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Nuclear Magnetic Resonance Volume 33 A Specialist Periodical Report Nuclear Magnetic Resonance Volume 33 A Review of...

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Nuclear Magnetic Resonance Volume 33

A Specialist Periodical Report

Nuclear Magnetic Resonance Volume 33 A Review of the Literature Published between June 2002 and May 2003 Senior Reporter G.A. Webb, formerly University of Surrey, Guildford, UK Reporters A.E. Aliev, University College, London, UK N. Asakawa, Tokyo Institute of Technology, Tokyo, Japan I. Barsukov, University of Leicester, UK A.C. de Dios, Georgetown University, Washington, DC, USA H. Fukui, Kitami Institute of Technology, Kitami, Japan E.F. Hounsell, Birkbeck College, London, UK C.J. Jameson, University of Illinois at Chicago, USA K. Kamienska-Trela, Polish Academy of Sciences, Warszawa, Poland C.L. Khetrapal, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India S. Kuroki, Tokyo Institute of Technology, Tokyo, Japan H. Kurosu, Nara Women's University, Nara City, Japan R.V. Law, Imperial College of Science, Technology and Medicine, London, UK R. Ludwig, University of Rostock, Germany S.J. Matthews, Imperial College of Science, Technology and Medicine, London, UK M. Monduzzi, University of Cagliari, Monserrato, Italy G.A. Nagana Gowda, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India and Indian Institute of Science, Bangalore, India 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 T. Watanabe, Aoyama Women's Junior College, Tokyo, Japan J. Wojcik, Polish Academy of Sciences, Warsaw, Poland T. Yamanobe, University of Gunma, Japan H. Yasunaga, Kyoto Institute of Technology, Kyoto, Japan

RSaC advancing the chemical sciences

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ISBN 0-85404-347-0 ISSN 0305-9804

A catalogue record for this book is availabe from the British Library Copyright 0 The Royal Society of Chemistry 2004

All rights reserved Apartjiom any fair dealing for the purposes of research or private study, or criticism or review as 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 Registered Charity Number 207890 For further information see our web site at www.rsc.org Typeset by Vision Typesetting, Manchester, UK Printed by Athenaeum Press Ltd, Gateshead, Tyne and Wear, UK

It is my pleasure to introduce Volume 33 of the Specialist Periodical Reports on NMR. This volume contains comprehensive coverage of the relevant literature appearing between June 2002 and May 2003. A minor change has occurred in the reporting arrangements for ‘NMR of Liquid Crystals and Micellar Solutions’ in that this will now be covered annually rather than biennially. Apart from this the pattern of coverage presented is that which has been established over a number of years in this series. It is a pleasure for me to express my gratitude to all members of the reporting team for both the comprehensive nature of their reports and for delivering them promptly.

G. A. Webb October 2003

V

Contents

Chapter 1 NMR Books and Reviews B y W. Schilf 1 2 3 4 5

1

Books Regular Reviews Series Edited Books and Symposia Reviews in Periodicals Reviews and Books in Foreign Languages

Chapter 2 Theoretical and Physical Aspects of Nuclear Shielding B y C.J. Jameson and A.C. de Dios 1 Theoretical Aspects of Nuclear Shielding 1.1 General Theory 1.2 A b initio Calculations 1.3 Semi-empirical 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 2.5 Absolute Shielding Scales References Chapter 3 Applications of Nuclear Shielding B y S. Kuroki, N . Asakawa and H . Yasunaga 1 Introduction 2 Shielding of Particular Nuclear Species 2.1 Group 1 ('H, 'H, 697Li,23Na,39K,87Rb,133Cs) 2.2 Group 2 (9Be,25Mg,43Ca, '37Ba) 2.3 Group 3 and Lanthanoids (45Sc,89Y,'39La,171Yb) 2.4 Group 4 (47349Ti) 2.5 Group 5 (Vand 93Nb) Nuclear Magnetic Resonance, Volume 33 0The Royal Society of Chemistry, 2004 vii

1 1 9 21 37

47 47 47 51 58 59 59 62 63 64 69 70

76 76 76 76 79 79 80 80

...

Contents

Vlll

2.6 Group 6 eSMo,lg3W) 2.7 Group 7 ("Mn) 2.8 Group 8 (S7Fe,99Ru) 2.9 Group 9 (59C0,"'Rh) 2.10 Group 10 (19'Pt) 2.11 Group 11 (63Cu,lo79lo9Ag) 9 199H g) 2.12 Group 12 (111,113Cd 2.13 Group 13 ("B, 27Al,7'Ga, 2039205Tl) 2.14 Group 14 ("C, 29Si,73Ge,'173119Sn, '07Pb) 2.15 Group 15 (14,1SN, 'lP) 2.16 Group 16 (170,33S,77Se,'"Te) 2.17 Group 17 (19F,35,37C1) 2.18 Group 18 ('He, lZ9Xe) References Chapter 4 Theoretical Aspects of Spin-Spin Couplings B y H. Fukui 1 Introduction 2 Fully Relativistic Calculation of Nuclear Spin-Spin Couplings 3 Nuclear Spin-Spin Coupling Density in Molecules 4 Proton Spin-Spin Coupling and Electron Delocalization 5 Density-Functional Linear Response Theory 6 Heavy Metal-Metal Coupling Constants 7 Hartree-Fock's Stability Problem 8 Other Ab Znitio Calculations of Spin-Spin Coupling Constants 8.1 Spin-Spin Couplings of the N H 3 . . . HzO Complex 8.2 Carbon-Carbon Coupling Constants in Compounds with Strained C-C Bonds 8.3 Spin-Spin Couplings in the Organolithium Compounds 8.4 Electric Field Effects on 'J(H,H) Spin-Spin Coupling Constants 8.5 H ydrogen-bond Transmitted Indirect Nuclear Spin-Spin Couplings 8.6 Substituent Effects on 'J(F, F) and 'J(F, F) Coupling Constants 8.7 Through-Space Spin-Spin Couplings 9 Other Density-Functional Calculations of Spin-Spin Couplings 9.1 Relativistic Density-Functional Calculations 9.2 Calculation of J(F, F) Spin-Spin Coupling Constants 9.3 Spin-Spin Coupling Constants in Formamide 9.4 'J(C,C) and 'J(C, H) Coupling Constants

81 82 82 82 83 83 84 85 89 95 96 100 102 104

118 118 118 120 121 123 124 127 128 128 129 130 131 131 132 133 134 134 135 136 136

ix

Contents

9.5 Solvent Effects on Spin-Spin Coupling Constants 9.6 Calculations of Spin-Spin Couplings as an Aid to the Conformational Analysis 9.7 Calculations of Spin-Spin Couplings in DNA or RNA References Chapter 5 Applications of Spin-Spin Couplings By K . Kamieriska-Trela and J . WLjcik Introduction New Methods One-bond Couplings to Hydrogen One-bond Couplings not Involving Hydrogen Two-bond Couplings to Hydrogen Two-bond Couplings not Involving Hydrogen Three-bond Hydrogen-H ydrogen Couplings Three-bond Couplings to Hydrogen Three-bond Couplings not Involving Hydrogen Couplings over More than Three Bonds and Through Space 11 Couplings Through Hydrogen Bonds 12 Residual Dipolar Couplings References 1 2 3 4 5 6 7 8 9 10

Chapter 6 Nuclear Spin Relaxation in Liquids and Gases By R. Ludwig

1 Introduction 2 General, Physical and Experimental Aspects of Nuclear Spin Relaxation 2.1 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 Solutions

136 137 138 139 146

146 147 150 153 159 160 161 168 172 173 174 174 178 192

192 194 194 198 199 200 202 204 206 207 210 212 215 215 216 217

Contents

X

3.4 Molten Salts 4 Nuclear Spin Relaxation in Gases 5 Self-diffusion in Liquids 5.1 Experimental and Theoretical Aspects 5.2 Selected Examples References Chapter 7 Solid-state NMR Spectroscopy A.E. Aliev and R.V. Law Introduction Reviews and Introductory Articles Theory and Numerical Calculations New Technique Developments 4.1 Spin 1/2 Nuclei 4.2 Quadrupolar Nuclei Distance and Angle Measurements by Solid-state NMR. Synergic Methodologies NMR Parameters: Experimental and Theoretical Studies 6.1 Chemical Shift and its Anisotropy: Spin 1/2 Nuclei 6.2 J-Couplings 6.3 Quadrupolar Nuclei 6.4 Troublesome Nuclei Applications 7.1 Structural Studies of Organic Solids 7.2 Dynamic Studies of Organic Solids by 2H NMR 7.3 Hydrogen-bonded Organic Solids 7.4 Structural Studies of Organometallic and Coordination Compounds 7.5 Dynamics Studies of Organometallic and Coordination Compounds 7.6 Cellulose and Related Materials 7.7 Amino Acids 7.8 Peptides 7.9 Proteins 7.10 Lipids and Membranes 7.11 Biomedical Applications 7.12 Coals and Carbonaceous Materials 7.13 Soils and Related Materials 7.14 Polymers 7.15 Glasses and Amorphous Solids 7.16 Cements 7.17 Microporus Solids and Related Materials 7.18 Surface Science and Catalysis 7.19 Inorganic and Other Related Solids References

218 219 220 220 222 223

233 233 234 237 24 1 24 1 245 247 250 250 253 253 254 255 255 258 259 260 262 263 264 265 267 271 271 272 273 274 279 28 1 282 285 287 293

xi

Contents

Chapter 8 Multiple Pulse NMR By I . Barsukov 1 Introduction 2 General Methods 3 NOE, Chemical Exchange, Relaxation and Diffusion 3.1 NOE aqd Chemical Exchange 3.2 Relaxation Rate Measurements 3.3 Cross-correlated Kclaxation Experiments 3.4 Diffusion Experiments 4 Coupling Constants Measurements 4.1 Scalar Couplings 4.2 Couplings Across Hydrogen Bonds 4.3 Residual Dipolar Couplings 5 Inverse Proton Detected Correlation Spectroscopy 5.1 Double-resonance Experiments 5.2 Isotope-filtered and Edited Experiments 5.3 TROSY 5.4 Heteronuclear Triple Resonance Experiments References Chapter 9 NMR of Proteins and Nucleic Acids By S.J. Matthews 1 Introduction 2 Application and Development of New Methodology 2.1 Automated Analysis 2.2 Dipolar Couplings 2.3 Transverse Relaxation Optimized Spectroscopy (TROSY) 3 Macromolecular Structures 3.1 Membrane Proteins 3.2 Protein Complexes 3.3 Nucleic Acid Complexes 4 Protein Relaxation and Dynamics 5 Protein Folding References Chapter 10 NMR of Carbohydrates, Lipids and Membranes By E.F. Hounsell

1 2 3 4

Introduction Drug Discovery and Combinatorial Chemistry Metabolomics, Metabonomics and I n Vivo Spectroscopy Membranes 4.1 Sphingomyelins and Glycolipids

306 306 306 307 307 307 308 309 310 310 310 311 314 314 314 315 316 324

326 326 326 326 329 333 335 335 338 342 344 348 351 357

357 357 358 359 359

Contents

xii

5 6

7

8 9

4.2 Lipids and Cholesterol 4.3 Proteins and Peptides within Membranes Extracellular Macromolecular Structure Ligand-Receptor Interactions Involving Glycosylated Molecules 6.1 Oligosaccharide-protein Interactions 6.2 Oligosaccharide Conformation and Their Role in Protein Function Natural Products 7.1 Parasites 1.2 Algae 7.3 Fungi 7.4 Mycobacteria 7.5 Bacteria and Vaccines 7.6 Plants Polysaccharides Materials Sciences References

Chapter 11 Synthetic Macromolecules B y H . Kurosu and T. Yamanobe 1 2 3 4 5 6 7

Introduction Primary Structure Liquid Crystalline Polymers Imaging Characterization of the Synthetic Macromolecules Dynamics of the Synthetic Macromolecules Polymer Blend of the Synthetic Macromolecules References

Chapter 12 NMR in Living Systems By M.J. W. Prior 1 General Applications and New Methodology 2 Cells 2.1 Microorganisms 2.2 Blood 2.3 Cultured Mammalian and Tumour 3 Plants Studies 4 Tissues 4.1 Brain 4.2 Eye 4.3 Heart 4.4 Liver 4.5 Muscle

360 36 1 362 364 364 365 366 366 367 367 368 368 371 374 376 377

386 386 386 405 405 406 412 416 420 439

439 440 440 44 1 44 1 442 442 442 444 448 448 449

...

Contents

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4.6 Tumour 4.7 Whole Organisms 5 Clinical Studies 5.1 Brain 5.2 Heart 5.3 Joints 5.4 Liver 5.5 Muscle References

45 1 45 1 451 45 1 456 456 456 456 459

Chapter 13 Nuclear Magnetic Resonance Imaging B y T. Watanabe

465

1 Introduction 2 Overview and Physicochemical Applications 2.1 Review 2.2 Catalyzed Reaction 2.3 Mesophase Formation 3 Instruments and Materials 3.1 Reviews 3.2 Imaging System 3.3 Magnets 3.4 Probe, Resonator, Coil and Transceiver 3.5 Optical Pumping System for Polarized Gases 3.6 Cryogenic Systems 3.7 Artifact via Implant Biomaterials 3.8 Temperature Measurement 4 Pulse Sequences and Data Processing 4.1 Pulse Sequences 4.2 Data Processing 5 Hyperpolarized Noble Gases, Gas Phase Imaging and Other Nuclei 5.1 Review 5.2 Hyperpolarized Nobel Gases 5.3 Imaging Via Other Gases 5.4 Other Nuclei 6 Dynamics: Diffusion, Flow and Velocity Imaging 6.1 Reviews 6.2 Data Processing 6.3 Diffusion 6.4 Flow in Suspensions and Colloids 6.5 Flow in Tubes 6.6 Gas Flow 6.7 Concentration Profiles in Columns 6.8 Porous Media 6.9 Miscellaneous

465 466 467 467 468 468 468 469 469 469 470 470 47 1 47 1 47 1 47 1 473 473 473 474 474 47 5 476 476 476 477 477 478 479 479 479 480

Contents

xiv 7 Polymer 7.1 Reviews 7.2 Characterization 7.3 Process Analysis 7.4 Polymer Gel Dosimetry 7.5 Tablet Disintegration, Swelling, Drug Release 8 Chemical Engineering and Industrial Application 8.1 Reviews 8.2 Process Analysis 8.3 Drying Processes 8.4 Coating Process 8.5 Softening and Melting Process - Coal and Coke 9 Plants, Trees and Woods 9.1 Reviews 9.2 Trees 10 Food 10.1 Review 10.2 Moisture Migration 10.3 Solid State Fermentations 10.4 Fat Contents, Distribution and Migration 10.5 Fruits and Vegetables 10.6 Tubers 11 In Vioo Application 11.1 Reviews 11.2 Gene Expression 11.3 Migration, Flow and Drug Release 11.4 Functional MRI

References

48 1 48 1 482 483 483 484 484 484 485 485 486 486 487 487 487 487 487 487 488 488 489 489 489 489 489 490 490 49 1

497 Chapter 14 Oriented Molecules B y K.V. Ramanathan, G.A. Nagana Gowda and C.L. Khetrapal 1 2 3 4

5 6

7 8 9 10 11

Introduction Reviews, Theory and General Studies New Techniques Dynamic NMR Studies Chiral, Smectic, Lyotropic and Polymeric Systems Relaxation Studies Orientational Order in Liquid Crystals Membranes and Molecules Oriented Therein Structure and Sheilding Tensor Studies of Small Molecules Quantum Computing Weak Ordering and Biomolecular Studies 11.1 Reviews 11.2 Orienting Media

497 498 500 50 1 503 505 507 508 509 5 10 512 512 513

xv

Contents

New Experimental Methodologies New Pulse Schemes Computational Methods Structure, Conformation, Orientation and Dynamics Studies References 11.3 11.4 11.5 11.6

Chapter 15 NMR of Liquid Crystals and Micellar Solutions By M . Monduzzi 1 Introduction 2 General Articles: Books, Models, Reviews 3 Liquid Crystals 3.1 Thermotropic Liquid Crystals 3.2 Lyotropic Liquid Crystals 4 Micellar Solutions 4.1 Micelles in Amphiphile-Solvent Systems 4.2 Solubilisation, Microemulsions and Emulsions References

515 518 519 520 523 531

53 1 533 534 534 531 545 545 550 556

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 n, 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 A

B

D

*J

Jr

J "K mi

hyperline (electron-nucleus) interaction constant (i) hyperfme (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 v,, v2 spin-rotation coupling constant of nucleus X (used sometimes in tensor form): c2=fee; + 2 c 3 . 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 &) nuclear or electronic g-factor magnetic field gradient element of matrix representation of H' 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("C, 'H) or additionally, the coupling path, e.g. J(P0CF) reduced splitting observed in a double resonance experiment rotational quantum number reduced nuclear spim-spin coupling constant (see the notes concerning "J) eigenvalue of I,, (magnetic component quantum number) equilibrium macroscopic magnetization of a spin system in the presence of Bo components of macroscopic magnetization

xvii

xviii

$(O) S

t

T

a

B

AJ An A6

4+ Au

Symbols and Abbreviations the number of average mol. wt. valence p 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 S, at nuclear A (i) singlet state (ii) electron (or, occasionally, nuclear spin) cJ I (iii) ordering parameter for oriented systems (iv) overlap integral between molecular orbitals elapsed time (i) temperature (ii) triplet state coalescence temperature for an NMR 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 , total dephasing time for M , or M,; (Tf)-' = (T;') + ( T i ) - ' decay time following 90,-~-90, pulse sequences spin-lattice and spin-spin relaxation time of the X nuclei in the frame of reference rotating with B , dipolar spin-lattice relaxation time mole fraction of compound atomic number of atom A (i) nuclear spin wavefunction (eigenfunction of I,) for a spin nucleus (ii) polarizability nuclear spin wavefunction (eigenfunction of I,) for a spin -f 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 = O otherwise) Dirac delta operator (i) time between field gradient pulses (ii) spectral width anisotropy in J (AJ = J - J,, 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 u(Au=ul-uL,for axial symmetry) (ii) differences in u for two different situations (i) susceptibility anisotropy (A =xl -xL, for axial symmetry (ii) differences in electronegatiiities 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)

-4


xix

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 0. components of ‘J parallel and perpendicular to a molecular symmetry axis diagrammatic contribution to u paramagnetic contribution to (J (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 (= e2qQ/h) carrier frequency in rad s-’ as for vi,ve vl, v2 but in rad s-l modulation angular frequency (in rad s-l) sample rotation (rad s-’)

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

xx


cross polarization Carr-Purcell pulse sequence. Meiboom-Gill modification chemical shielding anisotropy chemical shift imaging continuous wave cw digital-to-analogue converter DAC dipole-dipole (interaction or relaxation mechanism) DD distortionless enhancement by polarization transfer DEPT differential line broadening DLB dynamic nuclear polarization DNP double quantum DQ double quantum filter DQF exclusive correlation spectroscopy ECOSY extended Hiickel molecular orbital theory EHT electron-nucleus double resonance ENDOR equations of motion EOM electron spin resonance ESR exchange spectroscopy EXSY Fermi contact FC free induction decay FID fast low angle shot FLASH finite perturbation theory FPT Fourier transform FT gauge included atomic orbitals GIAO heteronuclear multiquantum HMQ homonuclear Hartman-Hahn HOHAHA higher random phased approximation HRPA improved depth selective single surface coil spectroscopy IDESS individual gauge for different localized orbitals IGLO INADEQU [ATE incredible natural abundance double quantum transfer experiment intermediate neglect of differential overlap INDO intermediate neglect of differential overlap calculations for spectroscopy INDO/S internuclear double resonance INDOR insensitive nuclei enhanced by polarization transfer INEPT infrared IR image selected in oioo spectroscopy ISIS lanthanide induced shift LIS local origin LORG lanthanide shift reagent LSR magic angle sample spinning MASS many body perturbation theory MBPT maximum entropy method MEM modified INDO MIND0 multiple quantum MQ multiple quantum coherence MQC multiple quantum filter MQF nuclear magnetic resonance NMR nuclear Overhauser enhancement NOE nuclear Overhauser enhancement spectroscopy NOESY nuclear quadruple coupling constant NQCC nuclear quadrupole resonance NQR pulsed field gradient PFG proton relaxation enhancement PRE quadrupole moment/field gradient QF quadrature phase detection QPD relativistically extended Huckel molecular orbital theory REX rotating frame Overhauser enhancement spectroscopy ROESY random phase approximation RPA self consistent perturbation theory SCPT spin dipolar SD spin echo correlation spectroscopy SECSY

CP CPMG CSA CSI

Symbols and Abbreviations SEFT SLITDRESS SOPPA SPI SPT SR TART TOCSY

uv

WAHUHA

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 T , imaging total correlation spectroscopy ultraviolet Waugh, Huber and HBberlen (cycle of pulses) zero quantum zero quantum coherence

(b) Chemical species* acetylacetonato acac adrenocorticotropic hormone (corticotropin) ACTH adenosine diphosphate ADP adenosine monophosphate AMP adenosine triphosphate ATP bovine serum albumin BSA cytidine monophosphate CMP cyclopentadienyl CP dodecylammonium propionate DAP 1,2-dimethoxyethane DME dimethylformamide DMF dimyristoyl-lecithin DML dimeth ylsiloxane DMS dimethyl sulfoxide DMSO deoxyribonucleic acid DNA 2,3-diphosphoglycerate DPG dipalmitoyl-lecithin DPI dipivaloylmethanato dPm diphen ylpicrylhydrazyl DPPH 2,2-dimethyl-2-silapentane-5-sulfonate (usually as the sodium salt) DSS di-t-butyl nitroxide DTBN N-@-ethoxybenzy1idene)-p-butylaniline EBBA ethylenediaminetetra-aceticacid EDTA ethylene-vinyl acetate EVA 1,1,1,2,2,3,3-heptafluoro-7,7-dimethyloctane-4,6-dionato fod 4,4'-bis(heptyl)azoxybenzene HAB hexameth ylphosphoramide HMPA p-n-hept ylox yazoxybenzene HOAB inositolhexaphosphate IHP potassium dihydrogen phosphate KDP N-@-methoxybenzy1idene)-p-butylaniline MBBA nicotinamide adenine dinucleotide (phosphate) NADH(P) N-methylformamide NMF p-azoxyanisole PAA pyrene butyric acid PBA poly(L-benzyl p-glutamate) PBLG phosphatidyl choline (lecithin) PC polychlorinated biphenyl PCB polydimethylsiloxane PDMS poly(methacry1icacid) PMA poly(methy1methacrylate) PMMA poly(oxymethy1ene) POM phosphatidylserine PS polytetrafluoroethylene PTFE poly(viny1chloride) PVC 'Lower case initials are used when the species is a ligand.

xxi


xxii PVF

PVP RNA SDS TAB TCNQ TFA THF TMS UTP

poly(viny1fluoride) poly(viny1pyrrolidone) ribonucleic acid (tRNA, transfer RNA) sodium dodecyl sulfate trimethylammonium bromide tetracyanoquinodimethane trifluoroacetic acid tetrahydrofuran tetramethylsilane uridine triphosphate

Amino-acid residues Ala alanine '4% arginine Asn asparagine ASP aspartic acid CYS cysteine Gln glutamine Glu glutamic acid GlY glycine His histidine HYP hydroxyproline Ile isoleucine

Leu LYS Met Phe Pro Ser Thr TrP TYr Val

1eucine lysine methionine phenylalanine proline serine threonin tryptophan tyrosine valine

1 NMR Books and Reviews BY W. SCHILF

1 R1

R2 R3

2

R4 R5 R6 R7

R8

Books R.A. Nyquist, ‘Interpreting Infrared, Raman, and Nuclear Magnetic Resonance Spectra’ Vol. 1, ‘Variables in Data Interpretation of Infrared and Raman Spectra’ Vol. 2, ‘Factors Aflecting Molecular Vibrations and Chemical Shifts of Infrared, Raman, and Nuclear Magnetic Resonance Spectra’, Academic, Orlando, Fla. 2001 I.R. Young, ‘Methods in Biomedical Magnetic Resonance Imaging and Spectroscopy, Volume 1 ’, John Wiley & Sons, Chichester, UK, 2000 I.R. Young, ‘Methods in Biomedical Magnetic Resonance Imaging and Spectroscopy, Volume Two’, John Wiley & Sons, Chichester, UK, 2000

Regular Reviews Series Accounts of Chemical Research, Vol. 35,2002 J.B. Lambert and G.O. Poinar Jr., ‘Amber: The Organic Gemstone’ p. 528 Vol. 36,2003 S.M. Weinreb, ‘Lepadiformine: A Case Study of the Value of Total Synthesis in Natural Product Structure Elucidation’, p. 59 Y. Kyogoku, Y. Fujiyoshi, I. Shimada, H. Nakamura, T. Tsukihara, H. Akutsu, T. Odahara, T. Okada and N. Nomura, ‘Structural Genomics of Membrane Proteins’, p. 199 D. Staunton, J. Owen and I.D. Campbell, ‘NMR and Structural Genomics’, p. 207 D.L. Bryce and R.E. Wasylishen, ‘Microwave Spectroscopy and Nuclear Magnetic Resonance Spectroscopy - What Is the Connection?, p. 327

Annual Reports on N M R Spectroscopy, ed. G.A. Webb, Academic Press Ltd., London, UK Vol. 47,2002 R 9 B. Wrackmeyer, ‘Application of 207PbNMR Parameters’, p. 1 R 10 H. Saito, S. Tuzi, M. Tanio and A. Naito, ‘Dynamic Aspects of Membrane Nuclear Magnetic Resonance, Volume 33 0 The Royal Society of Chemistry, 2004

2

R 11 R 12 R 13 R 14 R 15

R 16 R 17

Nuclear Magnetic Resonance

Proteins and Membrane-Associated Peptides as Revealed by 13CNMR: Lessons from Bacteriorhodopsin as an Intact Protein’, p. 39 E. Alberti, P.S. Belton and A.M. Gil, ‘Applications of NMR to Food Science’, p. 109 K.K. Laali and T. Okazaki, ‘NMR of Persistent Carbocations from Polycyclic Aromatic Hydrocarbons (PAHs)’, p. 149 Vol. 48,2002 K. Schmidt-Rohr and H.W. Spiess, ‘Dynamics of Polymers from Oneand Two-Dimensional Solid-state NMR Spectroscopy’, p. 1 M.A. Keniry and J.A. Carver, ‘NMR Spectroscopy of Large Proteins’, p. 31 C.L. Lean, R.L. Somarjai, I.C.P. Smith, P. Russell and C.E. Mountford, ‘Accurate Diagnosis and Prognosis of Human Cancers by Proton MRS and a Three-Stage Classification Strategy’, p. 72 A.T. Watson, J.T. Hollenshead, J. Uh and C.T.P. Chang, ‘NMR Determination of Porous Media Property Distributions’, p. 113 Y.R. Du, S. Zhao and L.F. Shen, ‘Nuclear Magnetic Resonance Studies of Micelles’, p. 145

Annual Reports on the Progress of Chemistry, Section B: Organic Chemistry, Royal Society of Chemistry Vol. 98,2002 R 18 C. Halkides and W.R. Thomas, ‘NMR Spectroscopic Methods’, p. 639 Annual Reports on the Progress of Chemistry, Section C: Physical Chemistry, Royal Society of Chemistry Vol. 98,2002 R 19 H. Weingaertner and M. Holz, ‘NMR Studies of Self-Diffusion in Liquids’, p. 121 Annual Review of Biomedical Engineering, Annual Reviews Inc., Vol. 4, 2002 R 20 R.M. Murphy, ‘Peptide Aggregation in Neurodegenerative Disease’, p. 155

R 21 R22 R 23 R 24

Annual Review of Biophysics and Biomolecular Structure, Annual Reviews Inc., Vol. 31,2002 R.J. Cushley and M. Okon, ‘NMR Studies of Lipoprotein Structure’, p. 177 I. Luque, S.A. Leavitt and E. Freire, ‘The Linkage between Protein Folding and Functional Cooperativity: Two Sides of the Same Coin?’, p. 235 J. Frank, ‘Single-Particle Imaging of Macromolecules by Cryo-Electron Microscopy’, p. 303 M. Ubbink, J.A.R. Worrall, G.W. Canters, E.J.J. Groenen and M. Huber, ‘Paramagnetic Resonance of Biological Metal Centers’, p. 393

Annual Review of Physical Chemistry Vol. 53,2002 R 25 I. Tinoco Jr., ‘Physical Chemistry of Nucleic Acids’ p. 1

1: N M R Books and Reviews

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R 26 E. Oldfield, ‘Chemical Shifts in Amino Acids, Peptides, and Proteins: From Quantum Chemistry to Drug Design’, p. 349 Chemical Reviews, Washington, DC, United States, Vol. 102,2002 R 27 H.C. Aspinall, ‘Chiral Lanthanide Complexes: Coordination Chemistry and Applications’, p. 1807 R 28 D. Parker, R.S. Dickins, H. Puschmann, C. Crossland and J.A.K. Howard, ‘Being Excited by Lanthanide Coordination Complexes: Aqua Species, Chirality, Excited-State Chemistry, and Exchange Dynamics’, p. 1977 Coordination Chemistry Reviews, Vol. 228,2002 R 29 G. Simonneaux and P. Le Maux, ‘Optically Active Ruthenium Porphyrins: Chiral Recognition and Asymmetric Catalysis’, p. 43 R 30 A. Bodor, I. Banyai and I. Toth, ‘Slow Dynamics of Aluminium-Citrate Complexes Studied by ‘H- and 13C-NMRSpectroscopy’, p. 163 R 31 A. Bodor, I. Banyai and I. Toth, “H- and 13C-NMRas Tools to Study Aluminium Coordination Chemistry-Aqueous Al(II1)-Citrate Complexes’, p. 175 R 32 K.M. Elkins and D.J. Nelson, ‘Spectroscopic Approaches to the Study of the Interaction of Aluminum with Humic Substances’,p. 205 R 33 W.R. Harris and L. Messori, ‘A Comparative Study of Aluminum (111), Gallium (111),Indium (111), and Thallium (111)Binding to Human Serum Transferrin’, p. 237 R 34 T.P. Flaten, ‘Aluminium in Tea. Concentrations, Speciation and Bioavailability’, p. 385 Vol. 232,2002 R 35 I. Turel, ‘The Interactions of Metal Ions with Quinolone Antibacterial Agents’, p. 27 Vol. 233-234,2002 R 36 S. Brydges, L.E. Harrington and M.J. McGlinchey, ‘Sterically Hindered Organometallics: Multi-n-Rotor (n = 5,6 and 7) Molecular Propellers and the Search for Correlated Rotations’, p. 75 R 37 J. Chan, Z. Huang, M.E. Merrifield, M.T. Salgado and M.J. Stillman, ‘Studies of Metal Binding Reactions in Metallothioneins by Spectroscopic, Molecular Biology, and Molecular Modeling Techniques’, p. 319 Vol. 235,2002 R 38 V. Chandrasekhar, S. Nagendran and V. Baskar, ‘Organotin Assemblies Containing Sn-0 Bonds’, p. 1 Vol. 236,2003 R 39 E. Tfouni, M. Krieger, B.R. McGarvey and D.W. Franco, ‘Structure, Chemical and Photochemical Reactivity and Biological Activity of Some Ruthenium Amine Nitrosyl Complexes’, p. 57 Current Drug Targets: Infectious Disorders, Bentham Science Publishers Ltd. Vol. 2,2002 R 40 G.L. Gilliland, A. Teplyakov, G. Obmolova, M. Tordova, N. Thanki, J.

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Ladner, 0. Herzberg, K. Lim, H. Zhang, K. Huang, Z . Li, A. Tempczyk, W. Krajewski, L. Parsons, D.C. Yeh, J. Orban, A.J. Howard, E. Eisenstein, J.F. Parsons, N. Bonander, K.E. Fisher, J. Toedt, P. Reddy, C.V. Rao, E. Melamud and J. Moult, ‘Assisting Functional Assignment for Hypothetical Haemophilus Influenzea Gene Products through Structural Genomics’, p. 339 Current H I V Research, Bentham Science Publishers Ltd., Vol. 1,2003 R 41 Y. Morikawa, ‘HIV Capsid Assembly’, p. 1 Current Medicinal Chemistry, Bentham Science Publishers, Vol. 9,2002 R 42 N. Pattabiraman, ‘Analysis of Ligand-Macromolecule Contacts: Computational Methods’, p. 609 R 4 3 K. Gharehbaghi, W. Grunberger and H.N. Jayaram, ‘Studies on the Mechanism of Action of Benzamide Riboside: A Novel Inhibitor of IMP Dehydrogenase’, p. 743 Current Medicinal Chemistry: Anti-Cancer Agents, Bentham Science Publishers, Vol. 2,2002 R 44 J. Jimenez-Barbero, F. Amat-Guerri and J.P. Snyder, ‘The Solid State, Solution and Tubulin-Bound Conformations of Agents that Promote Microtubule Stabilization’, p. 91 Current Opinion in Chemical Biology, Vol. 6,2002 R 45 S.J. Opella, T.M. DeSilva and G. Veglia, ‘Structural Biology of MetalBinding Sequences’, p. 2 17 R 46 A.M. Wolters, D.A. Jayawickrama and J.V. Sweedler, ‘Microscale NMR’, p. 711 Vol. 7,2003 R 47 S. Yokoyama, ‘Protein Expression Systems for Structural Genomics and Proteomics’, p. 39

R48

Current Opinion in Colloid & Interface Science, Elsevier Science Ltd., Vol. 7,2002 P. Griffiths and P. Stilbs, ‘NMR Self-Diffusion Studies of Polymeric Surfactants’, p. 249

Current Opinion in Drug Discovery & Development, PharmaPress Ltd., VOl. 5,2002 R 49 S.G. Buchanan, ‘Structural Genomics: Bridging Functional Genomics and Structure-Based Drug Design’, p. 367 Current Opinion in Structural Biology, Vol. 12,2002 R 50 D. MacDonald and P. Lu, ‘Residual Dipolar Couplings in Nucleic Acid Structure Determination’, p. 337 R 51 S.K. Burley and J.B. Bonanno, ‘Structural Genomics of Proteins from


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Conserved Biochemical Pathways and Processes’, p. 383 R 52 M. Akke, ‘NMR Methods for Characterizing Microsecond to Millisecond Dynamics in Recognition and Catalysis’, p. 642 R 53 L.K. Thomson, ‘Solid-state NMR Studies of the Structure and Mechanisms of Proteins’, p. 661 Current Organic Chemistry, Bentham Science Publishers Vol. 6,2002 R 54 P. Li, P.P. Roller and J. Xu, ‘Current Synthetic Approaches to Peptide and Peptidomimetic Cyclization’, p. 41 1 Current Pharmaceutical Design Vol. 8,2002 R 55 S. Sardari and D. Sardari, ‘Applications of Artificial Neural Networks in AIDS Research and Therapy’, p. 659 R 56 S.G. Buchanan, J.M. Sauder and T. Harris, ‘The Promise of Structural Genomics in the Discovery of New Antimicrobial Agents’, p. 1173 Current Protein and Peptide Science. Vol. 3,2002 R 57 D. Tolkatchev, A. Koutychenko and F. Ni, ‘DissectingFunctional Interactions in Coagulation Protein Complexes by Use of NMR Spectroscopy p. 275 R 58 L. Otvos and M. Cudic, ‘Post-Translational Modifications in Prion Proteins’, p. 643 Current Topics in Medicinal Chemistry (Hilversum, Netherlands), Bentham Science Publishers Vol. 2,2002 R 59 E.S. DeJong, B. Luy and J.P. Marino, ‘RNA and RNA-Protein Complexes as Targets for Therapeutic Intervention’, p. 289

R 60 R 61 R 62 R 63 R 64 R 65

Methods in Molecular Biology, Humana Press Inc., Totowa, NJ, United States, Vol. 173,2002 Calcium-Binding Protein Protocols, Vol. 2, Methods and Techniques, ed. H.J. Vogel R.D. Brokx and H.J. Vogel, ‘Proteolytic Fragments of Calcium-Binding Proteins’, p. 183 T.E. Clarke and H.J. Vogel, ‘Cadmium-113 and Lead-207 NMR Spectroscopic Studies of Calcium-Binding Proteins’, p. 205 T. Drakenberg, ‘Calcium-43NMR of Calcium-Binding Proteins’, p. 2 17 M.X. Li, D.C. Corson and B.D. Sykes, ‘Structure Determination by NMR: Isotope Labeling’, p. 255 T.K. Mal, S. Bagby and M. Ikura, ‘Protein Structure Calculation from NMR Data’, p. 267 J.M. Werner, I.D. Campbell and A.K. Downing, ‘Shape and Dynamics of a Calcium-Binding Protein Investigation by Nitrogen- 15 NMR Relaxation’, p. 285 Vol. 186,2002 Oxidative Stress Biomarkers and Antioxidant Protocols, ed. D. Armstrong

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R 66 A. Afzal, M. Afzal, A. Jones and D. Armstrong, ‘Rapid Determination of Glutamate Using HPLC Technology’, p. 111

R 67

R 68 R 69 R 70 R 71 R 72

R 73

R 74

NATO Science Series, 11: Mathematics, Physics and Chemistry Vol. 41, 2001 Magnetic Storage Systems Beyond 2000, ed. G.C. Hadjipanayis H. Szymczak and R. Szymczak, ‘Magnetic Measurement Techniques’, p. 225 Vol. 56,2002 Ring Opening Metathesis Polymerization and Related Chemistry K.J. Ivin, ‘ROMP and Related Chemistry: Past, Present and Future’, p. 1 A.M. Kenwright, ‘High-Resolution NMR and ROMP’, p. 57 Vol. 61,2002 New Trends in Intercalation Compoundsfor Energy Storage J. Conard, ‘Electronic Structure of Various Forms of Solid State Carbons. Graphite Intercalation Compounds’, p. 39 J. Conard and P. Lauginie, ‘Lithium NMR in Lithium-Carbon Solid State Compounds’, p. 77 C.W. Kwon, S.J. Hwang, A. Poquet, N. Treuil, G. Campet, J. Portier and J.H. Choy, ‘Nanocrystalline Materials for Lithium Batteries’, p. 439 Vol. 68,2002 Strengthfrom Weakness:Structural Consequences of Weak Interactions in Molecules, Supramolecules, and Crystals P. Sozzani, A. Comotti, R. Simonutti, S. Bracco and A. Simonelli, ‘Strength from Motion in Crystals. The Example of Supramolecular Adducts’, p. 319 Vol. 81,2002 New Kind of Phase Transitions: Transfarmationsin Disordered Substances S.R. Dillon and R.C. Dougherty, ‘Equilibrium Structural Model for Liquid Water: Evidence of Weak Continuous Phase Transitions in Aqueous Uni-Univalent Electrolyte Solutions’, p. 223

Nature Reviews Drug Discovery Vol. 1,2002 R 75 M. Pellecchia, D.S. Sem and K. Wuthrich, ‘NMR in Drug Discovery’, p. 21 1

R 76 R 77

R 78 R 79

Progress in Nuclear Magnetic Resonance Spectroscopy, ed. J.W. Emsley, J. Feeney and L.H. Sutcliffe, Elsevier Science B.V. Vol. 40,2002 P. Luginbuhl and K. Wuthrich, ‘Semi-Classical Nuclear Spin Relaxation Theory Revisited for Use with Biological Macromolecules’, p. 199 I. Bertini, C. Luchinat and G. Parigi, ‘Magnetic Susceptibility in Paramagnetic NMR’, p. 249 E.T. Ahrens, P.T. Narasimhan, T. Nakada and R.E. Jacobs, ‘Small Animal Neuroimaging Using Magnetic Resonance Microscopy’, p. 275 C. Mayer, ‘Nuclear Magnetic Resonance on Dispersed Nanoparticles’, p. 307


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Vol. 41,2002 R 80 M. Baldus, ‘Correlation Experiments for Assignment and Structure Elucidation of Immobilized Polypeptides under Magic Angle Spinning’, P- 1 R 81 R. Blinc and T. Apih, ‘NMR in Multidimensionally Modulated Incommensurate and CDW Systems’, p. 49 R 82 Z. Luz, P. Tekely and D. Reichert, ‘Slow Exchange Involving Equivalent Sites in Solids by One-Dimensional MAS NMR Techniques’, p. 83 R 83 R.Y. Dong, ‘Relaxation and the Dynamics of Molecules in the Liquid Crystalline Phases’, p. 115 R 84 C. Dybowski and G . Neue, ‘Solid State 207PbNMR Spectroscopy’, p. 153 R 85 B.M. Fung, ‘13CNMR Studies of Liquid Crystals’, p. 171 R 86 B.J. Stockman and C. Dalvit, ‘NMR Screening Techniques in Drug Discovery and Drug Design’, p. 187 R 87 J. Vaara, J. Jokisaari, R.E. Wasylishen and D.L. Bryce, ‘Spin-Spin Coupling Tensors as Determined by Experiment and Computational Chemist r y ’ , ~ .233 R 88 D. Frueh, ‘Internal Motions in Proteins and Interference Effects in Nuclear Magnetic Resonance’, p. 305 Progress in Polymer Science Vol. 27,2002 R 89 J. Lu, P.A. Mirau and A.E. Tonelli, ‘Chain Conformations and Dynamics of Crystalline Polymers as Observed in Their Inclusion Compounds by Solid-state NMR’, p. 357 Recent Research Developments in Agricultural & Food Chemistry, Research Signpost, Vol. 5,2001 R 90 Q. Teng, ‘NMR Structural Elucidation of Diterpene Derived Alkaloids’, p. 43 Recent Research Developments in Inorganic Chemistry, Transworld Research Network, Vol. 2,2000 R 91 A. Roodt and G.J.J. Steyr, ‘Iodomethane Oxidative Addition to Monocarbonylphosphine Complexes of Rhodium(1): Activation by N,S Bidentate Ligands’, p. 1 Recent Research Developments in Inorganic & Organometallic Chemistry, Research Signpost, Vol. 1,2001 R 92 L. Ciavatta, Equilibrium Studies on the Complexation of Cations with Orthophosphate Ions’, p. 83 Recent Research Developments in Lipids, Transworld Research Network, VOl. 5(Pt. l), 2001 R 93 J. Schiller, J. Arnhold, S. Benard, M. Mueller, M. Petkovic, 0.Zschoernig and K. Arnold, ‘MALDI-TOF Mass Spectrometry and 31PNMR Spectroscopy in Lipid Research’, p. 179

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Recent Research Developments in Non-Crystalline Solids, Transworld Research Network, Vol. 1,2001 R 94 J.G. Reynolds, ‘Hydrophobic Silica Aerogels’, p. 133 Recent Research Developments in Organic & Bioorganic Chemistry, Transworld Research Network Vol. 4,2001 R 95 M. Oda, T. Kajioka, Y. Kawamori, T. Uchiyama, S. Kuroda and N. Morita, ‘Spire[1H-azulenium-1,l’-cyclopropane] Ions; Their Synthesis, Characterization and Some Reactions’, p. 133 R 96 C. Marchiro, S. Davalli, S. Provera and G.H. Raza, ‘NMR Techniques in Drug Discovery’,p. 151 Recent Research Developments in Polymer Science, Transworld Research Network, Vol. 4,2000 R 97 T. Nakaya and Y.-J. Li, ‘Phosphatidylcholine Polyurethanes’, p. 53 Recent Research Development in Protein Engineering, Research Signpost, VOl. 1,2001 R 98 L. Patard, F. Duffieux, V. Stoven, E. Jacquet, 0. Pamlard and J.-Y. Lallemand, ‘Some Strategies to Undertake Structural Studies of Human Proteins’ p. 107

R 99

R 100 R 101 R 102

Specialists Periodical Reports, Royal Society of Chemistry Chemical Modeling: Applications and Theory: Vol. 2, senior reporter A. Hinchliffe, 2002 D. Pugh, ‘Electric Multipoles, Polarizabilities, Hyperpolarizabilities and Analogous Magnetic Properties’, p. 293 Spectroscopic Properties of Inorganic and Organometallic Compounds, Vol. 35, senior reporter G. Davidson, 2002 B.E. Mann, ‘NMR Spectroscopy in the Liquid and Gas Phase’, p. 1 B.E. Mann, ‘NMR Spectroscopy in the Solid State’, p. 112 K.B. Dillon, ‘Nuclear Quadrupole Resonance Spectroscopy’,p. 175

Studies of High Temperature Superconductors, Vol. 38,2002 Superconducting Magnesium Diboride, ed. A.V. Narlikar, Nova Science Publishers, Inc. R 103 V.P. Antropov, K.D. Belashchenko, M. van Schilfgaarde and S.N. Rashkeev, ‘Electronic Structure, Bonding and Optical Spectrum of MgB;, p. 91 Trends in Applied Spectroscopy, Research Trends, Vol. 3,2001 R 104 T. Hauet, J.P. Faure, H. Gibelin, H. Baumert, M. Eugene and M. Carretier, ‘A New Approach for Evaluation of Graft Function by Nuclear Magnetic Resonance Spectroscopy:From Experimental Studies to Clinical Applications’, p. 221 Trends in Food Science & Technology, Elsevier Science Ltd., Vol. 13,2002


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R 105 F. van de Velde, S.H. Knutsen, V.I. Usov, H.S. Rollema and A.S. Cerezo, “H and I3CHigh Resolution NMR Spectroscopy of Carrageenans: Application in Research and Industry’, p. 73 Trends in Heterocyclic Chemistry, Research Trends, Vol. 7,2001 R 106 A. Mucci, F. Parenti, L. Schenetti and C. Zanardi, ‘P-Functionalized a,a’-Conjugated Oligothiophenes with Alkylsulfanyl Groups: Synthesis and Characterization’, p. 55 3

Edited Books and Symposia

ACS Symposium Series, Vol. 8 12,2002 R 107 R. Nomura, H. Nakako, Y.Fukushima and T. Masuda, ‘Profile of the Helical Structure of Poly(Propio1ic Esters)’, p. 25 R 108 G.B. Fields, P. Forns, K. Pisarewicz and J.L. Lauer-Fields, ‘PeptideAmphiphile Induction of a-Helical and Triple-Helical Structures’, p. 117 R 109 US. Schubert, G. Hochwimmer and M. Heller, ‘Toward Functional Architectures via Tetrapyridine-Based Metallo-Supramolecular Initiators’, p. 163 Vol. 820,2002 R 110 H. Sillescu, R. Bohmer, A. Doss, G. Hinze, Th. Jorg and F. Qi, ‘Intramolecular Motions in Simple Glass-Forming Liquids Studied by Deuteron NMR’, p. 256 Vol. 827,2002 R 111 M.L. McKee, ‘Application of Theoretical Methods to NMR Chemical Shifts and Coupling Constants’, p. 135 R 112 J.A. Tossell, ‘AluminosilicateInorganic Compounds, Minerals, and Mineral Glasses: Connections Forget by Quantum Chemistry and NMR Spectroscopy’, p. 165 Vol. 830,2002 R 113 K. Shimizu, ‘Metabolic Systems Engineering Approach for Efficient Microbial Fermentation and Future Perspectives’, p. 8 Vol. 834,2003 R 114 H.N. Cheng and A.D. English, ‘Advances in the NMR Spectroscopy of Polymers: An Overview’, p. 3 R 115 R. Chujo, K. Fukutani and Y. Magoshi, ‘Estimation of Physical Properties of Archeological Silk with NMR Relaxation Time and FluctuationDissipation Theorem’, p. 83 R 116 P.L. Rinaldi, ‘Polymer Characterization by 3D Solution NMR’, p. 94 R 117 C.A. Bush, ‘Origins of Flexibility in Complex Polysaccharides’, p. 272 R 118 T.N. Huckerby, G.M. Brown, R.M. Lauder and LA. Nieduszynski, ‘Keratan Sulfates: Structural Investigations Using NMR Spectroscopy’, p. 289 R 119 N.D. Sachinvala, D.L. Winsor, W.P. Niemczura, K. Maskos, T.L. Vigo and N.R. Bertoniere, ‘Synthesis, Physical, and NMR Characteristics of di- and tri-Substituted Cellulose Ethers’, p. 306

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Advances in Cryogenic Engineering, AIP Conference Proceedings, Vol. 614, American Institute of Physics, 2002 R 120 M. Okata, H. Morita, J. Sato,T. Kiyoshi, H. Kitaguchi, H. Kumakura, K. Togano and H. Wada, ‘Bi-2212/Ag ROSAT Wire and the Magnet for High Field Applications’, p. 703 Advances in Flavours and Fragrances: From Sensation to the Synthesis, ed. K.A.D. Swift, in Special Publication - Royal Society of Chemistry, Vol. 277, Royal Society of Chemistry, 2002 R 121 M. Zviely, R. Giger, E. Abushkara, A. Kern, H. Sommer, H.-J. Bertram, G.E. Krammer, C.O. Schmidt, W. Stumpe and P. Werkhoff, ‘Application of Chromatographic and Spectroscopic Methods for Solving Quality Problems in Several Flavor Aroma Chemicals’, p. 39 Athens Conference on Coatings: Science and Technology, Proceedings, 27th, Athens, Greece, July 2-6, 2001, Institute of Materials Science, New Paltz, N.Y., 2001 R 122 E. Staring, A.A. Dias and R.A.T.M. Van Benthem, ‘New Challenges for R&D in Coating Resins’, p. 309 R 123 Th. Randoux, J.-Cl. Vanovervelt, H. Van Den Bergen and G. Camino, ‘Halogen-Free Flame Retardant Radiation Curable Coatings’, p. 353

R 124

R 125 R 126 R 127 R 128 R 129

Bioactive Natural Products, Part G, ed. Atta-ur-Rahman, in Studies in Natural Products Chemistry, Vol. 26, Elsevier Science B.V., 2002 Z. Jia, K. Koike, N.P. Sahu and T. Nikaido, ‘Triterpenoid Saponins from Caryophyllaceae Family’, p. 3 L.F. Szabo, ‘Some Aspects of the Chemistry of Secologanin’, p. 95 M. Liakopoulou-Kyriakides and D.A. Kyriakidis, ‘Crocus Sativus - Biologically Active Constituents’, p. 293 A. Evidente and A. Motta, ‘Bioactive Metabolites from Phytopathogenic Bacteria and Plants p. 581 P. Rasoanaivo, M.-T. Martin, E. Guittet and F. Frappier, ‘New Contributions to the Structure Elucidation and Pharmacology of Strychnos Alkaloids’, p. 1029 Y. Uda, Y. Ozawa and K. Yoneyama, ‘Occurrence of Biologically Active 2-Thioxopyrrolidines and 3,5-Disubstituted 2-Thiohydantoins from the Pungent Principle of Radish (Raphanus Sativus L.)’, p. 1073

Biological Systems under Extreme Conditions, ed. Y. Taniguchi, H.E. Stanley and H. Ludwig, Springer-Verlag, Berlin, Germany, 2002, R 130 L. Ballard and J. Jonas, ‘High-pressure NMR Spectroscopy of Proteins’, p. 75 R 131 M. Saito, ‘Accurate Calculations of Relative Melting Temperatures of Mutant Proteins by Molecular Dynamics/Free Energy Perturbation Methods’, p. 139 Biomedical Polymers and Polymer Therapeutics,[Proceedings of the Inter-


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national Symposium on Frontiers in Biomedical Polymers Including Polymer Therapeutics: From Laboratory to Clinical Practice], 3rd, Biwa Lake, Japan, M a y 23-27, 1999, ed. E. Chiellini, Kluwer Academic/Plenum Publishers, New York, N.Y., 2001 R 132 F. Chiellini, F. Petrucci, E. Ramucci and R. Solaro, ‘Polymeric Hydrogels in Drug Release’, p. 63 Bread Staling, ed. P. Chinachoti and Y. Vodovotz, CRC Press LLC, Boca Raton, Fla., 2001 R 133 R.R. Ruan and P.L. Chen, ‘Nuclear Magnetic Resonance Techniques’, p. 113 Cardiovascular Physiology in the Genetically Engineered Mouse (Second Edition), ed. B.D. Hoit and R.A. Walsh, in Developments in Cardiovascular Medicine, Vol. 238, Kluwer Academic Publishers, 2002 R 134 J.S. Ingwall, M.M. Javadpour and W. Miao, ‘31PNMR Spectroscopy of the Mouse Heart’, p. 151 Catalysis, ed. B. Viswanathan, S. Sivasanker and A.V. Ramaswamy, Narosa Publishing House, New Delhi, India, 2002 R 135 D. Srinivas, ‘Application of UV-Visible and Magnetic Resonance Spectroscopic Techniques in Heterogeneous Catalysis’, p. 145 Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging, ed. A.F. Merbach and E. Toth, John Wiley & Sons Ltd., Chichester, UK, 200 1 R 136 E. Toth, L. Helm and A.E. Merbach, ‘Relaxivity of Gadolinium (111) Complexes: Theory and Mechanism’, p. 45 R 137 E. Bruecher and A.D. Sherry, ‘Stability and Toxicity of Contrast Agents’, p. 243 R 138 J.A. Peters, E. Zitha-Bovens, D.M. Corsi and C.F.G.C. Geraldes, ‘Structure and Dynamics of Gadolinium-Based Contrast Agents’, p. 315 Chemistry of Organic Germanium, Tin and Lead Compounds, Vol. 2 (Part 1), ed. Z . Rappoport, John Wiley & Sons Ltd., Chichester, UK., 2002 R 139 H.C. Marsmann and F. Uhlik, ‘Further Advances in Germanium, Tin and Lead NMR’, p. 399 Chemistry of Organic Silicon Compounds, Vol. 3, ed. Z. Rappoport and Y. Apeloig, John Wiley & Sons Ltd., Chichester, UK., 2001 R 140 J. Schraml, ‘29SiNMR Experiments in Solution of Organosilicon Compounds’, p. 223 Comprehensive Toxicology, ed. I.G. Sipes, C.A. McQueen and A.J. Gandolfi, Elsevier Science B.V., Amsterdam, Netherlands, 2002 R 141 D.G. Robertson, M.D. Reily, J.C. Lindon, E. Holmes and J.K. Nicholson,

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‘Metabonomic Technology as a Tool for Rapid Throughput in Vivo Toxicity Screening’,p. 583 Encyclopedia of Nuclear Magnetic Resonance, Vol. 9, ed. D.M. Grant and R.K. Harris, John Wiley & Sons Ltd., Chichester, UK., 2002 R 142 A.K. Khitrin, B.M. Fung and G. McGeorge, ‘Heteronuclear Decoupling in Solids’, p. 91 R 143 G.E. Martin, ‘Microprobes and Methodologies for Spectral Assignments: Applications’, p. 98 R 144 F.G. Vogt and K.T. Mueller, ‘Orientationally Broadened NMR Spectra: Analysis by Special Processing Methods’, p. 112 R 145 G.A. Morris, ‘Reference Deconvolution’, p. 125 R 146 Z. Gan, ‘SatelliteTransition NMR Spectroscopy of Half-Integer Quadrupolar Nuclei under Magic-Angle Spinning’, p. 132 R 147 R.K. Harris and A.C. Olivieri, ‘Spinning Sideband Analysis for Spin-1/2 Nuclei’, p. 141 R 148 E.W. Randal, ‘Stray Field (STRAFI) NMR: Imaging in Large FieldGradients’, p. 150 R 149 D. Sakellariou and L. Emsley, ‘Through-Bond Experiments in Solids’, p. 196 R 150 D.J. Searles and H. Huber, ‘Accurate Determination of Nuclear Quadrupole Coupling Constants and Other NMR Parameters in Liquids from the Combination of Molecular Dynamics Simulations and ab initio Calculations’, p. 215 R 151 J.-P. Amoureux and M. Pruski, ‘Advances in MQMAS NMR’, p. 226 R 152 K. Takegoshi and T. Terao, ‘Determination of Three Dimensional Molecular Structures in Uniformly Labeled Solids Using Rotational Resonance in the Tilted Rotating Frame’, p. 252 R 153 L. Frydman, ‘Fundamentals of Multiple-Quantum Magic-Angle Spinning NMR on Half-Integer Quadrupolar Nuclei’, p. 262 R 154 R.E. Wasylishen, ‘Indirect Nuclear Spin-Spin Coupling Tensors’, p. 274 R 155 A. Naito and H. Saito, ‘Limit of Accuracy of Internuclear Distances Measured by REDOR’, p. 283 R 156 S. Caldarelli, ‘Local Field Experiments in Liquid Crystals’, p. 291 R 157 J. Autschbach and T.Ziegler, ‘Relativistic Computation of NMR Shieldings and Spin-Spin Coupling Constants’, p. 306 R 158 J.C. Facelli, ‘[NMR] Shielding Calculations’, p. 323 R 159 I.D. Campbell, ‘Protein Modules and NMR’, p. 436 R 160 J.B. Grutzner, ‘Applications of NMR in Carbanion Chemistry’, p. 48 1 R 161 J. Courtieu, P. Lesot, A. Meddour, D. Merlet and C. Aroulanda, ‘Chiral Liquid Crystal NMR: A Tool for Enantiomeric Crystals’, p. 497 R 162 C.A. Veracini and M. Geppi, ‘Chiral Smectic Phases: NMR Studies’, p. 506 R 163 M.J. Shapiro, ‘Combinatorial Chemistry: NMR Applications’, p. 514 R 164 S. Ando, R.K. Harris and U. Scheler, ‘Fluorine-19 NMR of Solids Containing Both Fluorine and Hydrogen’, p. 531


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R 165 A.M. Orendt, ‘Fullerenes and Related Molecules as Studied by Solid State NMR’, p. 551 R 166 F.G. Riddell, ‘Intramolecular Motions in Solid Organic Compounds’, p. 570 R 167 J.K. Harper, ‘Natural Products Structural Analysis Enhancements’, p. 589 R 168 S.B. Duckett and S.A. Colebrooke, ‘Parahydrogen Enhanced NMR Spectroscopic Methods: A Chemical Perspective’, p. 598 R 169 M.E. Smith, ‘Solid State Investigations of Less-Common Nuclei with Small Magnetic Moments’, p. 607 R 170 M. Pons and P. Bernado, ‘Supramolecular Chemistry’, p. 620 R 171 K. Schmidt-Rohr, E.R. de Azevedo and T.J. Bonagamba, ‘Centerband Only Detection of Exchange (CO-DEX): Efficient NMR Analysis of Slow Motions in Solids’, p. 633 R 172 L.M.K. Vandersypen, C.S. Yannoni and I.L. Chuang, ‘Liquid State NMR Quantum Computing’, p. 687 R 173 V.I. Yukalov, ‘Nuclear Spin Superradiance’, p. 697 R 174 X. Tang and Y. Wu, ‘Quasicrystalline Compounds: Metallic Glasses’, p. 729 R 175 C.R. Bowers, ‘Sensitivity Enhancement Utilizing Parahydrogen’, p. 750 R 176 C.M.V. Taylor and G.B. Jacobson, ‘Supercritical Fluids (and NMR)’, p. 788 Enzymes in Lipid Modification, ed. U.T. Bornscheuer, Wiley-VCH Verlag GmbH, Weinheim, Germany, 2000 R 177 T. Yamane, ‘Lipase-Catalysed Synthesis of Structured Triacylglycerols Containing Polyunsaturated Fatty Acids: Monitoring the Reaction and Increasing the Yield’, p. 148 Essays in Contemporary Chemistry, ed. G. Quinkert and M.V. Kisakuerek, Verlag Helvetica Chimica Acta, Zurich, Switzerland, 2001 R 178 C. Griesinger, ‘NMR Spectroscopy as a Tool for the Determination of Structure and Dynamics of Molecules’, p. 35 Evolutionary Algorithms in Molecular Design, ed. D.E. Clark, in Methods and Principles in Medicinal Chemistry, Vol. 8, VCH Verlagsgesellschaft mbH, 2000 R 179 B.C. Sanctuary, ‘Structure Determination by NMR Spectroscopy’, p. 195 Exotic Nuclei, International Symposium, Lake Baikal, Russian Federation, July 24-28,2001, ed. Yu. E. Penionzhkevich and E.A. Cherepanov, World Scientific Publishing Co. Pte. Ltd., Singapore, Singapore, 2002 R 180 D.L. Balabanski, G. Neyens, D. Borremans, N. Coulier, J.M. Daugas, S. Teughels, G. Georgiev, M. Lewitowicz, F. De Oliveira Santos and Yu. E.

