Nuclear Magnetic Resonance : Volume 29 [1 ed.] 9781847553850, 9780854043279

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Nuclear Magnetic Resonance Volume 29

A Specialist Periodical Report

Nuclear Magnetic Resonance Volume 29 A Review of the Literature Published between June 1998 and May I999 Senior Reporter G. A. Webb, Department of Chemistry, University of Surrey, Guildford, UK Reporters . 1. Barsukov, University of Leicester, UK A. C. de Dios, Georgetown University, Washington, DC, USA I?C. Driscoll, University College, London, UK D. Esposito, University College, London, UK H. Fukui, Kitami Institute of Technology, Kitami, Japan E. E Hounsell, Birkbeck College, London, UK C. J. Jameson, University of Illinois at Chicago, USA K. Kamienska-Trela, Polish Academy o f Sciences, Warszawa, Poland C. L. Khetrapal, University of Allahabad and Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India H. Kurosu, Nara Women's University, Nara City, Japan R. Ludwig, Universita t Do rtm und, German y G. A. Nagana Gowda, Indian Institute of Science, Bangalore, India M. Pfuhl, University College, London, UK M. J. W. Prior, University of Nottingham, UK K. V. Ramanathan, Indian Institute of Science, Bangalore, India W. Schilf, Polish Academy of Sciences, Warszawa, Poland M. E. Smith, University of Warwvick, Coventry, UK T. Watanabe, Tokyo University of Fisheries, Tokyo, Japan J. Wojcik, Polish Academy of Sciences, Warszawa, Poland M. Yamaguchi, Kao Corporation, Tochigi, Japan T. Yamanobe, University of Gunma, Japan

RSmC ROYAL SOCIETY

OF CHEMISTRY

ISBN 0-85404-327-6 ISSN 0305-9804 Copyright 0The Royal Society of Chemistry 2000 All Rights Reserved Apart from any fair dealing for the purposes of research or private study, or criticism or review cis permitted under the terms of the U K Copyright, Designs and Patents Act, 1988, this publication may not be reproduced, stored or transmitted, in any form or by any means, without the prior permission in writing of The Royal Society of Chemistry, or in the case of reprographic reproduction only in accordance with the terms of the licences issued by the Copyright Licensing Agency in the U K , or in accordance with the terms of the licences issued by the appropriate Reproduction Rights Organization outside the U K . Enquiries concerning reproduction outside the terms stated here should be sent to The Royal Society of Chemistry at the address printed on this page.

Published by The Royal Society of Chemistry Thomas Graham House, Science Park, Milton Road, Cambridge CB4 OWF, UK For further information see our web site at www.rsc.org Typeset by Computape (Pickering) Ltd, Pickering, North Yorkshire, UK Printed by Athenaeum Press Ltd, Gateshead, Tyne and Wear, UK

It is my pleasure to introduce Volume 29 of the Specialist Periodical Reports on NMR. Most of the present set of reports cover topics which will be familiar to readers of this series. However, after careful consideration and with a view to avoiding a certain amount of duplication it has been decided that the chapter on Conformational Analysis is to be discontinued. Much of the material which has appeared in this chapter in the past is covered in other chapters such as Applications of Spin-Spin Couplings, Multiple Pulse NMR and the reports on Natural and Synthetic Macromolecules. Another change introduced with this volume is the division of the chapter on NMR of Natural Macromolecules into two reports, one on NMR of Proteins and Nucleic Acids and the other on NMR of Carbohydrates, Lipids and Membranes. This latter topic is covered by Professor E. F. Hounsell whom I welcome to the reporting team. Four reporters are retiring from the team with this volume, all of whom have provided consistently good reports for a significant number of years. Those in question are Drs. M. Yamaguchi, P. C . Driscoll and M. E. Smith and Professor C . L. Khetrapal; I wish to express my thanks to them and to all of the other reporters for their support and devotion to this series of NMR reports. It is also a pleasure to record my thanks to the production staff at the Royal Society of Chemistry for their help in the realisation of this volume. University of Surrey Guild ford October 1999

G. A. Webb

Contents

Chapter 1

Chapter 2

Chapter 3

NMR Books and Reviews .By W . Schirf 1 Books 2 Regular Reviews Series 3 Edited Books and Symposia 4 Reviews in Periodicals 5 Reviews and Books in Foreign Languages

1

1 2 5 14 32

Theoretical and Physical Aspects of Nuclear Shielding By C. J. Jumeson and A . C . de Dios 1 Theoretical Aspects of Nuclear Shielding 1.1 General Theory 1.2 Ab Initio Calculations 2 Physical Aspects of Nuclear Shielding 2.1 Anisotropy of the Shielding Tensor 2.2 Shielding Surfaces and Rovibrational Averaging 2.3 Isotope Shifts 2.4 Intermolecular Effects on Nuclear Shielding 3 References

41

Applications of Nuclear Shielding By M . Yumuguchi 1 Introduction 2 Various Chemical and Nuclear Influences to Nuclear Shieldings 2.1 Computer Assisted Structural Assignment 2.1.1 Spectrum Simulation, Computer Assisted Assignments and Related Techniques 2.1.2 Nuclear Shielding Calculations 2.2 Stereochemical and Nuclear Shielding Non-Equivalence 2.2.1 Chirality Determination by Mosher’s and Related Methods 2.2.2 0ther Stereoc hemistry Determination 2.3 Isotope Effects 2.4 Substituent Effects 2.4.1 Proton Substituent Effects 2.4.2 Carbon and Heteroatom Substituent Effects 2.5 Intramolecular Hydrogen Bonding Effects and Related Effects

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Nuclear Magnetic Resonance, Volume 29 0The Royal Society of Chemistry, 2000 vii

41 41 54 58 58 65 70 71 78

85 85 85 85 86 87 87 88 89 89 89 90 90

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Contents

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2.6 Bond Anisotropy, Ring Current Effects and Aromaticity 91 2.7 Intermolecular Hydrogen Bonding Effects, Inclusion Phenomena and Related Effects 91 2.7.1 Proton and Heteronuclear Shifts 92 2.7.2 Cyclodextrins (CDs) 92 2.7.3 Other Molecular Recognition 93 2.8 Shift Reagent 93 2.9 Miscellaneous Topics 93 2.10 Reviews 94 3 Shieldings of Particular Nuclear Species 94 3.1 Group 1 ('H,2H,3H,6,7Li,23Na,87Rb,133C~)94 3.1.1 Hydrogen ('H) 94 95 3.1.2 Deuterium (2H) 3.1.3 Tritium (3H) 95 3.1.4 Lithium (6,7Li) 96 3.1.5 Sodium (23Na) 97 3.1.6 Rubidium (87Rb) 97 3.1.7 Caesium (133Cs) 98 3.2 Group 2 (9Be,25Mg,'37Ba) 98 3.2.1 Beryllium (9Be) 98 3.2.2 Magnesium (25Mg) 98 3.2.3 Barium (137Ba) 98 3.3 Group 3 and Lanthanoids (45S~,89Y,139La,'53E~) 98 3.3.1 Scandium (45Sc) 98 3.3.2 Yttrium (89Y) 98 3.3.3 Lanthanum ('39La) 99 3.3.4 Europium ( 1 5 3 E ~ ) 99 3.4 Group 4 (47747Ti) 99 99 3.5 Group 5 (51V,93Nb) 3.5.1 Vanadium (5'V) 99 3.5.2 Niobium (93Nb) 101 101 3.6 Group 6 (95Mo,183W) 3.6.1 Molybdenum (95Mo) 101 3.6.2 Tungsten (lX3W) 101 3.7 Group 7 (55Mn,99Tc) 102 3.7.1 Manganese (55Mn) 102 3.7.2 Technetium (99Tc) 102 3.8 Group 8 (57Fe,99Ru) 102 102 3.8.1 Iron (57Fe) 3.8.2 Ruthenium (99Ru) 103 3.9 Group 9 (59C~,103Rh) 103 3.9.1 Cobalt (59C0) 103 3.9.2 Rhodium (Io3Rh) 103 3.10 Group 10 (195Pt) 104 3.10. I Platinum 104 3.1 1 Group 1 1 (63C~,107,109Ag) 106 3. I I . I Copper (s3Cu) 106 3.11.2 Silver (107,109Ag) 106 3.12 Group 12 (67Zn,"1."3Cd 199H€9 106 3.12. I Zinc (67Zn) 106 7

Contents

ix 3.12.2 Cadmium (1117113Cd) 3.12.3 Mercury (199Hg) 3.13 Group 13 (11B,27A1,71Ga,203,205T1) 3.13.1 Boron ("B) 3.13.2 Aluminium (27Al) 3.13.3 Gallium (71Ga) 3.13.4 Thallium (203*205Tl) 3.14 Group 14 (13C,29Si,73Ge,117*1 19Sn,207Pb) 3.14.1 Carbon (13C) 3.14.2 Silicon (29Si) 3.14.3 Germanium (73Ge) 3.14.4 Tin (l17,119Sn) 3.14.5 Lead (207Pb) 3.15 Group 15 (14*15N,31P) 3.15.1 Nitrogen (l4,l5N) 3.15.2 Phosphorus (31P) 3.16 Group 16 (170,33S,77Se,125Te) 3.16.1 Oxygen (I7O) 3.16.2 Sulfur (33S) 3.16.3 Selenium (77Se) 3.16.4 Tellurium (125Te) 3.17 Group 17 (19F,35737C1) 3.17.1 Fluorine (19F) 3.17.2 Chlorine (35737C1) 3.18 Group 18 (3He,129Xe) 3.18.1 Helium (3He) 3.18.2 Xenon (129Xe) 4 References

Chapter 4

Theoretical Aspects of Spin-Spin Couplings By H . Fukui 1 Introduction 2 Origin of the Diamagnetic Term in Four-Component Relatavistic Calculations 3 Rovibrational Effects 3.1 Rovibrationally Averaged Spin-Spin Coupling of the Hydrogen Fluoride Molecule 3.2 Spin-Spin Coupling Surfaces in the Water Molecule, Oxonium Ion, and Hydroxyl Ion 4 Basis Set Dependence of Nuclear Spin-Spin Coupling Constants 4.1 Multiconfigurational Self-consistent Field Calculations 4.2 Second-Order Polarization Propagator Approximation Calculations 5 Other Ab Initio Calculations 6 Density Functional Theory Calculations 7 Semi-Empirical and Other Calculations 8 References

106 107 108 108 108

109 109 109 109 109 112 112 115

115 115 117 117 117 1 I8 118 118 119 I I9 1 I9 119 119 120 120 141 141 141 145 145 147 150

150 151

154 156 159 161

Contents

X

Chapter 5

Chapter 6

Chapter 7

Applications of Spin-Spin Couplings By K . Kamienska- Trela and Jacek Wojcik I Introduction 2 Methods 3 One-Bond Couplings to Hydrogen 4 One-Bond Couplings Not Involving Hydrogen 5 Two-Bond Couplings to Hydrogen 6 Two-Bond Couplings Not Involving Hydrogen 7 Three-Bond Hydrogen-Hydrogen Couplings 8 Three-Bond Couplings Between Hydrogen and Heteronuclei 9 Three-Bond Couplings Not Involving Hydrogen 10 Couplings Over More Than Three Bonds 11 Non-Typical Couplings 12 References Nuclear Spin Relaxation in Liquids and Gases By R . Ludwig I Introduction 2 General, Physical and Experimental Aspects of Nuclear Spin Relaxation 2. I General Aspects 2.2 Experimental Aspects 2.3 Relaxation in Coupled Spin Systems 2.4 Dipolar Couplings and Distance Information 2.5 Exchange Spectroscopy 2.6 Radiation Damping 2.7 Quadrupolar Interactions 2.8 Intermolecular Dipolar Interaction in Diamagnetic and Paramagnetic Solution 2.9 Slow Motions in Glasses 2.10 Models for Molecular Dynamics 3 Selected Applications of Nuclear Spin Relaxation 3.1 Pure Liquids 3.2 Non-Electrolyte Solutions 3.3 Electrolyte S o htions 3.4 Transition Metal Complexes 3.5 Molten Salts 4 Nuclear Spin Relaxation in Gases 5 Self-Diffusion in Liquids 5.1 Experimental and Theoretical Aspects 5.2 Selected Examples 6 References Solid State NMR By M . E. Smith 1 Introduction 2 Technique Development 2. I Theoretical 2.2 Experimental

165 165 166 171 174 182 183 185 193 198 200 200 204 217 217 219 219 22 I 222 224 226 228 229 23 1 233 235 236 236 237 237 238 238 238 240 240 24 1 242 25 1 25 1 252 252 254

xi

Contents 3 Carbonaceous Materials 3. I Coals, Pitches and Oil Shales 3.2 Fullerenes, Diamonds and Other Carbons 4 Organic Materials 4.1 General 4.2 Organometallics 4.3 Bio-organic 4.4 Liquid Crystals, Membranes, Bilayers, Cell Walls and Woods 5 Organic- Inorganic Materials 5.1 General 5.2 Soils and Humic Substances 6 Inorganic Materials 6.1 General 6.2 Silicates and Aluminosilicates 6.3 Microporous and Mesoporous Materials 6.3. I Silicate-Based Systems 6.3.2 Other Structural Studies 6.3.3 In-Situ and Surface Reactions 6.4 Glasses 6.5 Ceramics 7 Miscellaneous 7.1 General 7.2 Dynamics and Intercalates 8 References

Chapter 8

Multiple Pulse NMR By I. Barsukov 1 Introduction 2 Variation of the Radiofrequency Pulse 2.1 Selective Excitation/Inversion Pulses 2.2 Pulse Field Gradients 2.3 Solvent Suppression 3 Homonuclear Correlation Spectroscopy 4 NOE, Chemical Exchange and Relaxation 4.1 NOE and Chemical Exchange 4.2 Relaxation Time Measurements 4.3 Translational Diffusion Measurements 5 Inverse Proton Detected Correlation Spectroscopy 5.1 Genera1 5.2 Isotope Filtered and Edited Experiments 5.3 Scalar Coupling Constants Measurements 5.3.1 Quantitative J-Correlation 5.3.2 E-COSY 5.3.3 Spin-State Selective Experiments 5.3.4 Other 5.4 Heteronuclear Double Resonance Experiments 5.4.1 HSQC 5.4.2 HCCH 5.4.3 Other

257 257 258 259 259 260 262 265 267 267 269 269 269 272 274 274 276 277 279 280 28 1 28 1 282 283 316 316 317 317 317 318 318 319 319 32 1 323 324 324 324 325 325 326 327 328 328 329 330 33 1

Contents

xii

5.5 Heteronuclear Triple Resonance Experiments 6 References Chapter 9

NMR of Nucleic Acids and Proteins By P. C. Driscoll, D. Esposito and M . Pfuhl I Introduction 2 Proteins 2.1 Landmark Protein Structures 2.2 Protein-Ligand Complexes 2.3 Protein-Lipid Interactions 3 Nucleic Acids 3.1 RNA Psuedoknots 3.2 Double-Isotope Labelled DNA Molecules 3.3 Ion Binding to Nucleic Acids 3.4 Nucleic Acid Adducts 3.5 Nucleic Acid Ap tamers 4 Technical Developments 4.1 Transverse Relaxation-Optimised Spectroscopy (TROSY) 4.2 Scalar Coupling Across Hydrogen Bonds 4.3 Low Viscosity Solvents 4.4 Structural Restraints from Databases 4.5 J-Couplings 4.6 Software Developments 4.7 SAR-by-NMR 4.8 Resonance Assignments and Pulse Sequences 4.9 Macromolecular Hydration 4.10 Methyl Protonation in a Deuterated Background 4. I 1 Segmentally Labelled Proteins 4.12 Studies of a Protein Photointermediate 5 Measurements of Residual Dipolar Couplings 5.1 Background 5.2 Residual Dipolar Couplings in Structural Refinement 5.3 Liquid Crystalline Media for Partial Alignment 5.4 New Methods for Measurement of Residual Dipolar Couplings 5.5 Impact of Residual Dipolar Couplings on Macromolecular Structures 6 Study of Protein Dynamics Through Heteronuclear Relaxation Measurements 6.1 Methodological Developments 6.1.1 Study of Chemical Exchange 6. I .2 Improving the Accuracy of Relaxation Experiments and Their Interpretation 6.1.3 Relaxation Measurements Incorporating I5N CSA/Dipole Cross Correlation 6.1.4 Measurement of Side Chain Dynamics 6.1.5 Mechanistic Interpretation of the Dynamics Underlying Relaxation

332 336 340 340 34 1 34 1 343 344 346 346 346 348 349 350 35 I 35 1 354 356 357 360 36 1 363 363 365 366 368 369 370 370 37 1 372 375 376 377 377 377 379 38 1 382 383

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Contents

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6.2 Applications 6.2.1 Protein-Nucleic Acid Interactions 6.2.2 Protein-Protein Interactions 6.2.3 Enzyme Function 6.2.4 Protein-Metal Binding 6.2.5 Protein Structure and Stability 6.2.6 Protein Folding 6.2.7 Protein- Ligand Binding Effects 6.2.8 Redox Proteins 6.2.9 Dynamics in Multidomain Proteins 7 References Chapter 10

Chapter 11

Chapter 12

384 384 385 388 388 389 39 1 392 394 395 396

NMR of Carbohydrates, Lipids and Membranes By E. F. Hounsell I Introduction 2 Polysaccharides and Cyclodextrins 3 Carbohydrate-Based Natural Products 4 Membrane Studies, Glycoproteins and Gangliosides 5 Proteoglycans 5.1 Hyaluronan 5.2 Keratan Sulfate 5.3 Heparin and Haparan Sulfate 5.4 Chondroitan Sulfate 5.5 Other Studies of Uronic Acids and Sulfate Esters 6 Biomedical Applications 7 References

406

Synthetic Macromolecules By H. Kurosu and T. Yamanobe Introduction Liquid Crystals Primary Structure Characterization of the Synthetic Macromolecules Dynamics of the Synthetic Macromolecules Gels and Crosslinked Macromolecules Polymer Blend and Diffusion of the Synthetic Macromolecules References

419

Nuclear Magnetic Resonance Spectroscopy of Living Systems By M . J . W. Prior 1 General Applications and New Methodologies 1.1 Applications 1.2 Spectral Editing, Spectra Analysis, Localisation and Instrumentation 1.3 Intracellular Ions, Metabolites and pH 2 Cells 2.1 Reviews and General Methods 2.2 Bacteria 2.3 Blood 2.4 Mammalian

453

406 407 409 410 41 1 412 412 412 413 413 413 413

419 419 420 420 436 437 437 438

453 453 453 455 459 459 459 460 46 1

Contents

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3 4

5

6

Chapter 13

2.5 Plant 2.6 Reproductive 2.7 Tumour 2.8 Yeast and Fungi Plants and Algae Tissue Studies 4.1 Brain and Spinal Cord 4.2 Eye 4.3 Heart 4.4 Kidney 4.5 Liver 4.6 Reproductive System 4.7 Salivary Glands 4.8 Skeletal Muscle 4.9 Smooth Muscle 4.10 Tumour 4.1 1 Skin 4.1 2 Whole Animal Clinical Studies 5.1 Reviews 5.2 Brain 5.3 Heart 5.4 Liver 5.5 Muscle 5.6 Tumour 5.7 Adipose Tissue References

Nuclear Magnetic Resonance Imaging By T . Wutunabe 1 Introduction 2 General Aspects and Reviews 3 Instruments 4 Pulse Sequences and Data Processing 5 Solid State NMR Imaging 6 Other Nuclei 7 Diffusion, Flow and Velocity Imaging 7.1 Diffusion Tensor Imaging, Diffusion-Weighted Imaging and One-Dimensional Mapping 7.2 Flow, Velocity Profile, Velocity Imaging, Rheometry 7.3 Mass Transport, Absorption, Sedimentation, Precipitation 8 Porosity, Fluid Assisted Images 9 Polymers 10 Rock, Mineral, Clay, Coal 1 1 Food and Food Processing 12 Plant 13 In Vivo,Ex Vivo NMR Imaging 13.1 'H NMR Imaging (MT, Diffusion, Functional, and T2*-Weighted Imaging)

462 463 463 464 464 466 466 47 1 47 1 48 1 48 I 483 483 48 3 484 48 5 487 487 488 488 489 49 1 49 1 492 495 495 496 509 509 510 512 513 516 517 517 517 519 520 52 I 522 523 524 525 527 527

Con tents

Chapter 14

xv 13.2 Other Nuclei-NMR Imaging 13.3 Contrast Enhanced Imaging via Paramagnetic Agents 14 References

528 528 529

Oriented Molecules By C. L.Khetrapal, K . V. Kamanathan and G. A . Nagana Gowda 1 Introduction 2 Reviews, Theory and General Studies 3 Emerging Areas and New Techniques 4 Dynamic NMR Studies 5 Discotics, Lyotropics and Chiral Systems 6 Liquid Crystals in Confined Geometries and Polymeric Liquid Crystals 7 Membranes and Molecules Oriented by Them 8 Weak Ordering and Biomolecular Studies 9 Relaxation Studies 10 Molecular Order 11 Structure and Conformation 12 References

534 534 534 536 540 542 544 545 546 55 1 552 556 557

Symbols and Abbreviations

These lists contain the symbols and abbreviations most frequently used in this volume, but they are not expected to be exhaustive. Some specialized notation is only defined in the relevant chapter. An attempt has been made to standardize usage throughout the volume as far as is feasible, but it must be borne in mind that the original research literature certainly is not standardized in this way, and some difficulties may arise from this fact. Trivial use of subscripts etc. is not always mentioned in the symbols listed below. Some of the other symbols used in the text, e.g. for physical constants such as h or r, or for the thermodynamic quantities such as H or S, are not included in the list since they are considered to follow completely accepted usage.

Symbols hyperline (electron-nucleus) interaction constant (i) hyperfine (electron-nucleus) interaction constant (ii) parameter relating to electric field effects on nuclear shielding (i) magnetic induction field (magnetic flux density) (ii) parameter relating to electric field effects on nuclear shielding static magnetic field of NMR or ESR spectrometer r.f. magnetic fields associated with U I ,u2 spin-rotation coupling constant of nucleus X (used sometimes in tensor form): c2''/3(ci 2C2,). components of C parallel and perpendicular to a molecular symmetry axis (i) self-diffusion coefficient (ii) zero-field splitting constant rotational diffusion tensor components of D parallel and perpendicular to a molecular symmetry axis internal diffusion coefficient overall isotropic diffusion coefficient electric field eigenvalue of &or a contribution to 2) nuclear or electronic g-factor magnetic field gradient element of matrix representation of 2 Hamiltonian operator-subscripts indicate its nature nuclear spin operator for nucleus i components of I; (i) ionization potential (ii) moment of inertia nuclear spin-spin coupling constant through n bonds (in Hz). Further information may be given by subscripts or in brackets. Brackets are used for indicating the species of nuclei coupled, e.g. J(13C, ' H ) or additionally, the coupling path, e.g. J ( P 0 C F ) reduced splitting observed in a double resonance experiment rotational quantum number reduced nuclear spin-spin coupling constant (see the notes concerning t l J ) eigenvalue of Ijz (magnetic component quantum number) equilibrium macroscopic magnetization of a spin system in the presence of Bo components of macroscopic magnetization

+

xvi

Symbols and Abbreviations

xvii

the number of average mol. wt. valencep orbital of atom A fractional population (or rotamers etc.) element of bond-order, charge-density matrix electric field gradient (i) nuclear quadrupole moment (ii) quality factor for an r.f. coil valence s-orbital of atom A electron density in St! at nuclear A (i) singlet state (ii) electron (or, occasionally, nuclear spin) cf. I (iii) ordering parameter for oriented systems (iv) overlap integral between molecular orbitals elapsed time (i) temperature (ii) triplet state coalescence temperature for an N M R spectrum the glass transition temperature (of a polymer) spin-lattice relaxation time of the X nuclei (further subscripts refer to the relaxation mechanism) spin-spin relaxation time of the X nucleus (further subscripts refer to the relaxation mechanism) inhomogeneity contribution to dephasing time for M , or M y total dephasing time for M , or M y ; (T;)-’ = 75’ + ( T i ) - ’ decay time following 900-~-9090 pplse sequences spin-lattice and spin-spin relaxation time of the X nuclei in the frame of reference rotating with B I dipolar spin-lattice relaxation time mole fraction of compound atomic number of atom A (i) nuclear spin wavefunction (eigenfunction off,) for a spin - l / 2 nucleus (ii) polarizability nuclear spin wavefunction (eigenfunction of 1;) for a spin -‘/2 nucleus magnetogyric ratio of nucleus X chemical shift of a nucleus of element X (positive when the sample resonates to high frequency of the reference). Usually in p.p.m. Kronecker delta ( = 1 if i = j , and = 0 otherwise) Dirac delta operator (i) time between field gradient pulses (ii) spectral width anisotropy in J (A J = Jll - JI, for axial symmetry) population difference between nuclear states change of difference in 6 full width (in Hz) of a resonance line at half-height (i) anisotropy in a(Ao = (TII - 01,for axial symmetry) (ii) differences in (T for two different situations (i) susceptibility anisotropy (A, = X I I- X I , for axial symmetry (ii) differences in electronegativities relative permittivity permittivity of a vacuum (i) nuclear Overhauser effect (ii) asymmetry factor (e.g. in e2qQ/h) (iii) refractive index (iv) viscosity magnetic dipole moment permeability of a vacuum Bohr magneton nuclear magneton Larmor precession frequency of nucleus i (in Hz) (i) spectrometer operating frequency (ii) Larmor precession frequency (general, or of bare nucleus)

xviii

Symbols and Abbreviations frequency of ‘observing’ r.f. magnetic field frequency of ‘irradiating’ r.f. magnetic field shielding parameter of nucleus i (used sometimes in tensor form). Usually in p.p.m. Subscripts may alternatively indicate contributions to o. components of u parallel and perpendicular to a molecular symmetry axis diagrammatic contribution to u paramagnetic contribution to u (i) pre-exchange lifetime of molecular species (ii) time between r.f. pulses (general symbol) correlation time mean time between molecular collisions in the liquid state angular momentum correlation time pulse duration translational magnetic relaxation correlation time (i) magnetic susceptibility (ii) electronegativity (iii) nuclear quadrupole coupling constant (= e 2 q Q / h ) carrier frequency in rad s-l as for v,,vo,vl,vz but in rad s-’ modulation angular frequency (in rad s-’) sample rotation (rad s-’)

Abbreviations (a) Physical properties a.f. audiofrequency atomic unit a.u. a.m. amplitude modulation b.c.c. body-centred cubic c.m.c. critical micelle concentration , electron diffraction e.d. e.f.g. electric field gradient f.c.c. face-centred cubic f.m. frequency modulation h.c.p. hexagonal close-packed h.f. hyperfine i.d. inside diameter intermediate frequency i.f. liquid crystalline 1.c. molecular weight mol. wt. outside diameter 0.d. parts per million p.p.m. radio frequency r.f. root mean square r.m.s. super-high frequency s.h.f. ultra-high frequency u.h.f. analog-to-digital converter ADC average excitation energy approximation AEE acquire AQ ARP adiabatic rapid passage bilinear rotation decoupling BIRD coupled cluster polarization propagator approximation CCPPA carbon-hydrogen correlation spectroscopy CH-COSY CHESS chemical shift selection coupled Hartree-Fock molecular orbital calculations CHF chemically induced dynamic electron polarization CIDEP

Sym bo 1s and Abbreviations CIDNP COSY CP CPMG CSA CSI

cw

DAC DD DEPT DLB DNP DQ DQF ECOSY EHT ENDOR. EOM ESR EXSY FC FID FLASH FPT FT GIAO HMQ HOHAHA HRPA IDESS IGLO INADEQUATE INDO INDO/S INDOR INEPT IR ISIS LIS LORG LSR MASS MBPT MEM MIND0 MQ MQC MQF NMR NOE NOESY NQCC NQR PFG PRE QF QPD REX ROESY RPA

chemically induced dynamic nuclear polarization correlation spectroscopy cross polarization Carr-Purcell pulse sequence. Meiboom-Gill modification chemical shielding anisotropy chemical shift imaging continuous wave digital-to-analog converter dipole-dipole (interaction or relaxation mechanism) distortionless enhancement by polarization transfer differential line broadening dynamic nuclear polarization double quantum double quantum filter exclusive correlation spectroscopy extended Huckel molecular orbital theory electron-nucleus double resonance equations of motion electron spin resonance exchange spectroscopy Fermi contact free induction decay fast low angle shot finite perturbation theory Fourier transform gauge included atomic orbitals heteronuclear multiquantum homonuclear Hartman-Hahn higher random phased approximation improved depth selective single surface coil spectroscopy individual gauge for different localized orbitals incredible natural abundance double quantum transfer experiment intermediate neglect of differential overlap intermediate neglect of differential overlap calculations for spectroscopy internuclear double resonance insensitive nuclei enhanced by polarization transfer infrared image selected in vivo spectroscopy lanthanide induced shift local origin lanthanide shift reagent magic angle sample spinning many body perturbation theory maximum entropy method modified INDO multiple quantum multiple quantum coherence multiple quantum filter nuclear magnetic resonance nuclear Overhauser enhancement nuclear Overhauser enhancement spectroscopy nuclear quadrupole coupling constant nuclear quadrupole resonance pulsed field gradient proton relaxation enhancement quadrupole moment/field gradient quadrature phase detection relativistically extended Huckel molecular orbital theory rotating frame Overhauser enhancement spectroscopy random phase approximation

xix

xx SCPT SD SECSY SEFT SLITDRESS SOPPA SPI SPT SR TART TOCSY

uv

WAHUHA ZQ ZQC

Symbols and Abbreviations self consistent perturbation theory spin dipolar spin echo correlation spectroscopy spin echo Fourier transform slice interleaved depth resolved surface coil spectroscopy second order polarization propagator approach selective population inversion selective population transfer spin rotation (interaction or relaxation mechanism) tip angle reduced TI imaging total correlation spectroscopy ultraviolet Waugh, Huber and Hiiberlen (cycle of pulses) zero quantum zero quantum coherence

(b) Chemicnl species* acetylacetonato acac adrenocorticotropic hormone (corticotropin) ACTH adenosine diphosphate ADP adenosine monophosphate AMP adenosine triphosphate ATP bovine serum albumin BSA cytidine monophosphate CMP cyclopen tadieny 1 CP dodecylammonium propionate DAP 1,2-dimethoxyethane DME dimeth y 1formamide DMF dimyristoyl-lecithin DML dimeth ylsiloxane DMS dimethyl sulfoxide DMSO deoxyribonucleic acid DNA 2,3-diphosphoglycerate DPG dipalmitoyl-lecithin DPI dipivaloylmethanato dPm diphen ylpicryl hydrazyl DPPH 2,2-dimethyl-2-silapentane-5-sulfonate (usually as the sodium salt) DSS di-t-butyl nitroxide DTBN N-(p-ethoxybenzy 1idene)-p-butylaniline EBBA ethylenediaminetetra-acetic acid EDTA ethylene-vin yl acetate EVA 1,1,1,2,2,3,3-heptafluoro-7,7-dimethyloctane-4,6-dionato fod HAB 4,4’-bis(hepty1)azoxybenzene hexamethylphosphoramide HMPA p-n-hep tyloxyazoxybenzene HOAB inositolhexaphosphate IHP KDP potassium dihydrogen phosphate N-(p-methox y benzy 1idene)-p-but ylaniline MBBA nicotinamide adenine dinucleotide (phosphate) NADH(P) N-methylformamide NMF p-azoxyanisole PAA pyrene butyric acid PBA poly(L-benzyl p-glutamate) PBLG PC phosphatidyl choline (lecithin) polychlorinated biphenyl PCB PDMS polydimethylsiloxane poly(methacry1ic acid) PMA PMMA poly(methy1 methacrylate) poly(oxymethy1ene) POM phosphatidy lserine PS * Lower case initials are used when the species is a ligand.

xxi

Symbols and Abbreviations PTFE PVC PVF PVP RNA SDS TAB TCNQ TFA THF TMS UTP

polytetrafluoroethylene poly(viny1 chloride) poly(viny1 fluoride) poly(viny1 pyrrolidone) ribonucleic acid (tRNA, transfer RNA) sodium dodecyl sulfate trimethylammonium bromide tetracyanoquinodimethane trifluoroacetic acid tetrahydro furan tetramethylsilane uridine triphosphate

Amino-udic residues Ala alanine Arg arginine Asn asparagine Asp aspartic acid CYS cysteine Gln glutamine Glu glutamic acid GlY gl ycine His histidine HY P hydroxyproline Ile isoleucine

Leu LYS Met Phe Pro Ser Thr TrP TYr Val

leucine lysine methionine phen ylalanine proline serine threonin tryptophan tyrosine valine

1 NMR Books and Reviews BY W. SCHILF

Rl R2

R3 R4 R5

R6

R7

2

R8 R9

S. Braun, H.-0. Kalinowski and S. Berger, ‘150 and More Basic NMR Experiments: A Practical Course - Second Expanded Edition’, WileyVCH, Weinheim, Germany, 1998 H. Chihara and N. Nakamura, ‘Landolt-Bornstein Group 111: Condensed Matter; Volume 39: Nuclear Quadrupole Resonance Spectroscopy Data: Supplement to Volumes IW20 and IW3 1’, Springer, Berlin, Germany, 1998 H. Duddeck, W. Dietrich and G. Toth, ‘Structure Elucidation by Modern NMR: A Workbook, Third, Revised and Updated Edition’, Steinkopff, Darmstadt, Germany, 1998 J. Klinowski and W. Kolodziejski, ‘Solid-state NMR Techniques’, World Scientific, Singapore, 1998 W. Martienssen, ‘Chemical Shifts and Coupling Constants for Fluorine-1 9, Nitrogen-1 5. (Numerical Data and Functional Relationships in Science and Technology, New Series, Group 111: Condensed Matter; Volume 35: Nuclear Magnetic Resonance (NMR) Data, Subvolume B)’, Springer, Berlin, Germany, 1998 J. Petersson, J. Gonzalo, J. Kobayashi, V. Lemanov and V. H. Schmidt, ‘Dielectric, Elastic and Thermal Properties, Computer Simulations and NMR of Ferroelectrics and Related Materials. (Dedicated to Professor Dr. Horst Mueser in Celebration of his 70th Birthday’, Gordon & Breach, Amsterdam, Netherlands, 1998 U. Weber and H. Thiele, ‘NMR Spectroscopy: Modern Spectral Analysis’, Wiley, New York, N.Y., 1998

Regular Reviews Series

Accounts of Chemical Research, vol. 31, 1998 N. A. J. Van Nuland, V. Forge, J. Balbach and C. M, Dobson, ‘RealTime NMR Studies of Protein Folding’, p. 773, vol. 32, 1999 S. Mukamel, A. Piryatinski and V. Chernyak, ‘Two-Dimensional Raman Echoes: Femtosecond View of Molecular Structure and Vibrational Coherence’, p. 145

Nuclear Magnetic Resonance, Volume 29 0The Royal Society of Chemistry, 2000 1

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Annual Reports on N M R Spectroscopy, ed. G. A. Webb, vol. 35, Academic, London, UK, 1998 S. Szymanski, ‘Proton Tunneling Effects in Metal Hydride NMR’, p. 1 N. Asakawa, T. Kameda, S. Kuroki, H. Kurosu, S. Ando, I. Ando and A. Shoji, ‘Structural Studies of Hydrogen-Bonded Peptides and Polypeptides by Solid-state NMR’, p. 55 W. S. Price, ‘NMR Imaging’, p. 139 J. Grandjean, ‘NMR Studies on Interfacial Phenomena’, p. 217 Annual Reports on N M R Spectroscopy, ed. G. A. Webb, vol. 36, Academic, London, UK, 1998 Y. Yamamoto, ‘NMR Study of Active Sites in Paramagnetic Hemoproteins’, p. 1 H. Saito, S. Tuzi and A. Naito, ‘Empirical Versus Nonempirical Evaluation of Secondary Structure of Fibrous and Membrane Proteins by Solid-state NMR: A Practical Approach’, p. 79 C. I. Ratcliffe, ‘Xenon NMR’, p. 123 Annual Reports on N M R Spectroscopy, ed. G. A. Webb, vol. 37, Academic, London, UK, 1998 K. G. Orrell, ‘Dynamic Spectroscopy in Inorganic and Organometallic Chemistry’, p. 1 A. M. Gil and C . P. Neto, ‘Solid-state NMR Studies of Wood and Other Lignocellulosic Materials’, p. 75 B. J. Goodfellow and A. L. Macedo, ‘NMR Structural Studies of IronSulfur Proteins’, p. 119 A. Ramos and H. Santos, ‘NMR Studies of Wine Chemistry and Wine Bacteria’, p. 179 C. Dalvit and J-M.Bohlen, ‘Proton Phase-Semsitive Pulsed Field Gradient Double-Quantum Spectroscopy’, p. 204 Annual Review of Biophysics and Biomolecular Structure, vol. 27, 1998 A. Wlodawer and J. Vondrasek, ‘Inhibitors of HIV-1 Protease: A Major Success of Structure-Assisted Drug Design’, p. 249 K. H. Gardner and L. E. Kay, ‘The Use of 2H, 13C, I5N Multidimensional NMR to Study the Structure and Dynamics of Proteins’, p. 357 G. Varani and K. Nagai, ‘RNA Recognition by RNP Proteins During RNA Processing’, p. 407 Carbohydrate Chemistry, Specialist Periodical Reports, ed. R. J. Ferrier, The Royal Society of Chemistry, Cambridge, UK vol. 30, 1998 Anon. (UK), ‘NMR Spectroscopy and Conformational Features’, p. 316

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Chemical Reviews, vol. 98, 1998 V. T. D’Souza and K. B. Lipkowitz, ‘Cyclodextrins: Introduction’, p. 1741 H-J. Schneider, F. Hacket, V. Ruediger and H. Ikeda, ‘NMR Studies of Cyclodextrins and Cyclodextrin Complexes’, p. 1755 vol. 99, 1999 J. Y. Corey and J. Braddock-Wilking, ‘Reaction of Hydrosilanes with Transition-Metal Complexes: Formation of Stable Transition-Metal Silyl Compounds’, p. 175 T. Helgaker, M. Jaszunski and K. Ruud, ‘Ab Initio Methods for the Calculation of NMR Shielding and Indirect Spin-Spin Coupling Constants’, p. 293 Coordination Chemistry Reviews, vol. 174, 1998 M. R. McClure, K. V. Jung and J. H. Worrell, ‘High-Resolution NMR Analysis of Cobalt( 111) Complexes with 1, 8-Diamino-3, 6-dithiaoctane’, p. 33 vol. 176, 1998 S. Sato, 0. Takahashi and N. Furukawa, ‘Recent Progress in Hypervalent Organochalcogenuranes Bearing Four Carbon Ligands’, p. 483 V O ~178-180, . Pt.1, 1998 M. Fourmigue, ‘Mixed CyclopentadienyYDithiolene Complexes’, p. 823 C. Bremard, ‘Organometallic Chemistry of Group VI Metals in the Void Space of Zeolites’, p. 1647 J-M. Basset, F. Lefebvre and C. Santini, ‘Surface Organometallic Chemistry: Some Fundamental Features Including the Coordination Effects of the Support’, p. 1703 V O ~178-180, . Pt.2, 1998 E. Graf and M. W. Hosseini, ‘Cryptands Revisited. Design, Synthesis, Complexation Behavior and Structural Analysis of Borocryptands’, p. 1193 vol. 182, 1999 D. Rehder, ‘The Coordination Chemistry of Vanadium as Related to its Biological Functions’, p. 297 ’

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Current Medical Chemistry, vol. 5 , 1998 W. H. Gmeiner, ‘NMR Spectroscopy as a Tool to Investigate the Structural Basis of Anticancer Drugs’, p. I 15 vol. 6, 1999 A. Janczuk, J. Li, W. Zhang, X. Chen, Y. Chen, J. Fang, J. Wang and P. G. Wang, “a-Gal Oligosaccharides: Chemistry and Potential Biomedical Application’, p. 155 Current Opinion on Biotechnology, vol. 10, 1999 H. Lida, G. Jia and J. W. Lown, ‘Rational Recognition of Nucleic Acid Sequences’, p. 29

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P. A. Keifer, ‘NMR Tools for Biotechnology’, p. 34 G. C. K. Roberts, ‘NMR Spectroscopy in Structure-Based Drug Design’, p. 42 A. Watts, ‘NMR of Drugs and Ligands Bound to Membrane Receptors’, p. 48 J. M. Moore, ‘NMR Screening in Drug Discovery’, p. 54 Current Opinion on Chemistry and Biology, vol. 2, 1998 N. G. Walter and J. M. Burke, ‘The Hairpin Ribozyme: Structure, Assembly and Catalysis’, p. 24 M. J. Shapiro and J. R. Wareing, ‘NMR Methods in Combinatorial Chemistry’, p. 372 Current Opinion in Colloid & Interface Science, vol. 3, 1998 G. Lindblom and P-0. Quist, ‘Protein and Peptide Interactions with Lipids: Structure, Membrane Function and New Methods’, p. 499 Current Opinion in Drug Discovery & Development, vol. 1, 1998 H. Kubinyi, ‘Structure-Based Design of Enzyme Inhibitors and Receptor Ligands’, p. 4 H. Kubinyi, ‘Combinatorial and Computational Approaches in Structure-Based Drug Design’, p. 16 Current Opinion on Solid State & Materials Science, vol. 3, 1998 L. W. Jelinski, ‘Establishing the Relationship between Structure and Mechanical Function in Silks’, p. 237 I. Farnan, ‘Structure and Dynamics in Glassy and Molten Silicates’, p. 371 Current Opinion on Structural Biology, vol. 8, 1998 A. McGough, ‘F-Actin-Binding Proteins’, p. 166 S. A. Woodson and N. B. Leontis, ‘Structure and Dynamics of Ribosomal RNA’, p. 294 G. M. Clore and A. M. Gronenborn, ‘NMR Structure Determination of Proteins and Protein Complexes Larger then 20 kDa’, p. 564 V. Dotsch and G. Wagner, ‘New Approaches to Structure Determination by NMR Spectroscopy’, p. 619 D. A. Case, ‘The Use of Chemical Shifts and Their Anisotropies in Biomolecular Structure Determination’, p. 624 R. A. Laskowski, M. W. MacArthur and J. M. Thornton, ‘Validation of Protein Models Derived from Experiment’, p. 631 F. M. Marassi and S. J. Opella, ‘NMR Structural Studies of Membrane Proteins’, p. 640 vol. 9, 1999 J. Baum and B. Brodsky, ‘Folding of Peptide Models of Collagen and Misfolding in Disease’, p. 122

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Progress in N M R Spectroscopy,ed. J. W. Emsley, J. Feeney and L. H. Sutcliffe, vol. 32, 1998 M. Nilges and S. I. O’donoghue, ‘Ambiguous NOES and Automated NOE Assignment’, p. 107 A. P. M. Kentgens, ‘Off-Resonance Nutation Nuclear Magnetic Resonance Spectroscopy of Half-Integer Quadrupolar Nuclei’, p. 141 G. Wider, ‘Technical Aspects of NMR Spectroscopy with Biological Macromolecules and Studies of Hydration in Solution’, p. 193 C. H. Arrowsmith and Y-S.Wu, ‘NMR of Large (>25 kDa) Proteins and Protein Complexes’, p. 277 S. S. Wijmenga and B. N. M.Van Buuren, ‘The Use of NMR Methods for Conformational Studies of Nucleic Acids’, p. 287 vol. 33, 1998 P. Bachert, ‘Pharmacokinetics Using Fluorine NMR in Vivo’, p. 1 M. Luhmer and J. Reisse, ‘Quadrupole NMR Relaxation of the Noble Gases Dissolved in Simple Liquids and Solutions. A Critical Review of Experimental Data in the Light of Computer Simulation Results’, p. 57 A. Sodickson and D. G. Cory, ‘A Generalized k-Space Formalism for Treating the Spatial Aspects of a Variety of NMR Experiments’, p. 77 B. J. Stockman, ‘NMR Spectroscopy as a Tool for Structure-Based Drug Design’, p. 109 M. J. P. Ferreira, V. P. Emerenciano, G. A. R. Linia, P. Romoff, P. A. T. Macari and G. V. Rodrigues, ‘13C NMR Spectroscopy of Monoterpenoids’, p. 153 M. W. F. Fischer, A. Majumdar and E. R. P. Zuiderweg, ‘Protein NMR Relaxation: Theory, Applications and Outlook’, p. 207 J. B. Miller, ‘NMR Imaging of Materials’, p. 273 vol. 34, 1999 J. H. Kristensen, H. Bildsoe, H. J. Jakobsen and N. C . Nielsen, ‘Application of Lie Algebra to NMR Spectroscopy’, p. 1 M. E. Smith and E. R. H. Van Eck, ‘Recent Advances in Experimental Solid State NMR Methodology for Half-Integer Spin Quadrupolar Nuclei’, p. 159

Edited Books and Symposia

ACS Symposia Series R 73 F. A. Walker, ‘Advances in Single- and Multidimensional NMR Spectroscopy of Paramagnetic Metal Complexes’, 1998,692, 30 R74 D. F. Lawson, D. R. Brumbaugh, M. L. Stayer, J. R. Schreiffler, T. A. Antkowiak, D. Saffles, K. Morita, Y. Ozawa and S. Nakayama, ‘Anionic Polymerization of Dienes Using Homogeneous Lithium Amide (N-Li) Initiators, and Determination of Polymer-Bound Amines’, 1998,696, 77

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L. L. Landucci and S. Ralph, ‘Biomimetic Initiation of Lignol Dehydropolymerization with Metal Salts’, 1998, 697, 148 R76 D. Robert, E. Ammalahti, M. Bardet, G. Brunow, I. Kilpelainen, K. Lundquist, V. Neirinck and N. Terashima, ‘Improvement in NMR Structural Studies of Lignin through Two- and Three-Dimensional NMR Detection and Isotopic Enrichment’, 1998,697,237 R77 A. F. A. Wallis, ‘Structural Diversity in Lignans and Neolignans’, 1998, 697,323 R78 P. Burauel, A. Wais and F. Fuhr, ‘Soil-Bound Residues’, 1998,699, 177 R79 R. Tressll, G. Wondrak, E. Kersten, R. P. Kriiger and D. Rewicki, ‘Identification of Maillard Type Polymers with Antioxidative Activity’, 1998,702,209 R80 J. Feldthusen, B. Ivan and A. H. E. Muller, ‘Novel Functional Copolymers by Combination of Living Carbocationic and Anionic Polymerizations’, 1998, 704, 121 R8 1 K. J. Kramer, T. L. Hopkins and J. Schaefer, ‘Analysis of Interactable Biological Samples by Solids NMR’, 1998,707, 14 R82 S. Derenne, H. Knicker, C. Largeau and P. Hatcher, ‘Timing and Mechanisms of Changes in Nitrogen Functionality During Biomass Fossilization’, 1998,707, 243 R83 H. Knicker and I. Kogel-Knabner, ‘Soil Organic Nitrogen Formation Examined by Means of NMR Spectroscopy’, 1998,707,339 R84 X. X. Zhu, L. Masaro, J.-M. Petit, B. Roux and P. M. Macdonald, ‘Self-Diffusion of Solvents and Solute Probes in Polymer Solutions and Gels: The Use of a New Physical Model of Diffusion’, 1998,709,223 R85 L. Pettersson and K. Elvingson, ‘Studies of Vanadate-Organic Ligand Systems Using Potentiometry and NMR Spectroscopy’, 1998,711, 30 R86 V. Conte, F. Di Furia and S. Moro, ‘From the Speciation of Peroxovanadium Complexes in Aqueous Solution to the Chemistry of Haloperoxidases’, 1998,711, 136 R87 G. D. Rayson, L. R. Drake, H. Xia, S. Lin and P. J. Jackson, ‘Biologically Generated Materials for Metal-Ion Binding: Answers to Some Fundamental Chemical Questions’, 1999,716,260 R88 J. H. Fitzgerald, ‘Solid-state NMR Spectroscopy of Inorganic Materials’, 1999,717, 1 R89 R. M. Wenslow, K. Fiske and K. T. Mueller, ‘An Overview of SolidState NMR Correlation Experiments in Phosphate Glass Systems’, 1999,717,228 R90 M. E. Smith, ‘Solid-state NMR of Nitrides and Oxynitrides’, 1999,717, 377 R9 1 T. Liu, M. Ryan, F. W. Dahlquist and 0. H. Griffith, ‘Characterization of the Histidine Residues of B. Cereus Phosphatidylinositol-Specific Phospholipase C by NMR’, 1999,718,9 1 R75

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Analytical Methods in Food Authentication, ed. P. R. Ashurst and M. J. Dennis, Blackie, London, UK, 1998 I. J. Colquhoun and M. Lees, ‘Nuclear Magnetic Resonance Spectroscopy’, p. 36 Bioorganic Chemistry. Peptides and Proteins, ed. S. M. Hecht, Oxford University Press, New York, N.Y., 1998 C. W. Carter Jr., ‘Protein Structure’, p. 153 M. A. Massiah, P. R. Blake and M. F. Summers, ‘Nucleic Acid Interactive Protein Domains that Require Zinc’, p. 258,498 Borane, Carboborane, Carbocation Continuum, ed. J. Casanova, Wiley, New York, N.Y., 1998 G. K. S. Prakash, G. Rasul, A. K. Yudin and R. E. Williams, ‘Extension of the Borane-Carbocation Continuum to Cage Systems’, p. 147 J. W. Bausch and A. J. Tebben, ‘Computation Studies of NinoC4B7H Carboranes and Nido-%Vortex Baranes and Carboranes’, p. 217 T. Onak, ‘Applications of IGLO/GIAO NMR Computations of Carborane Systems: Products of Trimethylamine/Closo-CarboraneReactions; Carborane I3C Chemical Shifts’, p. 247 S. Hermanek, J. Machacek, J. Fusek and V. Blechta, ‘Importance of “B-*H Coupling Constants in Assigning the ‘B-Signals’, p. 367 Chemistry of Organic Silicon Compounds Vol. 2, Pt. 1 ., ed. Z . Rappoport and Y. Apeloig, Wiley, Chichester, UK, 1998 Y. Takeuchi and T. Takayama, ‘29SiNMR Spectroscopy of Organosilicon Compounds’, p. 267

Density-Functional Methods in Chemistry and Materials Science, ed. M. Springborg, Wiley, Chichester, UK., 1997 RlOO M. Springborg, ‘Some Recent Density-Functional Studies of Molecular Systems’, p. 43 Dicoordinated Carbocations, ed. Z. Rappoport and P. J. Stang, Wiley, Chichester, UK, 1997 RlOl H-U. Siehl, ‘NMR Spectroscopic Characterization [of Vinyl Cations]’, p. 189 Fundamentals of Adsorption - FOA6, ed. F. Meunier, Elsevier, Paris, 1998 R102 J. Karger and H. Schafer, ‘The Message of Pulsed Field Gradient NMR on Molecular Diffusion and Structural Confinement in Nanoporous Materials’, p. 1179

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Handbook of Liquid Crystals, vol. I , ed. D. Demus, Wiley-VCH Verlag GmbH, Weinheim, Germany, 1998 R103 C. Schmidt, H. W. Spiess, ‘Magnetic Resonance in Liquid Crystals’, p. 595 Handbook of Liquid Crystals, vol. 2A, ed. D. Demus, Wiley-VCH Verlag GnibH, Weinheim, Germany, 1998 R104 R. Blinc and I. Musevic, ‘Dynamic Properties of Nematic Liquid Crystals’, p. 170 Lipid Analysis in Oils Fats, ed. R. J. Hamilton, Blackie, London, UK, 1998 R105 B. W. K. Diehl, ‘Multinuclear High-Resolution Nuclear Magnetic Resonance Spectroscopy’, p. 87 Membrane Protein Assembly, ed. G. Von Heijne, Landes, Austin, Tex., 1997 R106 M. Montal and S. J. Opella, ‘Assembly Properties of Model Transmembrane Channel Peptides’, p. 39

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Methods for Structure Elucidation by High-Resolution N M R , Analytical Spectroscopy Library, vol. 8, ed. G. Batta, K. E. Kover and C. Szantay, Jr., Elsevier Science B. V., Amsterdam, Netherlands, 1997 S. J. F. Vincent and C. Zwahlen, ‘Selective Pulses in NMR’, p. 1 P. Bigler, ‘Multiple Selective Excitation: A Meth.od to Improve the Efficiency of Selective 1D and 2D Experiments’, p. 19 D. Uhrin, ‘Concatenation of Polarization Transfer Steps in 1D Homonuclear Chemical Shift Correlated Experiments. Application to Oligoand Polysaccharides’, p. 5 1 D. Canet and C. Roumestand, ‘The DANTE-Z Experiment’, p. 121 G. Otting, ‘Use of High Power Spin-Lock Purge Pulses in High Resolution NMR Spectroscopy’, p. 149 I. Pelczer and K. D. Bishop, ‘Optimal Acquisition and Presentation of HoMQC Spectra’, p. 187 G. A. Morris and H. Barjat, ‘High Resolution Diffusion Ordered Spectroscopy’, p. 209 A. D. Bain and G. J. Duns, ‘Chemical Exchange Measurements in NMR’, p. 227 N. Juranic, Z . Zolnai and S. Macura, ‘Homonuclear Two-Dimensional Cross-Relaxation Spectroscopy’, p. 265 G. A. Morris and H. Barjat, ‘Reference Deconvolution’, p. 303 D. Canet, P. Mutzenhardt and J. -B. Robert, ‘The Super Fast Inversion Recovery (SUFIR) Experiment’, p. 317 J. Kowalewski and L. Maler, ‘Measurements of Relaxation Rates for Low Natural Abundance I=1/2 Nuclei’, p. 325

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N A T O Advanced Study Institute Series, Series C, vol. 491, 1997 R119 J. Karger, ‘NMR Studies of Diffusion in Porous Solids’, p. 297 R120 M. A. Springuel-Huet, ‘lZ9XeNMR of Physiosorbet Xenon. Principles’, p. 331 R121 M. A. Springuel-Huet, ‘‘29XeNMR of Physiosorbet Xenon. Applications’, p. 349 vol. 510, 1998 R122 L. E. Kay, ‘The Development of NMR Methods to Study Protein Structure and Dynamics’, p. 285 vol. 519, 1999 R123 D. N. Reinhoudt, P. Timmerman and F. C. J. M. Van Veggel, ‘NonCovalent Synthesis and Characterization of Large Supramolecular Assemblies’, p. 51 R124 J. A. Ripmeester, I. L. Moudrakovski and C . I. Ratcliffe, ‘Gas Hydrates as Self-Assembled Templated Lattices’, p. 265 Series 3, vol. 49, 1998 R125 P. Panissod, ‘Structural and Magnetic Investigations of Ferromagnets by NMR. Application to Magnetic Metallic Multilayers’, p. 225 vol. 55, 1998 R126 V. A. Ivanov, E. A. Ugolkova and M. Ye. Zhuravlev, ‘Itinerant Electrons and Superconductivity in Exotic Layered Systems’, p. 39 1 Neocarzinostatin. The Past, Present, and Future of Anticancer Drugs. ed. H. Maeda, K. Edo and N. Ishida, Springer, Tokyo, Japan, 1997 R127 H. Takashima and Y. Kobayashi, ‘Solution Structure of Neocarzinostatin Determined by Homonuclear Two-Dimensional Nuclear Magnetic Resonance’, p. 83 Nuclear Methods in Mineralogy and Geology: Techniques and Applications, ed. A. Vertes, S. Nagy and K. Suvegh, Plenum, New York, N.Y, 1998 R128 K. Tompa, ‘Nuclear Magnetic Resonance in Geosciences’, p. 251 Phase Transitions in Complex Fluids, ed. P. Toledano and A. M. Figueiredo Neto, World Scientific, Singapore, 1998 R129 B. Halle and I. Furo, ‘NMR Studies of Lyotropic Liquid Crystals’, p. 8 1 R130 G. Chidichimo and F. P. Nicoletta, ‘Phase Transitions in Tilted Mesophases’, p. 271 R131 D. J. Pusiol, ‘Potential Applications of Field Cycling NMR SpinLattice Relaxation Dispersion Studies’, p. 41 1 pH and Brain Function, ed. K. Kaila and B. R. Ransom, Wiley-Liss, New York, N.Y., 1998 R132 J. W. Prichard, D. L. Rothman and 0.’A. C . Petroff, ‘Brain pH Measurement by Nuclear Magnetic Resonance Spectroscopy’. P. 153

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R133 R. A. Kauppinen and S. R. Williams, ‘Use of NMR Spectroscopy in Monitoring Cerebral pH and Metabolism During Systemic and Focal Acid-Base Disturbances’, p. 605 Polysaccharides. Structural Diversity and Functional Versatility, ed. S. Dumitriu, Dekker, New York, N.Y., 1998 R 134 K. Kajiwara and T. Miyamoto, ‘Progress in Structural Characterization of Functional Polysaccharides’, p. 1 Principles of Perinatal-Neonatal Metabolism (2nd Ed.), ed. R. M. Cowett, Springer, New York, N.Y., 1998 R135 D. M. Bier, ‘Methodology for the Study of Metabolism: Kinetic Techniques’, p. 3 Prions, ed. D. A. Harris, Horizon Scientific Press, Norfolk, UK., 1999 R136 R. Glockshuber, S. Hornemann, R. Riek, M. Billeter, G. Wider, S. Liemann, R. Zahn and K. Wuthrich, ‘Folding and Three-Dimensional NMR Structure of the Recombinant Cellular Prion Protein from the Mouse’, p. 1 Proceedings of an American Chemical Society Symposium held in Orlando, Florida August 25-30, 1996, [Interfacial Aspects of Multicomponent Polymer Materials], ed. D. J. Lohse, T. P. Russel and L. H. Sperling, Plenum, New York, N.Y., 1997 R137 N. Zumbulyadis and C. J. T. Landry, ‘Solid State NMR Studies of Polymer Interfaces’, p. 73 Proceedings of an ASI School held in Erice, Italy, 1997, [Pair Correlations in Many-Fermion Systems], ed. V. Z. Kresin, Plenum, New York, N.Y., 1998 R138 W. D. Knight, ‘Hyperfine Interactions in Metals’, p. 57 Proceedings of 17th Conference on Particle Acceleration 1997, ed. M. Comyn, Institute of Electrical and Electronics Engineers, New York, N.Y. 1998 R139 A. N. Zelenski, C. D. P. Levy, P. W. Schmor, W. T. H. Van Oers, G. W. Wight and G. Dutto, ‘OPPIS Development at TRIUMF’, p. 2781 Proceedings of the 24th European Peptide Symposium 1996, ed. R. Ramage and R. Epton, Mayflower Scientific, Kingswinford, UK, 1998 R140 D. Winkler, R. D. Stinger, B. Hoffmann, G. Zahn, W. Hohne, J. Rappsilber, P. Schmieder, H. Oschkinat and J. Schneider-Mergener, ’Elucidation of the Interaction of an Anti-TGFa Antibody with its Peptide Epitope Using Combinatorial Cyclic Peptide Libraries, Binding Studies, NMR, Computer Modeling and Flexible Docking Techniques’, p. 913

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R141 V. Wray, W. Blankelfeldt, K. Nokihara and S. Naruse, ‘Solution Structure Comparison of the VIPIPACAP Family of Peptides by NMR Spectroscopy’, p. 92 1 Proceedings of the First Asia-PaciJic EPRIESR Symposium, Hong Kong, 1997, [Modern Applications of EPRIESR: From Biophysics to Materials Science], ed. C. Z. Rudowicz, P. K. N. Yu and H. Hiraoki, Springer Verlag, Singapore, 1998 R 142 S. K. Misra, ‘Spin-Lattice Relaxation Times in Amorphous Materials as Effected by Exchange Interactions and TLS Centers’, p. 406 R143 J. Tritt-Goc, N. Pislewski and S. K. Hoffmann, ‘Investigation of the Ground and Metastable State of Nitroprusside Crystals by NMR and EPR Spectroscopy’, p. 487 R144 S. K. Hoffmann, W. Hilczer, J. Goslar and M. Gramza, ‘Minima in Temperature Variations of the Electron Spin Phase Memory Time TM Due to Molecular Motions - ESE Studies in Single Crystals’, p. 645 Proceedings of the First International Symposium, 1997 [Physical, Chemical and Biological Properties of Stable Water (IETM) Clusters], ed. S. Y. Lo and B. Bonavida, World Scientific, Singapore, 1998 R145 S. Y. Lo, ‘Survey of IETM Clusters’, p. 3 Proceedings of’ 1 7th International Conference on Cryogenic Engineering, ed. D. Dew-Hughes, R. G. Scurlock and J. H. P. Watson, Institute of Physics Publishing, Bristol, UK, 1998 R146 S. J. Welton, K. Cantrell, W. D. Markiewicz and S. W. Van Sciver, ‘Design of the NHMFL 900 MHz NMR Magnet Cryostat’, p. 875 Proceedings of the 2nd International Conference of Filled Polymers and Fillers, EUROFZLLERS 97, British Plastic Federation, London, UK., 1997 R147 V. J. Mcbrierty, ‘Elastomer-Carbon Black Filler Interactions: Their Characterization by Magnetic Resonance Techniques’, p. 67 Proceedings of the 2rtd International Conference, held in Bergamo in June 1996, [Nuclear Magnetic Resonance Spectroscopy of Cement-Based Materials]. ed. P. Colombet, A-R. Grimmer, H. Zanni and P. Sozzani, Springer, Berlin, Germany, 1998 R 148 D. Massiot, B. Touzo, D. Trumeau, C. Magnenet, V. Montouillout, P. Florian, C. Bessada, A. Douy, J-P.Coutures and J. Virlet, ‘New Progress in High Resolution 27AIand 1 7 0 Solid State NMR (MAS and MQ-MAS) of Aluminium Bearing Phases’, p. 89 R149 X. Cong, R. J. Kirkpatrick, J. L. Yarger and P. F. Mcmillan, ‘The Structure of Calcium Silicate Hydrate: NMR and Raman Spectroscopic Results’, p. 143

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Proceedings of the International Conference on Neural Networks, vol. 3, Institute of Electrical and Electronics Engineers, New York, N.Y., 1998 R150 P. J. G. Lisboa, N. M. Branston, W. El-Derady and A. Vellido, ‘Tissue Characterization with NMR Spectroscopy: Current State and Future Prospects for Application of Neural Networks Analysis’, p. 1385 Proceedings of 4th International Corference on Physical Chemistry ‘98, ed. S. Ribnikar and S. Anic, Society of Physical Chemists of Serbia, Belgrade, Yugoslavia, 1998 R 151 G. Gacic, ‘In Vivo EPR Spectroscopy: Principles and Applications’, p. 291 Proceedings of 9th International IUPA C Symposium on Mycotoxins and Phycotoxins, 1997, [Mycotoxins and Phycotoxins - Developments in Chemistry, Toxicology and Food Safety], ed. M. Miraglia, H. P. van Egmont, C. Brera and J. Gilbert, Alaken Inc., Fort Collins, Col., 1998 R 152 K. Sivonen, ‘Toxins Produced by Cyanobacteria’, p. 547 Proceedings of the International Wheat Quality Conference., ed. J. L. Steele and 0. K. Chung, Grain Industry Alliance, Manhattan, Kan., 1997 R153 P. A. Seib, ‘Wheat Starch as a Quality Determinant’, p. 61 Proceedings of the International Winterschool on Electronic Properties of Novel Materials, 1997, [Molecular Nanostructures], ed. H. Kuzmany, J. Fink, M. Mehring and S. Roth, World Scientific, Singapore, 1998 R154 A. Curioni and W. Andreoni, ‘Recent Progress in the Theoretical Approach to Azafullerenes’, p. 81 Proceedings of International Workshop, Benediktbeuern, Germany, 28-30 October 1996, ed. T. Von Egidy, F. J. Hartmann, D. Habs, K. E. G. Lobner and H. Nifenecker, World Scientific, Singapore, 1997 R 155 D. Forkel-Wirth, A. Burchard, M. Deicher, R. Magerle and U. Koster, ‘Solid-state Physics and PIAFE’, p. 293 Proceedings of the Second Royal Society - Unilever Indo - UK Forum in Materials Science and Engineering, [Dynamics of Complex Fluids], ed. M. J. Adams, R. A. Mashelkar, J. R. A. Pearson and A. R. Rennie, Imperial College Press, London, UK, 1998 R156 C. Schmidt, S. Muller and H. Siebert, ‘Deuterium NMR Investigations of Liquid Crystals During Shear Flow’, p. 188 Ribonucleases H , ed. R. J. Crouch and J-J. Toulme, INSERM, Paris, France, 1998 R157 S. Kanaya, ‘Enzymic Activity and Protein Stability of E. coli Ribonuclease HI’, p. 1

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RNA: Protein Interaction: A Practical Approach, ed. C. W. J. Smith, Oxford University Press, New York, N.Y., 1998 R158 S. R. Price, C. Oubridge, G. Varani and K. Nagai, ‘Preparation of RNA:Protein Complexes for X-Ray Crystallography and NMR’, p. 37 Separation of Fullerenes by Liquid Chromatography, ed. K. Jinno, Royal Society of Chemistry, Cambridge, UK., 1999 R159 K. Jinno, H. Ohta and Y. Sato, ‘Separation of Fullerenes by LC with Octadecyl Silica (ODS) Stationary Phases’, p. 49 Stable Isotopes: The Integration of Biological, Ecological and Geochemical Processes, ed. H. Griffiths, Bios Scientific Publishers, Oxford, UK, 1998 R160 J. Schleucher, ‘Intramolecular Deuterium Distribution and Plant Growth Conditions’, p. 63

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Structure, Motion, Interaction and Expression of Biological Macromolecules, Proceedings of Tenth Conversation in Biornolecular Structure & Dynamics, 1997, ed. R. H. Sarma and H. M. Sarna, Adenine Press, Schenactady, N.Y, 1998 N. B. Ulyanov, V. I. Ivanov, E. E. Minyat, E. B. Khomyakova, M. V. Petrova, K. Lesiak and T. L. James, ‘Immobile Slipped-Loop Structures of DNA in Solution’, vol. 1, p. 75 J. Careres-Cortes, H. Sugiyama, K. Ikudome, I. Saito and A. H.-J. Wang, ‘NMR Studies of the Anticancer Drug Popleomycin and Its Complexes with DNA’, vol. 1, p. 207 J. W. Lown, ‘Structural Studies on Minor Groove Binder: DNA Interactions’, vol. 1, p. 233 D. Fushman and D. Cowburn, ‘Studying Protein Dynamics with NMR Relaxation’, vol. 2, p. 63 P. Catasti, X. Chen, E. M. Bradbury and G. Gupta, ‘Structure-Function Relationship of the Insulin-Linked Polymorphic Region: Implications in Length Polymorphism and Gene Regulation’, vol. 2, p. 237

Structure and Transport Properties in Organized Polymeric Materials., ed. E. Chiellini, M. Giordano and D. Leporini, World Scientific, Singapore, 1997 R166 C. Boeffel and H. W. Spiess, ‘NMR Studies of Polymeric Liquid Crystals with Different Molecular Architecture’, p. 125 R167 N. J. Heaton and G. Kothe, ‘NMR Studies of Collective Motions and Viscoelastic Properties in Polymeric Liquid Crystals’, p. 161 The Surface Properties of Silicas, ed. A. P. Legrand, Wiley, Chichester, UK, 1998 R168 H. Hommel, A. P. Legrand, C. Doremieux and J-B. d’Espinose De La Caillerie, ‘Hydroxyl Groups on Silica Surfaces. NMR Spectroscopies’, p. 235

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Tunneling Systems in Amorphous and Crystalline Solids, ed. P. Esquinazi, Springer, Berlin, Germany, 1998 R169 G. Cannelli, R. Cantelli, F. Corder0 and F. Trequattrini, ‘Tunneling of H and D in Metals and Semiconductors’, p. 389-458, 571-591 Vitamin B12 and B12-Proteins,ed. B. Kraeuther, D. Arigoni and B. T. Golding, Wiley-VCH Verlag GmbH, Weinheim, Germany, 1998 R170 A. R. Battersby, ‘B12 Biosynthesis in an Aerobic Organism: How the Pathway Was Elucidated’, p. 47 R171 H. Bothe, G. Broker, U. Muller, I. Schall, S. Textor, B. T. Golding and W. Buckel, ‘Mechanisms of Coenzyme B 12-DependentCarbon-Carbon and Carbon-Oxygen Rearrangements’, p. 237

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Reviews in Periodicals

R172 J-L. Abad, A. J. Shallop, B. L. Gaffney and R. A. Jones, ‘Use of 13C Tags with Specifically 5N-Labeled DNA and RNA’, Biopolymers, 1998,48,57 R173 K. Akimoto, ‘Present States in Quality Evaluations of Horticultural Products’, J.Jpn.SOC.Hortic.Sci., 1998, 67, 1 171 R 174 K. Albert, ‘Correlation Between Chromatographic and Physicochemical Properties of Stationary Phases in HPLC: C30 Bonded ReversedPhase Silica’, TrAC, Trends.Anal. Chern., 1998, 17, 648 R175 K. Albert, M. Dachtler, T. Glaser, H. Haendel, T. Lacker, G. Schlotterbeck, S. Strohschein, L. H. Tseng and U. Braumann, ‘Online Coupling of Separation Techniques to NMR’, J. High Resolut. Chromatogr., 1999, 22, 135 R176 M. I. Altbach, A. J. Kim, X. Wu, G. L. Garner, F. R. Fronczek and L. G. Butler, ‘Applications of Solid-state NMR in Organometallic Chemistry’, Trans.Am. Crystallogr.Assoc., 1997,31, 39 R177 I. Ando, S. Matsukawa, A. Yamazaki, Y. Hotta and N. Tanaka, ‘Structures and Dynamics of Polymer Gels Observed by ‘H NMR Imaging and ‘H PGSE NMR Methods’, Wiley Polym.Networks Group Rev.Ser., 1998, 1.331 R178 C . Antz and B. Fakler, ‘Fast Inactivation of Voltage-Gated K+ Channels: From Cartoon to Structure’, News Physiol.Sci., 1998, 13, 177 R179 J. M. Aramini and M. W. Germann, ‘NMR Studies of DNA Duplexes Containing a-Anomeric Nucleotides and Polarity Reversals’, Biochem.Cell Biol., 1998, 76,403 R180 J. M. Aramini and H. J. Vogel, ‘Quadrupolar Metal Ion NMR Studies of Metalloproteins’, Biochem. Cell Biol., 1998,76,210 R 18 1 J. R. P. Arnold and J. Fisher, ‘Conformational Analysis’, Nucl. Magn. Reson., 1998,27, 370 R182 K. Asayama, Y. Kitaoka, G.-Q. Zheng, K. Ishida, K. Magishi,

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Y. Tokunaga and K. Yoshida, ‘NMR of High-T, Supercondoctors’, Int.J.Mod.Phys. B, 1998,12, 3207 A. Bacher, C. Rieder, D. Eichinger, D. Arigoni, G. Fuchs and W. Eisenreich, ‘Elucidation of Novel Biosynthetic Pathways and Metabolite Flux Patterns by Retrobiosynthetic NMR Analysis’, FEMS Microbiol. Rev., 1998, 22, 567 S. Bagby, C. H. Arrowsmith and M. Ikura, ‘New Perceptions of Transcription Factor Properties from NMR’, Biochem. Cell Biol., 1998, 76,368 A. D. Bain, ‘Blurring the Distinction between Slow and Intermediate Chemical Exchange’, Biochem. Cell Biol., 1998,76, 171 V. I. Bakhmutov and E. V. Vorontsov, ‘NMR Spin-Lattice Relaxation Approaches to Characterization of Transition Metal Hydride Complexes in Solution’, Rev. Inorg. Chem., 1998, 18, 183 M. A. Baldwin, T. L. James, F. E. Cohen and S. B. Prusiner, ‘The Three-Dimensional Structure of Prion Protein: Implications for Prion Disease’, Biochem.SOC.Trans., 1998,26,481 P. Barlow, ‘The Determination of Protein Structure by Nuclear Magnetic Resonance’, Protein (Stamford, Conn.), 1997,1,269 K . L. Behar, ‘Alternatives to Mass Spectrometry for Quantitating Stable Isotopes: Application of Nuclear Magnetic Resonance in Brain Metabolic Research’, Phurmacochem.Libr., 1997,26, 141 K. L. Behar, N. Sibson, D. L. Rothman, F. Hyder, J. Shen, G. F. Mason and R. G. Shulman, ‘NMR Studies of Cerebral Glutamate/ Glutamine Cycling and GABA Synthesis in Vivo’, z3CIgaku, 1998, 8,

44 R191 R. A. Bell and J. R. Kramer, ‘Structural Chemistry and Geochemistry of Silver-Sulfur Compounds: Critical Review’, Environ. Toxicol. Chem., 1999,18,9 R192 P. S . Belton, I. Delgadillo and A. M. Gil., ‘High-Field Solution State Proton NMR for Food Analysis’, Semin.Food Anal., 1998,3,223 R193 C. Bessada, V. Lacassagne, D. Massiot, P. Florian, J.-P. Coutures, E. Robert and B. Gilbert, ‘Strutural and Dynamic Approaches of Molten Salts by High Temperature Spectroscopies’, 2.Nuturforsch., A: Phys.Sci., 1999,54, 162 R194 S . M. Bjorge, ‘Identification and Characterization of Drug Metabolites Using Stable Isotope Techniques’, Pharmacochem.Libr., 1997,26,233 R195 B. Bleaney, ‘Magnetic Resonance, Nuclear Orientation and Antiferromagnetism’, Mol. Phys., 1998,95, 727 R196 K . Bobrowski, J. Poznanski, J. Holcman and K. L. Wierzchowski, ‘Long-Range Electron Transfer Between Proline-Bridged Aromatic Amino Acids’, Adv. Chem.Ser., 1998,254, 131 R197 R. Boehmer, ‘Nonexponential Relaxation in Disordered Materials. Phenomenological Correlation and Spectrally Selective Experiments’, Phase Transitions, 1998,65,211

