie des Membranes UMR 7592, Institut J. Monod, Université Paris VII, 2 place Jussieu 75251 Paris cédex 5 France. Multilamellar vesicles (MLVs) with a diameter of a few mm are generally used when investigating the physical state of lipid bilayers by broad-band solid-state NMR. Unilamellar vesicles are needed for some liposomes applications that require a well defined interior and exterior. Large unilamellar vesicles (LUVs) with a diameter of 200 or 100 nm can be obtained by extruding MLVs through polycarbonate filters. Unfortunately for broad band NMR, the 100 nm vesicles undergo spontaneous vesicle rotation which averages partially the anisotropy of the magnetic interactions in the case of 31P NMR or 2H NMR spectroscopy (Fenske, 1993). Thus, a lot of spectroscopic informations on the local order parameters are lost in LUVs. In addition these intermediate size vesicles cannot be used for high resolution spectroscopy and do not give in straightforward manner narrow peaks when using magic angle spinning (Traikia et al., 1997). In the case of dioleoylphosphatidylcholine (DOPC), MacDonald et al. (1994) have shown that repeated freeze-thawing results in the fragmentation of the MLVs into smaller vesicles. Here, using both 31P-NMR and electron microscopy, we show that cycles of freezing and thawing with lipid mixtures composed of DOPC and DOPA, progressively fragments the MLVs into an population of unilamellar vesicles of relatively large size. With ten cycles, the average diameter was around 1000 to 500 nm. The change in line-shape of broad band 31P-NMR spectra revealed a progressive appearance of small vesicles whose population increases as more cycles of freezing and thawing were applied. Similar studies were carried out with lipid mixtures composed of DOPC/DOPA/Lyso-PC and the same type of "very large" unilamellar vesicles (VLUV) were obtained. High resolution spectra were obtained by magic angle spinning (MAS). The chemical shift of DOPA was used to investigate the increased in lateral packing caused by the insertion of the cone-shaped lyso-PC molecule. References Fenske D. B., (1993) Chem. Phys. Lipids, 64, 143-162. MacDonald R.C. et al., (1994) Biochim. Biophys. Acta , 1191, 362-370. Traikia M., (1997) J. Magn. Res., 125, 140- 144.

Contribution to the conference:
NMR Crystallography of Inorganic Solids
F. Taulelle
The extremely rapid development of high resolution NMR of solid state allows to measure an always increasing number of information on the analyzed sample. In order to proceed to the geometrical description of an inorganic compound, its structure, it is necessary to measure a least 4N constraints, where N is the number of atoms contained into the unit cell of the considered structure. Three position coordinates and the nature of each atom are necessary, or any equivalent set of data. The actually NMR accessible data set allows to fulfill that requirement. The description on how to derive the structure out of the data set has to be described. It is first of all necessary to determine the compound's space group then its unit cell parameters. This conference aims at showing from examples how to determine the order of the space group. With the aid of the compound's graph, it will be shown how the sub ensemble of identical order is restricted to the compatible space groups. Then the NMR observed atoms are placed, and the unit cell parameters are derived. The space group proposed by diffraction can contain in addition to the elements of symmetry of the geometrical description (its static structure) elements of symmetry of motion (motion operations) or atoms distribution (displacement operations). The space group containing all those elements of symmetry is called the apparent space group, it contains as a sub group the geometrical space group. Comparison of structure obtained by diffraction and obtained by NMR allows to evidence a finer degree of description of the dynamic or the disorder. Diffraction and NMR used together, allow the emergence of a refinement method analogous to a Ritveld method on powders. At last, MAS, CPMAS, Double Quantum, RFDR and MQMAS as well as DOR NMR methods will exemplify ways of checking a structure obtained by diffraction mostly on microporous alumino- or gallo- phosphate compounds.

Contribution to the conference:
Dipolar Recoupling Enhanced by Amplitude Modulation of the Homonuclear Rotary Resonance Condition.
R. Verel, M. Baldus, M. Ernst and B.H. Meier
Efficient dipolar recoupling under high speed MAS is both of great importance and a challenge. The importance stems from the fact that the dipolar interaction contains valuable spatial information and the challenge is that MAS is designed especially to average out interactions which can be described by a second rank tensor, such as the dipole interaction. In this contribution we present a new homonuclear recoupling experiment, DREAM, based on the adiabatic variation around the HORROR condition. The experiment is shown to be insensitive with respect to offsets while at the same time remaining a highly efficient method for recoupling. A novel method, which incorporates the DREAM sequence, for filtering out coupled spins from a background of uncoupled spins as well as 2D homonuclear correlation spectroscopy on a larger spin systems serve as exapmles of applications.