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Penionzhkevich, ‘Experiments with Exotic Spin-Oriented Nuclear Beams and Examples of Nuclear Moment Measurements’, p. 404 Extended Abstracts - International Symposium on Non-Oxide Glasses and New Optical Glasses, 13th, Pardubice, Czech Republic, Sept. 9-1 3, 2002, (Part 2), ed. M. Frumar, University of Pardubice, Pardubice, Czech Republic, 2002 R 181 A. Monteil, M.A.P. Silva, D. Guichaoua, S. Chaussedent and M. Ferrari, ‘Molecular Dynamics in Glass’, p. 419 Functional Polymer Colloids & Microparticles, in Microspheres, Microcapsules & Liposomes, Vol. 4, Citus Books, 2002 R 182 A. Guyot, T. McKenna and M. Schneider, ‘Characterization of Colloidal Particles’, p. 39 R 183 A. Guyot, ‘Functional Colloids with Reactive Surfactants’, p. 77 Genome Science: Towards a New Paradigm?, ed. H. Yoshikawa, N. Ogasawara and N. Satoh, in International Congress Series, Vol. 1246, Elsevier Science B.V., 2002 R 184 T. Endo, ‘Mitochondria1 Protein Flux’, p. 27 Glycoscience, Vol. 2, ed. B.O. Fraser-Reid, K. Tatsuta and J. Thiem, Springer-Verlag, Berlin, Germany, 2001 R 185 Z.J. Witczak, ‘Properties [of Carbohydrates]’, p. 885 R 186 Z.J. Witczak, ‘Properties [of Oligosaccharides]’, p. 1445 R 187 Z.J. Witczak, ‘Properties [of Polysaccharides]’, p. 1883 Gums and Stabilisers for the Food Industry, ed. P.A. Williams and G.O. Phillips, in Special Publications - Royal Society of Chemistry, Vol. 11, Royal Society of Chemistry, 2002 R 188 S.W. Cui, ‘Application of Two Dimensional (2D) NMR Spectroscopy in the Structural Analysis of Selected Polysaccharides’, p. 27 Handbook of Applied Surface and Colloid Chemistry, Vol. 2, ed. K. Holmberg, John Wiley & Sons Ltd., Chichester, UK, 2002 R 189 M. Nyden, ‘Measuring Micelle Size and Shape’, p. 281 R 190 S.T. Hyde, ‘Identification of Lyotropic Liquid Crystalline Mesophases’, p. 299 R 191 U. Olsson, ‘Characterization of Microemulsion Structure’, p. 333 Handbook of Neurotoxicology, Vol. 2, ed. E.J. Massaro, Humana Press Inc., Totowa, N.J., 2002 R 192 D. Nutt and M. Daglish, ‘Structural and Functional Neuroimaging of the Effects of Opioids’, p. 397 Handbook of Organopalladium Chemistryfor Organic Synthesis, Vol. 1, ed.

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E.4. Negashi, John Wiley & Sons, Inc., Hoboken N.J., 2002 R 193 M. Ogasawara and T. Hayashi, ‘Chiral Pd(0) and Pd(I1) Complexes’, p. 103 Handbook of Polyelectrolytes and Their Applications, VoE. 2, ed. S.K. Tripathy, J. Kumar and H.S. Nalwa, American Scientific Publishers, Stevenson Ranch, CA, 2002 R 194 U. Scheler, ‘Determination of Effective Size and Charge of Polyelectrolytes by Diffusion and Electrophoresis Nuclear Magnetic Resonance (NMR),p. 173 Handbook of Porous Solids, Vol. 1, ed. F. Schueth, K.S.W. Sing and J. Weitkamp, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2002 R 195 D. Freude and J. Kaerger, ‘NMR Techniques’, p. 465 Handbook of Thin Film Materials, Vol. 2, ed. H.S. Nalwa, Academic Press, San Diego, CA, 2002 R 196 A. Naito and M. Kamihira, ‘Solid State NMR of Biomolecules’, p. 735 High Magnetic Fields, ed. C. Berthier, L.P. Levy and G. Martinez, in Lecture Notes in Physics, Vol. 595, Springer Verlag, 2001 R 197 M. Horvatic and C. Berthier, ‘NMR Studies of Low-Dimensional Quantum Antiferromagnets’, p. 191 R 198 G.A. Nikolaychuk, A.V. Lukashin, V.V. Matveev and I.V. Pleshakov, ‘Novel Magnetic Materials Based on HTSC-Ferrite Heterostructures and Co/Si02 Nanocomposites’, p. 203 R 199 D. Massiot, ‘High-Resolution Solid-state NMR’, p. 435 Humic Substances and Chemical Contaminations, Proceedings of a Workshop and Symposium, Anaheim, CA, United States, Oct. 26-27, 1997, ed. C.E. Clapp, Soil Science Society of America, Madison, Wis., 2001 R 200 M.H.B. Hayes and R.L. Malcolm, ‘Considerations of Compositions and of Aspects of the Structures of Humic Substances’, p. 3 R 201 W.L. Kingery, A.J. Simpson, F. Han and B. Xing, ‘Nuclear Magnetic Resonance Studies of Metal Interactions with Humic Substances’,p. 397 Industrial Polymers Handbook, ed. E.S. Wilks, Wiley-VCH Verlag GmbH, Weinheim, Germany, 2001 R 202 W. Hasse, ‘Phenolic Resins’, p. 1129 Innovation and Perspectives in Solid Phase Synthesis & Combinatorial Libraries: Peptides, Proteins and Nucleic Acids - Small Molecule Organic Chemistry Diversity, Collected Papers, International Symposium, 6th, York, United Kingdom, Aug. 31-Sept. 4, 1999, ed. R. Epton, Mayflower Scientific Ltd., Kingswinford, UK, 2001

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R 203 M. Grotli, C.H. Godtfredsen, J. Rademann, J. Buchardt, A.J. Clark, J.O. Duus and M. Meldal, ‘Physical Properties of Polyethylene Glycol (PEG)Based Resins for Solid Phase Organic Chemistry: A Comparison of PEG Cross-Linked and PEG Grafted Resins’, p. 109 Integrated Drug Discovery Technologies, ed. H.-Y. Mei and A.W. Czarnik, Marcel Dekker, Inc., New York, N.Y., 2002 R 204 P.A. Keifer, ‘The NMR ‘Toolkit‘for Compound Characterization’, p. 485 Internet for Cell and Molecular Biologists, ed. A. Cabibbo R.P. Grant and M. Helmer-Citterich, Horizon Scientific Press, Wymondham, UK, 2002 R205 F. Ferre, ‘From Sequence to Structure: An Easy Approach to Protein Structure Prediction’, p. 233 Introduction to Modern Methods of Quantum Many-Body Theory and Their Applications, ed. A. Fabrocini, S. Fantoni and E. Krotscheck, in Series on Advances in Quantum Many-Body Theory, Vol. 7, World Scientific Publishing Co. Pte. Ltd., Singapore, 2002 R 206 H. Godfrin, ‘The Magnetic Susceptibility of Liquid 3He’,p. 329 Layered Double Hydroxides, ed. V. Rives, Nova Science Publishers Inc., Huntington, N.Y., 2001 R 207 J. Rocha, ‘Solid-state NMR and EPR Studies of Hydrotalcites’, p. 193 Mechanical Spectroscopy I I , in Difusion and Defect Data-Solid State Data, Pt . B: Solid State Phenomena, Trans Tech Publications Ltd., Vol. 89, Zurich-Uetikon, 2003 R 208 S. Etienne, S. Elkoun, L. David and L.B. Magalas, ‘Mechanical Spectroscopy and Other Relaxation Spectroscopies’, p. 3 1 Membrane Transport and Renal Physiology, ed. H.E. Layton and A. M. Weinstein in I M A Volumes in Mathematics and Its Applications,Vol. 129, Springer-Verlag New York Inc., 2002 R 209 P.A. Knauf, ‘Asymmetry of the AE1 Anion Exchange System: Implications for Modeling the Physiological Rates of Chloride-Bicarbonate Exchange’, p. 85 Modern Coordination Chemistry, ed. G.J. Leigh and N. Winterton, Royal Society of Chemistry, Cambridge, UK, 2002 R210 B.T. Heaton, ‘NMR Studies of Metal Complexes and Clusters with Carbonyls and Phosphines’, p. 89 New Aspects in Phosphorus Chemistry I , ed. J.-P. Majoral, in Topics in Current Chemistry, Vol. 220, Springer-Verlag, 2002 R 21 1 M. Ehses, A. Romerosa and M. Peruzzini, ‘Metal-Mediated Degradation and Reaggregation of White Phosphorus’, p. 107


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Nitrogen Assimilation by Plants, ed. J.-F. Morot-Gaudry, Science Publishers, Inc., Enfield, N.H., 2001 R 212 E. Deleens, J.-F. Morot-Gaudry, F. Martin, A. Thoreux and A. Gojon, ‘”N Methodology’, p. 301 Novel Methods to Study Interfacial Layers, ed. D. Moebius and R. Miller, in Studies in Interface Science, Vol. 11, Elsevier Science B.V., 2001 R213 M. Schonhoff, ‘NMR Methods for Studies of Organic Adsorption Layers’, p. 285 On-Line L C - N M R and Related Techniques, ed. K. Albert, John Wiley & Sons Ltd., Chichester, UK, 2002 R 214 A. Preiss and M. Godejohann, ‘LC-NMR in Environmental Analysis’, p. 141 Peptides: The Wave of the Future, Proceedings of the Second International and the Seventeenth American Peptide Symposium, San Diego, C A , United States, June 9-14, 2001, ed. M. Lebl and R.A. Houghten, American Peptide Society, San Diego, CA, 2001 R 215 I. Daliani, T. Tselios, L. Probert, S. Deraos, T. Mavromoustakos and J. Matsoukas, ‘Rational Design and Synthesis of a Potent Amide-Linked Cyclic Analogue of MBP87-99 Based on 2D-NMR Studies and Molecular Dynamics’, p. 238 R 216 P. Cudic, J. Kranz, D.C. Behenna, R.G. Kruger, A.J. Wand and D. G. McCafferty, ‘Complexation of Peptidoglycan Intermediates by the Lipoglycodepsipeptide Antibiotic Ramoplanin: Structural Requirements for Intermolecular Complexation and Fibril Formation’, p. 5 12 R 217 J.S. Nowick, J.M. Cary, J.H. Tsai and W.A. Russu, ‘Triply-Templated Artificial P-Sheets’, p. 575 Pharmacokinetic Optimization in Drug Research: Biological, Physiocochemical, and Computational Strategies, [LogP2000, Lipophilicity Symposium], 2nd, Lausanne, Switzerland, Mar. 59,2000, ed. B. Testa, Verlag Helvetica Chimica Acta, Zurich, Switzerland, 2001 R 218 R. Fruttero, ‘NMR Spectroscopy for the Study of Drug-Phospholipid Interactions’, p. 465 Physical Phenomena at High Magnetic Fields - I V,Proceedings, Santa Fe, N M , United States, Oct. 19-25, 2001, ed. G. Boebinger, World Scientific Publishing Co. Pte. Ltd., Singapore, Singapore, 2002 R 219 A. Polkovnikov, S. Sachdev, M. Vojta and E. Demler, ‘Magnetic Field Tuning of Charge and Spin Order in the Cuprate Superconductors’, p. 258 R 220 M. Horvatic and C. Berthier, ‘High Field NMR in Strongly Correlated Low-Dimensional Fermionic Systems’, p. 37 1 PINSA-A, Proceedings of the Indian National Science Academy, Part A:

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Physical Sciences, Vol. 68, Indian National Science Academy, 2002 R221 F. Fabiola and V. Pattabhi, ‘The Structure Based Design of Peptide Motifs’, p. 251 Polarized Sources and Targets, Proceedings of the International Workshop, 9th, Nashville, I N , United States, Sept. 30 - Oct. 4, 2001, ed. V. P. Derenchuk and B. Von Przewoski, World Scientific Publishing Co. Pte. Ltd., Singapore, Singapore, 2002 R 222 G.R. Court, M.A. Houlden and D.G. Crabb, ‘High Precision Measurement of Target Polarization in Solid State Targets with NMR’, p. 111 R 223 T. Chupp, ‘Biomedical MRI with Laser Polarized Gases’, p. 321 Polymer Gels and Networks, ed. Y. Osada and A.R. Khokholov, Marcel Dekker, Inc., New York, N.Y., 2002 R 224 I. Ando, S. Kuroki, M. Kobayashi, C. Zhao and S. Matsukawa, ‘Structural and Dynamic Behavior of Polymer Gels as Elucidated by Nuclear Magnetic Resonance Spectroscopy’, p. 235 Polyoxometalate Chemistry, ed. M.T. Pope and A. Mueller, Kluwer Academic Publishers, Dordrecht, Netherlands, 2001 R 225 A. Proust and R. Villanneau, ‘Functionalization of Polyoxometalates: Achievements and Perspectives’, p. 23 R226 E. Cadot, B. Salignac, A. Dolbecq and F. Secheresse, ‘From the First Sulfurated Keggin Anion to a New Class of Compounds Based on the [M202S2I2+Building Block M = Mo, W’, p. 39 R 227 O.W. Howarth, L. Pettersson and I. Anderson, ‘Aqueous Peroxoisopolyoxometalates’, p. 145

R 228 R229 R 230 R 231

Porphyrin Handbook, Vol. 5 , ed. K.M. Kadish, K.M. Smith and R. Guilard, Academic Press, San Diego, CA, 2000 C.J. Medforth, ‘NMR Spectroscopy of Diamagnetic Porphyrins’, p. 1 F.A. Walker, ‘Proton NMR and ESR Spectroscopy of Paramagnetic Metalloporphyrins’, p. 81 G.N. La Mar, J.D. Satterlee and J.S. De Ropp, ‘Nuclear Magnetic Resonance of Hemoproteins’, p. 185 L. Banci, I. Bertini, C. Luchinat and P. Turano, ‘Solution Structures of Hemoproteins’, p. 323

Proceedings of International Symposium N UCEF 2001, Japan Atomic Energy Research Institute, 2002 R232 F.M.G. Wong, C. Stockman, R. Rechard and P. Brady, ‘Overview of Waste Form Degradation Process Models for Performance Assessment’, p. 333 Proceedings of the 4th International Symposium on Olive Growing, 2000, Vol. 2, in Acta Horticulturae, 2002,586, International Society for Horticultural Science


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R 233 N.A. Uccella, ‘The Secoiridoid Biphenols of Olea Europea L. Drupes and the Role of Their Metabolites’, p. 489 Proceedings of International Wire and Cable Symposium, 50th, 2001 R 234 V.M. Litvinov and A.A. Dias, ‘Characterization of Coating Systems on Optical Glass Fibers as a Whole by Solid State N M R , p. 267 Proceedings of SPIE - The International Society for Optical Engineering, [Developments in X-Ray Tomography I I I ] , SPIE - The International Society for Optical Engineering, 2002 R235 L.G. Butler, K. Ham, H. Jin and R.L. Kurtz, ‘Tomography at the Louisiana State University CAMD Synchrotron: Application to Polymer Blends’, p. 54 Progress in Heterocyclic Chemistry, Vol. 13, ed. G.W. Gribble and T.L. Gilchrist, Elsevier Science Ltd., 2001 R 236 D.M. Ketcha, ‘Five Membered Ring Systems: Pyrroles and Benzo Derivatives’, p. l l 1 Protein-Ligand Interactions: Structure and Spectroscopy, ed. S.E. Harding and B.Z. Chowdhry, Oxford University Press, Oxford, UK, 2001 R 237 L.-Y. Lian, ‘NMR Studies of Protein-Ligand Interactions’, p. 383 Refractory Organic Substances in the Environment, ed. F.H. Frimmel, Wiley-VCH Verlag GmbH, Weinheim, Germany, 2002 R 238 J. Lambert and U. Lankes, ‘Application of Nuclear Magnetic Resonance Spectroscopy to Structural Investigations of Refractory Organic Substances - Principles and Definitions’, p. 89 R 239 G. Abbt-Braun, U. Lankes, J.B. Jahnel, J. Lambert, H.-D. Luedemann and F.H. Frimmel, ‘Chemical and Spectroscopic Data of the Reference Samples - Comparison and Evaluation’, p. 302 Reviews in Mineralogy & Geochemistry, Vol. 44, ed. J.F. Baufield and A. Navrotskaya, Mineralogical Society of America, 2001, R 240 W.H. Casey, B.L. Phillips and G. Furrer, ‘Aqueous Aluminum Polynuclear Complexes and Nanoclusters: A Review’, p. 167 Sodium Channels and Neuronal Hyperexcitability, in Novartis Foundation Symposium, Vol. 241, ed. R.G. Bock and J.A. Goode, John Wiley & Sons Ltd. R 241 W.A. Catterall, ‘Molecular Mechanisms of Gating and Drug Block of Sodium Channels’, p, 206 So$ Matter: Complex Materials on Mesoscopic Scale, in Schrifen des Forschungszentrums Juelich, Materie und Material, Vol. 10, Forschungszentrum Juelich GmbH, 2002 R 242 G. Meier, ‘NMR, p. A2/1

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Soil Mineral - Organic Matter - Microorganism Interactions and Ecosystem Health,, ed. A. Violante, P.M. Huang, J.-M. Bollag and L. Gianfreda, in Developments in Soil Science, Vol. 28B, Elsevier Science B.V., 2002 R243 J.-M. Bollag, M. Strynar, M.-Y. Ahn and J. Dec, ‘Characterization of Enzymatic or Abiotic Immobilization of Xenobiotics in Soil’, p. 289 Solid-state N M R Spectroscopy, ed. M.J. Duer, Blackwell Science Ltd., Oxford, UK, 2002 R 244 O.N. Antzutkin, ‘Molecular Structure Determination: Applications in Biology’, p. 280

So lid Support 01igosaccharide Synthesis and Combinatorial Carbohydrate Libraries, ed. P.H. Seeberger, John Wiley & Sons, Inc. New York, N.Y., 200 1 R 245 W.-C. Haase, P.H. Seeberger and S.S. Pochapsky,. ‘Tools for ‘On-Bead’ Monitoring and Analysis in Solid-Phase Oligosaccharide Synthesis, p. 165 Speciality Chemicals in Mineral Processing, in Special Publication - Royal Society of Chemistry, Vol. 282, ed. D.R. Skuse, Royal Society of Chemistry, 2002 R246 M.R. Warne, N.L. Allan and T. Cosgrove, ‘Computer Simulation of Water Molecules at Mineral Surfaces’, p. 90 Stability and Shelf-Life of Food, ed. D. Kilcast and P. Subramaniam, Woodhead Publishing, Cambridge, UK, 2000 R 247 LA. Farhat, ‘Advanced Instrumental Methods: The Use of ‘H Relaxation NMR to Monitor Starch Retrogradation’, p. 129 Supramolecular Structure and Function, 7, [Proceedings of the International Summer School on Biophysics], 7th, Rovinj, Croatia Sept. 14-25, 2000, ed. G. Pifat-Mrzljak, Kluwer Academic/Plenum Publishers, New York, N.Y., 2001 R 248 V.V. Khramtsov, L.J. Berliner and T.L. Clanton, ‘New Approaches in Spin Labeling and Spin Trapping. Part Two: NMR Detects Free Radicals’, p. 107 R 249 L.J. Berliner and H. Fujii, ‘In Vivo Detection of Nitric Oxide: Combining EPR and NMR’, p. 131 Surface Alloys and Alloy Surfaces, ed. D.P. Woodruff, in Chemical Physics of Solid Surfaces, Vol. 10, Elsevier Science B.V., 2002 R 250 C.J. Baddeley, ‘Adsorbate Induced Segregation at Bimetallic Surfaces’, p. 495 Synthesis and Applications of Isotopically Labelled Compounds, Proceedings of the International Symposium, 7th, Dresden, Germany, June 18-22,


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2000, ed. U. Pleiss and R. Voges, John Wiley & Sons Ltd., Chichester, UK, 2001 R 251 A. Bacher and W. Eisenreich, ‘Application of Isotopes in Investigating Biosynthetic Pathways’, p. 619 R 252 G . Kollenz, W. Heilmayer and H. Sterk, ‘0-17 Labeling Studies on Molecular Rearrangements’, p. 644 Synthetic Peptides (2nd Edition), ed. G.A. Grant, Oxford University Press, Inc., New York, N.Y., 2002 R 253 G.A. Grant, ‘Evaluation of the Synthetic Product’, p. 220 Trends in High Pressure Bioscience and Biotechnology, in Progress in Biotechnology, Vol. 19, Elsevier Science B.V., 2002 R 254 D. Ishimaru, L.M.T.R. Lima, A. Ferrao-Gonzales, P.A. Quesado, L.M. Moiolino, J.L. Silva and D. Foguel, ‘Pressure Studies on Protein Folding, Misfolding, Protein-DNA Interactions and Amyloidogenesis’,p. 79 Unusual Structures and Physical Properties in Organometallic Chemistry, ed. M. Gilen, R. Willem and B. Wrackmeyer, in Organometallic Chemistry, Vol. 3, John Wiley & Sons Ltd., 2002 R 255 B. Wrackmeyer, ‘Unusual Nuclear Magnetic Shielding and Coupling Constants Related to Unusual Bonding Situations’, p. 51 R 256 V.I. Bakhmutov, ‘Deuterium Spin-Lattice Relaxation and Deuterium Quadrupole Coupling Constants. A Novel Strategy for Characterization of Transition Metal Hydrides and Dihydrogen Complexes in Solution’, p. 145 R 257 G.M. Bernard and R.E. Wasylishen, ‘NMR Studies of Ligand Nuclei in Organometallic Compounds. New Information from Solid-state NMR Techniques’, p. 165 Vasopressin and Oxytocin: From Genes to Clinical Applications, ed. D. Poulain, S. Oliet and D. Theodosis, in Progress in Brain Research, Vol. 139, Elsevier Science B.V., 2002 R 258 B. Mouillac, T. Sen, T. Durroux, G. Gaibelet and C. Barberis, ‘Expression of Human Vasopressin and Oxytocin Receptors in Escherichia Coli’, p. 163 4

Reviews in Periodicals

R 259 A.V. Akimov, A.V. Scherbakov, D.R. Yakovlev, W. Ossau, L.W. Molenkamp, T. Wojtowicz, J. Kossut, S. Tatarenko and J. Cibert, ‘Spin-Phonon Dynamics in Doped Magnetic Quantum Wells’, Physica B, Condens. Mat., 2002,316-317,41 R 260 K. Albert, M. Krucker, T. Glaser, A. Schefer, A. Lienau and D. Zeeb, ‘Hyphenated Techniques’, Anal. Bioanal. Chem., 2002,372,25

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R 261 T. Alemdaroglu, ‘Determination Methods for the Acidity of Solid Surfaces’, Communications de la Faculte des Sciences de 1’Universite d’Ankara, Series B, 2002,47,27 R 262 H. Amitsuka, M. Yokoyama, S. Miyazaki, K. Tenya, T. Sakakibara, W. Higemoto, K. Nagamine, K. Matsuda, Y. Kohori and T. Kohara, ‘Hidden Order and Weak Antiferromagnetism in URu2Si2),Physica B, (Amsterdam, Netherlands), 2002,312-313,390 R 263 J.-P. Amoureux and M. Pruski, ‘Theoretical and Experimental Assessment of Single- and Multiple-Quantum Cross-Polarization in Solid State NMR’, Mol. Phys., 2002,100,1595 R 264 N.K. Andrikopoulos, ‘Chromatographic and Spectroscopic Methods in the Analysis of Triacylglycerol Species and Regiospecific Isomers of Oils and Fats’, Crit. Reu. Food Sci. Nutr., 2002,42, 473 R 265 B. Antalek, ‘Using Pulsed Gradient Spin Echo NMR for Chemical Mixture Analysis. How to Obtain Optimum Results’, Concepts Magn. Reson., 2002,14,225 R 266 G.J. Arlaud, C. Gaboriaud, N.M. Thielens, M. Budayova-Spano, V. Rossi and J.C. Fontecilla-Camps, ‘Structural Biology of the C1 Complex of Complement Unveils the Mechanisms of Its Activation and Proteolytic Activity’, Mol. Immunol., 2002,39, 383 R 267 G.J. Arlaud, C. Gaboriaud, N.M. Thielens, and V. Rossi, ‘Structural Biology of Cl’, Biochem. SOC.Trans., 2002,30, 1001 R 268 F. Badulescu, A. Badulescu, S. Rogoz and R. Mustata, ‘Immunological Approach in the Diagnosis, Therapy and Prognosis of the Exocrine Pancreatic Cancer’, Rom. Arch. Microbiol. Immunol., 2001,60,5 R 269 B. Bago, P.E. Pfeffer, W. Zipfel, P. Lammers and Y. Shachar-Hill, ‘Tracking Metabolism and Imaging Transport in Arbuscular Mycorrhizal Fungi. Metabolism and Transport in AM Fungi’, Plant Soil, 2002,244, 189 R 270 V.V. Bardin and H.-J. Frohn, ‘(Po1yfluoroorgano)haloboranesand (Polyfluoroorgano)fluoroborate Salts: Preparation, NMR Spectra and Reactivity’, Main Group Met. Chem., 2002,25,589 R 271 V. Barone, 0.Crescenzi and R. Improta, ‘Computation of Spectroscopic Parameters in Vacuo and in Condensed Phases by Methods Based on the Density Functional Theory’, Quant. Struct-Act. Rel., 2002,21, 105 R 272 M.P. Beavers and X. Chen, ‘Structure-Based Combinatorial Library Design: Methodologies and Applications’, J . Mol. Graph. Model., 2002, 20,463 R 273 A.V. Belyaev, E.V. Renard, S.P. Khranenko, V.A. Emel’yanov and M. A. Fedotov, ‘State of Radiorhodium in High-Level Liquid Waste from Regeneration of Spent Nuclear Fuel’, Rudiochem., 2002,44,546 R 274 A. Bettio and A.G. Beck-Sickinger, ‘Biophysical Methods to Study Ligand-Receptor Interaction of Neuropeptide Y’, Biopolymers, 2001,60, 420 R 275 A. Bierzynski, ‘Methods of Peptide Conformation Studies’, Actu Biochim. Pol., 2002,48, 1091 R 276 T. Bizouarn, M. Althage, A. Pedersen, A. Tigerstrom, J. Karlsson, C.

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Johansson and J. Rydstrom, ‘The Organization of the Membrane Domain and Its Interaction with the NADP(H)-Binding Site in ProtonTranslocating Transhydrogenase from E.Coli’, Biochim. Biophys. Acta, 2002,1555,122 R 277 B. Bochicchio and A.M. Tamburro, ‘Polyproline I1 Structure in Proteins: Identification by Chiroptical Spectroscopies, Stability and Functions’, Chirality, 2002, 14,782 R 278 A. Bogdan and M. Kulmala, ‘Finely Divided Aqueous Systems: Fundamental and Application Aspects’, Curr. Top. Coll. Interface. Sci., 2002,5, 141 R 279 A.-K. Bouzier-Sore, M. Merle, P.J. Magistretti and L. Pellerin, ‘Feeding Active Neurons: (Re)emergence of a Nursing Role for Astrocytes’, J.Physiol., (Paris), 2002,96,273 R 2 8 0 H. Braunschweig and M. Colling, ‘The Chemistry of Borylene Complexes’, Eur. JJnorg. Chem., 2003,3, 393 R 28 1 D. Brinkmann and A.Y. Zavidonov, ‘Nuclear Spin-Lattice Relaxation in Cuprate Superconductors - Some New Approaches’, 2.Naturforsch., A , 2002,57,479 R 282 M.J.F. Broderick and S.J. Winder, ‘Towards a Complete Atomic Structure of Spectrin Family Proteins’, J.Struct. Biol., 2002,137, 184 R 283 G.A. Brooks, ‘Lactate Shuttles in Nature’, Biochem. SOC.Trans., 2002,30, 258 R 284 J. Brus, H. Petrickova and J. Dybal, ‘Potential and Limitations of 2D ‘H-’H Spin Exchange CRAMPS Experiments to Characterize Structures of Organic Solids’. Monatsh. Chem., 2002,133, 1587 R 285 S.F. Bureiko and G.S. Denisov, ‘Dynamics of Molecular Hydrogen Exchange in Hydrogen-Bonded Systems’, Pol. J.Chem., 2002,76,1177 R 286 M. Calichman, D. Hernandez-Rubio and C.W. Allen, ‘The Interaction of Cyclophosphazenes with Olefin Containing Exocyclic Groups’, Phosphorus Sulfur Relat. Elem., 2002,177,1811 R 287 I.D. Campbell, ‘Timeline: The March of Structural Biology’, Nut. Reu. Mol. Cell Biol., 2002,3, 377 R 288 T. Carlsson, ‘Survey of Methods for the Study of the Bentonite Microstructure and Its Relevance to Hydration’, VTT Tiedotteita, 2002,2132,l R289 D. Carmona, M.P. Lamata and L.A. Oro, ‘Half-Sandwich Complexes with Aminocarboxylate Ligands and Their Use as Enantioselective Hydrogen Transfer Catalysts’, Eur. JJnorg. Chem., 2002,2239 R 290 R. Carr and H. Jhoti, ‘Structure-Based Screening of Low-Affinity Compounds’, Drug Discou. Today, 2002,7,522 R 291 D.H. Chaplin and W.D. Hutchison, ‘Recent Trends in Low-Temperature Nuclear Orientation and Nuclear Magnetic Resonance on Oriented Nuclei’, Hyperjne Interact., 2001,136/137,239 R 292 Q. Chen, W. Kinzelbach and S. Oswald, ‘Nuclear Magnetic Resonance Imaging for Studies of Flow and Transport in Porous Media’, J.Enuiron. Qual., 2002,31,477

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Stimulated Insulin Secretion: Insights Gained from a Multidisciplinary Approach’, Diabetes, 2002,51, S389 R. Nomura, H. Nakako and T. Masuda, ‘Design and Synthesis of Semiflexible Substituted Polyacetylenes with Helical Conformation’, J . Mol. Catal., A: Chem., 2002,190, 197 M. Norin and M. Sundstrom, ‘Structural Proteomics: Lessons Learnt from the Early Case Studies’, Farmaco, 2002,57,947 W.A. Oleszek, ‘Chromatographic Determination of Plant Saponins’, J.Chromatogr. A, 2002,967,147 S.J. Opella, A. Nevzorov, M.F. Mesleh and F.M. Marassi, ‘Structure Determination of Membrane Proteins by NMR Spectroscopy’, Biochem. Cell Biol., 2002,80, 597 Y.F. Oprunenko, ‘High-Resolution and Cross-Polarization Magic Angle Spinning NMR Studies of Tricarbonylchromium Complexes with Polycyclic Aromatic Ligands’, Russ. Chern. Bull., 2002,51,907 J.D. Otvos, ‘Measurement of Lipoprotein Subclass Profiles by Nuclear Magnetic Resonance Spectroscopy’, Clinic. Labor., 2002,48, 17 1 J.D. Otvos, E.J. Jeyarajah and W.C. Cromwell, ‘Measurement Issues Related to Lipoprotein Heterogeneity’, Am. J.Cardiol., 2002,90,22i T. Pali and D. Marsh, ‘Structural Studies on Membrane Proteins Using Non-Linear Spin Label EPR Spectroscopy’, Cell. Mol. Biol. Lett., 2002,7, 87 S.B. Pandit, D. Gosar, S. Abhiman, S. Sujatha, S.S. Dixit, N.S. Mhatre, R. Sawdhamini and N. Srinivasan, ‘SUPFAM - A Database of Potential Protein Superfamily Relationships Derived by Comparing SequenceBased and Structure-Based Families: Implications for Structural Genomics and Function Annotation in Genomes’, Nucleic Acids Res., 2002,30,289 R.L. Paul, Z.R. Bell, J.S. Fleming, J.C. Jaffery, J.A. McCleverty and M.D. Ward, ‘Self-Assembly of Anion-Binding Supramolecular Cage Complexes’, Heteroatom Chern., 2002,13, 567 R.N. Perham, D.D. Jones, H.J. Chauhan and M.J. Howard, ‘Substrate Channeling in 2-0x0 Acid Dehydrogenase Multienzyme Complexes’, Biochem. SOC. Trans., 2002,30,47 S.J. Perkins, H.E. Gilbert, M. Aslam, J. Hannan, V.M. Holers and T.H.J. Goodship, ‘Solution Structures of Complement Components by X-Ray and Neutron Scattering and Analytical Ultracentrifugation’, Biochem. SOC.Trans., 2002,30,996 K.F. Petersen and G.I. Shulman, ‘Pathogenesis of Skeletal Muscle Insulin Resistance in Type 2 Diabetes Mellitus’, Am. J.Cardiol., 2002,90, 11G K.F. Petersen and G.I. Shulman, ‘Cellular Mechanism of Insulin Resistance in Skeletal Muscle’, J.Roy. SOC.Med., 2002,95, 8 W.-D. Pfeiffer, ‘Product Class 21: Annulated Isoselenazole Compounds’, Sci. Synth., 2002,11,93 1 R.P. Picone, D.J. Fournier and A. Makriyannis, ‘Ligand Based Structural Studies of the CB1 Cannabinoid Receptor’, J.Pept. Res., 2002,60,348


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R 447 M. Plazanet, A. Geis, M.R. Johnson and H.P. Trommsdorff, ‘Tunneling Systems in Molecular Crystals: Studies by Optical Spectroscopy and Neutron Scattering’, JLumin., 2002,98, 197 R 448 A. Polkovnikov, S. Sachdev, M. Vojta and E. Demler, ‘Magnetic Field Tuning of Charge and Spin Order in the Cuprate Superconductors’, Int. J.Mod. Phys., B, 2002,16,3 156 R 449 V.I. Polshakov, ‘Dihydrofolate Reductase: Structural Aspects of Mechanisms of Enzyme Catalysis and Inhibition’, Russ. Chem. Bull., 2001, 50, 1733 R 450 M.J. Potrzebowski, ‘What High-Resolution Solid-state NMR Spectroscopy Can Offer to Organic Chemists’, Eur. J.Org. Chem., 2003,8, 1367 R 451 M. Pouchard, C. Cros, P. Hagenmuller, E. Reny, A. Ammar, M. Menetrier and J.-M. Bassat, ‘A Brief Overview on Low Sodium Content Silicides: Are they Mainly Clathrates, Fullerenes, Intercalation Compounds or Zintl Phases?, Solid State Sci., 2002,4, 723 R 452 R. Powers, ‘Applications of NMR to Structure-Based Drug Design in Structural Genimics’, J.Struct. Funct. Genom., 2002,2, 113 R453 C.M. Preston, ‘Carbon-13 Solid-state NMR of Soil Organic Matter Using the Technique Effectively’, Can. JSoil Sci., 2001,81,255 R454 K. Prout, ‘Molecules in Motion: A Study in the Synergy of X-Ray Diffraction and Solid State NMR, Croat. Chem. Acta, 2002,75,817 R 455 M. Punkkinen, ‘I3C Nuclear Spin-Lattice Relaxation in CH3 Compounds’, Mol. Phys. Rep., 2001,31,92 R 456 K. Raghuraman, S.K. Mandal, T.S. Venkatakrishnan, S.S. Krishnamurthy and M. Nethaji, ‘Organometallic Chemistry of Chiral Diphosphazane Ligands: Synthesis and Structural Characterisation’, P.Indian AS.-Chem. Sci., 2002, 114,233 R 457 A. Ramos, A.R. Neves and H. Santos, ‘Metabolism of Lactic Acid Bacteria Studied by Nuclear Magnetic Resonance’, Antonie van Leeuwenhoek, 2002,82,249 R 458 Th. Randoux, J.-Cl. Vanovervelt, H. Van den Bergen and G. Camino, ‘Halogen-Free Retardant Radiation Curable Coatings’, Prog. Org. Coat., 2002,45,281 R 459 R.N. Rao, ‘Computer-Assisted Analytical Techniques in Quality Assurance of Fuels and Lubricants’, TrAC, Trend. Anal. Chem., 2002,21,175 R 460 T. Rehm, R. Huber and T.A. Holak, ‘Application of NMR in Structural Proteomics Screening for Proteins Amenable to Structural Analysis’, Structure, 2002, 10, 1613 R 461 N.V. Reo, ‘NMR-Based Metabolomics’, Drug Chem. Toxicol., 2002, 25, 375 R 462 S.M. Reutzel-Edens and J.K. Bush, ‘Solid-state NMR Spectroscopy of Small Molecules: From NMR Crystallography to the Characterization of Solid Oral Dosage Forms’, Am. Pharm. Reu., 2002,5, 112 R 463 T. Rezanka and J. Spizek, ‘Compounds Isolated at the Institute of Microbiology in 1989-2001 and Future Trends’, Folia Microbiol. (Prague, Czech Republic), 2002,47,587

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R 464 L.C. Robosky, D.G. Robertson, J.D. Baker, S. Rane and M.D. Reily, ‘In Vivo Toxicity Screening Programs Using Metabonomics’, Comb. Chem. High T.Scr., 2002,5,651 R 465 J.P. Rosenbusch, ‘Stability of Membrane Proteins: Relevance for the Selection of Appropriate Methods for High-Resolution Structure Determinations’, J.Struct. B i d , 2001,136, 144 R 466 S. Rudisser and W. Jahnke, ‘NMR and in Silico Screening’, Comb. Chem. High T.Scr., 2002,5, 591 R 467 M.I. Rybinskaya, A.Z. Kreindlin and A.A. Kamyshova, ‘Single-,Double-, and Triple-Charged Cations Based on Iron-Subgroup Decamethylmetallocenes’, Russ. Chem. Bull., 2002,51, 1616 R 468 K. Saalwachter and I. Schnell, ‘REDOR-Based Heteronuclear Dipolar Correlation Experiments in Multi-Spin Systems: Rotor-Encoding, Directing, and Multiple Distance and Angle Determination’, Solid State Nucl. Mag. Reson., 2002,22, 154 R 469 T.J. Sabo, S.R. Grguric-Sipka and S.R. Trifunovic, ‘Transition Metal Complexes with EDDA-Type Ligands - A Review’, Syn. React. Inorg. Met.-Org. Chem., 2002,32,1661 R470 M. Scandone and E. Melanson, ‘Knowledge Management of Spectral Data’, Am. Lab., 2002,34,24 R 471 M. Schnitzer, ‘The in Situ Analysis of Organic Matter in Soils’, Can. J.Soil Sci., 2001,81,249 R 472 J.D. Schrag, D.O. Procopio, M. Cygler, D.Y. Thomas and J.J.M. Bergeron, ‘Lectin Control of Protein Folding and Sorting in the Secretory Pathway’, Trends Biochem. Sci., 2003,28,49 R 473 M.D. Segall, ‘Applications of Ab Initio Atomistic Simulations to Biology’, J.Phys.-Condens. Mat., 2002,14,2957 R 474 D.S. Sem, L. Yu, S.M. Coutts and R. Jack, ‘Object-Oriented Approach to Drug Design Enabled by NMR SOLVE: First Real-Time Structural Tool for Characterizing Protein-Ligand Interactions’, J.Cel1. Biochem., 2001, (Suppl. 37), 99 R 475 G.L. Semenza, ‘Physiology Meets Biophysics: Visualizing the Interaction of Hypoxia-Inducible Factor la with p300 and CBP’, Proc. Natl. Acad. Sci. U.S.A.,2002,99,11570 R 476 L.S. Serafim, P.C. Lemos, C. Levantesi, V. Tandoi, H. Santos and M.A.M. Reis, ‘Methods for Detection and Visualization of Intercellular Polymers Stored by Pol yphosp hate- Accumulating Microorganisms’, J . Micro biol. Meth., 2002,51, 1 R 477 Y. Shachar-Hill, ‘Nuclear Magnetic Resonance and Plant Metabolic Engineering’, Matab. Eng., 2002,4,90 R 478 L. Shao, X. Lin and X. Shao, ‘A Wavelet Transform and Its Application to Spectroscopic Analysis’, Appl. Spectrosc. Rev., 2002,37,429 R 479 X. Shao, Z. Yu and L. Sun, ‘Immune Algorithms in Analytical Chemistry’, TrAC, Trends Anal. Chem., 2003,22,59 R 480 M.J. Shapiro, ‘Affinity NMR: A Powerful Method for Drug Discovery’, Am. Pharm. Rev., 2002,5,94


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R 48 1 A. Shimizu, ‘A Review on Metabolic Pathway Analysis with Emphasis on Isotope Labeling Approach’, Biotechnol. Bioproc. Eng., 2002,7,237 R 482 H. Shimizu, ‘Metabolic Engineering - Integrating Methodologies of Molecular Breeding and Bioprocess Systems Engineering’, J .Biosci. Bioeng., 2002,94,563 R 483 J.L. Silva, A.C. Oliveira, A.M.O. Gomes, L.M.T.R. Lima, R. MohanaBorges, A.B.F. Pacheco and D. Foguel, ‘Pressure Induces Folding Intermediates that Are Crucial for Protein-DNA Recognition and Virus Assembly’, Biochim. Biophys. Acta, 2002,1595,250 R 484 P.M. Singer, A.W. Hunt, A.F. Cederstroem and T. Imai, ‘What Has NMR Taught as about Stripes and Inhomogeneity?’, Los Alamos National Laboratory, Preprint Archive, Condensed Matter 4 Feb 2003, Avail. URL: http://xxx. lanl. gov/pdf/cond-mat/0302077 R 485 G.N. Sinyakov, A.M. Shul’ga, I.V. Filatov and K. Dzilinsky, ‘The Electronic Structure of Transient Species in the Photochemical Reduction of Porphyrins’, High Energ. Chem., 2002,36,255 R 486 G. Slosarek, ‘NMR of Peptides’, Mol. Phys. Rep., 2001,34,61 R 487 L. Spahr, P.R. Burkhard, H. Groetzsch and A. Hadengue, ‘Clinical Significance of Basal Ganglia Alterations at Brain MRI and ‘H MRS in Cirrhosis and Role in the Pathogenesis of Hepatic Encephalopathy’, Metab. Brain Dis., 2002,17, 399 R 488 H.W. Spiess, ‘Self-Assembly of Polymers from Advanced Solid State NMR and EPR Spectroscopy’, Polymer Preprints (American Chemical Society, Division ofPolymer Chemistry) [computer optical disk], 2002,43, 374 R 489 H.W. Spiess, ‘Nuclear Magnetic Resonance Spectroscopy in Macromolecular Science’, Macromol. Chem. Phys., 2003,204,340 R 490 H.W. Spiess and I. Schnell, ‘Supramolecular Structure and Function from Solid State NMR’, Polymer Preprints (American Chemical Society, Division of Polymer Chemistry) [computer optical disk], 2002,44,274 R 491 J.W. Sprengers, A.M. Kluwer, S. Gaemers and C.J. Elsevier, ‘High-Pressure NMR Spectroscopy: Some General Aspects and Applications’, Defect. Difus. Forum, 2002,208,283 R 492 A.K. Srivastava, M. Kamal, M. Kaur, S. Pandey, N. Daniel, A.K. Chaurasia and P. Pandey, ‘Terpolymerization: A Review’, J .Polym. Res., 2002,9.213 R 493 E. Staring, A.A. Dias and R.A.T.M. van Benthem, ‘New Challenges for R&D in Coating Resins’, Prog. Org. Coat., 2002,45,101 R 494 R. Stevenson, ‘The World of Separation Science: Separation Science Instruments / PITTCON ‘02 a Year of Evolutionary Advances in Separation Science’, Am. Lab., (Shelton, C T , United States), 2002,34,12 R 495 L.W. Sumner, P. Mendes and R.A. Dixon, ‘Plant Metabolomics: LargeScale Phytochemistry in the Functional Genomics Era’, Phytochemistry, (Elsevier), 2003,62,8 17 R496 D. Szabo, F. Ruff and A. Kucsman, ‘1,2-Oxathiole, 1,2-Oxathiin, 1,2Thiazole and 1,2-Thiazine Derivatives with Hypervalent Bonds and Close Contacts’, Targets Heterocycl. Syst., 2001,5, 199

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R 497 J. Takagi and T.A. Springer, ‘Integrin Activation and Structural Rearrangement’, Immunol. Rev., 2002,186,141 R 498 H. Takemura, ‘Azacalixarenes:Synthesis, Complexation, and Structures’, J.lnc1. Phenom. Macro. Chem., 2002,42,169 R 499 T. Timusk, ‘The Mysterious Pseudogap in High Temperature Superconductors: An Infrared View’, Los Alamos National Laboratory, Preprint Archive, Condensed Matter 19 Mar 2003, Avail. URL: http://xxx. lanl. gov/pdf/cond-mat/0303383 R 500 C. Toniolo, M. Crisma, F. Formaggio and C. Peggion, ‘Control of Peptide Conformation by the Thorpe-Ingold Effect (C-Tetrasubstitution)’, Biopolymers, 2001,60,396 R 501 M.R. Tosi, G. Fini, A. Tinti, A. Reggiani and V. Tugnoli, ‘Molecular Characterization of Human Healthy and Neoplastic Cerebral and Renal Tissues by in Vitro ‘H NMR Spectroscopy (Review)’,Int. J . Mol. Med., 2002,9,299 R 502 T. Tsuji, M. Ohkita and H. Kawai, ‘Preparation and Kinetic Stabilization of Highly Stained Paracyclophanes’, Bull. Chem. SOC.Jpn., 2002,75,415 R 503 G. Uccello-Barretta, R. Bernardini, F. Balzano and P. Salvadori, ‘Overall View of the Use of Chiral Platinum(I1) Complexes as Chiral Derivatizing Agents (CDAs) for the Enantiodiscrimination of Unsaturated Compounds by 195PtNMR’, Chirality, 2002,14,484 R 504 M. van Dongen, J. Weigelt, J. Uppenberg, J. Schultz and M. Wikstrom, ‘Structure-Based Screening and Design in Drug Discovery’, Drug Discou. Today, 2002,7,471 R 505 S.W. Vetter and E. Leclerc, ‘Novel Aspects of Calmodulin Target Recognition and Activation’, Eur. J . Biochem., 2003,270,404 R 506 G. Wang, ‘How the Lipid-Free Structure of the N-Terminal Truncated Human apoA-I Converts to the Lipid-Bound Form: New Insight from NMR and X-ray Structural Comparison’, FEBS Lett., 2002,529,157 R 507 J. Warwicker and C. Cole, ‘The Structural and Spectroscopic Basis for Modelling Prion Protein Interactions’, Spectrosc.-Int. J., 2001,15, 151 R 508 P. Waszczuk, G.-Q. Lu, A. Wieckowski, C. Lu, C. Rice and R.I. Masel, ‘UHV and Electrochemical Studies of CO and Methanol Adsorbed at Platinum/Ruthenium Surfaces, and Reference to Fuel Cell Catalysis’, Electrochim. Acta, 2002,47,3637 R 509 H. Watson, M.H. Kaunisto and J.B. Rosenholm, ‘Ureidosilanes on EGlass Fibres: Deposition and Surface Characteristics’, J.Adhes. Sci. Technol., 2002,16,429 R 5 10 A. Watts, ‘Direct Studies of Ligand-Receptor Interactions and Ion Channel Blocking (Review)’,Mol. Membr. BioE., 2002,19,267 R 511 J. Weigelt, M. Wikstrom, J. Schultz and M.J.P. Van Dongen, ‘SiteSelective Labeling Strategies for Screening by NMR’, Comb. Chern. High T.Scr., 2002,5, 623 R 512 T.J. Wenzel and J.D. Wilcox, ‘Chiral Reagents for the Determination of Enantiomeric Excess and Absolute Configuration Using NMR Spectroscopy’, Chirality, 2003, 15,256


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R 513 J.L. White, ‘NMR Imaging of Polymers, Blends and Composites: The Relevance of Chemical, Functional, and Dynamical Information at the Micron to Millimeter Length Scale’, Polym. Muter. Sci. Eng., [computer optical disk], 2002,87, 189 R 514 C.P. Whitman, ‘The 4-Oxalacrotonate Tautomerase Family of Enzymes: How Nature Makes New Enzymes Using a f3-a-p Structural Motif’, Arch. Biochem. Biophys., 2002,402,l R 515 R.W.T. Wilkins and S.C. George, ‘Coal as a Source Rock for Oil: A Review’, Int. J.Coal Geol., 2002,50,317 R 5 16 E. Williams, ‘Advances in Ultrahigh-Field NMR Technology: Proteomics and Genomics’, Am. Genom. Proteom. Technol., 2002,2,28 R517 I.D. Wilson, ‘Chromatography with Spectroscopy on Line, Can You Have It All? The ‘Hypernation‘ of HPLC with IR, UV and NMR Spectroscopy and Mass Spectrometry’, CAST, Chromatography and Separation Technology, 2001, 21’ 10 R 518 R. Winter, ‘Lasers and Levitation Allow a Look at Spectral Lines in Ceramics’, Mater. World, 2002,10,44 R 519 P. Woisel, G.G. Surpateanu and G. Surpateanu, ‘CycloimmoniumYlides. Structure and Electrocyclization Reactions’, Targets Heterocycl. Syst., 2001,5,461 R 520 J.C. Wright, ‘Coherent Multidimensional Vibrational Spectroscopy’, Int. Rev. Phys. Chem., 2002,21,185 R 521 Y. Xu, V.E. Yushmanov and P. Tang, ‘NMR Studies of Drug Interaction with Membranes and Membrane-Associated Proteins’, Biosci. Rep., 2002, 22,175 R 522 T. Yakovleva, A. Pramanik, L. Terenius, T.J. Ekstrom and G. Bakalkin, ‘p53 Latency - Out of the Blind Alley’, Trends Biochem. Sci., 2002,27,612 R 523 V.I. Yukalov, ‘Nuclear Spin Superradiance’, Los Alamos National Laboratory, Preprint Archive, Condensed Matter, Avail. URL: http://xxx. lanl. gov/pdf/cond-mat/02 12139 R 524 L. Zeng, L. Lu, M. Muller, E. Gouaux and M.-M. Zhou, ‘Structure-Based Functional Design of Chemical Ligands for AMPA-Subtype Glutamate Receptors’, J . Mol. Neurosci., 2002,19, 113

5

Reviews and Books in Foreign Languages

Chinese R 525 J. Chen, H. Zheng and Y. Zeng, ‘Recent Progress in Supercritical Water Theoretical Research’, Huaxue Jinzhan, 2002,14,409 R 526 J.-Y. Chen, H.-F. Zheng and Y.-S. Zeng, ‘Recent Progress of the Spectroscopic Study on Supercritical Water’, Guangpu Shiyanshi, 2002,19,285 R 527 L. Ding and C. Ao, ‘Application of Modern Nuclear Magnetic Resonance (NMR) Technique in the Creation of New Medicine’, Jingxi Huagong Zhongjianti, 2002, 32, 11 R 528 S. Ding, C.-L. Chen and C. Ye, ‘Quantum Computers: Principle, Poten-

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tial and NMR Implementation’, Huaxue, 2002,60, A47 R 529 X.-1. Du, Y.-h. Chen and L.-c. Tang, ‘Application of Nuclear Magnetic Resonance (NMR) Technology to the Studying on Coal Water Slurries’, Mei Huagong, 2002,30,13 R 530 X.-y. Guo, B.-z. Yan, H.-1. Guo, Q. Tian and Y. Liu, ‘Technique of Time-Resolved Chemically Induced Dynamic Polarization (CIDNP)’, Bopuxue Zazhi, 2003,20,91 R 531 A. He, R. Wang, B. Huang and S. Jiao, ‘High Trans-l,4-ButadieneIsoprene Copolymer’, Hecheng Xiangjiao Gongye, 2002,25,321 R 5 32 K. Hong, ‘Determination Method of Polyhydroxyalkanoates’, Weishengw u m e Tongbao, 2002,29,80 R 533 H. Hu, ‘NMR-Based Screening of Protein Inhibitors in Drug Discovery’, Yaoxue Xuebao, 2002,37,158 R 534 B. Li, J. Shi and G. Yang, ‘Cellulose-Based Chiral Stationary Phase in High Performance Liquid Chromatography’, Huaxue Tongbao, 2003,66, 169 R 535 X. Li, N. Li, K. Huang and M. Lin, ‘Spectral Characteristics, Biogenesis, and Biological Activities of Oligo-Stilbenes’, Yaoxue Xuebao, 2002,37,69 R 536 J.-c. Liu, C.-g. Chai and G.-z. Yang, ‘Advances in Determining the Absolute Configuration of Natural Products’, Huazhong Shifan Daxue Huebao Zirankexueban, 2002,36,64 R 537 Y.-1. Ren and J.-s. Yang, ‘Spectroscopic Features of Dibenzylbutyrolactone Ligands’, Bopuxue Zazhi, 2002,19,15 R 538 R.-W. Teng, P. Shen, D.-Z. Wang and C.-R. Yang, ‘Methods to Determine the Absolute Stereochemistry of Organic Compounds by NMR Spectroscopy’,Bopuxue Zazhi, 2002,19,203 R 539 W. Weng, J. Shen, J. Huang and Q. Zeng, ‘Chiral Discrimination on Cellulose and Cellulose Derivatives’, Huaxue Tongbao, 2002,65, W043/1 R 540 J. Wu and A. Qiu, ‘Analysis of Conjugated Linoleic Acid’, Zhonggua Youzhi, 2002,27,12 R 541 Y. Yu, Q. Zeng and H. Yao, ‘Advancement of Study on Inositol-Triphosphate’, Zhongguo Liangyou Xuebao, 2001,16,34 R 542 Z. Zhang, Y. Yang and H. Liu, ‘Progress in Solid-state NMR Studies of Electrode Materials for Lithium-Ion Batteries’, Huaxue Jinzhan, 2003,15, 18 R 543 X.-r. Zhou, Z.-m. Du, Z. Lin and J.-t. Lu, ‘Magnetic Resonance Studies on Lithium Intercalation into Carbons’, Dianhuaxue, 2003,9, 1 Danish R 544 U. Sidelmann, ‘NMR-Based Metabolic Analysis - New Technique in Drug Research and Development’, Dansk Kemi, 2002,83,23 French R 545 C.N. Cascaval and D. Rosu, ‘Behaviour of Para-tert-butylphenol Epoxyacrylic Resin in the Presence of Reactive Diluents’, Revue Roumaine de Chimie, 2001,46,671


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R 546 C. Delaurent and C.-P. Lienemann, ‘Analytical Chemistry and Spectroscopy at Pittcon 2002’’ Spectra Analyse, 2002,31, 15 R 547 N. Kosova and E. Devyatkina, ‘Soft Mechanochemical Synthesis: Preparation of Cathode Materials for Rechargeable Lithium Batteries’, Ann. Chim., (Paris, France), 2002,27, 77 R 548 S. Lehmann, ‘The Prion Protein’, J.Soc. Biol., 2002,196, 309 R 549 D. Massiot, ‘Recent Progress in Solid State Nuclear Magnetic Resonance for the Structural Characterization of Materials’, Spectra Analyse, 2002, 31,27 German R 550 M. Backhaus, D. Sandrock and W.A. Schmidt, ‘Imaging in Rheumatology’, Deutsche Medizinische Wochenschrift,2002,127, 1897 R 551 D.F. Braus, ‘Images of the Mind: Nuclear Spin Tomography Opens the First Insight into the Somatic Foundation of Mental Processes’, Medizinische Monatsschrift fuer Pharmazeuten, 2003,26,45 R 552 M. Bunzel, ‘Structural Elucidation of Dietary Fiber Carbohydrates’, Nachr. Chem., 2002,50,361 R 553 J. Engering and M. Jansen, ‘Preceramic Polyazanes via Sol Gel Route in the Ammono System and via Molecular Single Source Precursors’, 2.Anorg. Allg. Chern., 2003,629,913 R 554 R. Eymael and B. Blumich, ‘NMR for Process Control. Imaging and Localized NMR in Soft Matter’, Technisches Messen, 2002,69,340 R 555 C.D. Guibao, G. Liu and J. Zheng, ‘Structure Analysis of Proteins’, G I T Labor-Fachzietschrgt, 2002’46, 1106 R 556 P. Jelinek, H. Polzin and H. Polzin, ‘Structural Studies and Strength Properties of Sodium Silicate Binders’, Giesserei-Praxis,2003,2, 51 R 557 U. Klebe, L. Thiedecke and T. Stolcker, ‘Helium - A Special Element’, Gas Aktuell, 2002’63’4 R 558 W. Kremer and H.R. Kalbitzer, ‘High Pressure NMR Spectroscopy of Proteins’, BIOspektrurn, 2003,9, 142 R 559 S.E. Kulling, ‘Food Chemistry 2002’, Nachr. Chern., 2003,51,346 R 560 N. Nestle, T. Baumann and R. Niessner, ‘Patient Groundwater. Nuclear Spin Tomography in Hydrogeology’, Nachr. Chern., 2002,50,946 R 561 U. Neubauer, ‘Nuclear Magnetic Resonance Spectroscopy. On the Trail of Proteins’, C L B Chemie in Labor und Biotechnik, 2002,53,288 R 562 G. Schilling, ‘NMR Chromatography. DOSY’, G I T Labor-Fachzietschrijt, 2002,46, 1163 R 563 G. Schilling, ‘An Exote. Stable Pentamethylcyclopentadienyl Cation’, G I T Labor-Fachzietschrijt, 2002,46,8 18 R 564 G. Schilling, ‘Nobel Prize 2002. Nobel Prize for Chemistry. Analysis at the Highest Level’, G I T Labor-Fachzietschrijit, 2002,46, 1217 R 565 U. Schulte, ‘Nobel Prize for Chemistry 2002: Structure Analysis of Biological Macromolecules’,Deutsche Apotheker Zeitung, 2002,142’85 R 566 H. Schwalbe and H. Kessler, ‘900 MHz NMR Spectrometer in Munich and Frankfurt’, Nachr. Chem., 2003,51,412