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R198 K. L. B. Borden, ‘RING Fingers and B-Boxes: Zinc-Binding ProteinProtein Interaction Domains’, Biochim. Cell Biol., 1998,76, 351 R199 H. U. Borgstedt and C. Guminski, ‘Solubility Data. Gold-Lithium System’, Solubility Data Ser., 1996,64,84 R200 V. Boswick, J. L. Brodsky, F. Kepes, J-M. Neumann, A. Sanson and M. Garrigos, ‘Expression, Purification, and Characterization of Sss 1p, an Essential Component of the Yeast Secblp Protein Translocation Complex’, Protein Expression Purif., 1998, 13,423 R201 A. Boulares, C. Rodrigues, L. Rozes, M. Tessier and E. Marechal, ‘Preparation and Structure of Polyether-Block Containing Polymers’, Pure Appl. Chem., 1998,70, 1239 R202 G. J. Bowden, ‘A Review of the Low Temperature Properties of the Rare Earth Vanadates’, Aust. J. Phys., 1998,51,201 R203 L. Brammer, D. Zhao, W. Kwiatkowski and P. Sherwood, ‘Hydrogen Bonds Involving Transition Metal Centers: Information on Structures and Bonding from Different Characterization Methods’, Trans.Am.Crystallogr. Assoc., 1997,31, 69 R204 P. J. Bray and G. L. Petersen, ‘NMR and NQR Studies of Borate Glasses’, Z .Naturforsch., A: Phys. Sci., 1998,53,273 R205 D. Brinkmann, ‘Spin Gap, Electronic Crossover, and Charge Density Waves in Y-Ba-Cu-0 Superconductors’, 2.Naturforsch., A: Phys. Sci., 1998,53,488 R206 D. Brinkman, ‘The Mysterious Spin Gap in High-Temperature Superconductors. New NMR/NQR Studies’, Appl. Magn. Reson., 1998, 15, 197 R207 J. Brondum, ‘New Sensors and Techniques for Meat Quality Measurements’, Proc. Annu. Reciprocal Meat Con$, Am. Meat Sci. Assoc., 1998, 51,13 R208 E. B. Brouwer and J. A. Ripmeester, ‘Structural and Dynamic Properties of Solid Calixarenes’, Adv. Supramol. Chem., 1999,5, 121 R209 R. K. Brow, T. M. Alam, D. R. Tallant and R. J. Kirkpatrick, ‘Spectroscopic Studies on the Structures of Phosphate Sealing Glasses’, M R S Bull., 1998,23, 63 R210 A. D. Buckingham, ‘Molecules in Optical, Electric, and Magnetic Fields: A Personal Perspective’, Annu. Rev. Phys. Chem., 1998,49, xiii R211 M. Buhl, M. Kaupp, 0. L. Malkina and V. G. Malkin, ‘The DFT Route to NMR Chemical Shifts’, J. Comput. Chem., 1999,20, 91 R212 S. K. Burley and R. G. Roeder, ‘TATA Box Mimicry by TFIID: Autoinhibition of Pol I1 Transcription’, Cell (Cambridge, Mass.) 1998, 94,551 R213 A. Byczynska and J. Barciszewski, ‘The Biosynthesis, Structure, and Properties of Napin. The Storage Protein from Rape Seeds’, J.Plant Physiol., 1999, 154,417 R214 F. Caboi and M. Monduzzi, ‘On Microstructural Transitions of Lamellar Phase-Forming Surfactants’, Prog. Colloid Polym. Sci., 1998, 108, 153

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R215 A. M. Calafat and L. G. Marzilli, ‘Chiralities of Complexes of Bleomycin-Type Ligands, a Neglected Feature in Structural Studies Relevant to Anticancer Drug Action’, Comments Inorg. Chem., 1998, 20,121 R216 P. T. Callaghan, ‘Rheo-NMR: Nuclear Magnetic Resonance and the Rheology of Complex Fluids’, Rep. Prog. Phys., 1999,62, 599 R217 I. D. Campbell and A. K. Downing., ‘NMR of Modular Proteins’, Nut.Struct. Biol., 1998, 5(Suppl.), 496 R218 G. Chaboussant, M.-H. Julien, Y. Fagot-Revurat, M. Hanson, L. P. Levy, C. Borthier, M. Horvatic and 0. Piovesana, ‘Zero Temperature Phase Transitions in Spin-Ladders: Phase Diagram and Dynamical Studies of C U ~ ( C ~ H ~ ~ N ~ Phys. ) ~ CJ.~B,~ 1998,6, ’, Eur. 167 R219 H. Chao, D. L. Bautista, J. Litowski, R. T. Irvin and R. S. Hodges, ‘Use of a Heterodimeric Coiled-Coil System for Biosensor Application and Affinity Purification’, J. Chromatogr., B: Biomed. Sci. Appl., 1998, 715,307 R220 D. Y. Chirgadze, J. Hepple, R. A. Byrd, R. Sowdhamini, T. L. Blundell and E. Gherardi, ‘Insights into the Structure of Hepatocyte Growth FactorIScatter Factor (HGFISF) and Implications for Receptor Activation’, FABS Lett., 1998,430, 126 R22 1 S. H. Choh, ‘Magnetic Resonance Studies of Ferroelectric Sodium Nitrite; What’s New and Old?’, J.Korean Phys.Soc., 1998,32, S624 R222 C. Chuit and C. Reye, ‘Hypercoordinate P(II), P(III), and P(1V) Phosphorus Derivatives with Intramolecular Coordination by Donor Groups’, Eur.J.Inorg.Chem., 1998,12, 1847 R223 C. M. R. Clancy, V. F. Tarasov and M. D. E. Forbes, ‘Time-Resolved Electron Paramagnetic Resonance Studies in Organic Photochemistry’, Electron Paramagn. Reson., 1998, 16, 50 R224 J. B. Clark, ‘N-Acetyl Aspartate. A Marker for Neuronal Loss or Mitochondria1 Dysfunction’, Dev. Neurosci. (Basel), 1998,20, 27 1 R225 G. M. Clore and A. M. Gronenborn, ‘New Methods of Structure Refinement for Macromolecular Structure Determination by NMR’, Proc.Natl.Acad.Sci. U.S. A., 1998, 95, 589 1 R226 R. Corriu, ‘A New Trend in Metal-Alkoxide Chemistry: The Elaboration of Monophasis Organic-Inorganic Hybrid Materials’, Polyhedron, 1998,17,925 R227 S. F. J. Cox, ‘Muon Spin Relaxation Studies of Interstitial and Molecular Motion’, Solid State Nucl. Mngn. Reson., 1998, 11, 103 R228 L. K. Creamer, J. E. Plowman, M. J. Liddell, M. H. Smith and J. P. Hill, ‘Micelle Stability: K-Casein Structure and Function’, J. Dairy Sci., 1998,81,3004 R229 A. N. Davies, ‘A New NMR Data Standard for the Exchange and Archiving for Multi-Dimensional Data Sets’, Spectrosc.Eur., 1999,11, 14 R230 U. K . M. Decking and J. Schrader, ‘Role of Adenosina Kinaze and AMP Deaminase in the Regulation of Cardiac Purine Release’, Drug.Dev.Res., 1998,45,295

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R23 1 R. L. DeLeon, M. P. Joshi, E. F. Rexer, P. N. Prasad and J. F. Garvey, ‘Progress in Thin Film Formation by Laser Assisted Molecular Beam Deposition (LAMBD)’, Appl.Surf:Sci., 1998,127, 321 R232 A. Deville and B. Gaillard, ‘ESR and NMR in Non-Conducting Solids’, Key Eng.Mater., 1999,155,41 R233 K. B. Dillon, ‘Nuclear Quadrupole Resonance Spectroscopy’, Spectrosc. Prop. Inorg. Organomet. Compd., 1998,31, 197 R234 L. Dixit, ‘An Integrated Spectroscopic Approach for the Characterization of Petroleum Reforming Catalysts’, Appl. Spectrosc. Rev., 1998,33, 1 R235 C. M. Dobson and P. J. Hore, ‘Kinetic Studies of Protein Folding Using NMR Spectroscopy’, Nut. Struct. Biol., 1998, 5(Suppl.), 504 R236 B. Doudin, J. E. Wegrowe, S. E. Gilbert, V. Scarani, D. Kelly, J. P. Meier and J-Ph. Ansermet, ‘Magnetic and Transport Properties of Electrodeposited Nanostructured Nanowires’, ZEEE Trans. Magn., 1998,34968 R237 D. E. Draper and L. P. Reynaldo, ‘Survey and Summary: RNA Binding Strategies of Ribosomal Proteins’, Nucleic Acids Res., 1999, 27, 38 1 R238 P. C. Driscoll and S. M. Kristensen, ‘NMR of Natural Macromolecules’, Nucl. Magn. Reson., 1998,27,292 R239 A. Duda, T. Biela, J. Libiszowski, S. Penczek, P. Dubois, D. Mecerreyes and R. Jerome, ‘Block and Random Copolymers of c-Caprolactone’, Polym. Degrad. Stab., 1998,59,215 R240 C. Dybowski, ‘Solid-state Nuclear Magnetic Resonance’, Anal. Chem., 1998,70, 1R R241 H. J. Dyson and P. E. Wright, ‘Equilibrium NMR Studies of Unfolded and Partially Folded Proteins’, Nut. Struct. Biol., 1998, 5(Suppl.), 499 R242 W. L. Earl and C. T. Johnston, ‘Application of NMR Spectroscopy to the Study of the Chemistry of Environmental Interfaces’, IUPAC Ser. Anal. Phys. Chem.Environ. Syst., 1998,4, 25 1 R243 R. J. Elin, ‘Evaluating the Role of Ionized Magnesium in Laboratory and Clinical Practice’, Adv. Magnesium Res., 1997, 1, 525 R244 J. Englich, P. Novak and H. Lutgemeier, ‘Defects in Ferrites Studied by 57FeNMR’, Int. J.Mod. Phys. B, 1998, 12,609 R245 E. Farrannini and A. Mari, ‘How to Measure Insulin Sensitivity’, J.Hypertens., 1998, 16, 895 R246 M. A. Fedotov, ‘Nuclear Magnetic Resonance in Analytical Chemistry of Inorganic Substances in Solution’, J. Anal. Chem., 1999,54, 13 R247 J. Fiaux, C. I. J. Andersson, N. Holmberg, L. Buelow, P. T. Kallio, T. Szyperski, J. E. Bailey and K. Wuethrich, ‘13C NMR Flux Ratio Analysis of Escherichia coli Central Carbon Metabolism in Microaerobic Bioprocesses’, J. Am. Chem.Soc., 1999,121, 1407 R248 R. H. Fillingame, P. C. Jones, W. Jiang, F. I. Valiyaveetil and 0. Y. Dmitriev, ‘Subunit Organization and Structure in the Fo Sector of Escherichia coli F1Fo ATP Synthase’, Biochim. Biophys. Acta, 1998, 1365, 135

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R249 W. L. Fitch, ‘Analytical Methods for Quality Control of Combinatorial Libraries’, Mol. Diversity, 1999,4, 39 R250, H.-C. Flemming, J. Wingender and C. Mayer, ‘Extracellular Polymeric Substances - the Material Which Forms the Biofilm’, Meded. -Fac.Landbouwkd. Toegepaste Biol. Wet.(Univ. Gent), 1998,63, 1129 R25 1 A. P. Foucault and L. Chevolot, ‘Counter-Current Chromatography: Instrumentation, Solvent Selection and Some Recent Applications to Natural Product Purification’, J. Chromatogr., A , 1998,808, 3 R252 U. Friedrich, I. Schnell, S. P. Brown, A. Lupulescu, D. E. Demco and H. W. Spiess, ‘Spinning-Sideband Patterns in Multiplequantum Magic-Angle Spinning NMR Spectroscopy’, Mol. Phys., 1998, 95, 1209 R253 S. Gaemers and C. J. Elsevier, ‘Reducing the NMR Line Widths of Quadrupole Nuclei by Employing Supercritical Solvents’, Chem. Soc. Rev., 1999, 28, 135 R254 S. M. Gagne, M. X. Li, R. T. McKay and B. D. Sykes, ‘The NMR Angle on Troponin C’, Biochem. Cell Biol., 1998,76, 302 R255 Y. Galerne, ‘Characterization of Biaxial Nematic Phases in the Thermotropic Liquid Crystals’, Mol. Cryst.Liq. Cryst.Sci. Technol.Sect. A, 1998,323,211 R256 N. Gershenfeld and I. L. Chuang, ‘Quantum Computing with Molecules’, Sci.Am., 1998, 278, 66 R257 B. C. Gerstein, ‘Nuclear Magnetic Resonance and Surface Phenomena’, Adv. Spectrosc. (Chichester, UK), 1998,26, 34 1 R258 P. Gfrorer, J. Schewitz, K. Pusecker and E. Bayer, ‘On-Line Coupling of Capillary Separation Techniques with ‘H NMR’, Anal. Chem., 1999, 71,315A R259 E. L. Ghisalberti and I. M. Godfrey, ‘Application of Nuclear Magnetic Resonance Spectroscopy to the Analysis of Organic Archeological Materials’, Stud Conserv., 1998,43,215 R260 L. F. Gladden, ‘Applications of in Situ Magnetic Resonance Techniques in Chemical Reaction Engineering’, Top.Catal., 1999,8, 87 R26 1 B. A. Glowacki, ‘Niobium Aluminide as a Source of High-Current Superconductors’, Zntermetallics, 1999,7, 117 R262 M. Goez, ‘CIDNP Studies of Electron and Proton Transfer Reactions of Radical Cations’, Recent Res.Dev. Chem.Sci., 1997, 1, 59 R263 M. Goodman, M. Bhumralkar, E. A. Jefferson, J. Kwak and E. Locardi, ‘Collagen Mimetics’, Biopolymers, 1998,47, 127 R264 J. S. Goundarides, A. Chen and M. J. Shapiro, ‘Nuclar Magnetic Resonance Chromatography: Applications of Pulse Field Gradient Diffusion NMR to Mixture Analysis and Ligand-Receptor Interactions’, J. Chromatogr., B: Biomed.Sci. Appl., 1999,725,79 R265 M. N. Greco and B. E. Maryanoff, ‘Macrocyclic Inhibitors of Serine Proteases’, Adv. Amino Acid Mimetics Peptidomimetics, 1997, 1 , 4 1 R266 R. G. Griffin, ‘Dipolar Recoupling in MAS Spectra of Biological Solids’, Nat. Struct. Biol., 1998, 5(Suppl.), 508

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R267 P. Guntert, ‘Structure Calculation of Biological Macromolecules from NMR Data’, Q.Rev.Biophys., 1998,31, 145 R268 M. Hajek, M. Dezortova and V. Komarek, ‘H MR Spectroscopy in Patients with Mesial Temporal Epilepsy’, Magn. Reson.Mater. Phys., Biol.Med., 1998, 7,95 R269 J. M. Hakumaki, M. Ala-Korpela and R. A. Kauppinen, “ H Nuclear Magnetic Resonance Spectroscopy: Noninvasive Neurochemistry for Basic Research and Clinical Applications’, Curr.Top.Neurochem., 1997, 1, 59 R270 J. A. Hamilton, ‘Fatty Acid Transport: Difficult or Easy?’, J.Lipid. Res., 1998,39,467 R27 1 E. W. Hammond, ‘The Analysis of Lipids’, Adv.AppZ.Lipid Res., 1996, 2, 35 R272 M. Haniu and T. Arakawa, ‘Analysis of Disulfide Structures in Proteins’, Curr. Top.Pept. Protein Res., 1997,2, 115 R273 C. C. Hanstock and P. S. Allen, ‘Applications of Proton MRS to Study Human Brain Metabolism’, Neuromethods, 1999,33,347 R274 E. Hanusowska, I. Dovinova, I. Tkac and L. Novotny, ‘Application of NMR Spectroscopy in Biochemical Studies of Tumor Cells and Resistant to Anticancer Drugs: Minireview’, Neoplasma, 1998,45, 187 R275 K. Hatada, T. Kitayama, K. Ute and T. Nishiura, ‘Preparation of Uniform Stereoregular Polymer, Stereoblock Polymer, and Copolymer of Methacrylate and their Stereocomplex Formation’, Macromol. Symp., 1998,132,221 R276 G. Helmchen, ‘Enantioselective Palladium-Catalyzed Allylic Substitutions with Asymmetric Chiral Ligands’, J. Organomet.Chem., 1999, 576, 203 R277 R. Henderson, ‘Macromolecular Structure and Self-Assembly’, Novartis Found.Symp., 1998,213, 36 R278 P. Hewlett, G. Hunter and R. Jones, ‘Bridging the Gaps’, Chem.Br., 1999,35,40 R279 S. K. Hoffmann and J. Goslar, ‘Electron Spin Relaxation of Copper(I1) Ions in Diamagnetic Crystals’, Appl. Magn. Reson., 1998, 14, 293 R280 U. Holzgrabe, B. W. K. Diehl and I. Wawer, ‘NMR Spectroscopy in Pharmacy’, J. Pharm.Biomed.Anal., 1998,17,557 R28 1 S. W. Homans, R. A. Field, M. J. Milton, M. Probert and J. M. Richardson, ‘Probing Carbohydrate-Protein Interactions by High-Resolution NMR Spectroscopy’, Adv. Exp. Med. Biol., 1998,435,29 R282 M. Horvatic, C. Berthier, Y. Fagot-Revurat, N. Piegay, M. E. Hanson, G. Dhalanne and A. Revcolevschi, ‘NMR Study of the High Magnetic Field Incommensurate Phase of the CuGe03 Spin-Peierls System’, Physica B (Amasterdam), 1998, 246-247,22 R283 H. Hosoya, ‘Pascal’s Triangle, Non-Adjacent Numbers, and D-Dimensional Atomic Orbitals’, J.Math. Chem., 1998, 23, 169 R284 P. J. Houghton, ‘Chemistry and Biological Activity of Schuman-

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Niophyton Species and their Constituents’, Curr.Top.Phytochem., 1997, 1, 31 M. J. Howard, ‘Protein NMR Spectroscopy’, Curr.Biol., 1998,8, R331 G. Hubner, K. Tittmann, M. Killenberg-Jabs, J. Schaffner, M. Spinka, H. Neef, D. Kern, G. Kern, G. Schneider, C. Wikner and S. Ghisla, ‘Activation of Thiamin Diphosphate in Enzymes’, Biochim.Biophys. Acta, 1998, 1385,221 F. Humbert, M. Valtier, A. Retoumart and D. Canet, ‘Diffusion Measurements Using Radiofrequency Field Gradient: Artifacts, Remedies, Practical Hints’, J. Magn. Reson, 1998, 134, 245 P. M. Hwang and H. J. Vogel, ‘Structure - Function Relationships of Antimicrobial Peptides’, Biochem. Cell Biol., 1998,76, 235 K. Ishida, Y. Tokunaga, Y. Kitaoka, G.-Q. Zheng, K. Magishi, H. Mukuda, H. Tou, T. Mito and K. Asayama., ‘NMR Study of HighT, Superconductors and Related Materials’, Physica B (Amsterdam), 1999,259,511 A. A. Istratov and 0. F. Vyvenko, ‘Exponential Analysis in Physical Phenomena’, Rev. Sci. Instrum., 1999,70, 1233 G. Jeschke and M. Jansen, ‘High-Resolution 14N Solid State NMR Spectroscopy’, Angew. Chem., Int.Ed., 1998,37, 1282 J. Johansson, ‘Structure and Properties of Surfactant Protein C’, Biochim.Biophys.Acta, 1998,1408, 161 J. Jonas, L. Ballard and D. Nash, ‘High-Resolution, High-pressure NMR Studies of Proteins’, Biophys.J., 1998,75,445 A. H. Juffer, ‘Theoretical Calculations of Acid-Dissociation Constants of Proteins’, Biochem. Cell Biol., 1998,76, 198 S. R. Julian, A. P. Mackenzie, G. G. Lonzarich, C. Bergemann, R. K. W. Haselwimmer, Y. Maeno, S. Nishizaki, A. W. Tyler, S. Ikeda and T. Fujita, ‘Normal State, Superconductivity, and Quasiparticle Fermi Surface of the Strongly Correlated Oxide Sr2Ru04’, Physica B (Amsterdam), 1999,259,928 T. Kaibara, R. L. Tyson and G. R. Sutherland, ‘Human Cerebral Neoplasms Studied Using MR Spectroscopy: A Review’, Biochem. Cell Biol., 1998,76,477 Cz. Kapusta, P. C. Riedi and G. J. Tomka, ‘Magnetism of Permanent Magnet Materials and Related Compounds as Studied by NMR’, Handb.Magn.Mater., 1998,11,407 A. Karkin, V. Verkhovskii, V. Voronin, B. Goshchitskii and A. Mirmelstein, ‘Electronic States of High T, Superconductors Probed by Radiation Induced Disorder’, Stud. High Temp.Supercond., 1997,22,79 J. Karolin and L. B. A. Johansson, ‘Donor-Donor Energy Migration (DDEM) - a New Method for Examining Structure-Function of Proteins’, Trends Phys. Chem., 1997,6, 171 Y. Karzazi and G. Surpateanu, ‘Structure and Reactivity of Cycloimmonium Ylides’, Heterocycles, 1999, 51, 863

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R301 L. E. Kay, ‘Protein Dynamics from NMR’, Nat.Struct.Biol., 1998, 5(Suppl.), 5 13 R302 L. E. Kay, ‘Protein Dynamics from NMR’, Biochem.Cel1 Biol., 1998, 76,145 R303 L. S. Kegeles, T. J. Humaran and J. J. Mann, ‘In Vivo Neurochemistry of the Brain in Schizophrenia as Revealed by Magnetic Resonance Spectroscopy’, Biol. Psychiatry, 1998,44, 382 R304 P. A. Keifer, ‘New Methods for Obtaining High-Resolution NMR Spectra of Solid-Phase Synthesis Resins, Natural Products, and Solution-State Combinatorial Chemistry Libraries’, Drugs Future, 1998, 23, 30 1 R305 M. Keim, ‘Recent Measurements of Nuclear Moments Far from Stability’, AIP Con$ Proc., 1998,455, 50 R306 P. Khandelwal, N. N. Kuzma, S. E. Barrett, L. N. Pfeiffer and K. W. West, ‘Optically Pumped NMR in the Quantum Hall Regimes’, Physica B (Amsterdam), 1998,256, 113 R307 C. D. Kilts, ‘The Ups and Downs of Oral Lithium Dosing’, J.Clin.Psychiatry, 1998,59,21 R308 R. Kitamaru, ‘Phase Structure of Polyethylene and Other Crystalline Polymers by Solid-state 13C NMR’, Adv. Polym.Sci., 1998, 137,41 R309 C. Kleanthous, A. M. Hemmings and G. R. Mooret, ‘Protein Antibiotics and their Inhibitors’, Biochem. SOC.Trans., 1999,27,63 R310 L. H. Klemm, ‘Recent Interrelationships in the Chemistries of Thienopyridines, Benzopyridines, and Benzothiophenes’, Trends Heterocycl. Chem., 1997,5,37 R311 J. L. Koenig, ‘Spectroscopies I Have Known - Award Address: Philips ACS Award in Applied Polymer Science’, J. Appl. Polym. Sci., 1998, 70, 1359 R312 L. Koenig and S. Postel, ‘Fast Spin Finish Determinations with a Spectroscopic Method. Low-Resolution NMR’, Chem.Fibers Int., 1998, 48,500 R313 T. Koike, K. Ichikawa, H. Kasahara, T. Atsumi, A. Tsutsumi and E. Matsuura, ‘Epitopes on P2-GPI Recognized by Anticardiolipin Antibodies’, Lupus, 1998,7, S14 R314 S. J. Kok, N. H. Velthorst, C. Gooijer and U. A. T. Brinkman, ‘Analyte Identification in Capillary Electrophoretic Separation Techniques’, Electrophoresis, 1998,19,2753 R3 15 L. Konermann, ‘Protein Folding Studied by Electrospray Ionization Mass Spectrometry’, Sci. Prog. (Northwood, U K ) , 1998, 81, 123 R316 M. Kopf and H. G. Kilian, ‘Relaxation in the Glass Transition Regime Interpreted in Terms ot the Aggregate Model’, Acta Polym., 1999, 50, 109 R317 G. Kotovych, J. R. Cann, J. M. Steward and H. Yamomoto, ‘NMR and CD Conformational Studies of Bradykinin and its Agonists and Antagonists: Application to Receptor Binding’, Biochem. Cell Biol., 1998,76,257

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R318 J. Kowalewski, L. Maler and G. Widmalm, ‘NMR Relaxation Studies of Oligosaccharides in Solution: Reorientational Dynamics and Internal Motion’, J.Mo1.Liq., 1998, 78,255 R319 U. Kreutzer and T. Jue, ‘Metabolic Response to Oxygen Limitation in Arenicola Marina as Determined with the ‘H NMR Signals of Myoglobin’, Comp.Biochem.Physiol., Part A: Mol. Integr. Physiol., 1998, 120A, 127 R320 M. C. Krishna, P. Kuppusamy, M. Afeworki, J. L. Zweier, J. A. Cook, S. Subramanian and J. B. Mitchell, ‘Development of Functional Electron Paramagnetic Resonance Imaging’, Breast Dis., 1998, 10,209 R32 1 V. V. Krishnan, ‘Radiation Damping: Suryan’s Line Broadening Revisited in High Resolution Solution NMR’, Curr.Sci., 1998,74, 1049 R322 H. Kubinyi, ‘Structure-Based Drug Design’, Chim. Oggi, 1998,16, 17 R323 H. Kubinyi, ‘Chance Favors the Prepared Mind - from Serendipity to Rational Drug Design’, J. Recept. Signal Transduction Res., 1999, 19, 15 R324 H. Kurosu and T. Yamanobe, ‘Synthetic Macromolecules’, Nucl. Magn. Reson., 1998,27, 337 R325 M. Kussmann and P. Roepstorff, ‘Characterization of the Covalent Structure of Proteins from Biological Material by MALDI Mass Spectrometry - Possibilities and Limitations’, Spectroscopy (Amsterdam), 1998,14, 1 R326 N. N. Kuzma, P. Khandelwal, S. E. Barrett, L. N. Pfeiffer and K. W. West, ‘Ultraslow Electron Spin Dynamics in the Fractional Quantum Hall Regime’, Physica B (Amsterdam), 1998,256, 121 R327 W. R. LaCourse and C. 0. Dasenbrock, ‘Column Liquid Chromatography: Equipment and Instrumentation’, Anal. Chem., 1998,70, 37R R328 M. 0. Leach, M. Verrill, J. Glaholm, T. A. D. Smith, D. J. Collins, G. S. Payne, J. C. Sharp, S. M. Ronen, V. R. McCready, T. J. Powles and I. E. Smith, ‘Measurements of Human Breast Cancer Using Magnetic Resonance Spectroscopy: A Review of Clinical Measurements and a Report of Localized 31P Measurements of Response to Treatment’, N M R Biomed., 1998, 11, 314 R329 G. Le Caer and R. A. Brand, ‘General Models for the Distributions of Electric Field Gradients in Disordered Solids’, J.Phys. : Condens.Matter, 1998,10, 10715 R330 M. Leduc, P. J. Nacher and G. Tastevin, ‘Magnetic Resonance Imaging Using Polarized Noble Gases’, Laser Phys., 1998,8,799 R33 1 J-L. Lefaix and F. Daburon, ‘Diagnosis of Acute Localized Irradiation Lesions: Review of the French Experimental Experience’, Health Phys., 1998,75,375 R332 A. K-M. Leung, F-T. Chau and J-B. Gao, ‘A Review on Applications of Wavelet Transform Techniques in Chemical Analysis: 1989-1997’, Chemom.Intell.Lab. Syst., 1998,43, 165 R333 Y. Li, G. Jinliang and X. Song, ‘Copper(I1) 15-Crown-5-Coordination Compound with 3d,2 Ground State’, Tsinghua Sci. Technol., 1997, 2, 806

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R334 V. Liebers, V. van Kampen, S. Insringhausen-Bley and X. Baur, ‘Structure and Epitopes of Chi t l.Ol’, Znt.Arch. Allergy Zmmunol., 1998, 115,252 R335 P. Lonnecke and M. Meisel, ‘A Novel Method for the Synthesis of Polycyclic Phosphorus Chalcogenides’, Phosphorus, Sulfur Silicon Relat. Elem., 1997, 124 & 125,401 R336 K. L. Lutz, S. Bogdanowich-Knipp, D. Pal and T. J. Siahaan, ‘Structure, Function and Modulation of E-Cadherins as Mediators of Cell-Cell Adhesion’, Curr. Top.Pept. Protein Res., 1997,2,69 R337 I. Luyten and P. Herdewijn, ‘Hybridization Properties of Base-Modified Oligonucleotides within the Double and Triple Helix Motif’, Eur. J. Med. Chem., 1938,33, 5 15 R338 P. M. Macdonald, ‘2H-NMR and Polyelectrolyte-Surfctant Interactions: From Micelles to Monolayers to Membranes’, Colloids Surf.’, A, 1999,147, 115 R339 P. M. Macdonald, K. J. Crowell, C. M. Franzin, P. Mitrakos and D. J. Semchyschyn, ‘Polyelectrolyte-Induced Domains in Lipid Bilayer Membranes: The Deuterium NMR Perspective’, Biochem.Cell Biol., 1998, 76,452 R340 P. P. Mager, ‘Molecular Simulation of the Primary and Secondary Structures of the AP( 1-42)-Peptide of Alzheimer’s Disease’, Med Res.Rev., 1998, 18,403 R34 1 G. Majer, ‘Pulsed-Field-Gradient NMR Studies of Hydrogen Diffusion in Laves-Phase Hydrides’, Mater. Res. SOC.Symp. Proc., 1998, 513, 109 R342 K. L. Malisza, P. Kozlowski and J. Peeling, ‘A Review of in Vivo ‘H Magnetic Resonance Spectroscopy of Cerebral Ischemia in Rats’, Biochem. Cell Biol., 1998,76,487 R343 N. Manabe, ‘Histochemical and Structure-Biological Quantification of Muscle Physiological Properties’, Anim. Sci. Technol., I998,69, 8 I5 R344 B. E. Mann, ‘Nuclear Magnetic Resonance Spectroscopy’, Spectrosc.Prop.Znorg. Organomet.Compd., 1998,31, 1 R345 J. L. Markley, A. Bax, Y. Arata, C. W. Hilbers, R. Kaptein, B. D. Sykes, P. E. Wright and K. Wutrich, ‘Recommendations for the Presentation of NMR Structures of Proteins and Nucleic Acids’, Pure Appl. Chem., 1998,70, 117 R346 D. Marsh, T. Pali and L. I. Horvath, ‘Progressive Saturation and Saturation Transfer EPR for Measuring Exchange Processes and Proximity Relations in Membranes’, Biol.Magn. Reson., 1998,14,23 R347 A. G. Marshall, ‘Fourier Transform Ion Cyclotron Resonance Mass Spectrometry’, AZP Con$ Proc., 1998,430, 3 R348 Y. C. Martin, M. G. Bures and R. D. Brown, ‘Validated Descriptors for Diversity Measurements and Optimization’, Pharm.Pharmacol. Commun., 1998,4, 147 R349 L. G. Marzilli, P. A. Marzilli and E. Alessio, ‘Complexes of Lopsided N-Donor Heterocyclic Bioligands: Has the Electrostatic Effect of the

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N2CH Proton Been Overlooked in Metallobiochemistry?’, Pure Appl. Chem., 1998,70,961 L. Masaro and X. X. Zhu, ‘Pulsed Field Gradient NMR Spectroscopy in the Study of Self-Diffusion in Polymer Systems’, Can.J. Anal. Sci. Spectrosc., 1998,43, 81 K. A. Mauritz, ‘Organic-Inorganic Hybrid Materials. Perfluorinated Ionomers as Sol-Gel Polymerization Templates for Inorganic Alkoxides’, Muter. Sci. Eng., C, 1998, C6, 121 T. Mavromoustakos, ‘Drug-Membrane Interaction. An Example with the Pair of Anesthetic Steroids Alphaxalone and A 6-Alphaxalone’, Epitheor.Klin.Farmacol.Farmakokinet., Int. Ed., 1998, 12, 15 P. J. McDonald and B. Newling, ‘Stray Field Magnetic Resonance Imaging’, Rep. Prog.Phys., 1998,61, 1441 P. Mcdonald and J. Strange, ‘Magnetic Resonance and Porous Materials’, Phys. World, 1998, 11,29 C. R. Middaugh and R. Pearlman, ‘Proteins as Drugs: Analysis, Formulation and Delivery’, Handb.Exp. Pharmacol., 1999, 137, 33 E. Migirdicyan, B. Kozankiewicz and M. S. Platz, ‘High Resolution Fluorescence Spectroscopy: Determination of the Zero-Field Splitting Parameters of Excited Triplet States of Aromatic Carbenes’, Adv. Carbene Chem., 1998,2,97 V. A. Mikhalev, ‘Solvate Isotropic Shifts in NMR’, Russ.J. Gen.Chem., 1997,67, 1260 B. Mikhoval and H. Duddeck, ‘13C NMR Spectroscopy of Tri- and Tetracyclic Quinolizidine Alkaloids, Compilation and Discussion’, Magn. Reson. Chem., 1998,36,779 S. K. Misra, ‘Spin-Lattice Relaxation Times in Amorphous Materials as Effected by Exchange Interactions, Tunneling Level States (TLS) Centers and Fractons’, Spectrochim.Acta, Part A , 1998,54A, 2257 B. M. Moore 11, F. C.Seaman and L. H.Hurley, ‘Molecular Recognition of DNA by Ecteinascidin 743’, Ernst Schering Res.Found. Workshop, 1998,26,81 K. Nicolay, F. A. van Dorsten, T. Reese, M. J. Kruiskamp, J. F. Gellerich and C. J. A. van Echteld, ‘In Situ Measurements of Creatine Kinase Flux by NMR. The Lessons from Bioengineered Mice’, Mo1.Cell.Biochem., 1998,184, 195 P. E. Nielsen, ‘Structural and Biological Properties of Peptide Nucleic Acid (PNA)’, Pure Appl. Chem., 1998,70, 105 T. Nomura and T. Fukai, ‘Novel Methods of Structure Determination of Prenylated Phenols with ‘H- and 13C-NMR Spectra’, Int. Congr.Ser., 1998,1157,561 R. S. Norton, ‘Structure and Function of Peptide and Protein Toxins from Marine Organisms’, J. Toxicol., Toxin Rev., 1998, 17, 99 E. Novotny, S. Ashwal and M. Shevell, ‘Proton Magnetic Resonance Spectroscopy: An Emerging Technology in Pediatric Neurology Research’, Pediatr.Res., 1998,44, 1

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R366 J. M. Ordovas, L. A. Cupples, P. W. F. Wilson, C. Lahoz, D. Levy, J. D. Otvos, J. R. McNamara, E. Gagne, M. Hayden and E. J. Schaefer, ‘Advances in Cardiovascular Risk Prediction: New Biochemical and Genetic Markers’, Int. Congr.Ser., 1998, 1155,425 R367 G. Oz, D. L. Pountney and I. M. Armitage, ‘NMR Spectroscopic Studies of I=l/2 Metal Ions in Biological Systems’, Biochem.Cell Biol., 1998,76,223 R368 T. Parella, ‘Pulsed Field Gradients: A New Tool for Routine NMR’, Magn. Reson. Chem., 1998,36,467 R369 V. A. Paseshnichenko, ‘A New Alternative Non-Mevalonate Pathway for Isoprenoid Biosynthesis in Eubacteria and Plants’, Biochemistry (Moscow), 1998,63, 139 R370 E. Peggion, S. Mammi and E. Schievano, ‘Conformation and Interactions of Bioactive Peptides from Insect Venoms: The Bombolitins’, Biopolymers, 1997,43,419 R371 J . J. Pesek and M. T. Matyska, ‘Spectroscopic Characterization of Chemically Modified Oxide Surfaces’, Stud. Sur-Sci.Catal., 1999, 120A, 117 R372 P. E. Pfeffer, Y. Shachar-Hill, G. Becard, D. Rolin and D. D. Douds Jr., ‘Nutrient Transport and Metabolism in the Life Cycle of Arbuscular Mycorrhizae as Examined by NMR Spectroscopy’, Curr. Top. Plant. Physiol., 1998, 18, 187 R373 A. Piccolo and P. Conte, ‘Advances in Nuclear Magnetic Resonance and Infrared Spectroscopies of Soil Organic Particles’, IUPA C Ser. Anal.Phys. Chem.Environ.Syst., 1998,4, 183 R374 G. IS.S. Prakash, ‘Investigations on Intriguing Long Lived Carbodications’, Pure Appl. Chem., 1998,70,2001 R375 J. H. Prestegard, ‘New Techniques in Structural NMR-Anisotropic Interactions’, Nat.Struct. Biol., 1998, S(Suppl.), 517 R376 S. F. Previs and H. Brunengraber, ‘Methods for Measuring Gluconeogenesis in Vivo’, Curr. Opin.Clin.Nutr. Metab. Care, 1998, 1,461 R377 L. J. Prins, P. Timmerman and D. N. Reinhoudt, “on-Covalent Synthesis of Organic Nanostructures’, Pure Appl. Chem., 1998, 70, 1459 R378 M. J. W. Prior, ‘Nuclear Magnetic Resonance Spectroscopy of Living Systems’, Nucl.Magn.Reson., 1998,27, 386 R379 S. B. Prusiner, M. R. Scott, S. J. DeArmond and F. E. Cohen, ‘Prion Protein Biology’, Cell (Cambridge, Mass.), 1998,93, 337 R380 M. Pruski, C. Fernandez, D. P. Lang and J.-P. Amoureux, ‘Measurement of Interatomic Connectivities in Molecular Sieves Using MQMAS-Based Methods’, Catal. Today., 1999,49,401 R381 J. D. Puglisi and J. R. Williamson, ‘RNA Interaction with Small Ligands and Peptides’, Cold Spring Harbor Monogr.Ser., 1999,37,403 R382 E. Quaite-Randall and A. Joachimiak, ‘Purification of Chaperonins’, J. Chromatogr., B: Biomed Sci. Appl., 1999,722, 153 R383 H. M. Quiney, H. Skaane and I. P. Grant, ‘Ab Initio Relativistic

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Quantum Chemistry: Four-Components Good, Two-Components Bad!’, Adv. Quantum Chem., 1998,32, 1 K. D. Rector and M. D. Fayer, ‘Vibrational Echoes: A New Approach to Condensed-Matter Vibrational Spectroscopy’, In?.Rev. Phys. Chem., 1998,17,261 D. Reichert, G. Hempel and H. Schneider, ‘Experimental Artifacts in 2D-MAS Exchange Experiments: Non-Uniform Mixing Time within a Single 2D Run’, Solid State Nucl. Magn. Reson., 1998, 11,259 R. C. Reid and D. P. Fairlie, ‘Mimicking Extended Conformations of Protease Substrates: Designing Cyclic Peptidomimetics to Inhibit HIV1 Protease’, Adv. Amino Acid Mimetics Peptidomimetics, 1997, 1,77 M. D. Reily, A. M. Bokman, J. Offord and P. McConnell, ‘Nuclear Magnetic Resonance Spectroscopy of Peptide Ion Channel Ligands: Cloning and Expression as Aid to Evaluation of Structural and Dynamic Properties’, Methods Enzymol., 1999,294,92 G. Reuter and H.-J. Gabius, ‘Eukaryotic Glycosylation. Whim of Nature or Multipurpose Tool?’, Cell Mol. Life Sci., 1999,55, 368 L. Reven and L. Dickinson, ‘NMR Spectroscopy of Self-Assembled Monolayers’, Thin Films (San Diego), 1998,24, 149 C. Reymond, ‘Magnetic Resonance Techniques’, CERN [Rep.], 1998, 219 J. P. Richard, J. Crugeiras and R. W. Nagorski, ‘Imperatives for Enzymic Catalysis of Isomerization of Sugars and Sugar Phosphates’, J.Phys.Org.Chem., 1998, 11, 512 A. Rigamonti, F. Borsa and P. Carretta, ‘Basic Aspects and Main Results of NMR-NQR Spectroscopies in High-Temperature Superconductors’, Rep. Prog. Phys., 1998,61, 1367 S. P. Robinson, S. J. Barton, P. M. J. McSheehy and J. R. Griffiths, ‘Nuclear Magnetic Resonance Spectroscopy of Cancer’, Br.J. Radiol., 1997,70, S60 D. C. Roe, P. M. Kating, P. J. Krusic and B. E. Bruce, ‘High Resolution NMR Techniques in Catalysis’, Top Catal., 1998,5, 133 V. Saks, M. Aliev, P. D. Santos, M. Vendelin and 0. Kongas, ‘Mathematical Model of Energy Transfer in Hearts with Inhibited or Ablated Creatine Kinase System’, Magn. Reson. Mater. Phys., Med., 1998, 6,124 C. R. Sanders and R. S. Prosser, ‘Bicelles: A Model Membrane System for All Seasons?’ Structure (London), 1998,6, 1227 A. S. Sarma and N. C. Joshi, ‘Application of NMR Spectroscopy in Lubricant and Lubricant Additive Fields’, J. Sci. Ind. Res., 1998,57, 503 S. Saxena, D. Cizmeciyan and J. A. Kornfield, ‘Solid State 2H-NMR Studies of Segmental Dynamics in Polymer Blends’, Solid State Nucl. Magn.Reson., 1998,12, 165 J. Schaefer, ‘REDOR NMR of Biological Solids: From Protein Binding Sites to Bacterial Cell Walls’, Ernst Schering Res.Found. Workshop, 1998,26,25

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R400 R. Schatzler and M. Monkenbusch, ‘Use of Cold Neutrons for Condensed Matter Research at the Neutron Guide Laboratory ELLA in Julich’, Int. Top.Meet. Res. React. Fuel Manage., 2nd, 1998, 11 R40 1 A. Schellenberger, ‘Sixty Years of Thiamin Diphosphate Biochemistry’, Biochim.Biophys.Acta, 1998,1385, 177 R402 T. M. Schindler, ‘The New View of the Primary Cell Wall’, 2.PJanzenernaehr.Bodenkd., 1998,161,499 R403 V. H. Schmidt, Z. Trybula, N. J. Pinto and S. M. Shapiro, ‘Phase Coexistence in Proton Glass’, Phase Transitions, 1998,67,499 R404 H-P. Schrodel and A. Schmidpeter, ‘The 31P-ChemicalShift of Diphosphenes’, Phosphorus, Sulfur Silicon Relat. Elem., 1997, 129, 69 R405 R. P. W. Scott, ‘Tandem Liquid Chromatography Systems’, Chromatogr.Sci.Ser., 1998,78, 581 R406 M. Sekine, H. Tsuruoka, K-I. Shohda, T. Moriguchi and T. Wada, ‘New Strategies for the Chemical Synthesis of Biologically Important Nucleic Acid Derivatives’, Nucleosides Nucleotides, 1998, 17, 2033 R407 F. Sellschop and S. H. Connell, “‘Carbon!... and Its Analysis”’, Nucl. Instrum. Methods Phys. Res., Sect. B, 1998, 136, 1253 R408 F. Shahidi and U. N. Wanasundara, ‘Methods of Measuring Oxidative Rancidity in Fats and Oils’, Food Sci. Technol.(N. X ) , 1998,88, 377 R409 M. J. Shapiro, ‘Affinity NMR’, Am.Lab. (Shelton, Conn.), 1998,30,20 R410 R. G. Sharma, ‘Superconducting Magnet System Development Program at NPL’, Indian J. Cryog., 1995 (Pub.1997), 20,29 R411 H. Shinohara, ‘Endohedral Metallofullerenes: Structures and Electronic Properties’, Adv. Met. Semicond. Clusters, 1998, 4, 205 R412 E. S. Shubina, N. V. Belkova, E. V. Bakhmutova, L. N. Saitkulova, A. V. Ionidis and L. M. Epstein, ‘Problems of Unusual Hydrogen Bonds Between Proton Donors and Transition Metal Hydrides and Borohydrides’, Russ. Chem.Bull., 1998,47, 817 R413 G. Siligardi and R. Hussain, ‘Biomolecules Interactions and Competitions by Non-Immobilized Ligand Interaction Assay by Circular Dichroism’, Enantiomer, 1998, 3, 77 R414 H. Sillescu, ‘Heterogeneity at the Glass Transition: A Review’, J. NonCryst.Solids, 1999,243’8 1 R415 D. J. Siminovtch, ‘Solid-state NMR Studies of Proteins: The View from Static 2H NMR Experiments’, Biochem. Cell Biol., 1998,76,411 R416 E. D. Sloan Jr., ‘Physical/Chemical Properties of Gas Hydrates and Application to World Margin Stability and Climatic Change’, Geol. Soc.Spec. Publ., 1998,137,31 R417 D. L. Smith, ‘Local Structure and Dynamics in Proteins Characterized by Hydrogen Exchange and Mass Spectrometry’, Biochemistry (Moscow), 1998,63,285 R418 I. C. P. Smith and D. E. Blandford, ‘Diagnosis of Cancer in Humans by ‘H NMR of Tissue Biopsies’, Biochem.Cell Biol., 1998’76,472 R419 J. A. S. Smith, ‘Chapter 7. Nuclear Quadrupole Resonance and Relaxation’, Annu. Rep. Prog. Chem., Sect. C: Phys. Chem., 1998,94,259

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R420 S. P. Smith and G. S. Shaw, ‘A Change in Hand Mechanism for SlOO Signalling’, Biochem. Cell.Biol., 1998,76, 324 R42 1 V. I. Sokolov, ‘Fullerenes Coordinated to Transition Metals: Synthetic and Stereochemical Study’, Pure Appl. Chern., 1998,70,789 R422 R. J. Solaro and H. M. Rarick, ‘Troponin and Trypomyosin: Proteins that Switch on and Tune in the Activity of Cardiac Myofilaments’, Circ.Res., 1998,83,471 R423 P. Sompornpisut, ‘Structure Determination of Paramagnetic Proteins through NMR’, J.Sci.Soc. Thailand, 1998,24, 101 R424 J. Song and F. Ni, ‘NMR for the Design of Functional Mimetics of Protein-Protein Interactions: One Key is in the Building of Bridges’, Biochem.Cell Biol., 1998,76, 177 R425 F. D. Sonnichsen, P. L. Davies and B. D. Sykes, ‘NMR Structural Studies on Antifreeze Proteins’, Biochem.Cell Biol., 1998, 76,284 R426 V. Spitzer, ‘High-Resolution Nuclear Magnetic Resonance Spectroscopy of Fatty Acids and Lipids’, GIT Lab.J., 1998, 1,45 R427 J. F. Stebbins, ‘Silicate Melt Structure: Thermodynamic Interface vs. Spectroscopic Observation’, Mineral.Mag., 1998,62.4, 1445 R428 L. Stefaniak, ‘Investigation of some Intramolecular Hydrogen Bonds by Nitrogen Magnetic Resonance Methods’, Pol.J. Chem., 1999,73, 173 R429 P. Stilbs, ‘Component Separation in NMR Imaging and Multidimensional Spectroscopy through Global Least-Squares Analysis, Based on Prior Knowledge’, J.Magn. Reson., 1998, 135, 236 R430 P. Stilbs, ‘NMR Studies of Polymer-Surfactant Systems’, Surfactant Sci.Ser., 1998, 77,239 R43 1 M . Stocker, ‘Product Characterization by Nuclear Magnetic Resonance [in Zeolite Synthesis]’, Microporous Mesoporous Mater, 1998,22, 533 R432 T. Story, ‘IV-VI Semimagnetic Semiconductors: Recent Developments’, Acta Phys.Pol., A , 1998,94, 189 R433 M. Stutzmann, ‘Data on Hydrogen in a -Si:H from NMR’, EMIS Detarev.Ser., 1998, 19, 61 R434 T. Szyperski, ‘13C-NMR, MS and Metabolic Flux Balancing in Biotechnology Research’, QRev.Biophys., 1998,31,41 R435 M. Tabata, T. Sone and Y. Sadahiro, ‘Precise Synthesis of Monosubstituted Polyacetylenes Using Rh Complex Catalysts. Control of Solid Structure and n-Conjugation Length’, Macromol. Chem.Phys., 1999, 200,265 R436 M. Tanaka, A. Morimoto, K. Ishimori and I. Morishima, ‘StructureActivity Relation of Horseradish Peroxidases as Studied with Mutations at Heme Distal and Proximal Sites’, Pure AppLChem., 1998, 70, 91 1 R437 Y. Tanaka, M. Katahira, F. Nishikawa, T. Sakamoto, Y. Kurihara, S. Nishikawa and S. Uesugi, ‘Structural Investigation of HDV Ribozymes by NMR Spectroscopy’, Nucleic Acids Symp.Ser., 1998,39, 147 R438 C . P. Taylor, ‘Mechanisms of Action of Gabapentin’, Drugs Today, 1998,34 (Suppl. D), 3

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R439 C. Thessieu, K. Ishida, Y. Kitaoka, K. Asayama and G. Lapertot, ‘NMR Study of MnSi under Pressure’, Koatsuryoka no Kagaku to Gijutsu, 1998,7,487 R440 C. Thessieu, Y. Kitaoka and K. Asayama, ‘Magnetic Quantum Phase Transition in MnSi’, Physica B (Amsterdam), 1999,259, 847 R44 1 A. Thomson and K. N. Jeejeebhoy, ‘Muscle Function and Malnutrition’, Pediatr. Adolesc. Med., 1998, 8, 12 R442 J. A. Timbrell, ‘Biomarkers in Toxicology’, Toxicology, 1998, 129, 1 R443 T. Toida and R. J. Linhardt, ‘Structural Analysis of Heparan Sulfate and Heparan Sulfate Oligosaccharides’, Trends Glycosci.Glycotechnol., 1998,10, 125 R444 A. A. G. Tomlinson, ‘Characterization of Pillared Layered Structures’, J. Porous Muter., 1998,5,259 R445 Y. Tong, E. Oldfield and A. Wieckowski, ‘Exploring Electrochemical Interfaces’, Anal. Chem., 1998,70, 5 18A R446 E. Toth and A. E. Merbach, ‘Water Exchange Dynamics: The Key for High Relaxivity Contrast Agents in Medical Magnetic Resonance Imaging’, ACH - Models Chem., 1998,135, 873 R447 J. C. Traeger and R. Colton, ‘Electrospray Ionization in Inorganic Chemistry’, Adv. Muss Spectrom., [computer optical disc], 1998, 14, Chapter 29/637-Chapter 291659 R448 B. A. Trofimov and M. V. Sigalov, ‘N-Vinylpyrrolinum, Furanium and Thiophenium Ions’, MGCN, Main Group Chem.News, 1998,6,30 R449 M. Tsimidou, ‘Polyphenols and Quality of Virgin Olive Oil in Retrospect’, Ital. J. Food Sci., 1998, 10, 99 R450 V. V. Turov and R. Leboda, ‘Application of ‘H NMR Spectroscopy Method for Determination of Characteristics of Thin Layers of Water Adsorbed on the Surface of Dispersed and Porous Adsorbents’, Adv. Colloid Interface Sci., 1999,79, 173 R45 I J. A. Urbina and E. Oldfield, ‘Solid State Nuclear Magnetic Resonance Spectroscopic Approaches to the Study of the Molecular Organization of Biological Membranes’, Biomed. Health Res., 1998, 20, 113 R452 A. I. Usov, ‘Structural Analysis of Red Seaweed Galactans of Agar and Carrageenan Groups’, Food Hydrocolloids, 1998,12, 30 1 R453 L. C. M. Van Gorkom and A. Jensen, ‘Molecular Spectroscopy of Anionic Surfactants 11. Nuclear Magnetic Resonance Spectroscopy’, Surfactant Sci.Ser., 1998,73, 169 R454 C.-W. Von der Lieth, H.-C. Siebert, T. Kozar, M. Burchert, M. Frank, M. Gilleron, H. Kaltner, G. Kayser, E. Tajkhorshid, N. V. Bovin, J. F. G. Vligenthart and H.-J. Gabius, ‘Lectin Ligands. New Insights into their Conformations and their Dynamic Behavior and the Discovery of Conformer Selection by Lectins’, Acta Anat., 1998, 161,91 R455 W. von Philipsborn, ‘Probing Organometallic Structure and Reactivity by Transition Metal NMR Spectroscopy’, Chem.Soc. Rev., 1999,28,95 R456 A. Votres, K. Suvegh, M. Bokor, A. Domjan, T. Marek, M. Klapper, C. U. Chisholm. M. El-Sharif, K. Tomna. Z. Nemes-Vetessv and

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K. Burger, ‘Frontiers of Positron and Positronium Chemistry in Condensed Media’, J. Radioanal.Nucl. Chem., 1999,239,29 M. Waksmundzka-Hajnos, ‘Properties of Florisil and its Use in Chromatography’, Chem.Anal. (‘ Warsaw), 1998,43,301 B. A. Wallace, ‘Recent Advances in the High Resolution Structures of Bacterial Channels: Gramicidin A’, J. Struct. Biol., 1998, 121, 123 R. E. Walstedt, ‘Giant Static and Dynamic Magnetism in the La2Cu04 Family of Superconductors: A Progress Report’, J. Phys. Chem.Solids, 1998,59,2155 T. Walz and N. Grigorieff, ‘Electron Crystallography of Two-Dimensional Crystals of Membrane Proteins’, J. Struct. Biol., 1998, 121, 142 G-W. Wang, M. S. Meier, R. C. Haddon, J. P. Selegue, M. A. Lloyd and C. P. Brock, ‘Addition of Benzene to a Closed 5-6 Ring Fusion’, Proc.Electrochem.SOC.,1997,97-42,289 D. E. Warschawski, M. Traikia, P. F. Devaux and G. Bodenhausen, ‘Solid-state NMR for the Study of Membrane Systems: The Use of Anisotropic Interactions’, Biochimie, 1998,80,437 A. Watts, ‘Solid-state NMR Approaches for Studying the Interaction of Peptides and Proteins with Membranes’, Biochim.Biophys.Acta, 1998,1376,297 A. Watts, I. J. Burnett, C. Glaubitz, G. Grobner, D. A. Middleton, P. J. R. Spooner and P. T. F. Williamson, ‘Structural Discriptions of Ligands in their Binding Site of Integral Membrane Proteins at Near Physiological Conditions Using Solid-state NMR’, Eur.Biophys.J., 1998,28,84 E. B. Waygood, ‘The Structure and Function of HPr’, Biochem.Cel1 Biol.,1998,76, 359 P. A. Wender, Y. Martin-Cantelejo, A. J. Carpenter, A. Chiu, J. De Brabander, P. G. Harran, J-M. Jimenez, M. F. T. Koehler, B. Lippa, J. A. Morrison, S. G. Muller, S. N. Muller, C-M. Park, M. Shiozaki, C. Siedenbiedel, D. J. Skalitzky and M. Tanaka, ‘The ChemistryMedicine Continuum: Synthetic, Computer, Spectroscopic and Biological Studies on New Chemotherapeutic Leads’, Pure Appl. Chem., 1998,70,539 F. Westad and H. Martens, ‘Shift and Intensity Modeling in Spectroscopy - General Concept and Applications’, Chemom.Intell. Lab. Syst., 1999,45361 J. Wolber, I. J. Rowland and M. 0. L,each, ‘Intravascular Delivery of Hyperpolarized 129 Xenon for in Vivo MRI’, Appl. Magn. Reson., 1998, 15,343 J-L. Wolfender, K. Ndjoko and K. Hostettmann, ‘LC/NMR in Natural Products Chemistry’, Curr.Org. Chem., 1998,2, 575 V. Wray, K. Nokihara and S. Naruse, ‘Solution Structure Comparison of the VIPIPACAP Family of Peptides by NMR Spectroscopy’, Ann. N. Y.Acad. Sci., 1998, 865, 37 G. Wu, ‘Recent Developments in Solid-state Nuclear Magnetic

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Resonance of Quadrupolar Nuclei and Applications to Biological Systems’, Biochem. Cell Biol., 1998,76,429 K. Wuthrich, ‘The Second Decade [of NMR Spectroscopy Applied to Structural Biology] - into the Third Millennium’, Nut. Struct. Biol., 1998,5 Suppl.), 492 M. Yasuda, I. Shibata, A. Baba and H. Matsuda, ‘Selective Reactions of Organotin (IV) Enolates Using High-Coordination Methods’, Recent Res. Dev. Pure Appl. Chem., 1997, 1, 55 Z. Yu, T. Mohan, K. Erdmann and J. G.Verkade, ‘31-P NMR Analysis of Sulfur Removal from Coal Materials’, Prepr. Symp. -Am. Chem.Soc., Div.Fue1 Chem., 1998,43,905 R. C. Zamar, C. E. Gonzales and 0. Mensio, ‘Molecular Motions in Thermotropic Liquid Crystals Studied by NMR Spin-Lattice Relaxation’, Braz. J. Phys., 1998, 28, 314 L. A. Zemnukhova and R. L. Davidovich, ‘1219123Sb and *09Bi Nuclear Quadrupole Resonance Study of Complex Compounds of Antimony(II1) and Bismuth(II1) in the Temperature Range 77-500 K’, Z.Naturforsch., A: Phys.Sci., 1998, 53, 573 Y-Q.Zhang, ‘Natural Silk Fibroin as a Support for Enzyme Immobilization’, Biotechnol.Adv., 1998, 16,961 Reviews and Books in Foreign Languages

Chinese R478 Y. Chen and D. Yy, ‘Classification and Characteristics of y-Lactone and Tetrahydrofuran Moieties of Antitumor Bioactive Constituents of Annonaceae’, Yaoxue Xuebao, 1998,33,553 R479 C. Gao and J. Zhang, ‘Methods of Insulin Sensitivity Determination in Vivo’, Zhonghua Neifenmi Daixie Zazhi, 1997,13, 178 R480 J. Jiang, D. K. P. Ng, W. Liu, J. Xie and S. Sun., ‘Development in the Research of Homoleptic Sandwich-Type Phthalocyaninato Metal Complexes’, Huaxue Tongbao, 1999,2,2 R48 1 L. Liang, ‘Applying of NMR Hydrogen Spectra in the Orientation Effect of Aromatic Hydrocarbon’, Huabei Gongxueyuan Xuebao, 1997, 18, 141 R482 H. Liu, X. Liu and X. Xu, ‘Study on Titanium-Containing Oxidation Molecular Sieve Catalysts’, Dame H24nxue, 1999, 14, 10 R483 X. Liu and B. Li, ‘Recent Progress in Models of Coal Pyrolysis’, Meitan Zhuanhua, 1998,21,42 R484 Y. Luo, Z. Guo, T. Li and H. Shen, ‘Development of Application of Liquid-Crystalline Molecules in Analytical Chemistry’, Fenxi Huaxue, 1998,26,891 R485 S. Nakata, Y. Tanaka, S. Asaoka, T. Takatsuka and M. Nakamura, ‘Recent Advances in Applications of Solid-state NMR to Heteroge-

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neous Catalysis. I. Studies on Microporous Crystal Catalysts’, Shiyou Xuebao, Shiyou Jiagong, 1998,14, 36 S. Nakata, Y. Tanaka, S. Asaoka, T. Takatsuka and M. Nakamura, ‘Recent Advances in Applications of Solid-state NMR to Heterogeneous Catalysis. 11. Innovative Studies with New Solid-state Techniques’, Shiyou Xuebao, Shiyou Jiagong, 1998,14,27 H-L. Ren, Y. Yue and C-H. Ye, ‘Applications of Solid State NMR in Microstructure Studies of Glasses’, Wuji Huaxue Xuebao, 1999, 15, 151 J. Tian and 2 . Du, ‘Application of NMR in the Study of Apoptosis’, Junshi Yixue Kexueyuan Yuankan, 1998’22,309 J. G. Tian and Z. Du, ‘19F NMR in Biomedical Research’, Shengli Kexue Jinzhan, 1998,29,319 D-C. Wang, ‘Recent Progress in Structural Biology’, Shengwu Huaxue Yu Shengwu Wuli Jinzhan, 1998,25,396 Z. Wu, ‘Transition Metal Periodate Complexes’, Huaqiao Daxue Xuebao, Ziran Kexueban, 1998,19,255 H. Xie and X. Wu, ‘Ultra-Fast NMR Imaging and Its Future’, Wuli, 1997,26, 595 D. Yang, ‘Latest Developments in the Study of Small Particles by NMR’, Wuli, 1997,26,285 A-M. Yu, H-Z. Yang and Z-P. Zhang, ‘Solid Phase Organic Reactions and Analytical Techniques’, Youji Huaxue, 1998, 18, 386 C. Zhai, ‘New Development of NMR Ultra Microprobe’, Bopuxue Zazhi, 1998,15,465 A. Zhang, H. Wang and H. Shao, ‘Review on the 13CNMR Chemical Shifts of Alkaloids from Fritillaria’, Huaxi Yaoxue Zazhi, 1998, 13, 100 J. Zhang and G. Tian, ‘Isolation and Structural Analysis of Oligosaccharides’, Shengwu Huaxue Yu Shengwu Wuli Jinzhan, 1998,25, 114 Z. Zhang, J. Suo, X. Zhang and S. Li, ‘Recent Development on SilicaBased Mesoporous Molecular Sieves’, Huaxue Jinzhan, 1999,11, 11

Danish R499 I. Bjornsdottir, C. B. Knudsen and S. H. Hansen, ‘Hyphenated Analytical Techniques: HPLC-MS and Capillary Electrophoresis-Mass Spectrometry (CE-MS)’, Dan. Kemi, 1998,79, 12 R500 U. G. Sidelmann anf C. Cornett, ‘Directly Coupled HPLC-MS and HPLC-NMR-MS’, Dan.Kemi, 1998,79,14 French R5Ol E. Deleens, J.-F. Morot-Gaudry, F. Martin, A. Thoreux and A. Gujon, ‘15N Methodology’, Assimilation Azote Plant., 1997, p. 265, ed. J.-F. Morot-Gaudry, Institut National de la Recherche Agronomique, Paris, France R502 M. Farines, ‘Nuclear Magnetic Resonance: A Tool for Lipid Analysis’, Ol., Corps Gras, Lipides, 1998, 5,291

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R503 B. Gillet, S. Bleneau and J.-C. Beloeil, ‘Characterization of Phospholipids and Triglycerides by NMR’, Analusis, 1998,26, M26 R504 J. M. Lhoste, ‘Structural, Dynamic, and Interaction Studies of Proteins: The Example of Immunology’, Pathol. Biol., 1998,46, 103 R505 M. Malet-Martino, ‘NMR in Vitro: Metabolism and Pharmacokinetics of Drugs’, Pharmacocinet. Biodistrib. Med. :Ther.Genique Anti-sens, ed. J-M. Lhoste and C. Helene, INSERM, Paris, France, 1997, p. 93 R506 F. Mariette, A. Davenel, P. Marchal and B. Chaland, ‘Observation of Water in Dairy Processing by Using NMR and MRI’, Rev. Inst.Fr.Pet., 1998,53,521 R507 A. Quinsac and D. Ribaillier, ‘Perfecting the Quality Control of Oilseeds’, Ol., Corps, Gras, Lipides, 1998, 5,284 R508 A. Volk, ‘In Vivo and in Situ NMR: Application to the Study of Animal Models for Human Diseases’, Pharmacocinet. Biodistrib. Med.: Ther. Genique Anti-sens’, ed. J-M. Lhoste and C. Helene, INSERM, Paris, France, 1997, p. 115 German R509 K. Aitzetmuller and H. Herling, ‘13C-NMR Studies of Fats and Oils, Especially Animal Fats’, Schriftenr. Bundesminist.Ernaehr., Landwirtsch.Forsten, Reihe A: Angew. Wiss., 1998,469,23 R510 A. Burgath and H. Frey, ‘Hyperbranched Polymers. Macromolecular Trees Become Popular’, GIT Labor-Fachz., 1998,42,516 R511 H. Jancke, ‘NMR as Primary Analytical Measuring Method’, Nachr. Chem., Tech.Lab., 1998,46,720 R512 D. Marx, B. Engels, M. Buehl and P. Saalfrank, ‘Trends in Theoretical Chemistry in 1998’, Nachr. Chem., Tech.Lab., 1999,47, 186 R513 G. M. Milles, ‘Measuring and Testing Equipment in Plastics Processing’, Coating, 1998,31, 172 R5 14 G. Schwedt, ‘Structural Analysis’, CLB Chem.Labor Biotech., 1999,50,84 R515 H. C . Siebert, C. W. Von der Lieth and H. J. Gabius, ‘The Sugar Code. Oligosaccharides as Carrier of Biological Information’, Dtsch. Apoth. Ztg., 1999, 139, 272 Hungarian R516 G. Ferenczy, ‘Structure-Based Drug Design’, Acta Pharm.Hung., 1998, 68,21 R517 I. Laczko and M. Hollosi, ‘New Methods to Study the Secondary Structure of Polypeptides in Solution’, Magy.Kem.Lapja, 1999,54, 5 R518 G. Szalontai, ‘Solid State NMR Spectroscopy. Principles and Applications’, Magy. Kem.Lapja, 1998,53,469 Italian R 5 19 S. Bradamante, ‘Multinuclear Magnetic Resonance of Perfused Organs with Particular Reference to the Myocardium’, Rapp.ISTISAN, 1997, (97/40), 136

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R520 A. Ferretti, ‘Determination of Enzymic Activity in in Vitro Cell Systems (Homogenates and Extracts)’, Rapp. ISTISAN, 1997, (97/40), 144 R521 L. Guidoni, ‘NMR Spectroscopy in Biology and Medicine’, Rapp. ISTISAN, 1997, (97/40), 17 R522 R. Lodi, S. Iotti and B. Barbiroli, ‘Phosphorus NMR Spectroscopy for Evaluating Energy Metabolism in the Muscle and Brain of Humans’, Rapp.ISTISAN, 1997, (97/40), 170 R523 I. Messana, M. Castagnola, M. Orlando, L. Pennacchietti and B. Giardina, ‘Carbon- 13 NMR Experiments on Erythrocyte Metabolism’, Rapp. ISTISAN, 1997, (97/40), 53 R524 M. Paci and M. Sette, ‘NMR of Erythrocytes: NMR Spectra’ Rapp. ISTISAN, 1997, (97/40), 65 R525 R. Ricci, ‘Proton Magnetic Resonance Spectroscopy in Cerebral Pathology’, Rupp. ISTISAN, 1997, (97/40), 188 R526 V. Rizzo, M. Gigli and V. Pinciroli, ‘Analytical Characterization of Polymer-Drug Conjugates’, Chim.Ind. (Milan), 1998,80,901 R527 A. Rosi, ‘One- and Two-Dimensional Proton NMR Spectroscopy in Cell and Tissue Systems’, Rapp. ISTISAN, 1997, (97/40), 34 R528 M. Tassini, A. Vivi and G. Valensin, ‘Studies on Cellular Matabolism Using NMR Measurements. Introduction and Applications’, Rapp. ISTISAN, 1997, (97/40), 103 R529 Y-L.T. Ting, ‘Perfusion Systems for NMR Spectroscopy Studies in Biomedicine’, Rapp. ISTISAN, 1997, (97/40), 121 R530 R. Toffanin and P. Pollesello, ‘NMR Analysis of Phosphorylated Metabolites in Perchloric Acid Extracts: Use of Experimental *H-31P HMQC with a Gradient and Selective Impulse for Rapid and Accurate Identification’, Rapp. ISTISAN, 1997, (97/40), 160 R53 1 V. Viti, ‘Principles of NMR Spectroscopy’, Rapp. ISTISAN, 1997, (971 40172 R532 A. Vivi, M. Tassini and G. Valensin, ‘31P, 13C, ‘H, 23Na andS9Co NMR Spectroscopy of Perfused Organs’, Rupp. ISTISAN, 1997, (971 40), 81 Japanese R533 K. Akasaka, ‘New World of High Magnetic Field and High Pressure NMR’, Kagaku to Seibutsu’, 1998,36,423 R534 H. Aki and M. Yamamoto, ‘Thermodynamic Aspects of Multiple Inclusion Complexation between Barbiturates and 2-Hydroxypropyl-PCyclodextrin in Aqueous Solution’, Netsu Sokutei, 1998,25, 117 R535 H. Akutsu and T. Fujiwara, ‘Solid NMR and its Application’, Shirizu: Nyu BaioJjikkusu, 1997, 3, 142, ed. Y. Kyogoku and T. Tsukihara, Kyoritsu Shuppan, Tokyo, Japan R536 M. Demura, ‘NMR Structure Interpretation in 21st Century’, Kagaku (Kyoto), 1998,53,67 R537 K. Endo and M. Suhara, ‘Theoretical Spectra as Obtained by MO Calculations’, Bunseki, 1998, 12, 942

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R538 H. Fuji, ‘NMR of Heavy Electron System Anisotropic Superconductors - Parity and Knight Shift’, Bussei Kenkyu, 1998, 70, 146 R539 T. Fujiwara and H. Akutsu, ‘Solid-state Nuclear Magnetic Resonance’, Tanpakushitsu Kakusan Koso, 1999,44,502 R540 E. Fukushi and J. Kawabata, ‘CH-, CH2-, and CH3- Selected 2 DNMR Spectra and their Application’, Tennen Yuki Kagobutsu Toronkai Koen Yoshishu, 1998,40,517 R54 1 Y. Fukushi, ‘Development and Application of New Methods for NMR Structure Analyses Using Axially Chiral Reagents’, Nippon Nogei Kagaku Kaishi, 1998,72, 1345 R542 T. Hiaki, ‘Measurement and Prediction of Diffusion Coefficients in Liquid’, Kagaku Kogaku, 1998,62,440 R543 Y. Hirama, ‘Application of ‘H NMR to Quantitative Analysis. Determination of a Small Amount of Ordinary Water in Heavy Water and Particular Components in Human Urine’, Hokkaido Kogyo Gijutsu Kenkyusho Hokoku, 1998,71,23 R544 Y. Hirotsu and T. Ohkubo, ‘Medium Range Ordering in the Atomic Scale Structure of Amorphous Alloys’, Materia, 1998, 37, 684 R545 K. Hisatani, ‘Data Processing of Polymer Sequence and Stereoregularity Distributions Utilizing High-Resolution NMR’, Kin0 Zairyo, 1998,18,37 R546 Y. Horiguchi and K. Tanaka, ‘Advance in Analytical Technique for Surfactants’, Nihon Yukagakkaishi, 1998,47, 1123 R547 T. Iinuma, ‘Medical Imaging and Radiation - Recent Development and Prospects’, Hoshasen, 1998,24, 3 R548 A. Isogai, ‘Application of Stable Nitroxyl Radical Reagents to Cellulose Modifications’, Cellul.Commun., 1998, 5, 136 R549 K. Ito, Y. Ohba and T. Sone, ‘Synthesis and Properties of Calixarene Analogs Modified in the Macrocyclic Ring’, Nippon Kagaku Kaishi, 1999,4,217 R550 N. Iwasawa, ‘Fisher-Type Carbene Complexes in Organic Synthesis’, Yuki Gosei Kagaku Kyokaishi, 1998,56,413 R55 1 T. Kato, J. Murashita, T. Shioiri, N. Kato and T. Inubushi, ‘Clinical Applications of Functional Magnetic Resonance Spectroscopy for Measurement of Brain Matabolism’, No no Kagaku, 1998,20, 149 R552 E. Kawashima, K. Kamaike and Y. Ishido, ‘Efficient Synthesis of Nucleosides Labeled with Stable Isotopes and their Application to Structural Biology’, Yakugaku Zasshi, 1999, 119,299 R553 T. Kitao, ‘Contribution to Rheology through Studies on a Novel Series of Polymer Blend and as an Editor of JSRJ’, Nihon Reoroji Gakkaishi, 1998,26, 191 R554 K. Koizumi, ‘Separation and Analysis of Saccharides. On Mainly Oligosaccharides’, Kagaku to Kogyo (Osaka) 1998,72,476 R555 K. Koizumi, ‘Separation and Analysis of Saccharides: On Mainly Oligosaccharides’, Yakugaku Zasshi, 1999,119, 199

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R556 T. Kojima, ‘Application of Nuclear Magnetic Resonance to Investigation of Water in Food’, CA CS Forum, 1998, 18, 15 R557 K. Komatsu and T. Fukuhara, ‘Progress and Application of LC/NMR Experiment’, Bunseki, 1999,3,231 R558 T. Kusumi, ‘Development in Determination of Absolute Configuration by NMR’, Farumashia, 1999,35, 137 R559 K. Kuwata, ‘Studying of Intramolecular Movement of Proteins by NMR’, Shirizu: Nyu BaioJjikkusu, 1997, 1, 216, ed. H. Nakamura and F. Arisaka, Kyoritsu Shuppan, Tokyo, Japan R560 Y. Kyogoku, ‘Structural Biology and its Supporting Analysis Methods’, Shirizu: Nyu BaioJjikkusu, 1997, 3, 1, ed. Y. Kyogoku and T. Tsukihara, Kyoritsu Shuppan, Tokyo, Japan R56 1 M. Maeda, ‘Regioselectivity and Stereochemistry of the Synthesized Sulfated Polysaccharides’, CACS Forum, 1998, 18, 33 R562 H. Masuda, N. Ohata and 0. Yamauchi, ‘Self-Organization and SelfRecombination in Metal Complexes with Arginine or Glutamate’, Nippon Kessho Gakkaishi, 1998,40,21 1 R563 H. Murai, ‘Fundamentals and Advances in Photochemistry. From Gas Phase, Liquid Phase and Surface Photochemistry to the Photochemistry in Photosynthesis and for Global Environmental Concerns. Photochemistry in Liquid Phase. Spin Dynamics in Photochemistry. Study on Photochemical Reactions by the Observation and Operation of Electron Spins’, Kikan Kagaku Sosetsu, 1998,36, 102 R564 M. Murata, ‘Two Dimensional NMR’, Gendai Kagaku, 1998,329,32 R565 M. Murata and N. Matsumori, ‘Stereochemical Assignment for Acyclic Structures. NMR Analysis Based on Carbon-Proton Spin Coupling Constants’, Kagaku to Kogyo (Tokyo), 1998,51, 1083 R566 T. Nakai, ‘Organic Synthesis by Means of Trivalent Lanthanoids: Lanthanoid-Catalyzed Aldol Reactions and Michael Addition Reactions’, Kikan Kagaku Sosetsu, 1998,37, 81 R567 H. Nakanishi, T. Yamagaki and M. Hu, ‘Analysis of Bioactive Saccharide Chain Materials. Structure Study of Influenza Virus Receptor, Glyco-Antibiotics, Xylan and Xyloglucan’, Fragrance J., 1998, 26, 108 R568 Y. Nakatani and G. Ourisson, ‘Dynamic Analysis of Lipid Membranes’, Nihon Yukugakkaishi, 1998,47, 1083 R569 H. Nakayama, ‘Characterization of Inorganic Phosphate Compounds by Solid State High Resolution NMR’, Phosphorus Lett., 1998,32,22 R570 H. Nishimura, R. Takeda and T. Takahashi, ‘NMR to fMRI’, No no Kagaku, 1998,20, 113 R57 1 T. Nishinaka and T. Shibata, ‘How Does DNA Recognize Homologous Sites and Exchange Strands? Molecular Structure of DNA in Homology Search and Strand Exchange’, Tunpakushitsu Kakusun Koso, 1999,44,63 1 R572 K. Nomura, G. Suzukamo, M. Yamamoto and M. Itagaki, ‘New

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Homogeneous Nickel-Phosphine Catalyst System for the Efficient Dimerization of Propylene’, Sumitomo Kagaku (Osaka), 1998,1,99 M. Nomura, ‘How Far Does Chemical Structure of Coals Become Apparent?’, Kagaku to Kogyo (Tokyo), 1998,51,1579 K. Ogata, ‘Structure and Dynamics of the Transcription Factor, Myb, in DNA-Sequence Recognition’, Saikagaku, 1998,70, 1233 H. Ogiwara, T. Shintomi, T. Okamura, K. Asano, T. Ando and T. Kiyoshi, ‘Next-Generation Applied Superconductivity and Nb3Al Conductors’, Teion Kogaku, 1998,33, 597 S. Ohki and M. Kainosho, ‘New Aspects in Stable-Isotope-Assisted NMR Techniques. Application to the Structural Analysis of Larger Complexes between Proteins and Nucleic Acids’, Tanpakushitsu Kakusan Koso, 1999,44, 574. T. Ohkubo, ‘NMR Structural Analysis of Ligand-Receptor Complexes for Drug Design’, Farumashia, 1999,35, 19 T. Okawara, ‘One-Pot Preparation and Stereochemistry of Saturated Polyazapolyheterocycles’, Yuki Gosei Kagaku Kyokaishi, 1999,57,280 H. Saito, S. Tuzi and A. Naito, ‘Conformation and Dynamics of Membrane Proteins as Revealed by High-Resolution Solid-state NMR’, Maku, 1998,23, 162 S. Sato., ‘Study of Structure Analysis of Oil Shale and Fuel-Manufacturing Technology. 2. Grade of Oil Shale’, Nenryo Oyobi Nensho, 1998, 65,423 S. Sato., ‘Structure Analysis of Oil Shale and the Development of Fuel Production Technology. 4’, Nenryo Oyobi Nensho, 1998,65,590 S. Sato, ‘Study of Structure Analysis of Oil Shale and Fuel-Manufacturing Technology. 5’, Nenryo Oyobi Nensho, 1998,65,656 H. Shimahara and Y. Kobayashi, ‘NMR Study of Histidine in Catalytic Mechanism of Carbonic Anhydrase’, Kagaku (Kyoto), 1998,53,74 M. Shirakawa and K. Shimada, ‘Analysis Methods: NMR’, Shirizu: Nyu Baiojijikkusu, 1997, 3, 100, ed. Y. Kyogoku and T. Tsukihara, Kyoritsu Shuppan, Tokyo, Japan T. Takayama, ‘Modern Solid-state NMR’, Kanagawa Daigaku Kogakubu Kenkyu Hokoku, 1998,36,22 T. Takeda, A. Abe, T. Hiejima and H. Furuya, ‘Thermodynamics of Main-Chain Liquid Crystals-Characteristic Conformation of Spacers and the Role in Determining the Phase Transition Behaviors’, Kobunshi Ronhunshu, 1999,56, 175 K. Tatsumi, ‘Solid State 7Li Nuclear Magnetic Resonance Analysis of Lithium Intercalated in Carbonaceous Materials’, Tanso, 1999, 186,54 K. Tatsumi, T. Akai, N. Iwashita, S. Higuchi and Y. Sawada, ‘Lithium InsertionlExtraction Reactions of Carbon Anode Materials’, Osaka Kogyo Gijutsu Kenkyusho Hokoku, 1998,392, 1 M. Tatsumisago, ‘Development of Ion Conducting New Glasses’, Yoyuen oyobi Koon Kagaku, 1998,41,19 1 Y. Tezuka, ‘Structural Study on Polysaccharide Derivatives: New

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39

Developments by NMR Spectroscopy’, Sen’i Gakkaishi, 1998, 54, P26 1 S. Wada and T. Igarashi, ‘Analysis of DHA in Fish Oil by NMR Spectroscopy’, Kagaku to Kogyo (Osaka), 1998,72,469 H. Waki, ‘Analysis for the Structure and Function of Gangliosides’, Nihon Yukagakkaishi, 1998,47, 1023 T. Watanuki, ‘Organisms as Measured by Nuclear Magnetic Resonance Spectrometer’, Kanagawa-ken Eisei Kenkyusho Kenkyu Hokoku, 1998, 28, 1 K. Yamabe, ‘Magnetic Properties of Metals and Materials, and their Application’, Kinzoku, 1998, 68, 603 T. Yamamoto and T. Morikita, ‘Synthesis and Structural Regulation of .n-Conjugated Polymers Using Transition Metal Complexes’, Organomet.News, 1995,4, 125 Y. Yamamoto and K. Akiba, ‘Chemistry of Hypervalent Organic Compounds. Fundamental Aspects of Hypervalent Organic Compounds. Chemistry of Main Group Element Porphyrins’, Kikan Kagaku Sosetsu, 1998,34,93 Y. Yoshida, H. Hideyuki, M. Nagata, T. Sakagami and K. Yamada, ‘Development of Organic-Inorganic Hybrid Coatings by the Sol-Gel Technology’, Nepon Kagaku Kaishi, 1998,9, 571 Y. Yoshimura, T. Aono, Y. Ikeda, Y. Endou and H. Tokisue, ‘Development of Surface Oil Repellent Treatment Technique of a Slider in a Hard Disc Drive in Order to Prevent Seizure’, Materia, 1999,38, 172

Polish R599 M. Bochenska, ‘Amide-Group Ionophores in Ion-Selective Membrane Electrodes. Synthesis, Structure, and Properties’, Zesz.Nauk. Politech. Gdansk., Chem., 1998,38, 1 R600 J. Jarzyna, ‘Nuclear Magnetic Resonance Logging - New Effective Method for Evaluation of Reservoir Rock Properties’, Nafta-Gaz, 1998,54,215 R60 1 H. Krawczyk, ‘The Homoconjugation Effect’, Wiad.Chem., 1998, 52, 673 R602 N. Pislewski and A. M. Graviec, ‘Use of NMR in Studies of Cement Composites’, Cem.- Wapno-Beton, 1998,3, 171 R603 G. Slosarek, ‘Structural Analysis of High-Molecular-Weight Proteins’, Curr. Top.Biophys., 1998,22, A140 Por tugese R604 F. J . Luna and U. Schuchardt, ‘An Introduction to Pillared Clays’, Quim.Nova, 1999,22, 104

40

Nuclear Magnetic Resonance

Romanian R605 A. J. Petrescu and G. Turcu, ‘Characterization of Protein Structure, Dynamics and Ligands Interaction by NMR Methods (II)’, Stud. Cercet.Biochim., 1997,40, 111 Russian R606 N. A. Bragina and V. V. Chupin, ‘Method for the Synthesis of Deuterium-Labeled Lipids’, Usp.Khim., 1997,66, 1077 R607 I. I. Ivanova, ‘In Situ NMR Spectroscopy in Heterogeneous Catalysis: Achievements and Prospects’, Ross.Khim. Zh., 1998,42,67 R608 G. A. Lyakhov and K. F. Shipilov, ‘Thermal and Concentration Anomaly in the Number of Hydrogen Bonds in Associated Liquid Solutions’, Tr.Inst. Obshch.Fiz., Ross. Akad. Nauk, 1997, 54, 61 R609 L. G. Maslov, N. S. Evtushenko, A. N. Shchavlinskii and A. I. Luttseva, ‘Methods for Monitoring and Standardization of Pharmaceuticals Containing Gestagenic Hormones’, Khim. -Farm.Zh., 1998,32,45 Spanish R 610 M. I. Colombo, J. Zinczuk and E. A. Ruveda, ‘Studies on the Preparation of Chiral Intermediates for Synthesis of Biologically Active Natural Products’, An. Acad. Nac. Cienc.Exactas, Fis.Nat. (Buenos Aires), 1997, 47,59 R611 J. A. Pino and P. Borges, ‘Volatile Components in Spices. I. Methods for Isolation and Analysis’, Alimentaria (Madrid), 1999,301, 39

2 Theoretical and Physical Aspects of Nuclear Shielding BY CYNTHIA J. JAMESON AND ANGEL C. DE DlOS

1

Theoretical Aspects of Nuclear Shielding

1.1 General Theory - Theory development in this reporting period has focused on relativistic corrections to the shielding,1-7 and applications of the theory to particularly large spin orbit contribution^.^-^.^ In the Breit Pauli approximation, the dominant relativistic effects on nuclear shielding arise from three Hamiltonian corrections: the mass-velocity operator, the Darwin operator and the spin-orbit operator. In the shielding literature and in these reports, the effects resulting from the mass-velocity and Darwin operators are called scalar or spin-free relativistic effects. The Pauli-type Hamiltonian contains only first order relativistic effects, whereas higher order operators are neglected. As a consequence the Pauli Hamiltonian is variationally unstable. When used in a perturbation expansion, the highly relativistic core electrons especially of heavy elements cause difficulties since the assumption that p2 ,

(4.10)

i = C (KsjhThi - ~ : j h T h s ) .