Spectroscopy of Living Systems

Contribution to the conference:
Water diffusion and energy metabolism in hepatocytes-entrapping alginate gel beads as detected by NMR techniques.
Miccheli A., Capuani G.*, Tomassini A., Di Cocco M.E., Manetti C., Conti Devirgiliis L.**, Falasca L.** and Conti F.
Extracorporeal bioartificial liver using xenogenic hepatocytes has potential impact as a support therapy of acute and chronic liver failure, as a temporary bridge to transplantation. Such a device must satisfy the following requirements: 1) Immunoprotection by means of macromolecular cut-off avoiding interaction with the immunocompetent system, but allowing traffic of proteins such as albumin, either bound to toxic compounds or not; 2) restoring both cell-cell and cell-matrix intercellular organization, which allows to keep the differentiate functionality, both detoxificating and metabolic, of hepatocytes; 3) high cellular density, while assuring sufficient oxygen and substrate supply for cell viability and functionality. We developed a prototype of a fixed-bed bioreactor constituted by rat hepatocytes entrapped in alginate gel beads, and preliminary results obtained by MR techniques about diffusion as well as cell bioenergetics in the working bioreactor are the subject of this presentation. Hepatocytes were isolated from adult, male Wistar rats by using a two-step collagenase digestion. Cells were entrapped in alginate gel beads at different high cellular densities. Alginate beads containing rat hepatocytes were placed in a fixed-bed perfusion bioreactor and water diffusion in gel beads and cell energy metabolism were monitored respectively with H-1 MRI and P-31 MRS performed on a SMIS IVS spectrometer equipped with a 4.7 T, wide-bore horizontal magnet. Diffusion measurement were performed by using the standard PFG-SE sequence, while relaxation times were measured by IR and SE sequences. The apparent diffusion coefficient (ADC) of water in alginate beads is close to pure water values, while cell entrapping causes the ADC to decrease depending on cell density. T1 values in alginate gel beads do not vary significantly depending on the presence of hepatocytes, while T2 values decrease (350 msec vs. 210 msec without and with cells, respectively). P-31 MRS evidenced that ATP levels keep constant through the experiment (9 hrs.). Inter- and intra-cellular organization was evaluated by means of optical and electronic microscopy respectively. Our study shows that the prototype of hepatic bioreactor is well suited both for therapeutic applications as well as pharmacological studies. Results obtained jointly with MRI and MRS allow to evaluate substrate supply while monitoring cell viability and functionality. Further studies are in progress to rationalize, optimize and monitor the bioreactor performance over extended periods.

Contribution to the conference:
Metabolic fate of ([1, 2-13C]acetyl)-L-carnitine into the liver. A 13C NMR study
Puccetti C#, Aureli T*, Di Cocco M.E#, Ricciolini R*, Scalibasti M* and Conti F#
#Department of Chemistry, University "La Sapienza", Rome, Italy *Department of Biochemistry, Sigma-Tau Research Labs, Pomezia, Italy Introduction Carnitine is a naturally occurring molecule which is known to play an important role as carrier of fatty acids across mitochondrial membrane. Its acetyl-L-derivative, namely acetyl-L-carnitine, is the most prominent ester of carnitine in mammalian tissues and it has been thought to play pivotal role in the acetyl group trafficking. The isotopomeric distribution as determined by 13C NMR of the enriched compounds is a powerful tool in order to elucidate catabolic and anabolic processes inside cells. In the present study, using 13C NMR spectroscopy we have studied the fate of acetyl moiety of ([1, 2-13C]acetyl)-L-carnitine through different biochemical pathways in liver. Methods Fischer rats aged 6 months were fasted for 24 hrs. Freely moving rats were infused four hours with ([1, 2-13C]acetyl)-L-carnitine and then anaesthetized. Livers were immediately removed, frozen and extracted by modified Bligh-Dyer method1. 13C NMR spectra of aqueous and liposoluble phases were obtained with a Bruker AM 500 spectrometer. Results Doublets due to 13C-13C fragment in C4-C5 carbon atoms of glutamate, glutamine, glutathione and C1-C2 C3-C4 of succinate and 3-hydroxybutyrate were present in aqueous extracts. In the liposoluble phase 13C enrichments were observed only into cholesterol. Both 13C-13C doublets and 13C enrichments as singlet were reported into cholesterol carbon skeleton according to the known biosynthetic pathway2. Conclusions Our results showed that ([1, 2-13C]acetyl)-L-carnitine enters liver cells and feeds different acetyl-CoA pools. The 13C enrichments in C1-C2, C3-C4 of succinate are representative for an utilization of acetyl moiety of acetyl-L-carnitine via tricarboxylic acid cycle. The 13C-13C fragment found in C4-C5 of glutamate, glutamine, glutathione can be explained by the synthesis of these molecules from TCA cycle intermediates. ([1, 2-13C]acetyl)-L-carnitine also feeds the ketogenic acetyl-CoA pool as shown by the presence of 13C-13C fragment in C1-C2 and C3-C4 of 3-hydroxybutyrate. Finally a quantitative analysis of 13C labeling pattern into cholesterol carbon atoms revealed different amounts of 13C label between 13C-13C fragment and 13C enrichments as singlet. A possible explanation is that cholesterol is synthesized from two acetyl-CoA pools likely associated to mitochondrial and extramitochondrial compartments which are differently enriched by ([1, 2-13C]acetyl)-L-carnitine. 1. Miccheli A, Aureli T, Delfini M, Di Cocco ME, Viola P, Gobetto R, Conti F [1988] Cell.Mol.Biol. 34: 591-603 2. Likhodii SS, Cunnane SC [1995] Magn.Reson.Med. 34: 803- 813.

Contribution to the conference:
Advances in O-17 MRI and MRS

TBA

Contribution to the conference:
Structure and dynamics of metallodrug recognition
J. Parkinson