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R 567 F. Steiner and C. Huber, ‘Coupling Techniques in Chromatography’, Nachr. Chem., 2003,51,387 R 568 R. Voelkel and P. Hubler, ‘NMR Spectroscopic Methods in Polymer Physics’, GI T Labor-Fachzietschrft, 2002,46,784 R 569 G. Ziegleder, ‘Quality Assurance of Filled Chocolates Exemplified by Fat Migration’, Diskussionstagung - Forschungskreis der Ernaehrungsindustrie e. V., 2002,60, 65 Greek R 570 S. Papazacharias, G.A. Spyroulias, A. Gravanis and P. Cordopatis, ‘Corticotropin Releasing Hormone (CRH) a Contribution to the StructureActivity Relationship Study’, Pharmakeutike, 2002,15, 12 Hebrew R 571 T. Sternfeld and M. Rabinovitz, ‘3-D Aromaticity of Fullerenes: Changes in the Aromaticity of Fullerenes Following Reduction’, K himiya beYisra’el,2002,10, 3 Hungarian R 572 S. Gorog, ‘Some Results of the Analytical Research at Gadeon Richter Ltd. ’, Magyar Kemikusok Lapja, 2001’56,445 R 573 P. Sohar, ‘Hunting for Structures by Spectroscopy in the Jungle of Molecules’, Magyar Kemiai Folyoirat, 2002,108,277 R 574 C.I. Szntay and G. Balogh, ‘The Sensitivity of NMR Spectroscopy’, Acta Pharmaceutica Hungarica, 2002,72,106 R 5 7 5 G. Toth, ‘Recent Trends in NMR Spectroscopy’, Magyar Kemikusok Lapja, 2002,57,371 Japanese R 576 A. Abiko, ‘Boron-Mediated Aldol Reaction of Carboxylic Esters’, Yuki Gosei Kagaku Kyokaishi, 2003,61,26 R 577 K. Akasaka, ‘High Pressure NMR Expands the Protein Structure World’, Seibutsu Butsuri, 2002,42,206 R 578 H. Akutsu, ‘Nuclear Spin Relaxation’, Nippon Bunko Gakkai Sokuteiho Shirizu, 2003,41,45 R 579 H. Akutsu and T. Fujiwara, ‘Structural Analysis of Membrane Proteins by Solid-state NMR’, Tanpakushitsu Kakusan Koso, 2002,47,1144 R 580 I. Ando, S. Yokota, A. Sasaki, S. Koizumi, Y. Yamane, H. Kimura and S. Kuroki, ‘Development of Three-Dimensional High-Resolution NMR Microscopy and Its Application’, Kin0 Zairyo, 2002,22,33 R 581 T. Asakura, ‘Structure Determination of post and after Domestic Silkworm Fibering Using Recent Solid State NMR Method’, Kobunshi Kako, 2002,51,338 R 582 T. Endo, ‘Isolation, Purification and Characterization of Large-Ring Cyclodextrins, and Their Molecular Structures’, Hoshi Yakka Daigaku Kiyo, 2001,43, 7


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R 583 K. Fujita, H. Masui and S. Morimoto, ‘Researches on Automatization for NMR Measurement and Structural Elucidation’, Sumitomo Kagaku (Osaka, Japan), 2002,1,13 R 584 T. Fujiwara, ‘Molecular Structure Interpretation Using Torsion Angle Measurement by Solid-state NMR’, Kagaku (Kyoto, Japan), 2002,57,58 R 585 T. Fujiwara, ‘Nuclear Magnetic Resonance’, Nippon Bunko Gakkai Sokuteiho Shirizu, 2003,41,3 R 586 T. Fujiwara, ‘Chemical Exchange’, Nippon Bunko Gakkai Sokuteiho Shirizu, 2003,41,41 R 587 T. Fujiwara, ‘Fundamentals of Solid NMR, Bunko Gakkai Sokuteiho Shirizu, 2003,41,81 R 588 E. Fukushi, ‘Viewing Molecular Interactions by NMR’, Kagaku to Seibutsu, 2002,40,750 R 589 N. Harada and H. Taji, ‘Enantiomeric Excess Determination by Diastereomer Method Using NMR or MS Spectra’, Kagaku to Seibutsu, 2002,40,827 R590 H. Hatanaka, S. Koshiba, J. Kikuchi, T. Kigawa and S. Yokoyama, ‘High-Throughput NMR Structure Determination: The Present and the Future’, Tanpakushitsu Kakusan KOSO,2002,47,1038 R 591 F. Hayase, ‘Development of Technique for Quality Control by Biochemical Analysis of Color-Correlated Ingredients’, Meiji Daigaku Kagaku Gijutsu Kenkyusho Nenpo, 2000,42,108 R 592 S. Hayashi, ‘Analysis of Hydrogen Site and Diffusion Using Solid State NMR’, Kinzoku, 2002,72,649 R 593 T. Igarashi, ‘Methods for Monitoring Shellfish Toxins and Supply Programs for Standards’, Foods & Food Ingredients Journal of Japan, 2002, 198’6 R 594 T. Iijima, M. Mizuno, M. Suhara and K. Endo, ‘Molecular and ElectronSpin Dynamics in [M-(H20)6][AB6] as Studied by Solid State NMR’, Bunseki Kagaku, 2003,52,157 R 595 K. Ishida, H. Tou and Y. Kitaoka, ‘Spin-Triplet Superconductivity in Strongly Correlated Electron Systems. An NMR Perspective’, Kotai Butsuri, 2003,38, 179 R 596 N. Ishida, ‘To See Water Transfer in Plant Cells by NMR Microscopy. Visualizing Motion Speed and Flow Rate Employing Diffusion Measurement’, Kagaku to Seibutsu, 2003,41, 147 R 597 K. Ito, ‘Structural and Functional Analysis of Enzymes and Their Application to Clinical Analysis - Study on Pseudomonas Putida Formaldehyde Dehydrogenase’, Yakugaku Zasshi, 2002,122,805 R 598 T. Ito, ‘Measurement Methods and Measurement Parameter’, Nippon Bunko Gakkai Sokuteiho Shirizu, 2003,41, 121 R 599 M. Iwadate and H. Umeyama, ‘FAMS: A Homology Modeling Program’, Seibutsu Butsuri, 2002,42,282 R 600 Y. Iwasaki, ‘Interpretation of Peptide Adsorbed on Hydrophobic Polymer Surface’, Kagaku (Kyoto, Japan), 2002,57,52 R 601 Y .Izumi, ‘Searching Molecular Mechanisms of Multi Functional Protein

42

R 602 R 603

R 604 R 605

R 606 R 607

R 608 R 609 R 610 R 611

R 612 R 613 R 614 R 615 R 616 R617

R 618

R 6 19


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R620 H. Maeda, Y. Ito and S. Yokoyama, ‘Application of Superconducting NMR for Protein Structure Determination’, Teion Kogaku, 2002,37,2 R 621 N. Manabe, M. Sugimoto and H. Miyamoto, ‘Development of Chemical Substance Evaluation Method for Next Generation’, Kemikaru Enjiniyaringu, 2002,47, 110 R 622 N. Matsubayashi, C. Wakai and M. Nakahara, ‘Structural Analysis of Super Critical Fluid by NMR’, Chrinkai Ryutai no Subete, p. 23, ed. Y.Arai, Tekuno Shisutemu, Tokyo, Japan, 2002 R623 U. Matsushima, ‘Studies on the Preservation of Cut Carnations by Applying Non-Polar Gas’, Ryukyu Daigaku Nogakubu Gakujutsu Hokoku, 2001,48,1 R 624 T. Miyoshi and A. Kaito, ‘Application of Spin Diffusion NMR Methods to Nan0 Structures of Polymers’, Kin0 Zairyo, 2002,22,54 R 625 K. Nagase, Y. Kamiya, K. Hodumi and T. Miyakoshi, ‘Development of Naturally Drying Polymer Lacquer (II)’, Toso Kogaku, 2002,37, 129 R626 K. Nagayama, ‘Kurt Wuthrich, the Man and His Work’, Kagaku to Kogyo (Tokyo, Japan), 2002,55,1336 R 627 M. Nakano and A. Usuki, ‘Stereo- and Regiospecific Polymerization of Cyclic Conjugated Dienes Using Highly Active Nickel Catalysts’, Kobunshi Ranbunshu, 2002,59,356 R 628 Y. Nakashima, ‘Measurement of H 2 0 Self-Diffusion Coefficients in Clay Gels by Pulsed-Field-Gradient Nuclear Magnetic Resonance: A Review’, Nendo Kagaku, 2002,42,37 R 629 S. Natsuka, ‘Analysis of Glycan Expression by Fluorescence Labeling Method‘, Seibutsu Butsuri Kagaku, 2002,46,35 R 630 K. Ogata, ‘Sample Preparation for Structural Analyses; Focused on Protein-DNA Complexes’, Idenshi Igaku, 2002,6,86 R 631 S. Ohki, T. Terauchi and M. Kainosho, ‘Development of Highly Efficient and Highly Precise NMR Method for Protein Analysis in Genomics’, Kemikuru Enjiniyaringu, 2002,47,494 R 632 A. Okada, H. Takigawa, N. Iwata, M. Shirasaki, Y. Fujiwara and T. Sasaki, ‘Structure Analysis and Characterization of Synthetic Polymer Materials’ Sumitomo Kagaku (Osaka, Japan), 2002,4 R 633 M. Osawa and M. Ikura, ‘NMR-Based Approach for Drug Discovery’, Tanpakushitsu Kakusan Koso, 2002,47,948 R 634 H. Othake, X. Li, H. Takahashi and S. Ikegami, ‘Synthesis, Structure, and Application of Sugar Orthoesters’, Yuki Gosei Kagaku Kyokaishi, 2002, 60,206 R 635 K. Saito, ‘The Application of NMR Imaging to Industrial Materials’, Materia, 2002,41,878 R 636 T. Sano and Y. Oumi, ‘Dealurnination of Zeolite and Reinsertion of Metals into the Framework’, Zeoraito, 2002,19,133 R 637 Y. Sasaki and K. Ishii, ‘Structure of Molten Silicate and Aluminosilicate Steelmaking Slags’, Tetsu to Hagane, 2002,88,419 R 638 Y. Sasanuma, ‘Weak Hydrogen Bonds Formed by Polyethers’, Kobunshi Kako, 2002,51,218

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R 639 I. Shimada, ‘An NMR Method for the Determination of the Interface of Large Protein-Protein Complexes’, Tanpakushitsu Kakusan Koso, 2002, 47,1285 R 640 T. Suenaga, ‘Analytical Methods for Combinatorial Synthesis’, Yuki Gosei Kagaku Kyokaishi, 2002,60,454 R 641 K. Tachikawa and H. Kumukura, ‘MgB2 Tapes and Metallic Superconductors Update’, Oyo Butsuri, 2003,72, 13 R642 K.4. Takahashi and G. Kawai, ‘Two-step Dimerization of HIV-1 Genomic RNA’, Seitai no Kagaku, 2002,53,131 R 643 T. Takase, H. Tsuno, S. Fujiwara, K. Kotake and H. Kodama, ‘Automation of Heavy Solvent Substitution of NMR Sample’, Yuki Gosei Kagaku Kyokaishi, 2002,60,520 R644 H. Takemura, ‘Fluorine Atom Covalently Bonded to Carbon Atom: Properties as New Cation Donor’, Yuki Gosei Kagaku Kyokaishi, 2002, 60,963 R 645 S. Takeuchi, ‘An Overview on the Recent Progress of Quantum Computers’, Oyo Butsuri, 2002,71, 1367 R 646 T. Terauchi, S. Ohki and M. Kainosho, ‘Developing a New Approach for High-Throughput, High-Accuracy NMR Structural Analyses of Genomic Proteins’, Tanpakushitsu Kakusan KOSO, 2002,47,1045 R 647 T. Torizawa, T. Terauchi and M. Kainosho, ‘Recent Development in NMR Methods for Structural Biology’, Seikagaku, 2002,74, 1279 R 648 H. Tou, ‘Novel Superconductivity of Electron Doped Hafnium Nitride Li,(THF),HfNCl’, Kotai Butsuri, 2002,37, 779 R 649 H. Uehara, K. Yamanobe and T. Komoto, ‘Polyethylene Polymerization Power Morphology and Molecular Mobility’, Kobunshi Kako, 2002,51, 65 R 650 K. Ute, ‘Polymer Characterization by LC/NMR’, Bunseki, 2002,8,459 R 651 K. Ute, ‘LC-NMR and DOSY - New NMR Techniques for the Analysis of Polymer in Liquid‘, Kobunshi, 2002,51,824 R 652 J. Uzawa, ‘NMR for Mol. Structures’, Bunseki, 2002,8,444 R 653 S. Yamada, ‘Conformation Control of Organic Molecules by Intramolecular Interaction and Its Synthetic Applications’, Nippon Kessho Gakkaishi, 2002,44,240 R 654 A. Yamamoto, ‘Nuclear Magnetic Resonance Spectroscopy: The Recent Developments of Hardware: The Current Status of Cryogenic Probe’, Bunseki, 2002,6, 320 R 6 5 5 Y. Yamane, S. Kuroki and I. Ando, ‘Diffusional Behavior of Probe Molecules in Polymer Gels as Studied by Field-Gradient NMR Method’, Nettowaku Porima, 2002,23,211 R 656 T. Yamase, ‘Time-Resolved ESR-Spectroscopic Investigation of Polyoxometalate Photochemistry’, Shokubai, 2002,44, 340 R 657 M. Yashimasu, M. Honda, T. Mikawa, T. Shibata and Y. Ito, ‘NMR Approaches to Investigate Protein-Protein and Protein-Nucleic Acid Complexes’, R I K E N Review, 2002,46,32 R 658 I. Yasumatsu, ‘Structural Analysis of Protein by Using Public Databases


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and Personal Computers’, Kagaku ( K y o t o , Japan), 2002,57,42 R 659 K. Yoshimura, H. Sakai and S. Kambe, ‘Superconductivity in the Frustrated-Spin System Cd2Re207with Pyrochlore Structure’, Nippon Butsuri Gakkaishi, 2003,58, 34 Korean

R 660 Y.A. Kim, ‘Structural Study of Membrane Binding Protein by NMR’, Saenghwahak Nyusu, 2001,21,253 R 661 J.W. Korea and W.T. Lee, ‘Structural Genomics Study Using Nuclear Magnetic Resonance Spectroscopy’, Saenghwahak NYUSU, 2001,21,271 R 662 H.S. Won and B.J. Lee, ‘Structural Study of Protein-Nucleic Acid Composite by Nuclear Magnetic Resonance Spectroscopy’, Saenghwahak Nyusu, 2001,21,258 Polish R 663 D. Bauman, ‘Application of Spectroscopic Methods to Studies of Liquid Crystals’, Postepy Fizyki, 2002,53, 169 R 664 B. Czuprynski, J. Liszkowska and J. Sadowska, ‘Selected Issues in the Flammability of Some Plastics and Methods for Identification of Their Combustion Products’, Ekologia i Technika, 2001,9, 105 R 665 J. Kasperczyk, ‘Microstructure Analysis of Biodegradable Polyesters Obtained from Glycolide, Lactide, and A-Caprolactone Using High-Resolution NMR Spectroscopy’, Zeszyty Naukowe Politechniki Slaskiej, Chemia, 2001,145,l R 666 J. Milecki, ‘Chemical Synthesis of Oligoribonucleotides’, Wiadomosci Chemiczne, 2002,56,255 Portuguese R 667 A.A. de Sousa and A. Laverde, Jr., ‘Using Nuclear Magnetic Resonance Spectroscopy to Study Molecular Diffusion in Liquids: The DOSY Technique’, Quimica Nova, 2002,25,1022 Russian

R 668 L.A. Aleshina, S.V. Glazkova, L.A. Lugovskaya, M.V. Podoinikova, A.D. Fofanov and E.V. Silina, ‘Modern Concepts of Cellulose Structure’, Khimiya Rastitel’nogo Syr’ya, 2001, 1, 5 R 669 S.N. Barilo, D.I. Zhigunov, S.V. Shiryaev, P. Lemmens, V.P. Gnezdilov, Yu. G. Pashkevich, A.A. Zakharov, J.M. Trankvada and A.M. Galagurov, ‘Comparative Investigation of Growth Conditions and Subsequent Oxidation Treatment Influence on structural Phase Separation Character, Regulating of Spins and Holes and Superconductivity of La2Cu04+Single Crystals’, Poverkhnost, 2002,3,20 R 670 Z.R. Borsutskii and S.E. Il’yasov, ‘Studies of the Mechanisms of Magnetic Treatment of Petroleums Based on Results of Laboratory and Oil-Field Tests. 111. Study of the Mechanisms of Magnetic Treatment of Petroleums’, Neftepromyslovoe Delo, 2002,9, 38

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R 671 M. Panasenko, J. Arnhold and V.I. Sergienko, ‘Impairment of Membrane Lipids by Hypochlorite’, Biologicheskie Membrany, 2002,19,403 R 672 V.G. Shubin, ‘Carbocationic and Related Fluxional Rearrangements’, Khimiya: Metodika Prepodavaniya v Shkole, 2002,8,34 R 673 V.M. Sutjagin and A.A. Lyapkov, ‘Empirical Approach to Estimation of Reactivity Ratios of Vinyl Monomers in Radical Homo(co)polymerization’, Izvestiya Vysskikh Uchebnykh Zavedenii, Khimiya i Khimicheskaya Tekhnologiya, 2002,45,113 R 674 M.A. Zenkova, V.A. Petyuk, R.N. Serikov and V.V. Vlasov, ‘Mechanisms and Dynamics of Oligonucleotide Interactions with Natural RNAs’, Goryachie Tochki Supramolekulyarnoi Khimii, Materialy UchebnoMetodicheskoi Konferentsii ‘Opyt Vvedeniya Noveishikh Dostizhenii Supramolekulyarnoi Khimii v Uchebnye Programmy Srednikh I Vysshikh Uchebnykh Zavedenii’, Novosibirsk, Russian Federation, Aug. 6-1 2, 2001, ed. E.V. Boldyreva, Novosibirskii Gosudarstvennyi Universitet, Novosibirsk, Russia, 2002 Slovenian R 675 M. Vrbinc and F. Vrecer, ‘Pseudopolymorphism in the Development of Dosage Forms’, Farmacevtski Vestnik (Ljubljana, Slovenia), 2002,53,103 Spanish R 676 J.A. Bravo, ‘A Revision of Bidimensional(2D NMR) Nuclear Magnetic Resonance Spectral Methods’, Revista BoEiviana de Quimica, 2001,18,102 R 677 L. Garrido, ‘Nuclear Magnetic Resonance Spectroscopy of Solid-state Polymers’, Revista de Plasticos Modernos, 2002,83,485

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. - Widely accepted benchmarks for shielding calculations, invaluable for calibration of less accurate methods, especially for improvement of density functional methods, have been provided by the small molecule calculations by Jurgen Gauss using full configuration interaction or coupled cluster calculations. Thus, it is most appropriate that Gauss and Stanton have presented a comprehensive review of electron-correlated approaches for the calculation of NMR chemical shifts.' Benchmark investigations into the quantitative prediction of gas phase 13C absolute shieldings have been carried out by Auer, Gauss, and Stanton? They selected 16 molecules for which the absolute 13Cshieldings had been reported by Jameson and Jameson3based on the primary reference, I3COmolecule. For these molecules, the authors investigated basis set convergence, dependence on the choice of equilibrium molecular geometry, and zero-point vibrational corrections using the same basis set and correlation level of calculations for the force field as used for the shielding calculations. They neglected the rotational and vibrational contributions that would take their theoretical isolated vibrating molecule at 0 K to the experimental gas phase molecule at 300 K in the zero-pressure limit. Basis sets used for comparisons included triple zeta with one polarization function, quadruple zeta with two polarization functions, and so on, up to the largest set used, the uncontracted 15sllp4d3f/lOs4p3d set. Dunning's correlation consistent basis sets cc-pVTZ and cc-pVqZ were used for geometry optimization. In order to investigate electron correlation effects, shieldings are calculated at the Hartree-Fock self-consistent field (SCF), second order MarllerPlesset (MP2) perturbation theory, coupled-cluster singles and doubles (CCSD) and CCSD augmented by a perturbative treatment of triple excitations (CCSD(T)).In addition they computed density functional theory (DFT) nuclear magnetic shieldings using the BP86 functional4- and the B3LYP hybrid functional! Since the calculated shielding can be very sensitive to the molecular geometry chosen, they compared results using different theoretical geometries, eschewing experimental geometries altogether because the equilibrium geometries (re)are not well known for all the molecules considered, and in any case such Nuclear Magnetic Resonance, Volume 33 0 The Royal Society of Chemistry, 2004

47

48


‘experimental’ values of re are themselves found by making zero-point vibrational corrections on spectroscopic values of bond lengths (and bond angles). The required cubic force fields are computed by numerical differentiation of analytically calculated harmonic force fields with respect to the normal coordin a t e ~Likewise .~ numerical differentiation of the shielding surface with respect to the normal coordinates are carried out. The zero-point vibrational average shielding is then evaluated in the usual

(a)= o e

+ Xr

(aa/aQr)

Qr=O (Qr)

+ 1/2 &,,(a2a / a Q r d Q s )

Qr,Qs=O (QrQs)

where,

in which, 63, is the harmonic frequency of the rth normal mode and 4rstis the corresponding cubic force constant. The findings in the set of 16 benchmark molecules are as f01lows:~Using the same optimized geometry (the ones obtained using CCSD(T)/cc-pVTZ) for all levels of calculations being compared, the electron correlation contributions, assumed to be that obtained at the CCSD(T) level as the standard reference data, range from -0.4 ppm in CH3CHO to - 34.2 ppm in CO, that is, all deshielding. The exceptions are CH30H, CH3F, and CF4, which have electron correlation contributions of respectively +0.7, + 1.5 and + 12.4 ppm. The CCSD approximation tends to underestimate electron correlation effects; the MP2 method recovers most of the electron correlation contributions but tends to a slight overestimation of the latter. DFT/BP86 and DFT/B3LYP shieldings systematically deviate by a large amount from the CCSD(T) reference data, leading to uniformly too deshielded values (by - 10 to - 30 ppm) in each of the 16 13C benchmark nuclear sites. For most of the systems considered, the error with respect to the CCSD(T) reference is even larger than the corresponding deviation obtained at the Hartree-Fock level. Unlike for many other applications, DFT does not provide a systematic improvement relative to Hartree-Fock for absolute shielding calculation^.^ Where accurate experimental re geometries are available, CCSD(T)/cc-pVTZ geometries have errors in the range 0.002 to 0.003A in the bond distances and a few degrees in the bond angles. The MP2 structures using the same basis set are less reliable, with errors of up to 0.006-0.007A. The SCF geometries are assumed to be even less reliable. Basis set errors of 5-15 ppm can be attributed to the tzp set, smaller deviations for the qz2p set (1-5 ppm) compared to the large basis set 13s9p4d3f results. The latter is close to the basis set limit, as was shown by comparison with the even larger 15sl lp4d3f set, with remaining basis set error of ca. 0.5 ppm. Basis set errors are consistently of the same sign, improvement of the basis set leads to a decrease in shielding. Thus, accurate relative chemical shifts are often obtained with modest basis sets while accurate prediction of (absolute) nuclear magnetic shielding requires very large basis sets7 The zero-point vibrational corrections to the 13C shielding are all negative.

2: Theoretical and Physical Aspects of Nuclear Shielding

49

Hartree-Fock slightly overestimates while MP2 underestimates the vibrational corrections. Comparison of the theoretical values for the vibrating molecule at 0 K and the gas phase data at 300 K in the zero-density limit reveals a mean deviation of 1.6 ppm, with the theoretical values being still too shielded. The 13C in the CO molecule has only a small temperature dependence; the shielding difference between 0 K and 300 K is small." All these molecules have a nonnegligible temperature dependence arising from rotational and vibrational contributions; some have been measured experimentally.'o-12On the basis of rovibrational theory and the known signs of the derivatives of the 13Cshielding in these system, the latter is expected to be a deshielding contribution in all these 16 cases,I3 which explains in part the remaining small systematic differences between these theoretical and the experimental absolute shieldings in terms of signs and magnitudes (as reflected by the mean deviation of 1.6 ppm in the compariThe corresponding mean deviations for the DFT method are 6.2 ppm (DFT/BP86) and 7.3 ppm (DFT/B3LYP), higher than for all of the conventional methods, higher than Hartree-Fock SCF in particular. It is well known in the literature that standard DFT leads to an overestimation of the paramagnetic contribution and thus yields too low shielding constants. This is the reason why an empirical modification of the orbital energy denominator has been suggested in the sum over states method in 1994 by Malkin et aE.14and this 'fix' has been investigated by other authors as well.15* l6 Basis set enlargement and rovibrational corrections mainly affects the mean deviation of absolute shieldings; thus, it is found in many applications of 13Cchemical shift calculations for spectral assignments that reliable predictions of relative chemical shifts are usually possible at Hartree-Fock SCF and DFT level of calculations, especially with modest to small basis sets. The relative signs of the errors from neglect of electron correlation and basis set size lead to cancellation of errors. These observations apply to 13C,not necessarily to other nuclei. Despite the formal invariance of magnetic properties to gauge transformations, the values of some of these physical quantities do depend on the approximations used to calculate them. The relationships (sum rules) for gauge invariance of third order properties, that is, the response of a molecule in the presence of three perturbations: electric field, magnetic field and nuclear magnetic dipoles, are expressed in terms of second-rank response properties: the electric polarizabilities and the electric shielding at the nuclei. A numerical test has been carried out to determine the Hartree-Fock limit for a series of such third order response quantities in H2, F2,N2,CO, HCN, HNC, HCCH, H20, NH3, CH4, and HOOH molecules by Caputo and La~zeretti.'~ These investigations lead to recommendations for composing basis sets that more quickly approach the Hartree-Fock limit of the sum rules for these molecules. In practice, in calculations of these third order response properties, the basis set should contain diffuse polarization functions needed for good electric dipole polarizabilities, as well as functions with big exponents that yield accurate nuclear electric shieldings. Calculations of nuclear shieldings using CTOCD-DZ (continuous transformation of origin of the current density whereby the diamagnetic contribution to the current density is set to zero) have been formulated, implemented and applied

50


to benchmark molecules using large basis sets at the uncorrelated level by Lazzeretti and co-workers.18-20 In this reporting period, the first calculations of CTOCD-DZ nuclear shieldings at the MCSCF and SOPPA(CCSD) level have been reported.21 The results are compared with the correlated GIAO results using MPn and coupled cluster wave functions of Gauss et al. for the set of benchmark molecules: CH4, NH3, H20, HF, CO, N2, HCN, HCCH. (See Ref. 1 for a recent review of these calculations.) The CTOCD-DZ procedures make use of gaugeless basis sets. CTOCD-DZ computational schemes at the correlated level are practical alternatives to GIAO counterparts. Combined with the SOPPA and SOPPA(CCSD)methods, CTOCD-DZ procedures are competitive alternatives to the GIAO-MP2 approach. Basis set convergence for a given level of accuracy is slightly inferior for CTOCD-DZ than for GIAOs, but for accurate answers large basis sets are required in both methods.2’ The relation between a common gauge origin formulation and the GIAO formulation of the NMR shielding tensor has been traced out by Schreckenbach, starting from the GIAO formulation.22The derivations provide the terms which when added to the GIAO diamagnetic shielding lead to the diamagnetic shielding of the common gauge scheme. The latter, with the common origin centered at the nucleus is the one that has to be used in the relationship between the spin rotation tensor elements and the nuclear shielding tensor elements, when the absolute shielding is derived from experimental spin rotation tensors of isolated molecules, since the origin associated with the spin-rotation tensor is at the nucleus of interest. The diamagnetic and paramagnetic parts of the terms left out in the GIAO formalism but included into the common origin formalism are shown to be large contributions of opposite signs. This is part of the reason that GIAO (and other distributed origin schemes such as IGLO and LORG) provide more accurate results when using basis sets that are smaller than required for the Hartree-Fock limit. Sum-over-states density functional perturbation theory (SOS-DFPT) introduced by Malkin, Malkina, Casida and Salahub14in 1994 relies on the introduction of an ad hoc correction to the excitation energy that improves the calculation of the paramagnetic term in the shielding. Recently, Fadda et al. provide a theoretical justification for a new energy correction term that results in a local density approximation which the authors have labeled ‘ L O C . ~ They ’ . ~ ~ have tested the Loc.3 approximation in a series of 25 and compared their results with other SOS-DFPT local density approximations, and also with four functionals recently introduced and applied by Wilson and Tozer (MKS),25by Wilson, Amos and Handy (B3LYP0.05GGA),26 by Adamo and Barone (PBEO)?’ and Patchkovskii, Autschbach and Ziegler (SIC-VWN)?* for the same set of molecules. Performance of these functionals have been reviewed in previous volumes of this series. A two-component quasi-relativistic theory based on Douglas-Kroll-Hess (DKH) transformation of the magnetic potential has been developed by Nakatsuji and co-workers for nuclear magnetic shielding calculations. At first, they used only the spin-free parts of the DKH Hamiltonian, and the SO term and the magnetic interaction term remained in the Pauli form.29The relativistic correc-


51

tions to the zeroth order Hamiltonian were considered but the magnetic interaction term remained non-relativistic. In a recent paper, they have carried out the DKH transformation including the magnetic field and utilized the generalizedU H F formalism to describe the SO interaction and the magnetic field effect at the Hartree-Fock They used the Gaussian nucleus model to include the finite nucleus effect. In a subsequent paper, they introduced GIAOs in finite perturbation theory into this quasi-relativistic formulation and applied the approach (GIAO-FP-QR-GUHF) to hydrogen halides and mercury halides.31 Note that electron correlation has not been included in this formulation. For a comparison of the results from this new method with the DFT-ZORA method, see the table in Section 1.2 Fukui and Baba have also suggested a DKH transformation including magnetic vector potential, and used it for calculations of the nuclear magnetic shieldings in the hydrogen halide^.^^"^ Recently, they again carry out calculations on the same molecules using a zeroth order regular approximation (ZORA) method at the Hartree-Fock They compare their present ZORA HartreeFock results with the DKH results they previously obtained, and with the four component relativistic RPA calculations, also ~ n c o r r e l a t e dThe . ~ ~ZORA results underestimate the relativistic effects compared to the four-component RPA, which may be due to neglect of higher order relativistic effects beyond the zeroth order. Recent developments in the calculations of shielding in infinite networks make use of the theoretical framework for the description of a magnetic field under periodic boundary conditions developed by Sebastiani and Parinello, based on density functional theory.37 The strategy relies on the exponential decay of so-called localized Wannier orbitals, Kohn-Sham orbitals which have been maximally localized in space by means of a unitary rotation within the subspace of occupied orbitals. These orbitals are different from zero only in well-defined, finite regions of space. The approach is implemented in the CPMD suite of programs, a plane-wave pseudopotential In this reporting period, improvements in computational efficiency of this method permit the calculation of the proton spectrum of strongly hydrogen-bonded periodic systems such as proton-conducting crystals whose solid state spectra have been studied in the Spiess group.39 1.2 A6 initio Calculations.- The ability to calculate NMR shieldings for heavy elements like uranium is a particular strength of the two-component relativistic DFT-ZORA (zeroth order regular approximation) and DFT-QR (quasi-relativistic) methods. Schreckenbach has used two relativistic methods, ZORA and the quasi-relativistic method, to do calculations of 235Ushielding and also shieldings of ligand nuclei in diamagnetic uranium compounds. A large chemical shift range of at least 21,000 ppm has been predicted for 235Unucleus in uranium compounds ranging from uranyl complexes to organometallic and inorganic derivatives of UF6.40Very large spin-orbit relativistic effects were noted, but some parts of these effects cancel out in relative chemical shifts. This cancellation is an indication that these are core-type relativistic effects. Such core-type spin-orbit effects arise

52


because the spin-orbit induced spin polarization is picked up by the core-type s orbitals at the heavy uranium nucleus itself. The core-type spin-orbit is also considered to be responsible for at least part of the large constant offset between the calculated Pauli spin-orbit and ZORA spin-orbit absolute shieldings. While some scalar relativistic effects of the core are approximately included in the Pauli method, core-type spin-orbit effects are not. The latter are included in the all-electron ZORA calculations. Core effects are also responsible for the large difference between Pauli and ZORA shieldings that are calculated without spin-orbit effects, that is, in the scalar relativistic approximation. A large part of these core effects (not just the diamagnetic shielding of the core density, but the paramagnetic core-core and core-valence contributions) also cancels out when shielding differences are taken to find relative chemical shifts, but not completely. With DFT/ZORA the entire Periodic Table is becoming accessible to first principles studies of NMR parameters. Trends in the 235UNMR chemical shifts (relative to UF6, a convenient reference) in the series UF6-nLn molecules show all U nuclear sites to have positive chemical shifts (more deshielded) relative to UFs, and the I9F shieldings, for which the absolute shielding scale with multiple redundancies is available, are in excellent agreement with experiment!' 199Hgshielding tensors in methylmercury halides CH3HgX, (X = C1, Br, I) have been calculated using DFT/ZORA and the BP86 density functional!l Earlier calculations on these compounds using DFT/ZORA used a somewhat different gradient-corrected f ~ n c t i o n a lThe . ~ ~ calculated isotropic values are too shielded compared to experiment in every case and the shielding anisotropies are too large. Critical to the Hg calculations is not so much the particular GGA functional used but the inclusion of basis functions of sufficient flexibility in the near nuclear region, with Is exponents at least up to ca. lo4in the very vicinity of the nucleus.41The isotropic chemical shift trend (chemical shifts relative to Hg(CH&) and the anisotropy increasing in going from C1 to I is of course a result of the increasing magnitude of the spin-orbit contributions to the shielding in the series. Shieldings in CH3HgX,(X = Cl, Br, I) molecules have also been studied by Nakatsuji et al. using a two-component quasirelativistic Douglas-Kroll-Hess approach with GIAOs but not including electron correlation. 31 A comparison of the DFT/ZORA results 41 with the Douglas-Kroll-Hess QR approach (without electron correlation):' both using GIAOs, is shown in Table 1 for the subset of Hg compounds in common. The BP86 functional and triple-zeta polarized basis set V results from Ref. 41 are cited in the table. In many cases where theoretical results are presented only as isotropic chemical shift values, large differences between methods are hidden and systematic improvement of the theory can be hampered. In this instance, detailed comparison is possible because the authors provide the shielding results, not just the calculated isotropic shifts. We see in Table 1 that there is a systematic difference in the diamagnetic contributions, of the order of about 300 ppm higher in the quasi-relativistic approach. Setting this aside, there are significant differences in the spin orbit contributions calculated by the two methods. The paramagnetic term from DFT-ZORA is systematically larger negative for the olcomponent and also systematically less shielded for the


Table 1

53

Comparison of recent results for 199Hgshieldings ~~~

DFT-ZORA method (Ref. 41)

D K H method (Ref. 31) diam

param

9906 9894

SO

total

diam

param

SO

total

318 5777 -5920 7174

16001 11158

9620 961 1

246 3392 -6207 1997

13258 5401

9908 9890

433 5755 -4462 5410

16096 li838

9623 961 1

283 3249 -4885 2109

13154 6835

9910 9892

432 5764 -4671 7315

16106 12536

9624 9608

298 3260 -5008 2414

13182 7014

9912 9894

424 5669 -5133 8512

16004 13273

9626 9609

324 3220 -5422 2912

13171 7100

MeHgMe 0 1 1 01

MeHgCl <JII 01

MeHgBr 0 1 1 01

MeHgI 0 1 1 01

q component. There is a paramagnetic contribution to the parallel component in these cases because the molecules are not strictly linear: the hydrogen atoms are off-axis. The spin-orbit contributions are very different in both parallel and perpendicular components, with the DFT-ZORA providing smaller values compared to the quasi-relativistic approach. The 129Xe shieldings in several Xe fluorides, Xe03 and [XeCl]+ ion have been calculated using the DFT/ZORA method using basis sets up to triple zeta plus two polarization f~nctions.4~ The free Xe atom absolute shielding value 5660 ppm obtained here using DFT/ZORA may be compared with the best available relativistic (four-component Dirac-Fock) calculation using 1413 basis functions by Vaara and Pyykko,446938 ppm. The greatest problem encountered in this DFT/ZORA work is the geometry of the molecule (that is, the actual structure in solution) and accounting for the intermolecular effects that are included in the experimental results, especially in the case of the ions [XeF]+ , [XeF3]+ , and [XeCl]+ in superacid (HF/SbF5) solution. It had been postulated that [XeF]+ has the structure [FXe-F-XeF]+ . The spin orbit part was found to be fairly constant (a range of values of the order of 400 ppm) through the series of studied molecules, thus would subtract out in taking chemical shifts relative to neat liquid XeOF4, that is, except in the cases of the [XeF]' and [XeCl]+ ions, in which they are substantially larger than the spin-orbit part in XeOF4 by about 1500 and 500 ppm respectively. Given these difficulties, the authors find a linear correlation between the calculated and experimental values for seven points, that is, when [XeF]+ and [XeCl]+ ions, and XeF6 are not included: a slope of 1.02 and a regression coefficient of 0.996, a very slight improvement from the nonrelativistic results, which yield a slope of 0.96 and a regression coefficient of 0.994.43 However, this linear correlation hides a multitude of errors in the calculations which may be of the same order of magnitude as in XeOF4. Since the free Xe atom (dilute Xe gas) serves as a convenient primary reference, there is no reason to use chemical shifts relative to neat liquid XeOF4. A more comprehen-

54


sive study, with special attention to establishing the minimum energy geometry and comparing with gas phase results for a selected few molecules is required. The medium effects are themselves of interest, since it is well established that intermolecular shifts of the order of 300 ppm are observed for the Xe atom. In this present work, these medium effects are calculated for three systems: Xe-Xe, Xe-C6H6 and Xe-(C6H&, the results of which are discussed under Section 2.4. The 'I3Cd shieldings in some cadmium compounds were calculated using DFT and Hartree-Fock methods with the inadequate 3-21G and DZVP, or the modest Sadlej basis for the Cd atom.45Since it has been well known for more than a decade that at least a triple zeta plus polarization set is needed on the atom whose shielding is being calculated when GIAO or other distributed origin method is used, it is not surprising that only the Sadlej basis used in DFT gives reasonable results. The use of quantum mechanically optimized rather than experimental geometries and the inclusion of waters of hydration help to move calculated values closer to experimental chemical shifts. Since the Cd basis is insufficiently large, results of calculations get worse with increasing numbers of Cd atoms in the system. The 77Seshielding in the homologous series of eight-membered selenium sulfide ring molecules Se,Ss., were calculated using DFT/B3PW91 (Becke's three-parameter hybrid functional with Perdew/Wang 9 1 correlation) using SCF-optimized molecular g e o m e t r i e ~The . ~ ~ calculated relative isotropic shieldings permit unambiguous assignments of the spectra. Regularities in the contributions to the Se shielding permit a quantitative relationship to be derived between the chemical shifts and the relative locations of the Se and S atoms in the ring molecule. DFT/B3LYP calculations using 6-3lG(d,p) basis set on geometries optimized at the same level reveal that the 29Sinucleus at the positive charge center is highly deshielded, by - 190.6 to -404.7 ppm, for example, in sila-adamant-1-yl, bicyclohexyl, norbornyl, bicycle[2.2.2]octyl cations compared to the silane precursor.47 The I9F shielding has a well known absolute shielding scale anchored by several molecules in the gas phase. Calculations of I9F shieldings have been successfully carried out using fairly large basis sets with and without electron However, the I9Fshielding in solids of covalent or ionic fluorides presents some difficulties. The infinite periodic network calculations have not been done. The recent attempts to calculate 19Fchemical shifts in such solids have used cluster models, for example, [FA12F813for solid AlF3, [FMg3l5+, [FMg3F9I4-, and [FMg3FI1l6-for solid MgF2.50 Using standard Gaussian DFT/B3LYP and augmented correlation-consistent polarized valence triple zeta basis set for the F atom whose shielding is being calculated, smaller (double zeta) basis sets for the other F atoms, and relatively small basis sets for the metal atoms (Mg, Zn, Cd, Pb, Sn, Al, Ga, In), it was found that these 19Fshieldings are too deshielded while the values for alkaline earth metal fluorides are too shielded. No relativistic effects were included. On the other hand, the I9Fshieldings of uranium compounds calculated using DFT/ZORA are all found to be too shielded compared to experiment when medium effects are neglected.mFluorine


55

shieldings in fluorinated benzodithiadiazines have been calculated using DFT/B 3LY P.51 1 7 0 shieldings for selected S = O and C = O sites have been calculated by DFT,52,53 and the three oxygen sites in uranyl carbonate have been calculated by DFT/ZORA!' Nearly all the DFT results are systematically deshielded compared to the Hartree-Fock results for "N shieldings in three N sites, as well as all the 13Cshieldings, in l ~ m i f l a v i nThe . ~ ~ N5site is exceptional, it becomes dramatically shielded. Other I5N shielding calculations, using DFT/B3LYP7in benzot r i a ~ o l efluorinated ,~~ benzodithiadiazine~,~~ and derivatives of [salenMn(III)] complex56have been reported. Density functional calculations using B3LYP/6311G* of boron shieldings in model system [cIoso-CB~IH~~]ion are used to compare with experimental results in the solid silver salt.57 Carbon shielding calculations have been reported in various systems. In the stable isomers of fullerenes Cg0and Csg, DFT/B3LYP/6-31G method leads to predictions of 13C NMR 59 Since the geometry optimizations were carried out at a rather low level of theory, and since it is known from experience with c 6 0 that a high level of theory (MP2 using at least a triple zeta basis set) is required to get the molecular shielding and the carbon shieldings with any accuracy, it is doubtful whether the results from the present work are of much use. Furthermore, the carbon NMR spectra are very congested due to the large number of non-equivalent sites, so that any discrimination between isomers using I3CNMR would require rather fine distinctions that can not be provided by this level of calculations. CPMAS spectra of coumarins have been recorded and Hartree-Fock and DFT calculations using X-ray geometries lead to reasonable agreement between experimental data and theoretical results.60. Small basis sets 6-31G*, 6-31G(d), have been used in Hartree-Fock or DFT /B3LYP calculations of 13C shieldings in 2,4-dinitrofl~orobenzene,6~ in hydroxyl metabolites of a steroid (tib~lone):~ in pyramidalized a l k e n e ~C10-chloroterpenes,6S ,~~ in the antibiotics chloramphenicol, thiamphenicol, and their pyrrole analogs,66 . ~ ~ at this small basis and in tax01,~~ and of 'H shieldings in a double h e l i ~ e n eEven set level, in the taxol calculations 1185 basis functions were used and it was found that the CPU time for calculations using the GIAO method of gauge origin choice is greater only by a factor of 1.4 compared to using the LORG method of local origins.67On the other hand, when large basis sets are used, for example 950 basis functions for a-pinene (ten carbon atoms only), the cpu time ratio of using GIAO vs. LORG is 47.2. For this reason, Bour recommends going back to using the LORG method, which at one time was in popular use. The great difference in time has to do with the use of local origins at localized molecular orbitals, rather than having a gauge factor with each basis function. Moderately large basis sets were used for DFT/B3LYP calculations of carbon shieldings. For example, the 6-311 +G(2d,p) basis was used in ketenimine~,6~and bis- and tris(2thieny1)methinium and related cations," and 6-311 + + G** were used for thiophene, 3-methylthiophene, and ~elenophene.~~ Carbon shielding calculations using the 6-3 1l G * basis set were carried out for cations of benzoates,72and for epimeric methyl lithocholate and methyl iso-lithocholate m01ecules.~~ Results from calculations using large basis sets and various electron-corre+

56


lated methods applied to bicy~lobutane~~ are compared with the experimental values obtained from solution NMR and from solid state experiments on the molecule in an argon matrix. The results are shown in Tables 2 and 3. Although the correlation effects calculated at the MCSCF and CCSD approaches differ, they are small in all cases. A more significant contribution, neglected here, comes from the medium effects which contribute deshielding to the isolated molecule for which the calculations have been done. The calculated proton shieldings appear to be too shielded by about 0.2-0.5 ppm, about the right magnitude of the medium effects. In other words, the calculated proton values for the isolated molecule are very accurate. The best theoretical 13Cvalues, on the other hand, are still too deshielded by a few ppm when the deshielding medium effects in the argon matrix are taken into account. When the experiments are not corrected for deshielding medium contributions, as in Tables 2.2 and 2.3, the comparison between calculations and experiment appear more favorable. On the other hand, use of small basis sets at the SCF level can lead to too shielded calculated values due to inadequate description of the paramagnetic contributions, which then lead to apparently much better agreement with experiment due to the fortuitous

Table 2

Comparison of theoretical valuesfor the13C and ' H isotropic absolute shieldings in bicyclobutane molecule using various methods,I4 in ppm, and in argon against experimental vulues obtained in matrix.76

Method

Basis set

c1

c2

H5

H6

H7

SCF CCSD CASSCF CASSCF RAS-I1/2e Experiment"

Huzinaga 111 Huzinaga 111 Huzinaga I11 CC-PVTZ Huzinaga I11

198.61 197.18 202.25 203.53 202.54 204.7"

164.39 164.76 166.91 169.11 167.71 167"

30.54 30.26 30.59 30.5 1 30.50 30.232

30.59 30.32 30.56 30.53 30.43 30.090

3 1.69 3 1.44 31.61 31.56 31.55 31.101b

Experimental chemical shifts from Ref. 76 had been converted to absolute shielding using qs0 (13C,TMSref.) = 199.0 ppm by Ref. 74. However, the absolute shielding of o,,,(13C,TMSliq. sph.) = 184.1 ppm, measured against gas phase C O in the limit of zero density (Ref. 3), and the I3Cabsolute shielding scale defined by 13Cin CO molecule, not 199.0 ppm. The often quoted '199 ppm shielding for liquid TMS' is a persistent discrepancy in the literature. Highest level coupled cluster calculations by Auer and Gauss (Ref. 2) discussed at the beginning of this chapter show that with rovibrational corrections, high level calculations do reproduce the zero-density limit I3Cabsolute shieldings quoted in Ref. 3, to a standard deviation of 0.6 ppm and a mean deviation of 1.6 ppm. A change in the absolute shielding of the primary reference CO would change the mean deviation, but not the standard deviation for the entire set of 16 molecules. Therefore, absolute shieldings from experimental chemical shifts should be converted using the absolute shielding values G,,,('~C,TMSliq. sph.) = 184.1 ppm, or a,,,(13C,TMSliq. cyl. 11 B,) = 186.4 ppm, Experimental 1H chemical shifts from Ref. 75 have been converted by Ref. 74 to absolute shielding using qso(lH,TMS ref.) = 31.59 ppm (which they took from Ref. 77). 1H in TMS gas has an absolute shielding of 30.47 ppm based on also ('H, CH,, zero density limit) = 30.611 f0.024 ppm. (Ref. 78). The bulk susceptibility shift for TMS liquid at room temperature should make this more shielded by 2.3 ppm for a cylindrical TMS liquid sample parallel to B,, whereas the intermolecular interactions should make this less shielded by perhaps less than 1 ppm. An accurate conversion quantity absolute CT,,,(~H,TMS liq. cyl. 11 B,) is not available at the present time, and 31.59 ppm is not out of the question.

57


Table 3

Method

a

Comparison of calculated components of the 13C absolute shielding in ppm, against experimental tensors in bicyclobutane values obtained in Ar matrix.76 SCF Huzinaga I I I

CASSCF SCF Huzinaga 111 (Ref. 76)

Experiment”

184.90 158.06 150.22 164.39

187.26 158.24 155.23 166.91

189 161 156 168.7

179 164 158 167

159.16 216.51 220.16 198.61

163.05 220.74 222.97 202.25

167 223 225 205

176 218 220 204.7

Questionable conversion to absolute shielding is discussed in footnote (a) in Table 2

cancellation against the neglected medium corrections. The nuclear magnetic shieldings that can be compared directly, at least as differences between various nuclear sites, with experiment are those which are evaluated at nuclear positions, the nuclear sites for which the experimental NMR spectra are obtained. Molecular shielding at an arbitrary point in the molecule can be calculated, however, not necessarily at a nuclear All that is needed for the combined perturbation that gives rise to the shielding is a magnetic dipole moment, such as that of a neutron. Such calculated values are referred to by some authors as NICS (nucleus independent chemical shift). It has been shown that, when integrated over all positions, the molecular shielding mathematical surface leads to magnetizability times a factor involving only fundamental constants.80 The molecular shielding at centers of rings are of special interest and such calculated values are often provided as a measure of the qualitative labels: aromaticity or anti-aromaticity, for example in benz~thiadiazines.’~ Diamagnetic ring currents in porphycene (an isomer of porphin) have been calculated.81 The authors used the CTOCD-DZ (continuous transformation of origin of current density, the ‘diamagnetic zero’ version) method to map out the current densities and the complementary CTOCD-PZ2 (the ‘paramagnetic zero’ version) methods2 to calculate the shieldings at the nuclear positions and centers of rings.81The shieldings obtained in this work, even with the 6-31G** basis set at DFT/B3LYP-optimized geometries, are found to be in remarkably good agreement with experiment for the proton and carbon shieldings, particularly for the latter. The authors provide an analysis of the global current in the porphycene molecule, and a very nice overview of the various conventional definitions of aromaticity and anti-aromaticity parameters based on calculated molecular shieldings or NICS values at or just barely above the centers of the rings.81Three dimensional surfaces that consist of points having equal values of molecular shielding can be used as visual aids to provide some insight on the throughspace, i.e., direct (intermolecular) effects of the shielding on the nucleus of a

58


neighbor atom. This neglects any interactions between the electrons of the two molecules so can be invoked only for long range interactions. On the other hand, such surfaces have been used in an attempt to explain the difference in absolute isotropic shielding between an equatorial and an axial proton in cy~lohexane.'~ If the molecular shielding is calculated using a high level method and a large basis set, the results at the specific location of an axial proton, for example, will include all the contributions to the axial proton shielding in the isolated cyclohexane molecule. In a manner of speaking, some of these contributions are throughbond and others are through-space; they are all there and they do not have to be additive (separable into those two types of contributions). To attribute the shielding difference of axial and equatorial protons in the molecule entirely to differences in the through-space shielding contributions coming from other parts of the same molecule is misguided. A study of the basis set dependence of 13Cshielding tensors in a selected set of small moleculess4addresses the problem of basis set incompleteness by systematic studies using basis sets of the type cc-pVxZ which are a correlation consistent family of basis sets developed by Dunning, where x = double zeta, . . . up to 6-zeta, indicating the number of contractions used to represent Slater-type orbitals, and in these basis sets happens to also correspond to the highest angular momentum of the basis functions employed in the set. By doing calculations using GIAO at the Hartree-Fock level and also using the hybrid density functional method DFT-B3PW91 with increasing x, the authors observed the convergence limit for this basis set type for a set of molecules. They carried out the geometry optimization at the 6-311 + +G** or 6-31G* level in each method and did single point calculations of nuclear shielding. They show enhanced sensitivity of DFT to the basis set quality and reaffirm that DFT tends to overestimate the paramagnetic contribution. The chosen molecular systems have been treated at the highest levels of coupled cluster method; they should have compared with those calculations instead of comparing with experiments. Insights into steric effects are afforded by NMR chemical shifts. In mono- and disubstituted admantanes, 13Cchemical shifts are shown to be excellent probes for characterizing steric strains introduced by substitution with hydroxy, bromo, methoxy and acetamide groups on the adamantane ring.85 1.3 Semi-empirical Calculations.- MNDO calculations of molecular shielding at the centers of fullerene cages are reported for 153 fullerene isomers using geometries that are optimized using DFT/B3LYP 6-31G*.86The results are compared with SCF/3-21G calculations using the same geometries, and with the M. Saunders et ~ 1 He. NMR ~ results for 3Heencapsulated in some f~llerenes.'~~'' It is known that SCF calculations using small basis sets lead to results that are more shielded and fortuitously closer to experiment than using either SCF or DFT methods with larger basis sets, as illustrated recently for naked spin or 3He shielding calculations at the center of C60.89For the same geometries, the MNDO results are found to be typically smaller than the SCF values, although they reproduce the qualitative trends, and MNDO is found to be least reliable for the smaller strained fullerenes.


2

59

Physical Aspects of Nuclear Shielding

2.1 Anisotropy of the Shielding Tensor. - We begin this section by citing two excellent papers that illustrate successful applications of NMR shielding to structure determination. Petkova et al?’ have recently proposed a structural model for the 40-residue-amyloid peptide associated with Alzheimer’s disease. They based their model on data obtained from solid state NMR experiments, which included I3C and 15Nchemical shifts, torsion angles derived from finitepulse radio-frequency-driven recoupling (RFDR) and double quantum chemical shift anisotropy (DQCSA) experiments, and a number of intermolecular distance constraints drawn from measured 13C-13Cdipolar couplings. Applying similar strategies, a structure for a peptide fragment of transthyretin, also known to form amyloid fibrils, has likewise been proposed by Jaroniec et aL91These results are exceptional for they demonstrate structural studies on systems that are not amenable to either x-ray crystallography or solution NMR. Seeing how useful isotropic chemical shieldings are in the structure determination of fibrils, a full knowledge of the shielding tensor should enhance further our ability to investigate systems amenable only to solid state NMR. In this pursuit, Yao et ~ 1 continue to explore the dependence of the Ca chemical shift tensor on the torsion angles @ and q. By taking advantage of the modulation effect of the dipolar coupling on chemical shift anisotropy (CSA), the relative orientation of the shift tensor with respect to the C-H bond vector can be determined. Experimental results do indicate a greater sensitivity of the orientation of the tensor to the torsion angles and the experimentally determined orientation and magnitude of the principal components compare favorably with previous theoretical predictions. Ionic systems such as carbonates and thiocarbonates serve as excellent testing grounds for the embedded ion m e t h ~ d .In~ these ~ ? ~systems, ~ improved results for the calculated 13Cchemical shift tensors in comparison with experimental values are obtained when the calculations are performed with geometry optimization of the proton positions and the electrostatic crystal potentials. Chemical shielding tensors in nucleosides likewise continue to draw interest.” Using natural abundance samples, the 13Cand ”N shielding tensors have been recently measured for adenosine, guanosine dihydrate, 2’-deoxythymidine, and cytidine. It is interesting to note that theoretical calculations that incorporate electrostatic effects from the remainder of the infinite crystal in the form of a point charge array (the EIM model) are able to reproduce the experimental principal components without explicit quantum mechanical representation of hydrogen bonding partners of the molecule of interest. This is surprising for nucleosides that specifically use hydrogen bonding to form the helical structure in nucleic acids. Similar results are obtained for 13Cchemical shielding tensors in p-aminosalicylic acid, isoniazid, and pyrazinamide,96 where hydrogen bonding also influences the shielding tensor. The tensor orientations drawn from theoretical calculations have been compared against those of bridgehead and substituted trigonal sp2hybridized carbon atoms in polyaromatic hydrocarbons (PAHs). As in PAHs, the least shielded

.