(4.11)

i,S

Indices s, t and i, j are used for virtual and occupied orbitals, respectively. We write the perturbed Hamiltonian as HE= Ho

+ EHI

(4.12)

and assume that the zeroth-order wave function 10) is optimized for zero field condition, E = 0. We may make a Taylor expansion of the electronic energy in terms of the orbital parameters

(4.13)

144

Nuclear Magnetic Resonance

We used here the fact that K: = 0 corresponds to the wave function optimized with respect to the zeroth-order Hamiltonian Ho. That is, we note that both the K and ( a E / K , j ) K , oare zero at E = 0. The optimum orbital parameters K: for a fixed field-strength E are given by -EE\'I/E[~], the ratio of the gradient to the Hessian G2].We note that (4.14) at E = 0. The second-order properties are given by where EF1is defined as g2] the second derivatives of the electronic energy with respect to the perturbation parameter E at zero field. We have

We used here the fact that K: = 0 at E = 0. ((HI; HI)),=, is the polarization propagator4 at the random phase approximation (RPA) level for frequency independent properties. of eq. (4.15) corresponds to the The inverse Hessian matrix principal propagator P' of the response theory in a frequency independent case. Aucar et al.' used the response theory instead of the sum-over-states (SOS) formalism. They presented the following principal propagator in the relativistic domain which is based on a QED formulation:

(Ell)-l

The orbital rotation operator thereby splits into two classes

k=ke+kp.

(4.17)

The electronic block P,, originating from k,is identical to the relativistic nopair RPA propagator analyzed in the previous paper by Aucar and Odder~ h e d eIt . ~was shown that taking the nonrelativistic limit of the Pee block and neglecting the other blocks (that is, excluding f p ) gives the nonrelativistic RPA equations for the paramagnetic terms. By analogy with the one-electron case, it follows that the diamagnetic term comes from the nonrelativistic limit of the Ppp block in the full relativistic RPA equation. In the ee-block all spinors in the integrals included in the A,, and Bee matrices are of order unity: O(co). On the other hand, all integrals appearing in the ep- and pe- blocks are of order O(c-'). In the pp- block the integrals of (Zlij) type in the A , matrix are of order unity, while all the other integrals in A , and all integrals in Bpp are of order O(cP2).Aucar et al. investigated the effects of the five approximation

4: Theoretical Aspects of Spin-Spin Couplings

145

steps on the diamagnetic term from 'approximation o', the exact RPA expression in a finite basis, to 'approximation S, the expectation value expression ( ( ~ 1 ;H I ) ) ,

M

(e2/2me>(OIi2

~ ~ ) .

(4.18)

As an example they calculated the DSO contributions to the reduced nuclear

spin-spin coupling constants, 'K(X,H) and 2K(H,H), for the water analogous, i e . , XH2 model compounds (X=O, S, Se, and Te). They found that neglecting the Pep and Ppe blocks (that is, decoupling thepp-part of the propagator from the other parts) and using a diagonal approximation for the Ppp block gives only very small changes. Furthermore, the replacement of the diagonal elements of the Ppp block by -2mec2 gave a significant change for the X-H coupling for the heavier systems, but not for the H-H coupling.

3

Rovibrational Effects

For small closed-shell gas-phase molecules, magnetic properties can nowadays be calculated so accurately that the main discrepancies between theory and experiment may be expected to be due to rovibrational effects. During the last year, two have been published about the calculation of rovibrational effects on the nuclear spin-spin coupling constants. 3.1 Rovibrationally Averaged Spin-Spin Coupling of the Hydrogen Fluoride Molecule - For diatomic molecules, several methods have been developed for calculating rovibrational averages of molecular properties.8-'0 As one of the rovibrational averages of properties Astrand et aZe6calculated rovibrational effects on the spin-spin coupling constant of hydrogen fluoride with the approach based on a method for introducing an anharmonicity into the potential in the vibrational problem.' In the high-temperature limit, a rovibrationally averaged property (a) of a hereronuclear diatomic molecule is in this scheme obtained as12

where B is the rotational constant, w the harmonic frequency in wavenumbers, and T the temperature, and where we have truncated the expansion at second order. The derivatives are here given as 0' = (an/@)and 0" = (@n/8e2), respectively, where 5 = ( r - reH)/reH. The subscript effindicates the quantities evaluated at the effective distance renwhich was proposed by Astrand et a1.I1 The effective distance reg is defined as the bond distance providing the minimum of the effective potential, Vefkr),which is the sum of the potential energy, V, and the zero-point vibrational energy, V,,,, calculated from the harmonic vibrational frequency, w,at the bond distance, Y. h a n d et al." showed that the harmonic expansion around reg is superior to the expansion

146

Nuclear Magnetic Resonance

around the equilibrium distance, re, which corresponds to the bottom of the potential. Astrand et aZ.6 first considered the dependence of the coupling constant 'J(H,F) on the basis set and the treatment of electron correlation. Two kinds of basis sets were employed; the atomic natural orbital (ANO) sets of Widmark et ~ 1 . and ' ~ the correlation consistent basis sets of Dunning and his ~o-workers.'~-'~ For the A N 0 basis sets both the contracted and uncontracted function sets were tested. The contraction of the A N 0 basis led to dramatic changes in the FC term, which is related to the sensitivity of the FC term to the description of the charge distribution at the n u ~ l e u s . ' To ~ ~ investigate '~ the FC term in more detail, basis functions with large exponents which form a geometric series were added to the primitive A N 0 basis. To converge the correlated coupling constants to within a few tenths of a Hz (at the 1oooCAS4220 level described later), an extension of the primitive A N 0 basis with three s functions at the fluorine atom and five s functions at the hydrogen atom (ANO+F:3s;H:Ss) was required. They found that the ANO+F:3s;H:Ss basis set consisting of 125 basis functions is superior to the correlation consistent basis set aug-cc-pCV5Z including 256 primitive functions contracted to 236 basis functions. Since the A N 0 basis converged faster to the basis set limit than did the correlation consistent basis sets, only A N 0 sets were used in their remaining calculations. The effects of electron correlation at the complete active space selfconsistent-field (CASSCF) and restricted active space self-consistent-field (RASSCF) levels were investigated using the A N 0 [6~5p4d3j75s4p34basis set. The individual and total contributions to the isotropic spin-spin coupling constants are listed in Table 4.1 for the various correlated wave functions. The DSO term is small and negligible. The PSO term has a correlation contribution of about -13 Hz but appears to be converged to within 1 Hz for the two largest restricted active spaces (RASs). In contrast, the SD term increases by 9.5 Hz from H F to 1oooCAS4220 (becoming almost zero), but most of the correlation contribution is included already with this smallest CASSCF wave function. Here, the superscripts 1000 and 4220 mean the inactive and active spaces, respectively. For the FC term, the correlation effects are larger than for the other terms. The total correlation contribution is about -150 Hz. Moreover, the full-valence 1oooCAS4220 space recovers only about half of the correlation correction and the FC term appears to be converged only to within 3-5 Hz even for the two largest RASs. The effective geometry for rovibrational corrections was calculated using the A N 0 [6s5p4d3f5s4p3d] basis set and the 1000RAS2::! orbital space. Here, the superscripts and subscript 1000, 4220, and 6332 indicate the inactive, RAS2, and RAS3 orbital spaces, respectively. For the FC term, they used the extended ANO+F:3s;H:Ss basis and the 1oooRAS;'~~~8, orbital space. The rovibrationally averaged nuclear spin-spin coupling constant at 0 K, ( ' J ( H ,F))o, was estimated to be 510 f 10 Hz, in excellent agreement with the most recent experiment of 500 f 20 Hz.~O

4: Theoretical Aspects of Spin-Spin Couplings

147

Table 4.1 The correlation dependence of the 'J(H,F) spin-spin coupling terms (in H z ) calculated with the AN0[6~5p4d3fl5~4p3d] basis set. (Taken from re$ [6 J )

-0.10 -0.10 -0.11 -0. I 1 -0.05 -0.06 -0.05 -0.05 n.c.b n.c.b n.ch a

199.2 177.6 182.0 181.9 186.7 187.3 186.2 186.7 n.c.b nch n.c.h

-10.8 -1.30 -1.41 -I .44 -0.2 1 -5.69 -0.29 -0.1 1 n.c.h n.c.b n.c.b

487.5 419.9 363.2 364.2 352.3 360.3 350.2 348.1 344.6 341.5 342.6

675.8 596.1 543.7 544.6 538.7 541.9 536.1 534.6 n.c.h n.c.b n.c.h

The ANO+F:3s;H:Ss basis set was used. Not calculated.

3.2 Spin-Spin Coupling Surfaces in the Water Molecule, Oxonium Ion, and Hydroxyl Ion - The simplicity and importance of the water molecule have provided the impetus for the very large number of published reports of ab initio calculation of its structure and properties. However, almost all calculations of coupling constants of the water molecule have been carried out at the equilibrium geometry. Wigglesworth et aL7 calculated 0-H and H-H spin-spin coupling surfaces for the water molecule for the first time. Both the use of correlated wave functions and the inclusion of all four contributing terms (FC, SD, PSO, and DSO terms) are known to be essential for obtaining accurate values of spin-spin coupling constants. Wigglesworth et al. computed the coupling surfaces involving the four contributions at the second-order polarization propagator approximation with coupled-cluster singles and doubles amplitudes - SOPPA (CCSD)2* - level using a large basis set and a grid of forty-nine geometries on each surface. They found that the second-order polarization propagator approximation (SOPPA) based methods tend to give better couplings than the multiconfigurational random phase approximation (MCRPA)22 approach using medium size complete active spaces (CASs). The spectral representation of the polarization propagator4 taken at w = 0 is given by (4.20)

By using the propagator method**the three contributions of PSO, FC, and SD can elegantly be evaluated without explicit calculation of the excited states.

Nuclear Magnetic Resonance

I48

Displacements from the equilibrium geometry can be described using the symmetry coordinates Si. For the water molecule there are three such coordinates:

+ 4,

s 2 = a,

(4.21) (4.22)

s 3 = 2-ll2(q - Y2).

(4.23)

s 1 = 2--1/2(rl

and

In eqs. (4.21) and (4.23), rl and r2 denote extensions of the OH1 and OH2 bonds from their equilibrium bond lengths Y,, respectively, while a denotes an increase in the HIOH2 angle from its equilibrium value (defined to be less than 1SOo). Assuming the Born-Oppenheimer approximation to the second order in the symmetry coordinates, the 0-H coupling constant IJ(0,H) can be written ' J ( 0 , H ) ='Je(O,H) +JiSi 1

-k TJ33S:

+ J 2 S 2 + J3S3

+JI2Sls2

-k J 1 3 s I s 3

1

+yJii$

+ J23s2s3,

1

+5J22S,'

(4.24)

where 'J,(O,H) is the equilibrium value of 'J(0,H) and the other Js are molecular parameters. The H-H coupling constant 2J(H,H) is more symmetrical and its symmetry coordinate surface to the second order is

Equations (4.24) and (4.25) are easily transformed to the formulas for the internal valence coordinate surfaces. Wigglesworth et al. first calculated 'J(0,H) and 2J(H,H) for the water molecule at the equilibrium geometry. Then, they computed the coupling constants at different geometries and obtained the symmetry coordinate and internal valence coordinate coefficients of the spin-spin coupling surfaces for H20. In the 0-H spin-spin coupling surface of internal coordinates, the derivative J, = (aJ(0,H ) / a ~ l ) is~ more than tenfold greater than Js = (a l J ( 0 ,H ) / a r ~ ) ~That ) . is, stretching (or compressing) the 0-H1 bond by a small amount alters the coupling 'J(0,HI) by more than ten times the amount by which J(O,H2) is simultaneously affected. The unexpected differential sensitivity found in CH423724did not occur in the water molecule. For *J(O,H)and 'J(0,D) the bond stretching at first order was the dominant part of the nuclear motion correction with the second bending making an important contribution. For 2J(H,D) the second order bending was by far the largest contribution to the nuclear motion corrections although the other terms partially canceled this contribution. Using the force field by J e n ~ e nRaynes ~~ et al. obtained rovibrational average^.^^-^^ As expected, the bending terms contributed relatively more to the temperature dependence of the couplings for

4: Theoretical Aspects of Spin-Spin Couplings

149

Table 4.2 Coefficients of the temperature dependence of the spin-spin couplings for isotopomers of water as defined in eq. (4.26). (Takenfrom re$

VI) Coupling

Isotopomer

alHz

103b

'J(O,H)

~ ~

~ 1 7 0 ~ 1 7 0

~ ~

~ 1 7 0 ~ 1 7 0

-77.59 -77.69 -78.59 -78.66

1.285 1.226 1.356 1.302

'J(O,H) If (0,D)" 'J'(0,D)" 'J*(O,D) = (

H Z K-1

1 0 7 ~ HZ

K

2

-0.966 -1.571 -1.753 -3.067

y ~ / y x~ IJ(0,D). )

'J(O,H), 'J(O,D), and 2J(H,D). The calculated 'J(0,H) in H2170 of -77.22 Hz at 293 K is in very good agreement with Wasylishen and fried rich'^^^ observed value of -78.70(?0.02) Hz in cyclohexane at this temperature. The calculated 2J(H,D) in HD160 at 323 K of -1.233 Hz is close to a recent experimental value of - 1.114(f0.003) Hz in nitromethane-d3 observed by Sergeyev et aL3' at that temperature. Wigglesworth et al. found that the temperature dependences of 'J(0,H) can be fitted to an equation of the form ' J ( 0 ,H)/Hz

=a

+ bT + cT2,

(4.26)

where T is measured in K. The coefficients for the four isotopomers are given in Table 4.2. Sauer et al.32 calculated the nuclear spin-spin coupling constants of H 3 0 + and OH- ions, their temperature dependence, and isotope shifts by using RPA, SOPPA, and MCRPA methods. The RPA was neither able to reproduce the absolute values nor the geometry dependence obtained at the correlated levels. The SOPPA (CCSD) and MCRPA with a large active space gave very similar results for the absolute values as well as for the geometry dependence of the coupling constants and thus vibrational effects. They inferred, however, that the SOPPA (CCSD) results are more accurate than the MCRPA ones. For OH- a linear temperature dependence was observed with almost the same coefficient as in H 2 0 , whereas the temperature effects in H30+ were quite nonlinear. Experimental data are only available for H 3 0 + , where both the 'H and 1 7 0 NMR spectra were measured in 'magic acid' ~ o l u t i o n s . ~The ~ 9 observed ~~ values of ' J ( 1 7 0 ,H) = - 106 f 1.5 Hz34 at 258 K of the H20-HF- SbFS system in SO2 and 2J(H,H) = f 3 . 3 Hz33at 229 K of the H20-HS03F- SbFs system in SO2 were compared with their Calculated SOPPA (CCSD) values of 'J(170,H) = -124.5 Hz and *J(H,H) = -3.8 Hz at the same temperatures. They stated that the major differences between the calculated and experimental values are due to the inherent aggregation and solvent effects in the experimental data, which were estimated to be in the order of 15%.

150

4

Nuclear Magnetic Resonance

Basis Set Dependence of Nuclear Spin-Spin Coupling Constants

The standard basis sets of quantum chemistry have been developed for a flexible description of the valence region of the electronic system. However, to describe spin-spin couplings accurately, other regions are relevant since NMR measurements probe the electron density close to the nuclei, making the standard basis sets for ab initio calculations ill-suited for the computation of spin-spin coupling constant^.^^.^^ We have to use relevant basis sets for calculating spin-spin coupling constants. Another complication is that, for spin-spin couplings, Hartree-Fock (HF) level calculations are often meaningless: giving, for instance, results of the wrong order of magnitude or with the wrong sign.36A reliable analysis of basis set dependence should therefore be performed at the correlated level. We will introduce the recent three about basis set dependence of spin-spin coupling calculation. 4.1 Multiconfigurational Self-Consistent Field Calculations - Helgaker et al.37 investigated the convergence of nuclear spin-spin coupling constants with respect to the extension of the basis set and the active orbital space at the multiconfigurational self-consistent field (MCSCF) level for the H F and H20 systems. In order to investigate the basis set effects on the spin-spin couplings Helgaker et al. started their MCSCF calculations of couplings with the correlation consistent cc-pVXZ sets14 of 2 5 X 5 6. Next, they decontracted the s functions of the cc-pVXZ bases completely and obtained cc-pVXZ-su0 series. Finally, they improved upon the description of the cc-pVXZ-su0 sets by adding a sequence of n tight s functions with the exponents forming a geometric progression. The resulting sets were denoted cc-pVXZ-sun. All the calculations were performed using the DALTON program.40 The spin-spin coupling constant in HF was dominated by the FC and PSO contributions. The noncontact contribution (SD, PSO, and DSO contributions) converged easily. For the noncontact contributions to 'J(H,F) the ccpVQZ-suO basis was found to be enough. However, it was harder to achieve the convergence for the FC term and one needs to add a number of very tight s-type functions, as in the cc-pVXZ-sun (X 2 3) series, to improve the convergence of the FC term. In any case, the cc-PVDZ basis sets were too small and the triple-zeta or larger (X>3) correlation consistent sets must be used as the starting point. The noncontact and contact contributions to 'J(0,H) in H20 varied with the basis set in the same manner as for 'J(H,F). For 2J(H,H) in H20, the convergence pattern was rather different. As expected, the addition of core orbitals to oxygen had little effect, whereas augmentation with diffuse functions led to larger changes. For 2J(H,H), the total spin-spin coupling depended on three large contributions: negative FC and DSO terms and a positive PSO term. The DSO and PSO terms converged well and almost canceled each other out. However, the oscillations were observed for the FC contribution and the total 2J(H,H) appeared so poorly convergent. Their best total values of calculations were 542.60 Hz for 'J(H,F), -78.08 Hz for 'J(O,Hj, and -9.70 Hz for 2J(H,H), respectively. The experi-

4: Theoretical Aspects of Spin-Spin Couplings

151

mental values are 529 f 23 Hz for 1J(H,F),41-73.5 f 2.1 Hz for 1J(0,H),42 and -7.2 f 0.7 Hz for 2J(H,H),43respectively. Guilleme and San Fabian3*tried to find the efficient basis sets for calculating coupling constants with H and C nuclei. The FC term is very sensitive to the basis set and electron correlation effect,44 and usually constitutes the largest portion of the total spin-spin coupling constants. On the other hand, the noncontact contributions consisting of SD, PSO, and DSO terms are less sensitive to the size of the basis sets and to the electron correlation.44In order to get values of coupling constants close to the experimental ones, we must consider the use of large basis sets and the inclusion of electron correlation and rovibrational effects. However, the use of large basis sets is restricted to small size molecules. It is therefore very important to find small and good quality basis sets for calculating coupling constants in medium and large size molecules. It is known for spin-spin coupling calculations26 that (1) the addition of tight s type functions to the basis set is necessary to obtain reliable FC contributions, (2) polarization functions, dc and p H , are important, (3) only modest contractions are permissible, and (4) the inclusion of s or p c diffuse functions and tight pc functions in the basis set has a negligible effect. In order to evaluate the effect of the contraction on coupling constants, Guilleme and San Fabian first performed the H F level calculation for 'J(C,H) and 2J(H,H) in CH4 with both contracted and fully uncontracted basis sets. The FC contributions presented important variations when the basis sets were decontracted. They showed that the contraction effects came from the orbital energy variation and the change of the charge densities at the coupled nuclei. They stated that one should choose the contraction schemes in which the contracted basis set provides practically the same values of orbital energies and nuclear densities as those calculated with the uncontracted bases set. They finally obtained the BS8 basis set [9s5p3d/6s2p]as an efficient basis set for calculating the C-H and H-H coupling constants. They also proposed a smaller basis set BS8s [9s4pld/6slp] which can be used in larger molecules. Guilleme and San Fabian next calculated the spin-spin coupling constants in CH4 at the MCSCF level with different numbers of active orbitals and different numbers of excited electrons. They proposed a method of extrapolation approximation using three calculations to estimate the highly correlated FC contributions to the spin-spin coupling constants. Their best values obtained for lJ(C,H) and 2J(H,H) in CH4 are 120.63 Hz and -13.23 Hz, respectively. The experimental values for 'J(C,H) and *J(H,H) in CH4 are 125.30 Hz and -12.57 Hz, r e s p e ~ t i v e l y . ~The ~.~~ differences between the calculated and experimental values would have been diminished if they had included the rovibrational effects since an extension in the equilibrium bond length of methane will produce a change in the coupling constants and will shift both the 'J(C,H) and 2J(H,H)values to the positive direction.26

4.2 Second-Order Polarization Propagator Approximation Calculations - Ene. ~ ~the results for correlated calculations and basis set voldsen et ~ 1 presented dependence of the nuclear spin-spin coupling constants in HD, HF, H20,

Nuclear Magnetic Resonance

152

CH4, C2H2, BH, AlH, CO, and N2 at the level of SOPPA and SOPPA (CCSD). The indirect spin-spin coupling constant between two nuclei K and L consists according to R a m ~ e yof~the ~ following four contributions: the DSO term

the PSO term J

cr=x,y,z

the FC term

cr=x,y,z

and the SD term

(4.30)

where (4.3 1)

(eh/2me).Y K and "~r,are the nuclear + magnetogyric ratios of nuclei K and L, respectively. ?iK = 7; - I$ is the difference of the position vectors of electron i and nucleus K . 2j and 1 ; are ~ the spin and orbjtal angular momentum operators of electron i in unit Js, respectively. 1 ; ~is defined with respect to the position of nucleus K: liK = -&ih?iK x q. Equation (4.20) indicates that the three contributions of

pB is the Bohr magneton and defined as

-b

4: Theoretical Aspects of Spin-Spin Couplings

153

PSO, FC, and SD can be computed with the use of polarization propagator. The DSO contribution, on the other hand, is a ground state average value, although it is also possible to express it as a p r ~ p a g a t o r . ~ ~ In SOPPA and SOPPA (CCSD) the polarization propagator for static perturbation operators, P and Q, can be written as18949 ((Pi Q)),=o

=W2

-

+ W4,

(01[P, qq] 10)(l)D(o)*(01[qtqt,Q]lo)('),

(4.32)

(4.34)

where

qt and q are column vectors consisting of excitation and deexcitation operators, respectively. q t and q are their transposed row vectors. The matrices or vectors of A , B, C,D, T(P),and T ( Q )are given in the paper by Oddershede et al. l8 The superscripts (n)denote the order of the fluctuation potential. Following the same general rules as Helgaker et Enevoldsen et al.39 have developed basis sets for use in spin-spin coupling constant calculations. The basis sets were constructed from the correlation consistent basis sets of Dunning and his c o - ~ o r k e r s . ' They ~ ~ ' ~first ~ ~determined ~ a new basis set for C and H in CH4 at the RPA level calculation and next generated corresponding basis sets for all the other atoms of B, Al, N, 0, and F. The proposed basis sets are the spherical uncontracted Gaussian function sets of (lOs3pld) for H, (15s6p3dlf) for B, C, N, 0, F, and (20slOp3dlf) for Al. The new basis sets (denoted 11) were used to get the SOPPA and SOPPA (CCSD) results as well as the previous smaller basis sets (denoted I). The largest basis set effect of 84% was found in 1J(27Al,H)at the SOPPA (CCSD) level. With the exception of N2 they indicated that the correlation correction to the coupling constants was larger in the extended basis set 11. Comparing the four contributions to the coupling constants and their correlation dependence they showed that with the exception of the geminal or vicinal H-H coupling constants in H20, CH4, and C2H2, the DSO term gave a small contribution to the total coupling constants. There was a very small correlation effect on the JDsO term. To the JpsOterm there was a modest correlation contribution. It was most pronounced for the triply bonded molecules. For *J( lB,H)pso there was a large difference between SOPPA and SOPPA (CCSD): SOPPA (CCSD) was numerically larger by 66% then SOPPA. This is not surprising since SOPPA is known to have difficulties describing the near degeneracy effects in BH.51-54The contributions of the SD term obtained by SOPPA and SOPPA (CCSD) differed by at most 0.1 Hz except in one case. For the H F molecule the difference was 0.38 Hz. The main difference between the SOPPA and SOPPA (CCSD) numbers came from the FC contribution.

Nuclear Magnetic Resonance

154

Compared with experimental coupling constants SOPPA (CCSD) in general improved on the SOPPA results. For the majority of the coupling constants calculated the deviation from experiment was less than 2.5 Hz. It was shown that the SOPPA (CCSD) method serves as a cost efficient improvement over SOPPA for calculating reliable spin-spin coupling constants. However, the step necessary to obtain the correlation correction in the SOPPA (CCSD) calculation scales as @ rather than N5 in the SOPPA calculation. Analyzing contributions to the polarization propagator at the SOPPA level they found that the second-order W, term shown in eq. (4.34) is of little importance.

5

Other Ab Znitio Calculations

gal ass^^^ calculated the nuclear spin-spin coupling constants "J(X,Y) in the cyclooctyl (1,5-C8), cyclononyl (1,5-C9), and cyclodecyl (1,5-C10) cations (Scheme 4.1) at the ab initio level by the variational form of the equations-ofmotion (EOM) method, which was solved according to the particular set of approximations proposed by Shibuya and M c K o ~ . This ~ ~ ?method, ~ ~ which incorporates the main portion of the electron correlation, was shown to provide satisfactory results for a variety of hydrocarbon^.^^-^^ Geometries for the systems were fully optimized at the HF/6-31G** level of theory.

Scheme 4.1

The distinguished spectroscopic features of the medium-ring carbocations are the very high field *H chemical shift and the abnormally low coupling constant 'J(C,H) involving the p-hydrogen. The results of the ab initio EOM calculations are presented in Table 4.3. The most peculiar feature of the cycloalkyl cations shown in Table 4.3 is the remarkable difference (ca. 120 Hz) between the two 'J(C,H) coupling constants in the bridged skeleton. The coupling constant involving the p-hydrgen is anomalously low, while the coupling constant to the terminal bridge hydrogen has a normal alkanic value. The theoretical estimate of both 'J(C:H)s are in fair agreement with the experimental data for all three carbocations. Galasso showed that the large difference between the two coupling constants reflects a drastic difference in the s characters of the two bonds. The localization of the molecular orbitals is the following: C-H, sp2q21and C-H, sp6.65in the cyclooctyl cation, C-H, sp2.13 and C-H, sp7.17in the cyclononyl cation, and C-H, sp2*08and C-H, sp7.40in the cyclodecyl cation. Table 4.3 reveals that for both 'J(C,H)s the dominant contribution comes from the FC term and the noncontact terms provide far smaller contributions.

4: Theoretical Aspects of Spin-Spin Couplings

Table 4.3

155

J(C,H ) nuclear spin-spin coupling constants ( H z ) in ring carbocations. (Taken from ref: 55)

Molecule

1,5-C8

1,5-C9

1,5-C10 ~~

' J ( C ,HJ

FC DSO PSO SD Total Exp. FC DSO PSO SD Total Exp.

28.35 1.71 1.87 0.55 32.48 < 35 144.20 0.84 -0.27 0.07 144.84 147 & 10

27.73 1.78 1.90 0.68 32.09 < 35

27.74 1.88 2.04 0.99 32.65 < 32f5

144.44 0.88 -0.30 0.06 145.08 148 & 7

144.52 0.89 -0.34 0.06 145.13 l58&5

Ruiz de Azua et analyzed transmission mechanisms of the nuclear spin-spin coupling constant 'J(N,H) in NH3 based on the use of localized molecular orbitals (LMOs) and inner projections of the polarization propagator (IPPP) at the RPA level, namely, ab initio IPPP-CLOPPA (contributions from localized orbitals within the polarization propagator approach). The use of LMOs in quantum chemistry is a useful tool to obtain qualitative insights on many physico-chemical phenomena since the basic properties of a given molecular moiety are essentially determined by the local electronic distribution of that moiety. Within the polarization propagator (PP) formalism a%y comeonent of the spin-spin coupling tensor between nuclear spin vectors l~ and 1L can be expressed J(K, L),p = k

cc s,t

USi(K,a>Urj(L,m p - '

)s;,fj,

ij

a/3 E x, y , z, i,j E occupied LMOs,

s, t E virtual LMOs,

(4.36)

where (P1)sj,Q stands for the principal element of the propagator matrix P', Usi(K,a ) represents the elements of the perturbative Hamiltonian matrix due to the nuclear component Z K ~ and , k is the appropriate constant factor. For the dominant contribution J(K,L)rF P' is given by the triplet propagator matrix. The elements of the inverse propagator matrix P at the RPA level are given in this case by =( E ~ &j)G,tSo -

(stlji) - (sjlti).

(4.37)

The perturbator matrix U is defined as

Ruiz de Azua et al. calculated J(N,H) in NH3 as a function of HNH angle 9 with 6-31G** basis set which contains 30 basis functions. The 25 virtual

156

Nuclear Magnetic Resonance

LMOs were classified as follows: 11 polarization MOs, 2 LMOs centered on the N atom, and 4 LMOs on each N-H bond. The 11 polarization MOs, built up with the p AOs on the three H atoms and the d AOs on the N atom, have a remarkable property. They play an almost negligible role in the transmission of the 'J(N,H) coupling. This is not surprising since all polarization AOs have zero amplitude at the respective nuclear positions. Furthermore, an IPPP calculation in which these 11 MOs are excluded from the projection subspace yielded almost the same result as the all included calculation. This is particularly important to study extended systems where the inclusion of polarization AOs strongly increases the computational cost. Ruiz de Azua et al. showed that the leading pathways mainly producing the behavior of the total FC coupling in NH3 as a function of 8 are (N-H,N-H) and (N-H,N LP) coupling paths. The former corresponds to the sum of all terms in which both occupied LMOs are the N-H bond, and the latter to the sum in which one occupied LMO is the N-H bond and the other the N lone-pair (LP). Ab initio CLOPPA decomposition led to the results similar to those obtained with semi-empirical approaches for ' J c o ~ p l i n g s . ~ ~ ? ~ * Chakraborty and Chandra69carried out ab initio molecular orbital calculations of nuclear spin-spin coupling constants in PH,, PH3, PH,, and P2H4 at the self-consistent-field perturbation theory (SCFPT) Basis set dependence of all the four contributing terms was studied in order to find the criterion for the selection of basis sets to be employed for computing this property. They found that inclusion of bond-centered functions along with at least double zeta basis sets allows to reproduce the coupling constants quite satisfactorily. In all the cases studied uncontracted core basis functions yielded couplings which were in better agreement with experimental couplings than those obtained with contracted core functions.

6

Density Functional Theory Calculations

Density functional theory (DFT) has emerged as a tangible alternative to traditional ab initio methods since it is able to include electron correlation in a computationally efficient way that makes it possible to treat even large size molecules quantitatively. The use of DFT in spin-spin coupling calculations was pioneered by Malkin et a1.72773in an implementation based on Gaussian type orbitals (GTOs). Subsequently Dickson and Ziegler74 presented an implementation based on Slater type orbitals (STOs). In the last year Khandogin and Ziegler75 have published the DFT calculation results for nuclear spin-spin coupling constants in transition-metal systems. They presented results for metal-ligand one-bond reduced coupling constants, K(M,X), in 3d-, 4d-, and Sd-transition-metal carbonyl, 0x0, fluoro, and phosphin complexes. The two major contributions to the metal-ligand coupling constants came from the FC and PSO terms. The FC contribution was evaluated by a DFTfinite perturbation theory (FPT) and the PSO part by an uncoupled DFT-SOS approach. In the DFT-FPT calculation the lighter atom, namely, the ligand

4: Theoretical Aspects of Spin-Spin Couplings

157

atom was used as the perturbing atom. In the uncoupled DFT-SOS approach for the PSO term any influence of the current density on the exchange correlation energy was neglected. Compared with experiment, they indicated that the exceptionally large discrepancy of the calculated coupling constant in MnO; could be related to the error due to the neglect of current density. Only molecular orbitals with contributions from s-type orbitals on both the ligand and the metal center contributed to ' K ( M , m F C This . makes it possible to preclude in the FC term core and lone-pair orbitals and valence orbitals other than those in the totally symmetrical representation. Thus, the FC term can be viewed as a valence property, which allows the adoption of the frozen-core approximation for the calculation of the systems containing heavy atoms. Relativity enhanced the contributions in absolute values since the density of the ns and (n+l)s orbitals on the metal atom is increased. However, this might not result in a total increase in 'K(M,rnFCdue to the different signs of the individual contributions. The lK(M,X)psocontribution was attributed mainly to the couplings between the occupied orbitals of highest energy and the virtual orbitals of lowest energy. Onak et aZ.76calculated nuclear spin-spin coupling constants in 25 polyhedral boranes using DFT-FPT method. They considered solely the FC term."B NMR chemical shift calculations based on ab initio molecular orbital methods have provided powerful structural information for polyhedral boron-containing compounds. In contrast, there are few theoretical studies of nuclear spin-spin couplings in boron corn pound^.^^-^^ In view of the extensive application of coupling constant calculations to other molecular systems, this may seem surprising. Perhaps the most important reason is the extreme difficulty of obtaining accurate coupling constants in polyboranes since they exhibit exceedingly complex NMR s p e ~ t r a 1-84 . ~ ~Onak ? ~ et al. reported the computed results for coupling qonstants in polyhedral boranes which were in good agreement with the solution experimental data for a wide range of directly bonded, geminal, vicinal, and long-range couplings. It was shown that the FC contributions are dominant for these compounds. The DFT method using only the FC term proved to be very effective in the calculation of coupling constants in polyhedral boron compounds. However, caution should be exercised in the application to other systems, such as those containing fluorine, where the noncontact mechanisms are usually substantial. Bour and B ~ d e s i n s k yperformed ~~ nuclear spin-spin coupling constant calculations using the DFT-SOS expansion formula and compared the results with the DFT-coupled Hartree-Fock (CHF) calculations. The one-electron states and their energies were obtained from the Kohn-Sham (KS) equations.86 The accuracy of the DFT-SOS calculations for the couplings in nine small molecules (CH4, C2H6, C2H4, C2H2, H20, CH3F, N2, H2, and HF) was comparable with the DFT-CHF results but the coupling constants by the DFT-SOS method were uniformly underestimated. The SOS scheme has a merit that it is less limited by molecular size and can be applied for larger systems than the CHF method. The DFT-SOS method was tested for the calculation of H-H coupling constants in a-pinene, P-pinene, and camphor

Nuclear Magnetic Resonance

158

molecules. Relative magnitudes of most H-H coupling constants in these three compounds were correctly predicted, while their absolute magnitudes were typically smaller by factors of 2 to 3 than those found by the experiment. Stahl et al.87 calculated 13C-l 3C coupling constants in conformationally flexible hydrocarbons to obtain information about conformer equilibia in solution. They employed methods developed by Malkin et al.72 within the framework of 'DFT in conjunction with deMon Kohn-Sham p r ~ g r a m . ~ ~ - ~ ~ Coupling constants calculated for the individual conformers were Boltzmann weighted according to MM3 (molecular mechanics force field model 3) energies.91 Cuevas et ~ 1carried . ~out~DFT calculations for 'J(C,H) coupling constants in cyclohexane and 1,3-diheterocyclohexanes.The DFT calculation of 'J(C,H) coupling constants was done using the approach proposed by Malkin et ~ 1 . ~ Within ~ 1 ~this ~ methodology, 7 ~ ~ the total coupling constant is given as the sum of the three contributions of the FC, PSO, and DSO terms. The FC term is calculated by the DFT-FPT method and the PSO contribution is obtained using the DFT-SOS approach, and the DSO term is computed by numerical i n t e g r a t i ~ n . The ~ ~ . ~calculated ~ results showed no correlation between the 'J(C,H) values and the corresponding C-H bond distances. The direction of the Perlin effect,94 defined as the axial minus the equatorial one-bond C-H coupling constants, was correctly reproduced for all cases with the exception of one methylene group in 1,3-0xathiane. Conformational analysis of oligosaccharides has been based largely on the observation of inter-residue 'H-'H NOEs to assess the torsional preferences about their constituent 0-glycosidic linkages. In many cases, however, the number of measurable NOEs is smaller than that needed to establish solution conformation with reasonable ~ e r t a i n t y Consequently, .~~ the measurement of NMR parameters, such as 2J(COC), 3J(COCH), and 3J(COCC) values, that may be sensitive to linkage conformation and more easily interpreted than 'H-lH NOEs, is desirable. Bose et al.96 computed 3J(COCH) and 3J(COCC) coupling constants in some model compounds, mono- and di-saccharides, by the DFT-FPT approach. They obtained close agreement between the experimental and calculated 3J(COCC) values. They also proposed a simple Karplus type equation for trans-0-glycoside 3J(COCC) values and applied it to the reanalysis of trans-0-glycosidic couplings in 3C-labeled methyl P-lactoside and sucrose. Salsbury and Harris97investigated the spin-spin coupling constant of Xe-H and '29Xe- 13'Xe at various internuclear distances with the use of DFT, They considered only the FC contribution to the couplings. In the regions of interest, namely, at the internuclear distances for a cage molecule, 'J(Xe,H) was of the order of pHz and 'J('29Xe,'31Xe) was of the order of mHz. They indicated that these values are within the potential ability of experimentalists to measure with the techniques of optical pumping. Factors of 10000 or more enhancement in the xenon spin polarization are often seen with optical pumping. As a result, the small xenon coupling constants which one would expect in van der Waals complexes may be experimentally observable. The

'

159

4: Theoretical Aspects of Spin-Spin Couplings

observation of small Xe-H coupling constants is interesting as xenon often binds to biomolecules via van der Waals interaction.

7

Semi-Empirical and Other Calculations

Lobayan and Aucar9* calculated reduced coupling constants in molecules containing heavy atoms by using the CLOPPA-PM399 approach. They employed the PM3 scheme to extend the coupling calculation to nuclei belonging to atoms that were not parameterized for the modified neglect of diatomic overlap (MNDO)loO and AM1 (Austin model 1)lo1 methods. They got qualitative trends for coupling constants involving nuclei that are not parameterized within the MNDO and AM1 methods, i.e., for couplings in such as compounds containing atoms of As, Sb, Bi, Ga, In, and TI. However, they found quasi-instability problems in many of the compounds calculated. Kamienska-Trela et al.'02 calculated lJ(C,C) coupling constants in substituted diacetylenes, R - C' E C2 - C3 = C4 - H, and compared the computed values to the experimental ones measured by them. They used the intermediate neglect of differential overlap (IND0)-FPT method in their calculation. The obtained 'J(C3,C4) results revealed that in accordance with the experimental data the influence of substituents R is rather small but cannot be neglected. Its total range was smaller than 10 Hz; the smallest value of 176.5Hz was calculated for R = Li and the largest one, 184.4 Hz, for R = F. As expected, the largest substituent effect was found for lJ(C1,C2).The calculated values ranged from 62.5 Hz (R = Li) to 226.8 Hz (R = F). The influence of substituents upon lJ(C2,C3)was also large. The calculated values were from 129.3 Hz (R = Li) to 162.9 Hz (R = F). The INDO-FPT theoretical predictions for 'J(C2,C3) were in agreement with experimental results. The influence of substitutents on the 'J(C2,C3) coupling was additive. This additivity may be due to the fact that the lJ(C2,C3) values were extirely governed by the FC term. The graphical representation of the FC contribution vs. Pauling's electronagativity revealed the linear relationship. Furthermore, the calculated and experimental data strongly implied that the influence of substituents is not transferred along the conjugated triple bond system at least when 'J(C,C) couplings are considered. Kowalewski et al. lo4 investigated the intramolecular electric filed effects on 'J(C,H) coupling constants by using the INDO-FPT approach. They employed substituted salicylaldehydes (Scheme 4.2) as the model compounds. They showed that the particularly large electric field component along the C,-Hf bond, calculated for X = N02, accounts for a measured substituent effect of 7.36 Hz in 'J(C,,Hf). For other Xsubstituents a linear correlation was found between the calculated substituent electric field component along the C,-Hf bond and the substituent effect on 'J(C,,Hf). This result is in agreement with their theoretical calculation presented previously.' 0 5 Chirkina et al. lo6 evaluated 13C-13C spin-spin coupling constants of 20 tricycle[ 1.1.1.O1.3]pentanederivatives by the self-consistent perturbation theory (SCPT)-INDO procedure with preliminary optimization of the geometric ,

Nuclear Magnetic Resonance

160

Scheme 4.2

parameters. It was shown that the central C-C bonds in these compounds presented large coupling constants due to abnormally high s character ranging from 33 to 60%. This result suggests antibonding nature of the highest occupied molecular orbital localized on the central bond. Kuznetsova et al.'07 calculated 3C-13C coupling constants in 22 three- and four-membered heterocycles containing Si, 0, S, P, and N atoms in terms of the SCPT-INDO method with full geometry optimizations. Pathways of 3C-13C couplings were distinguished, and orbital hybridization of the C-C bonds were calculated. As the electronegativity of the hetroatoms increased, both geminal and vicinal contributions to the 13C-13C couplings became more positive and the direct path contribution to the couplings increased. They found a reliable linear correlation between 'J(CA,CB) and the product of the s percentages at CA and CB,(%sA)(%sB). Barbier et al. log applied the conformations and spectra (SC)-INDO rnethodlo9 to the evaluation of the J(H,H), J(C,C), and J(C,H) coupling constants of the following unsaturated compounds: ethylene, butadiene, hexatriene, benzene, s-cis and s-trans acrolein, trimethine- merocyanine, and streptocyanine. The CS-INDO approach includes the electron correlation effects up to the second order at the semi-empirical level. The effect of electron correlation on the computed FC contributions increased the ' J and 3J couplings and decreased the J couplings, sometimes leading to negative coupling constants. Indirect nuclear spin-spin coupling tensors in solids, dominated by the isotropic Ruderman-Kittel (RK) interaction' l o and the anisotropic pseudodipolar (PD) interaction," have been investigated thoroughly in metal' 12-' l 7 during the past three decades. However, systematic studies of spin-spin couplings in semiconductors are scarce. Patnaik et al.' l 8 calculated 207Pd-207Pdnuclear spin-spin coupling tensor in both n-type and p-type PdTe, which are degenerate narrow-gap semiconductors. The coupling constants calculated for carrier densities of the order of 10l8cmP3were found to be about four orders less than the corresponding value in metallic lead. They stated that although there are no experimental results for comparison, the order of magnitude appears to be reasonable.

'

'

4: Theoretical Aspects of Spin-Spin Couplings

8

161

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73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89

90 91 92 93

94 95 96 97 98 99 100 101 102 103 104

105

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0. L. Malkina, D. R. Salahub, and V. G. Malkin, J. Chem. Phys., 1996, 105, 8793. R. M. Dickson and T. Ziegler, J. Phys. Chem., 1996,100,5286. J. Khandogin and T. Zingler, Spectrochim. Acta, 1999, A55,607. T. Onak, J. Jaballas, and M. Barfield, J. Am. Chem. SOC.,1999,121,2850. V. Galasso and G. Fronzoni, J. Chem. Phys., 1986,85,5200. S. A. Perera, R. J. Bartlett, and P. von R. Schleyer, J. Am. Chem. Soc., 1995, 117, 8476. S. A. Perera, M. Nooijen, and R. J. Bartlett, J. Chem. Phys., 1996, 104, 3290. M. Nooijen, S. A. Perera, and R. J. Bartlett, Chem. Phys. Lett., 1997,266,456. T. C. Farrar, R. B. Johannesen, and T. D. Coyle, J. Chem. Phys., 1968,49,281. T. C. Farrar and G. R. Quinting, Inorg. Chem., 1985,24, 1941. R. C. Hopkins, T. D. Baldeschewieler, R. Schaeffer, F. N. Tebbe, and A. Norman, J. Chem. Phys., 1965,43,975. A. 0. Clouse, D. C. Moody, R. R. Rietz, T. Roseberry, R. Schaefer, and L. G. Sneddon, J. Am. Chem. SOC.,1973,95,2496. P. Bour and M. Budesinsky, J. Chem. Phys., 1999,110,2836. W. Kohn and L. J. Sham, Phys. Rev., 1965,140, 1133. M. Stahl, U. Schopfer, G. Frenking, and R. W. Hoffmann, Mol. Phys., 1997, 92, 569. A. St.-Amant and D. R. Salahub, Chem. Phys. Lett., 1990,169, 387. D. R. Salahub, R. Fournier, P. I. Mlynarski, A. St.-Amant, and J. Ushio, in Density Functional Methods in Chemisty, ed. J. Labanowski and J. Andzelm, Springer, New York, 1992, p. 77. M. Godbout, D. J. Salahub, J. Andzelm, and E. Wimmer, Can. J. Chem., 1992, 70, 560. N. L. Allinger, Y. H. Yuh, and J.-H. Lii, J. A m . Chem. Soc., 1989,111,8551. G. Cuevas, E. Juaristi, and A. Vela, J. Phys. Chem., 1999, A103, 932. V. G. Malkin, 0. L. Malkina, L. A. Eriksson, and D. R. Salahub, in Modern Density Functional Theory. A Tool for Chemistry, ed. J. M. Seminario and P. Politzer, Elsevier, Amsterdam, 1995. A. S. Perlin and B. Casu, Tetrahedron Lett., 1969,292 1 . S. W. Homans, Prog. N M R Spctrosc., 1990,22, 5 5 . B. Bose, S. Zhao, R. Stenutz, F. Cloran, P. B. Bondo, G. Bondo, B. Hertz, I. Carmichael, and A. S. Serianni, J. Am. Chem. Soc., 1998, 120, 1 1 158. F. R. Salsbury, Jr., and R. A. Harris, Mol. Phys., 1998,94,307. R. M. Lobayan and G. A. Aucar, J. Mol. Struct. (Theochem), 1998,452, 1 . J. J. P. Stewart, J. Comput. Chem., 1989, 10, 221. M. J. S. Dewar and W. Thiel, J. Am. Chem. Soc., 1977,99,4899. M. J. S. Dewar, E. G. Zoebisch, E. F. Healy, and J. J. P. Stewart, J. Am. Chem. Soc., 1985,107,3902. K. Kamienska-Trela, L. Kania, W. Schilf, and I. Balova, Spectrochim. Acta, 1999, A S , 817. R. McWeeny, in Coulson’s Valence, Oxford University Press, London, 1979, p. 163. D. G. de Kowalewski, V. J. Kowalewski, J. E. Peralta, G. Eskuche, R. H . Contreras, A. L. Esteban, M. P. Galache, and E. Diez, Magn. Reson. Chem., 1999,37, 227. C. G. Giribet, C. V. Vizioli, M. C. Ruiz de Azua, R. H. Contreras, J. J. Dannenberg, and A. Masunov, J. Chem. SOC.Faraday Trans., 1996,92, 3029.

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E. A. Chirkina, 0. R. Sergeeva, and L. B. Krivdin, Russ. J. Org. Chem., 1997,33, 975. T. A. Kuznetsova, N. V. Istomina, and L. B. Krivdin, Russ. J. Org. Chem., 1997, 33, 1620. C. Barbier, G. Berthier, I. Baraldi, and F. Momicchioli, J. Mol. Struct. (Theochem), 1998,433,231 . F. Momicchioli, I. Baraldi, and M. C. Bruni, Chem. Phys., 1982, 70, 161; 1983, 82, 229. M. A. Ruderman and C. Kittel, Phys. Rev., 1954,96,99. N. Bloembergen and T. J. Rowland, Phys. Rev., 1955,97, 1679. J. Poitrenaud, J. Phys. Chem. Solids, 1967, 28, 161. R. F. Walstedt, M. W. Dowley, E. L. Hahn, and C. Froidevaux, Phys. Rev. Lett., 1962,8,406. L. Tterlikkis, S. D. Mahanti, and T. P. Das, Phys. Rev., 1969, 178,630. L. Tterlikkis, S. D. Mahanti, and T. P. Das, Phys. Rev. Lett., 1968,21, 1796. S. J. Frisken and D. J. Miller, Phys. Rev. Lett., 1988,61, 1017. A. S. Oja, X. W. Wang, and B. N. Harmon, Phys. Rev., 1989, B39,4009. R. C. Patnaik, R. L. Hota, and G. S. Tripathi, Phys. Rev., 1998, B58,3924.

107 108 109 110 111 112 113 114 115 116 117 118

5 Applications of Spin-Spin Couplings BY KRYSTYNA KAMIENSKA-TRELAAND JACEK WOJCIK

1

Introduction

The material in this chapter covers the period from 1 June 1998 to 31 May 1999. It has been arranged as was done previously,' ie. according to (i) the increasing atomic number of the nuclei involved, and (ii) the number of the bonds separating them. However, sometimes it is difficult to define the coupling path or describe it in a typical way. This is especially true in the cases of the proton-proton couplings in transition metal dihydrides and dihydrogen complexes where the two-path mechanism should be taken into consideration, the nitrogen-nitrogen couplings across the hydrogen bond and the so-called through-space couplings. The latter couplings formally occur across five, six and even more bonds, but their values are unexpectedly large indicating that other than the throughbond mechanism should be taken into account. Therefore, a separate section in this review has been devoted to all the 'non-typical' couplings. Writing this review we have followed the recently published IUPAC2 recommendations with one notable exception, namely, the nucleus with a smaller mass is given first. This is in accord with the notation used by us in our previous reviews. For the sake of simplicity the following symbols were used throughout the paper: H for 'H, D - 2H, T - 3H, Li - 7Li, B - l*B,C - 13C,N 15N, 0 - 170,F - 19F, Si - 29Si,P - 31P,Mn - 55Mn,Cu - 65Cu,Ge - 73Ge,Se - 77Se,Y - 89Y,Nb - 93Nb,Tc - 9 9 T ~Rh , - lo3Rh,Ag - lo9Ag,Cd - l13Cd, In - l131n, Sn - '19Sn, Te - 125Te,I - 1271,W - 183W,0 s - 1870s,Pt - 195Pt,Hg 199Hg,TI - 205Tl,Pb - 207Pb.All the other isotopes are described explicitly. An excellent review devoted to ab initio methods for the calculation of NMR shielding and indirect spin-spin couplings has been published by Helgaker, Jaszunski and R ~ u dRecent .~ trends in theoretical chemistry including calculations of spin-spin couplings have been reviewed by Marx et aL4 A review on the llB NMR of carboranes and other heteroboranes with emphasis on the importance of JHBcouplings in assigning the "B-signals has been written by Hermanek et aL5 An interesting review on the stereochemical applications of Jcc couplings has been published by Krivdin and Zinchenko.6 An erroneous reference was assigned to this paper in ref. 7. Nuclear Magnetic Resonance, Volume 29 0The Royal Society of Chemistry, 2000 165

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A review on 29Si spectroscopy of organosilicon compounds has been published by Takeuchi and Takayama.8 A review on the 31Pchemical shifts and lJPpcouplings of diphosphenes has been written by Schrodel and S~hmidpeter.~ The indirect coupling between nuclei is important in investigating high temperature superconductors providing, among others, information on the possible origin of the high temperature transitions. The theory of the J-coupling in the cuprate superconductors and some novel relevant experiments have been discussed by Slichter et al.'O An elegant review has been written by von Philipsborn on the application of NMR spectroscopy to studies on the structure and reactivity of organometallic compounds containing transition metal nuclei such as 57Fe, Io3Rh, 1870s,51V,53Cr, 55Mn, 59C0 and 91Zr.The relevant coupling data have also been discussed. Nuclear magnetic resonance studies including J coupling data of crown ethers have been reviewed by Buchanan.12 A useful review on scalar couplings in structure determination of proteins has been published by Ejchart.13Ruterjans and c o - w ~ r k e r shave ' ~ reviewed 25 Karplus type equations corresponding to the six

s) in the paramagnetic protein cyanometmyoglobin.2 Europium ions have been used to enhance the alignment of a single stranded oligonucleotide, which forms a quadruplex structure in the presence of potassium. The observed H-' 3C residual dipolar couplings, ranging up to 70 Hz, have been used to obtain information about the alignment axis.3

'

5.2 Residual Dipolar Couplings in Structural Refinement - The exact nature of relationships between molecular structural features and their alignment properties are currently unknown. Since most molecules are not spherically symmetric, magnetic alignment is a rank-2 tensor rather than a vector, with different values for different orientations. A unitary value for the component of the alignment tensor parallel to the magnetic field corresponds to a perfect orientation of all molecules along that direction but, usually, substantial orthogonal components are present. The general expression for residual dipolar couplings of two interacting nuclei is

DiAB(ei, +i) = DaAB

+ 0.5 DrAB

where DaABand DrABare the axial and rhombic components of the molecular alignment tensor respectively, 6i and +i are the spherical coordinates describing the position of the ith interatomic vector ri in the principal axis system of the alignment tensor and the < > brackets denote motional averaging. The terms DaABand DrABcontain constants such as the gyromagnetic ratios of the two nuclei, the distance between the nuclei, the magnetic permeability of the medium, the magnetic field strength and the generalised order parameter S for the internal motion of the internuclear vector. This latter parameter is often taken as unity all over the macromolecule, introducing a negligible error and if a protein contains conformationally flexible residues these can be excluded from calculations. The use of residual dipolar couplings in molecular structural refinement requires the direct evaluation from the experimental data of the parameters in the above equation. It is not necessary to know the direction of the principal axis system of the alignment tensor in the coordinate axis frame, since it is treated as a variable during the structure calculation, Clore et al. have proposed ways to obtain the components of the tensor for an unknown macromolecular structure. One of the methods employed consists of a first

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evaluation of the axial component of the tensor from the experimental data and a calculation procedure that refines the structure against the measured residual dipolar couplings with a gradual change in the magnitude of the rhombic component of the tensor. The output results are a refined structure in addition to the components of the alignment tensor.137A more general method can be used where a '5N,'3C-double labelled sample is available and as a consequence many additional residual dipolar couplings are accessible. A histogram which reports the distribution of the normalised residual dipolar couplings for several internuclear vectors, approximates a chemical shift anisotropy powder pattern from which a direct estimation, in the absence of any a priori structural information, of the three component of the traceless molecular alignment tensor can be readily extracted from the lowest, highest and most probable values in the distribution.'38 If the three-dimensional structure of the macromolecule is known from some other sources, such as X-ray crystallography, a grid search or a simple Powell minimisation are common approaches in trying to minimise the differences between the experimental and calculated residual dipolar couplings and thereby extract the components of the alignment tensor.*39A fit using a linear combination of spherical harmonics by introducing the concept of vector space is a quick way to get an estimation for the magnitude and direction of the molecular alignment tensor together with a direct relation to the energies responsible for the molecular ~ r i e n t a t i o n . 'Recently ~~ Losonczi et al. have proposed a method of analysis based on Saupe order matrices calculated using the singular value decomposition method that allows the calculation of the alignment magnitude and the principal axis frame with a very small number of experimental data and a known ~tructure.'~'

5.3 Liquid Crystalline Media for Partial Alignment - The exploitation of residual dipolar couplings which are not close to zero as a result of molecular tumbling requires new ways to induce and/or enhance molecular alignment compatible with a retained resolution, sensitivity and simplicity in the NMR spectra. A tuneable degree of molecular alignment was first achieved by dissolving the target macromolecule in a liquid crystal medium containing magnetically oriented particles.'42 The medium consists of a mixture of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC). Over a substantial range of DMPC/DHPC molar ratios and at a temperature below the liquid crystalline phase transition the lipid mixture is in an isotropic state. Upon heating above the transition temperature the lipids form a liquid crystal phase consisting of disc-shaped particles, called bicelles (binary bilayered mixed micelles). The DMPC forms the plane of the disc whilst DHPC, which is mainly concentrated at the edges, controls the size of the discs. At a 1:3 DMPC/DHPC molar ratio the phospholipids form a ordered discotic nematic phase of liquid crystal corresponding to discs with a thickness of 40 and a diameter of 400 with a large magnetic susceptibility orienting with the plane parallel to the direction of the magnetic field.'43 The dipolar contribution is measured as the difference of J-splittings in the bicelle

A

A

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medium at 25°C’ when the solution is isotropic and only the scalar coupling contributes, and at 35°C where the medium is aligned and both dipolar and scalar couplings contribute. The liquid crystalline phase can be preserved even at low concentration of lipids (3% w/v) where the average spacing between two bicelle particles in which is much larger than the size of proteins and solution exceeds 400 nucleic acids typically studied by high resolution NMR. The concentration must be kept low enough to limit the solute ordering so that severe line broadening is avoided and the simplicity of the spectra is conserved. A concentration of bicelles that produces an alignment tensor in the order of will lead to residual dipolar couplings up to f20 Hz. The degree of alignment in the anisotropic medium is a function of macromolecular three-dimensional shape, the surface charge distribution and the density and degree of magnetic alignment of the b i ~ e l l e s In . ~general ~~~~~ the more asymmetric the molecule, the stronger will be the dipolar couplings. For many proteins no change in chemical shifts occurs upon addition of the lipids suggesting that protein shape is not distorted by transient interactions with the crystalline medium.142The small alignment due to the macromolecule’s own magnetic susceptibility is usually neglected and the molecular alignment can be considered to be independent of temperature in a narrow range around the bicellar transition point. Two protein structures have been refined with the introduction of residual dipolar couplings in the refinement obtained using a DMPC-DHPC bicellebased liquid crystal environment. The solution structure of the 101-residue inhibitor of HIV, cyanovirin-N, has been resolved by Bewley et al. using 334 residual dipolar couplings in the simulated annealing protocol.23The inclusion of N-HN, C‘-Hu, C W ’ , N-C’ and HN-C’ dipolar restraints increased the coordinate precision from 0.3 to 0.15 for the backbone atoms and from 0.54 to 0.45 for all heavy atoms. The atomic root mean square shift in the mean coordinates resulting from the inclusion of the dipolar couplings is 0.66 for the backbone atoms and 0.81 for all heavy atoms. Residual dipolar couplings were also used to refine the molecular structure of the cellular factor .~~ BAF responsible for protecting retroviral DNA from a ~ t o i n t e g r a t i o nBAF is a 21 kDa dimer with 89 residues per monomer. Its structure has been determined by using 1646 experimental NMR restraints per monomer including 250 N-HN, C‘-C’, N-C’ and HN-C’ residual dipolar couplings which provided direct information of the relative orientation of the monomers in the molecular coordinate frame. The measurements of ‘H-13C residual dipolar couplings in lipid-based bicelle solution was also exploited in the structural refinement of a 3C-enrichedtrisaccharide derived from a ganglioside.146 Despite the great excitement that this new method has provoked the practical aspects of its implementation are far from ideal. Bicelles are thermotropic and the temperature range over which the bicelle solution is stable is relatively narrow. The DMPC/DHPC lipids used are only stable over a limited pH range due to susceptibility to acid- and base-catalysed hydrolysis of ester bonds and the liquid crystal solution at neutral pH does not last for more than

A,

A

A

A

A

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Nuclear Magnetic Resonance

6 months even at - 80 "C. At acidic or basic pHs the hydrolysis of the liquid crystal phase becomes a serious problem and the alignment remains stable only over a time span as short as 18 hours. The concentration and type of cosolute also alter the stability of the nematic phase. Addition of salt increases the time necessary to reach alignment equilibrium and eventually phase separation occurs. 145 Although most proteins can be dissolved in high concentrations without any negative effect on the stability of the liquid crystalline phase, other proteins are not appropriate for study in this medium. These include proteins with a high binding affinity for membranes, or with hydrophobic regions, those that affect the chemical stability of phospholipids, or those that denature at temperatures close to the bicellar transition point. Examples where bicelles have proved to be unstable include enzyme I, the enzyme I-HPR complex, the N-terminal domain of HIV-1 integrase coordinated to cadmium, SIV gp41, and the Vct domain of a T-cell receptor and HIV p r ~ t e a s e . ' ~ ~ . ~ ~ ~ Several recent reports have focused on the improvement of bicelles stability. Doping of dilute bicellar solution with a small amount of charged amphiphiles can achieve an improvement in the stability and degree of alignment, extending the temperature range for these systems.148The stability of the nematic phase can be enhanced by using a small amount of tetradecyltrimethyammonium bromide (TTAB). 149 It is possible to obtain magnetically aligned bicelles at lower temperature by using different lipids. Ottiger and Bax tested mixtures of phospholipids such as l-myristoyl-2-myristoleoyl-sn-glycero-3-phosphocholine with DMPC and DHPC and obtained a bicellar transition temperature below 0 "C.145Ditridecanoylphosphatidylcholine(DTPC) mixed with DHPC gives a stable liquid crystalline phase over a temperature range between 22 and 50 "C. 145 For systems stable only at low temperature the measurement of the dipolar couplings in the isotropic phase in the presence of lipids is not accurate due to the high viscosity of the solution. It is better in this case to use a sample without lipids to measure the only scalar contribute to the coupling.145A mixture of dilauroylphosphatidylcholine (DLPC) and 3-(cholamidopropy1)dimethylammonio-2-hydroxyl- 1-propane sulfonate (CHAPSO) in water forms bicelles that become ordered at high magnetic fields over a range of temperature wider than bicelles formed by mixtures of DMPC and DHPC. Their application has been shown with the FK506 binding protein. I5O Large residual dipolar couplings can be measured with lipid concentrations as low as 5% w/v of a DLPCICHAPSO medium at a molar ratio of 4.2:l. This system can be applied to molecules which are only stable at low temperature since the bicellar transition point is at around 7°C. This latter orienting solution has also provided the basis for determining 66 lH-I5N residual dipolar couplings at 32°C for the N-terminal Dbl homology domain of the nucleotide exchange factor Trio that have been included as angular constraints for the corresponding bond vector in the protein structure calculations.28 The combination of equal quantities of cetylpyridinium chloride (CPCl) and n-hexanol in a 0.2 M NaCl solution produces a lamellar phase.151In a high

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magnetic field the magnetic anisotropy of the pyridinium ring is expected to lead to a magnetic alignment and the symmetry axis of the lamellae would be expected to be parallel to the applied field. Prosser et al. obtained residual dipolar couplings for a double isotope-labelled sample of human ubiquitin using this method.I5* By varying the concentration of the lamellae it was possible to obtain a scaleable degree of order for the protein. Improved bicelles have been prepared and characterised by Cavagnero et al. The system is composed of 1,2-di-O-dodecyl-sn-glycero-3-phosphocholine (DIODPC) and CHAPSO. At the optimum ratio of 4.3:l DIODPC/CHAPSO bicelles are formed and are stable at 25 "C in the pH range from 6.5 to l.0.152 The authors tested the system on the protein rusticyanin from Thiobacillus ferroxidans at pH 2.1. This system is useful for acidophilic proteins. 5.4 New Methods for Measurement of Residual Dipolar Couplings - Recently a new method to obtain molecular alignment has been proposed and has proved to be more generally applicable than bicelles. It requires the use of a colloidal suspension of filamentous bacteriophage or virus particles that are able to induce molecular alignment with both proteins and nucleic acids. Rod-like viruses are fully aligned at a magnetic field corresponding to 300 MHz and they remain in the aligned phase over a wider range of temperatures than bicelles. The extent of alignment of dissolved macromolecule can be easily scaled by changing the concentration of the virus particles, thereby modulating the magnitude of the resulting dipolar couplings. Filamentous viruses are extremely stable under physiological conditions and they can be produced in large quantities and to a high degree of purity. Suspensions of charged, filamentous virus particles, such as fd bacteriophage and tobacco mosaic virus (TMV) have been used to measure residual dipolar couplings at different pHs and temperatures for the 15N-labelled 56-residue streptococcal G protein.*47Residual dipolar couplings in the range of & 15 Hz and & 10 Hz were observed with a 28 mg mL-' solution of fd phage and a 50 mg mL-' of TMV viruses respectively. Hansen et al. used filamentous bacteriophage Pfl to align and measure residual dipolar couplings for a 15Nlabelled 29-nucleotide RNA hairpin containing the iron responsive element. 54 For all proteins and nucleic acids studied in these systems there appears to be little detectable effect on the rotational correlation time of the macromolecule, so standard NMR experiments are applicable and high-resolution NMR spectra can be obtained. 'H and 31Presonances from the phage particles are not observed and therefore they do not interfere with NMR spectra of the macromolecule. The system formed by phage and macromolecules in solution is stable indefinitely and neither the degree of alignment nor any measurable changes in the properties of the phage or protein/DNA/RNA are observed with time. The phage can be easily and quantitatively separated from the dissolved macromolecule by centrifugation which pellets the phage whilst leaving the macromolecule in the supernatant. These preliminary results show that the alignment induced by rod-shape 14771533154

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376

viruses represents a valuable and versatile method that has a more general applicability as compared to bicelles based environments. At the present no structure has been solved using filamentous bacteriophage nematic suspension but preliminary studies have been performed with both enzyme I and enzyme I-HPr complex, where previously a variety of lipid bicelles had proved to be unstable. 147 Another method to induce weak degrees of alignment in a macromolecule in a strong magnetic field has been proposed recently and uses disk-like purple membrane (PM) fragments. PM fragments comprise very stable twodimensional crystals of membrane protein bacteriorhodopsin (BR) with a diameter typically 0.75 pm and a thickness 49 The magnetic anisotropy of the fragments is defined by the membrane spanning helices of BR. The alignment of PM is complete and the normal to the membrane points in the direction of the applied magnetic field. The structure of BR within the PM is preserved for pH values from 2.5 to 10, ionic strengths of up to 5 M and a temperature range from -269 to 69 OC.I4O Unlike the liquid crystalline phase and like the filamentous bacteriophages there is no critical lower threshold for their concentration and no need to increase or decrease the temperature to obtain the aligned phase. The PM fragments are highly negatively charged and the alignment of proteins appears to be dominated by electrostatic interactions. Suitable molecules for the PM system are proteins at pH values above their PI and the negatively charged nucleic acids. 0.1-0.2Y0 w/v of PM are necessary to obtain an alignment of a similar magnitude to a 3% w/v lipid based bicellar solution and of a 25-28 mg ml-I of fd filamentous bacteriophage suspension. The direction of the solute alignment tensor obtained in the PM medium therefore is expected to be quite different from that in virus suspensions or bicelle based liquid crystal solutions. For bicelles the solute alignment appears to be dominated by steric interactions due to the uncharged nature of bi~e1les.I~~ Therefore the orienting force is stronger for PM fragments than for bicelles which explains why a much lower concentration is needed to induce the same magnitude of alignment. Residual dipolar couplings have been measured for 15N-labelled sample of human ubiquitin and the protein p53. 140 In the case of p53, due to the strong interaction with the negatively charged PM membranes, a small reduction in the transverse relaxation rate was observed which limits the applicability of the method. Measurements of residual dipolar couplings of the Va domain of a human T-cell receptor have been ~ e r f 0 r m e d . For l ~ ~ this latter protein no residual dipolar couplings could be obtained by using DMPCIDHPC bicelle-based liquid crystal solution. 147 1407155

A.