~

~

60


component for the sp2carbon in carbonates tends to align along the C-C bond of lowest n character, while the most shielded component lies normal to the sp2 plane. These trends, however, are not followed by the thiocarbonates. Shielding tensor calculations and measurements have been utilized to determine the relative stereochemistry of ambuic acid.97In addition, to bring forth a substantially closer agreement between calculated and observed principal components for the carboxyl carbon in this compound, a dimeric model involving hydrogen bonding between pairs of neighboring carboxyl groups is proposed. Analysis of I3C shielding components can furthermore lead to a detailed characterization of polymorphs?8 In 10-deacetyl Baccatin 111, observed tensor differences between polymorphs have been exhibited to arise from conformational differences in the cyclohexenyl ring and its substituents. I7Oshielding and electric field gradient tensors have been measured for three crystalline polymorphs of P205.99 The most useful parameter here turns out to be the electric field gradient on the oxygen nucleus as it correlates nicely with the bridging (P-0-P)bond angle. Although the isotropic shielding of "N is known to be almost invariant with aromatic substituents in nitrobenzene, the principal components have been shown to be especially sensitive to pura-substitution.lm However, no correlation is found between the shielding components and Hammett reactivity parameters. An application of shielding anisotropy to structural studies of oriented samples is illustrated in a work by Grage et aZ.lO'In this work, gramicidin A with the 5 position of its Trp residues labeled with I9Fis used. Prior knowledge of the I9Fchemical shift anisotropy, specifically its orientation with respect to the indole ring, and measurement of the I9F resonance provide an estimate for the side chain alignment of the Trp residues with respect to the channel's axis. The use of I9Ffor these systems is advantageous in terms of signal sensitivity over the use of deuterated samples. In this reporting period, shielding tensors combined with other NMR data such as electric field gradient and dipolar couplings continue to be of great utility in structure elucidation. Chemical shift tensors as well as quadrupolar and dipolar tensors of the nitrogen atom in sulfamic acid provide evidence in support of a Lewis acid adduct structure.'02A reduction in the N-S bond length, supportive of a Lewis adduct structure, is required to explain the lower quadrupole coupling constant observed. Since phosphorylation of hydroxyl groups of serine, threonine and tyrosine side chains are of great importance in enzymatic and cellular recognition, 13Cand 31Pshielding tensors have been closely examined in a series of 0-phosphorylated amino acids.lo3In this recent study, it has been demonstrated that hydrogen bonding is crucial in correctly reproducing the observed tensor components. A determination of the shielding tensor of the carbonyl carbon of Ala- 14 in bacteriorhodopsin, combined with rotational echo double resonance (REDOR) experiments aimed at measuring the distance between this carbon and the amide nitrogen of Ala-18, indicates that the a-helix structure remains intact in a lipid bilayer.lWThe shielding anisotropy has been correlated with inductive and coupling effects in a series of sulfonamide derivatives."' 31P shielding tensors have been measured for pentaphenylphosphinophosphonium tetrachlorogallate.'06 Similarities in the observed shielding


61

tensor orientation between the two chemical inequivalent P sites are noted. For example, in both cases, the most shielded component lies perpendicular to the P-P bond. "N shielding anisotropy has been observed for ammonium ions trapped in a crown ether cavity containing Ni(dmit),, where dmit = 1,3-dithiol2-thi0ne-4,5-dithiolate.'~~The observed chemical shift tensor is almost axial (-356, -353, -343 ppm with respect to nitromethane), suggesting an axially deformed structure for the trapped ammonium ion. 31Pshielding tensors have been measured for pentaphenylphosphinophosphonium tetrachlorogallate.'06 Similarities in the observed shielding tensor orientation between the two chemical inequivalent P sites are noted. For example, in both cases, the most shielded component lies perpendicular to the P-P bond. "N shielding anisotropy has been observed for ammonium ions trapped in a crown ether cavity containing Ni(dmit),, where dmit = 1,3-dithi01-2-thione-4,5-dithiolate.'~~ The observed chemical shift tensor is almost axial (-356, -353, -343 ppm with respect to nitromethane), suggesting an axially deformed structure for the trapped ammonium ion. Finally, axially symmetric 13C and 15N shielding tensors lend support to a linear structure for polymeric silver cyanide chains."* There is continuing interest in investigating shielding tensors of less common nuclei during this reporting period. Penner and Li109are recommending the use of silver methanesulfonate (AgS03CH3)as a standard for lo9Agsolid state NMR experiments. The methanesulfonate compound has a much smaller lo9Agshielding tensor span compared to silver lactate and silver acetate, resulting in a smaller loss in signal due to spinning sidebands. "B NMR shielding tensors have been measured for hexamethylborazine"' and decamethylcyclopentadienyl boron complexes."' In hexamethylborazine, the "B shielding tensor is estimated to have a span of about 55 ppm. On the other hand, the decamethylborocenium cation shows a span of 73 ppm and the tri-coordinated B in bis(pentamethy1cyc1opentadienyl)methylborane has an even larger span, 146 ppm. These measurements of "B shielding tensor are among the very few observed to date. The shielding components of 'I3Cd have been measured for cadmium histidinate and two different forms of cadmium formate.'12The tensor components of cadmium are shown to depend on coordination number, hydration, as well as the identity of neighboring ligand atoms. 27Aland 51VNMR data have been utilized to characterize aluminum orthovanadate (AlV04).'13The observed chemical shielding and electric field gradient tensors are found to be similar to other known orthovanadates, suggesting that the aluminum compound is assuming a similar structure. A large anisotropy (> 1000 ppm) is observed for 51Vin V02.114Although V is octahedrally coordinated in this compound, the V-0 bonds are found to be distinguishable, encompassing a range of 1.77 to 2.06 A. At a higher temperature ( > 340 K), a phase transition is observed, which leads to a more symmetric arrangement of V atoms, as indicated by a substantial decrease in the shielding anisotropy (175 ppm). It should be noted that there is a 7000 ppm change in the isotropic chemical shift accompanying this phase transition as it involves a change in electron pairing and hence dramatic Knight shifts. Ultrahigh spinning speed (50 KHz) afforded by a Samoson probe combined with a high magnetic field (19.6 T) has enabled the measurement of electric field

62


gradient and chemical shielding tensors for 93Nbin oxyfluoroniobates.' l 5 A large span, about 1000 ppm, is observed, indicative of a highly asymmetric site for the hexacoordinated N b atoms in these compounds. Chemical shielding anisotropies allow for a clearer distinction of various types of Si04 tetrahedra in calcium silicates.116As expected, the span of the 29Sishielding tensor strongly depends on the type of condensation. Isolated Si04 tetrahedra exhibit the smallest span while those with bridging 0 atoms show a much larger anisotropy. By examining various silicates, it appears that the 29Sishielding anisotropy depends strongly on the geometry of the Si04tetrahedron (how the Si-0 bond lengths differ from each other) and not on the types of counterions present. Il9Sn and 2"7Pbshielding tensors have been reported for triphenyl-substituted group14 cobalt(1) tetracarbonyl~."~ 125 Te shielding tensors have been measured for a series of para-substituted diphenyltellurium dichlorides.118Unfortunately, no correlation of the anisotropy with geometric parameters was apparent in this series of compounds. The presence of heavy elements introduces relativistic effects and thus, requires more sophisticated methods of calculations. By comparing experimental and theoretical shielding tensor components using DFT-GIAO, it is found that the perpendicular components of both 13Cand 15N shielding in cyanogen bromide are overestimated by about 30 ppm."' This estimate is obtained from the discrepancy seen between experimental and calculated results using DFTGIAO. 2.2 Shielding Surfaces and Rovibrational Averaging. - Xu and Case report on an extensive theoretical study of 15N and 13C chemical shifts in proteins and peptides.l2OPerforming DFT calculations on six different systems, various factors that can influence heavy atom chemical shifts in peptides have been investigated. These factors include terminal effects, hydrogen-bonding, backbone ($ and q)and side chain torsion angles (xl,x2,x3),neighborhood residue effects, and the charges on the side chain. From these calculations, a total of 2300 peptide conformations and 18,280 isotropic shifts have been collected into a database. Their program (called SHIFTS) which uses this database to predict chemical shifts from protein structures can be accessed at http://www.scripps.edu/case. An additive model has been assumed for the effects of various contributors to the chemical shift, which appears to be quite useful for amide 15Nchemical shifts, which depend on a multitude of factors such as hydrogen-bonding, backbone conformation, side-chain orientation, and neighbor residue effects. Although theoretical shielding surfaces for amino acids covering almost all of Ramachandran space have been constructed by various groups since the pioneering work of de Dios, Pearson and Oldfield,I2lCzinki et al. recently examined in detail the conformational space belonging to Type I and Type I1 Calculations have been performed on models containing Gly, Ala, Val and Ser residues since these amino acids tend to be found in the second position of p-turns. Correlation plots mimicking heteronuclear spectra reveal a clustering of resonances, which can be used as a quick identifying signature for p-turns in proteins.


63

Cycloolefin polymers and copolymers provide new classes of compounds where the conformational dependence of chemical shifts can be combined with a statistical averaging using the rotational isomeric states model (RISM), to arrive at the motionally-averaged shieldings. Carbone et ~ 2 l . lhave ~ ~ used molecular mechanics to systematically search for various energy minima of model compounds representing fragments in propene-norbornene copolymers. The 13C shieldings were then calculated at the MPWlPW91/6-311+ G(2d,p) level after optimizing further the geometries of the possible conformers at the B3LYP/631G** level. The chemical shifts averaged over the RIS populations enable the assignment of the complicated I3C spectra of isotactic propene-norbornene copolymers with mid-low norbornene content. Another case illustrating analysis of multiple conformers involves flexible diastereomeric compound^.'^^ Comparisons between calculated and experimental shifts in these compounds suggest that I3Cchemical shifts can be used to assess the relative stereochemistry. In contrast, some authors fail to consider the shielding surface (at least with respect to torsion angles). For example, a recent study's approach to the 13C chemical shifts in polymers, hydrogen-terminated oligomers (n = 1 to 3 repeat units) of ethylene, propylene, isobutylene, ethylene oxide, vinyl alcohol, and acrylonitrile are used as model systems.'25Hartree-Fock calculations of isotropic I3C shielding using 6-311 + G(2d,p) basis sets are carried out on a single conformer with energy minimized structure derived from DFT/B3LYP calculations. This approach is deficient in that no conformational analysis or statistical weighting over conformer populations is used, that is, no shielding surfaces are considered, only the single-point calculation at the global minimum. The individual results for each carbon type are plotted against l/n to observe the approach to large n. The extrapolated value of the isotropic shielding for each functional group is referenced to TMS and compared with the chemical shifts measured in the corresponding polymers by Spiess et ~ 2 1 . Although '~~ calculations in tetramers have not been carried out, and statistical weighting is not used, agreement with values of I3C chemical shifts in polymers is claimed to be reasonably good.

2.3 Isotope Shifts. - 1, 8-bis(dimethylamino)-naphthalene(DMAN) contains two amino groups close enough to share a proton. For this reason, DMAN acts as a proton sponge, exhibiting unusual acid-base properties. Since the protonated form of DMAN basically involves a proton equally shared by two amino nitrogens, interesting deuterium isotope effects on 15N, 13C, and 'H chemical shifts have been 0 b ~ e r v e d .DMAN l~~ also makes it possible to lose symmetry by introducing a substituent on a position in one of the naphthalene rings, which leads to two inequivalent tautomers. Normally, deuterium secondary isotope effects arise from a relatively shorter average bond length when a proton is replaced by deuterium. In the case of a proton sponge, to explain the isotope effects, one has to examine, in addition, the effect of introducing a deuterium to the equilibrium between tautomeric structures. The challenge therefore is not only to figure out how the shielding changes with the geometry upon deuterium substitution, but also to describe accurately the potential energies of these low barrier systems. Grech et ~ 1 . have l ~ ~described the observed deuterium isotope

64


effect on the NMR chemical shifts in protonated DMAN and its various symmetric and asymmetric derivatives by considering these systems as intermolecular hydrogen bonded with a fixed distance between donor and acceptor. To arrive at the observed shifts, one needs to consider a two-stage equilibrium model. This equilibrium model is supported by the fact that the isotope effects observed are proportional to the mole fraction of the protonated species. The isotope effects are therefore seen as a shift in equilibrium towards the more dominant form upon deuteration.

2.4 Intermolecular Effects on Nuclear Shielding.- The NMR spectrum can be dramatically perturbed by a paramagnetic center. Line widths as well as the positions of resonance signals can be drastically altered. The effects of an unpaired electron on the NMR spectrum are very much relevant to the study of transition metal complexes. In these systems, the spectrum of the ligand bound to the metal is very much dependent on the unpaired electron(s) on the metal. The observed chemical shifts in paramagnetic systems can be attributed to two terms: bobserved

=

bdiamagnetic

+

bhyperfine

where the diamagnetic shift corresponds to the values for the free ligand, whereas the hyperfine shift is primarily due to the presence of a paramagnetic center. Thus, if there are dramatic changes observed in the chemical shift upon binding to a metal, these can be attributed to the hyperfine shift. The hyperfine shift can be further decomposed into two contributions: ahyperfine

=

bcontact

+

adipolar

*

These two terms differ from each other in their mechanism. Contact shifts are caused by spin delocalization of the unpaired electrons through chemical bonds. The magnitude of these contact shifts is proportional to the Fermi contact spin density, which is a measure of the net imbalance between a and p spins at the site of the nucleus of interest. The spin density can either be positive or negative; therefore, it is possible to see contact shifts of opposite signs among various sites within one ligand. Dipolar shifts, on the other hand, result from a through-space interaction and are proportional to l/r3 , where Y is the distance between the paramagnetic center and the nucleus of interest. Dipolar shifts, being dependent only on positions and not chemical bonds, are expected to be independent of the identity of the nucleus. The Oldfield group has recently shown that it is possible to predict 'H, 13C,15N,I9Fchemical shifts in paramagnetic metalloprotein and metalloporphyrin systems.'**Using spin-unrestricted methods, the Fermi contact spin density on the site of the nucleus of interest can be determined. A graph of observed shifts versus the spin density yields an empirical scaling factor of 1.9 x lo7(S + l/T), where S is the total electron spin and T is the absolute temperature. The expected Curie law behavior is the obvious reason for choosing the 1/T term to analyse the data. This empirical value compares favorably with the theoretically expected factor, 2.4 x lo7 (S + l/T), indicating that the observed hyperfine shifts in metalloproteins and metalloporphyrin systems are indeed dominated by the Fermi contact term.


65

Liquid water continues to be a challenging example for the prediction of intermolecular effects on shielding. The highly dynamic nature of liquid water requires motional averaging over a distribution of hydrogen-bond geometries. To facilitate a detailed analysis of the proton shielding in liquid water, Modig and Halle'29 have measured the shielding anisotropy in liquid water for the temperature range 273-353 K. With deuterium isotope dilution, the dipolar mechanism for relaxation is effectively suppressed, making chemical shift anisotropy contribution to relaxation more significant. With a proton fraction of 0.0098 in a water sample, the contribution of the anisotropy to relaxation in a magnetic field of 18.8 T can become as large as 25%. Measurements of longitudinal relaxation times made at five different magnetic field strengths can therefore provide estimates for the chemical shift anisotropy. Results indicate that in the temperature range studied, the proton shielding anisotropy in water changes from 29 to 25 ppm. This range is about four times as large as the range observed for the isotropic shift. Ab initio calculations on a water dimer, a highly inadequate model, suggest that the greater sensitivity of the anisotropy lies in the opposite dependence of the principal components on hydrogen-bonding. For anisotropy, these effects enhance each other. On the other hand, for the isotropic value, they partially cancel. The availability of proton shielding anisotropies in liquid water at various temperatures combined with shielding surfaces as functions of hydrogen bond geometry afford an excellent route for the determination of moments and distributions of hydrogen bond length and angle in water as a function of temperat~re.'~' The construction of the shielding surface for this work involves nine liquid water configurations obtained from an ab initio molecular dynamics simulation of a cubic supercell containing 32 water molecules (a total of 576 proton shielding tensors). With this scheme, the average hydrogen-bond length (ROH)is shown to increase from 1.87 A at 273 K to 1.98 A at 353 K. The hydrogen bond angle becomes less linear, reaching 22" at 353 K, compared to 12" at 273 IS.It is comforting that the hydrogen bond geometries described for water in Ref. 130 are based on a cluster of 32 molecules, because Chesnut has shown that the use of small water clusters (dimer to hexamer) is not valid for modeling NMR shielding surfaces in liquid ~ a t e r . ' ~ Although ' agreement with experimental values in liquid water is reached for proton shielding associated with the optimum geometry of the hexamer, the oxygen shielding is still overestimated by about 25 ppm (using a cyclic hexamer). Previous work using a cluster of nine molecules leads to an oxygen shielding that is only 6 ppm away from experiment.'32Of course, such comparisons of the shielding in any single configuration of a large n-mer against experimental values in the liquid are spurious, since averaging over many configurations is really required in order to have a proper statistical average. The effects of hydrogen-bonding on chemical shifts are very important especially for biologically relevant molecules. For example, it has been recently demonstrated that formation of protein-protein complexes can be followed by observing the changes in the amide proton and 15N chemical shifts.'33 The perturbations are tabulated as chemical shift maps and are assumed to be primarily due to intermolecular interactions. Solving such complex structures by

66


X-ray crystallography or conventional NMR NOE methods can be overwhelming, but if the structures of the uncomplexed proteins are known and that conformational changes upon forming the complex are at a minimum, the structure of the complex can be determined solely from intermolecular distances drawn from 'H/ 15Nchemical shift maps, which summarize differences in 'H and "N NMR chemical shifts between complexed and uncomplexed forms. Clore and Schwieters applied this method successfully for three complexes, EIN-HPr, IIAG" -HPr, and IIAMt'-HPR.'33Proton shieldings have been used to derive the structure of benzoxazine 01igomers.'~~ Benzoxazine has tertiary amine and hydroxyl groups, which can form both intra- and intermolecular hydrogen bonds. With magic-angle spinning at 30 KHz, it is possible to resolve the proton signals and identify the resonances that are participating in hydrogen-bonding in the solid. Using DFT calculations, model structures for dimer, trimer and tetramers have been obtained. The structures show only intramolecular hydrogen-bonds in the trimer and tetramer and the chemical shifts obtained from these optimized structures agree with experiment. Similar to naphthalene amine described in the previous section, l-hydroxy2,4,5-triphenyl-1H-imidazole 3-oxide is also capable of a hydrogen-bond with a single-well potential. Sanchez-Migallon et al. have demonstrated by solid state MAS experiments and DFT calculations that the 0-He-0 intermolecular bonds in the solid have the proton in the middle of the two oxygen atoms resulting in a near chemical shift equivalence of the nitrogen sites.'35 Effects of hydrogenbonding and protonation on the 'H, 13C,and 15Nshieldings of imidazoles and pyrazoles have been examined in the solid GIAO calculations are shown to be capable of reproducing the experimental data for these systems. Lastly, the orientation of the 31Pshielding tensor in phosphorylated amino acids appears to be sensitive to hydrogen-bonding based on DFT-GIAO c a l c ~ l a t i o n s . ' ~ ~ Carbohydrates, similar to other biologically relevant molecules, can become so large that shielding calculations that incorporate electron correlation may no longer be feasible. The use of model systems which are fragments of the macromolecule but which include the geometric parameters (torsion angles, too) and hydrogen bonding partners and the influence of the remainder of the macromolecule via a field of point charges was pioneered by de Dios, Pearson and Oldfield.'*l Instead of a field of point charges, the remainder of the molecule may be represented by a lower level quantum mechanical treatment or molecular mechanics, as in Morokuma's ONIOM a p p r ~ a c h .Rickard '~~ et al. have examined the applicability of ONIOM to shielding calculations in predicting chemical shifts in P-D-gluc~pyranose.'~~ In this particular work, two ONIOM layers were used, in which a small model system containing the nuclei of interest is treated at the MP2-GIAO level of theory, and the rest of the molecule-using HF-GIAO. The fragment used is about half of the molecule and this has been found to be sufficient to describe the shieldings of the 13Cand 'H nuclei. For 1 7 0 , the model systems need to be slightly bigger to account for possible 1,3-diaxialinteractions. Electrostatic or electric polarization effects from intermolecular interactions have been included in shielding calculations by various reaction field type methods, some of which are illustrated by recent reports on polarizable continu-


67

um models (PCM) or embedded ion method (IEM). These methods do not include the electrons of the neighbor atoms in the shielding calculations, but attempt to include the entire (usually infinite) crystal or solvent system by representing the electrostatic part of the medium effects on shielding. Mennucci and Cammi have extended their integral equation formalism (IEF) model for medium effects on shielding to NMR shielding tensors for solutes in liquid ~rysta1s.l~~ The extension primarily involves the introduction of a permittivity tensor, representing the anisotropic liquid crystal solvent. The authors are quick to point out that their model is applicable only to systems where the medium effects to shielding are largely induced by electrostatic interactions. Polarizable continuum models such as IEF have seen increased use in shielding calculations. In a recent study of the nitrogen shieldings of N,N-dimethyla~etarnidine,'~'it has been pointed out that one needs to be careful when comparing the calculated values against experiment. Cyclohexane has been used as reference in most of these studies and it is assumed not to have any medium effects on the shielding. Non-polar solvents, such as cyclohexane, do affect shielding significantly, based on measured gas-to-solution shifts. It has therefore been suggested that a more appropriate comparison for PCM calculations would be results obtained using clusters or supermolecules.'40 With this comparison, it has been shown that IEF-PCM results are in agreement with those obtained using explicit solvent molecules. It has been demonstrated that a better description of the 'H shieldings in thiosemicarbazone ligands of Pd(I1) chloro complexes can be obtained via PCM-GIA0.14' In this particular case, there are several nuclear sites, thus, how well their relative positions from each other are reproduced provides an appropriate test for the PCM method. As described in Section 2.1 of this report, the alternative embedded ion method (EIM) is being successfully applied in repro98 ducing principal components of the shielding in the polycrystalline Another approach to intermolecular shifts makes use of cluster calculations, that is, the shielding is calculated in suitable clusters which model the structure of the solute surrounded by solvent molecules, as in the use of water n-mer clusters to model liquid water, as described above.'31 A recent application is to use geometry-optimized solute-solvent dimers as clusters to model the shifts observed in aceto-, propio, isovalero-, and pivalo- nitriles in aprotic solvents in the limit of infinite d i 1 ~ t i o n .Calculations l~~ were done only on dimers of acetonitrile with acetone and benzene. Since these 1:l complexes are too simplistic models for acetonitrile in solution, and since the calculations were not carried out in solutes which are longer and more branched, the observed 13Cand I5Nchemical shifts that suggest a trend of self association becoming weaker with heavier and more highly branched nitriles have not been supported by the calculations. A more complete approach to cluster calculations in the liquid phase is exemplified by the calculations carried out by Farrar and co-workers, in which partition functions are used to determine the populations of various cluster types and sizes at a specified temperature and the ab initio calculations are carried out in such clusters at their respective minimum energy g e ~ m e t r i e s . ' ~ ~ - ' ~ ~ '29Xecontinues to be used as a probe for porous materials, surfaces and proteins. Using enriched '29Xegas and optical pumping, 129XeNMR chemical

68


shifts have been measured for Xe atoms adsorbed on a carbon monoxide and an ethylidine covered Ir(ll1) surface.'46This is the first NMR experiment on a physisorbed species on a single crystal. The experiments combined the difficulties of doing NMR at very low concentrations with the difficulties of ultra-high vacuum techniques. Distinct resonances are observed at 139 ppm at 66.2 K and 164 ppm at 64.4 K for Xe on CO/Ir(lll). The resonances are consistent with different Xe coverage at these two temperatures, since the Xe chemical shift is an average that includes Xe interactions with the 0 atoms of the C O chemisorbed to the surface and Xe-Xe interactions, the latter becoming more pronounced at higher coverage at lower temperatures. The distinct resonance observed at 203 ppm (all values referenced to the gas signal) for Xe on CH3C/Ir(111)surface at 65 K clearly indicates that 129Xechemical shift can distinguish between the OC-Ir(s) and the CH3C-Ir(s) environments. When sufficient Xe has deposited on the crystal surface, the typical bulk Xe signal is observed, consistent with observations in pure solid xenon at the same temperature ( 61 K, for example). The signal from Xe atoms at the surface of bulk Xe is observed separately (an advantage provided by the use of hyperpolarized Xe) at 209 ppm whereas the Xe within the bulk Xe is at 321 ppm. These observations are consistent with the average number of Xe neighbors being fewer for a Xe atom at the surface of the bulk xenon than for a Xe atom within the bulk. have provided an additional system that illustrates the theoretiUeda et cal predictions made on Xe NMR line shapes for Xe confined in n a n ~ c h a n n e l s . ' ~ ~ The nanochannels are formed by dehydrated tris ethylenediammine cobalt(II1) chloride. Since the protons in this system can only be found along the walls of the channel, cross-polarization (CP) experiments from protons will selectively enhance the shielding component normal to the wall. Thus, by CP, it has been possible to confirm the orientation of the observed '29Xeshielding tensor. The use of '29XeNMR in characterizing binding sites in proteins is again illustrated in the case of maltose binding protein (MBP) from Esherichia c ~ l i . The ' ~ ~bindinginduced '29Xechemical shift appears to be very sensitive to the cavity structure so that it can discriminate between mutant and native proteins. 129Xelikewise serves as a good testing ground for theoretical calculations of intermolecular shifts for obvious reasons. It is therefore important to analyse how significant electron correlation effects are to its shielding. With DFT methods, it is now straightforward to introduce these effects to rare gas atom shielding calculations. B3LYP calculations show that the contribution of electron correlation to the '29Xeintermolecular shielding in Xe-Xe is 15%, in Xe-Kr is lo%, and in Xe-Ar is 15?40.'~'The chemical shift of '29Xein Xe@C60has been calculated at the B3LYP The calculated shielding with respect to a free Xe atom is - 181.58 ppm, which compares very well with the experimental value of -182.35 ppm. The good agreement here is attributed to the large basis set (24s19p15d9f) employed for Xe in the calculation. The carbon atoms in C6' are provided only a 6-311G* basis. Previous calculations using a smaller basis set on Xe produced only - 71.7 ppm.1513He shielding calculations for provide -0.7195 ppm, to be compared with experiment, -6.3 ppm. The discrepancy here lies in the description of the C6' molecule (basis set and level of theory) as


69

most of the intermolecular shielding of 3Hearises from the electronic structure of c 6 0 . A relativistic DFT-ZORA study of Xe shieldings in van der Waals complexes Xe-Xe, Xe-CH4, Xe-C6H6 and Xe-(C6H6)2has been reported by Bagno and Saielli?3The Xe-O[Si(OH)3]2 system was used to mimic the shielding of a Xe atom in a zeolite pore. For the van der Waals complexes, results differ slightly from previous works, but it is difficult to draw conclusions at this point with regard to the relative importance of electron correlation and relativistic effects since the basis set employed is not sufficiently large. Without a large number of properly chosen polarization functions on the Xe atom, the shielding response in all these van der Waals complexes will be too small at any level. See again the Xe@Cmexample mentioned above. Ring currents and aromaticity have been described in s-inda~ene,”~ in 1,3dehydro-silaadamantane di~ations,’’~ and in porphycene81during this reporting period. See Section 1.2 for a report on the analyses of global currents and their implications for shielding and magnetizability. Thermal and solvent effects on the chemical shifts of diamagnetic transition metal systems have been studied using a combination of B3LYP shielding calculations and molecular dynamics simulations. Car-Parrinello simulations were employed in reproducing the gas-to-solution shifts of ”Mn and 1 7 0 in an aqueous solution of ~ermanganate.’~~ In this work, it appears that a shortening of the Mn-0 bond length upon dissolution contributes to the change in shielding. A similar treatment has been applied to 57Fechemical shifts in hexacyanoferrate[II] and [Fe(CN)5N0]2.15’ Here, the dramatic sensitivity of the 57Feshielding on the Fe-C bond length is very evident. A recent review of nitrogen NMR spectroscopy of metal nitrosyls and related compounds makes note of the sensitivity of the nitrogen chemical shift to the bent orientation of the nitrosyl gro~p.”~ The metal surface effect on the shielding of a chemisorbed molecule is not easily observed due to lack of sensitivity and line broadening problems A favorable exception is when the surface areas are large and some motional narrowing is also present, such as in the case of chemisorbed species on nanoparticles in the solid state or suspended in a solvent. The effects of a gold nanoparticle on I3CNMR chemicals shifts in chemisorbed octanethiol have recently been r e ~ 0 r t e d .It l ~is~interesting to note that the chemical shift of the proximal 13Cis a sensitive function of the size of the Au nanoparticles. Metal surface effects on shielding of a chemisorbed species have been studied theoretically by Mauri et al.ls8They showed that electronic surface states have a strong effect on chemical shifts of atoms near the surface. Clearly, further chemical shift measurements on molecules attached to metal surfaces by chemisorption or physisorption would be of great interest. For this reason, the report on the ‘29XeNMR experiments on a single crystal Pt( 111)surface by Jansch et al. are eagerly anticipated.

2.5 Absolute Shielding Scales. - A nice retrospective review of how spin-rotation constants relate to shielding constants is provided by Bryce and Wa~y1ishen.l’~Wasylishen and Bryce have provided a long-awaited revised experimental absolute magnetic shielding scale for 170.160This revised scale is

70


based on a more precise measured value for the spin-rotation constant of 12C170.16'The new value reported for the absolute shielding of 1 7 0 in CO at the equilibrium geometry is - 56.79 & 0.59 ppm, and the thermal average at 300 K (rovibrationally averaged) is -62.74 0.59 ppm. This revision brings OF2 at -493.5 ppm, OCS at 87.5 ppm, N 2 0 at 180.1 ppm, C 0 2at 223.0, liquid water at 287.5 ppm, and water vapor at 323.6 pprn.l6' Interesting to note, the new thermal average isotropic absolute shielding value for I7Oin CO lies very close to the RASSCF predicted value of -62.3 ppm.162On the other hand, the CCSD(T) value at the equilibrium geometry is -52.9 ppm, which when corrected with a zero-point vibrational correction of - 5.73 and a further theoretical rovibrational thermal average from 0 K to 300 K by an amount -0.35 ppm leads to -59.3 ~ p m . The ' ~ ~remaining discrepancy between the -62.74 0.59 ppm (experimental) and -59.3 ppm (theoretical) values is now much smaller than that from a previous less precise spin-rotation constant for 170.The new experimental spin rotation constant for the v = 0 state is - 31.609 f 0.041 kHz,161while the vibrationally-averaged theoretical value calculated by Gauss and Sundholm for the same state is -31.32 ~ H z .The ' ~ ~difference between this high level calculations of the spin rotation constant and experiment is of course consistent with the difference between this new 'experimental' absolute shielding16' and the theoretical shielding value obtained by Gauss and S ~ n d h o l m . 'Theory ~~ has come very close to experiments in this challenging case. With the increased confidence in shielding calculations, Vaara and Pyykko have suggested the use of relativistic, large basis set shielding calculations for the establishment of nuclear magnetic shielding scales for rare gas atoms.44Their work provides the following absolute shieldings for the free atoms: He (59.93 ppm), Ne (561.3ppm), Ar (1274.3 ppm), Kr (3577.3 ppm), Xe (6938 ppm), and Rn (19630 ppm). These are improved values over the early calculations of Kolb et al. which were 21 ppm and 102 ppm larger for Kr and Xe atoms re~pectively.'~~

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3 Applications of Nuclear Shielding BY S. KUROKI, N. ASAKAWAAND H. YASUNAGA

1

Introduction

The aim of this report is to cover and introduce studies on the application of nuclear shielding (NMR chemical shifts) from 1 June 2002 to 31 May 2003. The shieldings of particular nuclear species are described in the following section according to their position in the periodic table. Although there is a great number of articles on NMR spectroscopy during the period of this review, the coverage of this chapter is restricted to widely available and common journals, which are published in English as a general rule, due to space limitation.

2

Shielding of Particular Nuclear Species

During the period covered by the report, the NMR spectra for most elements contained in molecules have been obtained in the course of physical, chemical, or biological investigation. The structure determination and related studies of natural products or macromolecules are excluded, and the review articles are given as necessity requires.

2.1 Group 1 ('H, 'H, 637Li,23Na,39K, s7Rb, 133Cs).- 2.1.1 Hydrogen ('H ) ( I =1/2). The anisotropic effects of the C-C single bond in ethane and various other X-C single bonds (X = OH, SH, NH2)were calculated as nuclear independent chemical shielding (NICSs) using gauge-including A 0 (GIAO) method.' GIAO method for calculating 'H and 13Cnuclear magnetic shielding tensors at both Hartree-Fock (HF) and DFT was applied to atrazine (2-chloro-4ethylamino-6-isopropyl-amine-s-triazine) and atrazine dimers.* The proton shielding constant and the shielding anisotropy of C6H6 - H-CX3,X = H, F, C1 and Br, complexes were calculated as a function of the intermolecular distance at the density functional theory (DFT) with the PW91 functional and HF level^.^ The configuration at C-3 of the 3a- and 3b-hydroxy metabolites of tibolone was studied by one- and two-dimensional 'H and 13CNMR spectroscopy combined with molecular m ~ d e l i n g The . ~ shielding tensors of the two molecules were computed by H F and DFT GIAO methods. Two Kemp's acid diamides were synthesized and their structure was studied by 'H NMR and computer simula*

Nuclear Magnetic Resonance, Volume 33 0 The Royal Society of Chemistry, 2004

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3: Applications of Nuclear Shielding

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t i ~ nThe . ~ change in geometry and charge distribution in thiophene, 3-methylthiophene and selenophene were studied by extended MO calculations using DFT, 'H and 13CNMR, and Raman.6 The structure of layered aluminosilicates was studied by 'H magic angle spinning (MAS) NMR.7 The chemical shift from water contribution gives valuable information on the acidic strength of the interlayer cation, correlations between the shielding of the protons of water, and the pKa. The 'H and 1 7 0 NMR isotropic shieldings and shielding anisotropies in small water clusters including between two and five water molecules were studied by H F and 2nd-order Moller-Plesset (MP2) approaches using GIAOs.* 2.1.2 Deuterium (2H ) (I = 1). The phase behaviour of the 1-[2H35]-~tearoyl-racglycerol ([2H35]-MSG)/dicetylphosphate(DCP) mixture and its interaction with P-lactoglobulin and lysozyme were studied by 2H and 31PNMR.9 These results suggest that interaction is facilitated by electrostatic interactions between the negative charged DCP and positive charged proteins. The 2H NMR Pakepowder patterns of perdeuterated isobutane-dloadsorbed on zeolite were studied and interpreted in terms of the motional behaviour of isobutane molecules." The 2HNMR spectroscopy was used to probe the dynamics of deuterated octadecylphosphonate (ODPA-d2) monolayers on nonporous Z r 0 2 powder at 200-340 K." The deuterium site occupancy and dynamics in ZrNiDl.oand ZrNiD3.0was studied by static and MAS 2H NMR spectroscopies.12The 2H NMR line splitting arising from deformed cis- l,Lt-poly(butadiene)network was investigated and the results were discussed according to the theoretical f r a m e ~ o r k . A ' ~ calculation methodology for 2H NMR spectra of grafted polyethylene melts was presented by analysing the atomistic configurations accumulated in the course of the end-bridging Monte Carlo (EBMC) ~irnulations.'~ 2.1.3 Lithium (677Li)( I = 1, 3/2). The local environments in a lithium-excess lithium manganese spinel (Li1.05Mn1.9504) were investigated by 6Li MAS NMR and the shift of its resonances due to manganese oxidation states was observed.'' On the other hand, the lithium local environments in a series of layered compounds such as Cr-doped LiCo02, Co-doped LiCr02, Cr-doped LiMn02, and the Li-rich material Li[Lio.2Cro.4Mno4]02 were studied by 6Li MAS NMR spectroscopy.'6 The fast ionic conducting Li2MgC14, Li2-,Cu,MgC&, Li2-xNaxMgC14, and Li2ZnC&were measured by 7Li MAS NMR including 1Dexchange spectroscopy (EXSY) and inversion recovery experiment^.'^ It was demonstrated that the 6Li MAS NMR spinning sideband is sensitive to the arrangements of the 1st cation coordination sphere of Mn ions around Li cations in lithium manganates.18 Chemically delithiated Lil -,C0O2 compounds were studied by 7Li MAS NMR and x-ray diffraction measurements. A large NMR chemical shift was observed for Li in the mixed-valence environment attributed to a Knight shift.19The 7LiNMR isotropic shift for LiMP04 (M = Fe, Mn, Co, Ni) in olivines was assigned to hyperfine coupling between the 7Li nucleus and the transition metal unpaired electrons.20The benzyllithium systems stabilized by different ligands (THF, PMDTA, TMEDA, 12-crown-4)were investigated by 7Li and 13Csolid-state MAS NMR with respect to quadrupole parameters and

78


chemical shifts.21A plastic Li/C battery and the insertion of Li into hard C fibers were studied by a continuous in situ static 7Li NMR.22 The amblygonite (LiA1PO4F)- montebrasite (LiA1P040H)solid solution was characterized by solid-state 6Li MAS, cross polarization (CP) MAS, rotational-echo doubleresonance (REDOR) NMR techniques, and powder and single-crystal neutron diffraction to study the effect of F, U, or OH s u b s t i t ~ t i o nThe . ~ ~6Liand 7LiMAS NMR spectra of 2.5-MeV electron-irradiated LiF crystals were measured in a field of 9.4 T observing the resonance line and Knight shift.24 2.1.4 Sodium (23Na)( I = 3/2). The solid borax (Na2B407-10H20) compound was employed as the example of 23NaNMR to discuss the magnetization transfer in the multiple-quantum MAS (MQMAS) NMR.25The novel technique was also applied in the 23NaNMR study of sodium metasilicate pentahydrate, sodium tungstate dihydrate, and sodium molybdate dihydrate. The 2D heteronuclear 23Na-1HM Q MAS technique with the excitation of 23Na3-quantum coherence was applied to Na2HP04 to check the advantage of it.26The hydration in Na20-3Si02glass was studied by 23NaMAS and 2D MQMAS NMR spectroscopy and it was found that 1D MAS spectra for the hydrated glasses with more than 8.4 wt% water consist of two signal^.^' A solid-state 23NaNMR study for the sodium cations in four hydrated disodium salts of nucleoside 5'-phosphates and ATP was made using one- and two-dimensional solid-state NMR techniques.28

2.1.5 Potassium (39K) ( I = 3/2). The binary metal clathrates of K/Si, Rb/Si, and Cs/Sn were synthesized and studied by 39Kand 29Sisolid state NMR and powder x-ray d i f f r a ~ t i o n .The ~ ~ information obtained from Knight shifts confirmed that the Cs/Sn clathrate is not conducting. Thermal behaviour of potassium sialate geopolymers, potassium polysialate and potassium sialate disiloxo, was studied by 39K,27Al, and 29SiMAS NMR.30 2.1.6 Rubidium (87Rb) ( I = 3/2). The critical statics and dynamics of the prototype incommensurate crystal Rb2ZnBr4was investigated by quadrupolar-perturbed NMR of 87Rb.31 The 87RbHahn echo NMR and 35ClHahn echo nuclear quadrupole resonance (NQR) measurements were made in the incommensurate (I) phase of Rb2ZnC14.32 The confirmation of the presence of modulation wave motion was obtained by 87Rb2D exchange-differenceNMR. The dependence of the rotor assisted population transfer (RAPT) enhancement on offset frequency for nuclei experiencing different quadrupolar couplings was exploited and applied to 87Rbspectra of Rb2S04.33

2.1.7 Cesium (133Cs) ( I = 7/2). Dehydrated Cs-Y and Cs-X zeolites were studied by variable temperature 133CsMAS NMR spectroscopy from 123 to 423 K and large 133Csshifts were observed at low temperatures due to 02-Csinteractions for 133Csresonances from cations in the super cage^.^^ The magnetic susceptibility and paramagnetic shift for central transition of 133CsNMR in CsCuC13 were measured in the temperature range of 4.2-300 K.35A series of Cs-phosphate


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glasses, xCs20(1-x)P205(0 < x < 0.60) were prepared and characterized by 133Cs and 31PMAS NMR.36 The '33CsNMR in a Cs2CoC14was measured for two mutually perpendicular crystal planes obtaining information on their quadrupole coupling constants and the asymmetry parameter.37The c S c 6 0 was studied by 133CsNMR and the quadrupole effects on 133Csconfirmed an inhomogeneous distribution of the charge between c 6 0 molecules.38 2.2 Group 2 ('Be, 25Mg,43Ca).- 2.2.1 Beryllium (9Be) ( I = - 3/2). The electron densities at the Be nucleus and 9Be NMR shieldings for both monomeric and oligomeric species of Be were calculated to assist in determining Be ~ p e c i a t i o n . ~ ~ Two bifunctional ligands, phenyl(carboxymethy1)phosphinate and P, Pfdiphenylmethylenediphosphinate, were tested as chelating agents of beryllium(I1) and the formation of chelating structure was studied by 9Be NMR spectroscopy!' The layered intermetallic compound ZrBe2(H/D),was studied by 2H and 9Be NMR using the 9Be- 2H spin-echo double resonance (SEDOR) technique!' The generation of pure quadrupolar stimulated-echo spectra was demonstrated for 9Bein a single crystal of triglycine fluoberyllate!2 2.2.2 Magnesium (25Mg) ( I = - 5/2). The 25MgNMR spectra and nuclear-spinlattice relaxation time ( T l )for MgB2 superconductor were measured and the quadrupole coupling frequency was obtained from the first-order and secondorder quadrupole perturbed NMR!3 The NMR line shift and nuclear spinlattice relaxation rate T1-' of 25Mgand "B were measured in superconducting MgB2.MThe isotropic Knight shift, TI, and electric field gradient for MgB2 were determined by 25MgNMR and the comparison between experimental data and theoretical predictions was made.45 2.2.3 Calcium ("Ca ) ( I = -7/2). The application of 43CaNMR in the study of calcium-binding proteins was reviewed and its results were compared with those obtained by 'H, 13C,and "N NMR!6 2.3 Group 3 (45Sc, 89Y,139La,'"Yb). - 2.3.1 Scandium (45Sc) ( I =7/2). Polycrystalline samples of ScRh3,ScRh3B,and their solid solution, ScRh3B, (O<x 27Al2D CPMAS (HETCOR) and 'H/"F 27AlREDOR solid-state NMR and by IR spectr~scopy.'~' 1 7 0 and 27Al NMR results for 170-enrichedsynthetic and natural layer silicates with significantlyimproved resolution of 0 sites including basal and apical oxygens using I7O 3QMAS NMR spectroscopy, were r e ~ 0 r t e d . l29Si, ~ ~ 27AlMAS NMR was used to characterize Laponite RD and synthetic saponites of variable interlayer charge.153Dicyclopentadienylaluminum alkoxides of general formula [Cp2Al-mOR12 (R = Me, Et , n-Bu, i-Bu, CH2t-Bu, s-Bu, CH2Ph, C6H4-4-t-Bu)were prepared, and were characterized by multinuclear NMR ('H, I3C, 27Al).'54 The (dimethy1amino)methyl compounds (Me2AlCHZNMe2)2, (Me2GaCH2NMe2)2, (Me21nCH2NMe2)2, [(Me2A1CH2NMez)( MeC1AlCH2NMe2)], CClAl(CH2NMe2)2]2,and [A1(CH2NMe2)3]2 were prepared by reacting LiCH2NMe2 with the corresponding element or organoelement chlorides. Characterization was undertaken by elemental analyses, NMR spectroscopy ('H, 13C,27Al),and mass ~pectrornetry.'~~ The substitution of A1 with Sc in the mixed Y A1 Sc garnets, (x = 0,0.05,0.5, 1.5), was studied for the first time using 27AlMAS, Y3S~xA15-x012 MQMAS NMR, and 45ScMAS NMR.49High-resolution solid state 29Si,27Al MAS and 27Al3Q-MAS NMR spectroscopic techniques were used to characterize highly crystallized ultrastable Y (USY) zeolites having different framework Si/Al ratios, obtained by multistep-temperature-programmed (MSTP) steaming at different temperature^.'^^ The Bhsted acidity of H-mordenite (H-MOR) and H-b with similar Si/Al ratios was characterized by conventional multinuclear solid-state NMR, and 'H/27AlTRAPDOR NMR have showed the presence of at


89

least two different types of Bksted acid sites in H-b.'57 27Al3QMAS 2-dimensional NMR was used to study the molecular-level organization of a clay pillared with the A113(heidi)63+ polycation, whose unusual structure is stabilized by hydroxyethyliminodiacetate (heidi) ligands.15* 2.13.3 Gallium ( 71Ga)(I= 3/2). Amorphous Na-Ga silicate materials were isolated after short periods of heating a gel of Ga203: Si02:4NaOH : 80H20at 100 C and 71GaMAS NMR spectra were recorded from the amorphous samples to provide information on the local Ga environment, and these show the presence of tetrahedral Ga, as in the crystalline zeolite, even in the samples prepared after the shortest period of heating.159Several complexes such as [MS2CNR3] (M = Ga or In; R = CH2CH20 CH2CH2,CH2CH2N(Me) CH2CH2) were prepared and were characterized by 71GaNMR chemical shifts and half line widths, which were influenced by the coordination number of Ga and the substituents on the ligand moiety.'60 2.1 3.4 Thallium (2031205Tl)(I = 1/2,1/2). 205Tllongitudinal relaxation rate measure-

ments were performed on several thallium(II1)complexes Tl(OH)n(H20)6-,('")+ (n = 1,2),T1(Cl)n(H20)m-n(3-n)+, Tl(Br),(H20),-,'3-n)+(m = 6 for n = 1-2, m = 5 for n = 3, m = 4 for n = 4), Tl(CN)n(H20)~.,'3-n'+ (m = 6 for n = 1-2, m = 4 for n = 3-4) in aqueous solution, at different magnetic fields and temperatures.'61A novel complex of monomeric Tl(II1) with the N donor ligand phenanthroline (phen) was prepared and characterized by multinuclear NMR ('H, I3C, 205Tl).The 3 complexes exist in equillibrium in DMSO and MeCN solution, which was proved by the 205TlNMR spectra.'62 2.14 Group 14 (13C,"Si, 73Ge,"73119Sn, 307Pb). - 2.14.1 Carbon (13C)(I = 1/2). Accurate, practical prediction of 13CNMR chemical shifts was achieved with a new 'H and 13CNMR system, CAST/CNMR, taking account of stere~chernistry.'~~ data for 13 nitrohistidine derivatives were reported, providing a diagnostic method for the elucidation of the N1- and the N3-substituted regiois~mers.'~~ Interpretation of I3C chemical shifts is essential for structure elucidation of organic molecules by NMR. An improved neural network approach was presented and its performance to that of commonly used approaches was compared.16' The boronium-carbonium ion continuum was extended to include hypercoordinated onium-carbonium dications and the isoelectronic oniumboronium cation analogs. Structures and I3C and "B NMR chemical shifts of the onium-carbonium dications and the corresponding isoelectronic and isostructural onium-boronium cations were calculated with the ab initio/GIAOMP2 method.'66 Artificial neural networks are capable of predicting the 13C chemical shifts of organic molecules nearly as fast as incremental methods while maintaining the accuracy of database methods. A recently developed neural network was applied to the screening of large sets of molecules obtained by structure generators in the process of automated structure e1~cidation.l~~ The authors showed that the combination of GIAO (B3LYP/6-31+ G*)and classical molecular mechanics (MM + ) calculations is an efficient procedure to reproduce

90


experimental results of solid-state 13C NMR chemical shift of alkoxy species adsorbed on different zeolites structures.168The 2D NMR-guided computer program COCON can be extremely valuable for the constitutional analysis of unknown compounds, if its results are evaluated by neural network-assisted 13C NMR chemical shift and substructure analyses. As instructive examples, data sets of four differently complex marine natural products were thoroughly investigated.'69 Upper Freeport coal was extended in the high yield at room temperature with a carbon disulfide/N-methyl-2-pyrrolidinone (CS2/NMP) mixed solvent. A solid-state 13C NMR spectrum was measured for the mixed-solvent insoluble residue.17' The NMR chemical shift of a three-dimensional polyacetylene crystal in the orthorhombic form was calculated by a combination of ab initio tight-binding M O theory and the sum-over-states method of the chemical shift theory within the STO-3G minimal basis set, in order to elucidate the inter-molecular interaction effect of the 13CNMR chemical shift and electronic structure, as associated with the crystallographic form. The effects of inter- and intra-chain on the 13C NMR chemical shift and the electronic structure were reasonably e~a1uated.I~' The series of the propargyl thioquinolines was prepared on the basis of the reaction of thioquinanthrene (1) (1,4-dithiin0[2,3-~:5,6-c']diquinoline) with sodium alkoxides. Some of these compounds were revealed good antiproliferative activity in vitro against the cells of human and murine cancer lines. 13C NMR spectra were measured for the studied compounds to examine the electronic properties-activity re1ation~hips.l~~ Ab initio and DFT methods were used to study the tautomers of barbituric acid in the gas phase and in a polar medium. In the gas phase, the tautomers were optimized at the HF/6-31G*, MP2/6-31G* and B3LYP/6-31G*, B3PW91/6-31G* levels of theory. The self-consistent reaction field theory (SCRF) at the HF/6-31G* level of theory was used to optimize the tautomers in a polar medium. The relative stability of the tautomers was compared in the gaseous and polar mediums. The ability of maximum hardness principle to predict the stable tautomer was studied. The 13C-NMR chemical shift for carbon atoms in the tautomers was calculated and the results were The low-affinity interaction between human serum albumin (HSA) and Diclofenac sodium (DCF) was studied using NMR techniques. Both 13C-NMR chemical shift and linewidth show that the dichlorophenyl ring in DCF molecule plays a primary role in its interaction with HSA.'74 2.1 4.2 Silicon (29Si)(l= 1/2). Lithium monosilicide (LiSi) was formed at high pressures and high temperatures (1.0-2.5G P a and 773-973K)in a piston-cylinder application. This compound was previously shown to have an unusual structure based on 3-fold coordinated silicon atoms arranged into interpenetrating sheets. In the investigation, lowered synthesis pressures permitted recovery of large (150-200 mg) quantities of sample for structural studies via NMR spectroscopy (29Siand 7Li),Raman spectroscopy and electrical conductivity rneasurement~.'~~ Density functional Born-Oppenheimer molecular dynamics (BOMD) is applied to study the evolution of geometry and 29SiNMR chemical shifts of Si(OH)4, which is the smallest zeolite p r e c ~ r s o r .The ' ~ ~ 29Sichemical shift tensors in both


91

symmetric and unsymmetric substituted disilenes were analysed by quantum chemical calculations.'77 Nanometer-sized faujasite zeolite (nanoNaX), synthesized with an organic-additive-free approach, was modified with organic functional groups. Raman scattering and solid-state 29SiNMR techniques were employed to study the organic components on the zeolites, the structures of the zeolitic frameworks, and the connections between the organosilyl groups and zeolitic silanol groups.'78 Powder and single-crystal X-ray diffraction, combined with 29SiMAS NMR measurements, was used to study the thermal expansion of siliceous zeolite ferrierite as it approaches a second-order displacive phase transition from a low-symmetry (Pnnm)to a high-symmetry (Immm) In order to unambiguously assign the 29Si-NMRchemical shifts of polydisilahydrocarbons containing structural units -CH2Sil(R1R2)Si2(R1R2)CHPh-, where R1= R2= Me or Ph, a model polymer viz., poly(tetramethyldisilyleneethy1ene) was synthesized through the dechlorination of 1,2bis(chlorodimethylsily1)ethaneusing potassium in toluene.18' 29Sichemical shift anisotropy (CSA) data were determined from 29SiMAS NMR spectra recorded at 14.1 T for a number of synthetic calcium silicates and calcium silicate hydrates.lg1Hydrosodalites and hydroxosodalites were analysed by 170,29Siand 'H MAS NMR in the fields of 17.6 and 11.7 T.lg2The X-ray crystal structures of four 1-(halodimethylsilylmethyl)-2-quinolinoneswere measured and used to model the reaction profile for nucleophilic substitution at Si. Similar structural correlations were performed in solution, the percentage Si-0 bond formation being obtained from the I3Cchemical shifts of the quinolinone carbons and the extent of pentacoordination from the 29Sichemical shift of the Si.lS3 1H/'3C/29Sitriple resonance 3D NMR combined with pulse field gradient (PFG) techniques was utilized for characterizing poly(dimethylsi1oxane) (PDMS), MD3MH.lS4 The ability of several ab initio models to predict experimental 29Si-NMRchemical shift was exarnined.ls5 A number of aminophosphines with bulky amino groups were prepared and their preferred conformation was determined by NMR spectroscopy in solution, by solid state 29SiNMR and x-ray crystallography186 The NMR properties of a smectite clay low in paramagnetic ions, and NMR experiments to detect organic material near the silicate surfaces with high sensitivity, were explored by 'H, 29Si,and 13CNMR.lS7 The structure of organicinorganic hybrid glass precursor Me2Si(OP:O(OH)2)2and low-melting glasses Sn0-Me2Si0-P205 was examined by 29Sistatic and MAS NMR, respectively.'88 A series of trihydrocarbyltin chlorides containing silicon, (R1R2MeSiCH2)3SnC1 (la-lj, R2 = Me; a R1 = Me, b Et, c Pr, d Bu, e Ph, f 4-MeC6H4,g 4-ClCsH4, h 4-MeOC6H4, i PhCH2, j 2-C4H3S;lk, R1 = R2 = Et) was synthesized. The crystal structure of l g was determined. The chemical shift of 29SiNMR resonance is in linear correlation with Hammet constant for aryl-substituted le-lh.189 Evidence for a three-coordinate silyl cation was provided by the crystal structure of [(M~S)~S~][H-CBI~M~~B~~]C~H~ (Mes is 2,4,6-trimethylphenyl). The downfield 29SiNMR chemical shift in the solid state (226.7 ppm) is almost identical to that in benzene solution and in gas phase calculations, indicating that threecoordination can be preserved in all phases."' Hot-filament CVD (HFCVD), a non-plasma technique, was used to prep. fluorocarbon-organosilicon

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copolymers from hexafluoropropylene oxide (HFPO) and hexamethylcyclotrisiloxane. The composition of the copolymer films was established from FTIR spectra and 19Fand 29SiNMR chemical shift assignment^.'^' Homogeneity and structure of organically modified polysiloxane networks prepared by sol-gel co-condensation, as well as location and nature of water molecules and silanol groups were studied by 1D and 2D solid-state 'H,29SiNMR.'92 High-resolution solid state 29Si,27AlMAS and 27Al3 4 MAS NMR spectroscopic techniques were used to characterize highly crystalline ultrastable Y (USY) zeolites having different framework %/A1 ratios, obtained by multistep-temperature-programmed (MSTP) steaming at different ternperat~res.'~~ Geometries and 29Si NMR chemical shifts were calculated for silanes SinH2n+2,n = 1, - - ,5, methylsilanes SiH,Me4-,, methox y silanes SiH,( OMe)4-,, and meth ylmethox ysilanes SiMe,(OMe)c,, n = 0, - ,4.194 a-Substituent effects on inversion barriers and NMR chemical shifts were studied on a set of silyl anions, [X3-,Y,Si]- (X, Y = H, CH3, and SiH3).195Two crystallographic distinct tetrahedral A1 sites in zeolite merlinoite were directly identified by ultra-high-field (18.8 T) 27AlMAS NMR spectroscopy. The experimental chemical shifts and peak areas indicate a preferential sitting of A1 in site TII. This non-random aluminum distribution was independently confirmed by ultra-high-field (18.8 T) 29SiMAS NMR spectros c ~ p y .Sic-C ' ~ ~ solid solutions powders were analysed by x-ray diffraction (x-ray diffraction) and 29SiMAS NMR.'97 The authors report ab initio MO calculations of 29Si NMR chemical shifts and relative stabilities of Si-polyalcohol monocyclic and spirocyclic complexes, from ethylene glycol (C2H602) to arabitol (C5H1205) with Si in quadra-, penta- and hexa-coordination (QSi, Psi, HSi), calculated at the HF/6-311+ G(2d,p)//HF/6-31G* l e ~ e 1 . l ~A~novel microporous crystalline silicoaluminophosphate of Si2A1P3013 was synthesized, templatefree, and characterized using XRD, BET, and solid-state 27Al,31P,and 29Si NMR.'99 A series of silylated carboxonium ions were prepared as long-lived species by treating triethylsilane and triphenylmethyl tetrakis(pentaf1uorophenyl)borate (Ph3C+B(C6F5)4-) with ketones, enones, carbonates, amides, and urea in CD2C12solution. Thus, they were characterized by 13Cand 29SiNMR spectroscopy at 195K.200The authors have studied short-range cation ordering across the diopside (CaMgSi206)-Ca-Tschermakpyroxene (CaA12Si06) (DiCaTs) solid solution in samples synthesized at 1673K and 2 GPa, for 24 h. Peak positions in 29SiMAS NMR spectra are sensitive to A1 substitution, both in the corner-sharing NN tetrahedral sites on the single chain and in one of the three NN octahedral M1 sites.201Tris(trimethylstanny1)amine and trimethylsilylbis(trimethylstanny1)amine (both ca. 13% "N labeled) were studied by 115/117/119 Sn-, 15N-,and 29SiNMR spectroscopy.202 *

*

*

2.14.3 Germanium (73Ge)(l = 9/2). A survey of development of NMR observation for 73Ge,'19Sn and 207Pbfor organometallic compounds between 1995 and 2000 is reviewed.203Emphasis was given to 72GeNMR spectroscopy and chemical shifts of organometallic compounds. 2.1 4.4 Tin (1179119Sn)(I = 1/2, 1/2). During the last decade, vinyl- and dienyltin


93

derivatives were extensively developed and used in organic synthesis. 'H NMR analysis of these compounds was the first analytical tool employed, together with '19Sn NMR, for the assignment of the E or Z stereochemistry of vinylstannyl derivatives. In this account, usefulness of 13CNMR as powerful tool for structural analysis of vinyl- and dienyltin compounds, was demonstrated.2M "B and '19Sn NMR (NMR) spectroscopies were used to determined the local environments of boron and tin in binary tin borate glasses of general composition xSnO ( 1-x)B203( 0 . 2 s ~ s O - 7 )Twenty-four .~~~ 2,2-di-n-butyl- and 2,2-diphenyl-6aza-l,3-dioxa-2-stannabenzocyclononen-4-ones, each having a transannular N -+ Sn bond, were prepared by one-step reactions of a-amino acids (la-1), salicylaldehyde(2),and either di-n-butyltin(1V)oxide (3) or diphenyltin(1V)oxide (4). The new methodology constitutes an easy, highly efficient one-step synthesis of diorganotin(1V) derivatives, such as 5a-1 and 6a-1, from iminic tridentate ligands without isolation of the Schiff bases. The structures of all the compounds have been established by a combination of 'H, 13C,15N,and '19Sn NMR spectroscopy, IR spectroscopy, mass spectrometry, and elemental analysis.206 Tris(2,4,6-triisopropylphenyl)stannylium tetrakis(pentafluorophenyl)borate, [Tip3Sn+ (TPFPB)], constitutes a free, tricoordinate tin cation according to its x-ray structure. Calculation of the '19Sn chemical shift is in good agreement with the observed value.207A survey of development of NMR observation for 73Ge, '19Sn and 207Pbfor organometallic compounds between 1995 and 2000.208Tin phosphate glasses, of general formula xSnO(1 - x)P2O5 (0.3 < x < 0.8), were prepared by conventional melt-quench techniques and their structures studied using 31P and '19Sn NMR.209 Water-soluble (3-methoxypropy1)stannanes (CH30CH2CH2CH2)xSnC14-x (x = 4 (1), 3 (2), 2 (3), 1 (4)) were prepared. Treatment of 3-methoxypropyl chloride with magnesium followed by reaction with SnC&yielded 1, which reacted with the appropriate amount of SnC14giving 2-4. For 3 and 4, which were isolated in the crystalline state, it was shown by X-ray diffraction that the tin atom was coordinated in distorted octahedral and trigonal-bipyramidal geometries, respectively ( i e . , the oxygen atoms of the 3methoxypropyl groups were coordinated to the central tin atom in both cases, forming chelates). For all compounds, structures in CDC13 and [d6]DMSO solutions were proposed on the basis of their 13Cand '19Sn NMR spectra.210The '19Sn and 19FNMR chemical shifts in alkali-metal hexafluorostannates were measured?" The metallostannylene compounds (q5-C5H5)(C0)3MSnC6H3-2,6Mes2 (Mes = C6H2-2,4,6-Me3;M = Cr (l), Mo (2), W (3)), (q5C5H5)(C0)3MSnC6H3-2,6-Trip2 (Trip = C6H2-2,4,6-Pri3;M = Cr (4,Mo (5),W (6)),and (q5-1,3-ButC6H3)MoSnc6H2-2,6-Trip2 (7) were synthesized by the reaction of the appropriate aryltin(I1) halide with the alkali-metal salt of the cyclopentadienylcarbonylmetalate. The compounds were also characterized by IR and UV-vis spectroscopy as well as 'H, 13C,and '19Sn NMR spectroscopy.212The structural characterization of organotin compounds that were grafted onto insoluble cross-linked polymers has necessarily been limited to elemental analysis, IR spectroscopy, and in a few instances, solid-state 'H, 13C,and '19Sn NMR spectro~copy?'~ Reaction of the Li triamidostannate [MeSi{ SiMe2N(p-T01)}3SnLi(OEt2)] with 0.5 molar equivalents of MC12 (M = Zn, Cd, Hg) in toluene

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afforded the corresponding heterodimetallic complexes [MeSi{SiMe2N(pT O ~ ) ) ~ S ~{ M ] ~= MHg, Cd, and Zn }. The different degree of metal-metal bond polarity is reflected in the I19Sn NMR chemical shifts of the complexes.214 Density functional theory (B3LYP) calculations were carried out for lI9Sn isotropic chemical shifts for a grouping C-N-SnMe3-N-C characteristic of solid organometallic corn pound^.^'^ Tetraalkyldistannoxanes of the type [{R2Sn(ON:C(Me)py)}20]2(R = Bu, Pr, Et and Me) were synthesized by the condensation reaction of RzSnO with 2-NC5H4(Me)C:NOHin 1:l molar ratio and characterized by elemental analyses, IR and NMR ('H, I3C and 'I9Sn) The reaction of a 1:l mixture of Ph2PbC12and ClSiMe2SiMe2Cl with HzS/NEt3 yielded the S-Si-Si-S-Pb cyclic SSiMe2SiMe2SPbPh2.Bicyclic Ph2Pb(S)2Si2Me2(S)2PbPh2 was obtained by similar treatment of a 2:l mixture of Ph2PbClzand C12SiMeSiMeC12.The corresponding Se compounds were obtained by reactions of mixtures of Ph2PbC12and methylchlorodisilanes with Li2Sein THF. All products were characterized by multinuclear ('H, 13C,29Si,77Se and 207Pb)NMR A series of para-substituted triaryltin(pentacarbonyl)manganese(I) compounds [(p-XC6H4)3SnMn(CO)5:11, X = CH3;111, X = CH30; IV, X = CH3S; V, X = F; VI, X = C1; VII, X = CH3S(02)]was reported for comparison with the known Ph analog I. IR data [v(CO)] as well as complete 119Sn/55Mn/'3C solution NMR results are given for I-VII. Chemical shifts, '19Sn vs. 55Mn,except I, correlate well, but have differing single parameter (SP)correlations, '19Sn vs. a1 and 55Mnvs. Nonrelativistic DFT calculations of the '19Sn chemical shift were presented for a large series of tetracoordinated Sn compounds, CH3SnRR'R", where R, R', R" are halogens, alkyl, halogenated alkyl, alkoxy, or alkylthio groups.219 Organotin tropolonato complexes R3Sn(trop)[l; R = Bu (b), Ph (c)], R2Sn(tr~p)2 [2; R = Me (a), Bu (b), Ph (c)] and RSn(trop)3[3; R = Bu (b), Ph (c)] were studied by single-crystal x-ray diffraction (lc, 2a,b, 3b), 13Cand '19Sn MAS NMR and '19Sn NMR in solution.220

2.14.5 Lead (207Pb)(I = 1/2). The group 14 1,3-diphosphacyclobutadienyl complexes [M(q4-P2C2But2)] (M = Ge, Pb) were synthesized by the reaction of [Zr(q 5-CsH5)2(PCBut)2]with GeC12 (dioxane) or Pb12 and fully characterized by multinuclear NMR spectroscopy and single-crystal X-ray diffraction studies. Structurally they can be viewed as 24-electron nido-5-vertex clusters. The significant solvent dependence of the 207Pbchemical shift of [Pb(q4-P2C2But2)]suggests the possibility of formation of an adduct with Lewis bases.221 Norbornyl cations of the group 14 elements Si + Pb (I +; wherein E = Si, Ge, Sn, Pb; R = Me, Et, n-Bu) were synthesized from substituted 3-cyclopentenemethyl precursors by intramolecular addition of transient cations to the C = C double bond of the 3-cyclopentenemethyl substituent (n-route to norbornyl cations). The norbornyl cations 4a (E = Si, R = Me), 4e (E = Si, R = Et), 4f (E = Si, R = Bu), 4g(E = Ge, R = Bu), 4h (E = Sn, R = Bu), and4i (E = Pb, R = Et) were identified by their characteristic 29Si,13C,and 207PbNMR chemical shifts.222A review of the study on 207Pbnucleus in solid materials using NMR spectroscopy. Calculations using the temperature-dependent chemical shifts of *


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the 207Pbresonance of Pb(N03)2were described.223207PbNMR parameters [chemical shifts, indirect nuclear spin-spin coupling constants, and relaxation times Tl, T2] were listed and discussed with respect to the structure and dynamics of lead compounds, focusing mainly on the liquid state.224

2.15 Group 15 N, 31P).- 2.15.1 Nitrogen ( l 4 9 l 5 N)(I = 1, 1/2). Directly detected ammine 14NNMR chemical shifts of 20 amminecobalt(II1) compounds were reported.225The effects of benzannelation, Ph substitution at the N atom, protonation at the carbene C, ionization, and the state of the carbene center (02 or n2) on the electronic structure, diamagnetic susceptibility, induced n-electron ring currents, the ‘H, 13C, and 14N chemical shifts, and the energies of the lowest electronic transitions of imidazol-2-ylidenes and their 0x0 and thio analogs were examined in the bound version of n-electron perturbation theory. The calculated and experimental data were A lecture on many challenges encountered in solid-state 14NMAS NMR spectroscopy, even at 14.1 T, using standard high-Q double resonance MAS probes. The topics include 14N quadrupole coupling and chemical shift anisotropy, 14NMAS NMR of nitrate ion, and phase transition in NH4N03.227 13Cand 14NNMR chemical shifts of the cyano group were observed for the solutions of four aliphatic nitriles (i.e. acetonitrile, propionitrile, isovaleronitrile and pivalonitrile) in ten aprotic A 14N solid-state NMR single crystal study was used to determine the 14Nquadrupolar, chemical shift, and 14N-lHdipolar tensors of sulfamic 3-Methylfurazans with N-containing substituents at position 4 were studied by ‘H, 13C,and 14N NMR spectroscopy.230The structure and relative energies of the tautomers of cytosine in gas phase and in different solvents were predicted using MP2 and density functional theory methods. Solvent induced effect on nitrogen NMR shielding of two dominant tautomers was calculated using density functional theory combined with polarizable continuum model and using the continuous set gauge transformation. Direct and indirect solvent effects on shielding were also calculated.231The isotropic chemical shift and the nuclear quadrupole coupling constant for 14Nwere obtained for 14 inorganic nitrates by solid-state MAS NMR measurements at two different field strengths, 9.4 and 11.7 T.23215N Chemical shielding and experimental techniques are re~iewed.2~~ Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions, which are experimental accessible through experiments performed on weakly and completely aligned samples. Importantly, the anisotropy of nuclear spin interactions results in a mapping of structure to the resonance frequencies and splittings observed in NMR spectra. Distinctive wheel-like patterns were observed in two-dimensional ‘H-”N heteronuclear dipolar/”N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples.234 The author describes the characterization of various carbohydrates using NMR spectroscopy. Such carbohydrates include acyclic, furanose, and pyranose and related systems, disaccharides, and oligosaccharides. ”N NMR spectroscopy was also applied to assess the impact of N-substitution on 15N chemical shift in N-derivatives of (l49l5

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2-amino-2-deoxy-(3-D-glucopyranose with d i p e p t i d e ~ This . ~ ~ ~review discusses recent progress in the investigation and use of 13C,15N,and 19FNMR chemical shifts and chemical shift tensors in proteins and model systems primarily using quantum chemistry (ab initio Hartree-Fock and density functional theory) techn i q u e ~ The . ~ ~implementation ~ of both the directly and the inversely detected 15N NMR techniques at the natural abundance level of the "N isotope was demonstrated for a diverse array of structural problems in organic chemistry.237The keto-enol tautomerization of a-diketones, isomerization of a-angelica lactone and the exact substitution pattern of pyrazines were discussed. The analytical methods used to determine the correct molecular structures of these aroma chemicals were gas chromatography (GC), GC-mass spectrometry and "NNMR spectroscopy, especially polarization-transfer for sensitivity enhancement, and 2-dimensional correlation 2.15.2 Phosphorus (31P)(I = 1/2).- The objective of this study was to investigate the self-association and DNA-binding properties of the DNA topoisomerases I (Topo I) and I1 (Topo 11) dual inhibitor: 6-[ [2-(dimethylamino)ethyl]amino]-3hydroxy-7H-indeno [2,l-c]quinoline- 7-one dihydrochloride (TAS-103), by 'HNMR and 31P-NMRspectroscopy, structure computation techniques, thermal melting study, and UV-Visible

2.16 Group 19 ( 1 7 0 , 33S,77Se,'25Te). - 2.16.1 Oxygen (I7O)(1=5/2). 1 7 0 NMR chemical shifts were measured for 23 liquid chemical compounds and their vapors at natural abundance of oxygen-17 and the temperature of 333K.240A multi-linear-regression analysis using the Kamlet-Abbout-Taft (KAT) solvatochromic parameters in order to elucidate and quantify the solvent effects on the 1 7 0 chemical shifts of three 4-dimethylsulfoximide-l,1,1 -trifluoro-3-buten-2ones was reported.241Single crystal X-ray structures (monoclinic space group P21) for Me 3-oxo-5P-cholan-24-oate and Me 3,12-dioxo-5~-cholan-24-oate were solved and compared with HF/6-31G optimized structures. 13C and I7O NMR chemical shifts of Me 3-oxo-5P-cholan-24-oate and Me 3,12-dioxo-5Pcholan-24-oate as well as the epimeric Me 3a-hydroxy-5P-cholan-24-oate and Me 3P-hydroxy-SP-cholan-24-oate were calculated (DFT/B3LYP/6-311G) and compared with the experimental values by linear regression analyses.242Ab initio calculations of the 1 7 0 electric field gradient (EFG) tensor, quadrupolar coupling const. (C,) and asymmetry parameter (q,), along with the 1 7 0 NMR isotropic chemical shift (biso)for the three crystalline polymorphs of P205were presented.243 Special high-temperature and -pressure multinuclear NMR equipments were constructed, and used for the measurements of I70-NMRchemical shift and TIin water over the range from liquid to supercritical (SC) states.244 The authors report 1 7 0 resolution-enhanced MAS NMR studies of the parae1ec.-antiferroelectric phase transition (at Tc = 373 K) of the model H-bonded compound squaric acid (H2C404).245 NMR spectroscopy is a relatively insensitive technique and many biomolecular applications operate near the limits of sensitivity and resolution. A particularly challenging example is detection of the quadrupolar nucleus 1 7 0 , due to its low natural abundance, large quadrupole couplings, and


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low gyromagnetic ratio.246The special multinuclear NMR equipments were constructed, and the measurements of 1 7 0 chemical shifts and TI in water were carried out over the range from liquid to supercritical (SC) states.247The oxidation of the trans, cis-(11) and trans, trans-epoxides (111) (first prefix refers to relative position of C4 carbonyl and C3’ aryl of the epoxide ring, second prefix describes relative position of C2 Ph and epoxide 0)of differently substituted (Z)-3-arylidene-1-thioflavan-4-ones with dimethyldioxirane (DMD) yielded the appropriate sulfoxides and sulfones. The structures were elucidated by the extensive application of one- and two-dimensional ‘H, 13Cand 1 7 0 NMR spect r o ~ c o p y .Hydrosodalites ~~* and hydroxosodalites were analysed by 170,29Siand ‘H MAS NMR in the fields of 17.6 and 11.7 T. The samples covered a range in Si-0-A1 bond angle of =23°.249With various concentrations of CaCl2 and MgC12 acquire solution below 1.0 mol/l, Raman spectra of water in the OH stretch region of 2500-4000 cm- and 1 7 0 NMR chemical shift of water were measured and the Raman spectra were d e ~ 0 n ~ 0 1 ~ t The e d ? two-layer ~~ ONIOM2(MP2GIA0:HF-GIAO) (our own n-layer integrated MO and molecular mechanics approach, in which a small model system containing the nuclei of interest was described at the MP2-GIAO level of theory, and the rest of the molecular-using the HF-GIAO method) ansatz was applied to the calculation of 13C,‘H, and 1 7 0 NMR chemical shifts in the 4C1 G + , 4C1 G-, 4C1 T, 1C4 G + , and 1C4 Gconformers of P-D-gluc~pyranose.~~~ 1 7 0 NMR parameters, both the chemical shifts and the quadrupolar parameters, were calculated for Si02 polymorphs using density functional theory with the generalized gradient-correlation PBE f~nctiona1.2~~ A new global analysis of EPR, 1 7 0 NMR relaxation and chemical shift and ‘H NMRD profiles with physical meaningful parameters for [Gd(DOTA)(H20)]- and for [Gd(DTPA)(H20)I2-in acquire solution was presented (DOTA = 1,4,7,10-tetraaza-1,4,7,10-tetrakis(carboxymethyl)cyclododecane; DTPA = diethylenetriamine-N,N,N‘,N’’,N”-pentaacetic Zeolite A provides a unique opportunity to explore the relation between structural parameters and 1 7 0 NMR isotropic chemical shifts because differences in site multiplicity for framework oxygens often permit unambiguous site a~signments.2’~ 2-Methyl-1,3-dioxolane in acetone-d6, Me acetate, and tert-Bu Me ether produced both the corresponding acetal hydrotrioxide (ROOOH) and the hemiortho ester (ROH) in molar ratio 15. Both intermediates were fully characterized by ‘H, 13C,and 1 7 0 NMR spectroscopy, and they both decomposited to the corresponding hydroxy ester at higher temperature^.^'^ Unusual behavior was observed in the study of the 170,13Cand ‘H NMR and IR spectra of crowded (1-adamanty1)alkylkef0nes.2~~ The IR absorption bands of the S:O and N-H stretching vibrations as well as the natural abundance 13C and the 1 7 0 NMR chemical shifts were measured for fifteen substituted benzenesulfonamides in CDC13 and a ~ e t o n e - d ~Relaxation .~~~ characteristics of Na+ and K + water clusters were studied by 1 7 0 NMR spectroscopy.2581 7 0 NMR signals of terminal and bridging oxygen atoms incorporated into dO transition-metal complexes differing in the composition of the coordination sphere were identified.259The environments for oxygen sites in crystalline V2O5 and in layered vanadia gels produced via sol-gel synthesis were studied using 1 7 0 MAS and 3QMAS

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NMR.260Ab initio, M O calculations were carried out to study the energetic stability and 1 7 0 NMR characteristics of 'triclusters' of composition [T(XO)(OH)2]O[Al(OH)3][T(OH)3](X = no atom, H + , Li+,Na+, K + ,Mg2+,or Ca2+ ; T = Si4+ or A13+).261 The gauge-included A 0 (GIAO) method was applied within the coupled Hartree-Fock (CHF) approximation to compute 13C, 1 7 0 and 33SNMR chemical shifts for solid OCS.262 The results of theoretical and experimental investigation of 1 7 0 NMR chemical shifts for a number of epoxidic compounds were reported.263The 1 7 0 NMR spectra of a number of unsatulated 5-membered cyclic acetals, 2-substituted 4-methylene-1,3-dioxolanesand their endocyclic isomers, 4-methyl- 1,3-dioxoles, were r e ~ o r d e d . 2The ~ ~ authors report a systematic solid-state 1 7 0 NMR study of free nucleic acid bases: thymine (T), uracil (U), cytosine (C), and guanine (G). Site-specifically 170-enriched samples were ~ynthesized:[2-'~0]thymine(l), [4-170]thymine (2), [2-'70]uracil (3), [4'70]uracil (4), [2-170]cytosine (5), and [6-'70]guanine monohydrate (6).265The 2D 1 7 0 (I = 5/2) 3Q MAS NMR powder spectra were obtained for orthoenstatite, clinoenstatite, and protoenstatite (MgSi03 polymorphs) and diopside (CaMgSi206).266 2.1 6.2 Sulfur (33S)(I= 3/3). Solid state 33SNMR spectra of a variety of inorganic sulfides were obtained at magnetic field strengths of 4.7 and 17.6 T. Spectra acquired with magic angle spinning show considerable improvements in sensitivThe 33SNMR signal of ity and resolution when compared with static gaseous carbonyl sulfide (COS) was monitored as a function of density for the first time.268 The IR absorption bands of the S = O and N-H stretching vibrations as well as the natural abundance 13Cand the 1 7 0 NMR chemical shifts were measured for fifteen substituted benzenesulfonamides in CDC13 and acetone-d6. For the unsubstituted compound the natural abundance 33SNMR chemical shift was also measured in acetone- d6 PM3 charge densities and bond orders were calculated for the completely optimized geometry.269The gauge-included A 0 (GIAO) method was applied within the coupled Hartree-Fock (CHF) approximation to compute 13C,170and 33SNMR chemical shifts for solid OCS.270 2.1 6.3 Selenium (77Se)(I= 1/2). An extensive library of 77Sechemical shifts were generated from the NMR measurements on substituted (phenylseleno)benzenes, including 33 new compounds. The variation in chemical shifts cover 265 ppm ranging from 446 to 181 ~ p m . The ~ ~ authors ' present a method for the first principles density functional calculation of relativistic all-electron NMR chemical shifts using pseudopotentials. They demonstrate its success for a range of Se and Te containing molecules?72The structures and spectroscopic properties of SenSX-n ring molecules were studied by the use of ab initio M O techniques and density functional techniques involving Stuttgart relativistic large core effective core potential approximation with double zeta basis sets for valence orbitals augmented by two polarization functions for both sulfur and selenium. The 77Se nuclear magnetic shielding tensor calculations were carried out by the Gaugeindependent A 0 method at the DFT level using Becke's three-parameter hybrid functional with Perdew/Wang 9 1 correlation.273 The 77SeNMR chemical shifts


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observed for 2-selenobenzyl halide derivatives (2-XCH2C6H4SeY; X = F, C1, Br; Y = CN, Cl, Br, SeC6H4CH2Br,Me) and the density functional theoretical calculation revealed that the strength of intramolecular nonbonded interactions between a divalent Se and halogen atoms decreases in the order, Se-F > Se-Clr Se * * Br.274Two types of clusters, [Ag(Se,P(OR),)], (R = Et, n = 00, la; R = Pri, n = 6, lb) and Agl&10-se)[se2P(oR)2]8 (2a, 2b for R = Et, Pri, respectively) were obtained from the reaction of stoichiometric amounts of Ag(MeCN)4PF6 and NH&e2P(OR)2in E t 2 0 at -20". These new Ag(1) clusters containing diselenophosphate (dsep) ligands were characterized by elemental anal., 77SeNMR spectroscopy, fast-atom bombardment mass spectrometry (FAB-MS) for 2, and, in the case of lb, and 2a.1/2Et20, by x-ray ~rystallography.~~~ Treatment of a-alkyl and apdialkyl homoallylic amines R 1R2C(NHBn)CH2CH:CH2with PhSeX (X = C1, Br, I), in CH3CN containing sodium carbonate produced mixtures of azetidinesand pyrrolidines. Some reactions were monitored by 77Se NMR at the beginning of the addition-cyclization Various diacyl selenides, diacyl diselenides and selenocarboxylates were synthesized by reaction of several acyl chlorides with LiAlHSeH. Reaction of diacyl chlorides with LiAlHSeH afforded cyclic selenoanhydrides. In the 77SeNMR spectra, the authors found that the chemical shifts of the diacyl selenides and the diacyl diselenides could facilitate their *

2.1 6.4 Tellurium ("'Te)(I= 1/2). The author present a method for the first principles density functional calculation of relativistic all-electron NMR chemical shifts using pseudopotentials and demonstrate its success for a range of Se and Te containing Tellurium(I1)dimethanethiolate, Te(SMe)2,and tellurium(I1) diethanethiolate, Te(SEt)2,were synthesized by reaction of Te02 and Te(OiPr)4with HSMe and HSEt, respectively. The '*'Te NMR chemical shift of Te(SR), largely depends on R (R = Me, Et, iPr, tBu) and shows a nearly linear correlation with the first ionization energy of the corresponding thiol HSR.279 Meso-Tetraphenyl-2 1-chalcogenaporphyrins 4-6(S,Se, and Te as 21-chalcogen atoms, respectively) and meso-tetraphenyl-21,23-dichalcogenaporphyrins7-10 [(S,S), (Se,S), (Se,Se),and (Te,S)combinations as 21,23-chalcogen atoms, respectively] were prepared by condensation of the appropriate 2,5-bis(phenylhydroxymethy1)chalcogenophene11 with (I) benzaldehyde, pyrrole, tetrachlorobenzoquinone (TCBQ), and B trifluoride etherate for the preparation of 4-6or (11) the appropriate 2,5-bis(1-phenyl-1-pyrrolomethyl)chalcogenophene 13, TCBQ, and B trifluoride etherate for the preparation of 7-10. The '25TeNMR spectrum of 6 gave a chemical shift of 6 834. Oxidation of 6 to oxotelluraporphyrin I11 gave a '25TeNMR chemical shift of 6 1045.21-Tellura-23-thiaporphyrin10 gave a 125TeNMR chemical shift of 6 1039, perhaps reflecting deshielding of the Te nucleus by the less than van der Waals contact with the S nucleus?80Diary1 ditellurides with and without coordinating amino/imino groups were synthesized by using either the Grignard route or the heteroatom-directed aromatic lithiation route. A correlation between the strength of Te ...N intramolecular interactions and thiol peroxidase activity reveals that the strong Te.. .N interactions reduce the thiol peroxidase activity of amino/imino-substituted ditellur-

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ides. A plot of Te ...N distances against "'Te NMR chemical shifts shows a linear correlation.281

2.17 Group 17 (19F,35737C1). - 2.1 7.1 Fluorine (19F)(I = 1/2). Solid state 19FMAS NMR studies of (3,3,3-trifluoropropyl)dimethylcholorosilane (TFS) probe molecules covalently bound to low surface area glass fibers were used for the quantitative analysis of hydroxyl sites on the surfaces of model E-glass fibers.282 The 19FNMR shieldings for several remotely substituted rigid polycyclic alkyl fluorides with common sets of substituents covering a wide range of electronic effects were calculated using the DFT-GIAO theoretical Based on structural information provided by recently reported crystal structures of rhodopsin, we present rationales for the regiospecific protein perturbation on the previously reported 19Fchemical shifts of the vinyl and trifluoromethylrhodopsins and their photo product^.^^^ A survey of possible structures for the Cb0F8 molecule was carried out, using both experimental (19FNMR data) and theoretical (structure-energy correlation) considerations to limit the number of isomers to be regarded as candidates for the previously isolated specie^.^" Anomalous tendencies in the I 9 F NMR chemical shifts of certain fluorine-containing, primarilyorganofluorine, compounds were explained.286Solution JSI-F and 2Jc-F NMR coupling constant and 19F-NMRchemical shift data were analysed in a series of pentacoordinate silyl monofluoride complexes used to model nucleophilic substitution at silicon.28719FNMR chemical shifts of CF4 in C 0 2 under extremely dilute concentrations were precisely determined at 299.8,314.4 and 328.9 K over a wide range of pressure between 0.4 and 33 MPa. The solvent-induced chemical shift, where the bulk magnetic susceptibility correction was made, was quantitatively expressed as a function of C 0 2 density.288The response of membraneassociated peptides toward the lipid environment or other binding partners can be monitored by solid-state 19FNMR of suitably labeled side chains.289The Il9Sn and 19FNMR chemical shifts in alkali-metal hexafluorostannates were measred.^^' Improved correlations between I9F-NMR chemical shifts in several fluorohalohydrocarbons and heats of formation, bond dissociation energies, group contribution to enthalpies of formation and enthalpies of formation of free radicals were given.291In this work the use of 1D and multidimensional 'H/13C/19F triple resonance 750 MHz NMR techniques for characterizing fluoropolymers were i l l ~ s t r a t e d . High-throughput 2~~ ligand-based NMR screening with competition binding experiments was extended to 19F Precise control of perfluoropolyether (PFPE) physical properties requires analytical tools, such as NMR (NMR) spectroscopy and gel permeation chromatography (GPC) to characterize their chain end groups, and molecular weight distribution. The 19Fchemical shifts for common perfluoropolyethers with six different types of end group are compiled within.294 The one-pot synthesis of nine novel 5-trifluoromethyl- 1,2-dimethyl-1H-pyrazolium chlorides from the cyclocondensation of 4-alkoxy-l,l,l-trifluoro-3-alken-2-ones [CF3C(0)CH:CRIOR, where R1 = H, Me, n-Pr, n-Hex, Ph, 4-MeCsH4, 4-FCsH4, 4-ClCsH4, 4-N02C6H4,and R = Me, Et] with 1,2-dimethylhydrazine, in quantitative yields, was reported. The 'H, 13C,19Fand 35ClNMR chemical shifts of the pyrazolium


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chlorides were described.295The state of SbF3, Sb302F5,MSb3Flo, MSb2F7, M3Sb4FI5,MSbF4, and M2SbF3,as well as H F and M F (M = Na, K, Rb, Cs, N&, and Tl), in a 0.25M aqueous solution at room temperature was studied by 19F NMR.296Using liquid state 'H, 2H and 19F NMR spectroscopy in the temperature range 110-130 K the authors have studied the H-bonded anions (FHhF- and (FH)3F- and their partially and fully deuterated analogs dissolved in the low-freezing Freon mixture CDFJCDF2Cl, in the presence of B&N+ as the c o ~ n t e r c a t i o n . A ~ ~simple ~ 19FNMR spectrometric method was proposed for the determination of the partition coefficients of fluorinated psychotropic drugs, trifluoperazine (TFPZ), flunitrazepam (FNZ), and flurazepam (FZ) between phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUVs) and water (buffer).298The 19FNMR spectra of three complexes each of two different pentafluorophenyl-substituted iron(II1) porphyrinates were studied and compared with the protonated phenyl-substituted iron p0rphyrinates.2~~The behavior of a new biphotochromic molecules formed by two naphthopyran moieties linked through a (Z)-ethenic double bond, was studied by 'H and 19FNMR spectroscopy?~The 31Pchemical shift (CS) tensors of the 1,3,2-diazaphospholenium cation and the P-chloro-1,3,2-diazaphospholenes(R = t-Bu, X = C1; R = Mesityl, X=C1) and the 31Pand 19F CS tensors of the P-fluoro-1,3,2diazaphospholene ( R = t-Bu, X = F ) were characterized by solid-state 31Pand 19FNMR studies and quantum chemical model calculations?01 The configuration of certain trifluoromethylated functional dienoates, aryldienoates and triethylnoates was presented by the measurement of their 13C NMR and 19F NMR chemical shifts, and their 3J(C-F), 4J(H-F) and through-space 'J(H-F) coupling constants.302Hot-filament CVD (HFCVD), a non-plasma technique, was used to prepare fluorocarbon-organosilicon copolymers from hexafluoropropylene oxide (HFPO) and hexamethylcyclotrisiloxane. The composition of the copolymer films was established from FTIR spectra and 19Fand 29SiNMR chemical shift assignments.303 The reaction of Xe difluoride with chloroform in PTFE-FEP, Pyrex and quartz tubes was studied using 19F and 13C NMR s p e c t r o ~ c o p y Fluoride .~~ ions in either solution or the solid state exhibit a wide range of 19FNMR shifts due to paramagnetic contributions from solvent interactions or ion-ion overlap. The only truly naked fluoride is the free gaseous ion. The MAS 19FNMR spectra of NMe4F and PMe4Fwere reported and exhibit 19F chemical shifts of -91 and -72 ppm, respe~tively.'~~ Chemical shifts, 8(19F),were recorded for solutions of LiBF4 in di-Et carbonate (DEC), di-Me carbonate (DMC), THF (THF), 1,2-dimethoxyethane (DME), y-butyrolactone (GBL), propylene carbonate (PC) and water.306 The 19F-NMR chemical shifts of the alkali metal fluorides and of monovalent tetraalkylammonium fluorides were measured under strictly anhydrous conditions.307Interaction between a cationic copolymer (acrylamide-trimethylaminoethylacrylate) P(AAm-TMA) and an anionic perfluorinated surfactant (lithium perfluorooctanoate) (LiPFO) was studied using 'H and 19FNMR.308 The halogen bonding between structurally different halo-perfluorocarbons (Rf-X, Rf = aliphatic or aromatic perfluorinated residue, X = I, Br, Cl) and heteroatom containing hydrocarbons (HC-D, HC = hydrocarbon residue, D = electron donor heteroatom) results in the formation

102


of Rf-X-D-HC complexes in the liquid phase. This formation strongly affects the 19FNMR spectra of the perfluorinated partners.30919FNMR chemical shifts of 2,3,4,5,6-pentafluorotoluene, chloropentafluorobenzene, and perfluoro(methylcyc1ohexane)in dilute carbon dioxide solutions were precisely determined at a fixed temperature of 314.3 K over a wide range of pressure from 1 to 35 MPa.310 Binary mixtures of fluorocarbon and hydrocarbon nonionic surfactants derived from the tris(hydroxymethy1)acrylamidomethane (THAM) were examined by 19FNMR spectroscopy and UV-visible spectroscopy in the presence of pinacyanol chloride as a p r ~ b e . ~This ' ' contribution compares the results obtained using two different charge models with those of the cluster model, when they were used to take into account the crystalline intermolecular interactions in the calculation of 19Fchemical shifts The behavior of a new biphotochromic molecule (CC-Z), formed by two naphthopyran moieties linked through a (Z)-ethenic double bond, was studied by 'H and 19F NMR spectros~opy.~'~ It was studied the efficacy of the tris2.17.2 Chlorine (35,37CE)(I=3/2,3/2). glycinatocobaltate(I1) complex ([C~(gly)~]-) as a shift reagent (SR) for chloride by 35ClNMR spectroscopy and compared to that of Co2+(aq). Due to the relatively low thermodynamic stability of [Co(gly)3]-, a 1:3 Co(II)/gly stoichiometric solution at physiological pH is approximately a 2:l mixture of [ C ~ ( g l y ) ~ ( H ~and O ) ~[C~(gly)(H~O)~l+ l .314The one-pot synthesis of nine novel Wrifluoromet hyl- 1,2-dimethyl-1H-pyrazolium chlorides from the cyclocondensation of 4-alkoxy-l,l,l-trifluoro-3-alken-2-ones [CF3C(0)CH:CR10R, where R1 = H, Me, n-Pr, n-Hex, Ph, 4-MeCsH4,4-FCsH4, 4-C1C&4,4-N02C&, and R = Me, Et] with 1,2-dimethylhydrazine,in quantitative yields, was reported. The 'H, 13C, I9F and 35ClNMR chemical shifts of the pyrazolium chlorides were de~cribed.~" 35ClNMR experiments and molecular dynamics modeling provide significant new insight into the structure and dynamics of the interlayer species in the layered double hydroxide (LDH) LiA12(0H)6Cl'nH20.316 This paper presents a 3sClNMR and XRD study of the structure and dynamical behavior of c104intercalated into the interlayers of Mg, A1 and Li, A1 layered double hydroxides ( L D H S ) . ~Despite ~ ~ the importance of the chloride ion in magmas and the fluids that separate from them, very little is known about at structural environments for Cl- in silicate glasses. The first solid-state 35ClNMR data for C1 in silicate and aluminosilicate glasses, made possible by the availability of very high (14.1 to 18.8 T) magnetic fields were presented.318 2.18 Group 18 (3He, '29Xe).- 2.18.1 Helium (3He)(I= 1/2). A 3He NMR resonance of c 6 0 6 - containing He was assigned to Hez at c62-, thus showing that c 6 0 can also accommodate two helium atoms. The 3HeNMR chemical shift of Hez at c60- is 0.093 ppm downfield from the already known resonance of He at c62-. In the reduced endohedral mono- and di-helium C70,the 3He NMR chemical shift of Hez at C706- is 0.154 ppm upfield from the peak of He at C706-.319 Structures of mono-doped fullerenes, CS9Xn and C59X(6mn)m (X = B", N+, P+, As+, Si), the isoelectronic analogues to c 6 0 and C60" with 60 and 66 n-electrons, were


103

investigated at the B3LYP/6-31G* level of density functional theory. The very distinct endohedral chemical shifts of the 66 n -electron systems may be useful to identify the heterofullerenes through their endohedral 3He NMR chemical shifts.320 2.18.2 Xenon ('29Xe)(l= 1/2). Zeolites NaY and mordenite were dealuminated by S i c 4and steaming methods and carefully characterized during and after the various synthetic stages by 27Aland 29SiMAS NMR and X-ray diffraction (XRD) to ensure sample quality. '29Xe NMR spectra for xenon adsorbed in NaX, siliceous Y, mordenite, and dealuminated mordenite were obtained for very low xenon loadings and as a function of t e m p e r a t ~ r e . ~The ~ ' '29Xe-NMRmethod was used for evaluation of the sizes of free volume elements in amorphous glassy materials-random copolymers of tetrafluoroethylene and perfluorodioxoles of different structures.322The authors report, for the first time, a reasonably good calculation of Xe shielding in a f ~ l l e r e n e Relativistic .~~~ contributions to the chemical shift of '29Xein Xe at C60were computed by means of a two-component relativistic d. functional approach.324The measurement of the '29XeNMR chemical shift as a function of density was reported.325The NMR properties (chemical shift and spin-spin coupling constants) of 12'Xe in covalent compounds and weakly bound complexes were investigated by DFT methods including relativistic effects.326129Xe NMR spectroscopy was performed on xenon adsorbates on a iridium single crystal surface.327Relationships among variations of microvoids and gas transport properties for miscible poly(2,6-dimethyl-1,4-phenyleneoxide) (PPO)/polystyrene (PS) blends in the glassy state were investigated by Xe sorpNew cryptophanes-223, tion, Xe permeation, and 129XeNMR -233, and -224 with C2 symmetry, bearing different linkers connecting the two cyclotriveratrylene units were synthesized following a multistep procedure. The formation of the xenon at cryptophane complexes was investigated by '29Xe The Xe shielding tensor surfaces for the Xe-Xe, Xe-Kr, NMR Xe-Ar, and Xe-Ne dimers were calculated as a function of separation, using gauge-including AOs (GIAO) at the Hartree-Fock level, and also using density functional theory with the B3LYP hybrid functional.330The cavity of the crystal part of poly(4-methyl-1-pentene) (PMP) was studied by gas permeation and '29XeNMR measurements of P M P membranes with various degrees of crystall i n i t ~ .The ~~~ gas permeability of poly[ 1-(trimethylsily1)-1-propyne](PTMSP) containing nanoparticulate fumed silica increases with increasing filler content. This unusual phenomenon was explored using 129XeNMR spectroscopy to examine the effect of filler on the free vol. of the PTMSP host Highpressure '29Xe NMR measurements were carried out on dehydrated (*)[ C ~ ( e n ) ~ ] Ctol ~study the pore size as well as the local structure about the confined Xe atoms.333Relationships among '29XeNMR chemical shift, Xe sorption properties, and density were investigated for poly(2,6-dimethyl-1,4-phenylene oxide)-polystyrene miscible blend system in order to evaluate the variation of microvoids by blending.334Platinum nanoclusters and mononuclear platinum complexes in zeolite KLTL were characterized by '29XeNMR spectroscopy at 100-296 K; the chemical shift increased with decreasing temperature, consistent


104

with xenon's increasingly strong interactions with the platinum.335 Xenonbinding sites in proteins have led to a number of applications of xenon in biochemical and structural studies. The authors further develop the utility of 129XeNMR in characterizing specific xenon-protein interaction~?~~ A general correlation for the 129XeNMR chemical shift-pore size relation (6 vs. D) in porous silica-based materials over the range 0.5-40 nm was demonstrated: 6 = 6s/(l + D/b), with 6s = 116 3 ppm and b = 117 8 for the 34 materials In this publication the authors report the first application of continuous flow hyperpolarized (HP) '29Xetechniques to studies of pore structure in ordered mesoporous materials.338Xe atoms in nanochannels of a crystal exhibit anisotropic NMR line shapes that are characteristic of the averaging shielding tensor; the line shape is a manifestation of the systematic variation of the observed component of the tensor with the orientation of the nanochannel axis in the static uniform external magnetic field. A method of calculating the Xe line shapes in nanochannels was presented.339The authors identify the formation of bound '29Xe- Xe molecules as the primary fundamental spin-relaxation process at densities 1)tensors of p-C&F2 can be considered to be at least of the MCSCF accuracy, only the one-bond F C coupling tensor remains the troublesome case for DFT. The performance of DFT was in general very satisfactory for the C-H and H-H coupling tensors. The B3LYP isotropic couplings were often closer to the experimental values than were the MCSCF isotropic couplings, in particular for large systems such as C6H6. For the anisotropic components of "J(C,H) and "j(H,H), B3LYP was clearly the most accurate functional; BLYP results were almost equal, whereas LDA was further off. With the recent implementation of the hybrid B3LYP functional, calculations of predictive quality for the J^ tensors are no longer restricted to small model molecules, opening up the possibility of studying the anisotropic components of f in large organic and biomolecules of experimental interest. Pecul and Helgaker57calculated the spin-spin coupling constants in ethane, methylamine, and methanol using DFT, CCSD (coupled-cluster singles-anddoubles) and MCSCF theory to test the performance of DFT against high level ab initio methods and experimental data. For each molecule, the Karplus curve was evaluated at the three computational levels. The comparisons with ab initio methods indicated that DFT reproduces the 'J(C,H), 'J(C,C), and 'J(N,H) well but is less accurate for 'J(C,N), 'J(O,H), and 'J(C,O) about onebond couplings. Although DFT performed well for the geminal couplings 2J(H,H)and 2J(C,H),the vicinal 'J(H,H) couplings were slightly overestimated. However, it was shown that DFT is sufficiently accurate for most purposes.

6

Heavy Metal-Metal Coupling Constants

Autschbach et al. reported DFT calculation results for one-bond Pt-Pt nuclear spin-spin coupling constants59and one- and two-bond Hg-Hg spin-spin coupling constants.60 Only recently the methodology for a reliable prediction of coupling constants for heavy nuclei based on first principles theory has become available because for their determination both a relativistic formalism and the inclusion of electron correlation are DFT is currently the method of choice for an investigation of properties of heavy metal complexes because of its reasonable accuracy and computational efficiency for many electron system. Researchers are now in the position to theoretically investigate problems regard-

125

4: Theoretical Aspects of Spin-Spin Couplings

ing heavy atom NMR which could not yet be soluved based on experimental data alone. 1J(195Pt, 195Pt)coupling constants have been studied for a long time,.66-69but they are not well understood. For chemically closely related complexes, variations in 'J(Pt, Pt) by an order of magnitude O C C UThese ~ . ~variations ~ ~ ~ ~ do not correlate with distances between the Pt centers. Examples are [Pt2(CO)6]2+, [Pt2C12(C0)2(PPh3)2], and [Pt2(CNCH3)6]2+,with small 'J(Pt, Pt) couplings of 551, 760, and 507 Hz, respectively, and on the other hand [Pt2C14(CO)2l2-and [Pt2Br4(CO)2]2-,with large 'J(Pt, Pt) values of 5250 and 4770 Hz, respectively. Autschbach et ~ 1 calculated . ~ ~ the 'J(Pt, Pt) couplings for the complexes [Pt2(C0)6]2+ (1) and [Pt2C14(C0)2]2-(2) as representatives for the groups of complexes with small and large 'J(Pt, Pt), respectively, in an attempt to reproduce and understand the difference. The Pt-Pt coupling constant of the less stable isomer 3 of complex 2 was also calculated for comparison. All computations were carried out with the Amsterdam Density Functional (ADF) program pa~kage.'~.''Autschbach e t al. employed their recently developed DFT method for the analytical calculation of nuclear spin-spin coupling c o n ~ t a n t sbased ~ ~ ' ~on ~ the relativistic zeroth-order regular approximation (ZORA).72The calculated 'J(Pt, Pt) values 873.6 and 6397 Hz for the complexes 1 and 2, respectively. These values fairly reproduce the experimental values of 550.9 and 5250 Hz for 1 and 2. The D2d-symmetricisomer 3 of complex 2 was found to be less stable by 155 kJ/mol in the computations. The calculated value for 'J(Pt, Pt) for 3 is - 963.4 Hz. Obviously, the coordination to the Pt-Pt fragment by CO in axial position reduces 'J(Pt, Pt) most effectively, because 2 and 3 differ qualitatively only in the placement of the strongly (CO) and less strongly (Cl-) interacting ligands in axial or equatorial position. Autschbach et u E . ~ ~ ] qualitatively explained the differnce between the 'J(Pt, Pt) values for 1 and 2. The pair of the occupied Pt 6s - Pt 6s bonding orbital a and the unoccupied Pt 6s - Pt 6s antibonding orbital o* yields a positive FC contribution to 'J(Pt, Pt) in the bare Pt22+fragment. In the complex 2, the same holds for the pair of 20 and 30* orbitals shown in Figure 1. The donation from the axial ligand a molecular orbitals (MOs) means that the antibonding Pt 6s - Pt 6s orbital a* mixes with the @ @ linear combination of the axial ligand a orbitals. The resultant new occupied orbital la shown in Figure 1 causes a negative contribution to 'J(Pt, Pt) because of the sign pattern of the Pt 6s combinations in la and 3a*. An increasing metal-ligand interaction decreases the positive 20-30" contribution to 'J(Pt, Pt) relative to that of the free

f'

CI CI

-Pt-Pt-

oc *-

,f'

Pt -Pt-

4

1 CI

1

2

3


126

Ligand 0 Ligand

(3

Pt 6s - Pt 6s

Figure 1

Qualitative symbolic molecular orbital ( M O ) diagram for the interaction of the Pt 6s orbitals of the bare PtZ2+ fragment with the axial ligand o MOs

Pt22+fragment. This qualitatively explains the differences of the ligand influence on the 'J(Pt, Pt) value between complexes 1 and 2. For heavy metal coupling constants, it is important to consider the interplay between relativistic effects and the ligands influence in order to rationalize the experimental results. In this context, relativistic effects magnify the influence of the ligands on the metalmetal bond. Scalar nuclear spin-spin coupling constants between mercury atoms yield the largest experimentally known coupling constants. Currently the 1J('99Hg,199Hg) of 284.1kHz of a complex of HgZ2+with the crown ethers, 18-crown-6 and 15-crown-5, reported in 2001 is the largest experimentally observed coupling constants.73Autschbach et ~ 1 . ~ carried ' out the two-component relativistic computation of nuclear spin-spin coupling constants 'J(Hg, Hg) and 2J(Hg,Hg) of the bare ions Hg;+ and Hg32+using the ZORA H a m i l t ~ n i a nand ~ ~ .the ~ ~ LDA Computed 'J(Hg, Hg) values in Hg22+and Hg32+ions were 941.5 and 242.9 kHz, respectively, and 2J(Hg,Hg) in Hg32+was 464.1 kHz. However, it has become clear that ligand and solvent effects must be explicitly considered if we want to understand and reproduce experimental NMR data for heavy metals by theoretical calculation^.^^^^^ When they computed 'J(Hg, Hg) for the 18crown-6-1 5-crown-5 complex of Hg22+with the use of the experimentally determined crystal structure of ref. 73, they obtained the result of 278.4 kHz which is in almost perfect agreement with the experimental value of 284.1 kHz. They showed that the one- and two-bond Hg-Hg coupling constants in Hgj2+is also strongly reduced in the presence of solvent molecules or counterions. It is clear that even subtle effects on the Hg-Hg bond due to the environment of the metal-metal fragments can result in drastic changes of IJ(Hg, Hg) and 2J(Hg,Hg). Two effects were suggested as the enviromental effects: coordination, i.e., the formation of more or less strong bond between the metal atoms and surrounding ligands, solvent molecules, or counterions, and polarization of the metal-metal fragment due to different coordination ligands. Both effects tend to reduce 'J(Hg, Hg) and 2J(Hg,Hg) in Hg22+and Hg32+ions.


7

127

HartreeFock's Stability Problem

The theoretical study of nuclear spin-spin coupling constants in compounds which are of interest for organic and inorganic chemistry is frequently plagued with problems of instabilities or quasi-instabilities (QIs) when the calculations are done at the H F or RPA level of approach. If the molecular system has a n-electronic framework, it is very likely that it will produce at least QI problems when calculations of J are done at that l e ~ e lThere . ~ ~ are ~ ~also some other model compounds for which J calculations cannot be done at HF level even when they are ~aturated.~' Previous works have shown that there are two general strategies to overcome this problem: (i) by using post-HF schemes or (ii) by applying a more crude approach. This is equivalent to say that one schould go in opposite directions including more or less electron correlation in the calculations. Gomez et al. developed a new m e t h ~ d o l o g yto~ ~calculate NMR-J parameters within response theory at RPA level of approach, where the principal propagator matrix can be expressed as a power series. The cause of HF instabilities or QIs at the R P A calculation of coupling constants becomes clear in this approach. The principal propagator matrix elements are given by mpia,jb

=

("A

(4.19)

k mB)&,,

where rn = 1 ( 3 ) for singlet imaginary (triplet real) type property and the + (-) sign is applied between ' A ( 3 A )and ' B ('B). The matrix elements for A and B are:

+

&i)GabGij 2(ajlib)-(ujlbi), Ei)bab&j - (ajlbi), ' B f a , j b = (ablji) - 2(ablq), 3Bja,jb = (ablji). 'Aia,jb

=

(&a -

3Aia,jb

=

(&a -

(4.20)

The indices i , j (a,b) denote occupied (unoccupied) MOs; Ei ( & a ) represents their corresponding orbital energies, and the symbol (ajlib)means the two-electronic integral 1)1$)(2)r-;~tpj( 1 N b ( 2 ) d q d T 2 . The singlet ('P) or triplet ('P) principal propagator matrix can be calculated as a power series,

Jv:(

(mPs)fa,jb = [ E - ' ( I -"NE - ')- '1ja,jb m

=(E-'

f: (.NE-')")

n=O

ia, j b

(4.21) where p stands for the number of terms considered for each series, E is the diagonal matrix built up from the difference of M O energies, and "N means the part of two-electronic integrals in eq. ( 4 . 2 0 ) . The first term of eq. ( 4 . 2 1 ) , obtained from n = 0, yields the Pople-Santry mode1,8O and the RPA approach corresponds to the matrix given by p = 00. The linear response theory for nuclear spin-spin coupling constant calculations at RPA level is equivalent to the finite perturbation theory (FPT)" and the self-consistent perturbation theory (SCPT).*2,83 In


128

both calculations, a QI problem manifests itself as a very low rate of convergence in the iterative procedure.84The maximum value of all ("N E-l)iqjbelements will be written as The convergence of the series is ensured when < 1.$:v' and 3uE:; for C2H4 are 0.371 and 0.964, respectively. It was observed that the singlet-type propagator elements converge to their RPA values quite fast, i.e., for p less than 10 the series was converged. On the other hand, the triplet-type principal propagator counterparts did need a lot more terms to reach their corresponding RPA values, i.e., 500 terms for C2H4 to reach a convergence.It was shown that when unsaturated model compounds have 3u;pixb less than 0.75, they will not be affected by QI problems. G6mez et ~ 1 proposed . ~ ~ a way to overcome QI problems, in which any of the two-electronic integrals, (a@) or (ablji) is removed from the (3N)ia7jb matrix element. They suggest that by this modification 3 max is reduced drastically, and NMR-J parameters close to the best theoretical values may be obtained.

mur;.

8

Imur$I

Other Ab Znitio Calculations of Spin-Spin Coupling Constants

8.1 Spin-Spin Couplings of the NH3*..H20 Complex. - Ab Initio studies of small hydrogen-bonded clusters provide increasingly accurate and reliable results. In particular, the computed structures and geometries of such clusters are in agreement with spectroscopic data. Accurate ab initio methods have been developed recently for the calculation of NMR spectrum parameters3There are many applications now, but most of these calculations are done at the SCF level and often only the shielding constants are considered. The SCF values provide a good qualitative estimate for shieldings, whereas for spin-spin coupling constants the SCF values are unreliable. This is due to the well-known failure of the SCF approximation - it does not describe properly the response to triplet perturbations. More accurate studies, using large basis sets and going beyond the SCF approximation, are performed primarily for small hydrogen-bonded c~mplexes.*~-*~ Janowski and Jaszunskigocomputed the nuclear spin-spin coupling constants for the ammonia-water complex. The NMR parameters of some small complexes involving N - H 0 hydrogen bonds have been previously studied in the literature. However, there was no systematic ab initio study of the simplest system, the NH3- - - H 2 0 complex, though its available results refer mostly to very different systems, such as complexes with oxigen atom acting as proton acceptor. Janowski and Jaszunski considered four sturctures of the NH3 - - HzO complex and computed their coupling constants with the MCSCF method and the aug-cc-PVTZ basis set." It was shown that for all the spin-spin coupling constants the change of the FC contribution due to the intermolecular interaction dominates. For the cis and trans conformers, the largest change of all the nuclear spin-spin coupling constants with respect to the monomer values was obtained for the 'J(H,b,O) coupling, in which Hobis the water hydrogen atom participating in the hydrogen bond. Fairly large values of intermolecular coupling constants across the hydrogen bond, e.g., - 2.51 Hz for IhJ(N,H,b)in trans geometry, were calculated. The value of 2hJ(N,0)in trans geometry was com*


129

puted to be -2.21 Hz. For two bifurcated geometries the intermolecular coupling constants were significantly smaller. On the other hand, a relatively large effect was computed for all the spin-spin coupling constants within the water monomer. Thus, although the water hydrogen atoms do not form a hydrogen bond in the bifurcation geometries, their interaction with the nitrogen atom of ammonia affects the compling constants in water. 8.2 Carbon-Carbon Coupling Constants in Compounds with Strained C-C Bonds. - Krivdin et aZ.92-94investigated the carbon-carbon indirect nuclear spin-spin coupling constants in compounds with strained C-C bonds. Carboncarbon coupling constants J(C,C) play a major role in structural elucidation of organic molecule^^^-^^ and stereochemical a n a l y s i ~ . ~The * ? ~range ~ of 'J(C,C) in organic compounds is about 300Hz (from -20Hz in bicyclobutanes to + 300 Hz in acetylenes), which demonstrates that carbon-carbon couplings are extremely sensitive to structural changes. The most obvious trend is the marked increase of 'J(C,C) with the s-character of the carbon-carbon bond between both coupled nuclei. The unusually low values of 'J(C,C) in three-membered rings could be mainly attributed to the reduced carbon-carbon bond s-character due to strained C-C bond. Furthermore, 'J(C,C) in three-membered rings could be sum of the contributions from the two physically quasi-independent coupling pathways, namely the sum of the genuine one-bond 'J(C,C)* and two-bond (geminal) 2J(C,C)*. 2J(C,C)* was estimated to be negative at about -(6~ ) H Z . ' ~Hence, ~ ' ~ ' 'J(C,C) in three-membered rings could be lowered by the negative 2J(C,C)*contribution. Krivdin et al.92calculated the 'J(C,C) coupling constants in the three three-membered ring compounds of cyclopropane, aziridine, and oxirane at the RPA, SOPPA (second-order polarization propagator a p p r o a ~ h ) , ' ~and ~ , ' ~DFT/B3LYP ~ levels. RPA is equivalent to HF-SCF and SOPPA is the development of the RPA method by means of replacing the H F wave function with that of Moller-Plesset (MP)104'105 at the second-order perturbation theory level. It was observed that all these three 'J(C,C) coupling constants are dominated by the FC contribution, regardless of the approach and basis set91~'06~'07 used in their calculation. The non-contact contribution amounts to only 3-4O/' of the respective FC term. The best SOPPA and DFT results were in a very good agreement with each other and with the experimental values, whereas calculations at the RPA level strongly overestimated the 'J(C,C) couplings. The medium-sized core-valence basis sets cc-p CVTZ and even cc-p CVDZ were found to perform fairly well at the SOPPA level for 'J(C,C) calculation. The best computed 'J(C,C) values for cyclopropane, aziridine, and oxirane were 14.30, 21.83, and 27.81 Hz, respectively, and their experimental values are 12.4, 21.0, and 28.0 Hz, respectively. K r i ~ d i ncarried ~~ out calculations for 'J(C,C) values of bicyclobutane (4), propellane (9,and spiropentane (6) at the RPA and SOPPA levels. The most intriguing fact about carbon-carbon coulings in the bicyclobutane skeleton is, of course, the negative sign of 'J(C,C) between bridgehead carbons, which reflects almost pure p-character of the bridgehead-bridgehead bond in bicyclobutane. The RPA calculations overestimated the values of 'J(C,C) on average by a factor

130

Nuclear Magnetic Resonance 5

4

5

6

of ca. 1.5-2 while the SOPPA level results were in very good agreement with experiment. The calculated bridgehead-bridgehead coupling constant in bicyclobutane was ca. - 14 H z and its experimental value obtained from a substituted & 0.2Hz.Io8 Krivdin estimated the ge bicyclobutane is -17.49 nuine one-bond bridgehead-bridgehead coupling constant 'J(C,C)* in bicyclobutane as - 14 -(- 6) x 2 = - 2 Hz, which gives ca. 10 % bond s-character or sp9-sp9in the formal sp" representation.