5.5 Impact of Residual Dipolar Couplings on Macromolecular Structures - It is very useful to be able to measure residual dipolar couplings in different environments where the driving forces for the alignment are different. It is possible, in these cases, from the evaluation of different orientation tensors, to lift the degeneracy in the determination of the internuclear vectors of coupled nuclei and at the same time to compare the orienting interactions. Due to its angular dependence, each residual dipolar coupling restraint constrains the

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position of the internuclear vector on a conical surface about the unique axis of the alignment tensor. Because the alignment tensor is a second-rank tensor its direction cannot be distinguished from its inverse, so that the dipolar couplings define two conical surfaces of possible bond orientations, in opposite directions. The measurement of residual dipolar couplings in different environments offers the possibility to triangulate internuclear vectors. This gives the prospect that the direction of such a vector in the space can be uniquely determined with a consequent possibility of determining local geometries with great improvement in the accuracy of the structure. Recently a modulation of the alignment tensor for ubiquitin has been reported using bicellar solutions with different composition. Doping bicelles with a small amount of a positively charged compound (cetyltrimethylammonium bromide, CTAB) caused a variation in the direction and magnitude of the alignment tensor with respect to that in the uncharged bicelle solution.156The comparison of different alignment tensors derived from different orienting environments also allows a direct investigation of the interactions and energy involved in the alignment. 140 The dependence of residual dipolar couplings with the inverse cube of the internuclear distance can also be used to achieve further structural information. With an accurate knowledge of a protein’s three-dimensional structure, residual dipolar couplings permit calculations of vibrationally corrected average bond lengths. 57 With this knowledge, residual dipolar couplings can be used to investigate on the effects of internal motions or distortion of geometries such as the small deviations from an ideal tetrahedral geometry suggested from the measurements of 13C-13Cand 13C-lH bonds residual dipolar couplings for methyl groups.158The approach based on the use of residual dipolar couplings derived from the small alignment of macromolecules in solution offers a wide spectrum of applications. Those described herein represent only a fraction of the potential that this new methodology offers, and which in the future we can realistically expect will be applied routinely to structural studies by NMR. Unfortunately, up to date, there has not been found any alignment system of universal applicability and much effort is required to achieve this goal.

6

Study of Protein Dynamics Through Heteronuclear Relaxation Measurements

6.1

Methodological Developments

6.1.1 Study of Chemical Exchunge

- In most applications of heteronuclear relaxation the problem of chemical exchange contributions to the line width is apparent. While this phenomenon provides highly interesting information about slow motions of potential biological relevance, for example to describe substrate recognition, it is often only characterised as a correction factor in the typical Lipari-Szabo ‘model-free’ analysis. Now further, very different

378

Nuclear Magnetic Resonance

approaches have become available to directly measure chemical exchange. For the best established ways - off-resonance spin lock and Carr-PurcellMeiboom-Gill (CPMG) pulses with variable delays - considerable improvements have been proposed. An improvement on the measurement of T1, relaxation times through off-resonance spin lock experiments was suggested. 59 It was demonstrated that a phase modulated adiabatic pulse is highly efficient at aligning the 15Nspins to the effective field. As such pulses are robust against mis-calibration and large offset differences this version for the implementation of off-resonance spin lock relaxation experiments might find widespread application. A modified version of measuring transverse relaxation rates with CPMG pulse trains is proposed to better characterise exchange contributions.160 Normally in-phase 15N magnetisation is maintained in the CPMG sequence even when long delays are used, e.g. with composite pulse decoupling in the delays. In the new 'exchange-compensated' experiment in-phase and antiphase 5N states are carefully mixed and maintained, alleviating the need for 'H decoupling during the CPMG sequence. This allows very simple measurements of transverse relaxation rates as a function of delay lengths for the detection of chemical exchange. A completely different approach to the identification of chemical exchange contributions to transversal relaxation rates in 15N relaxation studies is based on a correlation of residual dipolar couplings in the presence of bicelles with Tl/TZ ratios.16' This approach only works for proteins with a considerable asymmetric inertia tensor. Since it is assumed that the alignment of a protein to bicelles is mainly driven by nonspecific, steric interactions the alignment and the diffusion tensor should be strongly correlated. Hence, a correlation plot of residual dipolar couplings against T1/T2ratios should identify all residues undergoing chemical exchange as outliers. This method should also be helpful to find out if a protein of unknown structure has an asymmetric shape. A yet further experimental test for the presence of chemical exchange in 15N relaxation data relies on a comparison of 'H with 15N transverse relaxation rates in otherwise perdeuterated proteins. 162 The relaxation rates for 'H nuclei are measured with CPMG sequences, new ROESY-TI, and T1, (unlike) experiments, the latter leading to a substantial reduction in the dipolar interactions and thus in the relaxation rates. Further dampening of transversal 'H relaxation is achieved by only using the slowest relaxing doublet component, having the purpose of making exchange contributions, if present, as big as possible relative to any other relaxation mechanism. By comparison of transversal relaxation rates measured with CPMG sequences and TI, sequences for both 'H and 15Npotential candidates for exchange are selected from differences in the two relaxation rates. The simultaneous use of 'H and 15N transverse relaxation rates optimised for the detection of exchange has the main advantage to provide a more comprehensive coverage of a complete protein. As chemical exchange depends both on the presence of As-ms dynamics and a difference in chemical shift the use of 'H relaxation adds another dimension because of the different chemical shifts. In addition the dominating determinants for chemical shifts are considerably different for 15Nand 'H.

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6.1.2 Improving the Accuracy of Relaxation Experiments and Their Interpretation - The most demanding experiment in the analysis of 15N relaxation is the measurement of the transverse relaxation rate. The usually employed CPMG sequence has to be combined in an efficient manner with 'H decoupling to suppress the I5N-'H scalar coupling and interference of the dipolar and CSA relaxation mechanisms of the 15N. In addition, off-resonance effects can seriously degrade the performance of CPMG sequence and lead to errors in the measured relaxation rates. As it was shown that the B1 field inhomogeneity ' ~ ~ pulsecan contribute up to 5% to the uncertainty of the r n e a s ~ r e m e n tbetter sequences might be necessary. One proposal was the use adiabatic fast passage pulses. lo9 Adiabatic pulses in general are very robust in terms of off-resonance effects and therefore achieve the desired flip angle even at large offsets. It is also expected that B1 field inhomogeneity will have less adverse effects. Some new experiments also address the practical aspects of relaxation studies. While the traditional approach is centred on the measurement of twodimensional HSQC-type spectra it also has its limits in terms of resolution. A new approach was proposed for the measurement of a complete set of 15NT1, T2 and lH-15N NOE,164based on the intrinsically highly sensitive 3D HNCO experiment into which the appropriate relaxation elements were incorporated. It was demonstrated to work successfully on a protein as large as 44 kDa. Further improvements were demonstrated for the measurement of 5N and 13C' relaxation in larger proteins by the introduction of the accordion technique into a 3D HNCO experiment. 165 A straightforward extraction of relaxation parameters was demonstrated for a l5N,I3C labelled domain from tenascin. A number of new approaches have been put forward to improve the data analysis. The main efforts have been directed at a more sensible data selection and more robust statistical estimates of the final results. Partially in conjunction with the problem of the determination of T2 relaxation times a systematic study was performed on the determination of rotational correlation times z, based on 15Nrelaxation experiments.166A systematic bias to higher values was noted in z, determined from T I , T2 and heteronuclear NOE compared to values based on fluorescence data or T1 and NOE alone. It is assumed that zero-frequency dynamics, unrelated to chemical exchange, that are not accounted for in the Lipari-Szabo analysis are responsible for these observations. The molecular origin of these deviations resides in the basic assumption that the 15N CSA and I5N-'H dipolar axes are parallel while it is known that they form a small angle. To avoid this problem it is proposed to use only I5N TI and heteronuclear NOE for the determination. The calculation of z, is normally based on T1/T2 ratios for residues selected to avoid distortions from chemical exchange and large amplitude or intermediate time scale motions. While a variety of selection schemes are found in the literature it was noted that usually more residues are used in the calculation then are actually shown to be indeed unaffected by local mobility at the end of the analysis.167As this usually leads to an underestimation of z, a new selection method is proposed that uses an iterative estimation of z, from TJ"2 ratios

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and independently from NOES until the two values converge. It is demonstrated that using a z, value obtained from the new method allows the fitting of local mobility with simpler models. The poor correlation of published values for T, values with the corresponding molecular weights has led to the proposal to use the solvent accessible surface area (SASA) instead.168It is demonstrated that a better correlation exists between z, and SASA that between z, and molecular weight. It was demonstrated that it is possible to analyse I5N relaxation data for anisotropy in the rotational diffusion of proteins even if their structure is not known.169This method - based on statistical analysis of the expected distribution of relaxation rates - should provide additional evidence for a final analysis. As it is based on a statistical analysis pure 15Nrelaxation data might not be sufficient. With the emergence of better measurement and interpretation of 13Crelaxation this method is very promising. An alternative approach for the extraction of information about anisotropic rotational diffusion in the absence of structural data is based on the measurement of I5N relaxation at multiple magnetic field strengths.I7*Data collected at multiple fields allows to fit more parameters per residue which means that in the case of axially symmetric anisotropic rotational diffusion the angle of the N-H vector with the principal axis of the diffusion tensor can be fitted. The method was demonstrated to yield good results for molecules with substantial anisotropies (DII/DI >> 1 3 , here the C-terminal domain of DnaK. The impact of completely anisotropic rotational diffusion on the analysis of I5N relaxation data with the assumption of axial symmetry was studied.I7l In the case of the fully asymmetric cytochrome c2 from Rodobacter capsuZatus two minima were found in the fit of T I E 2 values to an axially symmetric model. The two minima corresponded to oblate and prolate shapes, respectively, with substantial differences in the ratio of the diffusion coefficients DII/DI D,1/DI. It is pointed out that a good fit to an axially symmetric model only might lead to erroneous interpretation of relaxation data with potentially severe consequences, e.g. when T1/T2 ratios are used in structure calculations. It is suggested that a fully anisotropic analysis be performed to ensure the adequacy of the chosen model for overall movement. A new approach for incorporating 15Nrelaxation data acquired at different field strengths into the Lipari-Szabo model-free analysis based on Bayesian statistics is presented.172This method is a generalisation of a previous attempt at obtaining better error estimates for relaxation data using a graphical method.173It is shown that the new statistical approach is not only capable of making relaxation data recorded at different fields available to the LipariSzabo analysis but also to include novel types of relaxation measurements, for example off-resonance spin-lock and CSNdipole cross-correlation rates or even the combination of relaxation data recorded for different nuclei. Furthermore, better error estimates are obtained which is an essential ingredient in the use of order parameters in the estimation of folding or binding entropies. An improved version of reduced spectral density mapping is demonstrated for the analysis of 13C relaxation data.174 The reduced spectral density

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approach demands less experimental data (only heteronucleus TI, T2 and NOE) based on the assumption that the spectral density curve is flat around J(wH), J(oH + ox) and J(oH - ox).While it is known that this approximation works well for 15N,it is demonstrated here that for 13C perturbations in some spectral densities, most notably J(o& do occur. A new fitting procedure is proposed to reduce the disturbances. The new method consists of an additional fitting procedure to a single Lorentzian. It is shown that the classical and the additional analysis produce upper and lower limits of the true value of the spectral density and thus allow an estimate of the deviation. 6. I . 3 Relaxation Measurements Incorporating 15N CSA/D@ole Cross Correlation - Simple experiments were proposed to measure interference rates between lH-15N dipolar and 15N CSA relaxation as well as between I3C' CSA and 13C7-15N dipolar relaxation. 175 The authors present approaches to correlate the transversal interference rates qxywith the structural features based on some simplifications. An extension of the measurement of longitudinal interference rates qz was recently demonstrated on a paramagnetic protein - HiPIP.176It is shown that by introducing an off-resonance irradiation of I5N it is demonstrated that the cross-correlation approach can be expanded to cover a broader range of spectral densities, in particular J(0). An approach is proposed to extract 15N CSA values from relaxation data through the use of the ratio of the CSAtdipole interference rate qxyand the transverse relaxation rate. 7x As this parameter is not dependent on any local motion the value of the CSA can be extracted from measurements at different fields. It is shown that in a protein the 15N CSA values cover a broad range from -125 to -216 ppm, a considerable deviation from the usual standard value of -160 ppm used in the analysis of * 5N relaxation experiments. Accurate, residue specific measurements of CSA values" might help in producing more accurate relaxation analyses in the future. A further complication in the analysis of I5N relaxation data might arise from a lack of collinearity of 15NCSA tensor and *H-'*Ndipolar axis. A small angle of 10-20 does exist between the two main axes while relaxation data are analysed with the assumption that the angle is rather 0 It is shown that this is a reasonable simplification only for isotropic tumbling molecules. As soon as D,,/D;, < 1.5 and at higher fields this approximation is no longer valid.179It was indeed reported in an independent study that the incorporation of a uniform angle different from zero significantly improved the analysis of 15N relaxation data measured for lysozyme.180Also these authors point out that larger effects are expected for more anisotropic molecules and higher magnetic fields. Also multiple-quantum states of 'H and I5N yield information about relaxation interference mechanisms and are potential sources to extract important parameters. The measurement of 'H CSA from lH-15N double quantum relaxation was recently demonstrated.181 The need for a better knowledge of CSA values and relative orientations in the analysis of I5N and 13C' relaxation data was addressed using quantum mechanical calculations in combination with molecular dynamics calcula17791

O

O.

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tion.182The authors claim that from a combination of quantum with classical mechanics calculations (density functional theory) based on a molecular dynamics trajectory good estimates of CSA values can be obtained. Such a determination of CSA values and the corresponding geometries could be performed explicitly for a protein for which relaxation data are measured and subsequently used in the analysis. The recognition of interference effects between chemical shift anisotropy (CSA) and dipolar relaxation mechanisms for nuclei as backbone "N has led to the development of a variety of new methods to overcome some of the classical obstacles connected with study and interpretation of heteronuclear relaxation. The precise measurement of transverse and longitudinal interference rates - qxyand qz- has been demonstrated to allow much more accurate interpretations than the measurements of autorelaxation rates.183Since the interference rates are independent of chemical exchange a comparison of interference rates with classical transversal and longitudinal relaxation rates lead to the unambiguous identification of exchange terms and better estimates for the overall tumbling time of the molecule. 6.1.4 Measurement of Side Chain Dynamics - A specific approach for the relaxation study of side chain PCH and PCH2 groups encompassing lH-13C heteronuclear NOE, 13C longitudinal relaxation rates and 13C-lH transverse dipole-dipole relaxation interference has been reported. 84 Using a specific motional model for side chain rotation around the angle the relaxation data for most residues in ribonuclease yielded good quantitative results. Another experimental approach to the analysis of side chain dynamics is based on the combination of 2H autocorrelation, i.e. T1 and TI,, and 'H-13C dipole-dipole interference rates in the relaxation of CH2 13C nuclei. 185 Measurements of 2H relaxation rates were performed on 50% deuterated proteins and CHD groups were selected in the pulse sequence. The results of both types of experiments as applied to an SH3 domain are in very good agreement. Relaxation data from both experiments are converted into order parameters showing a much broader dispersion than seen for the corresponding backbone values. Assuming that order parameters are dominated by side chain rotation they can be converted into explicit descriptions of side chain rotation limitations. In a similar approach 13C and 2H relaxation experiments measured at different field strengths for methyl group rotation in ubiquitin were compared.186. It was found that both methods yield exactly the same dynamic picture of side chain mobility. However, due to the different resonance frequencies of 13C and 2H different responses to different time scales were obtained. While 2H relaxation did not discriminate much over the picosecond to nanosecond time scale, the relaxation of 13C is more suited to resolve motions on different time scales. The two approaches thus provide complementary information for side chain motions. In a different approach, side chain dynamics in alternately 13C labelled thioredoxin have been analysed using 13C TI, T2 and lH-13C heteronuclear

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relaxation data.187It was found that the Lipari-Szabo model with two internal motional time scales can be applied for the analysis. Considerable correlations between backbone and side chain dynamics are found, e.g. crankshaft like connections of rotation about the x 2 angle and the parallel backbone axis (i.e. depending on xl). These correlations are stronger for more buried side chains. It is concluded that the presence of these extensive correlations make it necessary to map a much broader range of dynamical parameters for side chain and main chain mobility before more accurate values for configurational entropies can be obtained. To provide 'random coil' side chain dynamics parameters in analogy with the classical random coil chemical shift values a study was performed in which G-X-G synthetic peptides (for X all 20 common amino acids) where investigated. 88 The 13C and 3C-lH dipole-dipole cross correlated relaxation were measured and analysed using several different motional models. Angular fluctuations around the and \J/ bonds were found to be very similar to the side chain around 20". It was concluded that the rotation about x angles can be as restricted as around backbone angles for P-branched and aromatic side chains.

+

6.1.5 Mechanistic Interpretation of the Dynamics Underlying Relaxation - By measuring relaxation data for several rigidly connected atoms a more meaningful picture of protein dynamics can be obtained. A combination of backbone 15N and 13C' relaxation data was shown to allow an estimation of the anisotropy in the fluctuations of the peptide ~ 1 a n e .By l ~connecting ~ a number of peptide groups with similar characteristics correlated semi-local motions of an a-helix as a rigid unit were identified. Independently, the measurements of relaxation parameters of 5N and 3C' for the peptide plane- were used by another group in conjunction with molecular dynamics simulations to obtain a more detailed description of backbone movement^.'^^ The dependence of I5N and 13C' auto- and crosscorrelated relaxation on a variety of mechanisms and interactions allows a very efficient sampling of movements related to all three space axes. The authors were able to fit the experimental data to an analytic function describing the anisotropic motion of the peptide plane for most of the residues not affected by chemical exchange. Significant anisotropic motions of peptide planes were found for most residues that was not correlated to the type secondary structure element. It was also noted that the values used for the CSA and angles between the principal components of the CSA and dipolar interaction tensors had a large influence on the fitting results. The authors therefore point out the need for accurate values for these parameters to allow more meaningful interpretations of relaxation data in the future. A statistical survey of order parameters obtained from backbone 15N and methyl axis order parameters was performed. 1 9 1 Even though very similar distributions of S2 values for 15N and methyl were found in alanine, no strong correlation could be seen, presumably because of interference with the rotation about xl.As expected, methyl axis order parameter distributions broadened

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with increasing distance from the backbone. Surprisingly poor correlations between order parameters and structural parameters such as solvent accessibility and packing density were obtained. It was concluded that the dynamics features of a protein can not simply be predicted from the structure and that the cooperative nature of a protein fold might make itself felt particularly for dynamics on the picosecond time scale.

6.2 Applications 6.2.1 Protein-Nucleic Acid Interactions - The general trend of results on the dynamics of DNA binding proteins was a consistent presence of considerable conformational fluctuations on a broad range of time scales in sites essential for interactions with nucleic acids. Interestingly the extent of the conformational variations was strongly different ranging from essentially unfolded in the case of the DNA binding domain of GCN4 protein to some rather restricted very local fluctuations of loops in other cases. The backbone dynamics of the DNA binding protein HU from Bacillus stearothermophilus have been characterised using a hybrid approach between spectral density mapping and Lipari-Szabo model free a n a 1 y ~ i s . Analysis l~~ of overall tumbling indicated only small anisotropy (Di,/DI 1.15) which is explained by only a small contribution of the extended DNA binding arms to overall tumbling. These arms undergo local dynamics on an intermediate ns time scale with fairly low order parameters -0.3-0.6, smaller than the order parameters for ps time scale dynamics. These intermediate time scale dynamics are completely abolished upon binding to DNA. The dynamics of the basic leucine zipper of GCN4 has been studied by I5N relaxation experiments.193 Consistent with previous studies the N-terminal basic DNA binding region is unstructured in solution. Based on the dynamics analysis local cluster of higher order are identified, presumably stabilised by nascent helical conformations. Order parameters have been calculated and used to estimate the entropy changes associated with binding to DNA where a uniformly high degree of order in the entire protein is expected. The contribution from backbone disorder-order transitions is estimated to contribute -0.5 kJ K-I mol-' to the total entropy change, compared to -1.2 kJ K-' mo1-l as measured from calorimetric measurements. The authors point out that not only will the side chains, which were not part of the analysis, contribute to the total entropy change but also the extraction of order parameters solely from 5N autocorrelation experiments might not accurately reflect all possible fluctuations of the backbone. A comparative study using experimental I5N relaxation studies together with molecular dynamics and essential dynamics simulations was used to characterise the correlated motions in the DNA binding loops of the single strand DNA binding Protein from Pf3 ba~teriophage.'~~ Very good agreement is obtained between experimentally obtained backbone order parameters and S2 values calculated from a 1.2 ns molecular dynamics calculation. Strong

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correlations between the movement of DNA binding loops and loops in the interface of the dimeric protein are found and evaluated in detail using essential dynamics calculations. A mechanism is proposed in which the cooperativity of the DNA binding is explained through the correlated loop motions. The dynamics of the C-terminal DNA binding domain of human repair factor XPA was studied by 15N relaxation experiment^.'^^ A considerable degree of flexibility over a range of time scales was found in the DNA binding surface which forms a cleft in the C-terminal part. It is speculated that the flexibility night allow the protein to bind to a wide variety of DNA damage sites. The dynamics of the ribosomal RNA binding protein L9 were studied by 15N relaxation experiment^.'^^ The structure of L9 resembles that of calmodulin with a long central helix and small, globular subdomains at the ends. The fold stays rigid in solution as demonstrated by a good correlation of relaxation anisotropy and the shape of the structure. Small variations in z, between the N-terminal domain and the central helix and the C-terminal domain, respectively, indicated a small independence of movement. The putative RNA binding regions exhibit rapid local fluctuations on a ps time scale. The dynamics of free and DNA bound DNA binding domain of yeast transcription factor ADR1 was studied by 15Nre1a~ation.l~~ It was found that a stretch of more than 15 residues at the N-terminus which is completely unfolded in the free state forms a rigid fold in the complex with DNA. Clearly different values for z, in the two zinc fingers indicate independent rotational diffusion of the two subdomains while the Tl/T2 rates are much more uniform along the protein sequence in the DNA bound state. Additional widespread chemical exchange, particularly in the second zinc finger is identified by field dependent measurement of T2 values at several magnetic field strengths. 6.2.2 Protein-Protein Interactions - Analysis of 15N relaxation data has been used as an efficient tool in the characterisation of important aspects in proteinprotein interactions. A study of the EGF domain from heregulin a has demonstrated the dimeric state of the domain in solution.198In addition, mobile regions coinciding with functionally important parts were shown to be flexible on the ps as well as the p time scale. A good correlation of chemical exchange in segments of a protein important in interactions was found for an inhibitor of cell cycle kinase cdk4, ~ 1 9 ( 1 N K 4 ) .A' ~binding ~ mechanism of p19 to CDK4 involving loop segments of the inhibitor was proposed based on the dynamics data. As the authors also found a slightly too large value of z, reflecting the tendency of p19 to aggregate care has to be taken in the interpretation of the chemical shift data. Similar instances of chemical exchange correlated to parts of a protein implicated in interactions were found in a 9-cis retinoic acid receptor (RXR) DNA binding domain.200This study demonstrated the first use of a combination of a variation of delay length in CPMG sequences and off-resonance spin locks in the measurement of I5N TI,, relaxation times to cover a broader range of exchange times. All residues with chemical exchange were found to cluster

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in the second zinc finger which is assumed to interact with other zinc finger interaction domains when bound to DNA. In contrast, much less flexibility on the ps time scale was observed in a homologous protein, the all-trans retinoic acid receptor (RAR) DNA binding domain.201 The presence of varying degrees of ps time scale dynamics in the dimer interface is assumed to reflect differential abilities of members of the same family to adapt to different interaction partners. In contrast to the crystal structure, in solution 15N relaxation studies of human thioredoxin no dimerisation could be detected through analysis of the molecular rotational correlation time.202A monomeric version of the neutrophil activating peptide 2 (nap2) is characterised for its structure and 15N relaxation data.203Apart from considerable flexibility on the ps time scale chemical exchange is identified for a number of residues that form a contiguous region on the protein surface, amongst them the N-terminal residues which are not visible in the crystal structure of the tetramer. It is concluded that the widespread dynamics over several time scales reflects a diminished stability due to the absence of oligomer interactions. The ability of completely unfolded proteins to be biologically active by interactions with other proteins was characterised for a fibronectin binding protein from Staphylococccus a u r e ~ sSegmental . ~ ~ ~ fluctuations were found to extend over a length of about 7-10 residues. A similar situation was identified for a protein (4E-BP1) that binds to translation factor eIF4e and thus inhibits translation. Even though 4E-BP1 is fully active the measurement of 'H-15N heteronuclear NOES indicated high degrees of mobility along the entire polypeptide chain indicative of a completely unfolded state.205 The analysis of the correlation of oligomerisation as found in crystals and in solution was expanded to take into account detailed thermodynamic reasoning based on 15N relaxation data.206The measurement of 15N relaxation data for the Rous sarcoma virus M domain indicated high degrees of mobility on ps time scales in some surface loops. In some crystal structures of homologous proteins these loops are involved in formation of dimers and trimers which were interpreted as precursors in the formation of the icosahedral protein shell of the virus. No evidence of oligomerisation was found based on diffusion and 15Nrelaxation experiments. The authors use the expected change in local order of the loops involved in potential oligomerisation to calculate that the entropic cost could not possibly be compensated by new interactions in the oligomers. The effects of tetramer formation of the small peptide melittin were studied by 15N and 13C relaxation.207 Relaxation of both 15N and 13C on the unstructured monomer yields near identical rotational correlation times (-1.5 ns), order parameters and local effective correlation times; all in support of an unstructured peptide. Whilst the rotational correlation times extracted from I5N and 13C relaxation also agreed in the case of the helical tetramer (-4.3 ns) a systematic difference of 0.2 in the order parameter for HN and HCar were obtained. The authors postulate that formation of hydrogen bonds in the transition from unfolded monomer to structured, helical tetramer might be responsible for this variation. Assuming a certain degree of anisotropy for a helical tetramer, the different angles made in a very uniform fashion by

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backbone HN and HCa with respect to the principal axis of the diffusion tensor might be an alternative explanation. Slow, millisecond time scale fluctuations were identified from the analysis of 15N relaxation data on the bacterial response regulator S ~ O O F .The ~ ~ *regions where the chemical exchange is identified coincide with the binding sites for other proteins of the signalling pathway. The authors point out that the conformational variability reflected in the chemical exchange terms might be an important factor in allowing one and the same region to interact with three different proteins. The relative mobilities of the complementarity determining region (CDR) loops in s class I1 single-chain T-cell receptor were determined based on 15N relaxation measurements.16 It was found that the CDR3 loops in both Ig-type domains have significantly increased mobility on the ps time scale while all other CDR loops and the 0-sheet scaffold showed considerable rigidity. It is noted that the flexibility of the CDR3 loop is not caused simply by the length of the loop but by the amino acid composition, with a substantial preference for glycine. The authors speculate that high mobility of CDR3 is related to fine tuning since CDR3 is known to contribute only little to the stability of the complex. The anti-HIV chemokine VMIP-I1 has been studied by 15N relaxation experiments to determine its oligomeric state as well as to obtain indications to its broader receptor specificity.209For the ratio of T1/T2a value of z, (4.7 ns) was obtained that is strongly indicative of a monomer. In addition, most of the N-terminus, the usual site for oligomerisation in the family CC chemokines, is demonstrated to be highly flexible. Further dynamics on the ps-ms time scale in two loops together with the flexibility of the N-terminus is taken as an explanation for the unusual, broader receptor specificity of VMIP-I1 compared to other CC chemokiqes. Similar features are reported for a CX3C type chemokine domain from the membrane associated fractalkine.210 It is also found to be monomeric at low concentrations while at NMR concentrations variations of z, and diffusion coefficient were found in dependence of concentration. The most dynamic regions, the N-terminus on the ps and the 01/02 loop on the ps-ms time scale, were identified as receptor binding sites by titrations with a fragment of the receptor, consistent with the assumptions made for the VMP-I1 monomeric chemokine. A different approach to elucidate the problem of chemokine oligomeric state on the base of 15N relaxation data was undertaken by studying a series of N-terminal truncation mutants of macrophage inflammatory protein 1p.2'1 Removal of more than six residues lead to a monomeric protein at low salt. At physiological salt concentrations, however, all mutants were dimeric, indicating that the dimer is most certainly the biologically active species. The dynamics of a ubiquitin conjugation enzyme (E2) has been studied by 5N relaxation experiments to characterise its oligomerisation state and the dynamics of sites potentially involved in catalysis and substrate binding.212E2, a member of a family of structurally and functionally homologous enzymes, was found to dimerize with low affinity. A region with enhanced mobility on

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the ps and a region with enhanced mobility on the ps time scale are identified as linked to the recognition of substrate, specificity and catalytic activity. The C-terminal domain of human general transcription factor TFIIB was studied by 15N relaxation experiments at multiple field strengths.213The cyclin like repeat domains were found to be relatively rigid while the linker peptide was highly flexible. Substantial exchange broadening was found along the entire sequence. It is speculated that rearrangement of the relative domain orientation might be responsible since different relative orientations of the domains are seen in the crystal and NMR structure. The changes to the backbone dynamics of Hck SH2 domain upon phosphotyrosine peptide binding were studied by 15N relaxation experiments.214 Apart from the tendency of the protein to self-associate it was found that for most residues the order parameter increases upon peptide binding. 6.2.3 Enzyme Function - The dynamics of the wild type and a double mutant with enhanced enzymatic activity of lysozyme were studied using 15N relaxation experiments.215It was attempted to identify possible variations in local dynamics which were postulated as a potential reason for the enhanced activity of the mutant. Hardly any effect could be found for the free enzyme. In the complex with an inhibitor, however, variations in S2 values were seen for some residues in the active site. In general, the mutant showed greater flexibility over a broad range of time scales, from ps to ms. Considerable chemical exchange effects were found in the CD-loop. It is concluded that the increase of flexibility on ps-nis might be particularly relevant to the improved catalysis of the mutant since pl-oduct binding, induced fit and product release all take phce on this time scale. The effects of binding of a transition-state based inhibitor on the dynamics of a-lytic protease were studied by backbone 5N relaxation experiments.21 While regions with increased local mobility on the ps time scale are limited mainly to surface loops, regions with dynamics on a ps-ms time scale are centred around the active site. Binding of the inhibitor blocks only the ps-ms time scale movements in the active site. It is speculated that the ps-ms movements are caused by interconverting conformers of the active site and thus responsible for the broad substrate range of the enzyme. In an effort to better understand the events that lead to the re-synthesis of the scissile bond in serine proteinase inhibit01-s.~'~ The backbone dynamics of intact and cleaved inhibitors CMTT-I11 and CMTI-V as measured by 15N relaxation were compared. The dynamics of the newly formed ends showed considerable divergence between the two inhibitors, consistent with their position in the structure. It is concluded that the different end-mobilities are responsible for the different hydrolysis constants of the two inhibitors.

6.2.4 Protein-Metal Binding - The main focus of relaxation studies on metal binding proteins were members of the EF-hand family of calcium binding proteins. Several studies were performed on troponins. In a combined relaxation study of backbone 15N and side chain methyl 2H on the N-terminal

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domain of chicken skeletal troponin C order parameters of backbone NH vectors were determined for the apo and holo forms.218A difference in entropy loss upon binding between the two EF-hands of about 1 kcal mol-' is estimated, consistent with the different affinities of the two binding sites. Side chain order parameters derived from 2H relaxation of methyl groups shows that side chains that get exposed upon calcium binding are restricted in the apo form. It is speculated that the tuning of mobility might be a way Nature has employed to offset the exposure of hydrophobic side chains. The changes in dynamics in the regulatory domain of cardiac and skeletal troponin C were studied by 15N relaxation with the aim of dissecting the molecular base for the large differences in the mechanism.219It is found that in the cardiac isoform where only one of the two potential calcium binding sites is active the binding site gets more rigid leading to a drop in entropy of 2.2 kcal mol-l. An additional change in rigidity corresponding to a drop in entropy of 0.5 kcal mol-I is seen in the inactive site indicating the substantial degree of cooperativity in the EF-hand family of calcium binding proteins. The backbone dynamics of parvalbumin was studied in the calcium bound state by 15Nrelaxation.220The entire protein appears to be very rigid on the ps time scale with order parameters consistently > 0.65. This is in contrast to other calcium binding proteins - calmodulin and calbindin - which show greater degrees of mobility in the loop connecting the two EF-hands. The only significant instance of exchange broadening is found for one residue at a kink in helix D, close to the rigid linker loop between the two calcium binding sites. 6.2.5 Protein Structure and Stability - The apo form of cytochrome b5 was studied using 5N relaxation experiments at different temperatures and fields.221The results of the previous structure determination of the apo state are confirmed through the dynamics analysis: the heme binding region shows high degrees of flexibility over a broad range of time scales while the heme independent second hydrophobic core is as rigid as in the holo protein. Differential chemical exchange was found for the two halves of the protein, the unfolded part with ,z, > 300 ps and the folded part rex < 150 ps. Using newly developed off-resonance spin lock experiments to study 15N relaxation chemical exchange was identified in a large number of residues in a fibronectin type I11 domain.222 This led to the assumption of substantial whole-domain dynamics on the ps time scale, a potentially important aspect in the understanding of protein stability. Quite different results were obtained in a study of the dynamics of a fibronectin I11 domain from tenascin studied by 15N relaxation measurements with the aim of achieving a clear separation of relaxation contribution^.^^^ Identification of chemical exchange was mainly based on a combination of variable delay CPMG and off-resonance spin lock experiments which indicated the presence of considerable exchange contributions in a cluster of C-terminal residues. Despite the elongated shape of the molecule no anisotropic diffusion effects could be found for the residues located in the p strands. Only when loop residues were used could an anisotropic rotational diffusion be characterised

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(D1I/DI = 1.55). With the less accurate definition of loop structure, be it attributable to true dynamical averaging or simply lack of structural restraints, loop residues are usually not taken into account. The study of 15N relaxation data for the syrian hamster prion protein PrP(90-231) revealed the presence of a completely unfolded -20 residue Nterminal segment connected through a partly structured hydrophobic cluster to the main, stable core of the protein.224 It is assumed that these regions of differential stability might play a role in the aggregation of amyloids. A comparative 15N relaxation study was performed on collagen peptides corresponding to wild type and mutant sequences in brittle bone disease (or osteogenesis i r n p e r f e c ~ a ) It . ~ was ~ ~ demonstrated that the Gly/Ser mutation leads to a partial unravelling of the collagen triple helix. While in the wild type almost uniform values of J(O), J(oN) and J(0.87 oH)were obtained a steep N-C terminal gradient in J(0) and J(0.87 oH)was obtained in the mutant indicative of a C- to N-terminal folding gradient. The dynamics of several conformers in equilibrium of partially folded BPTI was studied by 15N relaxation experiments.226The BPTI variant with only the 14-38 disulfide bond present was shown to been an equilibrium of at least two states for which I5N relaxation parameters were obtained. The conformer known to be more structured had consistently higher S2 values almost as high as a well structured protein while the less structured conformer had values of S2 around 0.6. This is consistent with the slightly higher rotational correlation time of the less folded state indicating a less compact structure. Additional evidence for more flexibility comes from the observation of exchange broadening which is completely absent in the more compact conformer. The wild type and a Y35G mutant of BPTI were characterised by 15N relaxation experiments in the presence and absence of the 14-38 disulfide bridge with the aim to explore the mechanism underlying the previously reported line broadening around residues close to the 14-38 d i s ~ l f i d e The . ~ ~ ~removal of the disulfide in the wild type reduced the line broadening only a little, actually lead to an enhancement for one residue. In contrast, substituting Tyr35 with Gly led to substantial effects upon removal of the disulfide bond. It is assumed that an interaction of the tyrosine with the disulfide bridge is the cause of the line broadening in the wild type protein. New experiments and analysis methods for the study of protein dynamics from 13C relaxation data were applied to a synthetic, designed P-sheet peptide.228Even though the peptide presents an equilibrium of foldedhnfolded molecules considerable degrees of motional restriction were identified for residues around the turn. Of particular interest are the identification of strong correlation of motions about pairs of backbone angles, e.g. 4 and $ in the structured parts while less restriction and less correlation were found in the unfolded part. The dynamics of the carbohydrate portion of the monomeric form of the glycoprotein human chorionic gonadotropin (hCG) was studied by 13C offThe three inner GlcNAc resonance spin lock relaxation residues were found to be relatively rigid while also the residues at the end of

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the chain were limited in their conformations when compared to the sugar in solution. It was concluded that this close interaction of the carbohydrate chain might play an important role in stabilising the protein. 6.2.6 Protein Folding - The entropy associated with the folding of the Speptide of ribonuclease A was estimated from 15N relaxation experiments recorded on the free and bound ~ e p t i d eA. ~gradient ~~ of flexibility from the ends towards the centre of the peptide was found having a magnitude comparable to that of the entropy change upon binding to the protein. It is concluded that substantial parts of the polypeptide chain in an unfolded protein might be more ordered than usually expected due to transient nonspecific interactions. As a consequence the entropy cost upon folding would be smaller. The unfolded state of an immunoglobulin superfamily domain from twitchin has been studied at urea concentrations of 4.2 and 6.0 M.231A distinct turn like structural feature exists at the C-terminus at 4.2 M and is lost at 6.0 M. This region exhibits chemical exchange contributions as determined by 15N relaxation experiments which are assumed to be caused by conformational exchange between a range of equally stable turn conformations. It is assumed that this segment with residual structure might act as an initiation site for the folding. New experiments for the study of side chain 13Crelaxation data as described above185were applied to different unfolded stated of the SH3 domain from drkN.232This protein domain exists in an equilibrium between a folded and an unfolded state under native conditions. The relaxation study of side chain dynamics in the folded, the unfolded-native and the guanidinium hydrochloride unfolded state revealed the presence of a rather compact state in the native-unfolded protein as evidenced by rotational restrictions of hydrophobic side chains. The guanidinium unfolded state had only slightly less limited side chain rotations but the differences were strongly residue dependent. This result is interpreted based in the assumption that unfolded states induced by different environments might exhibit similar global characteristics but strong differences for individual residues. A good correlation of structure and dynamics were found for a folding intermediate of the Fe4S4 HiPIP protein.233While the C-terminal half retained most of its native fold the N-terminal half was not structured at all. The structural features (or lack thereof) were well represented by native-like order parameters S2 0.8 in the C-terminal half and S2 0.4 in the N-terminal half. The preservation of native structure is assumed to be brought about by the presence of the four iron-coordinating cysteine residues in this part of the sequence. The urea denatured state of a fibronectin type I11 domain was characterised by 5N relaxation studies and spectral density mapping analysis.234Residual structure was found for a small number of residues mainly based on interactions close in the sequence. Consistent mobility in these regions is dampened to the ns time scale. Interestingly, an explicit analysis of potential chemical

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exchange using off-resonance TI, experiments fails to reveal any slow motion on the ps time scale. This finding is in contrast to a number of unfolded states where chemical exchange was found for a number of residues implicated in early folding events. The absence of ps-ms motions in fibronectin type 111 domains might indicate that such interactions form much later in the folding process compared to other proteins. The denatured state of a fragment of staphylococcal nuclease was studied using 15N relaxation to better understand the question of long-range order in unstructured polypeptide chains.235Correlated variations in local dynamics were identified that were interpreted as evidence for transient interactions in a hydrophobic cluster. It is assumed that essentially the same interactions occur as in the native structure only with a substantially shorter lifetime. The stability and dynamic properties of apomyoglobin folding intermediates have been The results indicate a progressive restriction of backbone dynamics and the presence of a number of interconverting states prior to the final collapse to the native structure. A comprehensive characterisation of structure and dynamics was performed on the yellow photoactive protein from Ectothiohodospira ha20phiZa.l~'Some intermediates of its photocycle were identified and characterised using novel NMR experiments involving laser irradiation of the sample in a coordinated fashion, named 15N-SCOTCH.133A considerable degree of disorder and structural changes on the ms time scale where observed for the pB photointermediate in contrast to the crystal structure. 32 The apparently partially unfolded excited state exhibited differential refolding to the ground state with the regions distant from the chromophore restructured first. Further differences between X-ray structure and NMR data are found in the N-terminus which is a-helical in the crystal but disordered in the NMR structure with considerable fluctuations on a ps time scale according to 15N relaxation data. Interestingly this region is very unstable upon photoexcitation and tends to degrade.

'

6.2.7 Protein-Ligand Binding Effects - The impact of binding of inositol trisphosphate to the PH domain of P-spectrin was studied by 15N relaxation measurements.237For a number of residues ligand binding induces an increase in the order parameter S2. Consistent with a 9 ns molecular dynamics trajectory two time scales of motion are found in the ligand binding loops, of which only the slow one is dampened by ligand binding. A relaxation study of perdeuterated HIV protease dimer in complex with an asymmetric inhibitor was performed to allow an investigation of the two monomers independently which is not possible in the presence of symmetric inhibitors.238It was found that some residues in equivalent positions of the two monomers nevertheless displayed different relaxation behaviour, in particular at the tips of the flaps that close the active site. Here, the residues of one monomer have considerable exchange effects while the corresponding residues in the other monomer are unaffected. Interestingly, these residues form part of the binding site for the asymmetric inhibitor. The dynamics of yeast translation

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factor eIF4E bound to different nucleotides and in the presence of varying concentrations of CHAPS was studied.46There N-terminal -30 amino acids were found to be highly mobile, independent of nucleotide binding. Loops implicated in nucleotide binding are highly flexible and do not interact with CHAPS. In contrast, some loops are found to be stabilised in the presence of CHAPS. These loops also show chemical shift changes when CHAPS is titrated in the NMR sample. Binding of different nucleotides did not change the dynamics of the nucleotide binding site. Estimates for the overall correlation time of the protein are 11.8 ns for the free form, 15.9 ns in the presence of 25 mM CHAPS and 19.4 ns with 50 mM CHAPS indication that the CHAPS micelle is made of about 40 detergent molecules. The dynamics of human neutrophil gelatinase-associated lipocalin (HNGAL) were studied by 5N relaxation experiments at three different magnetic field strengths.239While most of the protein was found to be rigid on the ps time scale considerable exchange broadening was identified in the oloop and at the C-terminus. The C-terminus contains a free cysteine that is able to covalently link it to progelatinase B. The o-loop covers the open end of the barrel which in the case of HNGAL binds small bacterial derived peptides. The dynamics of the o-loop are proposed to be affected hy ligand binding so that cell surface receptors can distinguish the apo and the holo form of the protein solely by probing the dynamics of the loop. A 15N relaxation study was performed on the Fusariurn solani pisi cutinase with the aim to obtain insight into the dynamics behind the lack of interfacial activation.240 In contrast to the crystal structure where the active site is relatively open and the key residues for the oxyanion hole already in position the ligand binding site and the oxyanion residues show dynamics on the millisecond time scale. The authors speculate that the relaxation data indicate a openinglclosing movement of an a-helix in analogy with true lipases. The question of dynamic effects connected to ligand binding was subsequently addressed in a separate The complex of an inhibiting substrate analogue with the enzyme was studied by 15N relaxation. Even though the inhibitor had the tendency to decompose during the NMR experiments clear results were obtained relating to the effects of ligand binding on protein dynamics. The millisecond dynamics in the binding site is completely abolished while the residues of the oxyanion hole are now locked in position. Interestingly, the rotational correlation time for the complex is shorter than that for the free enzyme. The authors postulate that the flexible N-terminus of the proenzyme used in the study of the free protein that was not present in the complex might have a considerable influence on the rotational diffusion. The dynamics of the holo and apo-form of the biotin carboxyl carrier subunit of Escherichia coli were compared to elucidate the base for different protein interaction specificities of the two states.242Interestingly, only minor differences exist between the structures of the two states. In some regions of the backbone, however, small differences in the backbone dynamics are identified that are assumed to be sufficient to alter interaction specificities. A comparative study of 15N relaxation for two homologous fatty acid

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binding proteins, bovine heart fatty acid binding protein and porcine ileal lipid binding protein revealed unexpected discrepancies of members of the same protein family.243Considerable differences in local mobility were identified in the two proteins that were related to specificity and stability of ligand binding. In a similar manner backbone 15N and side chain 2H relaxation data were used to characterise dynamic differences between two other members of the same protein family, human adipocyte (A-LBP) and muscle fatty acid binding protein (M-FABP).244In general residues lining the binding pocket in A-LBP display a greater degree of flexibility over a broad range of time scales compared to M-FABP. It is speculated that the observed differences may play a role in ligand specificity. 6.2.8 Redox Proteins - A number of studies relating to the dynamics of various forms, e.g. oxidised vs. reduced, of important redox proteins appeared. In a 15Ndynamics study of rat microsomal cytochrome b5 the effects of oxidation/ reduction as well as the rotation of the heme group (A/B form) were It was found that the local environment of the heme facilitates rotation by being more flexible than the rest of the protein. The oxidised protein in both A- and B-form was found to be more flexible on the ps time scale than in the reduced state. The oxidised state also contained more residues showing chemical exchange in the range from 70-280 ps than the reduced state. It was concluded that the main difference between oxidised and reduced forms are on the level of the dynamics. The different mobilities reflecting the different entropies might be an explanation for the different redox potentials of the A- and B-form of cytochrome b5. The dynamics on the slower time scales might be important in molecular recognition processes. Similar results were obtained in an independent I5N relaxation study of the same protein.247 The oxidised state was found to be less ordered and the change in entropy as extracted from backbone order parameters to be around 70 J mol-' K-' which corresponds almost to the entire entropy difference for the oxidation as estimated from calorimetric data. Similar slow exchange that could be involved in protein recognition was identified in the high potential iron sulfur protein (HiPIP I) using off-resonance 5N relaxation experiments.248Marked chemical exchange contributions were found exclusively in a surface loop which is also known to be involved in crystal contacts in the X-ray structure. The dynamics of reduced and oxidised yeast Iso-1 cytochrome c were studied by I5N relaxation experiments.249 Uniform distributions of order parameter values around S2 0.8 were generally found with the only exception of loop B which indicated dynamics on the ps time scale. On average the order parameters were slightly higher in the reduced compared to the oxidised form which also had many more residues exhibiting exchange contribution. It was, however, pointed out that there was only poor correlation of these order parameter variations with the crystallographic B-factors of oxidised and reduced cytochrome c. The dynamics of Rhodobacter capsulatus cytochrome c2 have been charac-

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terised by 5N relaxation experiments.250The rotational diffusion was found to be completely asymmetric (principal components of the diffusion tensor ~ s-l) in good agreement with 1.41 x lo7 s-l, 1 . 5 7 lo7 ~ s-l and 1 . 8 3 lo7 hydrodynamic calculations based on the NMR structure. It is demonstrated that the use of an asymmetric model in the analysis of local dynamics results in a substantially simplified picture, in particular with regards to movements on an intermediate time scale while exchange contributions are only little affected. 6.2.9 Dynamics in Multidornain Proteins - A controversy arose about the solution structure of the HIV-I nucleocapsid protein (NC). This protein consists of two domains of the CCHC zinc knuckle type. While the structures of the individual knuckles were very similar, it was not clear whether the two would be interacting with each other. A study of "N relaxation revealed essentially independent rotational tumbling of the individual knuckle domains.251 Furthermore, the N-terminal domain exhibits a clearly axially symmetric anisotropic rotational diffusion while the C-terminal domain can be described by an isotropic motional model. Together with a few weak NOEs between the domains a picture emerged of largely independent domains with only transient interactions allowing a broad range of arrangements which is possibly important to fulfil its role, e.g. in genome recognition and packaging. Similar results regarding the interdomain flexibility of a related protein, the nucleocapsid protein of Mason-Pfizer monkey virus (MPMV), were found.252 Also in this case the linker between the two globular CCHC zinc-finger domains is flexible and the diffusion of the domains essentially independent. In contrast to the HIV-1 protein, however, the MPMV version exhibited a distinct motional behaviour of the two domains. Consistent with the paucity of NOEs in the N-terminal domain the relaxation data exhibited considerably stronger dynamical fluctuations in the N-terminus. These motions cover the entire time range with the slow ps components strongly suppressed at low temperature. Another example for the analysis of a two domain protein is the study of the ninth and tenth fibronectin type 111 modules of the mouse fibronectin attachment site? The analysis of I5N relaxation data gave an anisotropy for the whole molecule (D,l/DL = 1.7-2.1) well below that expected from the X-ray structure. Together with the results from the determination of the NMR structure a model is presented that depicts the module pair in an ensemble of varying interdomain angles form completely extended to bent in an angle of about 20". The consequence of this dynamic arrangement between the two domains is a flexibility in the positioning of the two cell attachment sites (the RGD and the synergy site) that are located on the two different domains. A related situation was seen for the relative domain orientation of I-band Igdomains in the giant sarcomeric protein titin studied by a combination of I5N relaxation and small angle X-ray scattering.253It was found that the sequentially connected domains have a preferentially adopted extended conformation with only small degrees of bending. The flexibility of the linker residues appeared to be limited as judged from I5N relaxation in a two-domain

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Nuclear Magnetic Resonance

construct. The main determinant for the multidomain flexibility appeared to be the length of the linker. The dynamics of the N-terminal LIM domain of quail cysteine and glycine rich protein (crp2) was studied by 5N relaxation experiments.254Considerable fluctuations in residues making the hydrophobic core are found on the intermediate time scale, suggesting a certain degree of flexibility in the centre of the protein. It is assumed that this explains the lack of definition of the relative orientation of the two zinc-fingers of the LIM motif in the NMR structure. The dynamics of cysteine and glycine rich protein (crp2) were analysed based on I5N relaxation experiments.255The two LIM domains in the -200 amino acid crp2 were found to have essentially the same dynamics features as in the isolated domains. No preferential orientation of the two domains relative to each other was observed. Instead, an about 50 amino acid long completely flexible linker was identified between the two domains indicating that the main function of crp2 is as an adapter. The effect of boundary selection on 15N relaxation data when cutting a single domain from a multidomain protein was demonstrated on a fibronectin type I11 domain from t e n a ~ c i n The .~~~ C-terminal extension of the protein from 90 to 92 residues is shown to have profound effects in the suppression of chemical exchange in regions of the protein close to the C-terminus in the structure. It is speculated that the interaction of the charge of the C-terminus with other local charges might lead to multiple conformers exchanging on a ps time scale as evidenced from 15N CPMG experiments. Furthermore the two additional residues form stabilising hydrogen bonds with the rest of the structure that could lock the C-terminus in a single conformation.

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D. Eliezer, J. Yao, H. J. Dyson, and P. E. Wright, Nature Struct. Biol., 1998, 5, 148-155. M. R. Gryk, R. Abseher, B. Simon, M. Nilges, and H. Oschkinat, J. Mol. Biol., 1998, 280,879-896. D. I. Freedberg, Y. X. Wang, S. J. Stahl, J. D. Kaufman, P. T. Wingfield, Y. Kiso, and D. A. Torchia, J. Amer. Chem. SOC.,1998,120, 7916-7923. M. Coles, T. Diercks, B. Muehlenweg, S. Bartsch, V. Zolzer, H. Tschesche, and H. Kessler, J. Mol. Biol., 1999,289, 139-157. J. J. Prompers, A. Groenewegen, C. W. Hilbers, and H. A. M. Pepermans, Biochemistry, 1999,38, 531 5-5327. J. J. Prompers, B. van Noorloos, M. L. M. Mannesse, A. Groenewegen, M. R. Egmond, H. M. Verheij, C. W. Hilbers, and H. A. M. Pepermans, Biochemistry, I999,38, 5982-5994. X. Yao, C. Soden, M. F. Summers, and D. Beckett, Protein Sci., 1999, 8, 307-317. C. Lucke, D. Fushman, C. Ludwig, J. A. Hamilton, J. C. Sacchettini, and H. Riiterjans, Mol. Cell. Biochem., 1999,192, 109-121. K. L. Constantine, M. S. Friedrichs, M. Wittekind, H. Jamil, C. H. Chu, R. A. Parker, V. Goldfarb, L. Mueller, and B. T. Farmer, Biochemistry, 1998, 37, 7965-7980. F. Arnesano, L. Banci, I. Bertini, I. C. Felli, and D. Koulougliotis, Eur. J. Biochem., 1999,260, 347-354. L. Banci, 1. Bertini, C. Cavazza, I. C. Felli, and D. Koulougliotis, Biochemistry, 1998,37, 12320-12330. B. Dangi, J. I. Blankman, C. J. Miller, B. F. Volkman, and R. D. Guiles, J. Phys. Chem. B, 1998,102,820 1-8208. L. Banci, I. C. Felli, and D. Koulougliotis, J. Biomol. NMR, 1998, 12, 307-318. J. S. Fetrow and S. M. Baxter, Biochemistry, 1999,38,4480-4492. F. Cordier, M. Caffrey, B. Brutscher, M. A. Cusanovich, D. Marion, and M. Blackledge, J. Mol. Biol., 1998,281, 341-361. B. M. Lee, R. N. DeGuzman, B. G. Turner, N. Tjandra, and M. F. Summers, J. Mol. Biol., 1998,279, 633-649. Y. Gao, K. Kaluarachchi, and D. P. Giedroc, Protein Sci., 1998,7,2265-2280. S. Improta, J. K. Krueger, M. Gautel, R. A. Atkinson, J. F. Lefevre, S. Moulton, J. Trewhella, and A. Pastore, J. Mol. Biol., 1998,284, 761-777. G. Kontaxis, R. Konrat, B. Krautler, R. Weiskirchen, and K. Bister, Biochemistry, 1998,37, 7 127-7134. R. Konrat, B. Krautler, R. Weiskirchen, and K. Bister, J. Biol. Chem., 1998, 273, 23233-23240. A. E. Meekhof, S. J. Hamill, V. L. Arcus, J. Clarke, and S . M. V. Freund, J. Mof. Biol., 1998, 282, 181-194.

10 NMR of Carbohydrates, Lipids and Membranes BYE. F. HOUNSELL

1

Introduction

NMR is used for both structural and conformational analysis of carbohydrate-based natural products, for artificial molecules which can be used in drug design and delivery, for vaccines, biosensors and glycoconjugates. Also in conjunction with crystallographic studies and molecular dynamics, these methods are being used to understand macromolecular assemblies at the cell surface involving lipid, carbohydrate and protein. The majority of the proteins in the outer layers of all organisms are glycosylated. Also here are other glycoconjugates such as glycolipids and glycosylphosphatidylinositols (GPI). A recent survey of the crystallographic literature' has catalogued the structural motifs of N-glycosylation (linked to Asn residues in the consensus sequence Asn.Xxx.Ser/Thr). Interestingly the oligosaccharides themselves showed a relatively restricted set of dihedral angles. Other studies have continued to use molecular dynamics to study the conformational space explored by rotation about the glycosidic In addition to understanding the conformations of glycolipids and glycoproteins at the cell surface, this type of analysis is important in the designing of vaccines against microorganisms. Analysis of many polysaccharides of bacteria, fungus and plants to be characterised this year can be searched by each journal with only a few being highlighted in this review. NMR is essential here for initial structural analysis and for conformational inferences, although some headway is now being made in crystallography, both in the presence and absence of carbohydrate binding proteins and a n t i b ~ d i e s . ~ Another major breakthrough has been in the analysis of lipids by NMRS and other physical methods and we are now being able to view complex networks of interactions at the cell surface, e.g. between glycoprotein, proteoglycans and lipids (discussed more fully in Section 4). Also the NMR of lipids has lead to a very practical application, i.e. their use to form liquid crystals for analysis of dipole-dipole interactions as pioneered recently by B ~ x .Analysis ~?~ of dipole-dipole interactions is particularly useful for oligosaccharides as it provides additional important conformational information to complement the relative scarcity of this from NOElROE measurementse8Dipole-dipole interactions measured in liquid crystals formed by DPCC and DMPC micelles of sialyl Lewis x and LNNT have provided conformational information, for Nuclear Magnetic Resonance, Volume 29

0The Royal Society of Chemistry, 2000 406

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e ~ a m p l e . An ~ ~ important '~ aspect of this analysis is its facilitation by isotopic labelling." 13Clabelling is carried out by chemical synthesis in the presence of 13C-glucosefollowed by a strategy of chemoenzymatic synthesis which rivals in ingenuity the early studies of Cornforth et al. for following metabolic pathways. Other isotopic labelling strategies include 2H'29'3and 15N.'4715 Conformational analysis is now being used in drug development together with X-ray crystallographic data on protein-carbohydrate interactions and combinatorial chemistry (Section 6). Carbohydrate binding proteins (CBPs) mammalian lectins such as selectins and antibodies. include plant lectins, In addition to X-ray crystallographic studies, of particular importance here are transfer NOE experiments.I8 Other methods include the analysis of J-modulated spin19 and measurement of three bond coupling constants20 in order to parameterise the Karplus equation, for example.2' Xu and Bush measured the long range carbon-carbon coupling constants in a uniformly enriched complex polysaccharide22 and Pozsgay et al. the 3J(CH) coupling constants of selectively 3C-labelled oligosaccharides by 2D J-resolved H NMR.23 Importantly, measurement of 2-bond 13CN-13Cspin coupling constants gave new measurements of coupling s i g n ~ . ~ ~ Several studies have been initiated to try and understand better the dynamic behaviour of di-oligosaccharides in solution in the presence and absence of metal ions and including solid state NMR.25-28Many of the studies are carried out on disaccharides such as sucrose, lactose, maltose and trehalose because of their industrial importance for which it is now deemed necessary to understand their dynamic solution behaviour. The mobility of maltose-water glasses was studied with 'H NMR.29 Kowalewski et al. have studied NMR relaxation of oligosaccharides in solution reorganisational dynamics and internal motion in a review on nuclear spin relaxation in small oligosa~charides.~~ Gustafsson et aL3' have compared solid state NMR and isothermal calorimetry in the assessment of the degree of disorder of processed lactose. Several additional reviews cover NMR in the context of other compounds and physicochemical method^.^^-^^ The applications of these methods in drug design are provided at the end of this review. First will be covered the use of NMR in plant polysaccharide analysis and inclusion complexes (Section 2), followed by natural product analysis (Section 3), lipids and membranes (Section 4). 16917

'

2

Polysaccharidesand Cyclodextrins

Plant polysaccharides are becoming increasingly important in neutriceuticals as well as in standard agricultural applications. Several derivatives of starch and cellulose have been investigated by NMR, e.g. hydroxymethyl starch36 and hydroxypropyl cellulose.37 Carboxymethyl-cellulose is one of several polymers that have been studied by dielectric relaxation spectroscopy (DRS)38 for studies of their molecular mobility in solid thin layers. Wickholm et al. have applied CP-MAS I3CNMR to the assignment of non-crystalline forms in cellulose I.39Chitosan, the de-N-acetylated version of chitin, after cellulose the

408

Nuclear Magnetic Resonance

most ubiquitous natural compounds, has in itself interesting properties and more so after chemical derivitisation as studied for example by solid state NMR.40 Other polysaccharides studied include l i g n i n ~ , ~ ~rhamnogalacturonans of wine,43 dextrin,44 ~ u g a r b e e tand ~ ~ the phosphate substitution pattern of phosphorylated p-( 1- 3 ) - g l u c a n ~ A . ~ ~better understanding of the properties of alginate solutions and gels has been achieved by quantitative magnetic resonance imaging47and of glycogen by steady-state magnetisation measurement with off-resonance irradiation4* and starch by time domain NM R ~ p e c t r o s c o p y ~ ~ and Several studies have been carried out at the monosaccharide of cyclodextrins discussed in more detail here. This is another important commercial area for oligosaccharides as inclusion complexes for various host molecules. Industrio-pharmaceutical applications include biosensors and drug delivery systems. To chose one potential application, Auzely-Velty et al. have synthesised and fully characterised by NMR cholesteryl-cyclodextrins and studied their insertion into phospholipid membranes by 31PNMR.53A range of guest molecules have been characterised recently. These include metal ions, lipids and others. Irwin et al. 54 have characterised cyclomaltoseoligosaccharide binding and solubilisation of hydroxy fatty acid matrixes in aqueous solution using caliorimetric titration and 13C NMR and reported processes driven by solvation phenomena. Other large molecular weight crosslinkers to cyclodextrins include epi~hlorohydrin~~ studied by solid state NMR spectroscopy CP-MAS in terms of 13C spin lattice relaxation and 'H spin lattice relaxation in the rotating frame. It was shown that the addition of water significantly increases the T1 values. Carbohydrate-carbohydrate interactions in water have been studied with a-cyclodextrins and glycophanes (cyclodextrin-cyclophane receptors containing two maltose units linked by 4-hydroxymethyl spacers) as model systems of carbohydrate-protein interactions and compared to a - t r e h a l o ~ eby ~ ~'H NMR and MM calculations. In another work, the structure of the bromide salt of 2-(3-dimethylaminopropyl)tricyclo[3.3.l.l]decan-2-olcomplexed with 0-cyclodextrin and ten water molecules was studied in the solid state by X-ray crystallography and in solution by NMR spectroscopy. The compound is a potent antimicrobial drug being investigated for its pharmacological properties in the complex57 Another is non-covalent associations of cyclomalto-oligosaccharides with trans-p-carotene for which 'H NMR experiments provided evidence of large aggregate . ~ ~the synthesis and CP-MAS analysis of formation.58 Jara et ~ 1 reported cyclodextrin host-guest inclusion compounds containing linear secondary alkylamines giving evidence of non-symmetrical location of the amine in the cyclodextrin channels. Amato et aL60 have compared high field NMR techniques with molecular dynamics simulations with guest 3-phenoxypyridine sulfate. T-ROESY experiments showed that a 1 :1 stoichiometry existed and revealed dipolar contacts between selected protons of the guest and inner protons of the hosts. The M D simulations with explicit water necessary in simulations of ionic systems? reproduced the experimental sets of 'H-'H contacts falling within 3.5 A (NOE effective) and thus provided reliable 942

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409

information on the relative host-guest alignments and geometries. Other inclusion complexes that are essentially non-carbohydrate in nature such as the calixerenes can also be functionlised with carbohydrate. Felix et a1.61used the Suzuki reaction to couple p-bromophenyl glycoside to boronic acid derivatives of n-propoxy-calix[4]arene to provide a deepened cavity. Specific targeting groups can also be added on the outside of cylcodextrins such as glycoclusters62 which have enhanced lectin binding a f f i n i t i e ~ . ~ ~ GlcNAc-terminated glycodendrimers form defined precipitates with soluble dimeric receptor of rat natural killer cells (sNKR-PIA)." The maltose binding protein of Escherichia coli has been studied by chemical shift assignment analysis of the protein-P-cyclodextrin complex.65

3

Carbohydrate-Based Natural Products

Another area for potential exploitation of NMR characterisation of oligosaccharide structures is in microbiology. The majority of compounds being analysed are those from bacteria, but fungi and algae have not been totally forgotten (next paragraph). Some types of oligosaccharide families can occur in different scenarios such as the rhamnofurans of plants and bacteria (Burkhold vietnamiensis).66 Others are specific to bacterial lipopolysaccharides (LPS) and 0-polysaccharides (capsular polysaccharides; CPS) and lipooligosaccharides (LOS).67The NMR data on LPS, CPS and LOS are too numerous to mention, but I will point you also in the direction of important work of Bock et al. on a new glycosidic linkage in b a ~ t e r i a . ~ NMR ~ 9 ~is~ essential in the structural analysis of bacterial components because of the diversity of monosaccharides utilised in both D and L forms. Who would imagine the following, for example, c h a r a ~ t e r i s e dby ~ ~'H and 13C NMR from the 1-81bacterial LPS of Burkholdeira cepacia: [4-amino-4-deoxy-f3-l-arabinosyl]-[ [D-g~ycero-a-~-ta~o-oct-~-u~opyranosy~ate]-~-[~]methy~-~-deoxy-~-man 2-ulopyranosid]onate. In the capsular polysaccharide of Rhodococcus equi, the monosaccharide 4-0-( 1-carboxyethyl)-mannose was characteri~ed.~'Novel structures also continue to be found in the lipid A portion of LPS such as in Neisseria rneningitide~.~~ A recent review of Knirel et ~ 1 shows . ~the~diversity of LPS oligosaccharides from the Proteus bacteria alone. Characterisation relies heavily on a combination of both 'H and 13C NMR. The latter shows characteristic chemical shift changes for particular linkages around the glycosidic ring which can be searched by a computer program CASPER update recently.74Many also are the studies on bacterial antibiotics such as erythromycin for which the solution structure has been elucidated using 'H and I3C NMR in organic and aqueous solution.75 Separovic et have characterised by NMR the structure of C-terminally tagged gramicidin channels and Q ~ i s t ~ ~ has acquired a natural abundance I3C NMR spectrum of gramicidin A in a lipid membrane under MAS conditions. Under slow sample spinning at 500 MHz the intensity of the spinning side bands from the 13C in the backbone carbonyls was used to determine the residual chemical shift tensor and this was

410

Nuclear Magnetic Resonance

consistent with a single stranded right-handed P-6,3 helix proposed for gramicidin A in lipid membranes. Besides bacteria, the following studies are of note on the related mycobacteria and mycoplasma. Wu et u Z . ~ in~ an extensive study carried out synthesis and NMR analysis of 3C-labelled oligosaccharides of the major glycolipid species of Mycobacterium Zeprae. The preparation and analysis is reported for example of the trisaccharide, 2-0-[4-0-(3,6-di-O-methyl-P-~glucopyranosyl)-2,3-di-O-methyl-a-~-rhamnosyl] -3-0-methyl-U-L-rhamnoside. The phosphatidyl-myo-inositol anchor of the lipoarabinomannans of M. bovis bacillus Calmette Guerin (BCG) were studied by Nijou et ~ 1 who. correlated ~ ~ structure with cytokine activation. The structures of several of the phenolic glycolipids of M. kansii have also been studied.80 Less well studied have been the Mycoplasma which contain other glycoconjugates such as a new phosphocholine-containing glycoglycerolipid of M. fermantuns.81In fungi, a novel non-reducing sugar P-GlcNAc-(1- l)-p-Man has been synthesised by the enzyme P-N-acetylhexosaminidase of AspergiZZus oryzae.82 The structures of polysaccharides and glycolipids of A. fumigatis grown in the presence of human serum have been e l ~ c i d a t e d .On ~ ~ the algal side, extensive NMR analysis has been carried out on seaweed f u c ~ i d a n s . ~ ~ , ~ ~

4

Membrane Studies, Glycolipids and Gangliosides

The analysis of lipids by high resolution NMR methods has been reviewed by Dieh1.86A growing number of important molecular events are being shown to involve the interactions between the proteins and glycolipids. One of the important properties of the bilayer is to orient and cluster glycolipid species, as strong interactions in biological systems are often achieved through multivalency arising from the simultaneous association of two or often many more receptors. The fluidity of the bilayer allows for correct geometric positioning of the oligosaccharide head group relative to the binding sites in the protein. This can be studied by NMR in model membranes.87However, our old rather static views of the lipid bilayer are quite literally being turned on their head,88-91i.e. NMR spectroscopy including CP/MAS and NOESY experiments and other physicochemical techniques have observed interactions between the polar head groups and the distal end of the hydrophobic tail showing that the lipids can loop within the membrane. The structure and function of biological membranes can be expected to be sensitive to the extent of hydrophobic matching between the length of the membrane-scanning part of intrinsic membrane proteins and the hydrophobic thickness of the lipid b i l a ~ e r Urbina . ~ ~ et aZ.93 give an historical overview of the development of NMR for the investigation of the static and dynamic structures of lipids and proteins in biological membranes with special emphasis on recently developed solid state NMR methods applicable to these systems. Topics include NMR studies of phospholipids, sterols peptides and proteins in model lipid bilayer membranes as well as biological membranes themselves.

10: N M R of Carbohydrates, Lipids and Membranes

41 1

On the industrial front again are the importance of the types of molecule discussed above as surfactants and many other compounds. For example, an NMR investigation of two-headed surfactant systems involving disodium 4-alkyl-3-sulfonatosuccinateshas been published.94 Many biological possibilities of lipid and glycolipid are increasingly being found, e.g. the thylalkaloid membrane lipids of Rhodopseudomonas v i r i d i ~and ~ ~ plant glycolipids which can be transferred to the bacteroid forms of Bradyrhizobium j a p o n i c ~ m . ~ ~ Lochnit et al. reported the structural elucidation using NMR and cytokineinducing activity of two zwitterionic glycosphingolipids derived from the . ~ ~ et al.98 have characterised porcine parasisitic nematode Ascaris S U U ~Toledo the sphingolipid cerebrosides of the mycopathogens of Paracoccidioides brasiliensis and Aspergillus fumigatus. There are many varieties of mammalian glycoconjugates being found too. Mikami et aZ.99 have published extensive characterisation of brain gangliosides having N-glycolylneuraminic acid as the sialic acid component. These were purified from the horse which has been known to harbour such structures from the ability to raise an immune response in serum sickness caused by transfusion of horse erythrocytes to humans who do not normally synthesise N-glycolylneuraminic acid although N-glycolylated structures do seem to appear sometimes as tumour-associated antigens. Yachida et al. have studied the stereochemistry of synthesised and natural plasmalogalactosylceramides from equine brain.lo0 Brocca et al. investigated the solution structure of the N-acetylated sialylated glycoshingolipid (ganglioside) GM 1carbohydrate moiety at the surface of a 102 kDa lipid-modified micelle by 'H-NOESY measurement at short mixing times. Measurements of the watedgangliosideOH proton chemical exchange rates suggested hydroxyl group involvement at position 8 of sialic acid in strong intramolecular interaction processes. lol This ganglioside in asialo form in liposomes was also studied for its interaction with the pilin surface protein of Pseudomonas aeruginosa. lo2

5

Proteoglycans

In addition to sialylated glycoconjugates in mammals, the major anionic forms are sulfated and by far the greatest amount of sulfate is found as part of the proteoglycans. These are classed together as a family of protein-oligosaccharide conjugates and also includes the non-conjugated hyaluronic acid (HA) or hyaluronan to give its correct title when cationated as found in nature. HA is polyanionic due to the alternating GlcA in its repeating disaccharide. The other PGs have additional sulfate groups on their alternating disaccharides (reviewed by Hounsell and Bailey in ref. 32). These are very high molecular weight polymers where cations and water are extremely important in determining their dynamic behaviour. In addition to their biological functions in this regard, proteoglycans have distinct regions of differing sulfation pattern and N-acetylation plus other variations which provide for functional recognition motifs. The proteins which interact specifically with them are a growing

412

Nuclear Magnetic Resonance

number and include such important biological molecules as antithrombin 1111033104, the interleukins (IL) IL2, 6 and 121°5), transforming growth factor b and its receptor and platelet factor 5.'06v107Here I describe the advances in the structural physicochemical studies which have been published in the past year which are relevant to our understanding of their nature and application. 5.1 Hyaluronan - The macromolecular characterisation, degradation, derivatisation and medical, pharmaceutical and other applications of HA have been reviewed recently.lo* Almond et al. O9 have studied the dynamic exchange between stabilised conformations predicted for HA tetrasaccharides by comparison of MD simulations and NMR data. These simulations confirmed the hydrogen bonds between the neighbouring sugar residues of HA in solution as proposed by Scottllo and predict that the hydrogen bonds are in constant dynamic exchange on the subnanosecond time scale. Thus no one hydrogen bond exists for an extended period of time explaining why NMR often fails to provide evidence for them. High values for 3JNHCH (approx 8 Hz) as experimentally found for HA were consistent with mixtures of both trans and cis conformations.

5.2 Keratan Sulfate - Several studies have appeared on the characterisation of the repeating sulfated polylactosamine sequences of keratan sulfate and including their various 0- and N-linked cores and peripheral substitutions from the group of Nieduszynski. 5.3 Heparin and Heparan Sulfate - A distinction between these two polym e r ~is an ~ important ~ ~ , ~one~ initially ~ based on the greater degree of sulfation of the former and differing susceptibility to specific enzyme degradation (heparinases and heparatinases). Functionally this has now become far more important, and in essence heparin is that found in large scale with anticoagulation properties whereas HS is produced by cells for specific recognition function. A recent article in Nature1'* went somewhat over the top to say that the major function is in interaction in the granules of mast cells with the chymases which cause the histamine response and in pathology allergy. This aspect has been researched for several years and is certainly true, but must take equal precedence with the interactions mentioned above, those or neutrophils in chemotaxis and probably many others. The race is on the find specific sequences which fulfill different functions so that some specificity for drug design can be determined. Regarding the first known function of polymers of sulfated GlcN-GlcNIdoA in anticoagulation, Duchaussoy et al. have publ i ~ h e d ' two ~ ~comprehensive ~'~~ papers on the hexasaccharide sequence able to inhibit thrombin and the assessment through chemical synthesis of the size of the heparin sequence involved in thrombin inhibition. The characterisation has provided extensive chemical shift information to add to that already in the literature (reviewed by Hounsell and Bailey32). One new area not mentioned above is the interaction of heparin with the NCl domain of collagens. This has been assessed by circular dichroism and NMR113 and brings us back to the

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whole concept of proteoglycan interactions in the extracellular matrix which can no longer be divorced from cell surface function.