7

8

9

K r i ~ d i computed n~~ 'J(C,C)values in the three polyhedranes of tetrahedrane (7), prismane (8), and cubane (9) using the SOPPA method. It was found that SOPPA performs perfectly well in combination with Dunning's correlationconsistent basis ~ e t s . ~ ' ~The ' * ~best ~ ~ calculated *~ 'J(C,C) values for tetrahedrane, prismane, and cubane were 12.51, 12.65, and 31.44Hz, respectivery, and the experimental values for tetrahedrane and cubane are 9.2 and 29.4 Hz, respectively. However, owing to the remarkable instability of prismane, no experimental measurements of 'J(C,C) have been performed either in prismane itself or in any of its known derivatives. 8.3 SpinSpin Couplings in the Organolithium Compounds. - The organolithium compounds are among the most widely used reagents in organic chemi~try.'~'It is because these species play a unique role in synthesis. Their tendency to aggregation in solution is well known, and the consequences of this feature on their reactivity have been the object of continuous studies. The recent increase of high-field NMR performances has made this spectroscopy the best current tool for the study of the solution state of such Still, determining the aggregation level of an organolithium is a difficult task. Bauer, Winchester, and Schleyer have compared the 'J(C,Li) constants for a series of alkyllithiums under various oligomeric forms."' They have deduced from this survey an empirical rule (called hereafter the BWS rule) 'J(13C,6Li) = (17 f


131

2)/nHz, where n is the number of lithium nuclei surrounding the carbon nucleus considered. It seems that the comparison of the experimental 'J(C,Li) or 'J(N,Li) values to the computed ones is very helpful to determine the aggregation state or the geometrical arrangement around the lithium. Parisel et ~ 1 . " ~investigated influence of the electron correlation, aggregation, and solvation on Li-C, Li-N, and Li-Li coupling constants at the H F and MP2 (second-order Marller-Plesset perturbation theory) levels of calculation. They found that electron correlation effects are not the dominant factors to get computationally correct coupling constants. Actually solvation and/or complexation, especially in the very close vicinity of the lithium atom, are/is the essential contribution (s). It was shown that the use of a continuous description of the solvent should be precluded. For example, the 'J(C,Li) value by the BWS rule for MeLi is 17 f 2 Hz, but its MP2 value using the continuous solvent was 27.5 Hz. However, the MP2 value of 'J(C,Li) for MeLi using a cluster model consisting of one Me20 and one MeLi was a much better value of 18.7 Hz. 8.4 Electric Field Effects on 2J(H,H) SpinSpin Coupling Constants. - When a molecule is in a non-gaseous environment, fields originating from localized charges and electric dipoles in the surroundings alter both the chemical shift and the spin-spin coupling constant. B~ckingham"~ first indicated this at 1960. Raynes and Ratcliffe"' worked out a number of unique derivatives in terms of the applied field for the nuclear magnetic shieldings. This problem still remains to be addressed for the spin-spin coupling, where the local symmetry of a two nucleus property has to be considered. Grayson"' calculated the electric field derivatives of the 2J(H,H) spin-spin coupling constants in the series of CH3F, CH3Cl, CH3Br, CH31, CH4, CH3Li, CH3Na, and CH3K. The derivative 8J/8FZ was computed, where F , is the electric field component in the z direction, and the z direction is along the principal axis of symmetry. The C-X heteroatom bond was placed in the positive z direction with the protons at negative z coordinates. aJ/aF, was calculated using the standard 2-point numerical differentiation formula. The field derivative 8J/aF, was calculated with both SCF and SOPPA approaches. It was shown that dJ/dF, was dominated by the FC contribution. All the values of 8J/aF, were positive in sign. The SCF values were too positive. The SOPPA values of d J / d F , for CH3F, CH3Cl, and CH3Br were 56.33, 56.78, and 56.68 Hz/au (electric field), respectively. 8.5 Hydrogen-Bond-TransmittedIndirect Nuclear SpinSpin Couplings. - The detection and characterization of intermolecular interactions, in particular hydrogen bond, are one of the basic goals of physical chemistry. In recent years, it has come to our knowledge that the intermolecular indirect nuclear spin-spin coupling constants can be measured to provide a unique direct experimental evidence for hydrogen-bond f ~ r r n a t i o n . ' After ~ ~ ' ~the ~ discovery of the intermolecular couplings, many theoretical ~ f u d i e s ~ ~ have ~ ~ ~been ~ ' ~undertaken "'~~ to correlate such hydrogen- bond- t ransmit ted couplings with the hydrogen- bond length and the hydrogen-bond en erg^,'^.^^"^^' or with the hydrogen-bond type.130 Pecul et ~ 1 . ' ~calculated ' the hydrogen-bond-transmitted indirect nuclear

132


spin-spin coupling constants for the systems of (NH3)2, (H20)2,and (HF)2and their charged counterparts N2H7+,H 5 0 2 +and , FHF- by linear-response theory using DFT/B3LYP, restricted-active space (RAS) MCSCF, and CCSD58approaches. They investigated the performance of DFT against the high level ab initio methods. As a general rule, it was shown that the reduced coupling constant K decreases with increasing hydrogen-bond length. However, there was one striking exception to this rule: in FHF-, the strongest complex, lhK(H,F)was much smaller than the corresponding constant IhK(H,O)in H 5 0 2 +In . all cases of X-H - Y, the lhK(H,Y)coupling constant changed its sign when the X-H - Y hydrogen bond is strengthened: it was negative in the neutral complexes and positive in the charged ones, at all levels of theory. This is to be expected since, when X is N, 0,or F nucleus, 'J(X,H) transmitted through a covalent bond had the apposite sigh of lhJ(H,Y)transmitted through a weak hydrogen bond. There was essentially no correlation between the reduced 2hK(X,Y)coupling and the X - Y distance. For example, the reduced 2hK(F,F)coupling constant in FHFwas fairly small, in spite of the short F - F distance in this anion. Interestingly, it was shown that 2hK(F,F)changes its sign when going from the strongly bound FHF- complex to the relatively weak bound (HF)2complex. A small absolute value of the coupling is not an evidence either of the large internuclear distance or of the small interatomic interaction. For the proton couplings, the performance of DFT deteriorated with the number of lone pairs on the heavy atom. For hydrogen-bond-transmitted nitrogen couplings, perhaps the most important ones in b i ~ c h e m i s t r y , ~DFT ~~'~~~'~~ seems to be sufficiently accurate. Pecul et al. confirmed the previous observations55,126,133 that CCSD is the only method which reproduces 2hJ(F,F)in an acceptable agreement with experiment. Of the methods used by them, the CCSD method was the most reliable one. However, CCSD had very high basis-set requirements. * *

* *

*

* *

8.6 Substituent Effects on 2J(F,F)and 3J(F,F) Coupling Constants. - J(19F,19F) spin-spin coupling constants (hereafter J(F,F))present some peculiarities which make them more diffecult to rationalize than other types of couplings. For instance, it has not been possible to establish an empirical correlation between vicinal couplings and the corresponding dihedral angles. Based on DFT calculations, it has recently been shown that the dihedral angle dependence for 3J(F,F) couplings is unusual, since noncontact contributions follow separate torsion angle dependence^.'^^ It appears that the fluorine lone electron pairs yield a very important negative contribution to the FC term for the tran~-~J(F,F). In general, coupling constants involving fluorine nuclei are dominated by noncontact contribution~.'~~ Another - ' ~ ~ unusual feature of J(F,F) couplings is that in many cases such interactions have been reported to be dominated by a through-space r n e c h a n i ~ m .It' ~is~ suggested that the behavior of 2J(F,F)and 3J(F,F)couplings, both in saturated and unsaturated compounds, may be governed by substituent effects acting selectively on the four different Ramsey terms (FC, SD, PSO, and DSO). In order to test this hypothesis, Barone et ~ 1 . carried ' ~ ~ out the SOPPA and DFT/B3LYP calculations of the four terms of *J(F,F)and 3J(F,F)coupling


133

constants. It is known that DFT-based approaches to calculating coupling constants fail for some couplings involving at least one F atom.36Thus, results obtained at both the SOPPA and DFT/B3LYP levels should provide an idea of how the latter method performs for J(F,F) couplings by contrast with an inherently superior method. Such comparisons are important because high level ab initio calculations are prohibitly expensive for polyatomic compounds. It was shown that the SOPPA methodology matches absolute experimental values reasonably well, whereas the DFT/B3LY P approach performs poorly in describing 2J(F,F)couplings. Fortunately, substituent effects were markedly better reproduced than total coupling constants. Aliphatic 2J(F,F)couplings were very sensitive to the electronegativity of substituents placed a to the CF2 group. The substituent effects on 2J(F,F)were dominated strongly by the sum of the noncontact PSO and SD contributions, though the former was much more important than the latter. Substituent effects on 2J(F,F)and 3J(F,F)couplings in fluorinated derivatives of ethylene were also dominated by non-Fermi contributions.

8.7 Through-Space Spin-Spin Couplings. - Nuclear spin-spin coupling is normally thought of as a probe of connectivities through covalent bonds only. Recently, it has been demonstrated that spin-spin coupling can also be transmitted through hydrogen bond (HB),'407141 as long as the two interacting moieties remain at a suitable distance for a time long enough for NMR detection. Furthermore, the unusually large long-range J couplings involving "F have been known for quite some time, and a through-space mechanism has been invoked in this connection, for example to explain 6J(H,F) and 'J(C,F) couplings in fl~oronaphtha1enes.l~~ An obvious extension of the above ideas predicts that spin-spin coupling might be detectable even in the case of dispersion-bound van der Waals complexes, For example, Pecul et al. calculated a surprisingly large coupling for the helium dimer (J(3He,3He) = 1.3Hz at the equilibrium dis~ t a n ~ e )and ' ~ ~the H F - CH4 van der Waals dimer (J(H,F) = ca. 4 H z ) . ' ~Previously, Bagno et al. predicted by means of DFT calculations that for two simple van der Waals dimers (methane-benzene and benzene-benzene), a small but non-negligibe intermolecular 13C- - 'H coupling (0.2-0.3Hz) may exist between nuclei belonging to nonbonded r n o l e ~ u l e s . Bagno ~ ~ ~ ~ 'et~ ~ all4' broadened the scope of this investigation to other van der Waals dimers involving methane, ethylene, and benzene. They used the RASMCSCF, SOPPA, and DFT methods for calculating coupling constants. In the range of intermolecular separations for which the interaction is stabilizing, weak couplings (0.1-0.3 Hz) were predicted for J(C,H), while the corresponding J(H,H) couplings were much smaller and negligible. SOPPA and RASMCSCF yielded essentially the same results. However, DFT systematically predicted J(C,H) to be 1.5 times larger than what was predicted by RASMCSCF and SOPPA, despite the high accuracy of DFT in calculating spin-spin couplings in covalent bonded hydrocarbon^.^^ They also performed DFT calculations for three large CH/n interacting compounds, and found the similar magnitude of through-space J(C,H) couplings. Bango et al.146investigated, by means of SOPPA and DFT calculations, the magnitude of through-space spin-spin couplings of J(C,H) and J(H,H) in CH/n * *

*

134


10

11

bonded van der Waals dimers involving acetylene, and in a structurally related covalent compound, 4-ethynylphenanthrene (10). Within regions where the interaction is stabilizing, J(H,H) couplings were very small for all complexes with the T-shaped arrangement between C2H2 molecule and C-H fragment. In the acetylene dimer (ll),J(H,H) showed a large dependence on the tilt angle a from the T-shape arrangement. J(H2,H3) increased from 0.03 Hz in the T-shaped arrangement up to 0.48 Hz for a slipped parallel configuration at the DFT level calculation. At the SOPPA level, J(H2,H3) increased from 0.17 Hz to 0.64 Hz. J(C,H) was also very small, but in the acetylene dimer 11 it showed a large dependence on the tilt angle a. At the DFT level, J(C3,H2) increased from - 0.1 1 Hz to 0.56 Hz. An interesting system for experimentally testing the through-space spin-spin couplings predicted for the model van der Waals complexes is 4-ethynylphenanthrene 10. In this molecule, a C-H aromatic bond is forced to lie close to the n system of the triple bond. The DFT calculation for 4-ethynylphenanthrene 10 showed that J(Hl,H2) is -0.11 Hz and J(Hl,C4) is - 0.35 Hz. The general behavior in 4-ethynylphenanthrene was qualitatively similar to that in the benzene-acethylene complex.

9

Other Density-Functional Calculationsof Spin-Spin Couplings

9.1 Relativistic Density-Functional Calculations. - The recent resurgence of activity and interest in noble gas chemistry draws attention to the multi-nuclear magnetic resonance for this field, in particula for xenon The most abundant (26.44%) NMR-active xenon nucleus, '29Xe,has a nuclear spin of 1/2 with a magnetic moment with magnitude slightly greater than that of 13C,which has made '29XeNMR studies feasible for a wide variety of xenon compounds. Furthermore, since most xenon compounds contain fluorine (19F;I = 1/2; natural abundance = 100 Yo),a wealth of spin-spin coupling constant data of J(129Xe,'9F)exists. Many polyatomic group 17 fluorides are isoelectronic or isovalent with xenon fluorides. A systematic investigation of the 3 tensors for the polyatomic group 17 fluorides has been made.35*52,65,137,'48 However, for a reliable calculation of the spin-spin coupling constants involving heavy elements such as xenon, iodine, and bromine, relativistic effects must be taken into account. Bryce and W a ~ y l i s h e nperformed '~~ the extensive calculation of the complete reduced coupling tensors, R, for the chlorine-fluorine, bromine-fluorine, iodine-fluorine,


135

and xenone-fluorine indirect nuclear spin-spin coupling constants using the relativistic zero t h-order regular approximation densit y-functional theory (ZORA-DFT) approach. Overall, the agreement between the calculated and experimental isotropic spin-spin coupling constants was excellent. It was demonstrated that a correlation exists between the number of lone electron pairs on the central heavy atom and the value of the PSO contribution to the reduced isotropic coupling constant, K . Furthermore, the value of the F C + S D term seems to increase approximately linearly with the separation between the central heavy nucleus and the fluorine nucleus for a given series of compounds. By considering both of PSO and FC + SD contributions to the total isotropic value, the experimental data can be explained. The alsolute sign of the coupling constants was assigned. The experimentalist should be aware that coupling constant values may have oposite signs for compounds which may seem to be closely related. It was shown that for a given set of isoelectronic and isostructural compounds, the value of reduced coupling constant K(X,F) (where X = Cl, Br, I, Xe) tends to increase as the atomic number of X is increased. 9.2 Calculation of J(F,F) Spin-Spin Coupling Constants. - Nonrelativistic theoretical studies broadly followed two different methodological approaches: post-HF methods and DFT approach. To obtain reliable results with post-HF met hods, it has been necessary to employ extensive computational resources even for small-sized structures. Therefore, systematic calculation of spin-spin couplings in medium-sized compounds with reasonable accuracy has been envisioned as a useful and complementary tool for experimental determinations. Barone et performed DFT calculation of J(F,F) spin-spin coupling constants in fluorinated pyridines. Prediction of J couplings for lone-pair-containing nuclei is a challenging task for any computational method, and DFT-based methods are not exception to this rule.38 However, recent preliminary DFT calculations of J(F,F)seem to be quite promi~ing.'~' Barone et aE. showed that experimental J(F,F) values in fluorinated pyridines are semiquantitatively reproduced by DFT calculation. It was shown that with few exceptions, the sum of the SD and PSO noncontact terms is larger than the FC contribution, even though in most cases the two noncontact values have opposite signs. The very large positive 4J(F4,F5)coupling constant (calculated value is 46.3 Hz and experimen(12)is dominated tal value is 45.8Hz) in 2-bromo-3,4,5,6,7,8-hexafluoroquinoline by a through-space mechanism, which has been discussed in many paper^.'^*^'^'

F

12

136


9.3 Spin-Spin Coupling Constants in Formamide. - All of the nuclei in formamide have NMR active isotopes: 'H, 13C,"N, and 1 7 0 , which lead to 15 different couplings. Hansen et calculated the 15 spin-spin coupling constants of formamide using a method combining FPT and DFT. Basis sets with decontracted s functions were used. Comparing the experimental values with the spin-spin coupling constants calculated at the largest basis set (6-311 + + G**) resulted in 0.8 Hz for B1LYP,'53,'54 and a mean absolute deviation of 2.6 Hz for B3LYP,44p45 4.4 Hz for NPW1PW91.15' 9.4 'J(C,C) and 'J(C,H) Coupling Constants. - Kamienska-Trela et ~ 1 . call ~ culated 'J(C,C) values for cyclopropene, its methyl and silyl derivatives, 1methylcyclobutene, 1-methylcyclopentene, and 1-methylcyclohexene to complement experimental data. A good agreement was observed between the experimental and calculated data. The results clearly showed that the ring closure and the related strain exerted upon the cyclopropene molecule only slightly disturb the electron structure of the double bond. The s characters of carbon bonds were estimated.

Q,/"

H -

13

14

Zaccari et al.lS7investigated the lone electron pair effect on 'J(C,H) couplings. The importance of lone pair orientation for 'J(C,H) couplings in a configuration like 13 has been known for a quarter of a 160 'J(C,H,) couplings are uniformly larger than 'J(C,H,) couplings in structures such as 14. The stereoelectronic interaction between a non-bonding electron pair and a C-H bond in a conformation as in 13 is frequently called *the anomeric effect'.'61However, since the interaction can be ascribed to a charge transfer interaction (CTI) from a non-bonding electron pair into a (C-H)* antibond, it can also be described as a negative hyperconjugation interaction (NHI).'62Zaccari et studied the NHI effect on 'J(C,H) couplings in the NH = CH2molecule by applying the natural localized molecular orbital (NLM0)'63procedure to the CTI from a lone electron pair into the antiperiplanar (C-H) antibond (n(N)+ (C-H)*). They showed that solvent effects, introduced by using the polarizable continuum model (PCM)'64*'65 or the molecular complex model, should be taken into account to improve the agreement between the calculated and experimental values of 'J(C,H,) and 'J(C,H,). invesSolvent Effects on Spin-Spin Coupling Constants. - Zaccari et tigated solvent effects on 'J(C,,HI) and 'J(C1,Hf)couplings in acetaldehyde (15). 9.5

*

~

4 : Theoretical Aspects of Spin-Spin Couplings

137

Two-bond coupling constants, 2J(X,Y),across a carbonyl carbon atom, C,, are known to be (provided the magnetogyric ratios of both coupled nuclei are of the same sign) and their absolute values are uncommonly large. Such features were rationalized in previous work'68 as originating mainly from the strong CTI between the carbonyl oxygen lone pair of purely p character, n(p), and the C,-X and C,-Y antibonds. This suggests that this type of coupling constants should be sensitive to solvent effects, which either enhance or inhibit such charge transfers. The former increases the corresponding two-bond coupling, while the latter decreases it. Solvent effects on 'J(C,,Hf) and 2J(C1,Hf) couplings were analyzed. It was shown that solvent effects tend to improve the agreement between calculated and experimental values. However, a continuum dielectric model had not enough flexibility as a molecular complex model for describing quantitatively solvent effects on spin-spin couplings.

15

Ruud et calculated solvent effects on J(C,H), J(C,C), and J(H,H) coupling constants of benzene using the PCM. The calculated solvent shifts were in good agreement with experimental observations when geometry relaxation was take into account. They proposed 157.46 Hz as a new experimental gas-phase value of the 'J(I3C,'H) spin-spin coupling constant of a vibrating benzene molecule at 300 K.

9.6 Calculations of Spin-Spin Couplings as an Aid to the Conformational Analysis. - Although the calculation of chemical shifts is an established tool of computational chemistry, that of spin-spin couplings has lagged behind somewhat owing to its intrinsically more complex nature. However, thanks to the advancements made in this field, namely, the recent development of the0ry,3,'~~ and the ready availability of increased computing power and code, this situation has rapidly changed. The stage has now been reached where meaningful and reliable calculations can provide synthesized spectra amenable to structrural analy~is.'~' Tahtinen et ~ 1 . used ' ~ ~ D F T calculations of spin-spin coupling constants to determine the preferred conformations of 10 compounds, viz., four 7amethyl octa(or hexa)hydrocyclopenta[d][1,3]oxazines, five 8a-methyl octa(or hexa)hydro[3,l]benzoxazines, and 8a-methyl hexahydro[ 1,3]benzoxazinone. Following geometry optimization, both the proton chemical shifts and the vicinal coupling constants were calculated. The agreement between the calculated and experimental vicinal coupling constants was found to be good. Cuevas and J u a r i ~ t icomputed '~~ 'J(C,H) coupling constants in 16 compounds of cyclohexane, four six-membered heterocycles, cyclohexanone, and ten cyc-

138


lohexanone derivatives to investigate stereoelectronic hyperconjugative effects. Both r(C-H) bond lengths and differences in 'J(C,H) coupling constants supported the validity of their models based on stereoelectronic effects. Furthermore, calculated r(C,H) bond lengths and 'J(C,H) coupling constants for C-H bonds participating in more than one hyperconjugative interaction showed additivity of the effects. Wu and Cremer'74 derived Karplus r e l a t i o n ~ h i p s ' ~for ~ ' ~all ~ 26 NMR spin-spin coupling constants J of the pseudorotating tetrahydrofuran (THF) molecule by expanding J as a function J(q,$) of the puckering amplitude q and the pseudorotational phase angle 4. For this purpose, the conformational potential V(q,$)and the nuclear spin-spin coupling constants "J(q,$) of T H F were calculated using DFT approach. The calculated coupling constants "J(q,$) were averaged with respect to $ to give average coupling constant ("J) values that can be compared with measured coupling constant " J data. The computed average coupling constant values ("J) were in reasonable agreement with measured "J values. T H F is one of the suitable modelsfor ribose. Wu and Cremer17' showed that functions "J($) calculated with DFT for a parenent molecule can also be used for derivatives of the parent molecule and helpful to determine their conformational probality distribution function p($).

9.7 Calculations of Spin-Spin Couplings in DNA or RNA. - It is well established that conformation, interactions, and other properties of DNA molecules depend not only on nucleotide sequence but also on solvent omp position.'^^^''^ NMR measurements'81 confirmed the existence of bound waters in the minor groove "spine of hydration"'s2 indicating that lifetimes of at least some water molecules are sufficiently long to be detected by NMR techniques. Sychrovsky et ~ 1 . ' ' ~theoretically investigated influence of water on isotropic one-bond 'J(X,H) and 'J(C,X), and two-bond 2J(X,H),(X = C,N) indirect NMR spin-spin coupling constants at DFT level by application of explicit and PCM solvent models. It was shown that sensitivity of the FC contribution twards the solvent inclusion is dominant, while solvent shifts of the other three contributions (PSO, DSO, SD) to the couplings are negligible. Consideration of a single water molecule led to a noticeable shift of the 'J(C,H) couplings; the largest shift of 6.1 Hz was found for 'J(C8,H8) coupling in guanine (16). Further inclusion of solvent water molecules decreased the mean absolute diviation between the calculated and experimental J constants from 1.7 to 1.1Hz for guanine, from 2.4 to 0.6Hz for cytosine, and from 2.3 to 1.6Hz for adenine.

k

A 16


139

The utilization of the torsion-angle dependence of vicinal scalar couplings for structural studies of organic molecules is based on the classical work of Kar~ l u s . ’ ~ In ~ - biomolecular ’~~ NMR, Karplus relationships can be used for the determination of the glycosidic torsion angle x that defines the orientation of the aromatic base with respect to the ribose (RNA) or 2’-deoxyribose (DNA) moiety in purine and pyrimidine nucleotides. Munzarova and Sklenar184analyzed the relationship between the glycosidic torsion angle x, the three-bond couplings 3J(C2/4,H1’) and 3J(C6/8,H1’), and the one-bond coupling ‘J(Cl’,Hl‘) in deoxyribonucleosides and a number of uracil cyclo-nucleosidesusing DFT. The influence of the sugar pucker and the hydroxymethyl conformation was also considered. The change in the sugar pucker from S to N decreased ’J(C2/4,Hl’) and 3J(C6/8,H1’),but increased ‘J(Cl’,Hl’) for the syn rotamers, whereas all of the trends were reversed for the anti rotamers. Most of theoretical ’J(C2/4,Hl’) and 3J(C6/8,H1’) couplings for uracil cyclo-nucleosides compared well with available experimental data. Theoretical ‘J(C1’,H1’)values were underestimated with respect to the experiment by ca. 10% but reproduced the trends in ‘J(Cl’,Hl’) us. x. Munzarova and SklenarlS5performed a series of 3J(C2/4,H1’) and 3J(C6/8,H1‘) calculations for deoxyadenosine (A), deoxyguanosine (G), deoxycytidine (C), and deoxythymidine (T) with x varying from -180” to 180”.A significant difference between purine and pyrimidine bases was found for 3J(C6/8,H1’). Hydrogen bonds (H-bonds) play a key role in both the stability and specificity of secondary and tertiary folding interactions in nucleic acids. The ability to directly observe individual hydrogen bonds and identify the participating atoms has provided valuable information about macromolecular structure and strength of interatomic interaction~.’~~ Luy et ~ 1 . performed ’ ~ ~ DFT calculations of 3hJ(H2,H3)coupling constants to predict the relative contributions of H-bond length and angular geometry to the magnitude of 3hJ(H2,H3)using a molecular fragment representation of an AU base pair. Although the DFT calculations did not reproduce the full range of magnitude observed experimentally for the 3hJ(H2,H3)coupling constants, the calculations predicted the correct sign and general trends in variation in size of these coupling constants. The calculations suggested that the magnitude of the coupling constants depends largely on H-bond length, but can also vary with differences in base pair geometry. The dependency of the 3hJ(H2,H3)coupling constant on H-bond strength and geometry may make it a new probe for defining base pairs in NMR studies of nucleic acids.

+

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5 Applications of Spin-Spin Couplings BY K. KAMIENSKA-TRELA AND J. WOJClK

1

Introduction

The material in this chapter covers the period from 1 June 2002 to 31 May 2003. It has been arranged as was done previously,'i.e. according to (i) the increasing atomic number of the nuclei involved, and (ii) the number of the bonds separating them. We follow the IUPAC2recommendations with one notable exception, namely, the nucleus with the smaller mass is given first. For the sake of simplicity the following symbols were used throughout the paper: H for 'H, D - 2H,T - 3H, Li - 6Li,Be - 9Be,B - llB, C - 13C,N - 15N,0 - 170,F - 19F,A1 27Al,Si - 29Si,P 31P,S - 33S,V - 51V,Mn - 55Mn,Fe - 57Fe,Co - 59C0,Cu - 65Cu,Se - 77Se,Br - 79Br, Y - 89Y,Nb - 93Nb,Mo - 9 5 M ~Ru, - 9 9 R ~Tc, - 9 9 T ~Rh , - lo3Rh,Ag - lo9Ag,CD 'I3Cd, Sn - '19Sn, Te- "'Te, I -1271,Cs - 133Cs, W - 183W,0s- 1870s,Pt-195Pt,Hg - 1 9 9g, ~ TI - 205Tl, P b - 207Pb.All the other isotopes are described explicitly. A special issue on NMR spin-spin coupling constants, their calculations and measurements has been published by International Journal of Molecular Science providing a good representation of the diversity of different research activities in this field.3 A progress on the application of theoretical methods to NMR chemical shifts and couplings has been written by McKee? and an extensive review on DFT and ab initio calculations of scalar couplings through hydrogen bonds and in van der Waals complexes has been published by Alkorta and Elguero,' who also collected the sources of experimental data on nhJXyscalar couplings. The problems connected with the question whether the existence of spin-spin couplings between a pair of atoms can be related to bond polarity have been reviewed by Bartlett et aL6 A review on synthesis, structural analysis and reactivity of 1,3-oxathiane derivatives has been written by Terec et aL7 A short review on gas-phase studies of spin-spin couplings has been published by Jackowski8 An extensive review which covers the literature prior to autumn 2001 on experimental and computational aspects of spin-spin coupling tensors has been published by Vaara et al.9 A review on experimental aspects of 29SiNMR in organosilicon liquids, which included solvent effects, determination of couplings, sensitivity enhancements Nuclear Magnetic Resonance, Volume 33 0 The Royal Society of Chemistry, 2004

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5: Applications of Spin-Spin Couplings

147

etc., has been published by Schraml.'O Two reviews have been written by Wrackmeyer. One covers chemical shifts and couplings of selected, rather unusual organometallic compounds," the other is devoted to the 207PbNMR spectroscopy.12 In his personal review on the history of structure elucidation of palytoxin Kishi13has underlined the importance of 3 J H H vicinal couplings as a source of structural properties of this compound. A review devoted to dipolar and scalar couplings in solid state NMR of qudrupolar nuclei has been written by J e r s c h o ~ , 'and ~ the solid-state NMR spectroscopic methods in chemistry are reviewed by this author, Laws and Bitter.'' They have pointed out that the recent technological breakthrough has made observations and utilizations of scalar couplings in polycrystalline samples possible. MacDonald and LuI6 have reviewed the triple use of residual dipolar couplings in nucleic acid structure determination. The review includes: refinement of the solution structure obtained using other NMR data, determination of the relative domain orientation and determination of the stoichiometry of homomultimeric complexes. Bax17has shortly reviewed opportunities provided by dipolar couplings for structure and internal motions determination in proteins. Koeniglg in his minireview has described the use of transferred dipolar couplings which allow one to study structure and orientation of the peptide ligands bound to membrane proteins. Opella et aE.I9 have reviewed the use of dipolar waves constructed from the PISEMA spectra in structure determination of membrane proteins. For a-helical peptide chains the waves describe the periodic wave-like variations of the magnitude of the heteronuclear dipolar couplings as a function of the residue number. They also provide information on the a-helix tilt. Two reviews have been published in Japanese. One, by Katahira;' refers to general aspects of spin-spin coupling, in the other, written by Takemura,21 problems connected with the interaction of the C-F bond with metal cations are discussed.

2

New Methods

A new technique which allows one to discriminate between 2 J H c and 3 / 4 J H c connectivities has been proposed by Sprang and Bigler.22The method has been demonstrated by using strychnine as a test example. Recently, the method designed to extract homo-and heteronuclear long-range couplings from spin echo experiments has been published by Mahi and Duplan.23In this method a set of signals is analysed in the time domain by an integration program. The authors have introduced substantial modifications into this technique which allow them to measure very small couplings in the presence of chemical exchange. It is of interest to note that spin-spin coupling is very difficult to measure in this situation, because the chemical exchange leads to an additional broadening of the individual signals.

148


A selective pulse sequence for the determination of long-range C,H spin couplings has been proposed by Findeisen and B e ~ g e r It . ~ is ~ based on the combination of a selective J-resolved spectrum with selective HMBC and displays the long-range spin couplings of one chosen carbon in the indirect dimension. A new optimised method for the measurement of the 3 J H H coupling between chemically equivalent vicinal protons has been reported by Glaser and coworker~.*~ Zwahlen and c o - w ~ r k e r s have ~ ~ , ~introduced ~ "N-COSMO-HSQC (Cosine Modulated HSQC) method26for rapid determination of homonuclear 3 J H N H a couplings in "N- labelled proteins and '3C-COSMO-HSQC27for determination of 3 J H H in unlabelled carbohydrates. Vogeli and Pervushin28have introduced [15N,'3C]DQ/ZQ-["N,'H]-TROSY-E.COSY experiment for the simultaneous measurement of intraresidual 3 J H N H a and sequential 4 J H N H a couplings in l3C,I5N labelled proteins. An effective strategy for the measurement of 3 J H H couplings in oligosaccharides has been proposed by Bendiak and c o - w ~ r k e r sThis . ~ ~ includes peracetylation of the sugar using doubly 13C-labelledacetyl groups and the use of PIP-TOCSY experiments. The iHN(CA),CO(a/P-J-C0HA)-TROSY experiment for the determination of 3JHaccouplings related to the dihedral angle cp in proteins has been proposed by Permi.30 Schwalbe and co-workers31have developed the T-HMBC experiment for the measurement of 3JHc couplings, which are very important for the conformational analysis of the glycosidic linkage in sugars. Lin and L i a have ~ ~ described ~ a sensitivity-enhanced SE-IPAP experiment designed for the measurement of ' J H N and IDHN couplings in proteins. Kozminski and c o - ~ o r k e rhave s ~ ~ presented a new J-HMQC-based technique for quantitative determination of 3 J H p couplings in DNA. Uhrin34has proposed a 3D HSQC-HSQMBC experiment with the increased resolution for the measurement of long-range proton-carbon couplings. The method for the accurate measurement of one- and two-bond J C N couplings of the backbone of 2H,13C,15N labelled large proteins has been described by Lohr and c o - w ~ r k e r s These . ~ ~ coupling values correlate well with the protein backbone w torsion angles. Marino and c o - ~ o r k e r have s ~ ~ introduced a new 2hJNN-HNN-E. COSY experiment designed for observation of the 3 h J H 2 H 3 scalar couplings in oligonucleotides. Bodenhausen and c o - ~ o r k e rhave s ~ ~ proposed the new method, HSQC-PEC2 (Heteronuclear Single Quantum Correlation with Phase Encoded Couplings and Partial Error Compensation), for the accurate measurement of dipolar couplings. The method allows the reduction of systematic errors caused by miscalibration of radio-frequency pulses and by relaxation effects. Brunner and c o - ~ o r k e rhave s ~ ~ presented a new TOCSY-based homonuclear 2D NMR experiment, MOCCA-SIAM, which allows the measurement of residual DIINHadipolar couplings of non-isotope enriched proteins. Wu and Bax39 have demonstrated a SS-HMQC2 (semiselective HMQC) method for the


149

measurement of D H H dipolar couplings in proteins. Uhrin and co-workersa have proposed a simple 1D directed COSY experiment for high precision measurement of n D H H couplings in oligosaccharides. Ding and Gronenborn4' have presented a novel sensitivity-enhanced (A,J)E.COSY HSQC experiment for the accurate measurement of ' D H N , 'DCN, and 2DHcdipolar couplings in proteins. Montelione and c o - ~ o r k e rhave s ~ ~ described an automatic strategy for rapid determination of the medium-accuracy structure of large deuterated proteins with NMR data including ' D H N and 'Dee dipolar couplings. JE-TROESY, a combined J - and TROSY spectroscopy for the measurement of ' D H C and 'DHNdipolar couplings in large '3C,'5N-labelled proteins and RNA molecules has been presented by Luy and Marin0.4~ Two 2D J-modulated HSQC-based experiments for the precise determination of small IDHCin natural abundance oligosaccharides have been designed by Pham et a1.44IPAP- and IPAP-relay- HCcH-COSY experiments have been introduced by Vallurupalli and Moore45to improve the measurement of ' D H C couplings in large RNA molecules. Bruschweiler and c o - ~ o r k e r have s ~ ~ presented a new strategy for the 'inverse' assignment problem. Using residual dipolar couplings, chemical shifts and a known 3D protein structure the authors have determined the NMR resonance assignment; in the case of ubiquitin a combinatorial optimisation algorithm has yielded 90% of the correct backbone resonances. In the paper by the same group4' the method for assessing structural heterogeneity in different alignment media has been proposed. The method is based on a principal component analysis of the covariance matrix of the dipolar couplings measured in six or more media. Zweckstetter and B ~ have x discussed ~ ~ the uncertainty in alignment tensors obtained from dipolar couplings caused by the uncertainty in the structure. They have found that the structural noise results in a systematic underestimate of the magnitude of these tensors. Rosevear and c o - ~ o r k e rhave s ~ ~ developed a new approach of derivation of structural restrains, ' D H N , in which a metalbinding tag is covalently attached at a unique site in the target protein. The authors have incorporated thiol-reactive EDTA at monocysteine derivatives of barnase thus permitting binding of Yb3+, Mn2+, Co2+ or Zn2+. The RDCs measured in these complexes were advantageous in structure determination of the protein. Pervushin and co-workers5' have outlined the perspectives of the use of residual IDCC couplings for structure determination of larger uniformly deuterated proteins. Now, these couplings have become available since the introduction of cryogenic probes makes sensitive detection of 13C resonances possible. Griffin and co-workers51 have described 3D TEDOR experiments for the measurement of weak l3C,l5Ndipolar couplings in double labelled proteins in solid phase. Schwieters et a152have announced the availability of the Xplor-NIH software package for biomolecular structure determination that utilizes J and D NMR observables. McAteer and Kennedy53 have found that X-Plor and Discover programs produce different final structures of DNA molecules despite using

150


identical distance and dihedral restraints. This force field dependence can be potentially overcome if residual dipolar coupling restraints are employed. Fermandjian and c o - ~ o r k e r susing ~ ~ simulations have shown that a small set of RDC constraints detectable at the natural abundance is sufficient to accurately derive the global and local DNA duplex structures when used in conjunction with NOE constraints. Tolmans5has proposed a novel method that allows the simultaneous extraction of both structural and dynamic information directly from RDCs. The method requires the collective use of RDCs measured in several oriented media. Using DFT calculations Bryce and W a ~ y l i s h e nhave ~ ~ evaluated the influence of the anisotropic indirect nuclear spin-spin coupling tensor on residual dipolar couplings. They have found that in model peptides these contributions to one bond RDCs are of the order of 0.5%. DFT calculations of indirect coupling tensors, J , have been performed by Lantto et aZ.57for a series of molecules such as acetylene, ethylene, ethane and benzene by the use of the linear response method. This is the first paper which reports the anisotropic components of J for a hybrid functional. An addendum to 'A practical guide to first-order multiplet analysis in 'H NMR spectroscopy' has been published recently by Hoye and Z h a ~ . ~The ' method allows one easily to extract couplings from the first-order 'H NMR spectra.

3

One-bond Couplings to Hydrogen

couplings have been used to characterize bis(dihydrogen)complexes [Cp*(Ru(H2)2(EPh3)]+(Cp* = C5Me5; E = Sb, the iron dihydride [Cp*Fe(dppe)(H2] BF4-,6' and the non-classical polyhydride complex [Cp*(PMe3)Rh(H)(H2)]+B(3,5-C6H3(CF3)2)4-.61 The l J H D and 'JHTcouplings have been measured for the latter compound, ca. 10 and ca. 70 Hz, respectively. Electronic mechanisms of intra and intermolecular J couplings in systems with CH--O interactions have been discussed by Giribet et ~ 1 The . known ~ ~ solvent dependence of *JHfCc and 2JHfc1 couplings in acetaldehyde has been studied by Zaccari et a1.63by the use of the DFT approach where the dielectric solvent has been taken into account with the polarizable continuum model. Studies on solvent effects on indirect spin-spin couplings, including ' J H C couplings in benzene by the use of the DFT-PCM approach have been performed by Ruud et Karplus relationships for a full set of J couplings (26) including those across one H-C and C-C bonds have been derived by Wu and Cremer6' for the pseudorotating tetrahydrofuran molecule. They expanded J as a function J(q, $) of the puckering amplitude q and the pseudorotational phase angle +. One-bond proton-carbon couplings are often used as a probe for steroelectronic hyperconjugative effects. Recently, the influence of these effects on the calculated carbon-hydrogen bond lengths and one bond JHc couplings in cyclohexane, six-membered heterocycles and cyclohexanone derivatives has been lJHD

+


151

studied theoretically by Cuevas and Juaristi.66They have found that the calculated YC-H and ' J H C for C-H bonds participating in more than one hyperconjugative interaction show additivity of the effects. A computational study of conformations and conformers of 1,3-dithiacyclohexane by the use of ab initio MO and DFT theories which included analysis of lJHC couplings has been performed by Freeman and Le.67Stereoelectronic substituent effects on intramolecular H-bonding in the crowded o-anisic acids have been studied by Schuster6*who applied the DFT method. The coupling values of ca. 163 Hz observed for the C1 carbon in the products of the reduction of cyclophane[2]metacyclo[2](2,7)pyrenophane and [7] (2,7)pyrenophane were typical of a strained sp3-hybridized carbon atom. This provided evidence that the benzene ring in the starting compounds is converted into a 'cyclopropano-cyclopentano' ring system.69 Afonin et al. have continued their studies on the electronic and spatial structures of heteroaromatic compounds in which analysis of lJHC couplings played an important role. This included heteroaryl vinyl ethers and sulfides," 2-(2acetyletheny1)-1-vinylpyrroles;' 2-(2-furyl)- and 2-(2-thienyl)pyrrole~.~~ A linear correlation between ' J H Ccouplings and the Charton steric parameter, v, for the alkoxy groups has been observed by Belarmino et ~ 2 1 in . ~their ~ studies on the effect of the alkyl group size on the mechanism of acid hydrolyses of benzaldehy de acetals. The E configuration of 5-chlor0-1,3-dimethyl- and 5-chloro-3-methyl-lphenyl- 1H-pyrazol-4-carbaldehyde has been assigned on the basis of the ' J H c coupling values ('J = 162.9 and 165.0 Hz, re~pectively).~~ Considerably larger ' J values, of ca. 177 Hz, are expected for the 2 configuration. 'JHC couplings have been applied by Katrizky et ~ 2 1 to . ~distinguish ~ the cis and trans isomers of 2-(benzotriazol-l-yl)-substitutedtetrahydrofurans, and by Perillo et ~ 1 in .conformational ~ ~ analysis of N-benzylimidazolidines. ' J H Cand 3 J H H couplings have been used by Lam and H s u to ~ ~characterize the left-handed polyproline type I1 helix. This secondary structure has been found by the authors for the peptide of the sequence YGRKKRRQRRRP, HIV-1 Tat protein RNA binding domain (47-58). 'JHC and 3 J H H couplings have been helpful in the characterization of different amounts and various types of bis-dinuclear molybdate complexes of octoses and their a l d i t ~ l s . ~ ~ Cremer and c o - ~ o r k e rexplored s~~ the structure of DNA hairpin using several heteronuclear couplings including 'JHC and 'JHN.Later, Sychrovskjl and coworkerss0 investigated theoretically at ab initio level the effect of water on one-bond and two-bond couplings in this molecule. The authors have obtained the largest improvement in the case of 'JH8C8couplings. The 'JHC couplings have already been shown to be directly correlated to the degree of agosticity as the coupling varies from its unperturbed value of ca. 150 Hz to as low as 70 Hz in the most strongly agostic complexes. A reduced 'JHc coupling of 106 Hz has been observed in an agostic f-element complex (2,6-iPr2C6H30)Sm[(p0-2,6-i-Pr2C6H3)(pMe)AlMe2]2.*' Recently, Lesage et ~ 1 have . ~shown ~ that 2D J-resolved solid-state NMR


152

spectroscopy allows one to determine the local structue of a heterogeneous catalyst, and to provide direct experimental evidence for the presence of an H-agostic bond. The 'JHC couplings of 109 and 159 Hz for the syn and anti isomers, respectively, of highly active rhenium-alkylidene olefin metathesis heterogeneous catalysts (see Figure 1) have been measured by the use of this method. These values have been used to determine the local structure of the alkylidene ligand in this complex. tBu

tBu I

I

SYn IJHC= 109 Hz

anti lJHC= 159 Hz

Figure 1

Sakharov et al.83have concluded on the basis of ' J H C couplings that the exocyclic bond in the coordinated ally1 fragment of the [(q3-fb C,oH15)Pd(PPh3)Cl]complex has a lower bond order than the cyclic one. Ab initio calculations have been performed by Janowski and J a ~ z u n s k for i~~ the NH3 H20 complex yielding the shielding constants, NQR and spin-spin couplings. The latter included ' J H N and 'JHOcouplings. The couplings in formamide have been calculated by Hansen et al.85using a method that combines finite perturbation and density functional theories (FPT and DFT). A comparison of ' J H N and ' J D N couplings for the non-symmetrical derivatives of proton sponges studied by Grech et a1.86has shown that the equilibrium shifts towards the dominant form upon deuteriation. A typical value of 89.5 Hz has been observed for ' J H N in I5Nlabelled thiobenc 1.5 Hz have been found in the 15Nenriched ~ a m i d e . '*J H~N = 102.2 and 'JHN benzohydroxamic acid, CsH5CONHOH.88 The magnitude of 'JHS, coupling is often used as a probe of the H-Si bonding in the field of silane Si-H bond a-complexes L,M(q2-H-SiR3).Usually electrondonating groups at the silicon atom effect higher 'JHS, values and, by implication, a stronger Si-H interaction. An increase of the ' J H S i coupling value upon the increase of the number of the chlorine atoms attached to the silicone atom has combeen observed by Dubberley et aLg9in the Cp(ArN)Ta(PMe3)(H)(SiR3) plexes where SiR3 = SiHMePh ( ' J H S i = 14 Hz); SiMezCl ( l J H S i = 33 Hz); SiMeClz ( ' J H S i = 40 Hz) and SiC13('JHsi = 50 Hz). These results provide for the first time a reverse correlation between the number of electron-withdrawing substituents at silicon and the silicon hydride couplings in transition metal silyl hydride comcoupling of 54.8 Hz has been found in ( T ~ ' - C ~ H ~ ) ( C O ) ~ M ~ H S ~ C ~ plexes. A JHsi and its value has been considered by Lichtenberger" from the point of view of


153

electron distribution and bonding in this complex. This value lies between the 'JHSi coupling of 370 Hz reported for HSiC13 and 20 Hz in ($CSH5)(CO)(PMe3)MnHSiC13. The negative signs have been calculated for all three couplings. A linear correlation has been exhibited between the Zx(SiX3)values and 'JHSi couplings measured by Freeman et aL91for a large series of the ruthenium silyl complexes containing Cp(PR3)Ru moiety. The magnitudes of the couplings increased as the electron-withdrawing ability of the substituents on silicon increased. A decrease of the ' J H a coupling value in sodium and potassium hydridosilylamides R2(H)Si-N(M)R' (M = Na, K) has been observed upon an increase of the ionic character of the M-N bond.92 lJHp couplings for non-quaternary (8-dimethylamino-napht-1-y1)phosphonium salts have been measured and analysed by Schiemenz et in order to obtain information about N-+P dative interactions. The Brarnsted acidity in ~ of H-mordenite and H-f3 zeolites has been studied by Kao et ~ 1 by. the~ use trimethylphosphine and trimethylphosphine oxide as 31PNMR probes. They came to the conclusion that both 31Pchemical shift and JHp coupling might be used to correlate with the average Brarnsted acidity in zeolites and thus their catalytic activity.

4

One-bond Couplings not Involving Hydrogen

The influence of the correlation, aggregation and solvation on ab initio computed Li-C, Li-N and Li-Li couplings has been studied by Parisel et The 13C spectrum of a solution of 8-lithio-1-methoxynaphtalenein THF at -100°C showed a septet for the lithiated carbon atom C8, 'J7LiC = 19.3 Hz indicating the existence of a dimeric aggregate of this compound in The 'JLiC couplings have been applied by Corruble et ~ 1 in .their ~ studies ~ on the aggregates between alkyllitium and chiral lithium amides. The presence of the lJLiN coupling of 2.7 Hz in a low-temperature spectrum of 2-(2dimethylaminoethy1)phenyllithium has confirmed that six-membered ring amine chelation takes place in the case of this compound.98A strong scalar coupling between 7Li and F (1J7LiF = 91 - 103 Hz) has been observed by Lehaire et in the LiF and the LiFHF complexes synthesized using the metallamacrocyclic receptors. A large 'JBeSi coupling of 51 Hz has been observed in CpBe(SiMe3)compound, a molecule containing an unprecedented beryllium-silicon bond.lW Simulations of 13CCP/MAS spectra of hexamethylborazine provided a ' J B c coupling of 57 Hz in this compound.lo1 'JBpcouplings between 55 and 63 Hz have been measured for a series of nido-carboranylmonophosphine-boraneadducts [NBu4] [7-PR2.BH3-8-R'-7,8C2B9HI0]-(R, R = Alk and/or = Ph).lo2It has been concluded on the basis of couplings that the nido-carboranylmonophosphines are these data and lJHB much better coordinating ligands than the corresponding closo-ones. +

154


A number of theoretical papers have been published on the spin-spin coupling calculations. This includes studies of the effect of substituents on 'Jcc couplings in methan- and ethanimine, methanal- and ethanaloxime performed by Provasi et al.,'03 an estimation of the equilibrium 'JCCvalues in ethylene by San Fabian et ~ l . , 'and ~ calculations of the couplings in ethane, methanol and methylamine performed by Pecul and Helgaker,Io5who compared DFT, MCSCF and CCSD results obtained for these compounds. Ab initio calculations of NMR chemical shifts and spin-spin couplings in cyclobutane have been performed by Jaszunski et ~ 1 . " ~Most of the J computed coupling values are in agreement with the experimental ones. It is of interest to note that the negative coupling between two bridgehead carbons is also very well reproduced ('Jcc = - 15.52 Hz has been computed). The results of non-empirical calculations of ' J C C couplings performed for strained polycarbocycles,107polyhedranes"' and bicyloalkane~'~~ have been published by Krivdin. It has been pointed out by Ruden et al."' that at the present level of electronicstructure theory, the differences between calculated and experimental spin-spin couplings could originate from the vibrational contributions to these constants. They have calculated these corrections using the hybrid density functional theory for a series of small molecules. The calculations have been performed, among others, for benzene including 'Jcc coupling, but agreement between the calculated and experimental values has not been meaningfully improved. A thorough experimental and DFT study on 'JCCcouplings in cyclopropene, its alkyl and silyl derivatives has been performed by Kamienska-Trela et al."' A full multinuclear magnetic resonance analysis of 2,4-dinitrofluorobenzene has been performed by Ariza-Castolo et ~ 1 . "yielding ~ a set of ' J c c , 'JHCand nJCF couplings. carbon in the The ' J c c coupling between the C = N carbon and aromatic Cipso X-C(Ph) = N - 0 moiety yields reliable information on the E/Z configuration in this fragment of N-alkoxybenzoimidimic acid derivatives provided that the substituent effect on the coupling value is taken into A 2 configuration has been assigned to (Z)-tert-b~tyldiphenylsilyl-['~N]-N-tert-butyldiphenylsilyl-oxybenzoimidate on the basis of the 'Jcc coupling between the C = N and aromatic Cipo carbons.@ A 13C INADEQUATE and HF-GIAO study on the 13Cenriched hydrofullerenes C60H2 and C6&&has been performed by Meier et ~ d . in ' ' ~order to identify ring currents in 1,2-dihydrofullerene, 13C MAS refocused INADEQUATE experiments have been carried out by Grasso et al."' on the polymer phase of cSc60 providing a complete and unambiguous assignment of the high resolution carbon-13 MAS spectrum. The INADEQUATE method has been used by Potmischil et aZ.'I6 to perform spectral assignments of a series of N-epimeric saturated six-membered azaheterocyclic amine oxides. Complexation of p-boronophenylalanine with 13C-enrichedfructose has been studied by Kitazawa and Y o ~ h i n o "by ~ the use of 'Jcc couplings. The data obtained have shown that BPA forms a complex with p-D-fructofuranose at the C2, C3 and C6 positions. coupling of 32.2 Hz and a 2Jc,14N coupling of 4.7 Hz have been A 1JC,14N


155

measured by Zhang et ~ 1 . 'for ' ~ nitroacetylene. The large value of the one-bond coupling has been taken by the authors as indicative of the charge symmetry about the nitrogen. They concluded that the acetylenic carbon and the attached oxygens of the nitro group have similar electron withdrawing properties. A ' J C N coupling has been determined for Troger's base and for its several precur~ors.'*~ Jackowski and co-workers have continued their studies on the chemical shieldings and couplings of various compounds in the gas-phase.12' They observed the dependence on the gas density of the 'Jcc, 'JcN, 2 J H and ~ 3 J H N couplings in the spectra of CH313C15N,120,122 and measured the ' J C F coupling in gaseous fluoromethane-d3 as a function of density.12' It has been found by Tormena et ~ 1 . that ' ~ ~ the ' J C F coupling in N-methylfluoroamides is sensitive to the F-CC = 0 orientations which allowed them, in combination with ab initio and DFT calculations as well as the solvation theory, to define both the interconverting rotamers of N-methyl-2-fluoroacetamide and N-methyl-2-fluoropropioamide and to obtain the rotamer energy differences in the vapour phase as well as in solution. A 13C,19FNMR analysis of an oxygen carrier, perfluorotributylamine, and perfluoropentanoic acid performed by Ribeiro and U m a ~ a h a r ayielded '~~ correct values of all the l J C F couplings in these compounds. 'Jcsi couplings have been measured by Lange and H e r ~ o g for ' ~ ~a series of bis(oligosilany1)chalcogenides [Me3Si),Me3.,Si12E, alkalimetaloligosilanylchalcogenolates (Me3Si),Me3.,Si-EMand oligosilanylchalcogenols (Me3Si),Me3-,SiEH (E = S, Se, Te). A 'JCpcoupling of 130.1 Hz and 3 J p C N of 8.9 Hz have been measured for 2-aminoethylaminomethanephosphonicacid.'26 Only a low temperature measurement (at - 100 "C) of [(C6H5)3Te][Ag(CN)2] has allowed Klapotke et to observe both, the 'JC,107Ag = 183 Hz and the 'JC,109Ag = 209 Hz couplings. A similar feature has been reported by Eujen et ~ 1 . ' ~ ' for K[Ag(CN)2] in D M F upon cooling below - 50 "C and explained in terms of the fast exchange mechanism. Measurements of 'JCAg couplings in 1,3-bis(2,4,6trimethylphenyl)-imidazo1-2-ylidene-silver(I)chloride performed by Ramnial et yielded 'JC,109Ag = 270 Hz and 'Jc,107Ag = 234 Hz. The couplings are considerably larger (by ca. 60 Hz) than those reported for the first silver(1)carbene complex, 1,3-bis(2,4,6-trimethylphenyl)-imidazol-2-ylidene-silver(1)triflateby Arduengo et al.l3' A 1JC,79Br coupling of 349 & 10 Hz has been determined by Molchanov and Gr~ff-Keller'~' for cyanogen bromide molecule by the use of relaxation data. Small 'JCSn(pb) couplings have been observed for 2,2-di-n-butyl-2-stannanorbornyl and 2,2-diethyl-2-plumbanorbornyl cations, 'Jcs,, = 26 and 'JCpb = 16 Hz, respectively providing evidence for the intramolecular interaction and the charge transfer between the element and the remote vinyl carbons in these The chemical shifts, 6('19Sn), and the 'JCSn couplings have been used to establish the coordination number of the tin atom and the geometry of its coordination sphere for a series of di-n-butyltin(1V) complexes with 4'/2'nitrobiphenyl-2-carboxylic acids,133and for twenty four 2,2-di-n-butyl- and 2,2-dipheny-6-aza-1,3-dioxa-2-stannabenzocyclononen-4-ones, each having a

156


transannular N+Sn bond and structure established by the use of multinuclear spectros~opy.'~~ The '.ICSncouplings measured for these compounds indicate that the pentacoordination of the tin atoms takes place in solutions. Evidence for the structure of a dinuclear tantalum dinitrogen complex shown in Figure 2 has been gained from the presence of a 'J" coupling of 16.6 H z . ' ~ ~

I

H,Si

\

[NPN] = (PhNSiMe,CH,),PPh Bu

lJNN = 16.6 Hz Figure 2

Numerous signs of 'JNP, 2Jcp,2Jsip couplings have been determined by Wrackmeyer et for a variety of aminophosphanes with bulky amino groups. 'JFSi and 2 J coupling ~ ~ data have been analysed by Bassindale et ~ 1 . inl ~a ~ series of pentacoordinate silyl monofluoride complexes used to model nucleophilic substitution at silicon. Clear evidence for the existence of the F-Si covalent bond in the [Si04/2F]unit in fluoride-containing as-synthesized STF zeolite has been provided by the use of fast spinning 29SiNMR spectra such as the refocused INEPT experiment which yielded a 'JFSi coupling of 165 H z . ' ~ ~ ' J F N b = 362 and 350 Hz have been observed for niobium oxyfluorides Cdpy4NbOFs and [pyH]2[Cdpy,(NbOF5)2, respectively in their solid-state I9F MAS NMR spectra.'39 NMR parameters including 'JFXe couplings in covalent compounds and weakly bound complexes have been studied by Bagno and Saielli.14' Relativistic effects have been taken into account and good agreement has been observed between the calculated and experimental results. 'JFP,couplings varying from 1525 up to 1865 Hz have been reported for cisand trans-(n-B~qN)~[PtF~(ox)~] and ( ~ - B u ~ N ) ~ [ P ~ F ~complexes (ox)] by Uttecht et They have also synthesized ( ~ - B U ~ N ) ~ [ and P ~ F(n-Bu4N)2[PtF2(ox)] ~] complexes with 'JFpt = 1747 and 1385 Hz, re~pective1y.l~~ lJMCo (M = Si, Sn, Pb) couplings have been extracted by Geller et ~ 1 . from l ~ ~ the solid-state 29Si,'19Sn, 207PbC P MAS NMR spectra of the group 14 metal(tetracarbony1)cobalt complexes Ph3MCo(C0)4. Wasylishen and co-workers have continued their studies on characterization of the indirect P-P spin-spin coupling and phosphorous chemical shift tensors by the use of solid-state 31PNMR spectroscopy. In the recently published work the compound studied was pentaphenylphosphinophosphonium tetrachlorogal-


157

late,'44 [Ph3P-PPPh2][GaCb]. Also a dissertation on this topic performed by Gee'45from this group has been published. A 'Jpc,, coupling of 1230 Hz has been observed in the MAS NMR spectrum of a copper iodide adduct of P4Se4,the hitherto unknown compound (cUI)3 P4Se4.146 The 'Jpc,, of 1.74 kHz has been observed in the solid P CPMAS spectrum of the centrosymmetric [(PCy3)Cu(m-X)2Cu(PCy3)]dimers with three-coordinate (PCuX2) copper. The corresponding coupling values in [ C U B ~ ( P C ~and ~ ) ~[Cur ] (PCy3)2] complexes were of ca. 1.2 kHz.14' The influence of a variety of substituents at the phosphorous atom on its electron-donating ability has been studied by the measurement of one-bond P-Se coupling for the corresponding selenides.14' The coupling values were in most cases of ca. 730 Hz. The 'JpSe couplings in the range of 1053- 1061 Hz have been found for a series of diselenides of new chiral phosphine-phosphites (P-OP). Analyses of the values of 'JpSe of the corresponding diselenides are in accordance with the expected lower-donor ability of the phosphite fragment, with respect to the phosphine group and with an increase of phosphine basicity after the substitution of phenyl substituents by methyl The large value of the P-Rh coupling, ' J p R h = 239 Hz, indicates a strong Rh-PPhs bonding interaction in the Cp*Rh(PPh3)= PMes* complex.'50An application of the dirhodium complex Rh2[(R)-MTPAI4 (Rh*; MTPA-H = methoxytrifluormethylphenylacetic acid, Mosher's acid) as a chiral solvating agent for the chiral recognition of various chiral monofunctional ligands, i.e. for the determination of enantiomeric ratios has been explored by Duddeck and co-workers.'sl Recently, they have obtained and characterized by the use of NMR the stable Rh2(MTPA)4phosphane adducts for which ' J p R h couplings were measured; l J p R h = 96.1 Hz for the PPh3 ligand and ' J p R h = 106.2 for a chiral phosphane PPh(Me)(NEt2).Their results represent the first example of P-chirality recognition by lo3RhNMR. A coupling between P and '11/113Cd nuclei has been observed in the terminal ligands only of binuclear cadmium dithiophosphate complexes, [Cd2{S2P(OR)2}4], in their high resolution solid-state spectra.lS2 A subtle but clear trend of 'JPwincreasing as more phosphine ligands are attached to octahedral tungsten sulfide clusters, W6SgL6-n (PR3), (n = 0 - 6; L = nonphosphine Lewis base ligands, PR3 phosphines) has been observed by Jin et a1.153 A linear dependence has been observed between the 'Jpptcoupling values and the basicity of the coordinated phosphane in the square-planar complexes of the type ~is-[Pt(Me)~(Me~S0)(PR~)l, where PR3 represents a series of isosteric tertiary phosphanes, and in the pyridine derivatives, ~is-[Pt(Me)~(py)(PR~)].~~~ 'Jpptcouplings of about 3200 Hz have been observed by Herberhold et al."' in platinum (11)bis(che1ate)complexescontaining both tri( l-cyclohepta-2,4,6-trienyl)phosphane, P(C7H7)3,and dichalcogenolato ligands. 'Jppt = 4350 Hz and 'JptHg = 1602 have been found for [Pt2Hg2(P2phen)3](PF6)2 (P2phen = 2,9-bis(diphenylphosphino)-l,10-phenanthroline)~s6 The P-Pt couplings observed for a novel [(dppe)2Pt2(m-BINOL)]2+ [OTf]-2 complex have been consistent with C = 0-Pt coordination ('Jppt = 3090 and 3690 Hz).'~'


158

A coupling lJSept= 366.2 Hz has been measured for cis-(nB u ~ N ) ~ [ P ~ ( S ~ C N ) ~where ( O X )ox ~ ]= the oxalato ligand.'58 An observation of a 'JRhSn coupling of 871.6 Hz has been used by Lutz et ~21."~ as evidence for the formation of a Rh-Sn bond in the mixed heterodimetallic complex [HC{ SiMe2N(2-MeOC6H4)}3Sn(Li)-Rh(q4-C8H12)(Cl)]. Two adducts containing the azide moiety, [HB(3,5-(CF3)2Pz)3]AgM(N3)[(nPr)2ATI], M = Ge and Sn and featuring unsupported silver-germanium and silver-tin bonds, respectively have been obtained by Dias and Ayers160: [HB(3,5(CF3)2Pz)3]= hydrotris(3,5-trifluoromethyl)pyrazolyl)borateand [(II-P~)~ATI] = N-(n-propyl)-2-(n-propylamino)troponiminate. 'J107i109Ag,Sn = 4866 Hz has been measured for the tin compound confirming its structure. The molecular structure of trimeric diphenyl chalcogenides (Ph2SnE)3,E = S, Se, Te, has been studied by Lange et ~ 2 1 . ' ~ 'by the use of NMR, and the results compared with those obtained earlier for the methyl compounds; for the selenium and tellurium derivatives the lJSnE couplings have been reported. The novel trialkyl(trichlorostanny1)platinum complexes [PtMe2(R)SnC13)(bipy)](R = allyl, Me, Bz) which have been obtained by ~~ a direct Sn-Pt bond in the solid state. The presence of Thoonen et ~ l . , 'revealed the Sn-Pt couplings in their '19SnNMR spectra, 'JSnpt= 3267 -4260 Hz indicates that the direct platinum-tin bond is maintained in solution. A small Pt-Pt coupling has been observed for ~ i s - [ P t ( N H ~ ) ~ ( N H c o - t B ~ ) ~ P t ( d m s o ) ~and ] ~ + ~is-[Pt(NH~)~(NHCO-t-Bu)~PtCl(dmso)]+ complexes, ('Jptpt = 809.2 Hz) and ('Jptpt = 1011.5 Hz), respectively indicating the presence of the Pt-Pt bond in these comp~unds.'~~(Figure 3)

= 809.2 Hz

Figure 3

Tong et ~ l . , who ' ~ ~ measured and analysed indirect spin-spin Pt-Pt couplings in a conducting, carbon-supported 8.8 nm platinum electrocatalyst, indicated that this approach can be the method of choice for investigating the spatial variation of the s-like Fermi level local density of states, D,(Ef,x). A linear correlation between the 205TlNMR chemical shifts and 'Jptmcouplings has been observed by Ma et ~ 2 1 . ' ~when ~ the data obtained for all Pt-T1 bonded compounds measured so far have been plotted. The equation obtained is: 'J Ptm = - 24.6 6(T1) + 92740. It is of interest to note that the couplings observed are very large; for example, ' J PtTI = 94.0,84.2 and 77.1 kHz have been found for [(NC)4Pt-Tl(phen),(solv)]+complexes (n = 1, 2, 3, respectively, phen = 1,lO-phenanthroline).


159

A theoretical study of the Hg-Hg couplings in Hg22+,H g t + and Hg22+-crown ' ~ ~experimental Hg-Hg complexes has been performed by Autschbach et ~ 1 . The couplings represent the largest couplings observed in NMR experiments so far; for example, ' J H g H g of 284.1 kHz was reported for Hg;+. The authors found on the basis of the DFT calculations that the bare ions would afford much larger couplings than those experimentally observed. The suggested upper limit is approximately of 0.9 MHz for Hg2+.

5

Two-bond Couplings to Hydrogen

Electric field effects on 2 J H H couplings in a variety of molecules have been studied by G r a ~ s 0 n . l ~ ~ For some 9-methyltriptycene derivatives for which the methyl rotation can be frozen on the NMR timescale, an apparent dependence on the temperature of the J couplings between the methyl protons has been detected by Szymanski and coworkers.'68The effect has been attributed to the occurrence of coherent quantum tunneling of the methyl rotor. Proton-proton couplings between the non-equivalent geminal protons have been observed by Burguete et ~ 1 .inl tetraaza-2,2'-biphenylophanes ~ ~ containing n = m = 1 (the smallest) and n = 2 and m = 3 (the largest) number of the ethylenic spacers. For compounds with n = 2, m = 1and n = 2, m = 2, the 2 J ~ ~ couplings have not been observed. This result has been used by the authors as evidence that the largest macrocycle is less flexible than the two smaller ones. The influence of heteroatom lone pairs on the magnitude of the geminal H-H couplings of adjacent methylene groups has been well established in a variety of nitrogen and oxygen heterocycles, including hexahydropyrimidines. Recently, they have been intensively used to characterize 1,3-di and 1,2,3-trisubstituted derivatives of N-arylhexahydropyrirnidine~.'~~ 2JHH couplings have been determined for 1,3-diamin0-2-hydroxypropaneat different pH, but no influence of it on this parameter has been " J H H (n = 2, 3) couplings have been determined for a variety of natural compounds. These are: new antranones isolated from the fungus Stachybotrys c h ~ r t a r u m ,five ' ~ ~rearranged abietane d i t e r p e n ~ i d s ,three ' ~ ~ labdane diterpenoids isolated from Leonotis ~cymifolia,'~~ pentacyclic triterpenes from the gum resin of Boswellia ~ e r r a t a and ' ~ ~ some selected bacterial ~ a r o t e n 0 i d s .A l ~set ~ of protonproton couplings which also included geminal couplings has been reported for sertraline hydrolide, an antidepressant and for a series of N-benzyl(piperidin or pyrr~lidin)-purines.'~~ The 2'3JHH couplings have been used to assign the 'H NMR spectra of several structurally very similar aminoglycosides, the major and minor components of the antibiotic agent gentamicin sulfate produced by fermentation of Micromonospora p ~ r p u r e a . ' ~ ~ A complete assignment of the 'H and 13Cspectra including 2 - 4 J H H couplings has been reported by de Arruda Campos et ~ 2 . for ' ~ four ~ 2-substituted 4a,8a-cisendo-5,8-methano-4a,5,8,8a-tetrahydronaphtalene-l,4-diones.Geminal and vicinal H,H couplings have been measured by Sobarzo-Sanchez et a2.181~182 for a

160


series of oxoisoaporphines and their derivatives. A complete NMR assignment including H,H couplings has been reported for a new condensed hexahydro1,3,5-triazine, the first example of the ketimine-type trimer.lg3 Geminal and vicinal proton-proton couplings in the methylene proton regions measured by the use of TOCSY spectra have been used by Hoodalg4andBrar et al. to characterize 4-vinylpyridine-Ig4and vinylidene chloride'85methyl acrylate copolymers. Recently, a configuration analysis method based on long-range proton-carbon couplings has been developed by Murata and co-workerdg6for the relative stereochemical determination of acyclic natural products. A similar approach has been applied by Kobayashi et ~ l . , 'who ~ ~ used 3 J H H and 2 7 3 J H C couplings to establish the structure of six new 26- and 27-macrolides, amphidinolides isolated from a marine dinoflagellate Amphidinium sp. The method has been extended by Williamson et ~ 1 . ' ~ 'to P-hydroxy carbonyl compounds (aldol products), and by Knust and HoffmandS9to conformational analysis of macrocyclic dilactones mimicking the pharamcophore of aplysiatoxin, a highly potent protein kinase C. The same approach in its modified form has been applied by other groups. Schulte and c o - w o r k e r ~have ' ~ ~ shown that both, the magnitude and sign of 2JHc coupling may be useful for the determination of the absolute configuration at a carbon centre in a fixed cyclic system. Extensive long-lasting studies on the toxins extracted from Adriatic blue mussels have been performed and recently reviewed by Ciminiello et aE.191 Among others they have solved the relative stereochemistry of a new cytotoxic polychlorinated sulfolipid by the combined use of 2'3JHC and 3 J H H couplings.'92 The same combination of the couplings and NOE/ROE data has been used by Cimino et to establish the correct configuration of sapinofuranone A, a phytotoxic molecule produced by three strains of Sphaeropsis sapinea, to sphinxolide; the parent member of a family of cytotoxins, and to the ascaulitoxin molecule. In the method applied by them the ab initio calculated set of couplings has been compared with the experimental data. 2 Jand~ 4 J H~N couplings have been measured in Zn(I1)-purine and -methylpurines complexes by Szlyk et Activation of dihydrogen by a system composed of ( B u ~ N ) [ I ~ B ~ ~ ( and C O )tin ~] bromide in various ratios has been studied by Permin and E i ~ e n b e r g . The '~~ structures of the dihydrides formed upon addition of dihydrogen have been assigned on the basis of hydride chemical shifts and the 2 J H S n values.The coordination of the tin atom and the geometry of its coordination sphere in a number of alkyltin(1V)toluenesulfonates, R , S ~ ( O S O ~ - ~ U ~ U - C ~(nH = ~ C2,H3;~R) ~=- ~ Alk) and ( ~ - B u ) ~ S ~ ( O S (X O ~=XCH3 ) ~ and CF3)have been elucidated by the use of the chemical shifts, 6('I9Sn),'JHSn and ' J C S n coupling^.'^^ The 2JHpt couplings of ca. 78 Hz observed in the [(PtMe3)2(~-mannitol)](BF4)2 complex are typical of the coordination of OH groups in the trans position to methyl ligands.19' ~

1

.

~

~

~

3

'

~

~


6

161

Two-bond Couplings not Involving Hydrogen

2Jcp, 'Jcp and 2JHpcouplings have been applied to establish the structure of the

[7-CpFe(CO)2-q2-nido-7,8,9-PC2B8Hlo] complex.'99The analogous set of data has been obtained for the isomeric symmetrical complex, [7-C~Fe(Co)~-q '-nido~,~,IO-PC~B~H~O]. Earlier theoretical predictions made by Szymanski that J couplings between magnetically equivalent, quadrupolar nuclei can be detectable in the presence of relaxation have just been confirmed experimentally by him and his coworker200 for water solution of selectively "N labelled sodium azide. A two-bond 14N-14N coupling has been estimated for this compound. Geminal and vicinal F-F couplings have been measured for iodotrifluorethylene dissolved in the nematic liquid phase by the use of the I9FSAS (switched angle spinning) -COSY experiment.2o' 2JsiNb couplings of 35.0 - 64.0 Hz observed in the spectrum of Rb4(Nb0)2(Si8021) are comparable to the value of 2 J F N b = 55 Hz for [Nb(PF&which was obtained from solution NMR. This is the first direct measurement of a heteronuclear two-bond coupling between a quadrupolar nucleus and a spin'/2 nucleus in the solid state.202 Two-bond P-P couplings have been measured for a series of cyclic triphosphazenes, and their influence on 'H and 13C NMR spectra of phosphorus substituents has been studied by Vicente et uL203 Novel terminal iridium phosphinidene complexes of the type Cp*(L)Ir = PR have been studied by Termaten et ~ 1 The. "P~ chemical ~ NMR shifts and 2 J p p couplings are diagnostic for the E and 2 forms. More shielded phosphinidene resonances and larger couplings are typical of the E isomers. A two-bond P-P coupling of 13.3 Hz has been observed in the platinum complex, [PtCl2(q2-Ph2POCH2CH2N(CH3)PPh2}], but not in a free ligand,205 which can be used as evidence that the coupling in the complex occurs through the P-Pt-P path. The cationic rhododendrimers containing up to 32 metal centres have been obtained by metalation of a series of chiral phosphine-functionalized poly(propy1eneimine)dendrimers and characterized by the use of NMR. 2 J p p of 32.9 and '.IpRhof 150,153 Hz have been observed for this largest chiral phosphine dendrimer catalyst studied to date.206 Through-bond phosphorus-phosphorus connectivities in crystalline and disordered phosphates have been determined by solid-state 2D 31Prefocused INADEQUATE NMR experiment~.~'~ A series of some sila-[ llferrocenophanes and a phospha-[ llferrocenophane has been studied by Wrackmeyer et ~ 1 . ~In' ~the case of 2,2,6,6-tetramethylpiperidin- 1-yl-phospha-[ llferrocenophane, [Fe(C5H4)2]P(tmp), the coupling 2 J p F e = 3.4 Hz has been measured for the first time. spin-spin couplings of 310 to 560 Hz have been measured for [TlHnDtpphln-, complexes formed between diethylenetriaminopentakis(methy1enephosphonic acid) and thallium(II1) from aqueous solution. The complexes are highly stable on the NMR time scale.209 233JpT1

2 9 9 10 11 18 20 27 27 29 29 29 30 39 41 42 42 47 55 55 55 60 68

3 0 structure CYCIO(LP-LP) (Sar7)desamino-and (MeAla7)desamino-vasopressinanalogues GPGMGPGMG, a model peptide of the spicule matrix protein PM27 ALPSYXXTYK (X = Hyp), the repeat decapeptide of Mytilus edulis foot protein-1 eledoisin a-GID, a novel a4/7-conotoxin from Conus geographus mersacidin, the type B lantibiotic; free, bound to lipid I1 and in DPC micelles [desHis’,desPhe6,Glu9]glucagonamide gm9a, a novel P-superfamily spasmodic conotoxin from Conus gloriarnaris [C/N] Np14 NZF, DNA encoding NZF domain from rat Np14 (580-608) [Ala’.”]kalata B1, an analogue of plant cyclotide kalata B1, a cyclotide from 0. afinis cycloviolacin 01, a cyclotide from V. odorata [N] PCI, a potato carboxypeptidase inhibitor CD-Phel2,AiblSICRH, a new analogue of human/rat corticotropin releasing hormone CnErgl (Ergtoxin), a HERG specific scorpion toxin crotamine, a N a + channel affecting toxin from Crotalus durissus terrificus venom NaD1, a new floral defensin from Nicotiana d a t a [NJ LEKTI domain 1 [C/N] [C28S] Xis (1-55),the phage encoded excisionase protein ATI, an ascidian trypsin inhibitor from Halocynthia roretzi Cd7-MT-nc, metallothionein of the Antarctic fish Notothenia coriiceps [C/N] ATT,, an Arabidopsis thaliana trypsin inhibitor

U

7 11 33

Peptides and proteins for which the solution structure has been calculated with ’ J H H

2 0 structure FKKGERL, a seven-residue type I1 p-turn from the src SH3 domain AcX2A702NH2,X = diaminobutyric acid, 0 = ornithine AFP, a type I antifreeze polypeptide from the shorthorn sculpin Myoxocephalus scorpius

Name

Table 1

5 10 32

20

C

21 11 20 38 14 20 47

C

10 6 7 6 9 15 13 22 26 13 19 29 33

b

239

238

237

236

235

am

$n

a

0

3

m

c a 233 234

Q

% 232

E2

2 215 23 1

230

230

229

228

221

226

225

224

223

222

22 1

220

219

218

210

216

Re$

a

the number of amino acid residues the total number of vicinal backbone and side chain proton-proton couplingsmeasured ' number not reported

-

LEKTI domain 6 :ytochrome c from Bacillus pasteurii, reduced 3 1apo-Znta(46-118),E. coli transport protein 3 1Zn(II)-Znta(46-118),E. coli transport protein 31Kv1.4 N(l-75), the tandem inactivation domain of the neuronal A-type potassium channel :C/N] PrP40 WW, the WW domain pair of the yeast splicing factor pre-mRNA processing protein :C/N] apo CopC, the metal free form of a cupredoxin-like protein :C/Nl pY from E.coli :C/N) DAF 2,3 (61-189),the second and third domain of human decay accelerating factor : C N EznM2E133QSOD, the copper free state of a monomeric form of superoxide dismutase : C N calerythrin from Saccharopolyspora erythraea

68 71 73 73 75 75 102 112 129 153 175 24 52 40 39 30 70 47 65 54 94 110 247

246

244 245

242 243

24 1

240

214 240

235

h

$2

9

I

7 co %. a

2.

z e 0;

=.

% K*

164

7


Three-bond Hydrogen-HydrogenCouplings

Among other NMR observables the 3 J H H couplings still remain an important source of structural information. A detailed analysis of thermal behaviour of vicinal proton-proton couplings allowed Kallenbach and co-workers210to trace the conformational change of the peptide from polyproline I1 type structure at 2 "C to a mixture of polyproline II/P-strand at 56 "C.Distributions of the values of the backbone angle cp in a coil state of protein have been analysed by the use of 3 5 H H by Avbelj and Baldwin.211 The conformational transition of Alzheimer amyloid P-peptide (12-24) to a p-structure in the pH range of 2.2 to 3.0 has been traced with 3 J H H couplings by Nakanishi and co-workers.212Nakahara and co-workers213have utilised this type of couplings in their analysis of conformational equilibria in the side chains of aspartic acid and aspargine. Recently, the solution structures of proteins have been determined by NMR to RMSD values in the range of 0.5 A for all backbone atom^.^'^,^'^ The list of peptides and proteins for which these couplings have been used in structure determination are listed in Table 1. 3 J H H along with 2 J H C and 3 J H C couplings have been extensively applied by He et in structural analysis of mannopeptimycins, novel antibacterial glycopeptides from Streptomyces hygroscopicus, LL-AC98. Griesinger and c o - w ~ r k e r have s ~ ~ ~used vicinal 3 J H H couplings to characterize a novel class of calixsugars. Sandstrom and c o - w o r k e r ~have ~ ~ ~used 3JCH,$)H couplings to monitor hydrogen bonds formation in oligosaccharides such as cyclodextrins and maltose. Conformational preferences of lignin P-0-4 model compounds have been studied by Mazeau and c o - w o r k e r ~ . ~ In~ these ' studies the experimental 3 J H H couplings have been rationalized with the computations performed with the CHARMM force field. An improved OPLS-AA force field for carbohydrates has been described by van Gunsteren and c o - w ~ r k e r sThe . ~ ~authors ~ have compared 3 J H H couplings of some sugars derived from molecular dynamics using improved force field with the experimental values. Both, calculated and measured 3 J H H couplings have been used by Bolton and c o - w o r k e r ~in~ ~the ~ assessment of the molecular dynamics structure of the oligonucleotide duplex (12 bp). More examples of sugars and nucleotides are given in Table 2. 3JH13 couplings have proved to be a useful tool for structure determination of non-natural non-peptide oligomers with a defined secondary structure. Guichard268has used these couplings as angle restraints to calculate the structure of N,N'-linked oligoureas. The structure of artificial porphyrin-based peptide receptor has been established by Schneider and c o - ~ o r k e r swith ~ ~ ~the help of 3 J H H couplings. Evaluation of 3 J H H couplings for products during oxidative cleavage of a benzylidene acetal has been used by Song and H o l l i n g s ~ o r t hfor ~ ~checking ~ a possible rearrangement mechanism. Vicinal proton-proton spin-spin couplings in alkanes, alkenes, their cyclic


Table 2

165

Nucleosides, nucleotides, oligonucleotides and carbohydratesfor which 3 J H H has been used as a structural parameter

Name

Re$

9-p-~-erythrofuranosyladenine and its carbocyclic analogues three d(TpXpT) where X = tetrahydrofuran or propyl, mimics of DNA damage [C/N] PEMV-1, the pea enation mosaic virus RNAl pseudoknot a-L-LNA/DNA duplexes (9 and 10 bp) dT-bulge, 5’-d-GCATCGAATAAGCTACG-3’and 5’-d-CGTAGCCGATGC-3’ dU-bulge, 5’-d-GCATCGAAUAAGCTACG-3’and 5’-d-CGTAGCCGATGC-3’ [C/N] the loop E region of the 5 S rRNA (68-108)from chloroplast ribosomes of Spinacia oleracia

254

carbohydrates a series of methyl 3-azido-2,3-dideoxy-a-~-lyxo-hexopyranosides di- and tetrasacharidic 0-acetylated C-glycosidescontaining 2,3-enopyranosyl system mono- and difructosyllactosucroses mycothiol bimane - MSmB, and two pseudodisaccharides- d-GI and AcGI deacetylase a novel pentasaccharide from Penicilliurn citrium pentasaccharide, the repeating unit of the 0-antigen polysaccharide from E. coli 0142 heparin-like hexa- and octasaccharide glycero-D-manno-heptoses from Mannheimia haemolytica serotype A 1 lipopolysaccharide

255 256 257 258

258 259

260 26 1

262 263

264 265 266 261

congeners and polybenzenoid hydrocarbons have been analysed by Bader and c o - w ~ r k e r from s~~~ the point of view of electron delocalization. They have shown that the Fermi contact term can be successfully modelled in terms of the exchange density and thus by the delocalization of the electrons between the basins of the associated hydrogen atoms. A good linear relationship has been observed by the authors between the 3 J H H couplings and the hydrogen-hydrogen delocalization indices. A full set of vicinal proton-proton couplings has been obtained for the tricarbonylchromium complex of 2,7-di-tert-butyl-trans-l2c, 12d-dimethylbenzoCe] dihydropyrene, and the results interpreted in terms of the effective aromaticity of the compound.272 A new method has been developed by Wu and Cremer273to determine the most stable conformations of puckered rings by a comparison of measured and calculated vicinal spin-spin couplings. The method called DORCO (determination of ring conformation) extensively uses the ring puckering coordinates to express the properties of a pseudorotating puckered ring. A complete analysis of ‘H NMR spectra of trans- 1,2-dichlorocyclopentaneand trans- 1,2-dibromocyclopentane has been performed by Zubkov et by the use of the total lineshape fitting algorithm VALISA developed by them. The obtained high ~

1

.

~

~

~

9

~

’

~

166


precision couplings have been applied to perform a conformational analysis of the compounds studied. Conformational equilibria of 2-halocyclohexanones~76trans-2-halocyclohexanols and their methyl have been studied by Abraham and coworkers by the use of 3 J H H coupling constants. Theoretical and solvation study has been performed for these compounds. A conformational analysis of cis-fused 7a(8a)-methyl octa(hexa)hydrocyclopenta[d] [1,3]oxazines and [3,1] benzoxazines has been performed by Tahtinen et al.278by the use of vicinal proton-proton couplings. Good agreement has been observed between the DFT calculated and experimental 3 J H H couplings providing a methodology for determining the conformational equilibria of such systems that is comparable in many respects with experimental approaches such as variable-temperature NMR. Pivnenko et ul.279-281have continued their studies on the configuration and conformation of the cyclohexanone derivatives by the use of 3 J H H couplings. The studied compounds included arylidene derivatives of 3R-methylcyclohexanone,281 2- [1-hydr oxy- 1'-(4-carbomet hox yphen y 1)-met h yl] -p-menthane3-one and some 1R,4S-2-arylidene-p-menthan-3-ones.280 Hopf and co-workers282have continued their studies on the conformation of substituted cyclophanes. Full bandshape analyses of the bridge proton signals of several pseudogeminally substituted [2.2]paracyclohanes yielded the vicinal H,H couplings which reflected the 1:l equilibrium between the two skew conformations of the bridges. An extensive use of 3 J H H couplings has been made by Sygula et al.283to establish conformational preferences in 1-alkyl- 1,2-dihydrocorannulenes, by to elucidate the structure of a fused Tsoleridis and Stephanidou-Stephanato~~~~ cyclopentanopyrazolidinol, and by Hoffmann et ~ 1 . ~to~ perform ' a conformational analysis of rneso-ter(1,3-dioxan-4-yls).The conformations of a large series and substituted tetof N-substituted-1,3,4,5-tetrahydro-lH-2-benza~epines2~~ rahydro- 1,4-ben~othiazepine$~~ have been assigned by Katrizky and co-workers on the basis of the magnitudes of H-H vicinal couplings. Pihlaja and cow o r k e r ~have ~ ~ used ~ , ~ 3 ~5 H ~H couplings to perform structural studies of the cisand trans-2-imino-l,3- and -3,l-perhydrobenzoxazinesand their 3- and 1-Nmethyl and of saturated 5H-pyrrolo[ 1,2-a][3,l]benzoxazinl(2H)-ones prepared from 4-oxopentanoic acid and cyclic amino Vicinal H-H couplings and ab initio quantum mechanics have been used by Kent et ~ 1 . ~to~ investigate ' solvent effects on conformational equilibria of but anedini t rile. The effect of pH on the rotational conformations of 1,3-diamino-2-hydroxypropane171and the conformations of 1,4-butanedioic acid as a function of solvent ~~' polarity in a series of alcohols has been studied by Roberts and c o - w o r k e r ~by the use of 3JHH couplings. The observed couplings were correlated with the expected couplings for the various possible staggered conformations in the attempt to determine the pattern of conformations for the parent compound and its conjugate acids. A simple 'H NMR analysis that permits the stereochemistry of f3-hydroxy ketones to be assigned by visual inspection of the ABX patterns for


167

the a-methylene unit of the P-hydroxy ketone in 'H NMR spectra has been published by Roush et al.292The method has been verified by application to a wide range of compounds. The relative configuration of the contiguous asymmetric centres of synthetic p-substituted tryptophans has been determined by the analysis of the vicinal proton-proton c0up1ings.l~~ A complete NMR set of data which included 3 J H H couplings has been obtained for several 6-substituted 2-(2-hydroxyaryl)benzoxazoles294from 2,2'-dihydroxydiaryl Schiff bases. The small vicinal coupling constants between two equivalent protons have been measured by the use of the C satellite band method295for two fullerene bis-adducts. Three new n-systems, benzocyclohepta[6,7-a]-, benzocyclohepta[6,5-a]- and benzocyclohepta[7,6-a]azulenylium ions, have been synthesized by Yamane et al.296and the vicinal couplings used to investigate how the conjugation mode as well as the theromodynamic stability are affected by the position of the benzoannulation. The solution conformation of a chiral phenyl-substituted dihydrofuroangelicin has been investigated by the use of molecular mechanics and DFT methods as well as the vicinal couplings.297 3 J H H couplings have been applied by Selivanov and Shavva298,299 to establish the spatial structure of modified analogues of steroid hormones. Using 3 5 H H couplings Kraszni et have determined the populations and site- and conformer-specific basicities of histamine rotamers in three distinct states of p r o t o n a t i ~ n and , ~ ~ conformer-specific partition coefficients of clenbuterol and amfetamine in octanol/water The precise 3 J H H coupling values of N-acetylaspartylglutamate have been derived by iterative computer fitting of their 'H NMR spectra measured at different pH, which yielded crucial information on the solution conformation of this molecule.302These data have been used for unequivocal determination of the presence of NAAG in the uric sample of a patient suffering from Canavan disease. The 'H chemical shifts and proton-proton couplings of 2-hydroxyglutaric acid and its lactone have been derived by Bal and Gryff-Keller303by the use of the total lineshape analysis. Determination of the level and absolute configuration of this compound in a urine sample is a method of diagnosing two metabolic diseases. Vicinal proton-proton couplings have been measured and used in elucidation of the structure of a variety of compounds isolated from natural sources. They include three terpene saponins isolated from the roots of Silene ~ u l g a r i secos~~~ adianane-type triterpenoids from Dorstenia brasiliensis ( M ~ r a c e a e ) ;15 ~ ~limonides, triterpenoids occurring in plants belonging to the Meliaceae and Rutaceae families,306some natural occurring furoclerodanes from Croton c a j ~ c a r ain,~~~ dole alkaloids extracted from Flustra f o l i a ~ e a and , ~ ~two ~ new cacalolides, 13aetoxy- and 13-acetoxy-14-oxocacalohastin,from Senecio b a r b a - j o h a n n i ~ . ~ ~ ~ Further examples include nocathiacin I, a cyclic thiazolyl peptide antibiotic isolated from Nocardia sponge that displays a potent activity against Grampositive bacteria,310the acetonide derivative obtained from the acid hydrolysate a1.3003301

168


of callipeltin A,311the P,P-carotene dication prepared by treatment of P,P-carotene with BF3ethereate;l2 and a semisynthetic derivative of epipodophyllotoxin, which is currently in clinical use as an important cancer chemotherapeutic agent.313The relations between the calculated and experimentally determined geometries of three sesquiterpene lactones, germacranolides have been established by the use of a modified Karplus relation.314Three cardenolides have been isolated from Calotropis procera and their spectra reported.315One of them was obtained in the form of the methoxy derivative which was formed during the workup in the presence of methanol. The 3 5 H H couplings have been used to elucidate the structure of a novel catechinic acid stereoisomer from basecatalysed reactions of ( +)-~atechin.~l~ Spectral assignments and reference 'H NMR data have been reported for the methylol derivatives of 2,4'- and 4,4'dihydroxydiphenylmethanes found in resol phenol-formaldehyde resins,317 pyrazole-fused P - c a r b ~ l i n e and s ~ ~three ~ model 2,4,6-tris(amin0)1,3,5-triazines?'~ An analysis of the intramolecular competition associated with the ring closing metathesis of ene-diene systems of differing chain length with a pair of ruthenium catalysts has been performed by P a q ~ e t t eAn . ~ ~assignment ~ of the geometry of double bonds has been made on the basis of vinyl proton chemical shifts and couplings. Several papers have been devoted to studies of the structure of various complexes. These are: cationic Ru (11) complexes containing a chelating q1:q6phosphinoarene ligand, [ R u C ~ ( L ) ( P P ~ ~ ( C H ~ ) ~[PF6],321 - ~ ~ - C ~a H novel ~ ] series of organometallic merocyanines combining the potent electron-donating [(CpFeC0)2(m-C:CH-)] fragment with tropylium-type acceptors322 and a [Pd(q3-PhCHCHCHPh)(phosphino-oxazoline)]+ complex with almost identical P- and N-trans influences.323 8

Three-bond Couplings to Hydrogen

Vicinal proton-carbon couplings, which, by analogy to vicinal proton-proton couplings follow the Karplus relationship, are becoming more and more extensively used in conformational analysis of a variety of compounds. They have been applied to study the stereochemistry of P-sulfonyl e n a m i n e ~and ~ ~ the ~ sulfoxide and sulfone derivatives of 3-arylidene-1-thioflavan-4-one e p ~ x i d e s , ~ ~ ~ to analyse the structure and conformation of 5-(1H-pyrrol-2-ylmethy1ene)substituted imidazolidine-2,4-diones and thia~olidine-2,4-diones:~~ and to obtain conformational charateristics of the monomeric model compound 1,2bi~(methy1thio)propane.~~~ Using the relationship 3 J H C trans > 3 J H C cis, configurational assignments based on the 3 J H C couplings have been made for some derivatives of 2 - ~ r o p e n o n ea, ~series ~ ~ of cyclic and acyclic a l d o n i t r o n e ~ and ,~~~ some 4-substituted 2-0~0-3-butenes.~~' The E , 2 and 2 configurations, respectively have been assigned to these particular groups of compounds. 3 J H H and 3 J H C couplings have been applied to establish the structure of a series of camptothecin-related akaloids extracted from hairy roots of Ophiorrhiza p ~ r n i l a . ~ ~ ~ Scalar 3 J H C and 3JHN couplings have been used by Pappu and Rose332in their


169

conformational studies on unfolded states of alanine peptides. The authors have found that the seven-residue peptide adopts the structure of polyproline I1 helix and its symmetry mate. The J-based configuration analysis method has been applied by Gerwick and c o - w ~ r k e r sto~ establish ~~ the structure and absolute stereochemistry of phormolide, a neu toxic metabolite from the marine cyanobacterium Phorrnidium sp. In this analysis 2’3JHH and, in particular, 3 J H C have been used. Munzarova and Sklenai.334have performed a DFT analysis of scalar, mainly 3JHc,couplings across the glycosidic bond in DNA. The modified CHARMM force field, Parm22/SUO1, has been applied by Eklund and Widmalm33sin molecular dynamics simulations of an oligossacharide. Calculated transglycosidic 3 J H C couplings have shown good agreement with those previously measured. The conformational behaviour of 3’-fluorinated furanoside rings has s ~the ~~ been explored in the series of ribonucleosides by Altona and c o - ~ o r k e ron ~ ~ with the aid of the PSEUROT6.3 program. basis of 3 J H H and 3 J couplings In Table 3 peptides and proteins and in Table 4 nucleotides and carbohydrates are listed for which heteronuclear vicinal couplings have been used as a structural parameter. A paper on systematic ”N NMR study of isoquinoline alkaloids, which also included estimation of three-bonds proton-nitrogen couplings has been published by Marek et a1.352 A series of phosphorylated P-hydrazones of the structure R2P(0)C(= NNHRF)CHR2/has been characterized by the use of 3 J ~3 J~F F ,and 3Jcpcouplings.353Some of these compounds exist as a mixture of 2 and E isomers, with the 2 form predominating. A good correlation between the values of 3 J H P and the H-C-0-P dihedral angles estimated from the calculated positions of the protons in the X-ray structure has been observed by Barloy et aZ.354for the binuclear Pt(I1) complex, [Pt2C12(PONOP)2](BF4)2,where PONOP = the new pyridinediphosphinite ligand. This allowed them to assign the four methylene signals in the ‘H NMR spectrum of this compound. The 3 J H C D couplings measured for Cd(~yclam)(ClO~)~ have been applied to calculate torsion angles by the use ofthe Karplus-type correlation between these couplings and the H-C-S-CD dihedral angles in protein^.^" 3-6JHSn couplings measured for a series of tetrakis(3-methoxypropy1)tin and (3-methoxypropy1)tin chlorides have been applied to estimate the degree of donor-acceptor bonding between the central tin atom and the oxygen atom of the 3-methoxypropyl s ~ b s t i t u e n t . ~ ~ ~ The *JHPt and 3 J H p t couplings have been used by Monsu Scolaro et aL3” to establish the geometrical configuration of monomethyl-Pt(I1) complexes of the type [Pt(DMSO)(L)Cl(CH3)]where L denotes a heterocyclic nitrogen base, a large variety of which has been used; for the trans (C,N) isomers average 2JHpt = 75 4 Hz and 3 J H p t = 36 _+ 4 Hz have been observed for the coordinated methyl and DMSO ligands, respectively. In the case of the cis (C,N) isomers, these values increase to ’JHpt= 83 f 2 Hz, and decrease to 3 J H p t = 26 f 3 Hz, which is due to the mutual exchange of ligands in the trans position to CH3and

3 19 82 d 106 124 16

1 6 93 96 107 135 233

Pt(I1)-allylglycine,Pt(I1)-S-methylcysteine, Pt(I1)-metionine and their sulfoxides Ga(II1)-azoverdin,a siderophore from Azomonas macrocytogenes ATCC 12334 [C/N] p67ph0"SH3(455-516)-p47Pho" (360-390) complex G16A-MNE1, a mutant of monellin [ C m T R X H E , a chimeric human (1-66)and E. coli (67-107)thioredoxin [C/N] KaiA, the circadian clock protein of Synechococcus elongatus [C/N] IIAMtl-HPr, the phosphoryl transfer complex

a

number of residues total number of vicinal couplings measured (homonuclear 3 5 H H couplings are also included if measured) types of heteronuclear couplings measured number not reported

m]

b

a

3JCC,3 JCN

3 J ~ 3 ~ J ~ 3Jcc , ~ ,

3JCC,3 JCN

JCC? JCN 3JHN

3JHN

2.3 JHPt

C

342 343

34 1

340

339

338

337

ReJ:

Peptides and proteins for which heteronuclear couplings have been used as a structural parameter in 3 0 structure calculations

Name

Table 3

a

the number of nucleotides type of vicinal heteronuclear couplings measured; 35,, homonuclear couplings have also been measured in most cases

carbohydrates P-~-Glcp-( 142)[ P-~-Glcp-(1-3)]-a-~-Glcp-Ome a series of gluco- and galactopyranosyl-glucoses

3, 144 14 15 23 34

a series of 2'-chloro-2',3'-dideoxy-3'-fluoro-~-ribonucleotides three novel zidovudine analogues thymidine 3'-O(N,N-diisopropyl-0-~-cyanoethyl-phosphoramidothioate)s a series of model 2',5'- and 3',5'-linked oligonucleotides [C/N] d(GCGAAGC) [C/N] d(GCGAAGC) an essential stem-loop of human telomerase RNA stem of SL1 RNA in HIV-1 [C/N] RNA aptamer (that binds HIV Tat) complexed with 2 argininamide molecules

1 1 1

a

3JHC

3JHC

JHP

3JH P

4JHP,

3JCP

3JHP, 1.2J 3JCP 1,2 J I,? HC, HN, JCC, 2,3 3 JHC, J H P

JCP

3JHC

JCF

b

''*JCN

35 1

335

350

349

348

l9 347

346

345

344

336

Re$

Nucleosides, nucleotides, oligonucleotides and carbohydrates for which heteronuclear vicinal couplings have been used as a structural parameter

Name

Table 4

:

2

c

2

3

x -.

0,

2. 9

I

3-

2.

172


DMSO. Rather large 3JHptcouplings of 92 - 98 Hz have been observed by Al-Najjar et al.358between the imino proton H-6 and platinum(I1) for a series of the novel ferrocenylimine complexes of Pt(I1) ethene of a general formula trans[PtClz(q2-C2H4)(imine)]where imine = N-alkyl- 1-(ferrocen-1yl)methanimine. This result can be used as an indication that a Pt-H-C interaction takes place in these compounds. 3JHpt couplings of 45 - 48 Hz have also been measured for Pt(I1) complexes containing q ~ i n o l i n e . ~ ~ ~

9

Three-bond Couplings not involving Hydrogen

Zhao and c o - w o r k e r ~have ~ ~ ~traced the isomerization of 0,O-phenylene pentacoordinated phosphoryl serine anhydride to ester with the help of 2Jcpand 3Jcp couplings. The conformational properties of a diastereomeric nucleoside-phosphorothioate pair have been studied by Gacs-Baitz and Kajtar-Pe~edy.’~~ Vicinal carbon-phosphorus couplings reflect the predominance of the e- conformation for the Sp isomer and the&‘conformation for the Rp isomer. The influence of the solvent and temperature on these vicinal couplings has been studied and the results interpreted in terms of the conformational shift towards the preferred et conformation. Solution conformations of 2-amino-l-hydroxy-2aryl(alky1)lphosphonic acids and their esters have been studied by Kafarski and c o - w ~ r k e rby s ~the ~ ~use ~ of ~ ~3Jcp, ~ 3JHH and 3JHp couplings. Rochon and have continued their studies on the platinum complexes. The vicinal carbon-platinum and proton-platinum couplings have been used to establish the structure of the aqueous products of the complexes cis and t r a n ~ - P t ( Y p y ) ~ ( Nwhere O ~ ) ~Ypy = pyridine and the configuration of the iodo-bridged Pt(I1) complexes { Pt(R2SO)I}2(m-I)2and of transPt (R2SO)pyrimidine)12. 364 Two P-phosp hino ylmeth yl aminophosphonium salts (bromide and tetrafluoroborate) have been studied by Lopez-Leonard0 et al.365 In the case of the fluoroborate a weak N-H - 0 intramolecular hydrogen bond (IMHB) is formed. A 3 J N p coupling of 1.5 Hz observed in the spectrum of the bromide appears to be a classical coupling across the covalent bonds and not a 3 h J N p in nature, i.e. across the IMHB. A series of the 1,2-difluorovinylgermanes Ph3GeCF= CFR (R = H, Me, n-Bu, t-Bu, Ph) has been synthesized by Brisdon et al.366 and their trans configuration has been assigned on the basis of the large 3JFF coupling values, of ca.128 Hz, observed for all these compounds. The torsional profiles of 1,2-difluoroethane have been studied by Kurtkaya et al.367as a function of 3JFF coupling. They used the DFT method in an attempt to clarify the reported by other authors lack of a correlation of this coupling with the F-C-C-F dihedral angle. The theoretical study performed by this group of authors has shown that all four terms (FC, DSO, PSO and SD) contribute significantly to this coupling and only the SD contribution follows a typical Karplus dependence. Vicinal F-Se couplings across a double bond of ca. 19 Hz have been reported for a series of fluoro(organylseleno)alkenes, and of ca. 4 Hz for alkanes.368 * *

173


A 3Jp.Mo-Mo-p coupling of about 16 f 1Hz has been found for the M o ~ ( O ~ C C H ~ ) ( U - D M O ~ ~ F )complex C ~ ~ ( Pwhere M ~ ~ o-DMophF )~ is the anion of N,N'-di(2-rnetho~yphenyl)formarnidine?~~ 10

Couplings over More than Three Bonds and Through Space

The through-space H-C coupling in acetylenic systems has been studied by Bagno et ~ 1 . ~by~ 'the use of the ab initio and DFT methods. DFT/FPT studies of the structural dependencies of long-range proton-proton couplings over four bonds in propanic and allylic systems have been performed by Barfield.371 Proton NMR spectroscopic evidence for a preferred [18lannulene substructure in carbaporphyrins has been derived from the magnitude of the 4 J H H couplings in the CH = C-CH3fragment.372 A set of nJHH (n = 2 - 4) has been reported by Constantino et al.373for fourteen cyclopentane derivatives; a relationship between the 4 5 H H coupling values and the calculated dihedral angle between the corresponding hydrogens has been recently reported by this group. The proton-proton couplings across three and four bonds have been reported for four biindenylidene isomers by Spiteller et The crucial argument in the assignment of the configuration of ( E ) and (Z)-4-phenyl-2-phenymethylidene-1,3-dithiolesresulted from the presence or not of 5 J H H and 5 J H C In each isomer these couplings are present or are too small to be observed depending on whether a zigzag shaped coupling pathway is available or not (see Figure 4).

Qsp H

-

Q S k

'

H

H

-

S

I' /

Z

5J,,
210 Recently, Kruk and Kowalewski211have developed a new theoretical model for nuclear spin relaxation in paramagnetic systems in solution. Fast rotational motion is included in the model, both as a source of modulation of the static zero-field splitting, which provides a mechanism for electron spin relaxation, and as an origin of the stochastic variation of the electron spin-nuclear spin dipole-dipole interaction leading to nuclear spin relaxation. At the limit of low magnetic field, the model is essentially identical to their earlier formulations, but new closed-form expressions are given for the inner- and outer-sphere relaxation at the high-field limit. Bertini et a1.212introduced paramagnetism-based constraints for the solution structure determination of metal-containing proteins. Relaxation rates, contact shifts, pseudocontact shifts, self-orientation residual dipolar couplings and, cross correlations between Curie relaxation and dipolar relaxation have been discussed in this respect. Assfalg et al.'13 discussed a structural model for a paramagnetic alkaline ferricytochrome c. Harris et al.214used paramagnetic cobalt(I1) as a probe for kinetic and NMR relaxation studies of phosphate binding and the catalytic mechanism of streptomyces dinuclear aminopeptidase. Gd(I1) based MRI contrast agents were investigated by Dunand et al.2'5 In particular the 2097

210


authors asked for the improved physical meaning in a combined analysis of EPR and NMR data. Paramagnetic metal centers [such as Fe-I11 found within ferriprotoporphyrin IX heme (FPIX)] exert through space effects on the relaxation rate of nearby proton spins that depend critically on the metal-proton distance. Leed et d 2 I 6 have measured these effects for all protons of several antimalarial drugs that bind to FPIX by systematically varying the drug:heme molar ratio in high field NMR experiments. Aime et aL217showed that the paramagnetic properties of lanthanides can be exploited to obtain information on specific parts of a protein surface. Owing to the high affinity of coordinatively unsaturated lanthanide complexes for oxygen donors, carboxylate groups can be used as preferential targets for the interaction.

2.9 Slow Motions in Glasses. - Studies of slow molecular motions in supercooled and glassy systems using NMR techniques has become more popular and the number of papers increased steadily during the past few years. In particular the homogeneous versus heterogeneous scenario for the dynamics of glassforming polymers was discussed intensively. In the heterogeneous scenario the nonexponentiality of the a-relaxation is attributed to a superposition of relaxation rates, whereas in the homogeneous scenario the nonexponentiality is intrinsic in nature. The dramatic slowdown of the a relaxation was often attributed to increasingly cooperative dynamics. This was described either as kinetic freezing (VFT equation218) or, alternatively, by the emergence of cooperatively rearranging domains (CRRs) within thermodynamic models.219 The model parameters were fitted via the temperature dependence of a-relaxation times,T,, available from, e.g., mechanical, dielectric, and NMR spectroscopy.220,221 Though the concept of cooperative molecular motion does not necessarily require structural heterogeneity (cf:the spin-facilitated king it was generally believed that, e.g., the CRRs are somehow related with spatial fluctuations resulting in a ‘distribution of correlation times’ that can explain the experimentally observed non-exponential relaxation functions. In this spirit, Anderson and Ullman 223 (following ideas of K a ~ z m a n ndeveloped ~ ~ ~ ) a theory of environmental fluctuations in which molecular reorientation rates can fluctuate between ‘slow’ and ‘fast’ depending on the value of a parameter qt)fluctuating with the environment. In other words, Debye-like rotation was augmented by environmental fluctuations resulting in a ‘composite Markov process’.225However, in addition to heterogeneous scenarios of this kind there were also homogeneous scenarios of the a-relaxation, e.g., models of fractal structure,226that could not be distinguished experimentally at the time they were proposed. In this situation, the ‘reduced 4D-NMR’ experiment introduced in 1991 by SchmidtRohr and S p i e s ~provided ~ ~ ~ a decisive progress. For the first time it became possible to probe the complex dynamics of molecular reorientation by multitime correlation functions. In particular, one can select a subensemble of ‘slow’ molecules and subsequently monitor its re-equilibration on a separate time scale. In the decade following this work several further experiments were devised that allow to probe the heterogeneity at the glass transition. Several reviews have

6: Nuclear Spin Relaxation in Liquids and Gases

211

been published describing this d e ~ e l o p m e n t . ~Sillescu ~ ~ - ~ ~et' aE.231 critically discussed the information that can be obtained from experiments with respect to the existence, the life time, and the length scale of dynamical heterogeneity in glass-forming liquids. The ability to select a dynamically distinguishable subensemble and observe its return to the full equilibrium ensemble is illustrated by examples from multi-dimensional NMR. Reinsberg et al.232 presented a comparison of the determination of the length scale of dynamic heterogeneities in different glass formers by means of a multidimensional 13C solid-state exchange NMR experiment. Results for poly(viny1 acetate) and glycerol have been reported. The existing data together with new results for o-terphenyl have been re-analysed in a slightly revised procedure. This revision is rationalised by computer experiments which are performed on hardsphere systems in analogy to the NMR experiment. Medick et a1.233 characterized the nature of the dynamical heterogeneities of the small component in a binary glass former and of a glass former confined in a zeolite matrix by applying 2D 2H NMR. In both cases the dynamics are described by a distribution of reorientation correlation times G(ln 'c) which is significantly broader compared to that of the corresponding neat system. Vogel et al.234studied the slow P-process of several organic compounds by recording dielectric and 'H NMR spectra below the glass transition temperature Tg.For the neat systems toluene, polybutadiene, cis-decalin and ethanol as well as for the binary glasses chlorobenzene/cis-decalin and toluene/2-picoline, the P-process manifests itself in very similar changes of the 2HNMR spectrum when varying the spatial resolution by increasing the interpulse delay t, in the solidecho pulse sequence. These findings indicate that for all considered glasses, in particular, for both components of the binary mixtures, molecular dynamics involved in the (J-process are highly comparable. Doss et al.235have studied details of the molecular dynamics in a series of pure polyalcohols by means of dielectric spectroscopy and 2H nuclear magnetic resonance (NMR). From glycerol to threitol, xylitol and sorbitol a systematic change in the dynamics of the primary and secondary relaxation is found. With increasing molecular weight and fragility an increase in the width of the a-peak is observed. Details of the molecular reorientation process responsible for the a-relaxation were exploited by two-dimensional NMR experiments. It is found that in the same sequence of polyalcohols the appearance of the secondary relaxation changes gradually from a wing type scenario to a pronounced p-peak. From NMR experiments using selectively deuterated samples the molecular origin of the secondary relaxation could be elucidated in more detail. Qiu and Ediger236performed a direct measurement of the length scale of dynamic heterogeneity in supercooled D-sorbitol using a multidimensional 13Csolid-state NMR experiment. Several models predict that the growth of this length scale were linked to the slowing of dynamics as the glass transition is approached from above. At 275 K ( T . + 7 K), 2.5 f 1.2 nm heterogeneities were detected in sorbitol. This result and recent results of similar measurements on glycerol, o-terphenyl, and poly(viny1acetate) were compared to various models. The reorientation dynamics of deuterated benzene and hexamethyl benzene as

212


additives to the glass former oligostyrene was studied by Medick et ~ 1 below . ~ ~ ~ the glass transition temperature T... By means of 2H NMR, analysed in the frequency and in the time domain, it was shown that the dynamics of the small molecules is governed by an isotropic large angle reorientation process, which is close to the random jump model. Furthermore, the dynamics was characterized by a broad distribution of correlation times. Wiench et ~ 1 . ~studied ~ ' a series of binary zinc phosphate glasses by means of two-dimensional 2D solid and 31P liquid state NMR. Cherry et al.239developed and used structural models of germanium glasses to explain bulk properties of glasses, including density, glass transition temperature and refractive index. The dynamic shear response of six glass formers from different substance classes with widely differing fragility was compared by Schroter and D ~ n t h . No ~ ~ 'correlation of the shear Kohlrausch exponent with fragility was observed. A comparison of shear with multidimensional NMR results on glycerol is compatible with dynamic heterogeneity in glass formers having mobile islands in a lesser mobile matrix. Schrooten et aE.241 characterized silver thioborosilicate glasses by NMR. The structure and magnetic properties of vanadium-sodium silicate glasses were investigated by Mekki et ~ 1 1 7 0. enriched ~ ~ Na20-P205 ~ glasses were measured by Zeyer et ~ 1 Sen. and ~ ~ ~ Youngmann24 studied the temperature dependent structural changes in fluorozirconate glasses and liquids by NMR spectroscopy. Structural studies of magnesium polyphosphate were performed by Walter et With the help of 27AlNMR Padro et al.246 investigated the water solubility mechanism in hydrous aluminosilicate glasses. Geogiev et al.247studied the self-organized phase of bulk PxSe 1-x glasses. Alkali and alkali-lead oxynitride phosphate glasses were meas~ ' et al.249measured spin-spin relaxation times to ured by Munoz et ~ 1 . ~Cosgrove investigate the mobility of poly-dimethylsiloxane polymers.

2.10 Models for Molecular Dynamics. - The liquid state of matter is of great importance in nature and technology. Almost all reactions in biological and chemical systems proceed in solution or liquid like environments. Therefore, it is of interest to develop further the existing models and theories for describing the molecular structure and dynamics of liquids. These models and theories should mediate a better understanding, for example, of the arrangement of the molecules relative to each other or of the dynamic behaviour of the molecules and thus of the route of chemical reactions in liquid systems. The model that is usually applied for the description of molecular reorientational processes in liquids is the rotational diffusion model. A particularly important experimental method to obtain information on the reorientational dynamics of molecules is the measurement of nuclear spin-lattice relaxation rates. Most of the experimental findings on the reorientation of molecules and molecular segments by means of nuclear relaxation rates were obtained in the extreme narrowing region where the product of the resonance frequency and the reorientational correlation time is much less than unity. Outside the extreme narrowing region the relaxation rates become frequency-dependent. Bloembergen, Purcell and Pound 14' employed in their study of relaxation effects on nuclear magnetic resonance phenomena the model of rotational diffusion by D e b ~ e . ~ ~ '


213

However, the interpretation of experimental relaxation data with the BPP model by Bloembergen, Purcell, and Pound was often hampered by the fact that the data could not be reproduced with this model when it was assumed that the reorienting molecules or molecular segments behave like totally or partially rigid b ~ d i e s . ~Thus, ~ ' , ~it~is~questionable whether the rotational diffusion model for rigid bodies is suited for the determination of molecular reorientational dynamics. H e r t ~introduced ~ ~ ~ , ~criteria ~ ~ by which it should be possible to prove the rigidity of the reorienting molecular entities. To explain the failure of the rotational diffusion model, several other models were developed, many of which were described in the comprehensive review by Be~kmann.2'~ One of the most successful attempts is the continuous distribution of correlation times by Cole and D a v i d s ~ n ?which ~ ~ found broad application in the interpretation of relaxation data from crystalline and glassy solids and viscous liquids. It is, however, a long-standing discussion whether the deviation from the BPP spectral density stems from the fact that there exists a distribution of correlation times or if the correlation functions are just not exponential. At least for highly viscous, glassy systems, the experimental results indicate a distribution of correlation times (cf: to the review by S i l l e s c ~ ~whereas ~ ~ ) , the situation in liquids is still not clear. Another way to describe the deviations from the BPP spectral density is the so-called model-free approach by Lipari and S ~ a b owhich ,~~~ corresponds to a discrete distribution of correlation times and is a generalization of the two-step model by Wennerstrom et al.256It was originally developed for taking internal motions in macromolecules into account without giving an explicit model when calculating the relaxation data.257Although the model-free approach was first applied mainly for the interpretation of relaxation data of macromolecules, it is now also used for fast internal dynamics of small and medium-sized molecules. D011e258studied the reorientational motion of the hydrocarbon 1,2,3,4-tetrahydro-5,6-dimethyl- 1,4-methanonaphthalene (5,6-Me2THMN) over a wide range of temperature by the evaluation of I3C spin-lattice relaxation rates and NOE factors. The data from measurements at 22.63,75.47, and 100.62MHz were fitted to spectral densities introduced by Bloembergen, Purcell and Pound (1948),Davidson and Cole (195l),and for the first time, by a new spectral density derived from a Tricomi correlation function recently introduced by Zeidler (1991). Wagner et al.259 measured the 19Fnuclear spin-lattice relaxation rate constants as a function of magnetic field strength for 1,12-diaminododecane labeled at one end with a nitroxide radical and at the other with a trifluoromethyl group. The magnetic relaxation dispersion profile (MRD) reports the spectral density function appropriate to the end-to-end correlation function for the doubly labelled molecule. After extrapolation to zero concentration to eliminate the intermolecular relaxation contribution to relaxation, the resulting intramolecular MRD profile was compared with several model approaches. The rotational model for the spectral density functions as included in the Solomon-Bloembergen-Morgan equations does not describe the data well. The earlier model of Freed260for nuclear spin relaxation induced by a freely diffusing paramagnetic co-solute is not rigorous for this case because the paramagnet is tethered to the

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observed nuclear spin and only a restricted space in the immediate vicinity of the nuclear spin is accessible for pseudo-translational diffusion of one end of the molecule with respect to the other. A generalization of the Torrey model for magnetic relaxation by translational diffusion developed by Nevzorov and Freed,261,262 which includes the effect of restrictions imposed by the finite length of the chain, describes the experiment within experimental errors. A simple modification of the Hwang-Freed that does not specifically include the dynamical effects of the finite tether also provides a good approximation to the data when the tether chain is sufficiently long. Kowalewski et al.264reported multiplefield (4.7, 9.4, 14.1 T) carbon-13 relaxation data for hexamethylenetetramine (HMTA) in the cryosolvent D20/DMSO at 243 K. Under these conditions, the reorientational motion of HMTA is outside of the extreme narrowing range and the relaxation data can be subjected to a quantitative interpretation. Because of the high symmetry of the HMTA molecule, the reorientation must be isotropic. Chenon and Werbe10w~~~ enlisted nuclear spin relaxation rates associated with cross-correlated, dipole-dipole interactions to help characterize the solution state dynamics of a small heptapeptide, deltorphin-I. A simple two-site jump model can be used to interpret the data obtained on two specific 13C labelled residues, D-alanine and glycine. The influence of temperature and solvent upon the observed dynamics is investigated. Similarly, relaxation rates associated with dipole-shielding anisotropy interferences were used to examine the magnitude and orientation of various chemical shielding tensors within the D-alanine and glycine residues. NMR relaxation experiments of isotopically labeled proteins provide a wealth of information on reorientational global and local dynamics on nanosecond and subnanosecond timescales for folded and nonfolded proteins in solution. Recent methodological advances in the interpretation of relaxation data have led to a better understanding of the overall tumbling behavior, the separability of internal and overall motions, and the presence of correlated dynamics between different nuclear sites, as well as to new insights into the relationship between reorientational dynamics and primary and tertiary protein structure. Bruschweiler266presented some new methods which are particularly useful when dealing with nonfolded protein states. An accurate description of global tumbling of a protein is essential for correct analysis and interpretation of internal dynamics and thermodynamics. The accurate fitting of global tumbling parameters is affected by the number of experimental relaxation data points available for analysis, the distribution of data points over the domain of the function describing the tumbling, the measurement error associated with the data, the error associated with use of an approximate functional form, and errors in the protein structure. Pawley et ~ l . ~ ~ ~ p r e s ean n t eanalysis d of the influence of these factors on the error in global tumbling parameters and the corresponding error in the calculated TI/T2 values. Model-free analysis of NMR relaxation data, which is widely used for the study of protein dynamics, consists of the separation of the global rotational diffusion from internal motions relative to the diffusion frame and the description of these internal motions by amplitude and timescale. Five model-free models exist, each of which describes a different type of motion. Model-free analysis requires the


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selection of the model which best describes the dynamics of the N H bond. D'Auvergne et demonstrated that the model selection technique currently used has two significant flaws, under-fitting, and not selecting a model when one ought to be selected.

3

Selected Applications of Nuclear Spin Relaxation

3.1 Pure Liquids. - Locci et a1.269showed that monoatomic xenon can be complexed in the proximal cavity of metmyoglobin. This study highlights the potential of 129Xe-relaxationrate measurements to obtain information on the xenon-metmyoglobin interaction. In another study 270 these authors monitored the mutarotation of D-glucose (see structure) using 129XeNMR spectroscopy. This is the first example of the use of xenon NMR spectroscopy to probe a configurational equilibrium in solution of a system which does not complex xenon. constructed special high-temperature and -pressure multiTsukahara et nuclear NMR equipments for the measurements of 170-NMRchemical shift and spin-lattice relaxation time (Tl)in water over the range from liquid to supercritical (SC) states. The chemical shift could be interpreted in terms of the extent of hydrogen bonding. Although the cleavage of hydrogen bonding of water proceeds continuously from liquid to SC conditions, the hydrogen bonding was found to still remain even under SC conditions. It was confirmed that the spin-lattice relaxation of 1 7 0 is mainly controlled by the quadrupole interaction and the TI values of 1 7 0 are related with the values of the molecular reorientational correlation time (TJ over the range from liquid to SC states. The chain dynamics in high molecular weight polymer melts observed from proton magnetic relaxation generates a reference frequency, closely related to the chain entanglement interaction. Observing polybutadiene, Addad and Guillerm0272reported on the slight molecular weight dependence of the segmental correlation time. presented quantum Cluster Equilibrium (QCE) Huelsekopf and theory for liquid 2,2-dimethyl-3-ethyl-3-pentanol (DMEP). The cluster equilibria that dictate phase composition are determined by the rigorous techniques of quantum statistical thermodynamics in the canonical ensemble, based on the ab initio partition function. The validity of the resulting QCE cluster distribution is demonstrated by comparison with experimental temperature dependent NMR hydroxyl proton chemical shifts and OH-stretch vibrational frequencies. The results indicate that liquid DMEP is built upon monomers and dimers only. calculated NMR properties such as chemical shifts and quadrupole coupling constants for liquid water, ammonia, hydrogen sulfide and phosphine. Farrar and Ropp274showed that experimental NMR measurements of the deuterium and oxygen relaxation times in neat water along with experimental/theoretical measurements of the deuterium and oxygen quadrupole coupling constants provide accurate values for the rotational correlation times of neat water over the liquid range. The results indicate that the motion of water is

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anisotropic. Weekley et ~ 1 described . ~ a~ nuclear ~ magnetic resonance (NMR) probe and spectrometer capable of investigating full intact wine bottles is described and used to study a series of Cabernet Sauvignons with high resolution 'H NMR spectroscopy. Selected examples of full bottle 13CNMR spectra are also provided. The application of this full bottle NMR method to the measurement of acetic acid content, the detection of complex sugars, phenols, and trace elements in wine is discussed.