5.4 Chondroitan Sulfate - Sugahara's group have continued to carry out comprehensive characterisation of chondroitin and dermatan sulfate core regions (reviewed by Hounsell and Bailey32)and backbone oligomers.' l4 The molecular dynamics of tetrasaccharide subunits of chondroitin 4-sulfate in water has been studied by others' l 5 and, more specifically, the conformational changes and anticoagulant activity of chondroitin sulfate following its 0-suifonation. l 6

'

5.5 Other Studies of Uronic Acids and Sulfate Esters - Tang et al.'17 have studied the molecular motions of GalA and GalA methyl ester measuring spinlattice relaxation and 13C CP-MAS. NMR studies have been extended in several cases with additional X-ray diffraction data. 1 8 v 1 l9

6

Biomedical Applications

In addition to the large amount of data on selectin-carbohydrate binding which can be accessed in the references quoted in Section 1 and which may yield effective therapies in inflammation and related disorders, the NMR of oligosaccharides, lipids and glycoconjugates have several other biomedical applications. NMR chemical shift analysis has been correlated with results of medical imaging by MRS of soft tissue tumours.'20 Other MRS studies include the differential diagnosis of low and high malignancy brain gliomas by analysis of the 'H NMR ganglioside ceramide resonance region.12' In a related area saccharide conjugates .are being used as contrast agents and therapeutic agents.122Self organising maps for the detection of human blood plasma lipoprotein lipid profiles on the basis of 'H NMR s p e c t r o ~ c o p yrevealed '~~ that clinically relevant lipid classifications can be obtained from computerised self organising map analysis of 'H NMR spectral data. The authors Wang et al. reported the 'H, 15Nand 13Cresonance assignments and secondary structure of apoliver fatty acid-binding protein. 124 Grosse et al. have assessed the potential targetting role for P-cyclodextrins to tumour cell membranes'25 and Live et al. have probed the cell-surface architecture through synthesis and NMR characterisation of structural motifs of tumour mucins.'26 Another success story in the carbohydrate field has been as enzyme inhibitors. Besides influenza virus neuraminidase, potent inhibitors of mannosidasel 27 and glucosidases'28 continue to be synthesised and their structure/activity relationships analysed.

7

References 1

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416 63

64 65 66 67

68 69 70 71 72 73 74 75 76 77 78 79 80

81 82 83 84 85 86 87 88 89 90 91 92

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10: NMR of Carbohydrates, Lipids and Membranes

93 94 95 96 97 98 99 100 101 102 103

104

105 106 107 108 109 110 111 112 113

114 115 116 117 118 119

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11 Synthetic Macromolecules BY H. KUROSU AND T. YAMANOBE

1

Introduction

NMR is an indispensable tool to characterize synthetic macromolecules. For synthetic macromolecules, NMR is used for several purposes; determination of composition, primary structure, conformation and crystal structure in the solid state, molecular mobility at molecular level, phase structure, imaging and so on. In this chapter, NMR work concerning synthetic macromolecules is reviewed by categorizing primary structure, liquid crystal, characterization in the solution and solid states, dynamics, gels and crosslinked, and blend. Saxena et al.' described recent advances of solid state 2D2 H NMR methods to study polymer blends. The selective labeling of individual blend components allows unambiguous determination of dynamics for each component in the blend. The 2D exchange method also provided the motional rate and the motional distributions. Macdonald2 reviewed 2H study of polyelectrolytesurfactant interactions. In phase separated polyelectrolyte-surfactant micelle complexes, 2H NMR can differentiate the loosely packed complexes from the tightly packed complexes. This method also reveals the dynamics of thermotropic phase transition. Stilbs3 produced a review about structure and dynamics of polymer-surfactant system based on NMR parameters such as chemical shift, relaxation times, line width, self diffusion and so on. Other reviews have been published about the development of NMR techniques to characterize polymer^,^ dynamics of liquid crystal polymer^,^.^ polymer interface^,^ elastomer-carbon black filler interactions,* monosubstituted polyacetylene^,^ polymerization of diene. lo

2

Liquid Crystals

Poly(4-(4-cyanobiphenyl-4-yloxy)butyl acrylate-co-acrylic acid)s were prepared by radical polymerization. These polymers form liquid crystalline phase if acrylic acid content is between 42 and 52 mol%. Temperature dependences of order parameter in the magnetic field were studied by wide line 'H NMR. Conformations of spacer group were investigated based on 13C NMR chemical shift for poly(ester imide) composed of 4-N-(carboxyphenyl) trimellitimide, 4-N-(carboxyethenylphenyl) trimellitimide), 4-hydroxy-N-(4Nuclear Magnetic Resonance, Volume 29

0The Royal Society of Chemistry, 2000 419

420

Nuclear Magnetic Resonance

hydroxypheny1)phthalimide and long aliphatic spacers.I2 In the frozen smectic phase, an alternating sequence of trans and disordered conformations is predominant. In the smectic phase, trans-trans conformation increased. The orientation and phase structure of liquid crystalline polymer in shear flow was investigated by NMR. Molecular motions in different phases were studied by solid state NMR for a series of polyesters composed of alkyl side chains and 4cyanobiphenyl. l 4 The reorientational process of liquid crystalline polymers by the static magnetic field and a dynamic electric field was investigated by placing a capacitor in the NMR probe for cyano- and biphenyl-containing polyacrylate. l 5 Simultaneous application of the electric field can rotate the director in every desired angle. Alignments and orientational behaviour were investigated for liquid crystalline polyacetylene through proton dipolar coupling. It was demonstrated that the liquid crystalline polyacetylene are uniaxially aligned by a magnetic field.l 6 For liquid crystalline polyester (4-hydroxybenzoic acid and 6-hydroxy-2-naphthoic acid), a director distribution in molten or nearly molten state was obtained by wide line 'H NMR and simulations. l7 CPlMAS study was carried out for poly(esterimide)s based on N-(4'-carboxyphenyl)trimellitimide and dihydroalkane. CP/MAS spectra revealed that the spacers longer than 12 methylene groups take all trans conformations up to 100°C. From 2H NMR spectra, the spacers are nearly immobile at - 180°C, but gain mobility upon heating. The mode of molecular motion is discussed.'* The conformation and dynamics of side chain liquid crystalline poly(viny1 ether)s composed of phenylbenzoate, stilbenzyloxy and 4-1 1 methylene spacer were assessed by NMR."

3

Primary Structure

NMR is one of the most powerful tools to characterize the primary structure of polymers, such as tacticity, regioregularity, end group, sequence distribution etc. Table 11.1 summarizes the papers in which NMR is used to characterize primary structure of polymers. 4

Characterization of the Synthetic Macromolecules

Solid state NMR is a powerful tool for characterizing the structure of macromolecules. Not only 13C NMR but also other nuclei, such as 15N, 29Si, 'H, 170, 129XeNMR etc., are widely used to obtain information about structure and mobility of macromolecules. The high-resolution solid state 13C NMR spectra of the a modification, modification and amorphous poly(ethy1ene naphthalene-2,6-dicarboxylate) have been measured by CP/MAS method. The chemical shift differences are explained in terms of different conformation and chain packing in the samples.328 Due to structural similarities, the polydiacetylenes of 1,1,6,6,tetraphenylhexadiynediamine(1) and 1,6-di-N-carbazoly1-2,4-hexadiyne( 11) are

I I : Synthetic Macromolecules

42 1

Table 11.1 NMR studies of primary structure of polymers ~~~

Polymer 6-carboxypullulan a-dextrin cis-isotactic 1,4-polypentadiene cis-polybutadiene copolyester:terephthalic acid,ethylene glycol, 9,lO-dihydro-10-(2,3-di(hydroxycarbonyl) propyl)-9-oxa-1- phosphaphenant hrene10-oxide end capped poly(2,5-benzophenone) epoxy epoxy epoxy epoxy ally1 sucrose, epoxy crotyl sucrose epoxy,(2S,3S)-3,4-epoxy-1,2-0-isopropylidene butane-diol,(2R,3S)-3,4-epoxy1,2-0isopropylidenebutane-diol it-poly(methy1methacrylate)-block-st-poly (methyl methacrylate) linear low density polyethylene-graft-poly (diethyl maleate) methoxy-terminated polysiloxane natural rubber natural rubber-graft-methyl methacrylate, natural rubber-graft-styrene nylon-6 organopol ysilazane oxazoline-terminated polystyrene phenol-urea resin phenolic resol resin poly((( 1S)- 1-( 1-naphthyl)- 1-phenyl-3,3dimethyldisiloxane-1,3-diyl)ethyIene)

Nucleus Contents

Ref.

substitution configuration tacticity, conformation characterization, epoxidation sequence distribution

20 21 22 23

25 26 27

H,Si C C

end group branch crosslink, cure mechanism crosslink sequence distribution end group

28 29 30

H

tacticity, block

31

H

graft

32

H,Si

end group configuration composition

33 34 35

degradation branch end group composition crosslink irregularity

36 37 38 39

H,C

H H

H C H

C H H,C C C Si

poly((2-fury1)methylenesulfide) H,C poly((p-nit ropheny1)acety lene) C poly((p-phenylene ethyny1ene)-alt-(2,5C thienylene ethynylene)) poly((R, S)-3-bu tyrolactone-co-4-butyrolactone) H ,C poly((R,S)-0-butyrolactone) poly((R)-3-hydroxybutanoate) poly((S)-lactic acid-alt-(S)valine) poly( 1,1,2-trimethylsilacyclobutane)

~~

C H H H

end group configuration regioregularity, irregularity sequence distribution, tacticity tacticity conformation configuration end group, molecular weight

24

40

41 42 43 44 45

46 47 48 49

422

Nuclear Magnetic Resonance

Table 11.1 contd. Polymer

Nucleus Contents

poly( 1,l’-ferrocenylene-alt-p-terphenyl-4,”‘ylene), poly( 1,l ’-ferrocenylene-alt-pquinquephenyl-4,4””-ylene),oly( 1,l‘ferrocenylene-alt-p-septiphenyl-4,4”””ylene) poly( 1,3-~yclohexadiene) poly( 1,3-~yclohexadiene) poly( 1,3-dioxepane) poly( 1,3-pentadiene)

H,C

sequence distribution

50

H H,C,Li H,C H,C

configuration configuration end group crosslink, cyclization, configuration branch

51 52 53

poly( 1,4,9-trioxaspiro(4,6)-9-undecanone-co- H,C E-caprolactone) poly( 1,5-dioxepan-2-one-co-L-lactide),poly C (1,5-dioxepan-2-one-co-~-caprolactone) 1,4poly( 1,8-octanedioxy-2,6-dimethoxyphenylene-1,2- ethenylene-1,4-phenylene1,2-ethenyIene-1,4-phenyIene-1,2-ethenyIene3,5-dimethoxy-1,4-phenyIene) poly(2,3,5,6-tetrahydroimidazo(2,l-b)(1,3) H oxazole) poly(2,3-bis(t rimeth ylsiloxy)but adiene), H,C poly(2,3-bis(tert-butyldimethylsiloxy) butadiene), poly(2,3-bis (trimethyl siloxy) butadiene-co-styrene) C poly(2-(( 1O-(4-hydroxyphenoxy)decyl)oxy) terephthalic acid-co-2-(( 10-((4’-hydroxy-1,l’biphenyl-4-y1)oxy)decyl)oxy)terephthalic acid) poly(2-(4-(6-(methacryloyloxy)hexyloxy)phenyl H azo) anthraquinone-co-p-methoxyphenyl-4(6-(methacryloyloxy) hexy1oxy)benzoate) poly(2-benzylidene-4,5-dicyano-1,3-dithiole) H,C poly(2-butylhexyl isocyanate) C poly(2-hydroxyethyl acrylate) H poly(2-hydroxyethyl acrylate-co-methyl H methacrylate) poly(2-hydroxyethyl met hacry late) H,C poly(2-methylene-1,3-dioxepane-co-ethyIene), H,C poly(2-mehylene-1,3-dioxepane-co-styrene) €4 , c poly(2-vinyl-4,4-dimethyl-5-oxazolone), poly(2-vinyl-4,4- dimethyl-5-oxazolone)-co(methyl methacrylate) poly(3,5-tricyclo[2.2.1.O]heptylene) poly(3-(2-((S)-2-methylbutoxy)ethyl) thiophene) poly(3-(6-methoxyethyl)-2,2’-bithiophene) H,C H,C poly(3-alkylphenylthiophene),poly( 3-(4dodecyl phenyl) thiophene) poly( 3- butyrolactone) H

Ref.

54

55

sequence distribution

56

sequence distribution

57

reaction mechanism

58

configuration, composition

59

branch

60

composition

61

conformation conformation molecular weight composition

62 63

64 65

reaction mechanism composition

66 67

sequence distribution, tacticity

68

branch regioregularity tacticity regioregularity, irregularity tacticity

69 70 71 72 73

11: Synthetic Macromolecules

H pol y(3-hexylthiophene) poly(3-hydroxyoctanoate-co-3-hydroxyundec- H 10-enoate) poly(4,4’-dihydroxydiphenylsulfone-co-4,4’- C difluoro benzophenone-co-4,4’difluorodiphenylsulfone) poly(4-(4-oxy-4‘-cyanoazobenzene)but-1-yl C methacrylate-co-mentyl methacrylate), poly(6-(4-oxy-4-cyano azo benzene) hex- 1-yl methacrylate-co-menthyl methacrylate), poly(8-(4-oxy-4’-cyanoazobenzene)oct-1-y I methacrylate-co-menthyl methacrylate) poly(4-(acryloyloxy)-3-methoxybenzal phenylamine-co-methyl methacrylate) poly(4-acetylpheyl methacrylate-co-glycidyl methacrylate) poly(4-nitropheyl methacrylate-co-methyl methacrylate) poly(4-tolydiphenylamine-paraformaldehyde), poly(N,N’-diphenyI-N,N’-bis (4-methylphenyl) - I ,4-phenylenediaminepara formaldehyde) poly(5,6-dicarbomethoxy)-norbornadiene), poly(5,6-di-exo-(carbomethoxy)norbornene) poly(5-tert-buty1)benzothiophene poly(6-(4-azamethano fullerenebenzoyloxy) hex y 1 met hacry late-co-t-buty I methacrylate) poly(6-henyl- 1,5-hexadiene),poly(7-methyl1,6-0ctadiene),poly(R(+)-5,7-dimethyl1,6-0ctadiene) poly(7-tetradecene oxide) poly(7-tetradecene oxide) pol y(8-octanolide),poly(8-octanolide-co-~caprolactone), poly(8-octanolide-co-12dodecanolide) poly(acety1iminoethy1ene)lipopolymer poly(acry1amide-co-acrylic acid-co-N-(1,ldimethyl-3-0x0buty1)acrylamide) poly(acry1amide-co-alkyl acrylate) poly(acry1amide-co-ethylacrylate),poly (acrylamide-co-butyl acrylate) poly(acry1amide-co-methyl acrylate) poly(acry1amide-co-vinyl acetate)

poly(acry1ate) poly(acry1ic acid)-graft-sawdust poIy(acry1onitrile-co-acrylic acid)

423 regioregularity composition

74 75

sequence distribution

76

tacticity

77

composition

78

composition

79

composition, sequence 80 distribution configuration 81

tacticity

82

characterization composition

83 84

end group

85

configuration reaction mechanism sequence distribution

86 87 88

end group sequence distribution

89 90

sequence distribution, compositional sequence distribution

91

sequence distribution composition, sequence distribution, configuration branch graft seauence distribution

93 94

92

95 96 97

424

Nuclear Magnetic Resonance

Table 11.1 contd. Polymer

Nucleus Con tents

poly(acrylonitri1e-co-glycidylmethacrylate) poly(acrylonitri1e-co-hexyl methacry late) poly(acrylonitri1e-co-methylacrylate) poly(acrylonitri1e-co-pentylacrylate)

poly(a-methoxy-3,6-endo-methylene1,2,3,6tetrahydro phthaloyl-5-fluorouracil),poly (a-methoxy-3,6-endo- methylene- 1,2,3,6tetrahydrophthalo yl-5-fluorouracil-coacrylic acid) poly(a-methoxy-exo-3,6-epoxy1,2,3,6tetrahydrophthaloyl- 5-fluorouracil) and its derivative poly(a-methylstyrene peroxide) poly(a-tocopherylmethacrylate) poly(amic acid) poly(amide-imide) poly(amideamine) poly(ary1 ether ketone amide) poly(ary1 ether ketone amide) poly(arylal1en-co-carbonmonoxide): phenylallene,(4-methylphenyl)allene, (4-methoxyphenyl)allene,(4-chlorophenyl) allene,(4-fluorophenyl)allene,(4-butylphenyl) allene, (4-tert-butylpheny1)allene poly(benzoxazine) C poly(benzy1 a-((1-phenylethoxy)methyl) C acrylate) poly(benzyl P-(2R, 3s)- 3-methy lmalate) C poly(P-methyl-P-propiolactone),poly(P-ethyl- C P-propiolactone) poly(bis(4-hydroxybuty1)terephthalate-co-bis H (2-h ydroxyethyl)terephthalate) poly(butadiene)-graft-polystyrene poly(buty1 acrylate) H,C poly(buty1ene oxide)-block-poly(ethy1ene oxide) poly(buty1ene terephtha1ate)-block-poly H,C (tetrame thylene glycol) poly(ch1oroprene)-graft-poly(methyl methacrylate) poly(copo1yether-block-polyamide) poly(cyc1opentene)

H,C C

Ref. 98

sequence distribution, composition, configuration sequence distribution tacticity, composition sequence distribution, composition composition

102

characterization

103

tacticity, end group tacticity conformation sequence distribution branch configuration conformation composition, end group, sequence distribution

104 105 106 107 108 109 110 111

crosslink tacticity

112 113

tacticity tacticity

114 115

sequence distribution

116

graft, branch end group composition, block

117 118 119

compostion, sequence distribution graft

120 121

end group configuration

122 123

99 100

101

I I : Synthetic Macromolecules

pol y(D, L-lact ide-co-ethylene methyl phosphate) H poly(di(4-vinyloxybutyl)succinate)-co- 1,5- bis H (maleimido)-2-methylpentane) poly(diethyl3-hydroxyglutarate) poly(difluorosily1enemethylene) H poly(dimethy1-co-methyl mercaptopropyl siloxane) Si poly(dimethylcyclosiloxane-codiphenylcyclosiloxane) poly(dimethy1 diphenyl silylene methylene) poly(dimethy1siloxane) H,Si poly(dimethylsi1oxane-co-ethyleneoxide) H poly(DL-lactide) H pol y(epi bromo hydrin) H,C poly(epichlorohydrin),poly(oxy- 1 -chloroC methylethylene),poly(oxy- 1 -chloromethyl ethylene-co-oxy- 1 rphenoxymethylethyleneco-oxy- 1 -methyleneethylene) H pol y(E-caprolactone) pol y(E-caprolactone),epoxy poly(s-caprolactone),multiblock copolyester H,C poly(&-caproIactone),poly(1act ide), poly(E-caprolactone-co-lactide) pol y(s-caprolactone) H pol y(ester-co-amide):silylated4-aminobenzoic H acid, 3,5-bisaminobenzoic acid, trisacety1 gallic acid chloride poly(ester-imide) H poly(ethene-co-norbornene) C poly(ethene-co-propene) C pol y(et hene-co-propene) C poly(ether amide) H,C poly(et her ket on) H H poly(ether urethane) poly(et hoxy-co-propoxy) C poly(ethy1ene glycol dimethacrylate) H,C poly(ethy1ene glycol) F poly(ethy1ene glycol)-block-poly(acrylonitrile), H,C poly(ethylene glycol)-block-poly(acry1amide) poly(ethy1ene glycol)-block-polylactide H poly(et hylene naphthalate-co-et hy lene H,C terephthalate) poly(ethy1ene oxide)-block-poly(buty1eneoxide) C poly(ethylene oxide)-block-poly(L-lactide), poly(ethy1ene oxide)-block-poly(L-lactideco-glycolide) poly(ethy1ene oxide-co-ethylene terephthalate) H

425 degradation composition, configuration branch conformation crosslink

126 127 128

sequence distribution

129

irregularity reaction mechanism composition end group crosslink composition

130 131 132 133 134 135

branch transesterification block sequence distribution

136 137

branch branch

140 141

branch composition sequence distribution regioregularity branch branch crosslink end group, sequence distribution branch end group block

142 143 144 145 146 147 148 149 150 151 152

block sequence distribution

153 1 54

block block,end group

155 156

composition

157

124 125

138

139

Nuclear Magnetic Resonance

426

Table 11.1 contd. Polymer

Nucleus Contents

poly(ethy1ene terephthalate), copolyester H poly(ethy1ene terephthalate), poly P (hydroquinone phenylphosphonate), poly(bis(4-hydroxypheny1)sulfone phenyl phosphonate), poly(4-hydroxyphenyl) sulfone methylphosphonate) poly(ethy1ene terephthalate-co-E-caprolactone) H poly(ethy1ene- 1-octene) C poly(et hylene-2,6-naphthala te-co-ethylene H terephthalare) C poly(ethy1ene-co-1-octen) poly(ethy1ene-co-butylenenaphthalene-2,6dicarboxylate) poly(ethylene-co-p-methylstyrene)

H C

poly(ethylene-co-propene) poly(ethylene-co-propylene)

C C

poly(ethy1ene-co-propylene-co1,4-hexadiene), C poly(et hylene-co-propylene-co-2-ethylidene5-norbornene),poly(ethylene-co-propyleneco-5,8-dicyclopent adiene) poly(ethylene-co-styrene) C poly (ethylene-co-vinyl acetate) poly(ethy1ene-co-vinyl alcohol) H poly(ethylene-glycol)-block-poly(acrylonitirile), H,C poly(ethy1ene glycol)-block-poly(acrylamide), poly(ethy1ene glycol)-block-poly(methy1 methacrylate), poly(ethy1ene glycol)-blockpoly(methacy1ic acid) poly(ethy1ene-iso-propyl phosphate) H,C,P poly(ethynylanthracene), poly H (et hyny lphenan t hrene) poly(exo-n-alkyl norbornene dicarboxyimide), H,C poly(end-n-ccty I-norbornene dicarboxyimide), poly(exo-n-alkyl norbornene)-block-poly(2,3-bis (trifluoromethyl) norbornadiene) poly(y-methyl L-glutamate)-block-polyurethane C poly(glyco1ide-co-s-caprolactone) H,C poly(glycidy1 methacrylate), poly(glycidy1 H methacrylate)-graft-polystyrene),poly(methy1 methacry late)-block-poly(glycidy1 methacrvlate)

Ref.

composition 158 transesterification, 159 sequence distribution

transesterification sequence distribution block, sequence distribution irregularity, sequence distribution composition

160 161 162

composition, sequence distribution sequence distribution composition, sequence distribution sequence distribution

165

sequence distribution composition, sequence distribution composition block

169 170

end group conformation

173 174

configuration, block

175

block composition, sequence distribution block,graft

176 177

163

164

166 167 168

171 172

178

427

11: Synthetic Macromolecules

poly(glycidy1 methacrylate)-block-poly(methy1 methacrylate)-block-polystyrene poly(glycidy1 phenyl ether) poly(hydr0xybutyrate-co-hydroxyva1erate)graft-styrene) poly(ibuprofen linked 2-hydroxyethyl methacrylate-co-methacrylic acid) poly(isobuty1ene) poly(isobuty1ene) poly(isobutylene)/poly(dimethylsiloxane) poly(isoprene)-block-poly(methacry1ic acid) poly(L-lactide-co-trimethylenecarbonate) poly(1actide) poly(1act ide) poly(lactide),poly(1actide-co-glycolide) poly(ma1eic acid) poly(ma1eic acid-co-styrene),poly(maleimideco-styrene) poly(methacry1ic acid) poly(methy1 methacrylate) poly(methy1 methacrylate) pol y(met hy 1 methacrylate) pol y(me t hyl met hacrylate) poly(met hyl methacrylate), polystyrene poly(methy1methacrylate), poly (macromonomer), polystyrene poly(methy1 methacrylate)-block-poly(acry1ate) H,C,P poly(methy1 methacrylate)-graft-poly(isobutene)H poly(methy1 methacrylate-co-dimethyl maleate) H poly(methy1 methacrylate-co-styrene) C poly(methy1 methacrylate-co-vinylidene H,C chloride) poly(methy1 vinyl ether)-block-fatty acid H poly(methylphenylsily1ene)-block-poly(methy1 methacry late) poly(met hylphenylsily1ene)-block-polystyrene poly(methyltriethoxysilane) Si poly(monoethyleneglyco1allenyl methyl ether- H co-N-vinyl pyrrolidone) poly(N-(n-(4-cyanobiphenyl-4’yloxy)alkyl)-7- H,C oxanorbornene-5,6-exo-dicar boximide) poly(N-ally1 morpholine-co-sulfur oxide),poly H (N-ally1 morpholine-co-styrene) pol y( N-isopropylacrylamide) H poly(N-substituted maleimide) C poly( N-vinylcarbazole) H

block

179

tacticity graft, composition

180 181

composition, drug linked polymer end group end group crosslink block composition tacticity tacticity branch, comb structure configuration sequence distribution

182

tacticity end group, molecular weight, tacticity tacticity, degradation tacticity tact icity end group tacticity, branch

183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199

block, reaction mechanism composition, branch composition end group tacticity

20 1 202 203 204

block, end group block

205 206

block characterization composition

207 208 209

conformation

210

sequence distribution

21 1

characterization tacticity tacticity

212 213 214

200

428

Nuclear Magnetic Resonance

Table 11.1 contd. Polymer

Nucleus Contents

Ref. ~

poly(norbornene),poly(norb0rnadiene) poly(norbornene-co-eth ylene) poly(oxirane-co-tetrahydro furan) C po ly(oxybenzoate-co-ethy lene terep ht hala te), C polycarbonate poly(oxyethy1ene-co-ally1 glycidyl ether), H poly(oxyethy1ene-co-ally glycidyl ether)graft-pol y styrene poly(oxyethy1ene-co-styrene) H poly(p-methylstyrene-co-styrene) H pol y(para-phen yleneet hyny1ene)-block-poly H (ethylene oxide) poly(pentylacry1ate) poly(phenylacety1ene) poly(pheny1acetylene-co-1-hexyne) POlY(propene) poly(propene-co- 1,5-hexadiene),poIy(propeneco-1,7- octadiene) poly(propene-co-ethene) C poly(propy1ene oxide)-graft-poly(ma1eic C anhydride) poly(propy1ene-co-ethylene) C poly(propy1ene-co-ethylene) C poly(propy1eneimine) C poly(sily1 ester) H,C,Si poly(styrene-co-3-methacryloyloxypropyltrimethoxysilane), poly(2((perfluorononeny1)oxyethyl met hacrylate))block-polystyrene-block-poly(2((perfluorononeny1)oxylethyl methacrylate)) poly(styrene),poly(methy 1 methacrylate), P poly(methy1 acrylate) poly(styrene)-block-poly(ethy1ene adipate)H block-poly(styrene) poly(st yrene)-block-polyisobutylene H poly(styrene-co-acrylamide) C poly(styrene-co-acrylamide) C poly(styrene-co-acrylicacid) poly(styrene-co-carbon monoxide) poly(styrene-co-maleic acid) C poly(styrene-co-methy acrylate-co-methyl vinyl ketone)

~

~ _ _

2 15

reaction mechanism, block configuration, end group, tacticity , sequence distribution composition transesterification

217 218

graft

219

graft composition block

220 22 1 222

tacticity configuration composition end group stereoselectivity

223 224 225 226 227

sequence distribution graft

228 229

sequence distribution sequence distribution tacticity degradation block

230 23 1 232 233 234

end group

235

block, sequence distribution block composition sequence distribution reaction mechanism tacticity sequence distribution end group

236

216

237 238 239 240 24 1 242 243

11: Synthetic Macromolecules

429

poly(styrene-co-methyl methacrylate) poly(styrene-co-methyl methacry late) poly(su1fur dioxide-co-diethyl diallylmalonate) poly(tert-butyl acrylate-co-methyl methacrylate), poly(tert-butyl acrylateco-2,4,5-trichlorophenyl acrylate),poly(tertbutyl acrylate-co-N,N-dimethyl-2-amino ethyl met hacrylate) poly(trans-4-acryloy loxyazobenzene) poly(trans-4-acryloyloxyazobenzene-co-methy l methacrylate) poly(viny1idene fluoride) poly(viny1 acetate) poly(viny1 acetate) poly(viny1 acetate-alt-butyl acrylate) poly(viny1 alcohol) poly(viny1 chloride) poly(viny1 chloride) poly(viny1 chloride) poly(viny1 chloride), poly(ethy1ene-co-vinyl chloride) poly(viny1 octanal acetal) poly(viny1 pivalate),poly(vinyl trifluoroacetate), poly(vinyl2,2-bis (trifluoromethylpropionate) poly(viny1idene chloride-co-butyl acrylate)

block copolymer end group end group composition, sequence distribution

244 245 246 247

tacticity composition, sequence distribution end group, telomerization tacticity tacticity sequence distribution degree of acetalization, esterification tacticit y degradation tacticity branch

248 249

255 256 257 258

tacticity tacticity

259 260

composition, configuration composition

26 1 262

tact icit y branch reactivity

263 264 265

conformation composition reaction mechanism characterization

266 267 268 269

transesterification

270

reaction mechanism dendrimer reaction mechanism

27 1 272 273

250 25 1 252 253 254

~

poly(viny1idene fluoride-cohexafluoropropylene), poly(viny1idene fluoride-co-chlorotrifluoroethylene), poly(viny1idene fluoride-cotetrafluoroethylene-hexafluoropropylene) pol y acrylamide pol y amide polyamide:3,5-diamino-l,2,4-triazole, acetic anhydride, aromatic anhydride pol yamidine polyaniline (undoped, doped) poly benzoxazine polybenzylsilsesquioxane, pol yphenylsilsesquioxane, polyphenethylsilsesquioxane polycaprolactone, poly(2,2-dimethyl trimethylene carbonate) polycarbonate polycarbosilane polydithiocarbonate

1 l,L

~.

430

Nuclear Magnetic Resonance

Table 11.1 contd. Polymer

Nucleus Contents

polyester resin : dimethyl terephthalate, H,C ethylene glycol, glycerol, tri(2-hydroxyethyl) isocyanurate polyether:3,3-bis(chloromethyl)oxetane, 1,3-dibromopropane, 2,7-dihydroxynaphthalene, 4,4’-dihydroxyazobenzene, 4,4’-dihydroxydiphenyl, 1,l-bis (4'-h ydroxyphenyl)cyclohexane polyet h y lene polyethylene polyethylene polyet hy lene polyet hy lene polyethylene,poly(ethylene-1-butene) polyethylene-block-poly(norbornene) poly isocyanide polyisoprene polylactide polylactide polyperfluoroether-silica hybrid polyphenol polypropene polypropylene polypropylene polypropylene polypropylene polypropylene polypropylene polypropylene polypropylene polypropylene polypropylene polypropylene polyrotaxane,poly(amide rotaxane) polysilane polystyrene polystyrene polystyrene polystyrene polystyrene polystyrene polystyrene

Ref.

composition

274

conformation

275

branch branch branch branch branch sequence distribution block, sequence distribution conformation degradation tacticity tacticity end group crosslink tacticity tacticity tacticity tacticity tacticity tacticity tacticity tacticity tacticity end group, tacticity tacticity, irregularity tacticity characterization branch end group tacticity tacticity end group end group end group, star block tacticity

276 277 278 279 280 28 1 282 283 284 285 286 287 288 289 290 29 1 292 293 294 295 296 297 298 299 300 30 1 302 303 304 305 306 307 308 309

11: Synthetic Macromolecules

polystyrene C polystyrene C F polystyrene,poly(methyl methacrylate) polystyrene-block-(poly(p-hydroxystyrene)H,C graft-poly(ethy1ene oxide))-block-polystyrene polystyrene-block-poly(ethy1ene-co-propylene) C polystyrene-block-polybutadiene H poly sulfide C polytetrafluoroet hylene F polyurethane C polyurethane polyurethane H polyurethane-block-polystyrene,polyurethane-H block-poly(methyl met hacrylate) polyvinylbenzoa te pyridine modified poly(viny1 chloride) resine:triphenylamine, formaldehyde, H,C butyraldehyde starch-graft-poly(methacry1ic acid-co-methyl H methacrylate) styrene-butadiene-styrene rubber, poly H,C (styrene-co-butadiene-co-styrene) urethane H

43 tact icity tacticity end group sequence distribution, graft, block tacticity, block block crosslink crosslink selectivity characterization end group block

314 315 316 317 318 319 320 32 1

tacticity ,irregularity composition configuration

322 323 324

sequence distribution, configuration, graft composition, conformation composition

325

310 31 1 312 313

326 327

a significant pair for comparison of fully crystalline macromolecules.13c CP/MAS measurements show that the data obtained for I1 are in accord with a previous report but those for I show a site-splitting of nonequivalent acetylenic carbons.329Solid state 13C and 15N NMR spectroscopy was used to study the structure and thermochemistry of nylon-6,6. REDOR, a solid state NMR experiment that permits the quantitative measurement of I3C-l5N labeled chemical bonds, was used to detect the formation and breaking of carbonnitrogen bonds due to polymerization and transamidation. 330 The spatial information is gained from a proton spin diffusion experiment combined with two 2D 13C exchange sequences via appropriate back and forth transfer of magnetization between 13C and 'H spins. For poly(viny1 acetate) at 10 K above the glass transition a length scale of 3 _+ 1 nm was detected.33' The 13C Pake doublet of the poly(phenylacety1ene) containing 5% [ 1,2-13C]phenylacetylene was observed by effectively removing the chemical shift anisotropies using a composite inversion pulse sequence in order to investigate the polymerization mechanism and the structure of poly(phenylacety1ene) synthesized using Rh(CCC6H5)(2,5-norbornadiene)[P(C6H5)3]2 initiator.332The conformational statistics of atactic polystyrene have been investigated by solid state NMR, on a sample labeled with 25% 13CH2groups. Double quantum pulse sequences are used to select statistically formed 13C-13Cspin pairs. The analyze of the spectra yields a trans content of 68%( The 29Si and

432

Nuclear Magnetic Resonance

13C CP/MAS NMR spectra and the spin-spin relaxation of the polysiloxaneimides doped with LiCF3S03 have been studied to probe the role of the salt.334 Benzoxazines derived from bisphenol-A, formaldehyde, and primary amines were characterized using 3C solid state NMR spectroscopy.335Several commercial and noncommercial, high and low density and ultraorientated polyethylene samples, as well as polyethylene samples with inorganic fillers, have been investigated by inversion-recovery CP/MAS 13C NMR. In all these samples two types of all-trans chains in orthorhombic crystalline domains are detected, which give two overlapping 13C lines with different line widths and different relaxation times. From the NMR relaxation parameters, it is concluded that one type of the crystalline chains can execute at room temperature 180" flips with a frequency in the kilohertz domain. The other crystalline chains are more rigid and probably are found in more perfect structures in which such chain flips do not occur or occur on a much slower time scale.336 Structural heterogeneties in polymers such as poly(viny1 chloride), segmented copoly(ether-ester) block copolymer, and nitrile rubber were analyzed by CP/ MAS 13C NMR. The results from rotating and laboratory frame spinlattice(T1 and TI,) relaxation behaviour provided a clear insight into the structural h e t e r ~ g e n e i t y .The ~ ~ ~effect of different synthesis routes on the chemical and molecular order of polyimides based on 4,4'-diaminotriphenylmethane and various aromatic dianhydrides was studied by solid state 13C NMR.338The combination of 13C solution and 13Csolid state NMR(CP/MAS and MAS) is used to reveal the relative amount of rigid(crystal1ine) PVC in two PVCIDOP(50150 wt.% of poly(viny1 chloride)/di-2-ethyl-hexylphthalate) samples with tacticities, a,of 0.52 and 0.575, respectively.339Detailed structures of methacrolein dimer were analyzed by two dimensional NMR and theoretical calculations(MND0 and ab initio DFT c a l ~ u l a t i o n ) .13C ~ ~ CP/ MAS NMR was used to determine whether defect sites of a poly(D-lactide) that contained 3% L-lactide as a stereo-defect were incorporated into the crystalline region or forced into the amorphous region of the polymer.341The microstructures of poly(styrene-co-acry1amide)s of varying compounds, prepared by solution polymerization of styrene, were studied by 13C NMR.342 Rapid MAS at temperatures above T, is developed as a method to probe the length scale of mixing and to identify specific intermolecular interactions in copolymers, blends and interfaces.343Capitalizing on the superior sensitivity of proton NMR, relatively rapid estimations of three parameters, namely, comonomer content, crystalinity, and long spacing, were determined for three ethylenehinyl alcohol copolymers using solid state proton NMR measurem e n t ~ Conformations .~~ of the powder samples of poly(L-alanine) with molecular weights of 356 Da (tetraalanine), 15,000 Da (PLA-200), and 23,600 Da (PLA-333) were characterized by 13C CP/MAS and 'H CRAMPS solid state NMR spectroscopy.345The bulk microphase structure of polystyrene and poly(methy1 methacrylate) block copolymer was studied by T1('H) and T1p( 'H) measurements.346 The erosion of a 1,3-bis(p-carboxyphenoxy)propane-sebacic acid poly(anhydride) copolymer by water vapor has been studied by 'H wide-line and 13C MAS solid state NMR spectroscopy. T1('H), T1,('H)

'

11: Synthetic Macromolecules

433

and line width data indicated that the polymer matrix became more rigid on erosion due to the formation of crystal degradation products.347Poly[(acrylonitrile)-rotaxa-(60-crown-20)] consists of a linear polyacrylonitrile backbone threaded by 60-crown-20 macro~ycles.~~C solid state NMR spectra of the free 60-crown-20 collected at room temperature reveal peaks attributed to crystalline and amorphous regions.348A novel approach is proposed that allows the evaluation of distribution functions of structural parameters, such as dihedral angles and orientations of molecular segments, from suitable NMR spectra in disordered solids.349Solution and solid state 'H and 13C NMR are used to analyze the stereochemical sequence distribution of polyactide. The effect of sequence distribution on polymer properties is discussed.350The application of the cyclic J cross-polarization(CYCLCR0P) NMR imaging pulse sequence for the acquisition of proton-detected 3C NMR images of elastomeric materials is described.351 Ambient-temperature 13C line width and T2(13C) data are measured for the natural abundance crystalline carbons of linear polyethylene under CW proton decoupling conditions and MAS and discussed the line width b e h a v i o ~ rCP/MAS . ~ ~ ~ 13CNMR analyses have been made for different frozen solutions of poly(viny1 alcohol) samples with different t a ~ t i c i t i e s . ~ ~ ~ Solid state 13C NMR analyses of the structure and chain conformation have been carried out for a main-chain thermotropic liquid crystalline polyether which was polymerized with 4,4 dihydroxy-a-methylstilbene and 1,9d i b r ~ m o n o n a n e High . ~ ~ ~ salt concentration polymer composite electrolytes, containing poly(ethy1ene oxide), LiI and nanoscale A1203 or MgO and, in some cases, ethylene carbonates and poly(methy1 methacrylate), were investigate by electric conductivity, DCS and both wide-line and high-resolution solid state 7Li NMR.355 The structure of perdeuterated polyaniline (PANI) chains interleaved between metal oxide layers in (PANI)0.24Mo03 aligned on glass slides was studied as a function of the orientation of the oxide host lattice using 1D and 2D synchronized MAS 2H NMR.356Polydiphenylsiloxane has been characterized by 29Si solid state NMR spectroscopy.357A difunctional 1,3-benzoxazine compound, 2,2-di-(3-phenyl-4-dihydro-1,3,2-benzoxazine) propane, derived from bisphenol-A, 5N-enriched aniline and formaldehyde was synthesized and characterized using 15N NMR and 13C NMR spectros ~ o p i e s . The ~ ~ *polymer-silica interaction in elastomeric materials composed of 1,4-cis-polyisoprene rubber and silica was studied by 13C and 29Si MAS NMR.359 The phase behaviour of poly(didecylsil1ane) was investigated by DSC and 29Si as well as 2H solid state NMR.360 Multinuclear solid state proton NMR has been used to study the domain structure in poly(styrene-bisoprene-b-styrene) triblock copolymers in clear and self-assembled polymer films.361The structure of the polysiloxaneimides(PS1) copolymers were characterized by IR, 29Si and I3C NMR and the dynamics in the interface of PSI copolymers were investigated by proton spin-spin relaxation measurements. 362 The structural and chemical features of alkenyl trialkoxysilane and trialkoxysilyl terminated polyisoprene grafting onto silica microparticles were detected and identified by solid state 13C and 29Si NMR.363 Sheet and tube organosilicon polymers were characterized by X-ray diffraction, IR, XPS and solid

434

Nuclear Magnetic Resonance

state 29Si NMR.364 High-resolution solid state 19F NMR spectra using 525kHz MAS was used to provide detailed bond sequencing for fluorocarbon thin films.365High speed 19F MAS NMR and 13C MAS NMR with 19F to 13C cross polarization allows spectroscopic identification of monofluorinated and geminally difluorinated carbon species in poly(carbon monofluoride). 366 Microstructural characterization of vinylidene fluoride based fluoropolymers was carried out using variable-temperature(VT) solid state 19F NMR spectroscopy with high speed MAS.367VT solid state 19F NMR spectroscopy with high speed MAS has been used to study copolymers of vinylidene fluoride and hexafluoropropylene and of vinylidene fluoride and chlorotrifluoroethylene, and terpolymers of vinylidene fluoride, tetrafluoroethylene, and hexafluorop r ~ p y l e n e Solid . ~ ~ ~state 19F NMR spectroscopy using fast MAS and high power proton decoupling has been employed to investigate the semicrystallinity and polymorphism of poly(viny1idene fluoride).36919F { 'H} NMR at 188.29MHz, including 'H+ 19F{'H) CP with high speed MAS, was used to study some fluoropolymers of the Viton type.370129XeNMR is used to study sorption sites in high density polyethylene, poly(4-methyl-1-pentene), and N a f i ~ n The . ~ ~structure ~ of a thermoresponsive polymer-grafted, lipid-based complex fluid was characterized by P NMR spectroscopy.372Reorientation processes in cyano- and biphenyl-containing liquid crystalline polyacrylate induced by the static magnetic field of the spectrometer and a dynamic electric field between the capacitor plates were investigated by solid state 'H NMR.373 H spin diffusion and small-angle X-ray scattering measurements were carried out for a series of polystyrene-polyisoprene block copolymers to characterize the microdomain structure.374 The polyimides LaRC-TPI, LaRC-SI and related model compounds were investigated using 13Cand 'H NMR.375A 4,4diphenylmethane diisocyanate (4,4-MDI)-based poly(ester)-poly(urethane) has been synthesized as selectively 2H labeled at the MDI methylene. The 2H NMR spectrum showed that this elastomer consisted of a rigid and a mobile component over a wide temperature range, with the mobile component increasing with temperature.376 Counter anion condensation in solutions of linear polyethyleneimine and branched polyethyleneimine was studied by 35Cl and 37Cl NMR relaxation as a function of charge density of the polycation, which is controlled by the degree of protonation of the polybase determined by the pH of the solution.377Structure of hydrogen-bonded peptides and polypeptides were studied by solid state NMR.3787379 The self-diffusion coefficient and 'H spin-spin relaxation time of n-alkane, n-C24H5o were measured in the rotator and liquid phases over a wide range of temperatures by means of pulsefield-gradient spin echo 'H NMR, in order to clarify the molecular motion of the alkane in the rotator and liquid phases.380The detailed structures of the crystalline and liquid crystalline phases of PB- 18 polyester composed of 4,4dihydroxybiphenyl and octadecanedioic acid were studied by 13C VT solid state NMR spectro~copy.~~' VT high-resolution 'H NMR experiments on four polyurea samples with different kinds of hard segments and soft segments were carried out in DMF solution.382129XeNMR spectroscopy was used to study the free volume changes associated with crosslinking of linear polymers and

1 1 : Synthetic Macromolecules

435

extensive chain branching in dendritic tree molecules.383'H relaxation data, determined via either wide-line 'H NMR or high-resolution solid state I3C NMR, were used to analyze the existence and extent of interphasic resions in methyl methacrylate-Bu acrylate block copolymers.384The equilibrium length scale of dynamic heterogeneities close to the glass transition was measured by a new multidimentional NMR experiment. The spatial information is gained from a proton spin diffusion experiment combined with two 2D 13C exchange sequences via appropriate back and forth transfer of magnetization between 13C and 'H spins. The length scale of 3 & 1 nm was detected for poly(viny1 acetate) at 10 K above the glass transition.385 CP/MAS 13C NMR data were obtained of frozen solutions of poly(viny1 alcohol)(PVA) with different tacticity to study intramolecular hydrogen bonding of PVA.386 Several poly(ester imide)s based on 4-N-(carboxyphenyl)trimellitimide, 4-N-(carboxyethenylphenyl) trimellitimide, 4-hydroxy-N-(4-hydroxyphenyl)phthalimide and long aliphatic spacers have been investigated by different solid state NMR techniques.387The commercial poly(viny1 acetate) bulk was studied by NMR in solution and solid state to understand the behaviour of this polymer and to obtain information on structure, sample molecular mobility and h e t e r ~ g e n e i t y .High ~ ~ ~ speed MAS solid state NMR has been used to characterize the structural changes in fluoropolymers resulting from high-energy electrons irradiation.389The high-resolution solid state 3C NMR spectra of the a-modification, P-modification and amorphous poly(ethy1ene naphthalene-2,6-dicarboxylate) were measured by employing CP/MAS to characterize structure and properties.390lH-l 3C nuclear magnetic cross relaxation of P(VDF73/TrFE27)near the Curie temperature Tc was investigated using a CP/MAS NMR method.391 Conformations of the powder samples of poly(L-alanine) with molecular weights of 356 Da, 15,000 Da and 23,600 Da were characterized by 13C CP/MAS and 'H CRAMPS solid state NMR spectroscopy.392 The shell materials core coverages of two-phase core-shell particles have been determined quantitatively by NMR spin-diffusion experim e n t ~ The . ~ ~characterization ~ of carbosilane dendrimers was carried out by 'H/13C/29Si triple resonance, 3D, and PFG NMR techniques.394 The 2D 29Si-29SiINADEQUATE experiment provides connectivity information in systems whose skeleton is composed entirely of Si-Si bonds. The structure of the first generation dendritic polysilane[(Me3Si)2MeSiMe&SiMe was characterized by this method.395The gradation of polyisoprene and polybutadiene in the presence of chloranil was studied by 'H NMR spectroscopy.396A size exclusion chromatograph coupled to a 750MHz 'H NMR spectrometer was used to analyzed molecular-weight distributions of isotactic PMMAs with Mn ranging from 3.27 x lo3 to 2.22 x 104.397The chain conformation of glassy PET was characterized by two-dimensional double-quantum NMR.398 The online detection of emulsion polymerization processes by means of solid state NMR spectroscopy is demonstrated for the first time using poly(buty1 acrylate) as a model system.399A full 'H and 13C NMR study through the use of one- and two-dimensional techniques (HMQC, HMBC, TOCSY and NOE-difference spectroscopy) was carried out on poly[3-(6-methoxyhexyl)-

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436

Nuclear Magnetic Resonance

2,2-bithi0phenyl].~~~ The barrier energy of the left and right handed helical conformations of poly(2-butylhexyl isocyanate), about 19 kcal mol- l, was determined through carbon resonances in the 13CNMR spectra.401A series of 3D NMR pulse sequences, utilizing pulsed field gradient (PFG) techniques, were developed for applications in the characterization of polymer structure .402

5

Dynamics of the Synthetic Macromolecules

NMR is a technique that allows the probing of the dynamics of specific magnetically active nuclei. The occurrence and rate of 180" chain-flip motions in the crystalline regions of two polyethylenes were studied using dipolar I3C NMR.403 The dynamics of the polycarbodimide was studied by I3C spinlattice relaxation times in the rotating frame (Tlp('3C)).404Analysis of chain dynamics and short-range order for elastomeric solid polyisobutylene and its copolymers with p-methylstyrene were carried out by solid state NMR.405The dynamics of amorphous aromatic polyesters consisting of poly(ethy1ene terephthalate), poly(ethy1ene isophthalate) and poly(ethy1ene 2,6-naphthalenedicarboxylate) were studied by solid state CP/MAS 13C NMR. Proton T2,I3C TI, and proton T1, decays were measured in particular, and the experimental data fitted to suitable model functions to determine best relaxation param e t e r ~ Molecular .~~~ motion of oriented copolyester comprising 73 mol% hydroxybenzoic acid and 27 mol% hydroxynaphthoic acid was studied by T1,( 3C) m e s ~ r e m e n t s . Segmental ~~~ dynamics of poly(styrene-b-2-vinylpyridine) in bulk and at the surface/air interface were studied using 2H wideline NMR.409The segmental motion of entangled random coil polymers were studied by pulsed gradient spin echo NMR.408The influence of entanglements on the chain dynamics in bimodal polyethylene melts were investigated on a microscopic length scale by PFG NMR.410Mobility of polyethylene chains in the orthorhombic and hexagonal phases were investigated by 'H and *H NMR and determine the rate and type of motion of the polyethylene chains both in two phases4* The properties of the pseudo-solid echoes were analyzed in order to estimate the contribution of the segmental dynamics of the polymeric chain to the relaxation function of the transversal rnagnetizati~n.~'~ The segmental dynamics of solid state poly(methylphenylsi1ane) were probed with 2H solid echo and 2D exchange NMR via a methyl-d3 2H NMR spectra at near room temperature were obtained to investigate the molecular motions of three high molecular weight (MW > 20000) poly(ferrocenylsi1anes) selectively deuterated at either the cyclopentadienyl rings or the side groups of silicon.4142Hsolid state quadrupole-echo NMR was used to study the effect of molecular weight on the segmental dynamics of bulk and adsorbed poly(meth~lacrylate)-d3.~~~ 7Li NMR study for the dynamics in polyether-urethane based solid polymer electrolytes were reported using T1 and T2 relaxation times me~urements.~'~

11: Synthetic Macromolecules

6

437

Gels and Crosslinked Macromolecules

'H pulse NMR and high-resolution solid state I3C NMR spectra of poly(viny1 alcohol)(PVA) gel were measured to clarify the structure and dynamics of the immobile component of the gel.417The unsaturated polyesters prepared from maleic anhydride and 2,2-di(4-hydroxypropoxyphenyl)propane and crosslinked at different temperatures with various amounts of styrene were studied by means of high resolution 13C NMR in the solid state(CP/DD/MAS) and DSC. Experimental and theoretical values of the TI,('H) and T, were determined.418The interpenetrating network structure of poly(methylviny1 siloxane) and poly(dimethylsi1oxane) was studied using I3C CP and cross polarization polarization inversion (CPPI) MAS solid state NMR on thermally cured samples.419The molecular conformational structure of isotactic polypropylenelo-dichlorobenzene gel was investigated by means of high resolution solid state 13C NMR spectroscopy. It was revealed from the resonance line of methylene carbon at the position generating one g-gauche effect that the gelforming crystal takes a 3/1 helical molecular conformation.420The dynamics of crosslinked poly(N- isopropylacrylamide) gel were studied by pulsed-gradient spin echo (PGSE) lH NMR, pulse 'H NMR and 'H NMR imaging. The selfdiffusion coefficients of HDO in D20 in the gels with various degree of swellings were determined by the PGSE 'H NMR method.421 The spatial distribution of 'H spin density and T2('H) of water molecules in a crosslinked poly(methyacry1ic acid) gel under an a.c. electric field were measured by 'H NMR imaging method.422The self-diffusion of molecules in stearyl itaconamide/N,N-dimethylacrylamide copolymer gel was discussed by PGSE * H NMR.423 Structure and dynamics of hydropolymer gel systems have been elucidated microscopically and macroscopically by means of H NMR imaging method and PGSE 'H NMR method.424Self-diffusion behaviour of solvents in polypeptide gel were investigated by PGSE 'H NMR.425Structure and dynamics of poly(viny1 alcohol) in the gel state were studied by solid-state 13C NMR and 'H pulse NMR.426 The temperature dependences of the associated fraction of PVC units were determined from measurements of integrated intensities of high resolution H NMR spectra in thermoreversible gels of PVC and chemical modified PVC in di-Et oxalate (DEO) and di-Bu ~ x a l a t e ( D B O ) .'H ~ ~double-quantum ~ solid state NMR spectroscopy under fast MAS is introduced as a new spectroscopic tool for the investigation of the structure and local chain dynamics of elastomers.428

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7

Polymer Blend and Diffusion of the Synthetic Macromolecules

Solid state NMR is a particularly useful for investigation of miscibility of polymer blends. Miscibility and specific interactions in poly( P-hydroxybutyrate-co-P-hydroxyvalerate)and poly(p-vinylphenol) blends were investigated by DSC, FTIR and high resolution solid state 13CNMR techniques.429

438

Nuclear Magnetic Resonance

The miscibility of LLDPE/polyamide-6 blends were investigated by solid state H and 3C NMR.430The miscibility, intermolecular interaction and thermal behaviour of poly(hydroxy ether of bisphenol A)/poly(ethyl oxazoline) blends were studied by DSC, FTIR and high resolution solid state 13C NMR technique^.^^' VT solid state NMR methods were used to determine that amorphous polyethylene is miscible with blends of poly(ethy1ene-co-1-butene) and poly(ethy1ene-co- octadecene).432 High resolution solid state NMR were used to study the miscibility of blends: poly (epichlorohydrin)/poly(N-vinyl-2p y r r ~ l i d o n e ) , poly(styrene-co-acrylonitrile)/poly(styrene-co-maleic ~~~ anhydride),434 poly(hydroxyether of bisphenol A)/poly(N-vinyl p y r r o l i d ~ n e ) ? ~ ~ poly(epichlorohydrin)/poly(N-vinyl-2-pyrrolidone),436 novolac/poly(&-caprol a ~ t o n e )Solid . ~ ~ ~state NMR spin diffusion experiments and 2D 'H -13C NMR experiments were used to study the phase structure, mobility and domain size of erucamide/isotactic poly(propy1ene) blends.438 The morphology of an ionomeric polymer blend consisting of an amino-silicone copolymer and Zn neutralized sulfonated polystyrene was studied by 13C and 129XeNMR and SAXS.439The characterization of polyamide-6 and lithium sulfonated polystyrene isomers blend was carried out by 7Li static and MAS NMR.*O Selfdiffusion of oligo- and poly(ethy1ene glyco1)s in poly(viny1 alcohol) aqueous solution was measured by PGSE technique.41 Xenon self-diffusion coefficients of organic organic polymers have been determined by PGSE NMR.442Force Rayleigh scattering (FRS) and PGSE NMR have been used to measure the self-diffusion coefficient of a poly(ethy1ene oxide)-poly(ethy1ethylene) diblock copolymer in the molten state.443Pulsed field gradient stimulated echo NMR was applied to study the diffusion of toluene in porous semicrystalline polyethylene Pulsed field gradient (PFG) NMR and microimaging experiments were performed to study the diffusion of liquid alkanes into a variety of semicrystalline polyethylene samples.445Chain diffusion in polyethylene and n-alkane crystals was studied by using 13CT1 measurements.446

8

References 1

2 3 4 5 6 7

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12 Nuclear Magnetic Resonance Spectroscopy of Living Systems BY M. J. W. PRIOR

1

General Applications and New Methodologies

1.1 Applications - A review of the use of I9F NMR in biomedical research has been produced with 18 references.' The application of NMR to the study of biomedicine has been reviewed with 42 references.2 A review has been produced, with 17 references, on the principles and applications of EPR spectroscopy to measurements of p02, redox metabolism, pharmacokinetics and of free radicals3 An investigation of the methods of data acquisition and analysis involved in the absolute quantification of metabolites from in vivo NMR data has been carried out in a multicentre c~llaboration.~ Protocols for in vivo localised 'H studies of human brain,5 multicentre analysis of 'H NMR by a single method and tests on localised NMR methods7 have been presented in subsequent papers. Furthermore, a comparison of methods for the quantification of NMR lines in the presence of overlapping, unwanted broad background features has been investigated by the same multicentre collaboration.*

1.2 Spectral Editing, Spectra Analysis, Localisation and Instrumentation The use of two-dimensional spectroscopy for studies in vivo has been reviewed with 45 reference^.^ A review of fast chemical shift imaging techniques has been produced which discusses the criteria for evaluating and optimising the sensitivity of these techniques for use in human brain studies.'O Perfusion systems for use in NMR experiments have been reviewed with 23 references and a review, with 45 references, has been produced on the use of 31P, 13C, 'H, 23Naand 59C0NMR in the investigation of perfused organs. An alternative scheme for 3C chemical-shift imaging has been proposed. Inverse detection of protons through metabolite imaging of lines in a spectrum (MILS) was proposed and demonstrated in a phantom. Spatially localised two-dimensional spectroscopy has been developed for in vivo use.12 In this technique, the conventional STEAM pulse was implemented for a 2D NOESY experiment by incorporation of the increment time tl. A method for obtaining'H spectra in vivo without water suppression has been developed. The technique is based on F1 oversampled J-resolved spectroscopy which allows Nuclear Magnetic Resonance, Volume 29

0The Royal Society of Chemistry, 2000 453

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Nuclear Magnetic Resonance

the water peak to be a reference and enables the detection of metabolite signals without baseline artefacts. The technique was demonstrated in phantoms and in ~ i v 0 . The l ~ observation of a double quantum filtered (DQF) signal of water in bovine sciatic nerve, bovine articular cartilage, rat tail tendon and rat brain has been reported. l 4 A localised double-quantum filter for the in vivo detection of glucose has been developed. The technique used PRESS localisation to detect glucose resonances in the 3.85 ppm region of brain 'H spectra with a 45% efficiency. It was also possible to simultaneously detect lactate with a 20% efficiency in 'H spectra of rat brain during global ischaemia.15A lactate editing method for 'H NMR has been developed using PRESS excitation which allows the observation of choline containing compounds (Cho), total creatine (Cr), N-acetylaspartate (NAA) and lipid singlets. Two T2-independent J-difference lactate editing schemes were presented which exploit the dependence of the in-phase intensity of the Me doublet upon the time interval separating two inversion (BASING) pulses applied to its coupling partner after initial excitation. Spectra were obtained from phantoms, the head and neck of volunteers and, a primary brain and a metastatic neck tumour.16 A lactate and P-hydroxybutyrate editing sequence has been proposed which has a refocusing pulse train, consisting of a semi-selective refocusing pulse and a selective inversion pulse, that yields pure-phase refocusing at the frequency of maximal excitation of the semi-selective refocusing pulse. The method was used to obtain spectra from the occipital cortex of juvenile epilepsy patients before and after ketogenic diet treatment. l 7 A process which uses an artificial neural network (ANN) for the automated analysis of data from single voxel 'H NMR experiments has been developed. Water-suppressed * H NMR spectra from 24 healthy volunteers were analyzed for Cho, Cr and NAA content using the ANN method or a lineshape fitting protocol. The methods were shown to given the same accuracy but the ANN method was more easily automated.lg A description of an iterative method for differentiating between known resonances and uncharacterised baseline contributions in NMR spectra has been presented.19 An automated method for the analysis of in vivo 'H NMR data and the reconstruction of metabolite distributions from spectroscopic imaging data has been described.20A spectral simulation method has been developed that generates a priori information for use in parametric spectral analysis.21 An improvement in spectral resolution with increasing field strength has been reported for 'H NMR spectra obtained in vivo from human brain (1.5 and 4.7 T) and, from rat and dog brain (9.7 T). Improvements in detection allowed the observation of changes in glutamine levels in patients with hepatic encephalopathy at 4.7 T compared with 1.5 T. Furthermore, glutamine H4 at 9.7 T was clearly resolved from glutamate H4 as were the peaks from yaminobutyric acid (GABA) and NAA. There was, also, a partial resolution of the peaks for creatine and phosphocreatine at 9.7 T.22The spatial distribution of the radiofrequency field produced by a surface coil has been i n ~ e s t i g a t e d . ~ ~ An ESR spectrometer and imager capable of detecting and imaging free

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radicals in biological objects has been developed24 and a multi-modality magnetic resonance system which can detected free radicals by conventional RF CW-ESR, sequential LODESR and PEDRI techniques has been built.25

1.3 Intracellular Ions, Metabolites and pH - A review, with 23 references, of the application of 7Li NMR to measurements of Li+ in vivo has been produced.26 The advantages and disadvantages of the use of I9F NMR to measure cytosolic Ca2+in isolated cells, perfused hearts, isolated aorta and in vivo has been reviewed.27A review, with 29 references, has been produced on the role of Mg2+ in laboratory and clinical practice. The review includes measurement of the concentration of Mg2+([Mg2+])by NMR.28 The relative contributions to the DQF and triple-quantum-filtered (TQF) spectra of 23Na+ bound to anisotropic and isotropic binding sites in the intracellular (15 mM) and extracellular (150 mM) sodium pools of human erythrocytes have been quantified for different haemocrits. The contribution of extracellular Na+ to the TQF signal dominated the spectrum at all haemocrits studied, whilst the contribution of the extracellular Na+ to the DQF signal was smaller.29A combination of 31Pand 23NaNMR has been used to measure the concentration of intracellular Na+ ([Na'li) in the perfused mouse liver. The resonances of di-methylphosphonate and LaDOTP5- were used to determine the total tissue space and intracellular liver volume, respectively. The infusion of the shift reagent TmDOTP5- into the liver allowed the resolution of intracellular and extracellular 23Na NMR signals and facilitated [Na+]i measurement^.^' 31P and 23Na NMR, with TmDOTP5-, has been used to measure [Na+]jand the volume of the extracellular space in perfused rat liver.31 The distribution of Li+ in the red blood cells and plasma of rats has been investigated with 7Li NMR using a shift reagent. At the lower dose (0.5 meq kg-') intracellular Li+ was dominant but as the dose was increased to the upper limit (10 meq kg-'), significantly larger amounts of Li+ accumulated in the plasma. The results suggest that 7Li NMR provides a reliable measurement of Li+ in red blood cells and plasma. The effects of the coadministration of drugs, such as thioridazine, on the intracellular and extracellular distribution of Li+ was also i n ~ e s t i g a t e d . ~ ~ A new analysis, based on the Wigner distribution method, for the determination of the concentration of intracellular Mg2+ ([Mg2+]i)from 31Pspectra has been proposed.33 The level of Mg2+ in skeletal muscle (gastrocnemius) and brain of normal volunteers has been assessed by 31PNMR. The range found in 22 - 46 year old muscle was 504 k 30 mM, whereas the level in brain was 369 k 51 mM. These levels showed no correlation with Mg2+levels assayed in plasma or erythrocyte^.^^ The effects of 500 mg of MgO day- for three weeks on the performance, clinical symptoms and Mg2+ content in various body compartments has been investigated in athletes. Supplementation did not affect exercise performance, neuromuscular activity, muscle related symptoms or the [Mg2'] in serum or any body compartment. It was also found that the [Mg2+]in the serum correlated with the [Mg2+]in erythrocytes and, the [Mg2+]in leukocytes correlated with the [Mg2+]in muscle when measured with 31PNMR.35

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A comparison has been made between the measurement of Mg2+ by 31PNMR and its assessment by ion-selective electrodes in serum, plasma and whole blood from healthy volunteers or patients with one of a number of disease states.36 The transport and [Mg2+]; has been investigated in the erythrocytes from 14 species. The total content of Mg2+ from rat, pig and cattle varied from 0.5 to 4.7 mM but the level of free Mg2+ measured by 31P NMR was nearly the same. In Mg2+-loadederythrocytes, the efflux of Mg2+ via the Na+/Mg2+antiport and the Na+-independent channel were highest in rodent and lowest in ruminant erythrocytes. The activity of the Na+/Mg2+ antiport was not related to the Mg2+ content of any erythrocytes but its activity was found to be depended on the K+ content and K+/Na+quotient in non-nucleated erythrocyte^.^^ The methods for the observation of individual metabolites in spectra, particularly from the brain, have been reviewed with 83 reference^.^^ The use of 13C NMR for the determination of metabolic activity in relation to somatosensory activation and neurotransmitter recycling has been reviewed with 20 reference^.^^ The use of neural networks for the automatic analysis of in vivo spectra has been reviewed with 27 reference^.^^ A review on the metabolism and role of NAA has been produced with 34 reference^.^^ A TCA cycle enzyme, citrate synthase 1, in Saccharomyces cerevisiae has been labelled by the incorporation of 5-fluorotryptophan and investigated with I9F NMR. A comparison of the 19F-signalsfrom the intact cell and cell-free lysates indicated that the enzyme was motionally restricted in the cell. The results are consistent with citrate synthase 1 being in a multienzyme complex in the cell.42 The effects of the interaction of lactate with serum albumin have been investigated with 'H NMR. In a test solution of lactate and alanine, containing 0.2 mM bovine serum albumin (BSA), there was a linear relationship between NMR visible lactate and the total lactate concentration over a range of 0.2 to 35 mM. However, 60% of the lactate was not visible to 'H NMR and the detected level of alanine remained unaffected. Furthermore, in a 0.1 mM solution of BSA only 25 to 30% of lactate was visible to 'H NMR.43 The serial precision of measurements of peak area ratios and water referenced metabolite peak areas in 'H spectra of the human brain has been investigated. Data were collected from the occipital lobe of normal volunteers using long echo time PRESS with a 2 s repetition time and a 136 ms echo time. Short-term in vitro precision was measured in a phantom and was determined by five repeated scans on five occasions. The mean coefficients of variance were NAA/Cho = 1.3%, NAA/Cr = 1.0%, Cho/Cr = 1.6%, NANH20 = 0.5%, Cho/ H 2 0 = 1.2%, Cr/H20 = 0.8%. Long term in vitro precision using 100 spectra acquired over 2 years was also very good; NANCho = 2.7%, NAA/Cr = 1.4%, Cho/Cr = 2.2%, NAA/H20 = 1.5%, Cho/H20 = 2.4%, Cr/H20 = 1.5%. Shortterm in vivo precision, determined by five repeated scans in a single scanning session on eight subjects, gave NAA/Cho = 5.2%, NAA/Cr = 3.0%, Cho/Cr = 6.6'/0, NAA/H2O = 1.4%, Cho/H20 = 4.9%, Cr/H20 = 2.7%. The long-term in vivo precision, measured by five repeated scans on eight subjects over 3

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months, was slightly worse with NAA/Cho = 5.2%, NAA/Cr = 4.8%, Cho/Cr = 7.7%, NAA/H20 = 2.5%, Cho/H20 = 6.4%, Cr/H20 = 3.8%. The high precision of the data was attributed to automatic techniques for voxel shimming, water suppression and peak area measurement^.^^ Measurements by I80-exchange analyses of net phosphoryl transfer by adenylate kinase and creatine kinase (CK), in conjunction with 31P NMR analyses of total unidirectional phosphoryl flux, have been used to investigate cellular bioenergetics in intact cells.45 31PNMR has been used to assess the oxidative function of heart and liver mitochondria which have been immobilised in agarose beads. In experiments using pyruvate plus malate as substrates, clear resolution of y-ATP, P-ADP and inorganic phosphate (Pi) signals were detected. The production of ATP was sensitive to hypoxia, ischaemia, carbonyl cyanide and p-(trifluoromethoxy)phenylhydrazone and was also depended on the ADP concentration in the media.46The forward and reverse fluxes through the CK reaction have been determined in isolated rat and bovine heart mitochondria using saturation transfer 31PNMR.47 A method for the quantitation of metabolites in the human brain has been developed. A 'H NMR method (i.e. PRESS localisation with a frequency selective water suppression) was used to detect NAA, Cr and Cho in a head shaped phantom to assess scanner performance and calibration accuracy. Comparison of these results with in vivo data suggested that reproducibility mainly depends on brain metabolite homogeneity and errors in localisation volumes.48The differences in the metabolites concentrations of grey and white matter detected in the human brain by 31P NMR have been assessed. Grey matter was found to have higher ratios of phosphocreatine (PCr) to ATP and PCr to total 31Psignal.49 A novel method for the determination of intracellular and extracellular muscle glucose concentrations in vivo has been developed using13C NMR in the rat. The method uses [l-13C]glucoseand [1-13C] mannitol (as an internal standard) to calculate glucose concentration after correction for intracellular and extracellular volumes.50 An investigation has been carried out into the discrepancies in reported values of human liver metabolite and pH values measured by 31PNMR. To assess the role of data processing three methods of data processing were applied to localised 31Pchemical shift imaging data from human liver. The three analysis methods were a manual method to define peak areas and, frequency domain curve fitting assuming Lorentzian or Gaussian line shapes. There were large differences in the metabolite peak areas but no differences in the pH values suggesting that integration methods can only account for differences in metabolite concentrations found in the literat~re.~~ 31P NMR has been used to investigate the intracellular and extracellular compartments of the brain Pi peak. Previously, it has been proposed that there are six Pi compartments in the brain which could be investigated by deconvolution of the Pi peak; one of these compartments was identified by the infusion of Pi into the blood. The infusion of 2-deoxyglucose-6-phosphate into the interstitial space by microdialysis resulted in a peak which could be deconvo-

458

Nuclear Magnetic Resonance

luted into 3 constituents. One of these peaks indicated a pH similar to that from previous results using the infusion of Pi. When neurone cellular energy metabolism was stressed, by induction of seizures following treatment with flurothyl, an increase in Pi was only seen in the compartment associated with a pH of 6.95 k 0.01 units. The infusion of ammonium acetate, which puts a metabolic load on astrocytes, resulted in an increase in Pi in the compartments at pH 7.05 k 0.01 and 7.15 k 0.02. It was proposed that the brain contains four intracellular and four extracellular compartment^.^^ The measurement of pHi of the brain, predominantly using3*PNMR, has been reviewed with many reference^.^^ A review of the use of NMR in monitoring cerebral pH and metabolism during systemic and focal acid-base disturbances has been produced with many references.54 The use of 6-fluoropyridoxol as an indicator of pHi and extracellular pH (pH,) has been assessed in the perfused rat heart. Following the infusion of 6fluoropyridoxolin into hearts,19F NMR detected two well resolved peaks indicating an PHi of 7.14 It 0.01 and a pHe of 7.52 k 0.02 on the basis of the chemical shift of 6-fluoropyridoxol relative to the external standard of sodium trifluoroacetate. During metabolic alkalosis, pHi remained relatively constant but respiratory challenge caused a significant increase in pHi. These measurements of pH were confirmed by measurements made with a pH electrode and 31PNMR.55 The use of histidine as a probe of PHi has been investigated in perfused canine and rat glioma cells subjected to ischaemia or to low pHe. The chemical shift of the histidine H2 proton, in cells incubated with 10 mM histidine, reported a pHi consistent with that obtained from the shift of Pi measured with 31PNMR. Measurements of pHi from histidine required less than 1 s of acquisition time.56The measurement of pHi in 'H NMR spectra of perfused cells and cat brain has been achieved using a new pulse sequence. This sequence avoids magnetisation transfer signal loses due to water saturation for solvent suppression and permits the observation of signals between 5 and 10 ppm from N-bound protons and aromatic C-H protons.57 The chemical shift of the peaks of ATP in the post-ischaemic rat heart has been used to estimate pHia5' A method for the determination of pHe and pHi from 31PNMR data has been developed. A microdialysis probe inserted into the rat brain allowed the introduction of 2-deoxyglucose-6-phosphateinto the extracellular space; pH, and pHi were determined from the chemical shift of 2-deoxyglucose-6-phosphateand Pi, respectively, relative to the shift of PCr. When rats were subjected to hypercapnia or hypocapnia, it was observed that pHe responded inversely to arterial Pco2 in a manner which could be described by the Henderson-Hasselbach equation indicating that the bicarbonate buffer system is dominant in the interstitial space. The response of pHi to arterial Pc02 was muted compared to the response of pH,. In ischaemic brain, induced by cardiac arrest, pHe decreased more rapidly in the first 5 min but there was no difference in pHe and pHi after 12 min.59

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459

Cells

2.1 Reviews and General Methods - The application of NMR and molecular genetics to the study of the control and organisation of cellular metabolism in mammalian and yeast cells in vivo has been reviewed.60 A review of the methods of isolated cell perfusion in NMR studies has been produced with 30 references.61 A method for the determination of intracellular water volume using from 35Cland 2H NMR measurements in cell free supernatant and cell suspensions has been presented.62 2.2 Bacteria - A review of the 13C-flux ratio analysis of central carbon metabolism in Escherichia coli microaerobic bioprocesses has been produced with many references.63 13C NMR has been used to monitor the synthesis of polymers in Rhizobium meliloti M5N 1 strain. Differences in polymer biosynthesis were observed as a function of the metabolic state of cells and the labelled carbon source used. In stationary phase cells polyhydroxybutarate synthesis was higher and gluconate synthesis was lower compared to that in cells in mid exponential phase, though, carbon source consumption and glycogen synthesis was complete. Cells grown with [l-13C]fructoseresulted in more label in exopolysaccharide and polyhydroxybutarate but less in gluconate compared to cells grown with [1-13C]glucose.64In a study of the metabolism of [' 3C]dichloromethane by Dehalobacterium formicoaceticum, 3C NMR of cell suspensions has detected the production of 3C-label in formate, the methyl group of acetate and minor amounts in methanol but no label in the carboxyl group of acetate. The results suggested that dichloromethane was converted to methylene tetrahydrofolate, of which two-thirds was oxidised to formate and one-third was used to produce acetate by incorporation of C 0 2 from the media.65 13C-edited 'H NMR has been used to assess the effects of osmotic stress on Methanococcus thermolithotrophicus. NMR was used to monitor substrate utilisation (sodium formate), methane production and, osmolyte synthesis and turnover under different conditions. Formate metabolism was slower in those cells grown in higher NaCl concentrations reflecting a slower growth rate; however, osmotic shock increased the rate of formate utilisation. Methane production varied little with NaCl concentration but larger changes were observed during osmotic shock. In the absence of osmotic stress, the turnover rates for the cellular osmolytes, a-glutamate, P-glutamate and NE-acetyl-P-lysineincreased with external NaCl concentration. Under hyperosmotic stress, there was net synthesis of a-glutamate but less P-glutamate and little N"-acetyl-P-lysine formation; in cells that were adapted to high NaCl concentrations the major osmolyte was N"-acetyllysine. In hypoosmotic shock, the turnover of P-glutamate and N"-acetyl-Plysine was selectively enhanced.66 The metabolism of [2,4-I3C]citrate has been investigated in Propionibacterium freudenreichii subspecies shermanii using' 3C NMR. In resting cells, the production of glutamate labelled in positions 2

460

Nuclear Magnetic Resonance

and 4 was observed; this labelling was faster in the presence of lactate or pyruva te .67 I5N NMR has been used to investigate the assimilation of NH4+ by various strains of Corynebacterium glutamicum in continuous culture. In conditions of excess NH4+ 30% of assimilated nitrogen was incorporated into glutamine by glutamine synthetase and 70% was assimilated by glutamate dehydrogenase. The assimilation of NH4+ by the glutamate dehydrogenase mutant of C. glutamicum was exclusively via glutamine synthetase with 30% of NH4+ being used for biomass synthesis. The lysine producing strain of C. glutamicum (MH20-22B)had reduced intracellular pools of the primary NH4+ assimilation products; glutamate and g l ~ t a m i n e . ~ ~ ~ ~ ~ The energy metabolism of E. coli in response to treatment with nalidixic acid, a topoisomerase I1 inhibitor, has been investigated with 31P and I9F NMR. The content of nucleoside triphosphates (NTP) in E. coli cells embedded in agarose threads was found to be 2.7 k 0.7 mM and was increased twofold by treatment with nalidixic acid. Removal of nalidixic acid after 40 min resulted in a decrease of NTP to control levels and allowed cell proliferation to resume. However, in E. coli ArecA cells, which are more sensitive to nalidixic acid, the elevation of NTP was not reversed.70 31PNMR has been used to investigate polyphosphate in the cyanobacterium Synechocystis sp. strain PCC 6308. Polyphosphate was detected after growth in sulfur-limited media containing excess phosphate. When the cells were treated with EDTA part of the broad polyphosphate peak appeared to shift downfield and become more narrow. The addition of Mg2+ resulted in the shifted polyphosphate peak to move back towards its original value. The results suggest that there are two pools of polyphosphate only one of which is accessible to EDTA.71 The uptake of 133Cs into Halomonas israelensis has been investigated ~ s i n g ' ~ ~NMR C s without a shift reagent. The chemical shift difference between 133Csin the extracellular and intracellular spaces was dependent on the salt concentration in the media. Cells grown on 0.2 to 4 M salt were transferred to media containing 25 mM CsCl and no potassium where the uptake of 133Cswas monitored.72 2.3 Blood - The application of 13CNMR to the study of l3C-labe1led glucose metabolism in erythrocytes has been reviewed with 3 reference^.^^ A review of the methods of 'H NMR and spectral analysis applied to the study of erythrocytes has been produced with 6 reference^.^^ The transport and redox cycling of ascorbic acid has been investigated in human erythrocytes using13C NMR. [1-13C]ascorbicacid and [2-13C]ascorbic acid displayed peak splittings indicating signals were from intracellular and extracellular environments. The membrane permeability of ascorbic acid, and for the reduction of dehydroascorbic acid were the apparent K, and V,, determined. Evidence was found for a dehydroascorbic acid transporter which is different to the glucose transporter that is also involved in the reduction of dehydroascorbic acid. Furthermore, a second reduction process appeared to

12: Nuclear Magnetic Resonance Spectroscopy of Living Systems

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take place e~tracellularly.~~ The transport of lysine and glutamate in erythrocytes from the Common Bushtail Possum (DactyZopsiZa trivirgata), the Eastern Gray Kangaroo (Macropus giganteus) and the Tammar Wallaby (Macropus eugenii) has been investigated with 'H NMR. The Tammar Wallaby has high levels of lysine in its erythrocytes and was found to have a non-saturable lysine transport system; the contrary was found in the other two species. The uptake of glutamate was found to be similar, and normal, in all species studied.76 The dependence of the pentose phosphate pathway (PPP), the glycolytic pathway and adenine nucleotide synthesis on [Mg2+Iiin the human erythrocyte has been investigated with 31PNMR.77The [Mg2'Ii at half maximal velocity of the PPP was 0.02 mM and flux through this pathway was reduced at higher values of [Mg2']; as flux through phosphofructokinase was increased; the [Mg2']i at half maximal velocity of phosphofructokinase was 0.16 mM. The [Mg2']i at half maximal velocity of phosphoglycerate kinase, which equals the ADP phosphorylation rate, was 0.27 mM which is within the physiological range. It was concluded that, although the flux through the PPP and glycolytic pathways were not dependent on [Mg2'Ii, the rate of ADP phosphorylation and the rate of adenosine nucleotide synthesis would be regulated by normal fluctuations in [Mg2']i. The response of Mg2+i to insulin and insulin-like growth factor I (IGF-I) in erythrocytes of patients with insulin resistance in essential hypertension has been assessed with 31P NMR. In normotensive subjects, incubation of erythrocytes with insulin, or IGF-I, resulted in timeand dose-dependent increases in Mg2+i.In hypertensive subjects, this response to insulin was blunted. The response to insulin in erythrocytes from all subjects was directly related to basal Mg2+i levels. However, when erythrocytes from hypertensive subjects were pre-incubated with IGF-I the response to insulin was no longer blunted.78 The temperature dependencies of the distribution of non-electrolytes between the intracellular and extracellular environment of human red blood cells has been investigated with 'H NMR.79 'H NMR has been used to investigate the role of glutathione and its interaction with membrane thiols in human erythrocytes.*O The effect of alternating weak electric fields on [Na+]iin human red blood cells has been investigated using 23Na NMR and a shift reagent. * * 2.4 Mammalian - Gradient, high resolution, magic angle spinning (MAS) 'H NMR has been used to record spectra from differentiated and undifferentiated NIH 3T3 F442A cells.82 The technique enabled clearer resolution of spectra and the use of diffusion weighting allowed compartment assignment and the determination of metabolite mobilities, Smaller, undifferentiated cells showed no evidence of lysis and remained viable after 2 h of MAS. However, 10-15% of the larger, lipid laden differentiated cells suffered lysis during MAS. The effects of infection with CZamidia psittaci has been investigated with 31P and 13C NMR in HeLa cells. Infection stimulated ATP production and glutamate accumulation with peak levels appearing at the mid point of the 2 day infection cycle. Furthermore, there was an increase in glucose consump-

462

Nuclear Magnetic Resonance

tion and lactate production, as well as an increase in glycogen a c c ~ m u l a t i o n . ~ ~ 'H NMR has been used in an investigation of the effects of eupatilin on Helicobacter pylori-induced leukotriene D4 in human neutrophils and gastric mucosal cells.84 Changes in the high energy phosphates (HEP) and pH of MOLT-4 cells in response to treatment with interferon-y have been recorded with 31PNMR.85 31PNMR has also been used to compare the response of immortalised human microglial cells (CHME-5) with the response of the monocyte cell line U937 to heat shock . A high level of PCr was observed in CHME-5 cells but no PCr was detected in the monocytes. Large, transient perturbations of the 31P spectra of CHME-5 cells was observed after heat treatment.86 A method has been described for the production of cultured primary rat neuronal cells immobilised in gel threads for use in NMR studies. After four days of incubation a population of morphologically differentiated neuronal cells with less than 5% astrocytes cells was p r ~ d u c e d . ~ NMR lP was used to measure changes in energy status and 13C NMR was used to monitor the metabolism of [ 1 3C]glucose to lactate, alanine, glutamate and aspartate for up to 24 hours.87

-'

2.5 Plant - 31PNMR has been used to investigate the metabolic changes in suspension culture of Catharanthus roseus upon elicitation by a yeast glucan preparation. Elicitation caused the induction of phenylalanine ammoniumlyase and the immediate reduction of phosphate uptake in oxygenated cells. Elicitation also caused a twofold increase in ATP but did not affect levels of Pi, phosphomonoesters (PME), UDP glucose and cytoplasmic pH.88 The response of cultured rice cells on exposure to the elicitor, N-acetylchitoheptaose, or exposure to propionic acid has been investigated with 31PNMR and other methods.89 The effects of y-irradiation on the uptake of Pi has been investigated in cultured spinach cells using3'P NMR. In non-irradiated cells Pi was taken up from the medium and transported into the vacuole. In the absence of extracellular Pi, the level of cytosolic Pi was maintained at the expense of the vacuolar store. When cells were irradiated with up to 5 kGy the uptake of Pi was unaffected; however, the movement of Pi into the vacuole was markedly affected with increasing doses of i r r a d i a t i ~ n . ~ ~ The transport, compartmentalisation and metabolism of homoserine has been characterised by I3C NMR in sycamore (Acer pseudoplantanus) and Echinochloa colonum plant cells. Homoserine entered sycamore cells at the maximum rate of 8 k 0.05 mmol h- g-l cell wet weight in competition with serine and threonine and was accumulated in the cytoplasm at a 4- to 5-fold greater concentration than in the vacuole. When sycamore cells were incubated with 100 pM homoserine 31PNMR detected the accumulation of phosphohomoserine in the cytoplasm at the rate of 0.7 pmol h-' g-' cell wet weight. The rate of metabolism of phosphohomoserine was 0.06 mmol h-' g-' cell wet weight and sustained threonine production. Homoserine was also accumulated by E. colonum plant cells, though intracellular homoserine levels were low, no phosphohomoserine was seen and large quantities of threonine were

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detected.91 IH and 13C NMR have been used to identify the metabolic solutes in one normal and two habituated sugar beet (Beta vulgaris L. altissima) cell lines obtained from the same mother strain. Spectra were recorded from living cell suspensions and perchloric acid extracts.92 The polyamine and alkaloid metabolism of transformed root cultures of Datura stramonium has been investigated with 15N NMR. After resuspension of cultures in spent growth medium the pools of [15N]glutamate and [15N]glutamine were depleted and there was an increase in 15N NMR signals from conjugated putrescines and hyoscyamine. The signal from conjugated putrescines passed through a maximum two days after the roots were resuspended. The transient accumulation of [ 5N]hydroxycinnamoylputrescineswas reduced when the de-differentiation of root cultures, to suspension cultures, was initiated by exposure to a-naphthaleneacetic acid and kinetin. Root cultures grown in the presence of inhibitors of putrescine biosynthesis (DL-a-difluoromethylarginine and DL-a-difluoromethylornithine) showed a marked reduction of conjugated polyamines and, hyoscyamine and DL-a-difluoromethylarginine also prevented the phytohormone-induced de-differentiation of root cultures.93 2.6 Reproductive - 'H NMR has been used to examine seminal fluid from men with spermatogenic failure and obstructive azoospermia. Four main metabolites were identified in seminal fluid; glycerophosphocholine (GPC), Cho, citrate and lactate. The ratios of Cho/citrate and Cho/lactate were significantly different between those with spermatogenic failure and obstructive azoospermia. When both groups had normal blood levels of follicle stimulating hormone there was a significant difference in the ratio of GPC/Cho between those with spermatogenic failure and obstructive azoospermia, and between different cases of spermatogenic failure.94The dynamic biochemical processes of incubated human seminal fluid samples have been investigated with 'H NMR. A variety of biochemical changes were observed including the enzymatic hydrolysis of phosphorylcholine to choline, the hydrolysis of polypeptides to amino acids and the conversion of uridine-5'-monophosphate to ~ r i d i n e . ~ ~

2.7 Tumour - A review of the application of NMR in biochemical studies of drug-resistant and drug-sensitive tumour cells has been produced with 79

reference^.^^ Two-dimensional purged correlation spectroscopy has been used to characterise immortalised mouse fibroblasts (NIH3T3) and two fibrosarcoma cell lines (WEHI-1 64 and MFS 8). Differences in triglycerides, cellular metabolites and cell surface fucosylation were observed.97 The effects of tumour necrosis factor (TNF) on the cellular metabolism of MCF7 cells has been investigated with 31P,13C and 2H NMR. In MCF7 cells, TNF induced cell cycle arrest and apoptosis. Immediately after TNF administration, 2H NMR detected the inhibition of [2H]choline uptake by 52% and, after 15 h, the activity of phosphocho1ine:cytidine triphosphate cytidyltransferase was enhanced twofold. During these changes, which led to a decrease in phosphocholine, the level of NTP and the rate of glycolytic flux

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Nuclear Magnetic Resonance

remained constant.98 31P and ‘H NMR has been used to investigate drugsensitive and, adriamycin- and mitoxantrone-resistant P388 murine leukaemic cells. Resistant cells had greater levels of HEP and higher levels of lactate. Metabolite levels in resistant cells were unaltered by drug treatment, whereas, in resistant cells there was a near total loss of HEP and a depletion phospholipid metab01ite.s.~~ The effects of taxol on the energy status of human oral cavity epithelioma KB cells and human HCT-8 cells have been investigated with 31P NMR. Perfusion of cells with taxol caused enhanced ATP peaks to be detected in the HCT-8 and, to a greater extent, in the KB cells. The increase in ATP reflected the level cytotoxicity of taxol towards each cell line. However, this result was not found when the cells were exposed to the same concentration of vincristine.loo The effects of glucose starvation on the uptake and metabolism of [ 5N]glutamate has been investigated in hybridoma and Sp2/0-Ag14 myeloma cells using H-detected 15NNMR. 7Li NMR, in combination with the shift reagent thulium- 1,4,7,1O-tetrazacyclododecane-N,N’,N”,N’”-tetramethylene phosphate (HTmDOTP4-), has been used to investigate Li+ in human neuroblastoma SH-SY5Y cells. The first order rate constant for Li+ influx and efflux in immobilised cells was 0.055 0.006 min-l and -0.025 5 0.006 min-l, respectively. The presence of Na+mediated Li+ transport was indicated by an increased influx and efflux in the presence of 0.05 mM veratridine. Measurements of relaxation parameters indicated that Li+ is immobilized to a greater extent in SH-SY5Y cells compared to Li+ in human erythrocytes.lo2

’

_+

2.8 Yeast and Fungi - 31PNMR has been applied to the measurement of pHi in brewing yeast and may provide useful information prior to fermentation.lo3 The phosphate trafficking and polyphosphate dynamics in wild type Saccharomyces cerevisiae and the vphl-1 mutant, which lacks polyphosphate, has been investigated with 31PNMR. Starved wild type yeast were shown to exclusively direct phosphate to the vacuole. Treatment dinitrophenol (DNP) or iodoacetate (IAA) affected cytosolic pH (pH,) and ATP but did not cause the metabolism of polyphosphate. However, alkalinization caused the rapid hydrolysis of polyphosphate to small polymers which then degrade to orthophosphate. Furthermore, this increase in Pi coincided with an increase in sugar phosphates and reacidification. When vphl-1 mutants were subjected to alkalinization the recovery of pH, was s10wer.l~~ The uptake of 133Cs into the‘ mycelium of the mushroom (Pleurotus nstreatus) has been investigated with 133CsNMR. Spectra obtained from the mycelium contained two resonances, of which the down-field shift peak probably reflected intracellular 33Csaccumulation.lo5

’

3

Plants and Algae

NMR studies of nutrient transport and metabolism during the life cycle of arbuscular mycorrhizae has been reviewed with 66 references.lo6

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31P NMR has been used to examine wheat, soya bean and mustard seeds during ripening. Signals were detected from PME, cytoplasmic and vacuolar Pi, phytate, NTP and nucleoside diphosphosugars. A broadening of signals was observed with maturation of the seeds.'07 The unidirectional steady state rates of several enzymes have been measured in hypoxic maize root tips using two-dimensional 31PNMR exchange spectroscopy. In a single spectrum ATP synthesis and hydrolysis was monitored along with the activities of UDPglucose phosphorylase, phosphoglucomutase, hexose-phosphate isomerase and enolase. When hypoxia was increased, by increasing the temperature, an increase in glycolysis was measured whilst the net flux through phosphoglucomutase and UDP-glucose phosphorylase toward carbohydrate synthesis was monitored.108 The level of Pi in the cytoplasm and vacuole of root tips and subapical root segments of pond pine (Pinus serotina Michx.) has been measured with 31PNMR. In root tips and seedlings grown in 100 mM Pi the cytoplasmic Pi content was 1.5 pmol cmP3 and that in the vacuole was 3.4 pmol ~ m - ~ In . Pi starved root tips vacuolar Pi could not be estimated and cytoplasmic Pi was 0.75 pmol cmW3.Similar results were obtained in subapical root segments.lo9 The early effects of glucose starvation on excised root tips of Zea Mays has been studied with 'H and 13C NMR. Root tips were pre-labelled with [ 1-* 3C]glucosesuch that carbohydrates and metabolic intermediates were close to steady state labelling but proteins and lipids were scarcely labelled. When glucose was removed phosphoenolpyruvate carboxylase flux stopped quickly and the contribution of glycolysis as the source of acetyl CoA for respiration decreased progressively. The enrichment of glutamate indicated that catabolism of proteins was delayed compared to that of lipids.110 The technique of cyclic J cross polarisation (CYCLCROP) which allows the detection of 13C-labelled metabolites with the sensitivity of 'H NMR has been used to study the carbohydrate metabolism in 6 day old castor bean (Ricinus communis) seedlings. The acquisition strategies and analysis of the effects of relaxation on CYCLCROP spectra have been discussed. The uptake of hexoses and their conversion to sucrose, and the transport of sucrose in the vascular bundles have been measured. The sucrose concentration in the phloem of individual bundles of the hypocotyl of R. communis seedlings has been measured with H NMR spectroscopic imaging. Sucrose concentrations were stable if the cotyledons were embedded in the endosperm or excised and incubated in 100 mM sucrose. When cotyledons were incubated in sucrose free medium the sucrose concentration in the phloem decreased steadily in some cases and oscillated in others. The oscillations were larger in the phloem at the apex of the hypocotyl compared to that in the base. Cutting the petiole of one cotyledon decreased the sucrose concentration in all bundles of the hypocotyl. Cutting the petiole and dividing the vascular ring at the cotyledonary node and at the root crown did not prevent this decrease. The results indicate that an equilibrium of translocated solutes between the eight bundles may occur by radial diffusion through the parenchyma of the hypocotyl. l 2 I3C NMR has been used to investigate the metabolism of [l-13C]glucosein

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Nuclear Magnetic Resonance

seedlings of Eucalyptus globulus, the fungus Pisolithus tinctorius and in their ectomycorrhizae. In uninoculated seedlings the label was mainly incorporated into sucrose and glutamine; the ratio of (C3+C2)/C4 of glutamine was nearly 1.O during the measured time course indicating equivalent contributions from phosphoenolpyruvate carboxylase and pyruvate dehydrogenase to the production of a-ketoglutarate. In free living P. tinctorius, most of the label was incorporated into mannitol, trehalose, glutamine and alanine whereas arabitol, erythritol and glutamate were weakly labelled. Furthermore, amino acid synthesis accounted for 43% of assimilated 13C-labeland, 42% of flux entering the TCA cycle was from anaplerotic CO;?fixation. In ectomycorrhizae, sucrose accumulation was reduced compared to uninoculated seedlings and 13C-label incorporation into arabitol and erythritol was nearly fourfold greater than in the free living fungus. The effects of electric fields on the physical state of cell-associated water in germinating morning glory seeds has been investigated with 'H NMR.' l4 In untreated, germinating seeds three water fractions were detected which had different TI relaxation times and chemical shifts. Treated and untreated cells were found to have the same water content but treatment with electric fields increased the fraction of water with the shortest T1 and decreased the fraction with the longest TI. The major, low molecular weight metabolites of the red alga Gracilariopsis lemaneiformis have been detected using high resolution MAS NMR. Floridoside was the major heteroside but digeneaside and isofloridoside were also detected. Other major components were isethionic acid, taurine and citrulline. The results were confirmed in cell extracts.'

'

4

Tissue Studies

The applications of NMR methods to the study of animal models of human disease have been reviewed with many references.

'

4.1 Brain and Spinal Cord - A review, with many references, of the application of 'H NMR to the study of cerebral ischaemia in the rat brain has been produced. The T2 relaxation times of brain metabolites have been assessed by 'H NMR before and after 20 min of global ischaemia in the rat brain. There were no changes observed in the T2 values of metabolites during occlusion and early reperfusion periods which indicates that T2 relaxation does not confound diffusion-weighted long-TE 'H NMR assessment of early brain ischaernia.Il8 19F NMR has been used to assess the apparent diffusion coefficient (ADC) in normal and globally ischaemic rat brain. The detection of 2-fluoro-2-deoxyglucose-6-phosphate in intracellular or extracellular spaces allowed the assessment of the ADC of these two compartments. There was no difference seen between the ADC of the intracellular and extracellular spaces in normal globally ischaemic brain. I 9

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The effects of hyperosmolar solutions containing 20% mannitol or 10% glycerol on the phosphorous metabolites of the gerbil brain have been investigated during ischaemia and reperfusion. Mannitol infusion caused a significant increase in pH compared to the glycerol- and saline-treated groups. There was a significant increase in PCr/Pi at 2, 4 and 8 h reperfusion with mannitol and at 6 h with glycerol infusion compared to controls. However, pATP/Pi was significantly raised in the glycerol-treated group at 2 and 8 h reperfusion but was only significantly raised in the mannitol-treated group at 2 h reperfusion.120 'H NMR has been used to measure various osmolytes in the rat brain following middle cerebral artery occlusion (incomplete focal ischaemia) or decapitation (complete global ischaemia). Measurements were made of amino acids (alanine, aspartate, y-amino butyric acid, glutamate, glutamine, NAA and taurine), methylamines (glycerophosphocholine), Cr and polyols (myo-inositol). The total osmolytes expressed as a sum of total amino acids, total methylamines or total polyols were significantly decreased following at 24 h of focal ischaemia but were unchanged following decapitation. Furthermore, the tissue water content was increased after focal ischaemia but not after decapitation suggesting a role for brain organic osmolytes in oedema following brain ischaemia.l2' In an investigation the effects of endogenous carnosine on rat brain during global ischaemia 'H NMR has been used to detect lactate, Cr, NAA and Cho. Carnosine was shown to reduced the level of lactate produced after ischaemic injury.'22 The effects of 3-hydroxybutarate (3HB) on PCr in rat brain slices following aglycaemic hypoxia has been assessed using3'P NMR. The substitution of 3HB for glucose in the incubation media caused a decrease in PCr in the adult rat brain slices only. In neonatal rat brain slices, 3HB had no effect on the recovery of PCr following aglycaemic hypoxia. In adult rat brain slices, the recovery of PCr was less than that in the neonatal rat brain slices but was improved when glucose together with 3HB was used in the recovery media.'23 31PNMR has been used to monitor the effects of dantrolene and 3-methoxybenzamide on the post-ischaemic recovery of neonatal rat brain slices. There was a 25% increase in HEP at the end of post-ischaemic recovery when dantrolene was introduced during the 0-40 min period of post-ischaemic reperfusion; this effect was not seen when dantrolene was introduced during the 40 to 120 min period of post-ischaemic reperfusion. However, the administration of 3-methoxybenzamide during the period of post-ischaemic perfusion, even with a 40 min delay, had similar effects as the early application of dantrolene. 124 The effects of oxidative stress have been examined with magnetic resonance imaging and 31PNMR in a rat model of anoxia followed by hyperoxia and, in l ~ ~NMR has been used to assess the a rat model of normobaric h y p e r 0 ~ i a . 31P effects of acute ischaemia on the mouse brain. Ligation of the common carotid artery and the vagus artery caused a decrease in PCr/Pi which was faster than the decline in P-ATP/Pi; cellular acidosis was also observed. Reperfusion partly reversed the effects of 5 min of ischaemia.'26 'H NMR has been used to examine the metabolites of the foetal lamb brain during hypoxia. Spectra

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contained similar metabolite peaks to those detected in human foetal brain and lactate was observed during h y p 0 ~ i a . The l ~ ~ levels of HEP and pHi have been monitored with 31P NMR in the neonatal rabbit brain during and following a 30 min period of hypoxia. Simultaneous recordings of EEG showed that, whilst pH and HEP decreased during hypoxia and subsequently recovered, brain function did not recover. 128 The PRESS pulse sequence, using CHESS for water suppression, has been used to record 'H NMR spectra of the rat brain before and after death. Spectra obtained with a 135 ms echo time contained peaks for NAA, Cr and Cho; lactate was also observed postmortem. These peaks were also observed in the spectra recorded with a 40 ms echo time along with peaks for glutamate/glutamine and lipids.129 31PNMR has been used in a study of the effects of anoxia on the energy status and pH in brain sheets of the western painted turtle. Within 30 rnin of anoxia PCr levels decreased to 40% and remained at that level whereas ATP gradually declined by 23% over the period of 110 min of anoxia. Furthermore, there was a decrease in pH from 7.2 to 6.6 over a period of 50 min of anoxia. Upon re-oxygenation, PCr, ATP and pH recovered to pre-anoxic values within 60 min. 130 31PNMR one-dimensional chemical shift imaging has been used to investigate phosphorous metabolism in the piglet brain during pentylenetetrazoleinduced seizures. In 2 and 14 day old piglets PCr/NTP was higher in the white matter compared to the grey matter, and this ratio was increased in the white matter of 14 day old compared to 2 day old piglets. The CK reaction rate increased in both areas between 2 and 14 days and decreased more during seizures in two-day-old piglets. During seizures in 14 day old piglets, PCr increased in the grey matter and decreased in the white matter. The level of NTP was relatively stable but more stable in white matter compared to grey matter.131The role of creatine and phosphocreatine levels in the brain of new born rats has been investigated with 31PNMR. Rat pups were injected with creatine for three days prior to exposure to hypoxia to induce seizures. Injections of creatine caused an increase in the PCr/NTP ratio and an increase in PCr and NTP recovery post hypoxia in 10 day old, but not in 20 day old rats. Furthermore, creatine reduced seizures and deaths in 10 day old rats but not 20 day old rats.132 An increased level of lactate has been detected in the hippocampus region of the rat brain during, and 24 hours after, kainic acid-induced seizures. The increase of tissue lactate was prevented by cyclohexamide which is protective against neural damage caused by kainic acid. There was an increase in NAA during the ictal period following kainic acid treatment and a decrease of NAA, reflecting neuronal loss, after seizures. 33 The effects of a 7 or 14 day infusion of 30% ethanol into the third cerebral ventricle of the rat has been investigated with 31PNMR. It was found that Mg2+i,fell by 33-39% and cytosolic phosphorylation potential (CPP) fell by 3 1 4 8 % in rats implanted with a ethanol-containing osmotic minipump whereas control rats showed a 20% decrease in Mg2+;and a 35% decrease in CPP. However, there was a 30% decrease in PCr in the treated rats. Following

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the systemic administration of 4g kg-' ethanol there was a 9- and 12-fold increase in haemorrhagic stroke mortality after 7 and 14 days, respectively, in rats with ethanol-containing i m ~ 1 a n t s . 31P I ~ ~NMR has been used to measure [Mg2'];, pH, PCr, ATP and Pi in the rat brain in a study of the effects of a diet deficient in Mg2+ on the response to ethanol. Four weeks of 3635% normal dietary Mg2+ caused a 40% loss of [Mg2']i and a rise in PCr and pH. Furthermore, reduced Mg2+resulted in a 100% mortality when animals where The effects of systemic Mg2+ on given a ED40 stroke dose of ethan01.l~~ cocaine-induced stroke in the rat has been investigated with 31PNMR. After the administration of a high dose of cocaine 40% of control animals died whereas, cocaine administration was lethal in 13% of animals which had been previously infused with MgC12. The infusion of Mg2+ prior to cocaine administration was also protective against a decrease in [Mg2']i, pH, PCrlATP and Pi/ATP associated with high-dose cocaine administration. The results suggest that the [Mg2'Ii and the level of PCr in the brain may be useful in the assessment of susceptibility to cocaine-induced stroke. 36 A miniature swine model for diffuse brain injury has been developed that replicates the inertial loading conditions associated with automotive accidents and causes axonal pathology without macroscopic damage. 'H NMR was used to detect a change in the NAA/Cr ratio when conventional MRI could detect no abn~rmalities.'~~ 13C NMR has been used to determine the rates of the TCA cycle and glutamine synthesis in the rat cortex over a range of synaptic activities. The results indicated that the stoichiometry between oxidative glucose metabolism and glutamate cycling in the cortex is close to 1:1 implying that the majority of cortical energy production supports synaptic glutamatergic neuronal activity.13*A new method has been introduced which has been used to measure the cerebral utilisation of [ 1-13]glucose.139 Measurements made in anaesthetised cats gave a maximal transport rate for glucose of 0.93 k 0.19 pmol g-' rnin-l, a cerebral metabolic rate of glucose utilisation of 0.22 k 0.03 pmol g- min- I , a normoglycaemic cerebral influx rate of 0.37 k 0.05 pmol g-I min-I and a hyperglycaemic t1/2 of 5.10 k 0.1 1 min-'. The labelling of NAA and N-acetylaspartylglutamate (NAAG) in the rat neocortex, hippocampus and cerebellum from [ 1-13C]glucosehas been demonstrated. Label was incorporated into NAA from acetate and aspartate whereas NAAG was labelled from glutamate. Low fractional enrichment of NAA (3%) compared to aspartate (20%) was observed. However, NAAG and glutamate had a fractional enrich13C NMR has been used to ment of 20.0% and 25.2%, respe~tive1y.l~~ investigate the metabolism of glucose in the brain transgenic mice overexpressing the gene for the liver-type subunit of phosphofructokinase; this gene is overexpressed in Down's syndrome. Although basal levels of blood glucose were similar in transgenic and control mice infused with [ 1- 13C]glucose,there was a slower clearance of glucose from the blood and a 58% faster initial rate of glucose utilisation in the brain of transgenic mice. The Cater rate of glucose metabolism was similar to the increase rate observed in Down's syndrome patients.

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A mathematical model of glutamate, glutamine and ammonia metabolism in the brain has been developed which allows the direct comparison of I3C and 15N data obtained in vivo. This model, in conjunction with 13C data obtained under normal and hyperammonaemic conditions, has been used to estimate the neurotransmitter cycling and detoxification components of glutamine synthesis. Furthermore, the simultaneous measurement of the rates of oxidative glucose metabolism and glutamate neurotransmitter cycling revealed a near 1:1 stoichiometry. The results of the study indicate that changes in cerebral glucose metabolism are coupled to neurotransmitter release.142 The metabolism of glutamate and glutamine under hyperammonaenic conditions in the monkey brain has been investigated with 3C NMR. Under hyperammonaemic conditions the incorporation of label from [l-13C]glucoseinto C4 of glutamine was accelerated without changes in the labelling of C4 of glutamate. Furthermore, under hyperammonaemic conditions labelling patterns from [2-I 3C]glucose indicated an increase in anaplerotic pathway of the TCA cycle in glia.143 15N NMR has been used to measure the rate of glutamate synthesis from leucine in the rat brain. The rate of increase in [15N]glutamatewas 0.7-1.8 pmol g-' h-' at a steady state brain [15N]leucineconcentration of 0.25 pmol g-l. The fractional enrichments of glutamate and leucine showed that leucine provided 50% of glutamate nitrogen under the experimental conditions. The results imply that at physiological leucine concentrations (0.1 1 pmol g- ') the rate of glutamate synthesis would be 0.35 - 0.9 pmol g g l h-I and that leucine would provide around 25% of glutamate nitrogen.'* The transport of ammonia and the synthesis of glutamine in the hyperammonaemic rat brain have been investigated with I5N NMR. The initial slope of the summed cerebral 15N-labelledmetabolites was used to determine the rate of ammonia net transport as 0.13 _+ 0.02 mmol min-' g-I during hyperammonaemia. The de novo glutamine synthesis, through anaplerosis and the action of glutamate dehydrogenase, was calculated to be 0.065 _+ 0.01 pmol min- g- and the rate of total glutamine synthesis was estimated to 0.20 _+ 0.06 pmol min-' g-', A large dilution was observed in the labelling of [2-'5N]glutamine which is consistent with the glutamate-glutamine cycle as an important pathway for neuronal glutamate repletion. 145 15N-labelled metabolites have been detected in the rat brain using the ISIS localisation technique in combination with nuclear-Overhauser-enhanced 15N NMR. Following an infusion of [ 5N]ammonium acetate the level of brain [5-'5N]glutamate was observed without signal contamination from blood [' 5N]urea. The labelling of, predominantly, brain ['5N]glutamate from ['5N]leucine was observed without contamination from [I5N]alanine signals in muscle. 146 The effects of Ca2+ and Mg2+on the detection of lipid signals in superfused cortical slices from neonatal rats has been investigated with 'H NMR. Increased lipid signals following sample preparation and superfusion were observed. These signals were reduced by 58% when slices were perfused with very low Ca2+ or Ca2+-freebuffer. Furthermore, the lipid signal was reduced by 47% when Mg2+ was raised to 10 mM and, reduced by 87% in high Mg2+, low Ca2+buffer.147

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47 1

Eye - The metabolism of rabbit lenses incubated with 5 to 40 mM glucose for up to 50 hours has been monitored with 31PNMR. At 10 to 40 mM glucose the polyol pathway was activated at a rate which was proportional to the concentration of glucose. The level of ATP was unaffected by the level of glucose used in the experiment^.'^^ 31P NMR has been used to investigate changes in a-glycerophosphate, phosphocholine, glycerophosphoethanolamine (GPE) and GPC after one week of alloxan treatment in streptozotocin-induced diabetic rats lens, hereditary cataract rat (ICR/f) lens, grey squirrel lens exposed to UV-A and the liver of partially hepatectomized rats. Similar increases in a-glycerophosphate and decreases in GPE and GPC were observed in all cases.149Changes in aldose and phosphorous metabolites in the rabbit lens incubated in 20 mM [l-13C]glucosehave been monitored with 13C and 31PNMR. Rapid increases in sorbitol, sorbitol-3-phosphate and aglycerophosphate were observed whereas, no changes in glucose, lactate and ATP were seen. When an aldose reductase inhibitor was added, sorbitol and sorbitol-3-phosphate were immediately reduced and a-glycerophosphate declined more slowly. Furthermore, there was a twofold increase in lactate though, the level of glucose and ATP were not affected.150 4.2

Heart - A review of the significance of studies of energy and nucleotide metabolism in the heart, which includes studies with NMR, has been produced for the fields of cardiology and cardiac surgery.151A mathematical model of energy transfer in hearts with an inhibited or ablated CK system has been reviewed with three reference^.'^^ The use of 31P, 23Na and 39Kfor the investigation of myocardial pathology and physiology has been reviewed with 37 references. 153 The use of 31PNMR for the characterisation of the cardiovascular phenotype of transgenic mice has been examined.154A new computer model of the response of myocardium to ischaemia and reperfusion has been produced. The method combines a pre-existing model (Oxsoft HEART) which contains highly developed dynamic descriptions of cardiac electrical activity with modelling of bioenergetic changes based on, and verified with, whole heart NMR. 55 The relationship between contractile state and [Na+]i in the isolate guinea pig heart has been investigated with 23Na NMR. Hearts were exposed to ouabain for 30 min during Langendorff perfusion and [Na+]i was measured with the aid of a chemical shift reagent. Treatment with ouabain was associated with a 2-fold increase in developed pressure but there was no significant increase in [Na+]i. The effects of chronic and acute hypokalaemia on the response of the heart to hypoxia and reperfusion have been investigated with 31PNMR. It was found that chronic and acute hyperkalaemia impaired contractility and the recovery of ATP in the heart. It was also observed that a combination of chronic with acute hypokalaemia had additive effects. 157 The measurement of left ventricular function, myocardial energetics (31PNMR) and the concentration of intracellular Ca2+ ([Ca2+]i)has provided evidence to support the hypothesis that reduced ATP hydrolysis impairs Ca2+-handlingin the perfused rat heart, thereby limiting the contractile reserve. 158 4.3

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Nuclear Magnetic Resonance

The effects of extracellular divalent cations on the [Na+]i in the perfused rat heart has been investigated with 23Na and 31PNMR. During a 30 min period of perfusion with Ca2+-freemedia there was no effect upon [Na+]i and during a subsequent period of ischaemia the rise of [Na+]iwas as expected. When hearts were perfused with media containing no Ca2+ and Mg2+ there was a tenfold rise in [Na+]i and during a subsequent period of ischaemia the rate of rise of [Na+]i increased further. It was demonstrated that the influx of Na+ occurred through L-type Ca2+-channelsand that the rise could be blocked by Mg2+and verapermil. 59 The contribution of cytosolic and mitochondria1 ion and energy changes to ischaemia-reperfusion injury in the isolated guinea pig heart have been investigated with 31PNMR. 160 The role of the Na+/Ca2+ exchanger on the outcome of ischaemiareperfusion injury has been investigated in the isolated mouse heart. Hearts from male and female transgenic mice, overexpressing the Na+/Ca2+exchanger protein, were subjected to 20 min of ischaemia and 40 min of reperfusion. Preischaemic left ventricular developed pressure (LVDP) was higher in the male transgenic mouse heart compared to the wild type. The postischaemic function in male transgenic hearts was lower than in male wild type hearts but there was no difference in the recovery of female transgenic hearts compared to female wild type hearts. However, the recovery of ovariectomized-female transgenic hearts was significantly lower than in wild type female hearts. The lower recovery of male transgenic and ovariectomized-female transgenic hearts was correlated with lower recovery of PCr and ATP.16' A comparison has been made on the effects of dobutamine, high perfusate calcium and the calcium sensitising agent N-hydroxy-5,6-dimethoxybenzo[b]thiophene-2-carboximidamide hydrochloride (ORG 3002) on the function and metabolism of the Langendorff-perfused rat heart. Contractility was affected in a dose dependent manner by dobutamine, high perfusate calcium and ORG 3002. There was no effects on myocardial oxygen consumption (MVo2) caused by ORG 3002 at the lowest concentrations despite a 50% increase in systolic pressure and a 17% increase in the force-time integral. Furthermore, dobutamine and high perfusate calcium caused a similar increase in systolic pressure and the force-time integral and, increased MV02 by 50% and 41%, respectively. However, there was no effect on the energy metabolism of the hearts, detected by 31PNMR, caused by dobutamine, high perfusate calcium or ORG 3002.'62 7Li NMR has been used to measure the flux of Li+, as a Na+ congener, in the perfused rat heart. Hearts were equilibrated for 32 min with 15 mM L.iCl prior to Li-efflux in Li-free media. Li+ efflux was biphasic and was dependent on heart rate. Cardiac arrest induced by 1 mM lidocaine or 20 mM KCl reduced the rate of efflux to a third of the control value. The application of increasing concentrations of carbachol gradually reduced the heart rate and revealed a linear relationship between heart rate and the rate of Li+ efflux. The efflux of Li+ from myocytes was mediated mainly by voltage-dependent Na+ channels. 163 23Na NMR, in conjunction with the shift reagent Tm(DOTP)5-, has been

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used to measure the intracellular Na+ concentration ([Na+]i) of perfused hearts during and after 20 min ischaemia. Ouabain pretreatment resulted in higher end-ischaemic [Na+]i and caused a rise in [Na+]i upon reperfusion. In control hearts dropped immediately on reperfusion indicating the activity of the Na+-K+ ATPase. When hearts were perfused with ouabain and EIPA there was no initial rise in [Na+]i indicating Na+-influx mediated by the activity of Na+/H+ exchange upon reperfusion. The effects of myocardial infarction on the Na+ content of the heart has been measured using 23Na NMR. The total myocardial Na+ content was found to have increased 3.2-fold one day after infarction. There was a 1.6-fold increase in the Na+-content of chronic scar tissue which allowed this region to be distinguished with 23Naimaging.16523Na NMR has been used to measure the T2 relaxation time of Na+ in the perfused rat heart during ischaemia and reperfusion. 166 39KNMR, in combination with the hyperfine shift reagent TmDOTP5-, has been used to measure the intracellular K+ concentration ([K+]i) in perfused guinea pig hearts. Approximately 23% of K+, detected by atomic absorption, was detected by 39KNMR in the heart. 39K NMR detected an increase in [K+Ii during ischaemia which returned to baseline levels during reperfusion. In K+depleted heart, where K+ was replaced by Rb+, there was no K+ detected by NMR during perfusion. However, during ischaemia a small peak for K+i was detected which subsequently disappeared during reperfusion. 167The activity of ATP-sensitive K+ channels in isolated rat hearts has been investigated with 87RbNMR to measure the efflux of Rb+; the energetic status of the hearts was measured with 31PNMR. The efflux of Rb+, following its removal from the perfusate, appeared to be monoexponential and independent of the concentration of intracellular Rb+ ([Rb+li). The rate constant for the efflux of Rb+ was halved by agents which depress excitability and heart rate such as lidocaine, carbachol and MgS04. However, a high concentration of extracellular K+ ([K+],) did not affect the rate of Rb+-efflux independently of the presence or absence of glibenclamide or bumetanide. A threefold increase in the rate constant of Rb+-efflux occurred in the presence of DNP, a high [K+Ie and bumetanide. This combination caused a decrease in PCr to lo%, a decrease in ATP to 15% and a sevenfold increase in Pi. The effects of DNP were reversed by glibenclamide.* The temperature dependence of monovalent cation fluxes in the isolated rat heart has been investigated with 87Rb, 7Li and 23NaNMR; pHi and HEP were monitored with 31PNMR. There was an incomplete efflux of Rb+ from the heart, with a fivefold increase in its rate constant, at 20" compared to 36 ". Glibenclamide completely depressed the rate of hypothermia-activated (20 ") Rb+-efflux. 7Li NMR showed a threefold increase in the Li+-efflux rate constant at 20" compared to 36". The rate constants measured with 87Rb and 7Li NMR at 10 ' were similar to those measured at 36 ". At 36 ",20 and 10 the values of pHi determined by 31PNMR were 7.05, 7.21 and 7.40, re~pective1y.l~~ The flux of mitochondria1 K+ in the perfused rat heart has been measured with 87Rb NMR. At 20" 87Rb NMR showed the existence of two Rb+ pools; these were assigned to the cytoplasm and to the mitochondria. After the washout of cytosolic Rb+, the mitochondria had a O

O

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Nuclear Magnetic Resonance

stable level of Rb+ for more than 30 min. Treatment with DNP stimulated the efflux of Rb+ from the mitochondria but this efflux was incomplete. However, treatment with saponin caused the rapid and complete efflux of Rb+ from the mitochondria. 170 The regulation of Mg2+i in the isolated perfused rat heart has been investigated with 31PNMR. 17' Prolonged application of isoproterenol caused a slow and significant decrease in [Mg2+]iwhich was restored by subsequent washouts. This decrease was antagonised by carbachol, except in the presence of atropine. A similar decrease in [Mg2+]iwas seen in the presence of a watersoluble derivative of forskolin (NKH477) which could, also, be antagonised by carbachol. No changes in the ATP concentration of the heart showed any correlation with changes in [Mg2']i. The relationship between the free energy of ATP hydrolysis (AGATP) and the energy required for the maintenance of the Ca2+-gradient across the sarcoplasmic reticulum (SR) has been investigated with 19F NMR in the perfused rabbit heart. 172 Perfusion of hearts with pyruvate or isoproterenol resulted in an increase in LVDP and an increase in the concentration of Ca2+ ([Ca2+])in the SR. The increase in the SR Ca2+-gradientcaused by pyruvate was matched by the increase in AGATPbut the increase in the SR Ca2+gradient caused by isoproterenol occurred despite a decrease in A G A T P . Ischaemia, which causes a decrease in A G A T p , resulted in a decrease of the energy required to maintain the SR-Ca2+ gradient that was not predicted by changes in A G A T p but was consistent with increased release of Ca2+ from the SR and increased SR-Ca2+ cycling. Furthermore, ischaemic preconditioning attenuated the fall in the energy required to maintain the SR Ca2+-gradient during a subsequent period of ischaemia such that, this fall was similar in magnitude to the fall in A G A T P . The effects of ischaemic preconditioning on pHi, [Na']i, [Ca2+]i,ATP and PCr in the newborn rabbit heart during ischaemia and reperfusion have been measured with NMR. Ischaemic preconditioning resulted in a higher pHi and lower [Na+]i and [Ca2+]i.Furthermore, ischaemic preconditioning resulted in decreased coronary resistance during reperfusion and improved recovery of LVDP. 173 The mechanism of pH preservation following ischaemic preconditioning has been investigated in the rat heart using 31PNMR. Hearts were subjected to either cyclic ischaemia or a1 -adrenergic stimulation by phenylephrine. Both protocols produced significant attenuation of endischaemia acidosis and this protection was prevented by blockade of protein kinase C. The accumulation of lactate was decreased by cyclic ischaemia but not by treatment with phenylephrine. Blockade of the Na+/H+ exchanger eliminated the attenuation of end-ischaemia acidosis in both groups. 174 31P NMR has been used to investigate the mechanism preservation of cardiac function through ischaemic preconditioning in the Langendorff perfused rat heart. The hearts were subjected to 30 min of ischaemia and 30 min of reperfusion with or without a 5 min period of ischaemic preconditioning. Ischaemic preconditioning improved the post-ischaemic recovery of LVDP and the post-ischaemic levels of PCr, Pi and A G A T p . However, there was no

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difference in ATP, PCr, Pi, pH or [Mg2+] during ischaemia in control compared to preconditioned hearts. During ischaemic preconditioning there was a threefold increase in the level of extracellular adenosine but a 75% decrease in extracellular adenosine during the subsequent period of ischaemia compared to controls. 75 31P and 23Na NMR has been used to study the mechanism of ischaemic damage during preservation of the isolated pig heart.176 The effects of hypothermic storage in University of Wisconsin (UW) or St. Thomas' Hospital (ST) solutions compared to storage in Krebs-Henseleit (KH) solution have been investigated in the rat heart. Cellular volumes were measured with 'H NMR of water combined with 59C0 NMR of the extracellular marker cobalticyanide and energy profiles were obtained with 31PNMR. Storage in ST solution reduced ischaemic swelling whilst storage in UW solution caused cellular shrinkage compared to the effects of KH solution. ATP levels and PHi were higher in hearts stored in UW compared to ST solution. Upon reperfusion, hearts stored in ST shrank and those stored in UW expanded resulting in similar intracellular volumes. Storage in UW solution, compared to storage in ST solution, resulted in improved postischaemic function and recovery of ATP.177 The effects of cardioplegic arrest on moderate hypertrophied rat hearts has been investigated with 31P NMR. Measurements were made in isolated perfused hearts during 40 min perfusion under normoxic conditions followed by infusion of ST cardioplegia and 90 min of ischaemia at 25 "C with infusions of cardioplegia every 30 min. Baseline concentrations of PCr, ATP and Pi were not different in the control compared to the hypertrophied hearts. After cardioplegic arrest, PCr increased more in control hearts and then declined at a greater rate in hypertrophied hearts. The level of PCr was transiently restored during each infusion of cardioplegic solution but PCr returned to baseline values in 2 min during reperfusion of control hearts, whereas, hypertrophied heart required 4 min to recover PCr levels. Furthermore, a greater deterioration of diastolic function was observed in hypertrophied hearts. 17* 31PNMR has been used to measure the effects of low-flow (7.5% of control) ischaemia on the pH and energy metabolism of the Langendorff-perfused ferret heart. The efflux of K+ was also estimated, using 86Rb NMR, from the efflux of Rb+ and was shown increase fivefold during ischaemia. A shortening of the action potential-duration also occurred during ischaemia. The efflux of K+ and the shortening of the action potential-duration were inhibited by glibenclamide, 5-hydroxydecanoate and a fall in pH from 7.14 to 6.83 when acidosis was not accompanied by a fall in ATP. Similar effects were seen when acidosis was induced with lactate or C02.179 The effects of fructose 1,6-bisphosphate (F16P) on the recovery from a 30 min period of ischaemia in the perfused rabbit heart has been investigated with 31PNMR. The infusion of F16P into non-ischaemic hearts caused an increase in glucose uptake and an increase in the levels of PCr, ATP and Pi. Following ischaemia, a 30 min infusion of F16P resulted in significantly higher developed pressure and lower diastolic pressure and, a higher level of PCr and greater

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Nuclear Magnetic Resonance

glucose uptake.18031PNMR has been used in an investigation of the effects of the rate of glucose transport on the tolerance of hypertrophied rabbit hearts to ischaemia and reperfusion. After aortic banding, myocardial glucose transporter content was assessed and functional recovery from ischaemiareperfusion was measured in isolated hearts. The rate of glucose transport was assessed by 31PNMR from the conversion of 2-deoxyglucose to 2-deoxyglucose6-phosphate. In hypertrophied hearts, there was reduced glucose transport, though there was no difference in glucose transporter content. Furthermore, hypertrophied hearts had a lower tolerance of ischaemia-reperfusion. Treatment with vanadyl sulfate in hypertrophied hearts increased glucose transport and improved the tolerance of ischaemia-reperfusion to that found in controls. Lactate production in vanadyl sulfate-treated hearts was increased during ischaemia.181 The effects of a novel voltage-gated Na+-channel blocker, 1-(5-phenyl-1,2,4thiadiazol- 3 - yl- oxypropyl) - 3 - [ N - methyl-N-[2- (3,4-dimethoxyphenyl)ethyl]aminolpropane hydrochloride (KC 1229l), on cardiac function, energy status, pH and Na+ homeostasis have been investigated with 31Pand 23Na NMR in the isolated perfused guinea pig heart during low-flow ischaemia and reperfusion. In well oxygenated hearts KC 12291 had no effect on LVDP. During ischaemia KC 12291 decreased contracture and improved the recovery of developed pressure during reperfusion. Although KC 12291 had no effect of the fall in PCr during ischaemia, it attenuated the decline in ATP and pH. During reperfusion, KC 12291 increased the recovery of PCr and ATP, and accelerated the recovery of pH. Furthermore, KC 12291 reduced the Na+ overload during ischaemia and enhanced Na+ recovery during reperfusion.* 82 The mechanism for the cardioprotective effects of the calcium channel blocker clentiazem during ischaemia and reperfusion has been investigated with 19FNMR and the intracellular Ca2+-indicator 1,2-bis(2-amin0-5-fluorophenoxy)ethane-N,N,N,'N'-tetraacetic acid (5FBAPTA). In the control group there was a significant rise in Ca2+ during 30 min of ischaemia and the first 5 min of a 30 min reperfusion. Following reperfusion, LDVP recovered to 63 & 7% of baseline. Pretreatment with clentiazem caused a 53 ? 9% fall in developed pressure but prevented any significant changes in [Ca2+];during ischaemia or reperfusion and allowed LVDP to recover to 87 k 8%. When the extracellular Ca2+ was raised to offset the negative inotropism of clentiazem, no beneficial effects of pretreatment with the drug were observed.183 The activities of two antianginal agents, diltiazem and propranolol, have been investigated in a closed chest canine infarct model using two-dimensional 31P chemical shift imaging. During a two hour left anterior descending coronary artery occlusion and, during the subsequent three weeks of reperfu~ i o n , ~ 'NMR P was used to monitored HEP and Pi; the infarct size was monitored with MRI. Although diltiazem appeared to reduce the impact of coronary occlusion on the level of HEP, there was no significant change in infarct size compared to controls. Treatment with propranolol, however, reduced the decline in pH during occlusion and reduced the extent of change in pH during early reperfusion. Furthermore, propranolol treatment was

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associated with a reduced infarct size. There was an inverse relationship between the change in pH during early reperfusion and the subsequent infarct size; this relationship was most significant after 1 hour of reperfusion. 184 The effects of carbon monoxide on perfused myocardium has been measured with 'H NMR to assess changes in the level of the myoglobin (Mb) Val E l l signal. On perfusion with CO the Mb02 signal at -2.76 ppm gradually disappears whilst the MbCO peak at -2.26 ppm emerges. At MbCO levels up to 76.8% saturation, myocardial O2 consumption remained constant though, the rate-pressure product dropped to 92% of the control level. At 87.6% Mb02 saturation, lactate formation had doubled and myocardial 0 2 consumption had begun to decline despite the fact that the C0/02 ratio was below the inhibition threshold for cytochrome oxidase activity and the high energy phosphate signals were unperturbed. The results indicate a role for Mb in directly regulating respiration. 185 The signals from the proximal histidyl NOH of deoxyhaemoglobin and deoxymyoglobin have been simultaneously detected in the rat myocardium. In normoxic conditions no deoxyhaemoglobin or deoxymyoglobin were detected but when inspired 0 2 was under 12% a peak from the a and p proximal histidyl N,H of deoxyhaemoglobin in erythrocytes was detected. 186 A mathematical model of the compartmentalised energy transfer in cardiac cells has been presented. The model was used for the interpretation of novel experimental data from 31PNMR determinations of the energy fluxes in the isolated hearts of transgenic mice with knocked-out CK isoen~ymes.~*~ 31PNMR has been used in an investigation of early changes in the control of mitochondrial function in myocardial ischaemia. Cardiac functional variables were also monitored by conventional means and the mitochondrial respiration variables were determined at the end of the protocol. The results indicated that alterations related to energy production control (by ADP and creatine) and to energy transfer are the earliest manifestations of damage to mitochondrial function during ischaemia. Furthermore, these alterations persist on reperfusion and could be responsible for an altered responsiveness of the mitochondrial function to energy demands.188A model system for manipulating the value of AGATP in normoxic-perfused rat hearts has been developed. Hearts were perfused with either no inhibitors, bromooctanoate (an inhibitor of poxidation), amino-oxyacetate (an inhibitor of the malate-aspartate shuttle) or a combination of these two inhibitors with or without butyrate. Three increasing levels of work demand were achieved by pacing hearts at 300 and 450 bpm and, at 450 bpm in the presence of dobutamine. The value AGATp was calculated from the HEP detected by 31PNMR.'89 A study of the dynamic adaptation to demands of cardiac oxidative phosphorylation has been carried out in the isolated rabbit heart u s i ~ i g ~NMR. ~ P The mean response time of cardiac mitochondrial 0 2 consumption was calculated from data obtained from measurements with oxygen electrodes. It was found that PCr and Pi change in the first few seconds after a quick increase in ATP hydrolysis caused by increased pacing but the mitochondrial 0 2 consumption changes with a much greater time constant. 190

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31PNMR has been used in a study on the effects of coronary underperfusion on adenosine 5’-nucleotide concentrations. Langendorff-perfused rabbit hearts were subjected to two periods of underperfusion (4-5% of baseline flow) separated by a 20 min period of reperfusion. Lactate efflux was similar in both periods of underperfusion but coronary adenosine efflux was reduced by 75% in the second period. There was a 46% fall in ATP during the first period but only a 10% fall in the second period despite a 75-78% fall in PCr in both periods of underperfusion. It was also shown that a 20 min period of underperfusion was required for this effect on the decrease of ATP. Analysis of the results by a mathematical model of the kinetics of myocardial energetics indicated that the down regulation of 5’-nucleotidase was responsible for the effect and suggests that this may be a protective rnechanism.lg1The effects of pH during ischaemia on the resynthesis of ATP during reflow have been assessed using 31P NMR in the hyperthyroid-rat heart. It was found that decreasing the pH from the normal ischaemic value of 6.7 to 6.4 lead to an increase in the accumulation of AMP (measured by chemical assay) during ischaemia and a 2.5-fold increase in ATP resynthesis on reperfusion. The results suggest that inhibition of 5’-nucleotidase by H+ may be responsible for the preservation of AMP and that this may provide a protective mechanism.192 The effects on the 31Pmetabolites in isolated perfused hearts caused by the allosteric modifier of haemoglobin 2-(4[[(3,5-Dimethylanilino)carbonyl]methyl]phenoxy)-2-methylpropionicacid (RSR 13) have been investigated with 31PNMR. In control hearts, perfused at 2.0 cm3 min-’, RSR13 had no effect on the 31P metabolites or the rate pressure product. In ischaemic hearts, perfused at 0.2 cm3 min-I for 30 min, RSRl3 improved the mechanical recovery upon reperfusion. Furthermore, RSR 13 reduced the rate of decrease in ATP in potassium-arrested hearts. 193 31P NMR has been used to assess the cardiac energetics of bioengineered mice which either lack the M isofonn (MCK-I-) of CK or lack the M isoform and mitochondria1 isoforms (M/MtCK-/-) of CK. Hearts from wild type, MCK-/- and M/MtCK-/- mice had comparable baseline function and similar increased rate-pressure product in response to increased heart rate and Ca2+ perfusion. However, M/MtCK-/- hearts had a 95% increase in the concentration of ADP ([ADP]), and a decrease in AGATP under the increased work load. In contrast to previous findings in the skeletal muscle of M/MtCK-/- mice, the hearts were able to hydrolyse and resynthesise phosphocreatine. 194 The hearts of wild type and cytosolic muscle CK knock-out mice have been studied with 31PNMR during pacing at 400 and 600 bpm and, under K+ arrest; 31PNMR saturation transfer was also used to measure the flux of CK. The levels of PCr and ATP in the knockout mouse hearts were not significantly different to those in the wild type hearts, however, there was a difference in the flux of CK. In knockout mice this flux was 4.8 ? 0.6 and 4.5 _+ 0.6 mM s - l at 400 and 600 bpm, respectively, which fell to 3.5 ? 0.4 mM s-l under K+ arrest. In control heart the flux of CK was 7.8 k 1.1 and 6.6 _+ 1.3 mM s- at 400 and 600 bpm, respectively, and decreased to 3.8 k 0.5 mM s-l during arrest.Ig5 The effects of dietary creatine on the function of the perfused rat heart has

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been investigated with 31PNMR. Whilst chronic increases of dietary creatine raised blood, liver and kidney creatine levels, there was no increase in the Cr in brain, skeletal muscle and heart. Furthermore, there was no effect on the mechanical function of hearts, the level of ATP, PCr and Pi, and no effect of the CK r e a ~ t i 0 n . I ~ ~ The effects congestive heart failure in the spontaneous hypertension and heart failure rat (SHHR) heart has been investigated with 31P NMR. Failing hearts had decreased PCr/ATP which was explained by a reduction in Cr. In pre-failure and compensated SHHR hearts, PCr was 5.1 to 5.3 pM g-' wet weight under basal conditions whereas in failing hearts, NMR detected PCr levels of 2.9 pM g-l wet weight. Compensated and failing SHHR hearts showed a decrease in PCr and an increase in Pi when the rate pressure product was i n ~ r e a s e d . '31P ~ ~ NMR has been used to investigate the bioenergetics of isolated, isovolumetric mouse heart preparations from mice with the missense mutation of arginine to glutamine at position 403 of the P-cardiac myosin heavy chain (aMHC403/+mice) which causes familial hypertrophic cardiopathy. There was no evidence of systolic dysfunction in the aMHC403/+ mice but diastolic function was impaired during inotropic stimulation. Under baseline conditions, aMHC403/+mice had lower PCr and increased Pi resulting in a lower calculated value of AGATP.Furthermore, unpaced hearts from aMHC403/+mice responded to perfusion with increased Ca2+ by a twofold decrease in heart rate compared to wild type hearts.19* The effects of age and hypoxia on the isolated Fisher 344 rat heart has been investigated with 31PNMR. Hearts from 24 to 25 month old rats had 30% lower heart rate and prevalent supraventricular arrhythmia, lower PCr and Cr content but normal ATP levels compared to hearts from 3 to 4 month old rats. During hypoxia ATP decreased similarly in hearts from younger and older rats but the decrease in ATP was 2-fold smaller in older hearts.19931PNMR has been used to assess the response of mature (2-4 month old) and aged (18-24 month old) rat hearts to hypoxia-reoxygenation or ischaemia-reperfusion. There was no differences detected by 31P NMR in aged or mature hearts during ischaemia or hypoxia, though, contracture tended to be higher in aged hearts. There was a reduced recovery of ATP, PCr, Pi and Mg2+in aged hearts during reperfusion or reoxygenation. Furthermore, aged heart had lower cytosolic free energy after either insult which reflected the lower recovery of post -insult contractile function .200 The effects of ethanol on the isolated rat heart have been investigated with 31Pand 19FNMR. Ethanol was found to produce a concentration-dependent biphasic haemodynamic change; 15 and 45 mM ethanol were stimulatory and 90, 135 and 170 mM ethanol were depressive. When hearts were treated with 2 90 mM ethanol Pi/PATP increased with no change in PCr/PATP, [Mg2+]ifell and the pH was more acidic. 19F NMR, using the Ca2+-indicator 5FBAPTA revealed that [Ca2+];increased at high levels of ethanol.201 The influence of a-ketoglutarate dehydrogenase (a-KGDH) activity on the balance between oxidative flux and transmitochondrial a-ketoglutardte (a-KG) exchange has been investigated in isolated mitochondria and perfused

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rabbit hearts.202In mitochondria, the value of K, for a-KGDH and a-KG efflux decreased at pH 6.8 compared to their values at pH 7. In isolated heart a decrease in K, was observed when PHi was decreased to 6.89 whilst the work load and respiratory rate were matched to control values by increasing [Ca2+] in the perfusate. I3C NMR spectra of hearts showed that the TCA cycle flux was the same at both values of Km but that the exchange between a-KG and cytosolic glutamate fell at the low Km. The effects of dichloroacetate on fatty acid oxidation and flux through pyruvate dehydrogenase has been studied in isolated perfused heart using 13C NMR. In non-ischaemic control hearts, dichloroacetate increased pyruvate dehydrogenase flux more than eightfold and significantly decreased the oxidation of acetoacetate and fatty acids. Treatment with dichloroacetate improved recovery of mechanical function after 30 min of ischaemia and 30 min reperfusion but this did not affect the flux through pyruvate dehydrogenase at the end of reperfusion when compared to untreated ischaemic hearts.203 13C NMR has been used in an evaluation of the TCA cycle flux in glucoseperfused hearts. The results were analysed using a new mathematical model which considers that the labelling of glutamate and aspartate carbons, by chemical and isotopic exchanges, are interdependent. The use of [U-’3C]glucose or [ 1,2-13C]acetate allowed the labelling of all glutamate and aspartate carbons. When [U-13C]glucose was used in the absence of insulin then aspartate, not glutamate, was the most abundantly labelled metabolite. Approximately twice as much aspartate was formed compared to glutamate indicating that aspartate transaminase is highly effective in the absence of insulin.204An investigation into the relationship between the affinity of glucose and 2-deoxyglucose for the glucose transporters and phosphorylation system has been carried out using 31P NMR in the perfused rat heart. It has been assumed in many studies that the affinity of this system for glucose, compared to 2-deoxyglucose, is constant. However, 31P NMR measurements of the increases in phosphorylated sugars revealed that the ratio of the affinity of the transport and phosphorylation system towards 2-deoxyglucose, compared to glucose, is lower when glucose uptake is stimulated by insulin or cardiac stunning.205 31PNMR has been used to compare the myocardial phosphodiesters (PDE) content of perfused, working hearts from three species of turtle: the hypoxia tolerant western painted turtle (Chrysemys picta belli), red-eared sliders (Trachemys) and the relatively hypoxia-sensitive softshell turtle (Trionyx spinifer). In Chrysemys hearts PDE constituted 24 & 8.6010 of total NMR signals, in Trachemys hearts PDE was 20.7 k 5.9% of the total and in Trionyx myocardium PDE was only 12.2 k 5.1% of the total 31Psignal. The majority of PDE in Chrysemys and Trachemys hearts was identified in extracts to be serine ethanolamine phosphodiester whereas, GPE was the dominant PDE in Trionyx myocardium which was also found to be the case in myocardial extracts from Kemp’s ridley sea turtles (Lepidochelys kempi).206 The hearts from Chrysemys and Trionyx have also been compared using 31P NMR to measure their response to either 240 min of anoxia, 90 min of global ischaemia

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or 240 min of global ischaemia followed by reoxygenation or perfusion. 31PNMR was used to measure PCr, ATP, Pi and pH. Results from the two types of myocardium during the 90 and 240 min periods of ischaemia suggested that the myocardium from Trionyx, which is the most sensitive to hypoxia in vivo, may have a greater tolerant to ischaemia in ~ i t r o . ~ ~ ~ Kidney - Volume selective 31PNMR has been used to assess renal injury in the rabbit kidney subjected to 1 h of ischaemia and 2 h of reperfusion in the presence of a new indeno-indole-derived lipid peroxidation - inhibitor (H290151). During ischaemia there was an decrease in ATP and pH with an increase in Pi. Two hours after reperfusion significantly higher P-ATP/Pi and pH were observed in kidneys pretreated with H290/5 1.208 4.4

4.5 Liver - 13C NMR has been used to observe the turnover of [4-13C]glutamate, [4-13C]glutamineand [2-' 3C]glutamate/glutamine in the liver of anaesthetised rats following the infusion of [2-13C]ethanol.The isotopic steady state analysis of 3C-labelling patterns was also performed on extracts.209The detection of ascorbic acid in the rat liver has been reported following the infusion of [I ,2-13C]glucose.210 The turqover rate of [5,6-13C]ascorbicacid was estimated as 1.9 k 0.4 nmol min- 'g- l. The effects of acute treatment with the hormone leptin have been assessed in the perfused mouse liver using13C NMR. In perfused liver from ob/ob or lean mice, acute treatment with leptin resulted in an increase of carbohydrate flux into glycogen and the rapid stimulation of fatty acid synthesis from acetyl CoA by the de novo synthesis route. However, chronic treatment with leptin was found to increase glycogen synthesis but decrease fatty acid synthesis.21 The effects of acute endotoxaemia on gluconeogenesis and phosphoenergetics in the rat liver have been investigated with 13C and 31P NMR. Lipopolysaccharide from E. coli was injected i.v. prior to an infusion of [3-13C]alanine.A decrease in ATP and an increase in Pi was observed in response to endotoxin treatment. Furthermore, the peaks from glucose C6 and glutamate/glutamine C2 were lower than those of the controls despite hyperglycaemia during endotoxaemia.212The synthesis of glycogen during pregnancy has been assessed by 13CNMR in rat following ad libitum or routine feeding regimes. There was no detectable glycogen synthesis from [ I-13C]glucosein 20 day pregnant and control (virgin) rats previously fed ad libitum. In 20 day pregnant rats fed by routine meal, glycogen synthesis occurred at the rate of 0.1 s-'. In 10 day pregnant rats, glycogen synthesis was maximal for rats fed by either regime.213 The use of phosphonates a markers of extracellular and intracellular space and pHi in the perfused liver have been studied using 31PNMR.214The inwash and outwash behaviour of phenylphosphonate (PhePo), 3-aminopropyl phosphonate (NPo) and methyl phosphonate (MePo) was compared with that of PCr, a marker of extracellular space, and with dimethyl methylphosphonate (MePoMe2) a marker of total water accessible space. There was a rapid distribution of MePoMe2 over the total liver volume approximately threefold greater than that accessible to PCr. Infusion of PhePo, NPo or MePo resulted

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in their rapid distribution over the extracellular space but they steadily spilled over into the intracellular space. Some residual amounts of these three compounds remained after washout following a prolonged infusion, The resonant frequency of residual PhePo was found to correspond to the pHi reported by Pi and responded to induced transient changes in pHi. The effects of ketamine and isoflurane anaesthetics on the energetic status of the liver during haemorrhage has been investigated in the rat using 31P NMR. Haemorrhage was simulated by the withdrawal of blood until mean arterial pressure had reached 40 mmHg and was maintained for 45 min. There was a greater decrease in P-ATP and increase in Pi in the ketamine- compared to isoflurane-anaesthetised rats.21 The role of bicarbonate in the regulation of pH in the isolated perfused rat liver has been investigated with 31P NMR. Removal of external HC03decreased proton efflux from 18.6 5 5.0 to 1.64 5 0.29 pmol min-I g- liver wet weight and decrease pHi from 7.17 k 0.06 to 6.87 k 0.06; inhibition of carbonic anhydrase further decreased proton efflux. Treatment with amiloride did not affect pH, though, proton efflux was amiloride-sensitive but treatment with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonicacid and amiloride caused a decrease in pH and proton efflux.216The effects of low temperature (4 "C) on the pHc regulation and buffering capacity in the isolated rat liver has been measured with 31PNMR. The cooling of livers from 37 to 4 "Cwith KH buffer at a pH of 7.35 caused an increase in pHi of 0.13 or 0.75 unit in the presence or absence of bicarbonate, respectively. At 4"C, in the presence or absence of bicarbonate, acute changes in the pH, did not affect PHi. However, intracellular loading with isobutyric acid or NH&l induced rapid changes in pHi. The buffering capacity of the liver at 4°C varied with PHi from 10 to 50 slykes; the largest value being observed at the pK value of the imidazole groups of intracellular proteins at 4 0C.217 The effects of a prostacyclin derivative (iloprost) has been investigated in the cold preserved pig liver using3'P NMR. Livers were collected using a clinical protocol and were subjected to cold storage in UW solution with, or without, iloprost. 31P spectra were collected every 2 min during hypoxia and cold reperfusion. In iloprost-treated liver the rate of P-NTP synthesis during reperfusion was 66.3% greater and resulted in 59.6% more P-NTP being detected at the end of reperfusion. The depletion of NTP was 32.6% less during storage in the prostacyclin-treated livers.21 A comparison has been made of methods used to assess metabolic activity in the hypothermic porcine liver. 31PNMR was used to measure the ratio of 0-ATP/total 31Psignal and the ratio of PME/Pi during ischaemia and reperfusion. The signals from ATP changed rapidly by an order of magnitude in a predictable fashion. However, the ratio of PME/Pi changed by a small amount in an unpredictable fashion. Measurements of PME/Pi may, therefore, be less useful in the assessment of liver function during recovery from hypothermic ischaemia compared to assessment from the more difficult measurement of the level of P-ATP.219The role of 31P NMR measurements of PVATP to assess dysoxia has been investigated in the pig liver.220

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19FNMR has been used to assess the metabolism of 5-fluorouracil(5FU) in KM mice compared to C57 mice.221The effects of the route of administration of 5FU has been investigated in the Wistar rat with 19F NMR. When 5FU was administered via the tail vein, the drug signal peaked in the first (5.5 min) spectrum and then gradually declined during the remainder of the hour of data collection. The catabolite a-fluoro-P-alanine (FBAL) gradually increased up to 33 min and then plateaued. The administration of 5FU by the hepatic portal vein caused a twofold increase in the intensity of first 5FU peak but the overall profile of 5FU and FBAL was the same as that obtained from administration via the tail vein route. When 5FU was administered via the hepatic artery, the intensity of 5FU in the first spectrum was the same as that in the first spectrum following hepatic portal vein administration but the intensity of 5FU remained high to the last spectrum. Furthermore, the intensity of the FBAL peak was less from the hepatic artery infusion than from the hepatic portal vein infusion. The level of the activated form of 5FU in the liver, following hepatic artery infusion, was more sustained than that following infusion by the other routes.222 4.6 Reproductive System - 31P NMR has been used to investigate the relationship between blood flow, smooth muscle contractile activity and high energy phosphate metabolism in the rat uterus. Small reductions in blood flow caused reductions in the contractions of the uterus, and reactive hyperaemia was absent in most preparations. Reductions in flow were also associated with significant reductions in ATP, PCr and pH and an increase in Pi.223

4.7 Salivary Glands - 'H NMR has been used in an investigation of the role of anions in the regulation of pHi and cell volume in perfused rat mandibular salivary glands. An initial rise in pHi, due to C1-/HCO3- exchange, and a secondary rise in PHi, due to activation of Na+/H+exchange, were detected by NMR. These changes followed a similar time-course to that of the reduction in cell volume.224 4.8 Skeletal Muscle - A review of the most recent findings from NMR flux measurements on CK activity in transgenic mouse models of the CK system has been produced with 56 references.225 The fluctuations in the metabolites of the rat gastrocnemius muscle have been investigated with 31P NMR. Using gated signal acquisition, an 11.3% decrease in PCr was detected within 16 ms after stimulation which corresponded to the time of maximal contraction. The level of Pi was seen to increase stoichiometrically whilst ATP remained constant. During a single twitch, PCr produced 3.1 p.mol ATP g tissue-' compared to 0.3 pmol ATP (g tissue)- for each twitch during steady state experiments.226The effects of muscle blood flow on the energy metabolism and contractility of rat femoral triceps muscles have been investigated with 31PNMR. Ligation of the femoral artery reduced the muscle blood flow ratio but did not effect the ratio of Pi/(Pi+PCr) during rest. However, the ratio of Pi/(Pi+PCr) was related to the

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reduction in the blood flow ratio during and after the exercise period; muscle tension was related to the blood flow ratio during exercise.227The Pi/PCr ratio of denervated rat muscle in response to succinylcholine treatment has been assessed with 31PNMR following treatment with dantrolene or vecuronium.228 The effects of CK deficiency in knockout mice has been studied with 31Pand 'H NMR. After 25 min of ischaemia, all muscles studied by 31PNMR were found to have maintained their ATP levels except those muscles with complete CK deficiency. In this latter group the level of ATP stabilized at 70% of baseline levels after 10 min of ischaemia. Furthermore, ischaemia caused a more rapid decline of pH, and to a lower value, in CK-deficient mice. During anoxia,'H NMR detected increases in the resonances for taurine in wild-type muscles.229 31PNMR has been used to assess the anaerobic metabolism of the isolated frog (Rana esculenta) gastrocnemius muscle at rest. Measurements were made with, or without, IAA poisoning. The results showed that the rate of PCr hydrolysis was greatest in the early period of anaerobiosis in the presence of IAA and was accompanied by a large accumulation of PME. The ratio of PCr to lactate suggested that glycogen was almost the sole substrate for glycoly~is.~~~ The role of 'H NMR in the assessment of muscle growth, dystrophy and glucocorticoid treatment has been investigated in limb and diaphragm muscle of normal and mdx mice. Treatment of dystrophy by glucocorticoids resulted in improved muscle phenotype and increased levels of taurine and creatine, particularly in limb muscle.'H NMR could also be used to discriminate between normal and mdx mouse muscle.231 The effects of safflower oil and fish oil on insulin-stimulated glycogen synthesis, glycolysis and pyruvate dehydrogenase flux in rat skeletal muscle has been investigated with 13C NMR. The incorporation of label from [ 1-*3C]glucoseinto [1-13C]glycogen,[3-13C]lactateand [3-13C]alanine was monitored in the hind limb of awake rats under euglycaemic/hyperinsulinaemic clamp. Safflower oil-fed rats were more insulin resistant and had a slower rate of glycolysis compared to control or fish oil-fed rats. No change in glycogen synthesis was detected in safflower oil-fed or fish oil-fed rats compared to controls. The triglyceride content of muscle was increased in the safflower oilfed rats and the ratio of pyruvate dehydrogenase flux to TCA cycle flux was decreased in this Smooth Muscle - The consequences of metabolic inhibition in smooth muscle isolated from guinea pig stomach have been investigated with 31PNMR. Following the application of IAA, spontaneous mechanical activity ceased within 10 to 20 min though, there was no effect on the resting membrane potential. The application of cyanide (CN), however, reduced mechanical activity without terminating it and slightly reduced the resting membrane potential. Glibenclamide did not affect the actions of IAA and, carbachol-induced contractions were reduced by the application of IAA.31PNMR detected a reduction in PCr and ATP to 60 and 80% of control 4.9

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values, respectively, whilst Pi was undetectable when spontaneous mechanical activity had been terminated by the application of IAA for 50 min. The application of CN for 50 min, however, caused a decrease in PCr and ATP to 30 and SO%, respectively, whilst Pi increased to 2.6-fold of control values. The results suggest that an ATP-sensitive mechanism is important in the generation of spontaneous mechanical activity but this does not involve ATP-sensitive K+-~hannels.~~~ The effects of hypoxia on the energy metabolism and pHi of resting porcine proximal intrapulmonary and superficial femoral arteries has been investigated with 31P NMR and chemical assay methods. The energy state and Cr was found to be lower, whilst the PHi was higher, in pulmonary compared to femoral artery segments; during hypoxia the energy state deteriorated more in femoral arteries. Furthermore, pHi fell in both tissues during hypoxia but pHi was always higher in pulmonary arteries. Reoxygenation of the tissues reversed the effects induced by h y p o ~ i a . ~ ~ ~ 4.10 Tumour - The effects of combretastatin A4 prodrug on implanted murine

tumours has been investigated with MRI and 31PNMR. Tumour necrosis and haemorrhage in response to treatment were detected by MRI. 31P NMR detected a decrease in NTP and pH with an increase in Pi following drug treatment.235In a separate study, the effects of combretastatin A4 prodrug was assessed in C3H mammary tumours in unanaesthetised mice using3'P and 'H NMR to detect changes in energy metabolism, and histology and MRI to determine tumour necrosis. There was a fall of 33% in the ratio of fl-NTP/Pi within 1 hour of treatment which remained for a further 2 hours. A small, significant, fall in pH was detected at 1 hour. In some tumours 'H NMR detected a rise in the peak at 1.32 ppm, predominantly from lactate. There was no tumour necrosis detected in tumours during the first three hours after treatment with combretastatin A4 p r o d r ~ g . ~ ~ ~ The T1 values and metabolite levels of Pi and P-NTP in BEX-t human melanoma xenografts have been measured following single dose radiation treatment of 5.0 or 15.0 Gy. Changes in T1 values were shown to correlate with changes in tumour water content following radiation treatment.237The effects of y-irradiation on lactate levels in EMT6 tumours has been measured by the 'H NMR selective multiple quantum coherence transfer method. After 10 and 20 Gy irradiation treatment, lactate levels decreased by 2 1% and do%, respectively, 48 h after treatment. No changes in lactate levels were observed in controls or tumours treated with 4 Gy irradiation in contrast to results previously obtained with the more radiosensitive RIF-1 tumours which responded to 2 and 4 Gy irradiation.238The effects of meta-iodobenzylguanidine (MIBG) on the pH of 9L glioma tumours has been investigated with 31PNMR. Fluorescence measurements were used to determine the effects of MIBG on intracellular electron acceptors, NADPH and flavoproteins in tumours and in vitro. Measurements made in vitro with a Clark oxygen electrode showed that MIBG inhibited oxygen utilisation. Changes in pH; were shown to occur before changes in fluorescence of NADPH and f l a v ~ p r o t e i n s . ~ ~ ~

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Magnetisation transfer has been used to attenuate the 'H NMR signals from F98 glioma tumours and contralateral tissue. Selective saturation of immobile metabolites was achieved by pulsed RF pre-irradiation and caused the attenuation of the CH3 signal from Cr in tumours. However, magnetisation transfer caused the attenuation of the CH3 and CH2 'H signals from creatine, the 3.44 ppm peak for taurine and the 3.78 ppm peak for glutamate and glutamine in contralateral brain.240 High resolution MAS 'H NMR has been used to examine tissue samples from patients with benign prostatic hyperplasia and malignant tumours. The results were compared to those from tissue extracts. Two-dimensional MAS NMR spectra were also used to identify metabolites. Marked differences, particularly in the lipid region, were observed in MAS NMR spectra of malignant compared to benign tissue. These differences were not apparent in tissue extracts.241Pulse-burst saturation recovery 19F NMR has been used to measure the spin-lattice relaxation rate of hexafluorobenzene as an indicator of tissue oxygenation. An intratumoural injection of hexafluorobenzene was administered to Dunning prostate adenocarcinoma R3327-AT 1 grown on pedicles in rats. The central region of tumours was found to have a p02 of 1.4-1.6 mmHg when rats breathed 33% oxygen or 95% 0 2 / 5 % C02. The periphery of the tumour had a p02 of 7.9-78.9 mmHg which increased significantly when 95% 0 2 / 5 % C02 was administered.242 19FNMR has been used to monitor the uptake and metabolism of 5FU in S180 tumour-bearing Kunning mice and B16 tumour-bearing C57 mice.243 The effects of 5-ethynyluracil, an inactivating inhibitor of dihydropyrimidine dehydrogenase, on the metabolism of 5FU has been studied with 19FNMR in vivo and in vitro. In extracts of liver, kidney and colon 38 tumours there was an increase in the cytotoxic fluoronucleotide (FNuct) metabolites and decrease in the metabolites of the elimination pathway. These findings were reflected in spectra obtained in vivo from colon 38 tumours.244The uptake and clearance of glucosylifosfamide and ifosfamide have been investigated with 31PNMR in rats bearing a prostate adenocarcinoma. The 31P signals from glucosylifosfamide and ifosfamide did not overlap with endogenous 31P signals and a rapid washout of glucosylifosfamidewas observed. No statistically significant changes were observed in pH or HEP.245The transverse and longitudinal relaxation rates of sodium borocaptate have been assessed with 'B NMR in horse serum. These values were then used in an evaluation of the quantitative detection of "B in tumour bearing mice. The level of llB in M2R melanomas, grown on the thigh, was shown to be significantly higher than in thigh muscle tissue though the kinetics of uptake and clearance were similar.246 The antitumour agent carboplatin has been detected in vivo ~ s i n g ' ~ NMR ~ P t at the site of injection. Experiments were carried out on four animals with doses of carboplatin ranging from 37.1 to 59.4 mg kg- body The metabolism of choline in breast cancer has been investigated with 2H, 13C and 31PNMR in MCF7 human breast cancer cells and tumours grown in nude mice. It was found that [1,2-13C]cholinewas transported into MCF7 cells and rapidly converted to phosphocholine which was accumulated to a high concentration. This accumulation did not affect the rate of synthesis of

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phosphatidylcholine. In mice, the infusion of [9-2H]choline resulted in a fivefold increase in the plasma concentration of choline and the gradual increase in the signal at 3.2 ppm indicating deuterated metabolites.248 'H NMR has been used to detect lactate turnover in rat C6 gliomas following steady state hyperglycaemia with [ 1-"C]glucose. Lactate measurements before the infusion of [l-13C]glucoseindicated that the majority of the lactate pool was metabolically active. Signals in the tumour from 3C-labelled glutamate were at least tenfold smaller than signals from lactate. However, in contralateral brain, the expected labelling of [4-* 3C]glutamate, [2-l3C]glutamate and [3 3C]glutamatewas observed.249 The metabolic changes that occur during growth of the mouse S 180 sarcoma have been characterized with 31P NMR.250The results showed that during growth there was an increase in Pi and PME and a decrease in PCr which was more rapid than the decrease in P-NTP. Furthermore, the tumour pH decreased with growth and was correlated to tumour size, PCr/Pi, P-NTP/Pi and (PCr'P-NTP)/Pi. 31P and 19FNMR have been used to measure the pHi and pH,, respectively, of SCCVII mouse t u m o u r ~ . A ~ ~decrease * in PHi and pHe were observed with growth. Furthermore, pHi was noted to fall before pHe and pHj was always lower than pHe. 'H NMR has been used to assess changes in the ADC of water, metabolites and macromolecules in the BT4C rat glioma in response to treatment with ganciclovir. In tumours transfected with the herpes simplex thymidine kinase gene, ganciclovir caused a 50% reduction in the ADC of Cho whilst there was an increase of 219% in the ADC of the rapidly diffusing water component; the relative fraction of this component was also increased from 87% to 94%. The ADC of the slowly diffusing water component and that of "H NMR-detected macromolecules remained unaltered.252 4.11 Skin - The effects of sulfur mustard on perfused human breast skin has

been investigated with 31PNMR.253 4.12 Whole Animal - The metabolic response to oxygen limitation in Arenicola marina, detected by myoglobin signals in 'H spectra, has been reviewed with 32 references.254 The method of using the adenylate energy charge as an index of an organism's metabolic state under conditions of imposed stress has been investigated in red abalone (Haliotis rufescens). A comparison was made between HPLC measurements of adenylate energy charge and 31P NMR measurements of free ADP in the assessment of the response of H. rufescens to hypoxia, pentachlorophenol (PCP) and sodium a ~ i d e31P . ~ NMR ~ ~ has, also, been used in a further study of red abalone exposed to hypoxia, azide or PCP. Hypoxia caused modest changes in phosphoarginine (PA) and Pi, a moderate, though not significant, change in [Mg2+]iand no significant change in pH. However, treatment with azide caused a decline in PA to 53% of reference values, a 10.66-fold increase in Pi and, a significant fall in pH which returned to control values after a 6 hour recovery period. A slight decrease in [Mg2']i

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was observed during exposure but this increased to 18% above reference levels by the end of the recovery period. Exposure to PCP resulted in similar effects on Pi and PA though the recovery was slower and The response of red abalone and black abalone (H. cracherodii) to PCP exposure in different sea water salinities has been investigated with 31PNMR. Upon PCP treatment PA levels declined and Pi levels increased faster in red abalone compared to black abalone. The disposition of PCP was also measured in extracts.257The effects of mild hypoxic challenge under different temperature conditions has been investigated in the common carp (Cyprinus carpio) using 31PNMR. Fish were acclimated to the optimal temperature of 25 ', the sublethal temperature of 33 ' or acclimated to 25 ' and then subjected to a temperature shock of 33 The level of ATP remained constant under all experimental conditions and the resting levels of PCr and Pi were comparable in all groups. When fish were subjected to hypoxia there was a decrease in pH and PCr whilst Pi increased. The recovery of PCr and pH was independent of temperature. In those fish exposed to temperature shock, the recovery of pH and PCr was slower. The resting pH decreased with increasing temperature and the extent of acidosis during hypoxia was more severe in those fish acclimated to 33 or subjected to temperature 31PNMR has been used to measure the pH of white muscle in a stenothermal Anarctic teleost (Harpagifer antarcticus) in response to different temperatures.259 The effects of pre-exposure to ammonia on the phosphorous metabolites of the anaesthetised goby (Pomatoschistus sp.) have been investigated with 31PNMR. No significant effects of the pre-exposure to ammonia was observed in 31Pspectra or measurements of oxygen consumption. Furthermore, no evidence of an affect on the level of 31Pmetabolites or respiration, cause by anaesthesia, was observed.260 O.

O

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Clinical Studies

5.1 Reviews - The development of human in vivo spectroscopy has been reviewed with 48 references.261 The application of NMR to the study of tumours in the clinical environment has been reviewed with many references.262A review of the application and contribution of NMR studies of the human brain to the understanding of brain dementia has been produced.263 The use of 'H NMR in cerebral pathology has been reviewed with 15 references.264A review of the application of 'H NMR to research in paediatric neurology has been produced with 94 references.265The application of functional magnetic resonance spectroscopy to the measurement of brain metabolism in patients with nervous system diseases and psychiatric disorders has been reviewed with 10 references.266The measurement of the neurochemistry of the brain, using NMR, in schizophrenia has been reviewed with many references.267A review of the use of 'H NMR in the assessment of neurochemistry for basic research and clinical applications has been produced with 211 references.268Developments in 'H NMR in

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relation to the study of human brain metabolism has been reviewed with 170 references269 and the methodologies involved in quantitative human brain NMR has been reviewed with 23 1 references.270 The use of 31PNMR for the investigation of human energy metabolism in muscle and brain has been reviewed with 59 reference^.^^' A review, with 29 references, on the use of NMR in the assessment of muscle function and energetics in relation to nutritional status has been produced.272 A review of the methods for the determination in vivo of insulin sensitivity, which include NMR applications, has been produced with 16 references.273Pre-clinical and clinical NMR studies of cancer has been reviewed with 93 references.274 31PNMR measurements of human breast cancer in the clinic, including a report of measurements of tumour response to treatment, has been reviewed with 106 references.275The application of 19F NMR to studies in humans and the methods for the improvement of 19F-signal detection have been reviewed with 186 references.276 Brain - The natural abundance 13C NMR signal from NAA has been detected in a 12 cm3 volume in the occipital lobe of healthy volunteers using proton-edited-carbon-observe NMR at 4 T.277The brain metabolite scylloinositol has been detected by 'H NMR in a healthy volunteer at a concentration of at least 300% above normal levels. The peak was confirmed by I3C NMR and its 'H NMR singlet characteristics. The results indicate that scylloinositol and myo-inositol metabolism may be regulated independently.278 Localised 13C NMR at 4 T has been used to measure the time course of labelling from *3C-glucosein the human visual cortex. Incorporation of label into C2, C3 and C4 of glutamine and of glutamate, and the incorporation into C2 and C3 of aspartate was observed. The labelling of GABA and lactate was detected for the first time in the human brain. A new model of cerebral glutamate metabolism was presented.279 Total brain NAA has been measured with 'H NMR in five women with an average age of 42 k 5 years. Non-localised non-echo 'H NMR was used to collect data from the head and from a phantom. The concentration of NAA in the brain was calculated using the volume of the brain obtained from magnetic resonance images.280 The effects of transient global amnesia on the metabolite concentrations in the hippocampus have been investigated by 'H NMR. During a typical episode, there were no differences in the levels of NAA, Cr, Cho and lactate compared to the spectra from the same subject two weeks later.281Localised 'H NMR has been used to investigate changes in brain metabolites in patients with HIV- 1-associated dementia compared to neuropsychiatrically unimpaired patients and normal volunteers. Spectra analysed from multiple voxels located in the fronto-parietal cortex and white matter at the level of centrum semiovale revealed a reduction in NAA/Cr and an increase in CholCr in patients with HIV- 1-associated dementia compared to normal volunteers. These changes in metabolite ratios of the cortical grey matter and subcortical white matter were of a similar order of magnitude to changes observed within the deeper white 5.2

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Nuclear Magnetic Resonance

matter of all patients.282 The effects of juvenile anorexia nervosa on brain metabolites have been assessed with 'H NMR in patients immediately after a period of excessive weight loss. Spectra from a voxel in the thalamus and in the parieto-occipital white matter revealed a higher Cho/Cr and a lower NAA/Cho ratio in the white matter of patients compared to controls.283 'H twodimensional chemical-shift imaging has been used to measure brain metabolite levels in patients with Alzheimer's disease. Spectra were obtained from areas of predominantly cortical grey matter, subcortical grey matter or white matter. In dementia patients, compared to controls, there was a decrease in the ratio of NAA/Cho, an increase in the ratio of CholCr and an increase in the ratio of myo-inositol to all other metabolites.284 The phenylalanine content in the brains. of patients with phenylketonuria has been assessed with 'H NMR. At blood levels of around 1.2 mM the brain phenylalanine content was 0.41 to 0.73 mM in patients compared to 0.15 mM in controls. Furthermore, patients were shown to have higher influx rates and lower consumption rates of phenylalanine compared to controls.285Localised 'H NMR has been used to assess the ratio of NAA/Cr and Cho/Cr in the pons and right or left lateral cerebellum of patients with schizophrenia. Significantly lower levels of NAA/ Cr were detected in the pons, but not in the cerebellum, of schizophrenic patients compared to control subjects.286 31P NMR has been used to assess two patients with mitochondria1 myopathy, encephalopathy, lactic acidosis and stroke-like episodes, It was found that the peak area of PCr detected in the occipital cortex during photic stimulation decreased and recovered thereafter.287 lH NMR has been used to assess the effects of thyroxin treatment in patients with congenital hypothyroidism. Patients had a reduced level of NAA/Cr and an increased level of Cho/Cr which tended to normalise with treatment. Abnormalities related to myelin maturation in the spectra of patients were reversible with thyroxin treatment despite initiation of therapy at ages beyond which abnormalities in myelinogenesis are considered irreversible. Hetero-geneity between the spectra from patients with congenital hyperthyroidism was observed.288The effects of vigabatrin on human brain levels of GABA have been assessed by 'H NMR. Following a single oral dose of vigabatrin the rate of synthesis of GABA was estimated to be 17% of the rate of the TCA cycle. As the daily dose of vigabatrin was increased up to 3g, the fractional elevation of GABA was the same in the brain and CSF.289 'H NMR has been used to assess the ratio of NAA/Cr in patients with amyotrophic lateral sclerosis treated with riluzole. In treated patients, there was an increase in the ratio of NAA/Cr from 2.14 k 0.26 to 2.27 k 0.24 compared to untreated patients where the ratio fell from 2.17 k 0.20 to 2.08 k 0.20.290Spin-echo volume-selective 'H NMR has been used to investigate the effects of treatment with acamprosate on the central nervous system of healthy volunteers. Acamprosate is used in the maintenance of alcohol abstinence following alcohol withdrawal in chronic alcoholism. Spectra obtained 20 min after infusion of acamprosate showed decreases in NAA and glutamate relative to those treated with a placebo. The results were consistent

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with those obtained by microdialysis measurement of glutamate in treated alcoholic rats. 29 5.3 Heart - The metabolites of CK (except ADP) and the rate of CK forward flux have been measured in the human heart. The quantification of, and the potential for imaging, PCr, ATP and Cr have been examined.292The use of cardiac gating in 'H NMR studies of the human heart has been investigated.293 A method for the improvement of the signal to noise in localised 31Pspectra of the heart and other organs has been developed 294 and implemented. The three-dimensional version of SLOOP (spectral localisation with optimal point-spread function) takes advantage of all a priori information to match the size and shape of the sensitive volume to the anatomical structure. Good localisation properties were demonstrated, in comparison to CSI and ISIS techniques, by the absence of signals from blood in heart spectra and the absence of PCr signals in liver spectra. There was a 30% increase in signal to noise of heart spectra using SLOOP compared to CSI. The technique also corrects for inhomogeneities in the B1 field from a surface coil such that no global saturation correction factor is required for the quantitation of spectra.295 The quantitative measurement of ATP and PCr in the human myocardium has been attempted by comparing the signal from an external standard with that from the myocardium using 31Pdepth-resolved surface coil spectroscopy. The region of interest was 15 mm diameter disk with a 25 mm thickness but this region was estimated to be within 22 to 66 g of myocardium. There was no difference observed between age-matched control myocardium and that from patients with hypertension. However, there was a significant difference between control PCr and ATP levels and those from patients with hypertrophic c a r d i ~ m y o p a t h y .The ~ ~ ~technique of 31P NMR has, also, been developed in a separate study to allow the detection of phosphomonoester signals from the hearts of patients with hypertrophic c a r d i ~ m y o p a t h y . ~ ~ ~ Furthermore, this method was applied to the examination of the anterior left ventricular myocardium of asymptomatic patients with hypertrophic cardiomyopathy ( ~ 1 4 )and aortic stenosis (r1=12).~~* A peak for phosphomonoesters, not observed in controls, was detected at 6.9 ppm in 7 hypertrophic cardiomyopathy and 2 aortic stenosis patients. It has also been found that asymptomatic patients with hypertrophic cardiomyopathy had a lower PCr to ATP ratio and patients with severe hypertrophy of the interventricular septum had a significant increase in Pi/PCr.299

Liver - The mechanism of inhibition of glycogenolysis by insulin and glucose has been investigated with 13C NMR. Flux rates of hepatic glycogen synthase and phosphorylase were measured in overnight-fasted subjects under one of four hypoglucagonaemic conditions: hyperglycaemic/hypoinsulinaemic, euglycaemic/hyperinsulinaemic, hyperglycaemic/hyperinsulinaemic or euglycaemic/hypoinsulinaemic. Under these conditions it was found that hyperglycaemia or hyperinsulinaemia inhibit net hepatic glycogenolysis, primarily 5.4

492

Nuclear Magnetic Resonance

through inhibition of glycogen phosphorylase flux or stimulation of glycogen synthase flux, respectively, and that this inhibition and stimulation are not necessarily coupled or co-ordinated in a reciprocal fashion. The results suggest a role for hepatic glycogen cycling as a mechanism by which insulin inhibits net hepatic glycogenolysis and endogenous glucose production in humans under euglycaemic conditions.300 The metabolism of 5FU in human liver has been measured with 19FNMR. Nine patients received 5FU in combination with methotrexate, leucovorin or levamisole. The time constant for the disappearance of 5FU varied from 5 to 17 min whilst the time constant for the appearance of the major catabolite, FBAL, varied from 7 to 86 min. The co-administration of methotrexate, leucovorin or levamisole did not appear to affect the pharmacokinetics of 5FU.30119FNMR has been used to assess the pharmacokinetics of 5FU in the tumours of 103 patients with either breast, colorectal or other tumour types. Measurable signals were detected in 99 patients and of these, 51 patients had a half-life (t1/2) of 5FU in the tumour of more than 20 min. There was a correlation between a tumour t1/2 of 5FU greater than 20 rnin and the responsiveness to therapy. Furthermore, patients with a tumour t1/2 of 5FU below 20 min did not respond to therapy.302 5.5 Muscle - The effects of plasma free fatty acids (FFA) on the uptake of glucose into the muscle of healthy volunteers has been measured with 31PNMR. The rates of whole body glucose uptake were similar for up to 140 min in subjects with low (0.01 mmol dmP3), basal (0.35 mmol dmP3) and elevated (1.44 mmol dm - 3, plasma FFA under euglycaemic/hyperinsulinaemic clamp. The presence of high plasma FFA blunted the rise in muscle glucose-6phosphate (G6P) concentrations by 45 min of exposure. At 180 min, the concentration of muscle G6P was lower in the high and basal FFA In a separate study of the effects of plasma FFA on glucose t r a n ~ p o r t , ~ and lP 13C NMR have been used to measure muscle G6P and glucose levels, respectively. The rates of whole body glucose uptake were 50-60% lower in the lipid-infused subjects compared to glycerol-infused subjects. Furthermore, there was a similar decrease in glucose oxidation and glycogen synthesis following lipid infusion which was associated with a 90% decrease in the increment of muscle G6P levels.13C NMR measured a significantly lower intracellular glucose level following lipid infusion.30413C NMR has been used to investigate the effects of lipid or glycerol infusions on the resynthesis of glycogen in the plantar flexion muscle of healthy volunteers during recovery from 90 min of moderate exercise. The rate of glycogen resynthesis in the glycerol group was faster during the first, compared to the second, hour of recovery. The level of glycogen synthesis was lower in the lipid-infused group compared to the glycerol-infused group during recovery.305 The transport of glucose in human skeletal muscle has beep investigated usingI3C and 31PNMR to measure muscle intracellular free glucose concentrations and the rate of glycogen synthesis. Measurements were made during hyperinsulinaemic clamp at three plasma glucose concentrations: euglycaemia

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(6 mM), mild hyperglycaemia (10 mM) or high hyperglycaemia (16 mM). A small, significant amount of free intracellular glucose was observed under the hyperglycaemic conditions. Glycogen synthesis rates were significantly higher under mild hyperglycaemia compared to euglycaemia and were significantly higher under high hyperglycaemia compared to mild hyperglycaemia.306 31P and 13C NMR have been used in an investigation of the effects of adrenaline on muscle glycogenolysis and insulin-stimulated muscle glycogen synthesis in humans. An infusion of adrenaline of 1.2 pg m-2 body surface area min-' in the presence of slight hyperinsulinaemia (13 pU crnA3)or basal insulin levels (7 pU cmP3) did not cause changes in muscle G6P or glycogen, though there was a 35% reduction in Pi. In the presence of a euglycaemic-hyperinsulinaemic clamp, there was an initial rise of G6P to 160% but with the infusion of adrenaline G6P levels were reduced by 300/.30731P and I3C NMR have been used in an investigation of the mechanism of insulin resistance in obesity. Measurements of muscle glycogen synthesis and intramuscular G6P showed that in obese women under hyperglycaemic-hyperinsulinaemic clamp glycogen synthesis was reduced to 70% and G6P failed to increase.308The relationship between muscle lipid content and insulin resistance has been investigated with 'H NMR. The measurement of the lipid content of muscle in biopsy samples is known to be related to insulin resistance. However, this finding has been confirmed with 'H NMR estimates of intramyocellular lipid contents of the soleus muscle in non-diabetic, non-obese humans.309 The response to exercise of the gastrocnemius muscle of 11 patients with insulin-dependent diabetes mellitus has been measured with 31PNMR. At rest, two patients had low PCr levels, two had high Pi levels and the remaining were within normal limits. Following exercise, five patients had a slow rate of recovery of PCr, five patients had a slow rate of recovery of Pi and eight patients showed a defective recovery of pH.310 31P NMR has been used to determine the effects on muscle bioenergetics of growth hormone deficiency (GHD). The effects on muscle bioenergetics of growth hormone treatment, with recombinant human growth hormone, were also determined. However, there were no differences observed in the bioenergetics of muscles in GHD patients compared to controls. Furthermore, differences in muscle strength between patients and controls, and changes in muscle strength following growth hormone treatment, could be accounted for by changes in muscle rnas~.~'' The kinetics of oxygen uptake and the kinetics of PCr during the adjustment to, and recovery from, plantar flexion exercise has been measured in moderately active older (66.9 years) and younger (27.5 years) individuals. The kinetics of oxygen uptake were similar in the two groups as were the kinetics of PCr detected by 31PNMR.312The energy metabolism of the gastrocnemius and soleus muscles during voluntary and electrically induced contractions has been investigated with 31P NMR. Anaerobic ATP turnover in soleus and gastrocnemius muscles was slower during continuous voluntary contraction compared to that during continuous electrically induced contraction. There was no significant difference in anaerobic ATP turnover during electrically

494

Nuclear Magnetic Resonance

induced and voluntary brief contractions, but the turnover of ATP was significantly faster in the gastrocnemius muscle compared to that in the soleus muscle. During continuous voluntary contraction ATP turnover was slower in the gastrocnemius muscle compared to that in the soleus muscle.313The energy metabolism of tibialis anterior muscles has been investigated in control subjects, trained endurance runners and trained sprinters. Peak torque per unit muscle volume of the dorsiflexors was higher in sprinters compared to other subjects. When subjects performed work at 50% maximal exercise for 50 s under cuff-arrested circulation to produce anaerobic conditions, the ATP turnover in controls and endurance runners increased rapidly at 20 s compared to initial values and then increase progressively afterwards. However, the ATP turnover in the muscle of endurance runners remained relatively constant.314 The influence of repeated ischaemia and reperfusion on the human calf muscle has been investigated with 31P NMR and infrared spectroscopy to estimate changes in oxidative and glycolytic metabolism; 23Na NMR was to estimate tissue edema.^'^ 31P NMR has beer, used to detect an undershoot of free ADP, Pi and the value of AGATP to below resting values during recovery from severe ischaemic exercise. The results have implications for the understanding of the mechanisms of control of oxidative phosphorylation. 316 31PNMR has been used to investigate some aspects of human bioenergetics: the transport of Pi from the cytosol to the mitochondria, the role of Coenzyme Q in the efficiency of oxidative metabolism in muscle and brain, and the functional relationship between cytosolic free Mg2+ and cell bioenergetics in the The functional and metabolic status of muscles in patients with fibromyalgia has been assessed with 31PNMR. Functional status was determined from the PCr/Pi ratio, phosphorylation potential (PP) and total oxidative capacity (Vmax) compared to healthy subjects. Patients had lower than normal PCr and ATP levels and PCr/Pi ratios in the quadriceps muscles at rest. Values for PP and V m a x were significantly reduced during rest and exercise.31831Pand 13C NMR has been used to investigate a purely muscular form of phosphorylase-kinase deficiency. The results showed this condition could be distinguished from other clinically similar metabolic m y ~ p a t h i e s . ~ ' ~ The effects of adenylosuccinase deficiency on muscle energy metabolism of a patient, before and after exercise, has been investigated with 31P NMR. At rest, the subject had a decreased PCr/Pi ratio and ATP was depleted after mild exercise. Furthermore, the rate of resynthesis of PCr was markedly slower compared to controls.320 The effects of malnutrition on the bioenergetics of skeletal muscle have been investigated with 31P NMR. In malnourished subjects with an average body mass index of 16.4 kg m-2, the ratio of PCr to ATP was 2.28 0.27 compared to 3.1 k 0.15 in controls; the ratio of Pi to ATP was 0.33 k 0.04 compared to 0.48 0.03 in controls. There was no difference in the ratio of PCr to Pi between malnourished and control subjects.321The effects of alcoholism on muscle energy metabolism has been investigated with 31PNMR in conjunction with near-infrared light absorption to assess the levels of o ~ y h a e m o g l o b i n . ~ ~ ~ 31PNMR has been used in a study of the effects of creatine supplementation _+

+_

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on muscle metabolism in young (30 k 5 years old) and middle aged (58 k years old) healthy volunteers. Subjects performed two bouts of single-leg knee extension exercises for 2 min and a third bout to exhaustion. The middle aged group had lower resting PCr levels compared to the young group (35.0 k 5.2 and 39.5 k 5.1 mmol kg-', respectively) and a lower resynthesis rate (18.1 k 3.5 against 23.2 k 6.0 mmol kg-l min-', respectively). After 7 days of creatine supplement, the resting PCr rose by 15% in the young and by 30% in the middle aged group compared to controls; the mean initial PCr resynthesis rate and time to exhaustion were also the same in both creatine-treated The effects of glucose or Mg2+-loading on the pH and [Mg2']i of human calf muscle has been investigated in healthy volunteers using 31PNMR. Following a standard glucose tolerance test or, an i.v. load of 4g MgS04, no significant changes in pH or [Mg2']i were observed in 31Pspectra.324In a further study, the effects of a Mg2+-load on the pH and [Mg2+]i of calf muscle was investigated with 31PNMR in patients with essential hypertension, with or without insulin resistance. Whilst no changes in [Mg2']i were found following loading there was a slight, but significant, decrease in pH in patients with essential hypertension.325 Localised 'H NMR using the STEAM sequence, with a long mixing time, has been used to investigate lactate levels in muscle during breaks between periods of exercise. The method was tested using perfused rabbit biceps brachii muscle which allowed metabolic changes to be followed before and after cessation of muscle perf~sion.~lP NMR was used to detect a decrease in HEP and pH. A linear relationship between lactate levels and pH was noted. In human subjects a marked increase in the lactate peak occurred in response to exercise at peak anaerobic 5.6 Tumour - The concentration of citrate in the pathologic prostate gland has been correlated to the water T2 relaxation time for the same tissue. The results indicate that these measurements may be used in the differentiation of normal and benign diseased tissue from prostatic carcinoma.327A comparison has been made of 'H NMR-detected citrate levels in prostate cancer, benign prostatic hyperplasia or normal central gland. Lower ratios of citrate/Cho+Cr and citrate/lipids were consistently found in tumours compared to benign prostatic hyperplasia or normal central gland.328 'H NMR spectroscopic imaging has been used to investigate the distribution of lactate in tumours and brain tissue of 18 patients with cerebral gliomas. In the majority of the cases the lactate content of the peritumoural oedema was lower than that in the tumour margin or centre. This suggests that the lactate in the peritumoural oedema originates in the tumour. In 3 of the 18 cases studied, lactate was higher in the peritumoural oedematous tissue compared to the tumour region and it was suggested that this was the result of ischaemia caused by the oedema .329

5.7 Adipose Tissue - The measurement of the composition of adipose tissue by in vivo 13CNMR or gas-liquid chromatography of biopsy samples has been

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Nuclear Magnetic Resonance

investigated in volunteer subjects. The results from measurements made by 13C NMR were shown to be highly reproducible but could significantly differ from the results obtained from biopsies. Analysis revealed that the discrepancies arose because of problems with either technique, though NMR assessment of adipose tissue would still be useful for long-term, repeated measurements.330

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13 Nuclear Magnetic Resonance Imaging BY TOKUKO WATANABE

1

Introduction

NMR is now frequently the technique of choice for the determination of chemical structure in solution and in solids and mobility at the molecular level in all phases. The research literature in the subject is vast and ever-increasing. NMR imaging has been developed as a powerful tool in medical diagnostics at the early stage, but the technique is also receiving increasing attention in material science because it is non-invasive and because of the abundance of different contrast parameters which reflect the molecular properties of the samples. The number of papers concerning NMR imaging is increasing in variety of the field as well. This review covers the progress in the field of NMR imaging (NMRI), NMR microimaging or microscopy over a period from June 1998 to May 1999 and is a continuation of the last three years'. The topics were limited to mainly basic and technical aspects and the non-clinical research field, such as physicochemical, biological, physiological, pharmaceutical, geological, environmental, and industrial applications carried out by employing a high-resolution NMR machine or a horizontal MRI machine for animal use. A lot of papers on a clinically oriented MRI by using a whole body MRI machine have been published in the period, but almost all of such papers have not been picked up because of their clinical or medical interest. A recent technical development of the whole body MRI machine, however, allows us to observe microimaging of a tiny part of a human body, which has been included in this review. In some cases the technical development for imaging methods or pulse sequences precede in the clinical MRI machine rather than in the highresolution NMR machine because of the severe, specific demands for using with patients. Such technical developments can be available in the highresolution NMR machine and they have been partly picked up. Journals that concentrate on the development and applications within the field include Journal of Magnetic Resonance, Magnetic Resonance of Medicine, Journal of Magnetic Resonance Imaging, Magnetic Resonance Imaging, NMR in Biomedicine, Magnetic Resonance in Chemistry, Journal of Computer Assisted of Tomography, as well as more clinical publications, Radiology, American Journal of Roentgenology, American Journal of Neuroradiology, Neuroimage and Investive Radiology. Theoretical aspects of the field are often documented in Medical Physics and hardware developments regularly make an appearance ~~

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Nuclear Magnetic Resonance, Volume 29 0The Royal Society of Chemistry, 2000

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in the Review of Scientific Instruments. Concepts in Magnetic Resonance which presents fundamental aspects of the technique and Magnetic Resonance Quarterly which contains review articles of various topics of the field are recommended from the educational view point. The abstracts from the Annual Meeting of International Society for Magnetic Resonance in Medicine (ISMRM) is recommended as a means of deriving concise, up to date information on developments within the subjects.

2

General Aspects and Reviews

Parameters and symbols for use in NMR have been recommended based on the IUPAC (International Union of Pure and Applied Chemistry) recommendation 19972. Many articles do not contain sufficient information for experiments to be repeated elsewhere, and there are many variations in the usage of symbols for the same physical quantity. The present recommendation aims to provide simple check-lists that will enable such problems to be minimized in a way that is consistent with general IUPAC formulation. The document presents recommended notation or use in journal publications involving a significant contribution of NMR spectroscopy. The recommendations are in two parts: (1) experimental parameters and (2) a list of symbols to be used for quantities relevant to NMR2. The rethinking of the assumption behind its standard picture, which is leading toward substantially improved pulse sequences, permitting measurements of entirely new parameters, and extending the range of sample sizes and molecular sizes accessible by NMR, has been proposed and substantial improvements have been predicted3. The experimental signals are large and demonstrated applications include improving contrast in magnetic resonance imaging and removing inhomogeneities without removing chemical shifts. The task is not so much to see what no one has yet seen, but to think what nobody has yet thought about that which everybody sees. The use of gradients for providing spatially dependent information has been reviewed with 344 references, which covers non-clinically oriented imaging, especially high resolution imaging ( i e . , NMR microscopy), the types of experiments that are currently feasible and their applications4. In this article a conceptual introduction to making imaging, a discussion of the building blocks of imaging pulse sequences, the physical phenomena that produce contrast in images, the pulse sequences that can be used to incorporate these sources of contrast into the image are described. The technical and hardware aspects of imaging are presented as well as a discussion of artifacts and complications in imaging. A review with 288 references on recently induced or improved NMR imaging techniques and their applications includes linenarrowing techniques, large gradient techniques, three-dimensional imaging, NQR imaging, magnetization-grid rotating-frame imaging (MAGROFI), use of spin-polarized * 29Xe, fast low-angle shot (FLASH) imaging, and magnetic resonance force microscope imaging5. The principles of contrast in solid state

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NMR imaging6, basic filters (relaxation times, chemical shift selectivity, field distortion from susceptibility differences, and relaxation on multiple excitation for manipulation of the dipole-dipole interaction in solids) for preparation of magnetization7 and contrast in solid-state NMR imaging* were discussed as a series. Exploitation of spectroscopic dimensions for generating contrast in the imaging is addressed as are possibilities for generating contrast by temperature variation, electric fields, and contrast agents*. Solid state NMR imaging and its spatial information are discussed and reviewed with applicationsg. NMR measurements of flowing materials are reviewed with emphasis on applications to multiphase flows with a brief presentation of NMR physics and a discussion on experimental considerations related to flow measurements'O. Both imaging and non-imaging NMR as well as topics such as earth's field NMR and rapid imaging are covered. Specific topics are tagging and time-offlight, phase measurement of velocity, diffusion, turbulence, and calibration and validations. Recent applications are reviewed in the areas of sedimentation, suspension flows in Couette and pipe geometries, rheometers and viscometers, liquid-liquid multiphase flows, porous media flow, granular flows, and turbulencelo. Application to flow and diffusion processes, stress, strain, and orientation imaging, gels, forms, elastomers, polymers, skin tissue, at cryogenic temperatures, and to chemical waves are discussed. NMR imaging in material science is reviewed with 23 references in German' l . Development of NMR imaging based on viscometric technique using gradient NMR for characterizing fluid materials under steady tube flow conditions has been reported12. The distributions in microscopic molecular reorientations and macroscopic coherent motion can be imaged by appropriate techniques. A method for investigations of surface-near volume elements of arbitrarily shaped samples is also shown. Various solid state NMR approaches and underlying principles for studying polymer interfaces were reviewed with specific examples of application (28 references) 3. Structures and dynamics of hydropolymer gel systems were elucidated microscopically and macroscopically by means of NMR imaging and pulse-field gradient spin echo NMR spectroscopy with a short review of 13 reference^'^. An introduction to NMR spectroscopy and imaging in relation to applications in the field of environmental science and engineering is given15.Then, the use of NMR is illustrated and reviewed in studies of biodegradation and biotransformation of soluble and solid organic matter, removal of nutrients and xenobiotics, fate of heavy metal ions, and transport processes in bioreactor systems. Prospects for in vivo NMR methods in xenobiotic research in plants is described with reviews in relation to: (i) the characterization of the effects of xenobiotics on the metabolism of plants and plant cell suspensions; (ii) the direct detection and their degradation products in vivo; and (iii) the spatial location of xenobiotics and their derivatives at the subcellular and tissue 1evelsl6. Component separation in NMR imaging and multidimensional spectroscopy through global-squares analysis has been reviewed and suggested as a

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potentially powerful tool in functional MRI17. In the in vivo oriented fields reviews are described on technical topics such as intravascular delivery of hyperpolarized ‘29Xenon for in vivo MRI18, recent advances of in vivo MRI with laser-polarized noble gases19, role of high field MR systems in neuroscience20,and in diseases2’.

3

Instruments

A practical cryogenic probehead applicable for NMR microscopy at 7 T was developed22.The superconducting coil is composed of a YBa2Cu307 (YBCO) layer on a LaA103 substrate and consists of a continuous flow cryostat with room temperature bore for the sample. The sample can be positioned in the center of the coil. To evaluate the gain in sensitivity, spin-echo images are presented which compare the YBCO resonator to a room temperature and a cryogenic Cu resonator. The potential of superconducting coil for NMR microscopy was estimated by theoretical comparison of a YBCO resonator with a microscopic solenoid with three winding. For NMR skin imaging a multi-turn transmission line resonator made from a sandwich YBaCuOSapphire-YBaCuO has been developed23.The use of high temperature superconducting materials at low temperature with small-sized antennas considerably improved the image quality at the surface of the body. A new developed head and body holder for performing functional MRI in fully conscious animals has been reported24. A NMR stray field imaging (STRAFI) method was developed to obtain a high field gradient. A novel variant is presented in which an additional magnetic field sweep coil is used to sweep the region of resonant frequency through a stationary sample25. The technique using an integral superconducting field sweep coil as part of a spectroscopy magnet and a room temperature field sweep coil mounted inside on imaging magnet is detailed. The results of one-dimensional profiles of ‘Perspex’ and poly(tetrafluor0ethylene) samples from both field sweep coils are compared favorably with ‘conventional’ STRAFI. A simple arrangement for suppressing convection in NMR probe has been tested experimentally by diffusion experiments to detect the onset of convection and by ‘H temperature imaging26. A convenient new ‘H NMR thermometer, CH2Br2 dissolved in a nematic thermotropic liquid crystal, is presented26. Some different magnetic field measuring techniques using NMR, and the conditions to achieve such measurements, are described with the fundamental main characteristics of the NMR magnetic measurements, such as accuracy, independence of field direction and zero temperature ~oefficient~~. Recent NMR application, used in various domains such as MRI and accelerator-magnet alignment, are also described. Dedicated equipment has been designed for solid state chemical shift imaging in the spatial direction coinciding with the rotation axis of a 13C CP/ MAS probe2! It involves a two-turn B1 gradient coil whose plane is perpendi-

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cular to that axis and a lH-13C doubly tuned saddle coil used for normal spectroscopic operations. The two-dimensional diagram with 3C chemical shift in one dimension and the space variable in another dimension is provided. For an extension of the solid state NMRI based on magic angle in the rotating frame (MART) line narrowing approach experimental details, especially regarding probe coil design, recently improved, are discussed and some results are presented29.The modified magic angle is able to yield TI, maps of large band polymers with remarkable contrast sensitivity and without contrast parameter alteration referable to the narrowing procedure. A low field (21 G) laser polarized 3He gas MRI system was designed for potable use for diagnosis of lung disease, as well as imaging of voids in porous media and within metallic systems30. A versatile high-pressure autoclave for NMR imaging is described, which allows measurement in any horizontal NMR imager using magnetic field coil system with an inside diameter of more than 70 mm31. Any sample with diameter up to 28 mm and a length of about 200 mm can be investigated. The autoclave is constructed for operating pressures up to 10 MPa and temperature controlled between 10 and 60°C. A real time NMR image reconstruction and display system for MRI was developed using high-speed personal computer and optimized for the 32 bit multitasking Microsoft Windows 95 operating system32. When the Pentium CPU was at 200 MHz clock frequency, the reconstruction time for one 128 x 128 pixel image was 48 msand that for the image display on the enlarged 256 x 256 pixel window was 8 ms. This system demonstrated the ability of the real time system for three fast imaging sequences (FLASH, multi EPI, and one-shot EPI). RF coil assemblies and the appropriate instrumentation for combining proton NMR imaging with LODESR imaging have been described. This system has enabled us to collect sequential images from the same sample using both methods33.

Pulse Sequences and Data Processing

4

Improved pulse sequences DIFN (DIFferentiation by N pulses), 90 - z (1) 180 - z (1) . . . 180" - z (n), with optimized time intervals z (1) for T1 measurement and contrast enhancing in NMRI are presented34.35. The DIFN pulse sequences showed better sensitivity to TI than the well-known pulse sequences, saturation recovery, and more reliable information of image intensity than the inversion recovery pulse sequence because the NMR signal does not change its sign. Adiabatic pulses are limited for NMRI applications due to the lack of slice-selective excitation capability, although it is useful in generating uniform spin nutation in the presence of inhomogeneous R 1field. A scheme is presented that allows single-shot, arbitrary flip-angle, and sliceselective excitation with only four adiabatic half-passage segments36. The technique is demonstrated via computer simulation and experimental tests on a phantom. O

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The relaxation time in the rotational frame, TIP,is sensitive to the molecular s. A motion characterized by correlation times ranging between lov2 and TI, filter for the NMRI of solid polymer is presented37.The method allows us to obtain a good spatial resolution without the need of strong field gradient and without the effects related to the spatial encoding manipulation of the spin system. Proton TI, dispersion imaging is a recently described method for indirect detect of I7O. In order to circumvent high SAR (i.e.,exceeding current limitation in specific absorption rate) in clinical implements an off-resonance proton TI, dispersion imaging is offered through the use of low-amplitude off~ . and off-resonance TI, resonance spin-locking pulses (yB1 < 300 H z ) ~ Ondispersion images exhibited similar 70-based image contrast for 70-enriched tissue phantoms. An alternative scheme in acquiring 13C spectroscopic images using inverse detection via polarization transfer through protons was proposed and experimentally verified by a phantom using a heteronuclear multiple-quantum coherence technique39. The signal enhancement, spectroscopic encoding via constant time, and improvement of the time efficiency of the constant time method via optimization by singular-value decomposition analysis constitute a characteristic feature. In some cases, these features yield better sensitivity than the conventional 3DFT technique without sacrificing the most important attribute of 13C, the enormity of the chemical shifts and higher spatial resolution by shifting the emphasis of signal encoding from a spectral-resolution-first viewpoint to one that favors spatial resolution. The cyclic J cross-polarization (CYCLCROP) NMR imaging pulse sequence for the acquisition of proton-detected 13C NMR images was described40. In CYCLCROP a series of two polarization transfers in the sense 1H-'3C-'H is applied before imaging in order to select a specific IH nucleus J-coupled to a 13Cnucleus and filter out all other 'H coherences. In multicomponent systems this technique can be used to acquire selective images of one of the components by suitable selection of the cross-polarization transfer pair of CH, nuclei. An important advantage of CYCLCROP imaging turns out to be its selectivity even in the case of completely unresolved 'H lines, as they are often encountered in polymer proton spectra. Selective determination of elastomer distribution in multicomponent systems was demonstrated. The CYCLCROP sequence was applied to in vivo study of carbohydrate transport and metabolites in seedling (see Section 12). Flip angle dependence of the localized single-voxel 'H NMR spectroscopic sequences STREAM and PRESS using numerically optimized Shinnar-Le Roux (SLR) and conventional sinc R F pulses was evaluated4l. The voxel profiles from MR images of the selected voxels for both sequences using the SLR RF pulses are identical, unlike the case for the sinc R F pulses. Localized water suppressed 'H NMR spectra recorded from the frontal gray matter in healthy volunteers (n = 3) showed less lipid contamination using the SLR R F pulses compared with the sinc pulses. Theoretical and experimental results for NMR imaging measurements of powder materials using the +1/2 to - 1/2 transition of half-integer spin nuclei

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in the presence of a very large second-order electric quadrupolar broadening were presented4*.An ‘effective spin-1/2’ formalism is developed to account for additional effects due to the presence of quadrupolar interactions comparable in size to the Zeeman interaction. A large (7.9 mT cm-’) and rapid (30 ps) pulsed linear gradient field was used to obtain images in the limit ?HI. DECRA (Direct Exponential Curve Resolution Algorithm), which was presented as a fast method to resolve a series of NMR mixture spectra by the present authors, was applied to two series of MR i m a g i n g ~ one ~ ~ ; is based upon T2 relaxation and the other is based upon T1 relaxation. A transformation is introduced to enable the application of DECRA to a TI series. The theoretical problem of how to describe apparent image spin density under conditions of restricted diffusion, given any general gradient sequence, is intrinsically complex. A simple approach to calculating the signal and the corresponding density in NMRI experiments is demonstrated by means of an impulse-propagator method based on matrix multiplicationM. Good agreement is found between theoretical predictions and the results of microimaging experiments on water trapped in rectangular pores whose wall are spaced by 100 pm along the read direction. The stochastic transport theory was experimentally verified by NMR imaging45. A modified stimulated echo pulse sequence was used to make velocity images in an aperiodic heterogeneous porous medium and the velocity covariance was constructed from measurements of fluctuating velocity. Use of prior knowledge with regard to the number of components in an image or NMR data set makes possible a full analysis and separation of correlated sets of such data17. A diffusional NMR microscopic image set can readily be separated into its components, with the extra benefit of a global least-squares fit over the whole image of the respective diffusional rates. The computational approach (CORE processing) is also applicable to various multidimensional NMR data sets. A new technique for obtaining water-oil capillary pressure curves, based on NMRI of the saturation distribution in flooded cores, is presented and applied to that for North Sea chalk46. This paper presents the NMR technique and the procedure for calculating the pressure distribution in the sample, especially inhomogeneous samples, where an irregular saturation profiles produced are interpreted in terms of variation in permeability, porosity, and capillary pressure. Surface NMRI of large electromagnetically active systems, for example geophysical application, was theoretically considered47.When the conductivity grows to the point where the electromagnetic skin depth becomes comparable to the sample size, significant diffusive retardation effects occur that strongly affect the signal. Accounting for these, it is shown that the TI may in principle be inferred directly from the diffusive tail of the signal. For obtaining an absolute metabolite quantification by in vivo NMR spectroscopy European multi-center trials have been organized and the main results for four projects are reported; namely, I. Introduction, objectives and activities of a concerted action in biomedical research, 11. Multicentre trial of protocols for in vivo localized proton studies of human brain, 111.

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Multicentre 'H MRS of the human brain addressed by one and the same data-analysis protocol, and IV. Multicentre trial on MRSI (Magnetic Resonance Spectroscopic Imaging) l o ~ a l i z a t i o nThe ~ ~ . difference between the experimental and theoretical spatial response function of a narrow tube with water is used for a localization test for MRSI. From this difference a quantitative performance parameter is derived for the relative amount of signal within a limited region in the field of view. Results of a European multi-center trial show that this approach is suited for assessment of MRSI localization performance. Automated spectral analysis of in vivo 'H MRS49 was applied to the spectroscopic imaging and the reconstruction of metabolite d i s t r i b ~ t i o n ~ ~ . i

5

Solid State NMR Imaging

Technical and analytical developments of solid state NMR imaging strongly attract attention in the field of materials, especially polymers. As mentioned in the sections 2, 3 and 4, various solid state NMR approaches have been done with a view to better sensitivity, better contrast, better spatial and temporal r e ~ o 1 ~ t i o n ~For ~ preparing ~ ~ ~ ~ magnetization ~ ~ ~ ~ ~ ~ in~ imaging ~ * ~ ~advanced - ~ ~ . Biters are described, which are based on combinations of different filters for introducing combined parameter weights - including spin diffusion weights and on multiple-quantum coherences8. The relaxation time in the rotating frame T1 is observed for space-resolved low frequency dynamics in entangled polymers37,and TI, maps of large band polymers29. Mechanical deformation in rigid polymers was investigated by two-dimensional solid-state NMR imagingS1.Variation in local molecular mobility in cold-drawn rigid polycarbonate samples is visualized by using a magic-echobased 2D solid state NMRI sequence that has been supplemented by a T2 relaxation-filter sequence for probing mobility contrast. Characteristic regions of chain immobilization caused by the mechanical treatment are mapped by NMR parameter images. A very interesting application of solid state NMRI was reported, that is MRI of laser-polarized 129Xein the solid phase at low t e m p e r a t ~ r e ~Due ~ . to the high xenon density in the solid phase and the enhanced spin polarization, it is possible to achieve high intensity and spatial resolution of the image. Signals were observed from xenon films solidified onto the glass container walls and not from an enclosed chili pepper. It is a new idea to use polarized xenon not in the gas phase but in the solid phase. Three-dimensional solid-state 31P NMR projection imaging was used to evaluate the bone mineral density53.Multiple gradient directions covering the unit sphere are used in an efficient spherical polar to Cartesian interpolation and Fourier transform projection reconstruction scheme to image the threedimensional distribution of phosphorus within the specimen. Comparison of solid state NMRI with dual energy X-ray absorptiometry (DXA), gravimetric analysis, and chemical analysis of calcium and phosphorus demonstrates good quantitative accuracy.

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517

Other Nuclei

Application of other nuclei to NMR imaging must be a big challenge, except for some nuclei. The number of the papers on this topics has not increased during this period. An effort and/or idea has been brought into the development of other nuclei's NMR imaging. In this section articles reviewed are in the fields except in vivo (see section 13 for the application to in vivo systems). Noble gases with and without a laser p~larization,'~C-,'~O-,~~P-NMR imaging has been reported, but the number is not so many. In most of applications, such as 3He and 129Xe,the gases have been induced into the porous systems. Diffusion measurement or relaxation time measurement of the gases in the pores is performed more than NMR imaging. Pore-structure determinations of silica aerogels by 129XeNMR spectroscopy and imaging were reported54. Xenon exchange and diffusion measurement was also discussed for the determination of the pore structure. The combination of 129XeNMR spectroscopy and the 'H NMR imaging are often used to estimate a heterogeneous distribution both at the nanometric and macroscopic scales55. MRI of thermally polarized samples (e.g. H20) is not practical at the very low field such as 2.1 x T. MRI of laser polarized 3He gas is possible at such low field30. Low-field noble gas MRI for portable use will have novel scientific, engineering, and medical applications. As mentioned above, laser polarized 129Xein solid phase was used at low t e m p e r a t ~ r e ~ ~ . 13C chemical shift imaging via inverse detection of protons through a metabolite imaging of lines in a spectrum (MILS) (or inverse SLIM) technique was reported as an alternative technique in acquiring 3C spectroscopic images39.Proton-detected 13C imaging was applied to selective determination of elastomer distribution in multicomponent systems40. Proton spin-lattice relaxation times in the off-resonance rotating frame were measured in 1 7 0 enriched tissue phantoms and on- and off-resonance T1 dispersion imaging was implemented at 2 T using a spin-locking preparatory pulse cluster appended to a standard spin echo sequence38. One-dimensional projections of 63Cu in Cu20 powder and in a sample containing Cu20 and YBa2C~306.7 were presented as examples for NMR imaging using second order quadrupole broadening resonance42.

7

Diffusion, Flow and Velocity Imaging

7.1 Diffusion Tensor Imaging, Diffusion-Weighted Imaging and One-Dimensional Mapping - Diffusion tensor imaging (DTI) of biological systems (control and mechanically injured spinal cord of a rat in vitro and in vivo) are ~ ~ . measureobtained on 6.4 T and 9.4 T magnetic resonance m i c r o ~ c o p e The ment of the spin echo NMR in the earth's magnetic field demonstrated the usefulness of the method for diffusion- and flow-weighted imaging57.Due to a weak Larmor field, the usual definition of the magnetic field gradient must be replaced by a generalized formula valid when the strength of a nonuniform

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magnetic field and a Larmor field are comparable. Generalized calculation of NMR imaging edge effects arising from restricted diffusion in porous media is demonstrated by means of an impulse-propagator method based on matrix mu1tiplication4. Pulsed field gradient (PFG) NMR and microimaging experiments were performed to study the diffusion of liquid alkanes into a variety of semicrystalline polyethylene (PE) samples. The results highlight the importance of the crystal phase in controlling the diffusion process in terms of both the geometric impedance imposed by the presence of impenetrable crystals and their effect on the mobility of the polymer chains comprising the amorphous material through which the penetrants migrate5*. Water diffusion into the polymer electrolytes (Pb, Zn)(CF3S03)2 (PEO), was studied by one-dimensional mapping of properties such as a spin density and relaxation times59.The water absorption in the materials studied can be classified as Fickian diffusion and water is absorbed mainly by the amorphous regions. Hydrogels are being developed for application in areas such as controlledrelease of drugs, in intraocular lenses and for water sorption. Detailed studies of the diffusion of water were carried out in hydrogel polymers, copolymers and IPN (Interpenetrating Polymer Network)s based on hydroxyethymethacrylate (HEMA) and other less hydrophilic monomers, such as tetrahydrofurfuryl methacrylate60, poly(viny1 alcohol) (PVA) based samples6', hydroxypropylmethylcellulose (HPMC) tablets62. One-dimensional profiles weighted to the variation of water content and diffusion-weighted profiles provide the supporting evidence for the hypothesis that phenomena such as reptation are important near the glassyhubbery interface of polymers during dissolution, while the diffusion gradually changes to Zimm type near the rubberyholvent interface6'. For the HPMC tablets the reason for an overestimation of the total polymer weight calculated from polymer concentration distributions in the swollen matrices by NMRI was traced to the presence of air bubbles in the swollen matrix62.Internal flow process of water in polymers was studied by 'H-NMR microscopy at 500 M H z ~ ~ . In order to detect water movement during the extrusion of pastes, water distribution using MRI was analyzed and the influence of water content and the speed of the ram on water movement was assessed as extrusion process progressed. Two types of water movement, vertical and radial, were detected? Heterogeneous distribution both at nanometric and macroscopic scales caused by coking of an HY zeolite pellet was shown by combined use of '29Xe NMR and 'H-imaging NMR55. This distribution depends upon the direction of the flow of reactant. Occurrence of diffusion of dimethylpentane through both uncoked and coked sections of the pellet was observed. The fluid exchange of methanol for liquid C02 at a pressure of about 7 MPa at 15 "C in Si02 alcogels was observed on line by using a p r ~ b e h e a dspecially ~~ designed as an autoclave65.The process of drying of alumina and titania porous catalyst supported pellets impregnated with various liquids was studied by detecting one-dimensional liquid concentration profiles66.A simple experiment is proposed that yields an empirical relation between the detected profile intensity

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and the actual liquid content, which allows one to correct the profiles for the inevitable T2-weightedeffects. 7.2 Flow, Velocity Profile, Velocity Imaging, Rheometry - Velocity profiles of water and 0.5 wt.% (consistency) wood pulp suspension flowing through a circular pipe in the vicinity of a 1 : 1.7 tubular expansion were measured67. Both laminar and turbulent flows for water, and upstream and downstream from the expansion plane for pulp suspension flow are investigated. Flow images are provided for various flow rates and axial positions with respect to the expansion plane. Fiber floc network was disrupted and the fiber reflocculation length is proportional to the bulk flow rate. A mean flocculation time of approximately 510 ms was determined for this pulp fiber consistency. A concentric cylinder Couette flow system was experimented by NMR imaging6*. Velocity image in porous media is a big target for NMR imaging45.69-71. Two NMR velocity imaging methods based on the pulsed field gradient stimulated echo technique have been evaluated for the measurement of fluid flow in porous media. A theoretical formulation for data interpretation is presented and demonstrated with experimental data for water flow in a glassbead pack and sandstone samples69.NMRI data measured two-phase displacement experiments on porous rock samples are used in an inverse problem methodology to estimate the two-phase flow functions relative permeability and capillary pressure70. A precise agreement between measured data, which also includes pressure drop across the core sample, and the data simulated by using the estimated flow functions is found, indicating accurate flow function estimates. The direct three-dimensional measurement of flow in rough-walled water-saturated rock fractures is presented. Rock-water interfaces and water flow velocities, effects of wall morphology on flow patterns inside a typical rock fracture have been in~estigated~l. The features discussed in this study emphasize the strong heterogeneity and the highly three-dimensional nature of the flow patterns in natural rock fractures and consequently the need for threedimensional flow analysis. NMR imaging based viscometric technique using pulsed gradient NMR was developed for characterizing fluid materials under steady tube flow condit i o n ~ ~By * . simultaneously measuring velocity profiles and pressure gradients it is possible to characterize complex fluids locally. Shear viscosity-shear rate data, ranging from one to over two decades of shear rate, are provided for five fluids that exhibit both Newtonian and shear thinning characteristics. Velocity profile measurements with a velocity resolution of 1 mm s-' and radial resolutions, which provided from 22 to 110 spatially resolved velocity data points accurately characterized fluids for shear rates >0.1 s - l . A modified version of the shear-induced migration model to predict transient torque reductions in torsional startup flows and transient pressure drop reductions in capillary developing flows has been reported73. In this paper special attention was devoted to the relationship between the evolution of the driving forces and various mechanisms that contribute to the shear-induced migration of the

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suspended particles. A narrow-gap, temperature-controlled Couette flow rheometer has been developed to study fluid velocities within the annular gap between two concentric cylinders by NMRI and ~ e l o c i m e t r y ~ ~ . Dispersive flow in chromatographic column has been i n v e ~ t i g a t e d ~ ~ - ~ ~ . Pulsed field gradient multi-echo and turbo-echo imaging75,or a combination of pulsed field gradient NMR and NMR was used. By using the pulsed field gradient turbo-echo sequence spatially resolved displacement imaging can be accelerated by a factor of 1675. The positive effect of homogeneous radial packing on flow velocity and dispersion is demonstrated. Small heterogeneous of only a few percent are shown to cause changes of the dispersion coefficient of up to 50% in radially compressed columns packed with porous silica beads7s. A discrete spatial and temporal motion of the molecules of a fluid percolating through the packed bed of a dynamic radial compression column was determined76.The local axial dispersion coefficient, fluid velocity and external porosity of the bed were estimated in the resolution of 250 pmx250 pmx2500 pm (thickness) per voxel. Convective and diffusional transport in chromatographic column packed with totally porous support materials, silica gels, were studied77.Here, stagnant zones exist in the particle pores and diffusional mass-transfer limitations between fluid molecules diffusing in the intraparticle pore network and flowing in the interparticle void space are detected quantitatively. 7.3 Mass Transport, Absorption, Sedimentation, Precipitation - NMRI is used to study rates of sedimentation on several non-Newtonian fluids7*. Sedimentation is most rapid at the beginning of the process and grows steadily slower in time. Two-dimensional images of vertical section of suspension showed clearly the development of a nonhomogeneous microstructure during sedimentation in a viscoelastic fluid. The phase behavior of D20 solutions of phosphine-polyacrylic acids (PPAA) which belong to a widely used class of threshold inhibitor, was studied in presence of Ca2+ ions and in a column packed with quartz sand79. Profiles of the distribution of phosphin-PPAA during flow of the solutions inside the sandpack are obtained by TI-weighted spin-echo images and a precipitation phenomenon are obtained by spatially resolved T2 estimation. No adsorption occurred under the conditions adopted, whereas precipitation was visualized at the interface areas between the different solutions. The phenomenon was very slow and mainly governed by diffusion of the chemicals79.NMR micro-imaging has been used to investigate concentration polarization phenomena in membrane filtration of colloidal silica suspensions using a single tubular microfiltration membrane, with the feedstock fed to the inner lumen of the membrane and the filtrate removed from the outer shell sideg0. T1-weighted signal intensity of the solvent protons clearly exhibit details of the formation and dissipation of the silica particle concentration polarization layers at the surface of the membrane in response to changes in trans-membrane pressure difference and feedstock crossflow rate. The flow profile of the polarization layer presented in this paper provides direct experimental evidence for fluidity and motion of concentration polarizaI

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tion layers, an assumption which has been invoked for the development of some theoretical models but which has not previously been confirmed experimentally80. The tendency for selected organic compounds to sorb to sediments has been studied. These studies, however, have not permitted the observation or measurement of advection/diffusion processes and the breakdown of organics within sediments. MRI was used as a means of monitoring movement of oil within a series of estuarine sediments, thus offering a method of assessing the harming potential of oils in nearshore environments8*.

8

Porosity, Fluid Assisted Images

The spatial dependence of the droplet size distribution in the cream layer of oil-in-water emulsions (containing various fractions of gum xanthan thickener dissolved in water) was determined by pulsed field gradient spin-echo NMR restricted diffusion analysis and NMR imaging82. lH-13C cyclic J crosspolarization POSEm is introduced as a technique for this purpose in cases of selective observation of the oil component (or other carbohydrate constituent). The experimental results are compared with a recent model of the creaming process due to Pinfield et al. (1994). Theoretical aspects of the estimation of pore and mass fractal dimensions of porous media, such as porous catalyst support pellets, on the macroscopic scale were studied by spin density and TI contrasted NMR images. This method is suggested as a way of selecting a suitable model to simulate the structural properties of real porous materials over length scales from 0.01-10 mm83. Macroscopic disorder in a porous glass was characterized by NMR mi~roimaging~~. Intensity fluctuations in one-dimensional NMR imaging profiles were obtained and an approach to characterize some aspects of the macroscopic randomness was developed. Statistical properties of the distribution of peak separations between the intensity fluctuation are shown to reveal information about the pore size and the pore-to-pore distance in porous media. The approach developed to characterize the macroscopic randomness in these porous glass filters should prove generic for the study of other random materials. NMR relaxation and q-space diffusion measurements have been used to probe the microscopic water distribution in a variety of starch-based systems, including packed beds of native starch granules with varying water contents, starch gels, and freeze-dried starch gels85.The q-space data for the granular beds is compared with a variety of theoretical models and conforms best to unbounded diffusion in a low dimensional space. In order to know more about internal structure of industrial or civil engineering materials in relation to their macroscopic behavior, MRI coupled with an analysis of respectively freezing and relaxation effects was applied86.A mathematical treatment of data obtained by these techniques directly provides spatial maps of the different moments of the pore size distribution.

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Water molecular dynamics in porous building materials such as a hardened cement and mortar were characterized by proton NMR Three different types of water molecules (physically bound, chemically bound, and porous trapped) were observed in a hardened Portland cements7. The loss of the physically bound water by heating at 105°C proved to be a diffusioncontrolled process, and a Pake doublet observed is a result of the oscillation of the water molecules with hindered molecular motions due to their entrapment in the cement pores. T2-weighted imaging showed that the distribution of the physically bound water inside the cylindrical sample formed a doughnut shape after overnight soaking the dried samples in water. NMR microscopy, T2 mapping, self-diffusion constant measurement and magnetization transfer experiment of water penetration into glass-ionomer cements (chemically and light cured samples) were performedss. The distinction in internal structure of the two materials is clearly visible after few days when the water has reached the centers of both samples. Variations in pore size through the sample are responsible for signal inhomogeneities of chemically cured samples. For mortar moisture profiles and relaxation were measured during water absorptions9. The effect of the drying treatment on the microstructure and the water absorption was investigated.

9

Polymers

Reviews on polymer materials are summarized in section As mentioned in section 4 on pulse sequences and section 5 on solid state NMR, polymers in the solid state are widely targeted by NMRI. TI, filter for the solid state NMR imaging is applied to space-resolved low frequency dynamics in entangled polymersg0. Molecular motion and orientation distribution in melt-processed, fully aromatic liquid crystalline polyesters were studied via rigorous simulation of wide-line spectral lineshapes and via a extension of rigorous NMR spectral deconvolution to recently developed solid state NMRI sequencesg1. The interaction of the morpholgy with the dielectrical aging inside the polyethylene insulation system has been subject of many investigations. The aged specimens were investigated with the NMR imaging especially with regard to morphological changes in electric treeing which is the last phase of the dielectric aging processg2. For this purpose we need information on morphological features with a size of a few nanometers. The morphological information can then be gained by comparing the experimental result with analytical and numerical solutions of the diffusion equation for different boundary conditions reflecting the morphology. The size of the crystal and the amorphous regions and the interface between them are examined. Shrinkage of crosslinked poly(methacry1ic acid) gel under application of an alternating electric field was demonstrated by the spatial distribution images of 'H spin density and 'H spin-spin relaxation time of water molecules93. The translational behavior of water molecules in the gel during shrinkage processes is discussed. 2531'7'4.

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The number of papers concerning inhomogeneous elastomeric materials is i n ~ r e a s i n g ~ ~By* selecting ~ ~ - ~ ~ the . CH methine proton of polyisoprene in the CYCLCROP NMR40 edited 'H images of commercial natural abundance cispolyisoprene, na-PI, in the presence of a second elastomeric materials were obtained, whose 'H coherences were completely edited out. With 3C-enriched polyisoprene, 3C-PI, CYCLCROP was employed to record images of the spatial distribution of PI in mixtures of PI with polybutadiene, PB, and poly(hydroxyoctanoate), PH040. The detection of interfacial layers in elastomer, EPDM, has been recorded by NMRI using spin- and gradient-echo technique for differentiating between voids and inhomogeneities inside the elastomer by characteristic changes of the magnetic s~sceptibility~~. Layer-like structures were observed, which result from folding of the material during the mixing process. Interface coupling of silica in Zn activated, S-vulcanized cis-polyisoprene (75% cure state)95 and molecular mobility in carbon-black-filled, TBBS/Svulcanized cis polyi~oprene~~ were studied by NMRI. NMRI showed an increase in the T2 relaxation times and a decrease in the proton spin density as the filler load increases in the former case95. In the distribution of T2 in the carbon black filled rubbers, two T2 relaxation times are observed, reflecting the existence of the hard regions adjacent to the crosslinks or filler particle and soft regions distant from such rigid component^^^. The aging of rubber filler materials was studied by common spin-echo NMRI and a parameter-selective NMRI (material properties The decay of the echo-magnetization is discussed on the basis of a single chain model with a distribution of dipolar interactions. This model is based on the influence of a very fast but anisotropic local motion as well as larger and slower motions, which are able to diminish the residual dipolar interaction. The behavior of crumb rubber materials, natural tire rubber and styrenebutadiene tire rubber, immersed in a high acid-content asphalt and heated at 70 "C, was studied by NMR 3D imaging. Images showed swelling of the crumb rubber materials in the presence of the asphalt, whilst they showed only a small loss of signal with heating and hardly any swelling in the absence of asphaltg8.NMR images were obtained on asphaltene precipitation in asphalt which was dissolved in toluene and titrated with isooctane to the point of onset of flocculation. Images showed that a layer of material formed at the solvent interface which was different from the asphaltenes or the m a l t h e n e ~ ~ ~ .

'

10

Rock, Mineral, Clay, Coal

NMRI of 'H has been applied to hydrous mineralslOO~lO1. The three-dimensional imaging of hydrogen-bearing samples (stilbite, agate, and enhydros) from geothermal fields was obtained for the first timeloo.In order to overcome the problem caused by very short T2 relaxation time of 'H nuclei, probe heads and pulse sequences were newly developed. 'H-NMRI was performed for the measurement of self-diffusion coefficients of water molecules in two smectite

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samples (saponite and montmorillonite) with 96.6 wt.% of water at 23.8 OCIO1. Eight NMR images were obtained with systematically changing the strength of field gradient pulses and the averaged diffusion coefficient was statistically calculated from 225 diffusion data for each samples (2.06 x lop9 and 1.52 x lop9 cm2 s-' for saponite and montmorillonite, respectively). The trend in utilization of NMR in studying petroleum reservoir rocks is NMR relaxation and NMRI'02-'04. In the relaxation study T1 and T2 relaxation times and the spin density of 'H nuclei (water and oil) are measured and the response of the hydrogenated fluids are related to the properties of the rock-fluids system. In NMRI the NMR signal is codified spatially in one, two and three dimensions for nondestructive imaging of the distribution of fluids present in the Chemically-induced pore surface modification on rock cores was evaluated by NMR relaxation time and NMRI'03. Chemical modification of pore surface properties due to active components of crude oils could significantly affect production process. In this paper, carbonate core plugs subjected to aging treatments in crude oil was examined by monitoring of solid/liquid interface alteration and the results were compared with a result by Cryo-SEM. Polyanionic cellulose, xanthan and polyglycol are additives of water based drilling fluids. In laboratory experiments the influence of these additives were tested with crude oil and water saturated sandstonelM. Changes depending on the reaction of these additives with the rock were determined by NMR, NMRI and computer tomography. Irreversible changes of the rock were found resulting in a flux inhibition of the hydrocarbons. A new carbonization process with high temperature preheating has been studied by solid state NMR and NMR imaging methods to examine the mechanism for improving the coking property of coallo5. 11

Food and Food Processing

The heat and moisture treatment of starch is important to food manufacture. In cereal processing, grains must be boiled or steamed for the starch to gelatinise and be digestible. NMRI has been used to study the cooking (boiling Mapping of the distribution of water on the and steaming) central cross section of wheat grains was demonstrated for the boiling and steaming of intact grainslo6. The different results for the two processes imply that different mechanisms control the influx of water in each case, and provide important data for modeling these processes. For steamed wheat grains the moisture content distributions by NMRI, the time evolution of the moisture content by a gravimetric measurement and the grain temperature by thermocouple measurement are examinedlo7.A prediction from a model proposed for the mass and heat transfer are compared with experimental data, and the values of water activity coefficient, heat and mass transfer coefficients are extracted and compared to values in the literature where availablelo7. Using a specially designed holder, NMR image of water distribution and structural changes in cooked rice grains were obtained, even when up to 10 grains were

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studied simultaneously108.NMR relaxation and Q-space diffusion measurements have been used to prove the microscopic water distribution in a variety of starch-based systems and the experimental results were compared with a variety of theoretical predictionslog. The paper concludes with a novel method for probing pore size distribution in freeze-dried starch gels by infusion of acetone. The formation of ice during freezing of foods, such as potatoes, carrots, peas, corn, and chicken legs, was examined with an image resolution of 350 pm by monitoring a reduction in spatially located NMR signal intensity' lo. Internal structures were differentiated by water or fat content by NMR imaging. A method for measuring the viscosity of polysaccharide model meal using the known relationship between relaxation times and polysaccharide concentration in solution in vitro is presented with a aim to develop a method for monitoring digesta viscosity in vivo, using EPI to capture the motion of the gastro-intestinal lumen1l l . 12

Plant

Novel information has been generated by in vivo NMR studies of both agrochemicals and heavy metals, but a lack of generality in the methods makes it difficult to extrapolate from one successful application to the next. In vivo NMR spectroscopy has been shown to be informative for analysis of a xenobioticaly perturbed metabolic pathway and for probing the partitioning of paramagnetic metal ions between the cytoplasm and the vacuole. In contrast NMR imaging techniques have been little used for xenobiotic research in plants and have been shown to be capable of monitoring the uptake and translocation of paramagnetic ions in plants. Prospects for in vivo NMR methods in xenobiotic research in plant are described16. A particular strength of the use of NMR microimaging in research on plants is its ability to perform non-invasively repeated measurements on the same specimen. Some applications of NMR microimaging and EPR spectroscopy to the study of disease and senescence processes in plant organs, especially olive specimens, are presented112. The method for imaging and spectroscopy based on CYCLCROP technique40 was used in the first to in vivo NMR study of monitoring of carbohydrate transport and metabolites in caster bean seedlings (Ricinus communis L.)'13. In this paper, a full analysis of the effect of relaxation on the signals generated from small J-coupled spin systems by the CYCRCROP sequence is given and the high sensitivity of the sequence is demonstrated. In the in vivo study of six days old caster been seedling, the uptake of labeled hexoses, supplied in solution to the cotyledons, and their conversion to sucrose were measured, as well as the transport of this sucrose in the vascular bundles. By *HNMR spectroscopic imaging a sucrose concentration was measured at 70 min intervals in the phloem of individual bundles of the hypocotyl of Ricinus seedlings under several conditions' 14. It was observed

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that the sucrose concentration stayed fairly constant in all bundles for more than 7 hours if the cotyledons were embedded in the endosperm or excised and incubated in 100 mM sucrose. It was also described that a function equilibration of translocated solutes between the eight bundles may occur within the 1 hour measuring interval by radial diffusion through the parenchyma of the h ypocotyl. Fruit maturity was studied by NMRI in kiwi fruit115 and blackcurrant fruit'? Quantitative NMRI was used to determine relaxation changes (TI, T2) at regular intervals during growth and ripening of kiwi fruit (Actinidia deliciosa var deliciosa). Results imply that relaxation parameters determined from NMRI may not be appreciably influenced by processes that cause solution components to vary dramatically, even though these changes are reflected in the relaxation properties of the juice itself. Possible reasons for this are discussed with regard to the impact of cell structure and magnetic field strength on relaxation processes' 15. A comparative study by 3DNMR micro-imaging, low temperature scanning electron microscopy (LTSEM) and conventional resin histology was presented for the development of blackcurrant fruit from flower to maturity'I6. The NMR images derived from 2D and 3D datasets showed the previously unreported growth of arillar tissue to the extent that they almost completely occlude the locular cavity. Embryos taken from late maturation phase seeds of Phaselous vulgaris cv. Seminole prior to seed desiccation (35-45 DAA) can be induced to germinate in the absence of water by exogeneous ethylene1l7. The hypothesis that, in vivo, quiescence at this developmental stage is induced and maintained by sequestration of water within the cotyledons was discussed according to the changes of NMR imaging. Water uptake and water transport is a big topic in intact plant. A fast method applying FLASH sequence for quantitative NMRI of flow velocities in intact plants is described with a purpose to observe dynamic changes of flow velocity in the xylem of plants after fast changes of environmental conditions"*. Compared to other flow NMR imaging sequences, the imaging time was reduced by a factor of 6 with comparable signal-to-noise ratio. A complete flow measurement consists of a set of 8 different flow weighted images with a total acquisition time of 3.5 min for the spatial image resolution of 47 x 188 pm2. The intensity of tissue hydration in the barley kernel without physical dissection and its effect on metabolic activity was examined by a high resolution NMRI technique' 19. Significant differences in the rate of water uptake were observed between different tissues of the kernel, and between the small and large barley kernels. The moisture diffusivity of Jonagold apple flesh and skin was estimated based on NMR images obtained from drying experiments120. The average flesh diffusivity was calculated to be 10.3 x lo-" m2 S Kand I did not change significantly during ultra-low oxygen storage and skin diffusivity was calculated as 1.32 x 10- m2 s The potential of NMRI to study how plant organs penetrate the soil under the influence of gravitropism, mechanical impedance and thigmotropism was discussed'21. This paper shows for the first time that gravitropic experiment

13: Nuclear Magnetic Resonance Imaging

527

can be performed in artificial soil using NMRI as a convenient and suitable recording method. 13

In Vivo, Ex Vivo NMR Imaging

13.1 'H NMR Imaging (MT, Diffusion, Functional, and TL-Weighted Imaging) - Metabolites in proton exchange with water were detected via the water proton signal using saturation transfer techniques in model systems and biological tissues' 22. Using a difference imaging technique between a control irradiation above and below the water resonance, a chemical exchange of the kidney was calculated. These studies demonstrate that these metabolites can be detected and imaged via the water proton using the signal amplification properties of saturation transfer in the presence of water/macromolecular magnetization transfer. Diffusion-weighted MRI (DWI) and perfusion-weighted MRI (PWI) provide real time and dynamic data on the evolution of a thrombotic cerebral lesion with and without t h r o m b ~ l y s i s 'in~ ~the purpose of investigation of the biophysical changes of a new rat model of cerebral artery thrombotic. Combined NMR imaging and spectroscopy have been applied to mouse brain during focal cerebral ischemia * 24. The acquisition of high quality diffusionweighted, perfusion-weighted dnd T2-weighted images is demonstrated and complemented by measurements of H volume-selective spectroscopy and spectroscopic imaging (SI). This is the first report on both SI data and perfusion-weighted imaging on mouse brain. The isolated perfused rat hippocampal slices were studied using 600 MHz narrow bore spectrometer by NMR imaging of anisotropic water diffusion125. The obtained data support the hypothesis that the decrease in the ADC (apparent diffusion coefficient) of brain water following an ischemia attack is caused by cell swelling. Effect of oxidative stress on brain damage was detected by MRI and in vivo 31P-NMR126.Using T2-weighted imaging of proton, brain intensity decreased following the treatment suggesting water loss or free radical production. EPR spin trapping method confirmed production ,of free radical from anoxic-anoxia hyperoxia treated animals126. To define the myoarchitecture of the intact mammalian tongue, NMR techniques have been utilized to assess the location and orientation of muscle fiber bundles through measurement of the direct-specific diffusional properties of water molecules127. Somatosensory evoked potentials (SEP) and T,*-weighted NMRI were recorded simultaneously during somatosensory stimulation of rat to investigate the relationship between electrical activation of the brain tissue and the signal intensity change in functional NMRI12*.The feasiblity of simultaneous, artifact-free recording of T2*-weighted images and of evoked potentials is proved. An application of microimaging for 3-dimensional structure study of trabecular bone is presented with elementary types of image presentationt2'.

528

Nuclear Magnetic Resonance

Solid bone in itself does not produce any significant NMR intensity and natural lipids and/or added 70% EtOH has been used as a signal source. Threedimensional spin-echo and three-dimensional chemical shift selective spin-echo pulse sequences were applied. The basic usage of NMR microimaging for materials characterization is introduced. Cartilage formation in a hollow fiber bioreactor was studied by NMR microscopy130.

13.2 Other Nuclei-NMR Imaging - The apparent concentration of lithium (Li+) in vivo was determined to be 10.1 mM for muscle and 4.2-5.3 mM for various brain regions under dosing conditions used by 7Li NMR imaging at 4.7 T with inclusion of an external standard of known c~ncentration'~'. The calculated visibilities among the five rats studied suggested essentially full visibility (96%) for Li+ in muscle, and somewhat reduced visibility (74-93'%0)in the various brain regions. Novel approach to measuring tumor oxygen tension in vivo was developed based on 19F NMR spin lattice echo planar imaging (EPI) relaxometry of hexafluorobenzene (HFB) and feasibility of monitoring dynamic changes in regional tumor oxygenation (Local p02) was examined * 32. Extra- and intracellular apparent diffusion in normal and globally ischemic rat brain was evaluated via 19FNMR133. The field of polarization-based magnetic resonance imaging has expanded rapidly in the past few years. A polarization and delivery system of 129Xethat is currently in use in the fringe field of a MRI magnet has been con~tructed'~ to realization of this new modality of medical imaging. A review with 25 references on some current work directed towards delivery of optically polarized Xe for in vivo MR application has been publishedI8. The high solubility of Xe in blood and lipids suggests a variety of in vivo MR applications, for instance perfusion measurements or functional MR studies. Three-dimensional 31P NMR was used to evaluation of bone mineral density in solid state projection imaging53, and to high-energy phosphate metabolism in injured rat heart in spectroscopic imaging134.The effect of combretastatin A4 prodrug on perfusion and the levels of 31Pmetabolites in an implanted murine tumor were investigated for 3 h after the drug treatment using MRI and MRS135. The feasibility of monitoring intracellular sodium changes was investigated in an isolated rat heart during a variety of interventions for Na+ loading by using 23Na TQF (triple quantum filtered) NMR without a chemical shift reagents (SR)136.It is concluded that 23NaTQF NMR without using SR offers a unique and non-invasive opportunity to monitor alterations of intracellular sodium. 13.3 Contrast Enhanced Imaging via Paramagnetic Agents - Paramagetic or superparamagnetic complexes have been widely used for diagnostic MRI in order to obtain enhanced contrasts between tissues and/or high signal-to-noise ratio. In the field covered in this review, however, such contrast agent is not so often used. Three different types of example are shown below. For details the journals indicated in the Introduction should be consulted.

13: Nuclear Magnetic Resonance Imaging

529

Tumor-sprouted vessels are greater in both number and diameter in comparison to their healthy counterpart, A novel technique based on magnetic susceptibility contrast mechanisms that are sensitive to varying sizes of blood vessels is presented to measure differences between the relaxation rates ( 1/T2 and 1/T2*)in a rat glioma model and normal cerebral cortex. The difference between relaxation rates precontrast and postcontrast agent injection, DR2 and DR2*, were measured for an intravascular equiliblium contrast agent (MION) at various echo-times. The ratio of (AR;/AR$ is a useful quantitative metric of in vivo tumor vascular morphology137. A biocompatible, dextran coated superparamagnetic iron oxide particle was derivatized with a peptide sequence from the HIV-tat protein to improve intracellular magnetic labeling of different target cells' 38. Labeled cells were highly magnetic, were detectable by NMR imaging, and could be retained on magnetic-separation columns. The human transferrin receptor (hTfR) has been used as a model molecule target to direct therapeutic agents to tumor cells and to shuttle drugs across the blood-brain-barrier. Receptor expression and regulation can be visualized by NMR imaging, when the receptor is probed with a sterically protected iron containing magnetic hTfR probe139. This research provides proof of the principle that it is possible to image receptor gene expression and regulation and it demonstrates a possibility of imaging gene transfer in vivo.

14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

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P. J. McDonald, E. Ciampi, J. L. Keddie, M. Heidenreich, R. Kimmich, Phys. Rev., E., 1999,59, part B, 874. S. P. Rigby, Chaos Solitons Fractals, 1998,9, 1519. L. Lacelle, S. Lacelle, C. G. Fry, Can. J. Chem. Rev., 1998,76, 1998. B. P. Hill, J. Godward, C. E. Manning, J. L. Biechlin, K. M. Wright, Magn. Reson. Imag., 1998, 16, 557. P. Coussot, Magn. Reson. Med., 1998,16, 621. P. S. Wang, M. M. Ferguson, G. Eng, D. P. Bentz, C. F. Ferrais, J. R. Clifton, J. Mater Sci., 1998, 12, 3065. 0. Jarh, A. Sepe, P. Jevnikar, N. Funduk, R. Toffanin, V. Miynarik, Nucl. Magn. Reson. Spectrosc. Chem. Based Materials, [Int. ConJ], 1998, 1998, 369. L. Pel, K. Hazrati, K. Kopings, J. Marchand, Magn. Reson. Imag., 1998, 16, 525. A. Gargaro, F. De Luca, E. De Vite, G. H. Raza, B. Maraviglia, Colloids Surf.’, A , 1998,140,321. M. Gentzler, S. Patil, J. A. Reimer, M. M. Denn, Solid State NMR, 1998, 12,97. P. Blumler, N. Paus, G. Salge, Proc. IEEE Int. Con$ Conduct. Breakdown Solid Dielectr., 6th, 1998, 168. Y. Hotta, T. Shibuya, H. Yasunaga, H. Kurosu, 1. Ando, Polym. Gels Networks, 1998,6, 1. P. Bluemler, V. Litvinov, H. G. Dikland, M. Van Duin, Kautsch. Gummi Kunstst., 1998,51, 865. C. M. Hill, J. L. Koenig, Proc. Annu. Meet. Adhes. SOC.,1998,21, 144. M. Mori, J. L. Koenig, J. Appl. Polym. Sci., 1998,70, 1385. M. Knoergen, U. Heuert, H. Menge, H. Schneider, Angew. Makromol. Chem., 1998,261-262, 123. F. P. Miknis, L. C . Michon, Fuel, 1998,77, 393. F. P. Miknis, A. T. Pauli, L. C. Michon, D. A. Netzel, Fuel, 1998,77,399. Y. Nakashima, S. Nakashima, D. Gross, K. Weiss, K. Yamauchi, Geothermics, 1998,27,43. Y. Nakashima, F. Mitsumori, S. Nakashima, M. Takahashi, Appl. Clay Sci., 1999, 14, 59. E. Causin, A. Brancolini, Ind. Min. (Rome), 1997, 18, 12. G. Maddinelli, R. Vitali, Magn. Reson. Imag., 1998, 16,669. C. Van Der Zwaag, F. Stallmach, J. E. Hanssen, E. Soergaard, R. Toennesen, DGMK Tagungsber., 1998,9801,81. K. Saito, M. Hatakeyama, M. Matsuura, K. Kato, I. Komaki, J. Iron Steel Inst. Jpn., 1999, 85, 195. A. G. F. Stapley, T. M. Hyde, L. F. Gladden, P. J. Fryer, Int. J. Food Sci. Technol., 1997,32,355. A. G. F. Stapley, K. A. Landman, C. P. Please, P. J. Fryer, Chem. Engin. Sci., 1999, 54, 965. A. K. Horigane, H. Toyoshima, H. Hemmi, W. M. H. G. Engelaar, A. Okubo, T. Nagata, J. Food S c i , 1999,64, 1. B. P. Hills, J. Godward, C. E. Manning, J. L. Biechlin, K. M. Wright, Magn. Reson. Imag., 1998, 16, 557. W. L. Kerr, R. J. Kauten, M. J. McCarthy, D. S. Reid, Food Sci. Technol., 1998, 31, 215. L. Marciani, P. Manoj, B. P. Hills, R. J. Moore, P. Young, A. Fillery-Travis, R. C . Spiller, P. A. Gowland, J. Magn. Reson., 1998, 135,82. B. A. Goodman, S. M. Glidewell, Phyton (Horn, Austria), 1997,37,81.

83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104

105 106 107 108 109 110 111

112

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113 M. Heidenreich, W. Kockenberger, R. Kimmich, N. Chandrakumar, R. Bowtell, J. Mugn. Reson., 1998,132, 109. 114 J. Verscht, B. Berbhard, J. Koehler, W. Koeckenberger, A. Metzel, A. Haase, E. Komor, Plant (Berlin), 1998,205, 132. 115 C . J. Clark, L. N. Drummond, J. S. MacFall, J. Sci. Food Agric., 1998,78, 349. 116 S. M. Glidewell, B. Williamson, G. H. Duncan, J. A. Chudek, G. S. 0. Hunter, New Phytol., 1999, 141,85. 117 D. W. Fountain, L. C. Forde, E. E. Smith, K. R. Owens, D. G. Bailey, P. T. Callaghan, SeedSci. Rex, 1998,8, 357. 118 M. Rokitta, U. Zimmennann, A. Haase, J. Magn. Reson., 1999, 137,29. 119 X. S. Yin, E. J. Kendall, W. J. Fernets, G. S. Laycock, G. D. Abrams, S. R. Abrams, Proc. Congr. - Eur. Brew. Conv., 26th, 1997, 127. 120 E. Verstreken, P. Van Hecke, N. Scheerlinck, J. DeBaerdemaeker, B. Nicolai, Magn. Reson. Chem., 1998,36, 196. 121 F. Antonsen, A. Johnsson, C. Futsaether, J. R. P. Krane, A. Johnsson, New Phytol., 1999,142, 59. 122 V. Guivel-Scharen, T. Sinnwell, S. D. Wolff, p. S. Balaban, J. Magn. Reson., 1998, 133, 36. 123 Q. Jiang, Z. G. Zhang, R. L. Zhang, J. R. Ewing, G. W. Divine, P. Jiang, M. Chopp, Fibrinolysis Proteolysis, 1998, 12, 33. 124 D. Hesselbarth, C. Franke, R. Hata, C. Bribker, M. Hoehn-Berlage, NMR Biomed., 1998, 11,423. 125 D. L. Buckley, J. D. Bui, M. I. Phillips, T. Zelles, B. A. Inglis, H. D. Plant, S. J. Blackband, Magn. Reson. Med., 1999,41, 137. 126 M. D. Noseworthy, T. M. Bray, Free Radical Biol. M e d , 1998,24,942. 127 R. J. Gilbert, T. G. Reese, S. J. Daftary, R. N. Smith, R. M. Weisskoff, J. van Wedeen, Am. J. Physiol., 1998,275, G363. 128 G . Brinker, C. Bock, E. Busch, H. Krep, K. A. Hossmann, M. Hoehn-Berlage, Magn. Reson. Med., 1999,41,469. 129 J. Timonen, L. Alvila, P. Hirva, T. T. Pakkanen, J. Mater Sci., 1998,9, 187. 130 K. Potter, J. J. Butler, C. Adams, K. W. Fishbein, E. W. McFarland, W. E. Horton, R. G. S. Spencer, Matrix B i d , 1998, 17, 5 13. 131 R. A. Komoroski, J. M. Pearce, J. E. 0. Newton, J. Magn. Reson., 1998,133,98. 132 R. P. Mason, S. Hunjan, D. Le, A. Constantinescu, B. R. Barker, P. S. Wong, P. Peschke, E. W. Harn, P. P. Antich, Int. J. Radial. Oncol., B i d , Phys., 1998, 42, 747. 133 T. Q. Duong, J. J. H. Ackerman, H. S. Ying, J. J. Neil, Mugn. Reson. Med., 1998, 40, I. 134 M. Van Kienlin, C. Rosch, Y. Le Fur, W. Behr, F. Roder, A. Haase, M. Horn, B. Illing, K. Hu, G. Ertl, S. Neubauer, Magn. Reson. Med., 1998,39, 731. 135 D. A. Beauregard, P. E. Thelwall, D. J. Chaplin, S. A. Hill, G. E. Adams, K. M. Brindle, Br. J. Cancer, 1998,77, 1761. 136 V. D. Schepkin, I. 0. Choy, T. F. Budinger, D. Y. Obayashi, S. E. Taylor, W. M. DeCampli, S. C. Amartur, J. N. Young, Magn. Reson. Med., 1998,39, 557. 137 J. Dennie, J. B. Mandeville, J. L. Boxerman, S. D. Packard, B. R. Rosen, R. M. Weisskoff, Magn. Reson. Med., 1998,40, 793. 138 L. Jesephson, C. H. Tung, A. Moore, R. Weissleder, Bioconj. Chem., 1999, 10, 186. 139 A. Moore, J. P. Basilion, E. A. Chiocca, R. Weissleder, Biochim. Biophys. Acta, 1998,1402,239. I

14 Oriented Molecules BY C.L. KHETRAPAL, K.V. RAMANATHAN AND G.A. NAGANA GOWDA

1

Introduction

The report on the topic of oriented molecules is published in alternate volumes of this series and the present one covers the literature published during the biennial period ending with May 1999. The complete literature on the subject can be traced back by referring to volume 27' of this series and the references therein. In the previous report' it was pointed out that the field is undergoing rapid changes and that developments are taking place in new directions. Four areas where the field has a lot of scope for future developments were identified. These are the discovery of liquid crystals with low order parameter, orientation of molecules by high magnetic field, natural abundance 2H NMR and techniques for spectral simplification. Of these, the last three areas mentioned here have shown only moderate progress during the period under report. This is perhaps so as developments in these fields depend to a great extent on the developments in instrumentation such as availability of still higher magnetic fields and better sensitivity than what is available at the present time. On the other hand, the area of the discovery and use of liquid crystals of low order parameter, particularly for the study of large biological molecules is making rapid progress and is becoming one of the major techniques in biomolecular NMR. Consequently, in this report this area is covered under a separate section. The literature available on the subject of oriented molecules is presented here under the following headings: (1) reviews, theory and general studies, (2) emerging areas and new techniques, (3) dynamic NMR studies, (4) discotics, lyotropics and chiral systems, (5) liquid crystals in confined geometries and polymeric liquid crystals, (6) membranes and molecules oriented by them, (7) weak ordering and biomolecular studies, (8) relaxation studies, (9) molecular order and (10) structure and conformation.

2

Reviews, Theory and General Studies

A review containing 164 references on the study of solute orientational order from molecular shapes has been published.2 The experimental and computaNuclear Magnetic Resonance, Volume 29 0The Royal Society of Chemistry, 2000 534

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535

tional methodologies to be adbpted for the study of the spectra of oriented molecules has been pre~ented.~ Application of the modified 2D Z-COSY t e c h n i q ~ e ~as9 an ~ aid in the analyses of spectra of oriented molecules has been dealt with, with examples.6NMR studies of lyotropic liquid crystals have been re~iewed.~ It contains 128 references and describes the application of NMR line shapes and relaxation rates for the study of structural variations at phase transitions and for obtaining information about the elastic properties and interactions of surfactant aggregates. A review of NMR studies of polymeric liquid crystals with different architecture is available.8 The deuterium quadrupole coupling tensors have been calculated for the methyl halides CD3X (X=F, CI, Br and I) using an ab initio m e t h ~ d The .~ value for methyl chloride has also been obtained from liquid crystal NMR. The proton decoupled 13C spectra of 2,2'-difluorobiphenyl in the isotropic and oriented phases have been obtained and analyzed.I0 The spectrum of each 13C nucleus corresponds to the X part of an ABX type spectrum, with A and B being the two fluorine nuclei. It has been shown that the corresponding chemical shifts and spin-spin coupling constants can be obtained provided all the transitions are detected and that the intensities and frequencies of transitions are used in the analysis. The variable angle spinning experiment has been used to get the sensitive range for the dipolar couplings. The method has also been used to obtain the absolute signs of the scalar couplings. 13C-13C spin-spin coupling constants and 'H and 13C nuclear shielding tensors determined experimentally and by theoretical ab initio calculations are reported for ethane, ethene and ethyne." The experimental anisotropies of the C-C coupling tensors AJK and also the combination J,,,,, - Jcc,yy for ethene were derived from sets of anisotropic couplings analysed from the 'H and I3C NMR spectra of molecules partially oriented in liquid-crystalline environments. Both harmonic vibrations and structural deformations arising from the correlation of vibrational and reorientational motions have been taken into account. The calculated and experimental values are in agreement with each other. It is observed that the contribution of the anisotropic part of J,, to experimental couplings is small and can in general be omitted. The discrepancy in the 'H and I3C chemical shift anisotropy values for chloroform determined using the ENEMIX and other methods12 has been shown to arise from the use of reference compound and not because of the hydrogen bonding between chloroform and the cyanide group of the solvent used.I3 The NEMIX method,I4 on the other hand, has been shown to give more consistent results, though support for the role of hydrogen bonding in this system is sought to be provided by ab initio and model calculation^.'^ A numerical procedure which permits a formal distribution of collective fluctuation modes to be derived from field-cycling NMR-relaxometry experiments on ordered systems has been reported.16The method helps to distinguish true order fluctuation modes from local reorientation mechanism and serves the elucidation of modified or limited director fluctuation modes in the case of liquid crystals in pores and lyotropic systems. Deuterium NMR study of the biaxiality of the nematic phase of 4-[3,4,5-tris-

536

Nuclear Magnetic Resonance

(4-dodecyloxybenzyloxy) benzoyloxy]-4'-(4-dodecyloxybenzoyloxy)1,l'-biphenyl has been made.17 It has been concluded that within experimental error the biaxiality parameter is zero and that the phase exhibits essentially a uniaxial symmetry. A novel method to study biaxiality in liquid crystals has been proposed and its application demonstrated in 1,4"(p-terphenyl)-bis [2,3,4tri(dodecyloxy)benzal]imine. * The method involves the determination of the molecular geometry, for example the H-C-H bond angle in a CH3 group, of a molecule such as acetonitrile or methyl iodide dissolved in the system studied and comparison of the result with values determined from studies in several other known uniaxial liquid crystals. Significant difference in the values in the two cases can then be attributed to the biaxiality of the system studied, in the absence of other factors such as solvent effects. Results of such a study combined with 2H-NMR study of molecules such as benzene-d6 and dimethyl sulfoxide-d6 dissolved in 1,4"-(p-terphenyl)-bis [2,3,4-tri(dodecyloxy)benzal]imine indicate the occurrence of biaxiality in this system.

3

Emerging Areas and New Techniques

This section covers areas which hold the potential for future developments as well as techniques which are already established but undergoing further improvements. The need for observing 2H NMR spectra in natural abundance, as reported earlier,19.20is indicated by the fact that the molecular order may be significantly altered by isotopic substitution as pointed even at the early stages of the development of the field.21y22Recently this has been emphasised by the study of 4'-propyl-4-(3,4-difluorophenyl)bicyclohexane,for which the quadrupolar splittings in the 2H NMR spectrum, obtained from a sample with natural abundance deuterium, are shown to be considerably different from those obtained from specifically deuterated samples.23 Proton-decoupled natural abundance deuterium NMR has been used to visualize chiral molecules oriented in organic solutions of poly (y-benzyl-L-glutamate) (PBLG) for the first time (Figure 14.1).24 The chiral discrimination is observed through measurements of the deuterium quadrupole splitting differences and the differential ordering effects could be measured with sufficient sensitivity in a magnetic field of 5.87 T. The potential of the approach and the advantages of using higher magnetic field are also discussed. There are three reports on the orientation of molecules by high magnetic fields. Fine structure in the 500 MHz and 750 MHz 'H NMR spectra of hydrogenated fullerenes C60H2 and C60H4 have been observed which are attributed to partial alignment of the molecules in the magnetic field.25726 At 750 MHz, a splitting of 0.30 Hz results from the residual dipole-dipole interaction between the two magnetically and chemically equivalent protons in C60H2. Similarly a splitting of 0.74 Hz is observed for C60H4.The anisotropic relaxation and cross correlations between different relaxation mechanism have been considered for understanding the observed unequal peak heights, in the latter case. Magnetic field induced alignment of the transition metal dihydrogen complexes of [Os(H2)(PPh3)2(bipyridyl)(CO)]

o/-

537

14: Oriented Molecules

H

OH

H

H

CH3

CDH 4 doublets

@-d (om)

~,,,..~,,,..,,.,',,.,,,,,.'.,, 500 400 300 200

HZ

I

I

I

I " " ' " " I " ' " " " I ' " ~

100

0

-100

Figure 14.1 Natural abundance *H-{'H} NMR spectrum of (IfI)-phenethyl alcohol dissolved in PBLG at 320 K. The peaks arising from the deuterons in the ortho, meta and para positions of the benzyl group are defined by the symbols 4 - d (o,m) and 4 - d ( p ) , respectively. In the insert, eight lines associated with the ortho and meta deuterons of the R and S benzyl groups are shown. (Reproduced with permission from Tetrahedron: Asym., 1998,9, 187 1)

(OS02CF& and its analogue with phenanthroline in place of bipyridine has been observed.27The splitting of the HD resonance of the deuterated analogue at several magnetic fields up to 750 MHz has been obtained leading to the estimation of the dipolar coupling DHDand the determination of the H-H distance in both the complexes. Neat phases of quaternary phosphonium halides with three n-octadecyl chains and a shorter alkyl chain have been examined by deuterium NMR and have been found to exhibit nematic liquid crystalline behaviour within certain temperature ranges.28 Proton and deuterium NMR spectra of dichloromethane and dimethyl sulfoxide dissolved in these systems have also been obtained. The important feature of these thermotropic liquid crystalline phases is that the order parameters of the neat phases as well as those of the dissolved molecules are small. This indicates possible use of such phases for obtaining first order spectra. In the rest of this section, use of multiple pulse and multiple quantum techniques, efficient methods for spectral simplifications and analysis and the use of electric fields for achieving desired molecular alignment are reported. The single and multiple quantum NMR spectra of 1,3-dichlor0-2-ethenylbenzene as solute have bee0 obtained in two different nematic liquid crystals and

538

Nuclear Magnetic Resonance

-5000 1

0

f : 5000

Hz

Figure 14.2 Two-dimensional 13Cspectrum of ( a ) the aliphatic carbons and (b) aromatic carbons of EBBA recorded at 75.47 MHz and at 318K obtained using dipolar oscillations enhanced by the Lee-Goldburg decoupling. The horizontal axes represent 13C chemical shifts and the vertical axes correspond to oscillation frequencies, from which dipolar couplings can be obtained. (Reproduced from Liq. Cryst., 1999,26, 17).

analysed to yield the dipolar couplings and chemical shift difference^.^^ The structural information and the order matrix have been obtained. The dihedral angle between the ethenyl group and the aromatic ring as well as the C-C=C angle have been estimated. Automated analysis of the complex spectra of molecules in the anisotropic media has an advantage over the traditional

14: Oriented Molecules

539

method of analysis in that the former method enables saving considerable amount of time and effort. Some of the programs belonging to this category are DANSOM30and DAISY.31The existing algorithms for such analysis have been improved to treat very complex NMR spectra of molecules dissolved in nematic solvents.32The main options added to the original algorithm are a wider choice of smoothing functions, the use of principal component regression method and the possibility of selecting molecular coordinates and order parameters as the variables of the problem. The efficiency of the procedure has been tested on sixteen molecules with spectra of increasing complexity. The method was found to be powerful for analysing rigid solutes, but the efficiency steeply decreased when applied to a rigid system with a bad guess of the geometrical parameters or with a very large number of independent spectral parameters. Lee-Goldburg decoupling has been applied to enhance dipolar oscillations observed in cross polarisation experiments on liquid crystals (Figure 14.2).33 The experiment has been utilized for obtaining order parameters of the liquid crystal N-(4’-ethoxybenzylidene)-4-n-butylaniline(EBBA) at different temperatures and the core order parameters thus obtained have been compared with those obtained from the 2H NMR spectra. A method to assign deuterium NMR spectra using deuterium to carbon cross-polarization has been proposed.34The assignment of deuterons, made on the basis of the chemical shift of the attached carbon is demonstrated for the case of 4-n-pentyl-4(-cyanobiphenyl (5CB). The theory for the above experiment describing nuclear crosspolarization from spin- 1 to spin- 1/2 is generalized to the experiments in which the amplitude of the radio-frequency field is swept, which allows efficient cross-polarization over the entire width of the spectrum of a spin 1 nucleus.35 The evolution of the spin system can be understood in terms of individual quasiadiabatic population exchanges, allowing the prediction of the effects of the various experimental parameters on the efficiency of cross-polarization. The efficiency of the broad band polarization step in this experiment has been demonstrated and the cross-polarization dynamics studied.36The heteronuclear C-D couplings are shown to be measurable by direct inspection of the 2D spectra. A new method named dynamic director spectroscopy has been proposed for measuring the isotropic and anisotropic chemical shifts in ordered systems having high viscosities such as liquid crystalline polymers.37In this method, the sample initially aligned in a magnetic field is rotated by a known angle and the spectrum is recorded. The plot of peak position and the angle which the director makes with the magnetic field yields the required information. A new and efficient broad band decoupling sequence for use with liquid crystals is reported.38The effects of several new homonuclear decoupling sequences such as FSLG-2, MSHO-3, BHOT-4 on the I3C spectra of liquid crystals and molecules dissolved in them have been studied and the results compared with those of similar studies on some of the already known sequences.39 It is observed that for benzene dissolved in two-liquid crystalline solvents BLEW-48 sequence gives the best spectral resolution. This sequence is also found to be preferable for the 2D method of proton-encoded local field

540

Nuclear Magnetic Resonance

(PELF) spectroscopy to study bulk liquid crystals. The use of MREV-840 or Flip-Flop- 1 sequences during t -period in a I9F COSY experiment on a dissolved in a nematic solvent has been sample of 1,3-dichloro-4-fluorobenzene made, leading to a F1-projection which provides a first order spectrum.43The analysis yields reduced ‘H-19F couplings. The reduction factors have been obtained by analysis of the usual 1D spectrum and compared with values predicted by the average Hamiltonian theory. Numerical simulations have been used to investigate differences between the observed and the predicted values. The proton NMR spectrum of partially deuterated liquid crystal 5CBd15 has been recorded with deuterium decoupling.44This leaves strong peaks from protons in the undeuterated sites and also weak resonances due to coupling to those protons at low abundance at the deuterated sites. It has been shown that the weak resonances can be separated from the strong by recording the spectra while spinning the samples at a series of angles with respect to the magnetic field. Proton two-dimensional multiple pulse experiments have been carried out on the nematic phase of the liquid crystal 4’-methoxybenzylidene-4n-butylaniline (MBBA) to separate proton dipolar couplings from the chemical shifts and to observe spin diffusion between protons with different chemical shifts.45The data have been analysed using an additive potential model and the twist angles of the two phenyl rings from the -CH=N- plane have been determined. The use of multiple-pulse techniques to extract proton chemical shift information for static liquid crystalline samples is presented.46 The orientation of the liquid crystal could be manipulated by the application of electric fields. The application of an orienting electric field in order to rapidly switch the director orientation and study the reorientation process in a magnetic field is discussed and demonstrated for the case of the liquid crystal 5CB and a side chain liquid crystal p ~ l y m e r . ~ ~ ? ~ ~ y42

4

Dynamic NMR Studies

This section covers reports of studies on molecular dynamics and director dynamics in meso-phases due to influences of external agencies such as sample rotation and internal thermal motion. Selectively and perdeuterated samples of bromocyclohexane, dissolved in a liquid crystalline solution have been studied by dynamic 2H NMR spectroscopy (Figure 14.3).49From variable temperature and 2D exchange experiments the assignment of spectral lines in terms of axial and equatorial conformers and their relative populations have been obtained. The molecular order parameters of both the species have been estimated and the activation enthalpy for the ring inversion process has been estimated from the line shapes at different temperatures. The study of magnetohydrodynamics of a chiral nematic liquid crystal made from a mixture of psly-y-benzyl-lglutamate (PBLG) and deuteriated chloroform has been made by deuterium NMR.50 The observed changes in the spectral patterns with increasing spinning speed together with observations made earlier51-53 on systems which

54 1

14: Oriented Molecules

I

40

I

1

20

1

I

0

I

I

-20

I

1

-40

a4/24& Figure 14.3 2 0 exchange deuterium N M R spectrum of an oriented mixture of bromocyclohexane-1-dl and 10% bromocyclohexane-cis-2-dl, recorded at 238 K with a mixing time of 3 ms. a and e refer to the axial and equatorial conformers. (Reproduced with permission from Liq. Cryst., 1999,26, 759).

have their magnetic susceptibility anisotropy close to zero indicate the existence of a second critical speed which is not predicted by theories so far. The behaviour of the mesophase directors when a sample with a negative diamagnetic susceptibility anisotropy is rotated in a magnetic field about an axis inclined at an angle p to the magnetic field has been studied leading to the determination of the critical speed and the twist viscosity ~ o e f f i c i e n tThe .~~ molecular dynamics in the chiral nematic phases of an antiferroelectric liquid crystal (S)-44 1-methylheptyloxycarbonyl) phenyl 4’-octyloxybiphenyl-4 carboxylate has been investigated by solid state 13C NMR spectro~copy.~~ The angular dependence of the critical magnetic field for the unwinding of the Sm-C* helix of a ferroelectric liquid crystal has been measured by deuterium NMR.56The alignment of directors produced by the competing magnetic and rotational torques has been monitored by the effect produced on the deuterium quadrupole splittings of the five alkyl chain and one biphenyl core sites in 5CB.57 The field-induced director relaxation rate and the twist viscosity have

Nuclear Magnetic Resonance

542

been found to be independent of the site monitored, in contrast to 13CNMR measurements reported earlier.58

5

Discotics, Lyotropics and Chiral Systems

There are three reports of studies on discotic columnar mesophases and two reports on lyotropic liquid crystals. The rest of the studies reported here are on chiral systems. The mesomorphic properties of four members of the homologous series of dimers, alkandioic acid bis [pentakis(n-heptanoy1oxy)phenyll esters [(C&13C00)5 C60C(O)(CH2)(,-2,,2]2 with n = 6, 8, 12 and 14 (referred to as T6,T8, T12, and T14 respectively) are reported.59 The four compounds exhibit with increasing temperature the phase sequence crystalline+P+ isotropic where the phase P is crystalline for T6 and Tg and columnar discotic for T12and T14,as confirmed by X-ray measurements. 2H and 13CNMR have been used to obtain the dynamic properties of the benzene core and side chain. The structure of the mesophases could be identified as columns of stacked monomeric units. The mesomorphic properties of trifluoroacetic acid complexes with hexa-a1kyloxyt riphenylenes and with hexa-a1ky 1oxyt ribenzocyclononene have been investigated by means of optical microscopy, differential scanning calorimetry, X-ray diffraction and 2H NMR spectroscopy.60It has been suggested that the observed enhancement in the mesomorphic properties is due to formation of the oxonium ion complexes. 13C NMR studies of benzene dissolved in a columnar liquid crystal formed by octa-o-decanoyl-pcellobiose are reported.61 From the spectral line shapes the director distribution functions and the molecular order parameter of the solute have been estimated. An unexpected increase of the order parameter with increased temperature is observed which is explained as due to an exchange process between different solvation sites in the mesophase. Macroscopically oriented silicate-surfactant liquid crystals have been obtained by slow cooling of lamellar and hexagonal mesophases from the isotropic phase through the isotropic-anisotropic phase transition in a magnetic field of 11.7 Tesla and the 2H NMR spectrum is used to quantify the degrees of orientational order.62 Lamellar or hexagonal domains are shown to adopt different orientations according to the diamagnetic susceptibilities of different organic additives present in the mixtures, thus providing a handle on the macroscopic alignment of these liquid crystals by adjusting their composition. The alignment of lyotropic liquid crystal formed by hexadecyltrimethylammonium bromide with D20 has been studied by the use of 2H NMR, as a function of their relative concentrations and the dependence of the alignment time on concentration, temperature and the strength of the magnetic field has been determined.63The addition of sodium salicylate was found to change the orientation of the micelles from an orientation parallel to the magnetic field to one that is perpendicular to it. Line-shape simulations have also been carried out to estimate the rate of exchange between free D20 molecules and those bound to the micelles.

14: Oriented Molecules

543

High resolution spectra of cholesteric liquid crystals have been obtained by off-magic-angle spinning.64Using the average potential theory it is shown that the pitch axes of a cholesteric liquid crystal with a positive (negative) magnetic susceptibility anisotropy are aligned along the spinning axis for the angle of spinning larger (smaller) than the magic angle without any distortion of the helical structure, thus removing the line broadening due to the anisotropies of chemical shifts and quadrupole couplings, while retaining the information of the line positions. The results of the detailed theoretical analysis are verified by 3C NMR experiments on a cholesteric liquid crystal p-ethoxybenzyl-p-[(S)-2methylbutyl]aniline and cholesteric mixtures of cholesteryl chloride and MBBA. Due to the growing importance of asymmetric synthesis and its use in the pharmaceutical industry, the development of new and convenient NMR methods for enantiomeric analysis is being actively explored. A large number of chiral molecules including a case of axial chirality has been studied by using proton decoupled 13C NMR at natural abundance by orienting them in organic solutions of poly-y-(benzyl-L-glutamate) (PBLG).65 The differential ordering effects of the medium helps to discriminate between the enantiomers through the chemical shift anisotropy differences and an accurate measurement of enantiomeric excess could be made. The method offers a powerful alternative to the existing enantiomeric analysis techniques. Natural abundance deuterium auto-correlation 2D NMR experiments of enantiomers oriented in PBLG have also been proposed to identify the quadrupolar doublets and to study chiral discrimination.66 Differential ordering effects are analysed in homologous series of 22 chiral secondary aliphatic alcohols dissolved in PBLG/dichloromethane liquid crystal solvent, in terms of the quadrupolar splittings of the deuterons at their chiral centre measured as a function of PBLG concentration and t e m p e r a t ~ r eThe . ~ ~ results are interpreted qualitatively in terms of the structure and chirality of the molecules. The use of 19F-NMR and trifluoroacetic anhydride as an achiral derivatizing agent is demonstrated for enantiomeric excess measurement through chiral liquid crystal NMR.68 The -CO-CF3 group has been shown to allow good enantiomeric discrimination based on both chemical shifts and dipolar splittings. Examples involving amines, amino acids and alcohols have been presented. A quantitative description of the discrimination of the two faces of molecules such as ethanol containing a prochiral methylene group oriented in an organic solution of PBLG is reported.69 From the analysis of 'H, 13C and 2H NMR spectra, it has been shown that five non-zero independent order parameters, instead of three required for a system with a C, symmetry, are necessary to describe the molecular ordering of ethanol in this medium correctly, thereby demonstrating that the symmetry ordering of a prochiral molecule is broken. It has also been shown that such studies yield the sign and magnitude of the geminal scalar coupling between the methylene protons which has been obtained for series of linear alcohols and compared with values derived by the isotopic substitution method. Data from an earlier 13C NMR study7' where the hydrochloride of decyl-L-proline was doped into an amphiphilic nematic

544

Nuclear Magnetic Resonance

liquid crystal with the achiral host detergent being tetradecyltrimethylammonium bromide has been used to determine the rotamer populations in this system and the twisting powers of the individual trans and cis r ~ t a m e r s . ~ ~ Several a-dideuterated dodecanoyl amino acids have been used as chiral dopants in amphiphilic cholesteric liquid crystals and studied using 2H NMR.72 The variation in the deuterium quadrupolar splitting is attributed to molecular reorientation which is not related to the bulk chirality twist. Racemic mixtures of enantiomers of borocryptates of caesium and ammonium ions dissolved in a nematic liquid crystal have been studied by NMR of quadrupolar nuclei such as l l B , log, 133Csand 14N.73From the multiplicity of the observed quadrupolar splittings, the chirality of the compounds has been studied.

6

Liquid Crystals in Confined Geometries and Polymeric Liquid Crystals

Due to their technological applications and interesting physio-chemical properties, study of these systems has become an attractive topic of research in recent years. Crystalline and liquid crystalline phases of PB- 18 polyester composed of 4,4’-dihydroxybiphenyl and octadecanedioic acid have been studied by 3C NMR spectro~copy.~~ Measurements of proton cross-relaxation rate in the polymer dispersed 4’-pentyl-4-cyanobiphenyl(5CB) droplets have been performed by the selective magnetisation inversion NMR technique.75It has been shown that the cross relaxation rate depends strongly on the type of liquid crystal-polymer dispersion and that it is considerably larger for the pure liquid crystal 5CB than that reported for the commercial mixture (MERK E7) in the same epoxy matrix. Orientational order and dynamic properties of the nematic liquid crystal 5CB confined to the cavities of porous glass have been investigated by means of proton and carbon-13 NMR line shape studies and broad band dielectric spectro~copy.~~ It is observed that the dynamics of mesogenic molecules in the free volume of the pores of size 5 nm is as fast as in the free bulk phase. In addition, a boundary layer of molecules with reduced mobility covers the walls of the pores. Isotropic-nematic transition as well as transition to the crystalline phase are found to be considerably suppressed. Orientational order and mobility of nematogens adsorbed in nanoporous glass have been investigated by ‘H and 13C NMR and broad band dielectric spectro~copy.~~ For the mesogen 5CB, quantitative measurements of the order parameter in cavities of different diameter have been reported. The degree of orientational order of a liquid crystalline side chain polymer, thermotropic polyacrylate, in its nematic phase on lecithin monolayers has been studied by proton NMR.78 The alignment of the mesogenic unit is obtained from the angular dependence of the linewidth. The correlation length of the decay of the orientational order parameter into the bulk has also been estimated. The phase behaviour of liquid crystals 5CB and octylcyanobiphenyl (8CB) confined to controlled porous glass of different void sizes has been studied using deuterium NMR.79A detailed theoretical analysis of the origins of the temperature shift

14: Oriented Molecules

545

and the change of the charactek of the isotropic-nematic and nematic-smectic A phase transitions is given. The influence of the surface treatment on the details of anchoring has also been studied. A study of orientational order via 2H NMR and high resolution ac calorimetry on the liquid crystal 8CB confined to randomly oriented and inter-connected macroporous and microporous glasses has been reported and substantial modifications to the N-I and SmA-N transitions have been observed.80 Different results in terms of ordering are seen depending on whether the host material length scale is larger or smaller than the nematic correlation length. Polyacetylene substituted by liquid crystalline side chains and its monomer have been investigated by 13C N M R spectroscopy and their orientation and dynamics have been studied in the liquid crystalline state.81A deuterium NMR study of the ordering of the 8CB liquid crystal confined to the controlled pore glass matrix is reported.82 A method based on NMR relaxometry has been presented to study surfaceinduced order in the isotropic phase of confined liquid crystals.83 From an increase in the deuterium transverse relaxation time due to the molecular diffusion between the weakly ordered surface region and disordered area in the rest of the cavity, the magnitude of the surface order parameter So could be obtained. The method has been applied to a polymer dispersed liquid crystal to obtain a temperature independent So of magnitude 0.08, which indicates that only partial wetting of the surface occurs.

7

Membranes and Molecules Oriented by Them

This section deals with the work reported on membranes and molecules oriented by them. Studies in which the ordering is large requiring the use of solid state NMR methodologies are reported here. Literature dealing with weakly ordered systems and their applications are discussed in the following separate section due to their importance in studies of large biomolecules. Two lipids, the phospholipid DMPC carrying two geminal fluorine labels in one chain and a doubly fluorinated sterol, which are arranged in liquid crystalline bilayers, have been used as models to elucidate the use of 19F N M R as a probe for investigating local molecular structure and order.84Employing the Carr-Purcell-Meiboom-Gill multipulse sequence to measure the homonuclear dipolar couplings, order parameters for the anisotropic motion of the lipids have been determined. Besides measuring the distance dependent term of the dipolar coupling in powder samples, angular information has been obtained from measurements on the oriented membranes. A numerical method that uses the shape of the 2H NMR spectra for determining the orientational order parameters of partially oriented multilamellar vesicles in high magnetic fields in the presence of an unknown non-random distribution of orientations is reported.85 An experimental spectrum of a phospholipid-ether mixture in water is analysed as an example leading to a proposed ellipsoidal shape for vesicles with a ratio of semi-axes of approximately 3.4. 2H NMR spectroscopy has been used to investigate the orientation of the retinylidene chromophore of

Nuclear Magnetic Resonance

546

bacteriorhodopsin employing an oriented sample of purple membrane on glass slides.86A Monte Carlo method has been developed to accurately simulate the 2H NMR lineshape due to the distribution of bond angles and the orientational disorder of the membranes. The solutions obtained from 2H NMR have been combined with additional constraints from linear dichroism and electron cryomicroscopy to obtain the allowed orientation of retinal in the membrane structure. Bovine rhodopsin containing 11-cis-retinal, specifically deuterated at its methyl group at the C19 or C20 position has been uniaxially oriented in DMPC bilayers and its 2H NMR spectrum obtained.87 Angles for the individual labeled chemical bond vectors leading to an overall picture for the three-dimensional structure of the polyene chain of the chromophore have been obtained. Complete resolution of the amide resonances in three-dimensional solid-state NMR correlation spectrum of a uniformly I5N labeled membrane protein in oriented phospholipid bilayers is reported.88 ‘H chemical shift, IH-l5N dipolar coupling and 15N chemical shift associated with the amide resonances have been obtained which provide sufficient angular restrictions for protein structure determination. Perdeuterated indole-d6 and Nmethylated indole-d6 were solubilized in lamellar liquid crystalline phases or 1,2-dialkylcomposed of either 1,2-diacyl-glycero-3-phosphocholine/water glycero-3-phosphocholine/waterand were studied by 2H NMR.89 The molecular ordering was determined from the deuterium quadrupole splittings on the macroscopically aligned lipid bilayers. The results suggest that the tryptophan analogs are solubilized in the interfacial region of the bilayer and are discussed in terms of the hydrogen bonding in the case of indole and the lipid carbonyl groups. The use of solid state NMR methodology for providing structural constraints from uniformly aligned samples in lamellar phase lipid environment is illustrated with the example of gramicidin A channel structure at high r e s o l u t i ~ n .The ~ ~ application of magic angle spinning to uniformly aligned biomembrane samples is reported.” The method is shown to provide significant resolution improvements for protons even at a spinning speed of 220 Hz in a magnetic field of 9.4 T and has been applied to a lipid and a membrane protein. A new method for the preparation of oriented biological samples based on the preparation of small unilamellar proteolipid vesicles has been reported along with the NMR results.92The effect of gramicidin A on the dynamics of two model membranes in the lamellar and in the hexagonal phases using 31P NMR has been studied.93 2H NMR spectra of oriented bilayers made of DMPC and deuterated cholesterol have been used to obtain very accurate quadrupolar splittings for eight C-D bonds of cholesterol, which has enabled a re-evaluation of the data published and led to the estimation of the molecular order parameter to be 0.95 0.01.94

8

Weak Ordering and Biomolecular Studies

Several approaches are being attempted to obtain a weak alignment of molecules, such as the use of high magnetic fields95,the use of thermotropic

547

14: Oriented Molecules

liquid crystals with low order ~ a r a m e t e rand ~ ~the ? ~ use ~ of a paramagnetic ion to increase the overall magnetic susceptibility anisotropy of the molecules.98 Another possibility is based on using lipid bilayer fragments that have come to be known as 'bicelle~',~~ which are formed as liquid crystalline arrays, when prepared at 20-30 weight YOlipid to aqueous buffer using mixtures of long chain phospholipids such as dimyristoyl phosphatidylcholine (DMPC) and dihexanoyl phosphatidylcholine (DHPC). loo It has been shown that one can dilute such bicelle medium by a factor of five or six to produce cooperatively oriented homogeneous medium with spaces between bicelles large enough to accommodate soluble proteins and that the level of orientational order can be tuned to yield a favourable residual dipolar splitting by adjusting the concentration of the bicelles in the mixture.'" Distances and angles derived from 'H-15N,1HQ-13Ca and 13Ca-13C dipolar couplings for human ubiquitin in such a medium are shown to be in very good agreement with the crystal structure data of the molecule. The bicelles have been shown to have a negligible effect on the rotational diffusion of the protein, ubiquitin as judged by the 15N TI, values of the backbone amides relative to those in isotropic aqueous solution.lo2 The above method as well as other novel techniques available for structure determination of larger and more complex biomolecules by NMR have been reviewed.lo3 The simultaneous measurement of H-' 5N, H- 3C', 5N- 3C' dipolar couplings in a perdeuterated 30 kDa protein dissolved in a dilute liquid crystalline phase has been demonstrated.lo4 It has been shown that, in high magnetic fields, the "N line-narrowing obtained by constructive use of relaxation interference between 15N-'H dipolar coupling and 15N chemical shift anisotropy permits accurate measurement of even the small couplings between 13C' and 'HN and the one-bond dipolar coupling between lHN-15N could be derived from the data set. Measurements of dipolar couplings for methine, methylene and methyl groups in weakly oriented macromolecules have also been re~0rted.l'~It has been shown that the measured side chain dipolar couplings are in good agreement with an ensemble of NMR structures calculated without the use of the dipolar couplings. Residual one-bond 13C-13C and 13C-lH dipolar couplings of methyl groups in weakly aligned human ubiquitin have been measured with high accuracy. lo6 Experimentally, the ratio between 13C-'H and 13C-13Cdipolar couplings is found to be -3.17f0.03. Assuming a static conformation of the methyl group, rapidly spinning about its 3-fold symmetry axis, this ratio corresponds to an aveage C-C-H bond angle of 110.9+ 1 degrees, which is larger than the ideal tetrahedral value of 109.5 degrees. Data indicate that the geometry of the various methyl groups is quite uniform, but that small (less than or equal to 1 degree) deviations of the angle between the C-C vector and the axis connecting the methyl carbon to the geometric center of the three methyl protons may occur. The use of the residual dipolar coupling in concert with backbone relaxation rates to identify conformational exchange has been reported. lo7 A procedure is presented in which complementary in-phase and antiphase components in a 2D-NMR spectrum with J couplings are recorded.lo8 The sum and difference of these spectra

'

'

' '

548

Nuclear Magnetic Resonance

contain only the up-field and the downfield components of a doublet, making it possible to measure the J splitting directly. The method enables dipolar couplings to be obtained by measurements in the isotropic and oriented phases and has been demonstrated for the case of the protein ubiquitin. A triple resonance pulse scheme for recording 13Cu-1Hone bond dipolar couplings in 15N-13Clabeled proteins has been proposed.109The experiment makes use of the methodology in which the protein ubiquitin is dissolved in a dilute solution of bicelles which orient in the magnetic field above a critical temperature. To make use of the information regarding the dipolar couplings available from the alignment of macromolecules in an aqueous dilute liquid crystalline medium, the magnitude of the axial and rhombic components of the alignment tensor must be known;110the rhombic component of the alignment tensor is the difference in the degree of alignment along two orthogonal axes in a plane perpendicular to the axial direction. Unlike the case of the small molecules,111,112 for globular proteins a shape which is not distorted by the liquid crystalline medium is assumed. By also assuming that the internuclear vectors are randomly distributed with reference to the alignment tensor, it is shown that a histogram of the ensemble of normalised residual dipolar couplings approximates to a power pattern from which the magnitude of the axial and rhombic components of the tensor are extracted, without any prior structural information. The applicability of this method has been demonstrated using synthetic data derived for four proteins and experimental data on ubiquitin. To ascertain the magnitude of the axial and rhombic components of the alignment tensor and to simultaneously refine the structures, a calculational strategy that makes use of simulated annealing refinement against the residual dipolar couplings in combination with a grid search has been proposed.l13 Anisotropy of the molecular magnetic susceptibility arising out of binding a protein to DNA has been utilized to achieve weak alignment of a complex of the DNA-binding domain of the transcription factor GATA-1 with a sixteen base pair oligodeoxyribonucleotide, in magnetic fields of 8.5 T and above. l 4 The use of a large number of residual dipolar couplings between 'H and 13C and 'H and 15N as powerful restraints in determining the structures of such magnetically oriented macromolecules is demonstrated. Highly accurate one bond N-HN, C'-Hu, C'-C' and C'-N and two bond C'-HN dipolar couplings have been measured in I3C/l=N-enriched ubiquitin oriented in mixtures of DMPC, DHPC and CTAB.'I5 Using the data, calculation of the relative vibrationally corrected ayerage bond lengths have been carried out. Using C'N bond length of 1.329 A as a reference, the relative C'-C', N-HN and C*-H* distances have been calculated. While the C(-C(distanceof 1.526 is found to be in agreement with crystal structure studies, the N-HN and Cu-Hudistances are considerably longer which is attributed to fast librations of these bonds which contribute to a reduction of the observed dipolar couplings. One bond J couplings between C'-Hu at two different magnetic fields for the case of 13C labeled protein ubiquitin have been measured.lI6 The field dependence of the J coupling has been shown to arise out of the residual dipolar couplings due to the weak alignment of the protein in the magnetic field. It has been shown that

'

A

14: Oriented Molecules

549

the N-H dipolar couplings of myoglobin117are in reasonable agreement with the static crystal structure, provided the magnitude and orientation of the magnetic susceptibility tensor are used as variables. Use of singular value decomposition as a method for calculating the Saupe order matrices, principal axis frames and order parameters has been demonstrated, even when a very limited set of experimental data is available.119 Analysis of lH-l 5N dipolar couplings, measured in a two-domain fragment of the barely lectin protein, is used to illustrate the computational method. Heteronuclear NMR studies on selectively 13C and 5N labeled N-myristoylated peptide (Myrl5) in a magnetically oriented membrane array has been carried out.120 Residual dipolar couplings between 13C-13C, 15N-'H and l3C-I5N pairs have been obtained and interpreted using order matrix calculations to yield orientational and dynamic information for four separate, rigid amide planes. The results indicate that the amphiphathic peptide interacts with the bilayer in a mode that is consistent with an a-helix having its axis parallel to the membrane surface. The structure of the protein rat apo-SIOOB (PP) has been recalculated using a large number of constraints derived from dipolar couplings that were measured in a dilute liquid crystalline phase.I2' The use of dipolar couplings have been shown to remove much of the uncertainty in nOebased structures, as well as ambiguities regarding the conclusions on the Ca2+induced conformational change required for target protein binding. Various NMR constraints used for the refinement of macromolecular structure have been reviewed.122It has been shown that refinement against residual dipolar couplings obtained by dissolving the macromolecules in low ordered liquid crystals provide long range constraints that are not accessible to other solution NMR parameters. Homonuclear proton-proton correlation spectroscopy has been applied to an oligosaccharide oriented in a dilute bicelle medium.123 Potential application of such a technique has been illustrated with data on a trisaccharide. Measurement of residual dipolar couplings for 3C enriched oligosaccharides in a dilute liquid crystalline medium has been reported. 24 The long range conformational restraints provided by the measurements have been used in dynamical simulated annealing calculations. Deuteron NMR spectroscopy has been employed to characterise the concentration dependence of orientational order in DMPCIDHPC bicellar solutions with molar ratios of DMPC/DHPC = 3.3, 2.7 and 2.3.125A dilute liquid crystal which is robust in terms of sample conditions, temperature and buffer concentration and which is inert in terms of binding of aqueous membrane associated proteins has been reported. 126 This lyotropic liquid crystal consists of a 2-50h (w/w) aqueous solution of an equal weight mixture of cetylpyridinium chloride and n-hexanol in 200 mM NaCl. An alternative to the use of bicellar liquid crystalline phases has been proposed which is the use of the nematic phase of a colloidal suspension of rod-shaped viruses.127It is shown that these viruses are highly suitable for obtaining moderate degrees of alignment of macromolecules for the purpose of measuring residual dipolar couplings. Tunable degrees of alignment of macromolecules by addition of magnetically aligned Pfl filamentous bacteriophage

550

Nuclear Magnetic Resonance

b

a

109.8 II

-93 Hz

I

/

I I I I

110.7

-85Hz i

I I

I

G25

I

I II

111.6 PPm

G6 1

I I I I

I I

,

1

I I II

$

112.5

I

; -94 Hz

i -107 HZ I

II

I

113.4

1

10.2

1

10.0

9.8

10.0

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PPm

9.8

Figure 14.4. 2 0 ("N, ' H ) HSQC spectra corresponding to glycines 25 and 61 of bovine apocalmodulin with and without H decoupling during t2 and t l , respectively (a) in isotropic phase and (b) in the presence of phage p f l . Separation between the cross peaks along Fl axis is different in (b) due to the contribution from residual dipolar couplings. (Reproduced with permission from Nature Struct. Biol., 1998,5, 1065).

'

as co-solute has been proposed. 12* This phage induced alignment technique has been used to study 'H-*H,'H-13Cand 'H-15N dipolar coupling interactions in a DNA duplex and RNA hairpin and several proteins including thioredoxin and apocalmodulin (Figure 14.4). Alignment of water soluble biomacromolecules in the presence of purple membrane has been reported.129The extent of the alignment is tunable by the concentration of purple membranes and by the salt which reduces the strength of the electric interaction between solute macromolecule and the membrane. The induced alignment of solute molecules is shown for the two proteins, ubiquitin and p53, with residual dipolar onebond 'H-''N couplings of the order of 20 Hz. A novel bicelle system composed of 1,2-di-o-dodecyl-sn-glycero-3-phosphocholine and 3-(chloramidopropy1)dimethylammonio-2-hydroxyl- 1 -propane sulfonate has been suggested for orienting macromolecules over a wide temperature range at low pH and its utility demonstrated by the measurement of large residual dipolar couplings for the protein rusticyanin from thiobacillus ferrooxidans at pH 2.1. 130 The issues that are involved in the use of bicelles for weak alignment of solute molecules with magnetic field, particularly in the context of the weak alignment

14: Oriented Molecules

55 1

of biological molecules for the purpose of extracting the dipolar couplings between pairs of 'H, 13Cand 15N nuclei, are discussed in terms of the stability of the liquid crystals, the degree of alignment, salt concentration, temperature and pH.13' The problems associated with the use of lanthanides for the study of phospholipid membranes and membrane proteins by magnetic field alignment . ~NMR ~ ~ ~studies ~ ~ ~ of myristoyland means of solving them are d i s c ~ s s e d2H ated peptide have been used to illustrate how magnetically aligned neutral and acidic phospholipid bicelles can be used to characterize the ordering and mode of binding of peptides to membranes. 34 Doping dilute bicelle solutions with small amounts of charged amphiphiles is shown to substantially improve their stability and degree of alignment in the magnetic field as well as extend the temperature range of orientation for these systems.135The use of a mixture of dilauroyl phosphatidylcholine and 3-(cholamidopropyl)dimethylammonio-2hydroxyl- 1-propane sulfonate in water which forms disc shaped bicelles that become ordered at high magnetic fields over a wide range of temperatures is suggested and illustrated for the FK506 binding protein. 136 Europium ions bound to DNA have been shown to assist the magnetic alignment of DNA, leading to observation of proton-carbon dipolar couplings, which have been used to determine the positions of the alignment axes relative to the position of the binding sites of the paramagnetic europium ions.'37 The occurrence of water which displays a residual 170-quadrupolarsplitting in a model biological system and in an excised tissue sample has been demonstrated. 13* The resulting 170-NMR spectra are shown to have the characteristics predicted in computersimulated I = 512 NMR spectra.

9

Relaxation Studies

Deuterium NMR study of quadrupolar splittings and spin-lattice relaxation times as a function of temperature at two different Larmor frequencies on haxakis(n-hexy1oxy)triphenylene is reported. 39 The additive potential method is used to model the quadrupolar splittings and the order parameter tensor is determined. The rotational diffusion constants for the spinning and tumbling motions of the molecular core are also estimated. Deuterium spin-spin relaxation studies at 46 MHz in the perdeuterated liquid crystal 4-n-pentyl-cyanobiphenyl have been reported and the spectral densities for various carbon sites determined. I4O Model parameters that include rotational diffusion constants, internal jump constants and the pre-factor for director fluctuations have been determined as a function of temperature. The deuterium spin-spin relaxation times in the nematic phases of p-methoxy-d3-benzylidene-dl -p-n butyl-d9aniline (MBBA-d13) and 4-n-pentyl-dl l -4'-cyanobiphenyl-d4 (5CB-d 15) have been measured using quadrupolar echo pulse trains and the spectral densities at different sites derived and used to obtain insights on director fluctuation^.'^^ A numerical procedure has been proposed to derive a formal distribution of collective fluctuation modes from experimental field-cycling NMR relaxometry

552

Nuclear Magnetic Resonance

data of ordered systems and the method has been demonstrated for the case of potassium laurate/l -decanol/D20 system.142 Deuterium longitudinal (T1Z) and quadrupolar (T1Q) spin-lattice relaxation times and quadrupolar splittings have been measured for a mixture of perdeuterated 4-n-hexyloxy-4’-cyanobiphenyl (60CB) and 4-n-octyloxy-4’-cyanobiphenyl (80CB) at two different frequen~ i e s . The ’ ~ ~mixture having 28 wt% of 60CB shows nematic, smectic A and reentrant-nematic phases. The additive potential method is used to construct the potential mean torque using the quadrupolar splittings. The dynamic and static behaviours of the mixture is found to be similar to that of pure 60CB. The results indicate that 60CB and 80CB tend to form dimers, the degree of dimerization being inferred from the dynamics of the 60CB molecules. 14N NMR measurements have been carried out in the D phase of liquid crystals belonging to the family 4’-n-alkoxy-3’-nitrobiphenyl-4-carboxylic acids. 144 Relaxation measurements carried out on the single peak observed in this phase indicated the existence of two relaxational processes and suggested that the molecules act as a dimer. Two mixtures of cetyltrimethylammonium bromide and formamide which form lyotropic mesophases have been studied using 2H and 14N-NMR spectroscopy at different temperature^.'^^ The 2HNMR lineshapes of deuterated formamide are explained with a model in which formamide is moving fast compared to its nuclear quadrupole coupling constants in uniaxial domain oriented in different directions. The orientation parameters and the average location and orientation of the constituents among the micelles are discussed. A detailed study of phenyl ring dynamics and spin relaxation in a highly ordered main chainhide chain liquid crystal polymer has been carried out with models for the different motional processes discussed with reference to experimental measurements of the deuterium spectral densities.146 Proton spin-lattice relaxation dispersion measurement has been carried out by means of the fast field cycling NMR technique in the smectic-C phase of 4,4’bis-heptyloxy-azoxy-benzeneand a bimolecular structure associated with the elemental liquid crystalline unit has been proposed. 147 Simultaneous analysis of the deuterium NMR spin relaxation rates as a function of both frequency and sample orientation have been used to obtain dynamic models for lipid bilayer~.’~* The frequency dependence of the longitudinal proton spin relaxation time has been measured using standard methods and the field-cycling technique. The utility of the methods to study collective motions and bending rigidity in multilamellar system of lipid and surfactant bilayers is illustrated. 149 Jeener-Broekaert pulse sequence has been combined with fast field-cycling technique to study dipolar relaxation times in the low Larmor frequency regime on thermotropic nematic liquid crystals and the order fluctuations of the director is attributed as the relaxation mechanism for this process. 150 10

Molecular Order

2H NMR spectra from the pentadeuterated aromatic ring of a series of linear benzyl-d5-alkyl ethers, with alkyl chain length from 1 to 15 carbon atoms have

14: Oriented Molecules

553

been obtained by incorporating these molecules into type I1 cationic nematic lyotropic liquid crystals.151 Using the 2H quadrupole splittings, the two order parameters that describe the average orientation of the aromatic ring with respect to the direction of the magnetic field have been estimated. The results indicate that the aromatic ring plane of benzyl methyl ether is oriented nearly parallel to the bilayer surface and that the aromatic ring progressively incorporates into the superstructure as the alkyl chain length increases. A discussion on the location and orientation of the aromatic ring of the guest molecules and the symmetry and solvation capabilities of the head groups is also presented. Molecules with similar size and shape have been used to investigate the effects of dipole and quadrupole interactions on the average orientation of solutes in nematic liquid crystals152.Order parameters predicted from strong, short-range repulsive forces coupled with interactions between the solute quadrupole and the average electric field gradient felt by the solute are shown to be consistent with experimental values. Nine planar molecules have been used as probes to investigate the -mechanism of orientational ordering in the nematic phase. 153 Orientational order parameters have been derived from proton dipolar couplings. The data are interpreted in terms of an orientational potential, for which a model is presented and compared with other models suggested earlier. Tritium NMR study has been carried out on T2, HT and DT isotopomers of dihydrogen dissolved in different nematic phases, including a zero-electric-field gradient mixture and ab initio calculations have been performed to reproduce the observed dipolar couplings.154 Two contributions to the solute orientation have been identified, one of them being the interaction between the mean solvent electric-field gradient and the solute quadrupole moment and the other contribution has been modeled with a phenomenological mean field interaction and its effects studied. 2H-NMR measurements of orientational order and spectral densities in the nematic, smectic A and smectic B phases of p-hexyloxybenzylidene-p(-fluoroaniline deuterated in the benzylidine methine group and in the aniline ring are r e ~ 0 r t e d . lThe ~ ~ system exhibits a high order of the aromatic core in the smectic phases. Assignment of the 13C spectral lines of the antiferroelectric liquid crystal 4-[( 1-methylheptyloxy)-carbonyll-phenyl 4-[4-(octy1oxy)biphenyllcarboxylate has been reported. 56 With a trifluoromethyl group attached to the chiral centre, the 19F-13Cdipolar couplings were measured to assist in the assignment. The principal elements of the shielding tensor have also been obtained and the orientational order parameter in the SA phase evaluated. The order parameters of various molecular segments in a liquid crystal can be obtained from the measurement of C-H dipolar coupling constants which is accomplished using 2D 13C-NMRtechniques such as the use of separated local field spectroscopy (SLF) in combination with variable angle spinning (VAS)' 57 or by the use of transient oscillations in cr~ss-polarisation.~~~ The use of such 2D techniques has the requirement of prolonged spectrometer time. On the other hand the measurement of chemical shift which also can indicate local order is less time consuming. However, in order to use the chemical shift

554

Nuclear Magnetic Resonance

information effectively, the correlation between I3C chemical shifts and order parameters of the aromatic rings obtained by the SLFlVAS method for nine classes of liquid crystals in their nematic and smectic phases have been obtained. 159 The parameters in the linear correlation provide a convenient way to obtain the order parameters from direct measurement of 13Cchemical shifts for other homologous members of these classes of liquid crystals. The orientational ordering of six liquid crystalline compounds belonging to the homologous series of 4-n-alkoxy benzylidene-4'-n-alkylanilines and 4,4'-di-nalkyldiphenyldiacetyleneshas been studied at different temperatures by 2D 13C NMR with variable angle spinning. 160 A linear correlation between the chemical shifts of the carbons in the aromatic cores and the order parameters was observed, thus providing additional data base of order parameters versus 13Cchemical shifts. Influence of large lateral substituents on the nematic liquid crystalline properties has been studied by I3C NMR.16' The compounds contain four rings in main core and a lateral hexyloxy chain and a lateral aromatic branch modified by different substituents at the meta or para positions. From the 13C chemical shift and the measurement of the order parameters, the orientations of the lateral chains and their influence on the molecular shape and orientation are derived. The orientational ordering behaviour of four compounds containing a lateral alkoxy chain has been investigated by 2D SLFlVAS technique and 13C chemical shift measurements.162 It was observed that the first methylene fragment in the lateral chain adopts a cis conformation and the chain is aligned parallel to the long molecular axis. The terminal substituent near the lateral chain was also found to have a great influence on the ordering of the para-disubstituted aromatic ring. Laterally substituted liquid crystalline compounds containing four rings in the core with two lateral substituents on the same side of one of the inner rings have been studied by the 2D variable angle spinning technique and by following 13C chemical shift as a function of t e m p e r a t ~ r e . The ' ~ ~ results indicate that the two lateral substituents are folded back along the mesogenic core. The mean width of the molecule is substantially increased. However, the core order is not influenced by the type and position of the substituents. Mesogens containing four rings in the main core and six aliphatic chains at the terminal and lateral positions have been examined by the 2D dipolar oscillations technique and 13C chemical shift measurements.164The direction of the jump in the I3C chemical shift at the nematic-isotropic phase transition and the bond order parameter of the methyleneoxy group indicate that two lateral alkoxy substituents are folded back along the mesogenic core and these chains are more ordered than the ones folded along the terminal chain. Laterally dialkylated nematogens containing a short terminal polyoxyethylene chain have been studied by I3C NMR in the liquid crystalline phase and are found to show a wide nematic range.165The order parameters of the oxyethylene unit and the field-induced I3C chemical shifts do not have a linear correlation, indicating that the proportion of gauche- and trans-conformations is temperature dependent. A liquid crystal containing four rings in the main core, two lateral hexyloxy

14: Oriented Molecules

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chains and a terminal dioxyethylene methyl ether chain has been investigated?" The orientations of the lateral chains and the dioxyethylene chain have been obtained and the order parameters measured. The orientational ordering of a series of ferroelectric liquid crystals 4 [(2S,3S)-3-methyl-2-halopentanoyloxy]-4-hexyloxybiphenyls with the halogen atom being fluorine, chlorine or bromine and their racemates has been studied by two-dimensional 3C-NMR spectroscopy.* 67 Carbon-proton dipolar coupling constants and the order parameters of the biphenyl core segments and of each carbon-proton bond in the aliphatic chains were measured as a function of temperature. The results show that the substitution of different halogens on the chiral chain affects the carbon-proton bond order parameters of the entire chiral chain, while the orientational ordering of the rest of the liquid crystal molecule remains unchanged. The results also indicate that there is negligible change in the mesogen conformation at the SmA-SmC* transition. A chiral centre has been introduced in the lateral alkoxy chain of mesogens containing four rings in the main core and the folding of each branch has been studied by 13C NMR.'68 Compounds belonging to the homologous series of trans-1(trans-4-alkylcyclohexyl)-2-(4-chlorophenyl)-difluoroethylenehave been investigated by X-ray diffraction and one- and two-dimensional 13C NMR.169The 13C spectra which are complex due to coupling to two 19F nuclei have been analysed by combining chemical shift anisotropy and variable angle spinning studies. The order parameters of the two conjugated molecular segments have been obtained and their temperature dependences have been studied. Four compounds each containing four aromatic rings in the main core and a lateral N,N-dialkylated aminomethylene fragment on one of the inner rings have been studied by 13C NMR.170 Three of these compounds containing two butyl, hexyl or octyl chains grafted on the lateral nitrogen atom exhibit an enantiotropic mesophase and the mean conformations of the aliphatic chains and aromatic rings have been studied. Upon heating in the nematic phase or in the isotropic melt all the compounds give a new solid phase. The solid formed has been identified as a disubstituted 2H-indazole which is found to exhibit a larger nematic range stable at high temperature. Six members of a new homologous series of achiral banana-shaped molecules have been synthesised and studied by optical microscopy, differential scanning calorimetry, NMR spectroscopy and X-ray diffraction in their smectic phases.17' From the 13C NMR studies, an orientational order parameter of 0.8 has been estimated and the conformation of the molecules has been derived. High resolution 13C NMR study of an antiferroelectric liquid crystal, 4-[( 1-methylheptyloxy)carbonyl]phenyl 4-[4-(octyloxy)biphenyl]carboxylate has been studied.17* The structures of both the chiral and achiral chains have been studied and it is shown that the average direction of the chiral chain deviates from the molecular long axis by 43", while that of the achiral chain extends along the long molecular axis. The conformation around the C,-C,. bond of the alkyl spacer of a,o-bis [(4,4'-cyanobiphenyly1)oxyl decane has been evaluated in the solid and the liquid crystalline states.'73v'74In the 13CNMR spectra, a large change in the 13Cchemical shift was observed on

556

Nuclear Magnetic Resonance

going from the isotropic to nematic phase due to the uniaxial alignment of the molecule. From the 13C chemical shift tensor components determined in the solid state, the order parameters along the molecular axis, the ring order parameters and the order parameter for the C,-C, axis have been determined at different temperatures in the mesophase. Two structures have been proposed for the intercalated smectic phases formed by non-symmetric liquid crystal dimers in which different mesogenic groups are linked by a flexible spacer and have been distinguished by following the deuterium quadrupole splitting as a function of time even as the directors align in a magnetic field.175The orientational order of the intercalated phases has also been studied by EPR and 2H NMR s p e c t r o ~ c o p y . ~ ~ ~

11

Structure and Conformation

The molecular structure and conformation of methyl acrylate have been determined by analysing the 'H NMR spectrum with I3C satellites of the molecule dissolved in liquid crystal ZLI 1132.177The dipolar couplings have been corrected for molecular vibrations. The correlation between reorientational motion and internal rotation has also been taken into account by using the mean external potential described in terms of the bond interaction tensors. The molecular structure of y-picoline has been determined in the gas phase and in liquid crystals by electron diffraction and 'H NMR, respectively, and by the combined use of rotational constants, vibrational frequencies and ab initio calculations.178Liquid crystal solvents used were ZLI-1132, EBBA and a mixture of these two. The ratios of the inter proton distances were determined from the dipolar couplings corrected 'for molecular vibrations. The differences between the ratios for the three different solvents as well as between the ratios were determined earlier'79 and now are less than 1% whereas the differences between the ratios in the mesophase and the corresponding ratios in the gas phase are less than 2%. The proton spectrum of a sample of o-fluoroacetophenone dissolved in nematic liquid crystalline solvent has been obtained and analysed to yield a set of ten independent dipolar couplings.lgOThese have been analysed to obtain a conformational distribution of the bond rotational angles about the C-CF bond and about the ring-C bond and a correlation between the respective barrier to rotation. The structure of 2,2'-difluorobiphenyl has been determined in the solid phase by X-ray diffraction and in the liquid crystalline phase by NMR.18' While in the solid phase the molecules are all in a single conformation having the two fluorine nuclei in a syn-arrangement, in the liquid crystalline medium, the syn- and anti-forms are present in the approximate ratio of 0.58 : 0.42, The dipolar couplings between carbons and fluorine in the fluorinated biphenyl groups in the two monofluorinated nematic liquid crystals I35 and 152, together with a mean field theoretical model, has been used to determine the shape of the potential governing rotation about the inter-ring bond.Ig2For rotation about the aromatic ring to alkyl chain bonds, the minimum energy corresponds to when the chains are

14: Oriented Molecules

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orthogonal to the rings. The model also predicts that the conformer probability for the molecules in the nematic phase is substantially different from those in the isotropic phase at the same temperature. The ethylene complexes Os2(C0)&-q',qf-C2H4) and Os(C0)4(q-C2H4) have been studied by 'H NMR in the nematic phase of liquid crystalline solvents.18313C labeling has been used to obtain l3C-IH dipolar couplings in addition to the intramolecular geminal, cis and trans 'H-'H dipolar couplings. The absolute bond angles and the relative bond lengths for the ethylene portion have been derived. Deuterium substitution has also been made to demonstrate the use of liquid crystal NMR as a stereochemical probe. The conformational probabilities for rotations about the ring-oxygen bonds in 1,2dimethoxy-(DMB) and 1,2,3-trimethoxy-benzene(TMB) have been determined by comparing observed and calculated proton dipolar couplings obtained by the analysis of the *H NMR spectra of the samples dissolved in nematic liquid crystalline solvents.184 The calculated couplings are obtained by averaging over the conformations which are obtained by taking 5 " steps in bond rotational angles with the sterically hindered conformers being eliminated by including a steric term in the bond rotational energies. The results obtained are compared with conformational distributions obtained by analysing the same data with a rotational isomeric state model from which it is concluded that this model produces a reasonable approximation to the correct distribution for DMB, but not for the more sterically hindered TMB.

12 1

2 3 4

5 6 7 8

9 10 I1

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