Non-Electrolyte Solutions. - It is a challenge to predict the self-diffusion coefficients of the different components in a fluid mixture from the corresponding values of the pure components. Theories concerned with this problem are due to Thorne and E n ~ k o gJaccuci , ~ ~ ~ and McDonald277and Enninghorst, Wayne and Zeidle~-.~~' Recollecting the development for pure fluids, the Boltzmann expression for self-diffusion in hard-sphere gases had to be extended to higher densities in order to apply it to the liquid state. Whereas Enskog could extend the theory to denser gases, the real improvement for liquids was done by Alder279and later authors280-282 using computer simulations on hard spheres. Finally it turned out that the hard-sphere model is too simple and coupling of translation with rotation required introduction of a roughness parameter283which was connected to the attractive part of the Lennard-Jones potential by but a more recent investigation makes this connection This is along the same line as with the structure of dense liquids which is determined essentially by the repulsive and not by the attractive potential, therefore it is rather the deviation from the sphere that is responsible for the coupling. Experimental determinations of the roughness parameter indicate that it depends onthe molecular size and on the temperature.286Jaccuci and McDonald277applied the same corrections to the Thorne-Enskog mixture diffusion coefficients as are appropriate to correct the Enskog result for the single-component hard-sphere fluid. In a previous self-diffusion study on the cyclopentane with isopentane mixture Enninghorst et al.278found it necessary to extend Jaccuci and McDonald's theory by introducing roughness parameters also for the mixture. The coupling acts between like and unlike molecules, and for the latter the Lorentz-Berthelot mixing rule was employed. Jacob and Zeidler287investigated another mixture: cyclohexane and benzene in equimolar composition. Since the resolution was sufficient to differentiate between the protons of both components, it was possible to obtain the single self-diffusioncoefficients for benzene and cyclohexane in the mixture. The results are discussed on the basis of different theories (ThorneEnskog, Jacucci-McDonald, Enninghorst-Wayne-Zeidler) applicable to mixtures. Vittadini et al.288studied the molecular mobility of water in a microbiological media containing complex and heterogeneous mixtures of cellulose, I-sorbose, and orange serum broth (OSB)using 'H and 170high-resolution NMR. Presence of liquid or solvent water had a profound effect on a marked increase in T2 relaxation time observed in sorbose-containing samples. 1 7 0 NMR data were not composition dependent, while 2HNMR data were highly sorbose dependent. Ariza-Casteolo et ~ 1 . ~completely ' ~ assigned the high-resolution NMR spectra

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217

2,4-dinitrofluorobenzene. The one-bond and long-range coupling constants were determinated and the signs were obtained by means of two-dimensional correlation spectroscopy. The spin-lattice relaxation times of 13Crevealed their anisotropy components and it was evident that the dipole-dipole contribution of fluorine was an efficient mechanism for 13Crelaxation. Molchanov and Gryff-Kelle?” determined the temperature dependence of the correlation time describing reorientation kinetics of cyanogens bromide in CDC13 solution on the basis of the linewidths of the 14NNMR signal. It has been found that the longitudinal spin relaxation of the 15Nnucleus occurs by shielding anisotropy and spin-rotation mechanisms, whereas for the 13C nucleus these mechanisms are of lesser importance. In the latter case the scalar relaxation of the second kind due to carbon-bromine coupling is the predominant relaxation mechanism. Dawson and Wallen291reported previously that the nuclear magnetic resonance (NMR) 14N spin-lattice relaxation times (TI) of CH3CN in CH3CN-H20 mixtures directly correlate with the solution viscosity when scaled for temperature in this common chromatographic mobile phase system. Then they demonstrated that the 2HTI relaxation times also correlate with viscosity, contrary to a previous report. This establishes 2H TI relaxation times as a useful means of measuring changes in solution viscosity in CH3CN-H20mixtures. Nagashima et al.292studied Water-in-carbon dioxide (W/C) microemulsions stabilized by an ammonium carboxylate perfluoropolyether surfactant with NMR diffusion and relaxation methods with the aim of obtaining information on the dynamics of this system, as well as aiding in the design of new surfactants that can form stable microemulsions in C02. Nuclear magnetic resonance spectroscopy is frequently used for structural characterization of humic substances (HS). However, the study of relaxation phenomena of HS, including spin-lattice and spin-spin relaxation, has received little attention. Wang et aE.293investigated proton spin-lattice relaxation times (T,) of humic acids (HA) in deuterated 0.5 M sodium hydroxide (NaOD) and dimethylsulfoxide (d(6)-DMSO) at various concentrations, temperatures, and pHs. Takamuku et aE.294studied aqueous mixtures. of cycloethers, 1,4-dioxane, 1,3dioxane, and tetrahydrofuran (THF) by using large-angle X-ray scattering, small-angle neutron scattering, and NMR relaxation techniques. 3.3 Electrolyte Solutions. - Rhee and Kim295formulated the stable iron ferrite nanoparticles by using direct coprecipitation of an aqueous solution of iron salt and a tetramethylammonium hydroxide (TMAOH) solution. These super paramagnetic nanoparticles were found to have uniform sizes of about 7 nm and showed no coalescence in the aqueous solution for a few months. The relaxation times, TI and TZ, of the hydrogen protons in the aqueous solution of these nanoparticles were measured using a nuclear magnetic resonance (NMR) spectrometer at room temperature over a wide range of concentrations of the nanoparticles in the aqueous solution. The inverses of the relaxation times were observed to directly depend on the concentration of nanoparticles.

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Okroyan et al.296studied the relaxation characteristics of the 1 7 0 nucleus in aqueous clusters of Na+ and K + by nuclear magnetic resonance (NMR) spectroscopy. The influence of the viscosity and pH value of the solution on the spin-spin relaxation of 1 7 0 nucleus was investigated. Tian et ~ 1 measured . ~ a~ series of amino acids and carboxylic acids by 13CNMR spectroscopy. The results showed that addition of 3M MgC12,led to shortening of relaxation time and 13C NMR integral area of samples was well proportional to the number of carbon atoms. So MgC12 is proposed as an efficient relaxation agent for analysis of amino acids and some carboxylic acids. Fumino et al.298studied pressure effect on the spin-lattice relaxation rates of 'H and 1 7 0 nuclei for heavy water molecules in MgC12and CaC12aqueous solutions. The same effects were also measured heavy water molecules in alkali earth chloride aqueous solutions.299Ding et a1.300reported combined theoretical and experimental NMR studies on alkali ion-cryptand interactions which affect charge transport in liquid electrolytes. Encapsulation of the cation by a cryptand cage can significantly improve the ionic conductivity, by reducing the fraction of time in which the cation is bound to its counterion. He et aL3*' separated saccharides derivatized with 2-aminobenzoic acid by capillary electrophoresis and their structural consideration by nuclear magnetic resonance. Adebahr et aL302 performed multinuclear pulsed field gradient NMR measurements and theological viscosity measurements on three series of polymer gel electrolytes. The gels were based on a lithium salt electrolyte swollen into a copolymer matrix comprising an acrylate backbone and ethylene oxide side chains. Tambelli et al.303reported a 'H and 7Li NMR lineshape and spin-lattice relaxation time investigation of a polymer electrolyte obtained from the polymerization of ethylene glycol and citric acid with lithium salt. The 7Li data indicated that the lineshape and spin-lattice relaxation were affected by the coupling of its quadrupolar moment to the surrounding electric field gradients. Ruso et aL304studied the self-association of n-hexyltrimethylammonium bromide (C(6)TAB)in aqueous solution by static and dynamic light scattering and NMR spectroscopy in the presence of added electrolyte. Li and He3()'introduced multidimensional electrophoretic NMR (ENMR) to determine structures of coexisting proteins and protein conformations in solution. Signals of different proteins are separated in a new dimension of electrophoretic flow according to their characteristic electrophoretic mobilities. The electrophoretic interferograms have been generated in the flow dimension in two approaches by incrementing either the amplitude or the duration of the electric field. The ENMR method of incrementing the duration of the electric field, however, introduces severe signal decays due to molecular diffusion and spin relaxation, limiting the effectiveness of the method. In this study, an improved method of constant-time multidimensional ENMR (CT-ENMR) has been proposed and successfully tested. Molten Salts. - Structural and dynamic approaches of molten salts were '~ liquid and polymer studied by several authors. Nakagawa et ~ 1 . ~investigated gel electrolytes for lithium batteries composed of room-temperature molten salt

3.4

~


219

doped by lithium salt. Ma et al.307designed two new chiral ionic liquids and characterized them by NMR spectroscopy. Egashira et ~ 1 . ~studied ~ ' the effect of the coexistence of anion species in imidazolium cation-based molten salt systems. Molten salt hydrates proved to be alternative solvents to cellulose. To investigate the reasons for this dissolving ability, information about the solventcellulose interactions is essential. Brendler et aL309used 13CHR NMR as well as 7LiNMR to obtain further insight into this problem. Carper et al.310solved the NMR rotational correlation equations for dipolar relaxation between 'H and either 'I9Sn, 195Pt,199Hgor 207Pbfor viscous solutions using the R2/R1 dipolar ratio. The calculated correlation times at each field strength have been fitted to pairs of polynomials. Sun et ~ 1 . ~developed ~ ' novel lithium polyelectrolyte-ionic liquid systems. The results of 7Li-NMR showed that lithium cations in the copolymer system are mobile whereas in the homopolymer, only a fraction appears to be mobile.

4

Nuclear Spin Relaxation in Gases

Gas-phase NMR has great potential as a probe for a variety of interesting physical and biomedical problems that are not amenable to study by water or similar liquid. However, NMR of gases was largely neglected due to the low signal obtained from the thermally polarized gases with very low sample density. The advent pf optical pumping techniques for enhancing polarization of the noble gas 3He and 129Xehas bought new life in this field, especially in medical imaging where 3Helung inhalation imaging is approaching a clinical application. However, there are numerous applications in materials science that also benefit from the uses of these gases. The sensitivity of conventional nuclear magnetic resonance (NMR)techniques is fundamentally limited by the ordinarily low spin polarization achievable in even the strongest NMR magnets. However, by transferring angular momentum from laser light to electronic and nuclear spins, optical pumping methods can increase the nuclear spin polarization of noble gases by several orders of magnitude, thereby greatly enhancing their NMR sensitivity. Goodson7 reviewed the principles and magnetic resonance applications of laser-polarized noble gases. The enormous sensitivity enhancement afforded by optical pumping can be exploited to permit a variety of novel NMR experiments across numerous disciplines. Many such experiments are reviewed, including the void-space imaging of organisms and materials, NMR and MRI of living tissues, probing structure and dynamics of molecules in solution and on surfaces, NMR sensitivity enhancement via polarization transfer, and low-field NMR and MRI. Berthault and Desvaux3'* used NMR of laser-polarized xenon to probe the dissolution behaviour of the noble gas in different liquids. The dissolution and self-relaxation rates are extracted via a macroscopic model, and comparison of the decay rate of the xenon magnetization in deuterated and non-deuterated solvent pairs allows the determination of the pure dipole-dipole contribution to

220


relaxation. Rice and Raftery313studied the production of hyperpolarized Xe via spin exchange with optically pumped Rb atoms at 47 000 G under high-pressure conditions. From variable pressure and temperature studies, the spin exchange efficiency was found to be pressure dependent and lower than comparable low-field measurements. Hyperpolarized gases ('29Xeand 3He) are being used increasingly in both MRI and NMR spectroscopy studies. However, it has been shown that carrier agents are required to preserve the long relaxation times of gases in biological fluids. Oregioni et aL314showed that optimised gas transport can be achieved through controlled TI and T2 measurements of '29Xegas at equilibrium, using the steady-state free precession method (SSFP). Desvaux et aL315 studied the magnetization transfer from laser-polarized xenon to protons with spin-diffusion quenching. Ledbetter and R ~ m a l i sinves.~ ' ~ tigated nonlinear effects of long-range dipolar interactions in a spherical sample of hyperpolarized liquid '29Xe.Using two high-T-c SQUID detectors they directly measure the evolution of the magnetization gradients. For small initial r.f. tip angles the authors observe an increase in the transverse relaxation time T2*by a factor of 5 and coherent oscillations of magnetization gradients. For large tip angles they observed an exponential growth of the magnetization gradients and demonstrated a gain in sensitivity to magnetic field gradients by a factor of 10. Mortuza et aL3l7explored gas mixtures with high xenon densities for continuous flow spin-exchange optical pumping. It was shown that the 129Xe-NMR signal increases significantly with increasing xenon partial pressures up to about 200 kPa, despite a decreasing spin-polarization. There is currently a debate on whether or not the transverse relaxation time is finite at low temperature in spin-polarized Fermi liquids. The experimental results so far have been contradictory. Buu et aL318presented NMR measurements carried out in 6.2% 3He-4Hemixture at 11.3 T down to 3.5 mK. A careful analysis of their data yielded an anisotropy temperature T-a = 6.11mK, which is smaller than the values reported earlier. In a second paper319the authors described a model for calculating spin-wave modes and spin-echo signals in 3He4Hemixtures. Suarez et a1.320performed a gas-phase dynamic NMR study of the internal rotation in N-trifluoroacetylpyrrolidine. Kinetic parameters were found to be lower in the gas phase than in solution. Jackowski and W i l ~ z e k ~ performed ~' new measurements of NMR spectral parameters for acetonitrile in the gas phase. Penner et al.321studied the structure and dynamics of ND3BF3in the solid and gas phases.

5

Self-Diffusionin Liquids

5.1 Experimental and Theoretical Aspects. - Pulsed gradient spin echo NMR is a powerful technique for measuring diffusion coefficients. When coupled with appropriate data processing schemes, the technique becomes an exceptionally valuable tool for mixture analysis, the separation of which is based on the molecular size. Extremely fine differentiation may be possible in the diffusion


221

dimension but only with high-quality data. For fully resolved resonances, components with diffusion coefficients that differ by less than 2% may be distinguished in mixtures. For highly overlapped resonances, the resolved spectra of pure components with diffusion coefficients that differ by less than 30% may be obtained. In order to achieve the best possible data quality one must be aware of the primary sources of artefacts and incorporate the necessary means to alleviate them. Antalek323described the origin of these artefacts, along with the methods necessary to observe them. Jeener324discussed macroscopic molecular diffusion in liquid NMR. His paper has three independent parts. First, Torrey’s treatment of molecular diffusion in NMR was directly extended to polyatomic molecules by the introduction of a position-dependent average density operator. Second, some improvement was proposed for the study of 11 abnormal’ diffusion by spin-echo experiments, by directly answering the following question: ‘Which statistical properties of the molecular motion are directly measured in simple spin-echo experiments’? Third, a simple numerical technique was proposed for the prediction of the effects of diffusion in spin echo experiments of the CRAZED type, in which the relevant dipolar field inhomogeneities have the spatial periodicity of the magnetization helix. Mair et al.325reported initial NMR studies of xenon gas diffusion in model heterogeneous porous media and continuous flow laser-polarized xenon gas. Both areas utilize the pulsed gradient spin-echo (PGSE) techniques in the gas phase, with the aim of obtaining more sophisticated information than just translational self-diffusion coefficients. Diffusion coefficients provide uniquely detailed and easily interpreted information on molecular organization and phase structure. They are quite sensitive to structural changes, and to binding and association phenomena. in particular for liquid colloidal or macromolecular systems. De Souza and L a ~ e r d edescribed ~*~ the principles of diffusion measurements in liquids by pulsed magnetic field gradient spin-echo (PFG-SE) NMR spectroscopy. The important PFG-SE technique DOSY is presented and discussed. This is a non-invasive technique that can provide individual multicomponent translational diffusion coefficients with good precision in a few minutes, without the need for radioactive isotopic labelling. Todica and analysed different methods of investigation of the spin-echo attenuation, in the NMR pulsed field gradient experiments, in order to optimise the measurements of the self-diffusion coefficient of the small molecules of simplest liquids. A particular algorithm of representation of the experimental data was proposed in order to eliminate the effect of the residual gradient. Sun et al.328introduced a new method that suppresses the background gradient in pulsed gradient stimulated echo measurements. Buckley et al.329presented novel applications of fast self-diffusion measurement. Difftrain (Diffusion train), which uses successive stimulated echoes from a single excitation pulse where a portion of the available magnetisation is recovered for each echo, is used to measure self-diffusion by varying the observation time. Thrippleton et ~ 1 . ~ ~ ’ presented a new experiment for the single-scan measurement of diffusion coefficients. The principle is to introduce a spatial variation in the parameters of a

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conventional pulse sequence, so that all of the scans required to determine some physical parameter can be recorded simultaneously from different parts of the sample. Fur0 and D ~ i n s k i reviewed ~~' experiments for the determination of anisotropic diffusion. Experiments of this kind preferably require oriented samples and/or orientation-dependent spin coupling and/or magnetic field gradients in different directions. When applying pulsed field gradient (PFG) NMR experiments to determine the molecular mobility characterized by the diffusion coefficient, it is crucial to have control over all experimental parameters that may affect the performance of the diffusion experiment. This could be diffusion measurement in the presence of magnetic field transients, internal magnetic field gradients, either constant or spatially varying, convection, mechanical vibrations, or in the presence of physical restrictions affecting the diffusion propagator. Sorland and A k ~ n e diss~~~ cussed the effect of these parameters on the diffusion experiment. It is shown that the tracer diffusion and self-diffusion coefficients of liquids are in a simple linear relation with a constant coefficient, which depends on only the molecular size ratio and the mass ratio of the solute and the solvent molecule. With experimentally determined tracer diffusion and self-diffusion coefficients, the relation can be used for estimating the molecular sizes of polyatomic molecules. Rah et ~ 1 . could show that the relation of diffusion coefficients is an excellent means to estimate molecular sizes from the data on diffusion coefficients measurable by various methods including NMR techniques. The CPMG sequence has been extremely useful for efficient measurements of NMR signal, spin-spin relaxation, and diffusion, particularly in inhomogeneous magnetic fields, such as when samples are outside the magnet and r.f. coil. Song334 described a method to classify the CPMG coherence pathways and illustrated the nature of these types of pathways. This classification showed that direct echo and stimulated echoes are the major contribution to the CPMG signal.

Selected Examples. - Regan et ~ 1 performed . ~ ~a PGSE ~ NMR diffusion study of the self-association of N-methylacetamide in carbon tetrachloride. N-Methylacetamide (NMA) is known to self-associate in solution through the formation of hydrogen bonds. This behaviour, and the presence in the molecule of an amide bond, make it an interesting model for study since the oligomer can be considered to be a protein analogue. The aims of the present study were to estimate the thermodynamic parameters of the self-association process, through the measurement of the diffusion coefficient of NMA in carbon tetrachloride (CC4),and to predict the population distribution of oligomers. as a function of the concentration of NMA. Sucrose and trehalose in aqueous solutions have been studied by EkdawiSever et ul.336Jacob and Zeidler287investigated self-diffusionin binary systems at high pressure. A high-pressure probe head was constructed for this purpose. The results were discussed on the bases of different theories. Fang et ~ 1used . PFG ~ ~ NMR technique to measure the self-diffusion coefficients of two surfactants cetyltrimethylammonium bromide (CTAB) and Triton X-100 (TX-100) in their mixed aqueous solutions. Ludemann and Chen [3381 measured transport prop-

~

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erties of supercritical fluids and their binary mixtures. The diffusion of water in liquid and supercritical carbon dioxide has been studied by Xu et aE.339 Evertsson et ~ 1 . ~ measured ~’ NMR self-diffusion of the monooleoylglycerol/poly ethylene glycol/water L-3 phase. Swallen et ~ 1 . presented ~ ~ ’ a direct measurement of self-diffusion of a single-component glass-forming liquid at the glass transition temperature. De Luca and characterized a hyperbranched polyester measuring diffusion coefficients. Kwak et al.343measured macroscale mutual-diffusion coefficients of phosphate ions in dextran gels. The data could be fitted to Fick’s law. Measuring diffusion coefficients by nuclear magnetic resonance spectroscopy is a useful way to study binding, because changes can be detected when a small ligand interacts with a macromolecular target. Diffusion coefficients can be miscalculated, however, due to magnetization transfer between the receptor and ligand. This transferred nuclear Overhauser effect (trNOE) disrupts the observed signal decay due to diffusion as a function of the experimental diffusion time. Since longer diffusion times also selectively edit free ligand signal, the measured diffusion coefficients become biased toward the fraction of bound ligand. Despite this discrepancy, under these experimental conditions, the trNOE selectively influences the measured signals of binding ligands and can be used to gain insight into ligand-protein interac. ~ these ~ phenomena. ~ Novikov et ~ 1 . ~studied ~ ’ diffutions. Lucas et ~ 1 discussed sion of large large single-charge ions in aqueous solutions. Walker et ~ 1 determined the rotation-diffusion tensor orientation from 13C-’Hcross-relaxation rates for a chlorotoluene.

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

R. Ludwig, in ‘Nuclear Magnetic Resonance’, ed. G.A. Webb, Specialist Periodical Reports, The Royal Society of Chemistry, Cambridge, 2003, vol. 32, p.201-237. N. Murali and V. Krishnan, Conc. Magn. Reson., 2003,17A, 86. D. Frueh, Prog. Nucl. Magn. Reson. Spectrosc., 2002,41, 305. D.A. Case, Acc. Chem. Res., 2002,35,325. M Akke, Curr. Opin. Struc. Biol., 2002,12, 642. J.G. Kempf, J.P. Loria, Cell Biochem. Biophys., 2003,37, 187. B.M. Goodson, J . Magn. Reson., 2002,155, 157. R.W. Mair and R.L. Walsworth, A p p l . Magn. Reson., 2002,22, 159. I.V. Koptyug, and R.Z. Sagdeev RZ, Usp. Khim., 2002,71,672. I. Bertini, C . Luchinat, and G. Parigi, Conc. Magn. Reson., 2002,14,259. H. Sillescu, R. Bohmer, A. Doss, G. Hinze, T. Jorg and F. Qi, ACS Sym. Ser., 2002, 820,256. Q. He, W. Richter, S. Vathyam, and W.S. Warren, J . Chem. Phys., 1993,98,6779. W.S. Warren, W. Richter, A.H. Andreotti, and B.T. Farmer 11, Science, 1993, 262, 2005. A. Ahn, W.S. Warren, and S. Lee, J . Magn. Reson., 1997,128, 114. A. Ahn, S. Lee, and W.S. Warren, Mol. Phys., 1998,95,769. P. Brokaert, A. Vlassenbroek, J. Jeener, G. Lippens, and J.-M. Wieruszeski, J . Magn. Reson. A, 1996,120,97.

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7 Solid State NMR Spectroscopy BY A.E. ALIEV AND R.V. LAW

1

Introduction

The aim of our contribution is to outline important recent advances achieved in the area of solid-state NMR based on the literature published between July 2002 and June 2003. A very large number of works making use of solid-state NMR techniques has been published during this period, however, not all of these publications have been included in this report. Specifically, over 800 references that included solid-state NMR and related terms either in the title or in the keywords were initially retrieved for the preparation of this report, and 375 of these were selected. In choosing sources for this report we have aimed at highlighting publications that use solid-state NMR as the only or the primary technique for structural and/or dynamics studies of solid materials, as well as those that have applied the technique in an innovative manner. In selecting references, we have also aimed at illustrating diversity of problems and subject areas covered by modern solid-state NMR applications. The following section 2 of the report includes review articles on both general and specific aspects of solid-state NMR spectroscopy and its applications. Publications on theoretical and experimental developments that are of general methodological interest are arranged in sections 3-6. A few general trends can be noticed regarding developments of the solid-state NMR in the last year. In terms of theory (section 3), Floquet treatment is becoming a widely accepted approach for describing a wide range of solid-state NMR phenomena, especially when it comes to overcoming the limitations of the average Hamiltonian theory. With regard to experimental developments (Section 4), specific aspects of the solid-sate NMR of uniformly l3C-labe1ledsolids, triple resonance experiments for sequential assignments in peptides and proteins, as well as satellite transition MAS NMR of quadrupolar nuclei have received close attention of various groups. In respect of structure determinations (section 5), some striking examples comprise re-refinement of X-ray structure based on REDOR measurements, use of distance and torsional angle constraints from solid-state NMR for the 3D structure determination of peptides, as well as combined solid-state NMR and powder diffraction analysis for the molecular structure determination, Among various quantum-mechanical techniques used for the calculations of the NMR parameters and their anisotropy in the solid state (Section 6), the large-scale success Nuclear Magnetic Resonance, Volume 33 0 The Royal Society of Chemistry, 2004

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of the density functional theory (DFT) is notable. Many publications have demonstrated that additional computational efforts, involving DFT or similar type of calculations, significantly improve both the quality and the level of interpretation of solid-state NMR data. Finally, applications of solid-state NMR are included in section 7, which has been divided into subsections depending on the class of the materials studied, as well as the nature of the solid-state NMR studies undertaken. Over 250 references are included in the applications section 7, compared to approximately 100 references in the previous sections 2-6, thus the level of detail for many references in section 8 is restricted.

2

Reviews and Introductory Articles

Solid-state NMR spectroscopic techniques, such as CP, MAS, multiple-pulse sequences, homo- and heteronuclear decoupling and recoupling experiments, M Q spectroscopy and dynamic angle spinning, as well as their applications to structure determination have been reviewed by Laws et al.' As outlined by the authors, modern solid-state NMR spectroscopic techniques not only produce spectra with a resolution close to that of solution NMR spectra, but also provide valuable information about anisotropic interactions, which are often unavailable for liquid samples. New applications of 1D and 2D solid-state NMR spectroscopy in structural studies of organic solids have been reviewed by Potrzebowski? Particular subject areas covered in this article include recent developments in the studies of polymorphism and pseudo-polymorphism, as well as new advances in the NMR studies of strong and weak hydrogen bonding in the solid state. In addition, the use of solid-state NMR techniques for distinguishing between enantiomers and racemates has been presented. In another review of general interest solid-state NMR spectroscopy and its application in analytical chemistry has been de~cribed.~ Recent experimental and theoretical research involving the nuclear spin-spin coupling tensor and the indirect coupling between two magnetic nuclei in closed-shell molecules has been reviewed by Vaara et aL4 This review covers literature published prior to autumn 2001. In another related article applications of theoretical methods to NMR chemical shifts and coupling constants have been reviewed by McKee.' Recent progress on accurate and reliable prediction of these NMR parameters has been summarised. The GIAO calculations of absolute shieldings and their relationship with experimental chemical shifts for aromatic heterocycles have been presented by Alkorta et ~ 1 Conformational . ~ analysis and tautomerism, as well as solid-state and solvent effects have been examined. Jerschow has reviewed dipolar and scalar couplings in solid-state NMR of quadrupolar nuclei7 In the case of asymmetric electron distribution, quadrupolar interaction very often overshadows dipolar and scalar couplings. A summary of how these interactions manifest themselves in quadrupolar NMR and how

7: Solid State N M R Spectroscopy

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they can be exploited for resonance assignment and structure elucidation is presented. Studies of slow exchange processes involving equivalent sites in solids by 1D MAS NMR have been reviewed by Luz et aE.' In ordinary experiments the exchange is measured by magnetisation transfer under MAS conditions and spinning sideband manifolds are selectively saturated. However, special 1D magnetisation methods are needed for measuring spin exchange between equivalent sites. Review of these methods and their mathematical description for quantitative analysis has been the main purpose of the article by Luz et al. In addition, the spin diffusion effects have also been considered. Solid-state NMR experiments under periodic modulation by fast MAS and r.f. pulses have been reviewed by Hafner and D e m ~ o Understanding .~ of signal encoding by spin interactions under fast MAS conditions and interferences between MAS and pulses is prerequisite for new pulse sequences. This review discusses corresponding strategies and techniques with particular emphasis on homonuclear decoupling techniques for line narrowing in solids. Experimental and theoretical aspects of the M Q MAS experiment have been discussed in the review by Goldbourt and Madhu.'' This review addresses the development of various pulse schemes aimed at improving the signal-to-noise ratio and anisotropic line shapes. Representative spectra are shown to underscore the importance and applications of the M Q MAS experiment. Computer simulations of solid-state NMR spectra have been presented by Eden in a series of three detailed article^."“^ The first article" discusses the basics of the writing of computer programs for simulating solid-state NMR experiments with static and rotating samples and gives an account of the relevant NMR theory needed. The concept of irreducible spherical tensors has been reviewed and their use for the construction of Hamiltonians that represent various NMR interactions has been outlined. The second article'* explains how the theory may be implemented for simulating NMR line shapes of a single crystal in a static and rotating sample. The general building blocks of a computer program for numerically calculating NMR signals have been discussed and computer algorithms for carrying out the simulations have been presented. Finally, in the third a r t i ~ l evarious '~ aspects of simulations of NMR responses from powders have been discussed. The underlying equations for powder averaging have been derived and orientational symmetry in solid-state NMR has been exploited to achieve more efficient calculations. Explicit computer code is given for simulation of NMR spectra from powders containing two homonuclear spins-1/2 in a static sample and a heteronuclear two spin-1/2 system under MAS conditions. The data acquisition and processing of the wideline chemical shift NMR spectra of solids have been reviewed by Van Bramer et al.14The article highlights the importance of developing procedures to minimise inherent line shape distortions for the proper determination of NMR chemical shift parameters from the wideline spectra. Techniques for the analysis of spin-echo spectra broadened by chemical shift dispersion have also been discussed.

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Different aspects of solid-state 207Pbspectroscopy - calculation of chemical shift and spin-spin coupling tensors, relationship between structure and NMR parameters, wideline and MAS techniques and their applications - have been presented by Dybowski and Neue." Blinc and Apih have reviewed the NMR spectra of multidimensionally modulated incommensurate systems.16Unlike periodic crystals, NMR spectra of incommensurate crystals reflect the large number of inequivalent nuclear sites and instead of sharp lines characteristic frequency distributions are observed. Specific features of the NMR spectra of the incommensurate solids and their dependence on electric field and thermal fluctuations are presented. Solid-state NMR studies of the structure and mechanisms of proteins (including membrane proteins and disease-related protein aggregates) have been reviewed by T h o m p s ~ n . Progress '~ in the development of NMR and biochemical methods for the complete structure determination of small (lo6Hz above room temperature. The quadrupole coupling of 6.5 kHz remained unchanged even at 360 K for the ND4+ ion. The anisotropic tensor of 15Nchemical shift suggested that the rotating ammonium ion is slightly deformed in the cavity of 18-crown-6.

7.6 Cellulose and Related Materials. - Complete assignment of the C P MAS 13CNMR spectrum of I3C-enrichedcellulose 1111 has been r e p ~ r t e d . By ' ~ ~comparison of the C P MAS 13CNMR spectrum of Cladophora cellulose with those of the 13C-enriched celluloses, 13C resonance lines of the cellulose 1111 were completely assigned for the first time. 'H and 13Cspin diffusion NMR analyses have been undertaken to establish the microfibril structure of bacterial cellu10se.'~~ Rather large splitting of the C4 resonance line into the downfield (C4,,) and upfield (C4")lines in C P MAS 13C NMR spectra were observed for native cellulose. In order to reveal reasons behind this unusual splitting, 13Cand 'H spin diffusion measurements have been conducted by using different types of bacterial cellulose samples. In 13C spin diffusion measurements, the C4Dresonance line was selectively inverted by the Dante n-pulse sequence and the I3C spin diffusion was allowed to proceed from the C4Dcarbons to other carbons including the C4ucarbons. The I3C4-enriched bacterial cellulose sample was used in these experiments. Using additional NMR experiments, it was found that the C4" carbons may exist as structural defects probably due to conformational irregularity associated with disordered hydrogen bonding of the CH20Hgroups in the microfibrils.

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In a related work, C P MAS 13CNMR and electron diffraction study of the bacterial cellulose structure affected by cell wall polysaccharides has been presented.'4gThe experimental findings suggested that the uronic acid in the polysaccharides prevents interactions with cellulose leading to alterations of the structure of the cellulose crystal. Characterisation and comparison of two ligno-cellulosic substrates by 13CC P MAS NMR, XPS, conventional pyrolysis and thermochemolysis have been ~ e p 0 r t e d .The l ~ ~ combined study highlighted the limitation of some of the above methods when applied to ligno-cellulosic materials. The average lateral fibril and fibril aggregate dimensions and the crystallinity of the cellulose in a spruce kraft pulp have been investigated by C P MAS I3C NMR in combination with spectral fitting.15' Cellulose isolated by chloritedelignification and acid hydrolysis from fractions enriched in surface material and long fiber fractions enriched in bulk material exhibited no major differences in either lateral dimensions or crystallinity index. Cell wall material was isolated from the outer pericarp of kiwifruit at harvest and at several ripening stages following a postharvest ethylene treatment.'51 Solid-state 13CNMR spectra showed no evidence for changes in the nature of the cellulose crystallites or the polysaccharides adhering to crystallite surfaces even in cell wall material isolated from fruits in which cell wall dissolution was extreme. Spin relaxation experiments showed that pectin retained in the cell wall became softened in the early stages of ripening, prior to solubilisation later in ripening. Fruit firmness was directly proportional to the amount of non-cellulosic matter that remained sufficiently rigid to respond to the C P pulse sequence. The molecular mobility of a starch sample has been characterised by 13CC P MAS NMR and by 'H T1, measurement~.'~~ Mango fruits were also analysed by delayed contact time experiments and the 13Cdecays showed that the polysaccharides are heterogeneous and contain one rigid domain that controls the relaxation parameter.

7.7 Amino Acids. - An interesting experimental approach to the subject of According to chirality of amino acids have been presented by Wang et fundamental physics, the weak force, which is one of the four forces in nature, is the only universal chiral force. It is believed that the weak force can make one enantiomer slightly more stable than the other. In molecules the weak interactions include short-range parity-violating forces, which only take place in the nuclei. The authors have assumed that as the NMR chemical shift is dependent on the electronic environment at the nucleus site, then the chemical shift may also be parity non-conservation free and in two different enantiomeric forms a nucleus, which is itself chiral, may be in a diastereomeric environment. Secondorder phase transition effects have been discussed in conjunction with chirality and it has been suggested that an amplification mechanism based on quantummechanical tunnelling could give rise to a second-order phase transition. In order to understand the transition mechanism, the temperature dependence of 'H CRAMPS solid-state NMR and 13CC P MAS spectra of D- and L-alanine crystals from 295 K through to 220 K have been measured.


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L-Glutamic acid, which exists as two polymorphic solid phases (aand P) with different molecular conformations, has been studied using variable-temperature 2H NMR.'54 For both phases the 2H NMR line shapes were simulated successfully in terms of a 3-site n/3 jump motion of the -ND3+group. The activation energy for the reorientation of the -ND3+group was higher for the a phase (47 kJ mol-') than far the P phase (34 kJ mol-l). The T1(2H)data were also consistent with the 3-site jump motion of the -ND3+ group. The differences in the dynamic properties of the -ND3+groups between the two polymorphs were rationalised directly in terms of different local environments in the crystal structures, particularly concerning the different hydrogen bonding geometries involving the ND3+groups. 13CMAS NMR experiments on solid L-tyrosine-ethylester with 13Cin natural abundance and in fully 13C-enriched form have been The phenyl rings were found to undergo JC flips with a rather low activation energy of 50 kJ mol-'. In addition, the ester groups were afflicted by dynamic disorder at all temperatures accessible to MAS NMR experiments. Homonuclear ZQ 13Crecoupling experiments on fully 13C-enriched L-tyrosine-ethylester were carried out at low temperature. These experiments reproduced those structural features, known from single crystal X-ray diffraction, that are defined by short-range 13C-13Cinteractions but fail to uniquely characterise the complete molecular conformation defined by intermediate-range l3C-I3Cinteractions. 1 7 0 MAS NMR has been applied to characterise the bonding arrangements of L- and D- glutamates in the solid At 14.1 T highly structured and very similar NMR spectra were observed for L- and D-glutamic acidSHC1. Lines from all 4 oxygen sites were readily distinguished and assigned. These 1 7 0 NMR spectra were very different from the previously reported 1 7 0 spectrum of the D,L-form. 1 7 0 NMR from monosodium L-glutamateaHC1 was recorded using double angle rotation and MQ MAS NMR to provide resolution of 5 different sites.

7.8 Peptides. - 2D 13C-13Cexchange spectra have been used to measure constraints on supramolecular structure of amyloid fibrils with uniformisotopic labelling of multiple residues.lS7It has been shown that these 2D spectra can also be used for establishing the degree of supramolecular structural order in amyloid fibrils that has not been otherwise available. A structural model for amyloid fibrils formed by the 40-residue P-amyloid peptide associated with Alzheimer's disease has been proposed, based on a set of experimental constraints from solid-state NMR.'58 It was found that residues 12-24 and 30-40 adopt P-strand conformations and form parallel sheets through intermolecular hydrogen bonding. Residues 25-29 contain a bend of the peptide backbone that brings the two P-sheets in contact through sidechain-sidechain interactions. A single cross-p unit is then a double-layered P-sheet structure with a hydrophobic core and one hydrophobic face. Direct experimental identification of non-P-strand conformations in amyloid fibrils has been reported based on the results of solid-state NMR measurements on amyloid fibrils formed by the 40-residue P-amyloid peptide, prepared syn-

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thetically with pairs of 13Clabels at consecutive backbone carbonyl The measurements probed the peptide backbone conformation in residues 24-30, a segment where a non-P-strand conformation has been suggested by earlier sequence analysis, cross-linking experiments and molecular modelling. Data obtained with the advance 2D MAS techniques, indicated non-(j-strand conformations at G25, S26 and G29. Wideline and MAS I4N and 31PNMR have been used in combination with differential scanning calorimetry and circular dichroism to investigate the association between amyloid-(j(1-40) peptide and membranes with different electrostatic surface potentials.160 The intermolecular hydrogen bond interactions in polyglycine (PG)/poly(Lvaline) (PLV) blend have been studied through high-speed frequency-switched Lee-Goldburg I3C-lH HETCOR NMR experiments.161The spectral assignment of the polypeptide blend was made by using multiple proton cross-peaks in the HETCOR spectra. Intermolecular correlation peaks between PG(P-sheet) and PLV(P-sheet) appeared in the HETCOR spectra with long contact times. From these experimental results, it has been established that intermolecular hydrogen bond interactions C = 0 H-N between the PG(P-sheet) and PLV(P-sheet) are formed. Protein folding and stability has been well studied in solution, but little is known about solid-state environments, such as the core of a folded protein, where peptide packing interactions are the dominant factor in determining structural stability. Hence, the relative stability of a-helix and P-sheet secondary structure in the solid state has been investigated using poly(L-alanine) (PLA) as a model system.'62 I3C C P MAS NMR was used to determine the backbone conformation of solid powder samples of 15 kDa and 2 1.4 kDa PLA before and after various sample treatments. It was found that reprecipitation from helixinducing solvents traps the a-helical conformation of PLA, whereas grinding converts the secondary structure of PLA to a final steady-stare mixture of 55% P-sheet and 45% a-helix. On the other hand, cooling the sample in liquid nitrogen or subjecting it to high pressure had no effect on secondary structure. The results were consistent with the (3-sheet structure being approximately 260 J mol-' more stable than the a-helix structure in solid PLA. Isotopically labelled and unlabelled P-peptides with geminal dimethyl substitution in 2-position of each residue have been investigated using 2D MAS and TEDOR/REDOR NMR technique^.'^^ 2H, 1 3 c, 15N and 31PNMR techniques have been applied to study protegrin-1, which is a P-sheet antimicrobial peptide found in porcine leukocyte^.'^^ Its mechanism of action and the orientation in lipid bilayers were investigated by 2H NMR, which indicated that at high concentrations protegrin-1 destroys the orientational order of the aligned lamellar bilayer. The conformation of the lipid headgroups in the unoriented region was significantly altered, as seen from the 31Pspectra. These observations indicated that protegrin-1 disrupts microbial membranes by breaking the extended bilayer into smaller disks. The I3Cand I5N chemical shifts of Val-16 labelled protegrin-1 indicated that the P-strand axis is tilted by 55" from the bilayer normal while the normal of the P-sheet plane is 48" * * *


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from the bilayer normal. This orientation favours interaction of the hydrophobic backbone of the peptide with the hydrophobic core of the bilayer and positions the cationic Arg side chains to interact with the anionic phosphate groups. Solid-state NMR was used to study the interaction between melittin and the peptide inhibitor.165Melittin is a cytolytic peptide whose biological activity is lost upon binding to a six-residue peptide, Ac-Ile-Val-Ile-Phe-Asp-Cys-NH2, with which it forms a highly insoluble complex. As a result, the structural analysis of the interaction between the two peptides is difficult. The NMR results indicated that the inhibitor binding site in melittin is near Leul3, Leu16 and Ile17. On the basis of these data it was proposed that the inhibitor binds to melittin in the vicinity of Ala15 to Trpl9 and prevents insertion of melittin into cell membranes by disrupting the helical structure. Supporting evidence for this model was produced by determining the distance between the [l-"C] of Leu13 in melittin and the [3-13C] of Phe4 in the inhibitor using REDOR. I3C C P MAS NMR spectra were recorded for polylysine and polyglutamic acid adsorbed on a synthetic montmorillonite clay in acidic medium.166The spectra showed that these polypeptides, which exhibit a mixture of a-helical and random coil conformations in the bulk, tend to unfold and adopt a more extended random coil structure on adsorption on the phyllosilicate. Finally, solid-state 13CNMR techniques have been used for conformational characterisation of (Val-Pr~-Gly-Val-Glyk.'~~ 7.9 Proteins. - Potentially, solid-state NMR have a crucial role in structural investigations of those proteins that do not easily form crystals or are not accessible to solution NMR, such as amyloid systems or membrane proteins. A protein structure determined by solid-state MAS NMR has been presented by Castellani et aZ.16*13Cand "N resonance assignments formed the basis for the extraction of a set of distance restraints. These restraints were derived from proton-driven spin diffusion spectra of biosynthetically (site-directed) labelled samples. A set of differently labelled samples of the a-spectrin SH3 domain was prepared, which enabled a semi-quantitative interpretation of cross-peak intensities. The set consisted of a uniformly l3C-labe1led and two biosynthetically site-directed 13C-enrichedsamples, obtained by growing bacteria on [1,3-13C]glycerol and [2-'3C]-glycerol. Isotope labelling allowed the observation of longrange distance correlations up to 7 A.The calculated global fold of the a-spectrin SH3 domain was based on 286 inter-residue 13C-13Cand six I5N-l5Nrestraints, all self-consistently obtained by solid-state MAS NMR. The proposed NMR procedure is expected to be widely applicable to small membrane proteins that can be expressed in bacteria. 13C C P MAS NMR spectra of [1-'3C]-Pro labelled bacteriorhodopsin and P50G, P91G and P186A mutants under fully hydrated condition have been de~cribed.'~~ Well-resolved 13Csignals were distinguished by C P MAS NMR, which were assigned to Pro50, 91 and 186 residues in the inner part of the transmembrane a-helices. It was found that these Pro 13CNMR signals were appreciably broadened at temperatures below 233 K as a result of superposition of a variety of frozen conformers of the transmembrane a-helices exhibiting

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dispersion of chemical shifts. This meant that the dynamic behaviour of bacteriorhodopsin as viewed from Pro residue is very similar to that of ordinary amino acid residues such as Ala, Val, Phe, etc. The role of proline residues in the photocycle of bacteriorhodopsin has been addressed using solid-state NMR 13Cand "N chemical shifts from X-Pro peptide NMR chemical shifts were assigned based on the REDOR difference spectra of pairwise labelled samples. The effect of time and spatial averaging on 15Nchemical shift/'H-''N dipolar correlation spectra (PISEMA spectra) of a-helical membrane peptides and proteins has been in~estigated.'~~ Three types of motion were considered: librational motion of the peptide planes in the a-helix, rotation of the helix about its long axis and wobble of the helix about a nominal tilt angle. A 2 ns molecular dynamics simulation of helix D of bacteriorhodopsin was used to determine the effect of librational motion on the spectral parameters. For the time averaging, the rotation and wobble of this same helix are modelled by assuming either Gaussian motion about the respective angles or a uniform distribution of a given width. For the spatial averaging, regions of possible 15Nchemical shift/'H-''N dipolar splittings were computed for a distribution of rotations and/or tilt angles of the helix. The computed spectra showed that under certain motional modes the 15Nchemical shift/'H-''N dipolar pairs for each of the residues do not form patterns, which mimic helical wheel patterns. As a result, the unambiguous identification of helix tilt and helix rotation without any resonance assignments or on the basis of a single assignment may be difficult. 'H and 13CMAS NMR evidence for pronounced ligand-protein interactions involving the ionone ring of the retinylidene chromophore in rhodopsin has been p r e ~ e n t e d .Complete '~~ 'H and 13Cassignments of the 11-cis-retinylidene chromophore in its ligand-binding site were determined with ultra high field MAS NMR. Native bovine opsin was regenerated with uniformly 13C-labelled1l-cisretinal. From the labelled pigment, 13Ccarbon chemical shifts were obtained by using 2D MAS homonuclear correlation experiment. The 'H chemical shifts were assigned by 'H-I3C 2D dipolar correlation spectroscopy with phasemodulated Lee-Goldburg 'H decoupling. The data indicated nonbonding interactions between the protons of the methyls of the retinylidene ionone ring and the protein. These nonbonding interactions were attributed to nearby aromatic acid residues. Furthermore, it has been shown that binding of the chromophore involves a chiral selection of the ring conformation, resulting in equatorial and axial positions for 16-CH3and 17-CH3. Site-directed high-resolution solid-state I3C NMR techniques have been applied to study [3-13C]-Ala, [ 1-13C]-Vallabelled pharaonis phoborhodopsin (ppR or sensory rhodopsin 11) incorporated into egg phosphatidylcholine b i 1 a ~ e r . I ~ ~ Seven 13CNMR signals from transmembrane a-helices were resolved for [3-"C] -Ala-ppR at almost the same positions as those of bacteriorhodopsin. I3CCP MAS experiments combined with 2D 13C-'HWISE and *H NMR have been applied to study an a-keratin sourced from equine h00f.I~~ The results revealed a strong dependence of molecular conformation and molecular dynamics on the degree of hydration of the material. In particular, dehydration results in


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a much more rigid and ordered structure, with a loss of a-helical components in the structure and breaking of cysteine disulfide linkages. 31PNMR has been used to study molecular structure and dynamics of single crystals of a protein by NMR spectro~copy.'~~ The sensitivity of the measurements was sufficient to detect the NMR signal from individual phosphorus sites in a 0.3 mm3size single crystal of GTPase Ras bound to the nucleotide GppNHp. The NMR spectra obtained were discussed in terms of the conformational variability of the active centre of the Ras-nucleotide complex. It was concluded that, in the crystal, the protein complex exists in 3 different conformations. MAS NMR spectra of a powder sample of Ras-GppNHp showed a splitting of one of the phosphate resonances and thus confirmed this conclusion. Furthermore, the MAS spectra provided evidence of a slow, temperature-dependent dynamic exchange process in the Ras protein crystal. 15N-'H d i ~ o l a r / ' ~chemical N shift correlation experiments have been used to investigate the uniformly 15N-labelledand both "N-Val- and 15N-Leu-labelled M2 protein from influenza A.176The spectra supported a transmembrane helix in M2 protein having a tilt angle of 25". In addition, the spectra suggested that the tetrameric protein forms a symmetric or at least pseudosymmetric bundle consistent with data obtained by electrophysiological measurements and substituted cysteine scanning mutagenesis experiments. 13CC P MAS NMR techniques have been used to study two different types of spider silk from two Australian spider species.'77A comparison of 13CT1 and 'H T1, relaxation data of the Ala Ca, Ala Cp, Gly Ca and carbonyl resonances revealed subtle differences between dragline and cocoon silk. I3C TI, and 'H T1 relaxation experiments showed significant differences between silks of the two species with possible structural variations. Comparison of data from different sources revealed that differences in molecular mobility of dragline silk exist between species. Relaxation studies performed on wet (supercontracted) and dry silks showed that the degree of hydration affects relaxation properties and hence changes in molecular mobility are correlated with functional properties of silk. Conformational study of silk model polypeptides (Ala-Gly)12by solid-state NMR has been r e ~ 0 r t e d . The l ~ ~ intrinsic 'H, 13Cand 15NNMR chemical shifts of Ala and Gly residues, characteristic to the silk I and silk I1 forms in Bombyx mori were measured by 13Cand "N C P MAS, 'H CRAMPS and 'H-I3C HETCOR. In a related work 13C NMR has been applied to elucidate heterogeneous structure of silk fibers from Bombyx m01i.l~~ 2D spin-diffusion spectrum of a model Ala-Gly polypeptide under off-MAS condition was simulated for the estimation of the torsion angles. The multicomponent 13CMAS signal of the Ala side chain was deconvoluted to yield a quantitative description of up to five different structural motifs in the native silk fiber. 13CC P MAS NMR and wide-angle X-ray scattering have been employed to study Bombyx mori silk fibroin.'gOThe sequential model peptides containing Gly-Ala-Ala-Ser units selected from the primary structure of Bombyx mori silk fibroin were prepared and their secondary structure was studied. The 13Cisotope labelling of the peptides and the 13Cconformation-dependent chemical shifts were used for this purpose. It was found that the Ala-Ala units adopt antiparallel

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P-sheet structure locally and the introduction of one Ala-Ala unit in (Ala-Gly)15 chain promotes dramatical structural changes from silk I (repeated p-turn type I1 structure) to silk I1 (antiparallel P-sheet structure). The role of Tyr residue in the peptide chain was also studied and was found to induce a locally disordered structure. Native pig articular cartilage has been investigated by 13CCP MAS NMR at 17.6 T.'*l CP MAS spectra were dominated by resonances from rigid collagen, while only low-intensity signals from the glycosaminoglycans were observed. The spectral resolution of collagen fibrils in native cartilage was somewhat higher than for isolated collagen fibrils from bovine achilles tendon investigated for comparison. This was confirmed qualitatively by 'H-lH wideline separation spectra that showed much lower line widths for cartilage collagen compared to isolated collagen. The strength of 'H-13C dipolar couplings was measured providing a motionally averaged dipolar coupling value for each resolved signal. These scaled couplings were converted to molecular order parameters for the C-H bond vector. According to the results obtained, the higher mobility of collagen in cartilage is due to the high water content and collisions with the is0t ropicall y mobile glycosaminoglycans. I3C C P MAS NMR has been used to examine fibrillation of a human calcitonin mutant at the position of Aspl 5 and to reveal the effect of the electrostatic interaction of Aspl 5 with charged side chains.'82The secondary structures of fibrils and soluble monomers in the site-specific 13C-labelledsamples were reported. Solid-state NMR spectroscopy has been applied to establish the structure of the coat pr~tein.''~ The atomic resolution structure determined by NMR differs from that previously determined by X-ray fiber diffraction. Most notably, the 50-residue protein is not a single curved helix, but rather is a nearly ideal straight helix between residues 7 and 38. In addition, new NMR results included the complete sequential assignment of the 2D polarisation inversion spin-exchange of the MAS spectrum of a uniformly "N-labelled 50-residue protein. The calculation of the 3D structure of the protein from orientational restraints with accuracy equivalent to an r.m.s. deviation of ca. 1 A has also been reported. Solid-state 'H and 13CNMR study of the hydration of a high molecular weight wheat glutenin subunit has been ~ n d e r t a k e n . 'The ~ ~ effect of the presence of disulfide bonds on the hydration behaviour of the subunit has been investigated by a comparison of the unalkylated and alkylated forms of the protein. Hydration is known to induce partial plasticisation of the protein so that some segments become more mobile than others. The I3C CP MAS spectra of the samples in the dry state and at two hydration levels were used to monitor the protein fraction resisting plasticisation. Conversely, 13C single pulse excitation and 'H MAS experiments were used to gain information on the more plasticised segments. The molecular motion of the two protein dynamic populations was further characterised by I3CTI and 'H TIP,T2and T1relaxation times. The results suggested that hydration leads to the formation of a network held by a cooperative action of hydrogen-bonded glutamines and some hydrophobic interactions. The presence of disulfide bonds was observed to promote easier


27 1

plasticisation of the protein and the formation of a more mobile network. 7.10 Lipids and Membranes. - Solid-state NMR techniques have been used to probe the interactions of cholesterol (Chol) with bovine brain sphingomyelin (SM) and for comparison of the interactions of Chol with dipalmitoylphosphatidylcholine (DPPC), which has a similar gel-to-liquid crystalline transition temperat~re.''~ Multinuclear 'H, 13Cand 31PMAS NMR yielded high-resolution spectra from multilamellar dispersions of unlabelled brain SM and Chol for analysis of chemical shifts and line widths. In addition, 2H NMR spectra of oriented lipid membranes with specific deuterium labels gave information about membrane ordering and mobility. As inferred from 13C chemical shifts, the boundaries between the ordered and disordered liquid crystalline phases were similar for SM and DPPC. No evidence for specific hydrogen bonding between Chol and the amide group of SM was found. The 2H NMR powder line shapes and relaxation times, TI and T2,of the liquid crystal La, the intermediate Pv and the gel Lv phases of DPPC/2H20 system have been analysed.ls6 The water structure and dynamics of the lipid/water interfaces of DPPC in the hydration regime, where all water molecules are associated to the interface, were described in terms of orientational order parameters and correlation times. The line shape of the ripple phase (Pv) was analysed assuming model parameters of the gel or liquid crystalline phase. The line widths in 14N MAS NMR spectra clearly indicated slow dynamics of the polar headgroup in the ripple phase. The results indicated that the headgroup hydrations of the ripple and liquid crystalline phases are similar, while the acyl chains are still in their gel state in the ripple phase. 2H and 31Psolid-state NMR studies have been undertaken to investigate cholesterol dynamics in membranes of raft comp~sition.'~~ On average, the axis of anisotropic rotation of cholesterol was found to be nearly perpendicular to the membrane plane in all of the systems investigated. It has been suggested that cholesterol could be in the fast exchange regime in the NMR time scale (milli- to microseconds), between two or more membrane regions of different dynamics. 13CMAS NMR studies of the location of (+)-totarol, a diterpenoid bioactive molecule, in phospholipid model membranes have been reported.18*13C T1 relaxation times of both the phospholipid and ( +)-totaro1 molecules in the presence of Gd3+were measured to obtain information on molecular distances. The results indicated that (+)-totaro1 is situated in the upper region of the membrane, with its hydroxyl group located in the vicinity of the phospholipid acyl chain and nearly perpendicular with respect to the phospholipid acyl chain axis. Orientationally dependent "N chemical shifts and 'H-"N-dipolar couplings were measured to investigate binding of the myristoylated protein to aligned phospholipid bi1a~ers.l~~ It was demonstrated that the protein adopts a similar structure in both its myristoylated and non-myristoylated forms and that the structure is not grossly distorted by its interaction with the phospholipid bilayer surface. 7.11 Biomedical Applications. - Solid state NMR studies have been used to

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characterise the polymorphism of roxifiban and quantify the purity of the different p o l y m ~ r p h s .C' ~P~MAS was used to study the apparent equilibrium solubility of indomethacin and measure the degree of short- and long-range The polymorphism of clarithromycin was investiagted by solid state NMR. Two polymorphs, If and 1, of clarithromycins indicated characteristic resonances of C1 carbonyl carbon at 176.2 and 175.2 ppm, respectively. Quantification of the two forms was carried I3C and "N CP MAS have been carried out on sepia melanin and its derivatives and compared to model compounds. C P contact time dependence and NQS experiments were performed on the melanin samples. The 15NCP MAS spectra of the melanin samples confirmed the presence of indole and pyrrole units in the melanin polymer chain.'9323Na NMR has been carried out on four hydrated disodium salts of nucleoside 5'-phosphates: 2'-deoxycytidine 5'-monophosphate (dCMP), 2'-deoxyguanosine 5'- monophosphate (dGMP), 2'-deoxyuridine 5'-monophosphate (dUMP), and adenosine 5'-triphosphate (ATP). The chemical shifts of these nucleosides were compared to those obtained via quantum mechanical calculations.'94The TI of 23Naand 31Pof a solid sample of Na-DNA under carefully controlled conditions of relative ambient humidity have been described. The observed substantial changes of the NMR parameters were related to the water-induced transitions between the different.'95Previous3'PCP MAS and (direct polarization) DP MAS NMR spectroscopy studies of native bone and of the isolated crystals of the calcified matrix, synthesized by osteoblasts in cell culture, identified the major phosphate components of the mineral phase. The present study demonstrated that the major phosphate components identified by 31PMAS NMR in the very earliest stages of mineralization are protein phosphoryl groups which are not complexed with ~ a 1 c i u m .Hydroxyapatite l~~ has been studied by using a 2D solid state correlation experiments. Results on several mammalian species show that the bone crystal OH- is readily detectable; a rough estimate yields an OHcontent of human cortical bone of about 20% of the amount expected in stoichiometric hydr~xyapatite.'~~ 7.12 Coals and Carbonaceous Materials. - A method for the deconvolution of 13CNMR spectra of coals has been developed. The spectrum of coal was resolved into 24 peaks which were classified into nine types of carbon-functional group. This analytical method can be applied to all ranks of coal from lignite to anthra~ite.'~' Peat char samples produced by heat treatments under inert atmosphere were characterized through 13C solid-state NMR. A simplified model is presented to explain the influence of these particles on the 13CD P MAS NMR spectra of the heat-treated peat samples, showing their capability to generate SSB much stronger than the ordinary SSB due to chemical shift anisotropy in pure carbon mate~ia1s.l~~ Condensed solids from three aerosols resulting from heating anthracene in a drop-tube furnace with a helium atmosphere at high temperatures were studied by 13Csolid-state NMR and by ESR methods. The 'H T1,value of 17.6 s allowed the principal values of the 13Cchemical shift tensors to be measured by the FIREMAT experiment.200The chemical structure of an Estonian kerogen was evaluated using a simulation of I3CMAS NMR spectrum


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and compared to model compounds?" A coal tar Pitch used in manufacturing of graphite electrodes was individually modified with various silicon- bearing additives yielding mixtures that were pyrolysed (carbonized) and examined by 29SiMAS NMR.202Solid C6' was stored in NO under high pressure and the gas molecules N O were found to diffuse into the octahedral interstitial sites in its fcc crystal lattice. Its 13CNMR MAS spectra composed of a primary resonance at 143.7 ppm, accompanied by two minor peaks shifted 0.4 and 0.8 ppm downfield, re~pectively.~'~ Temperature dependent I9F NMR measurements of solid C60 intercalated with AsF5and SbFS acceptor species have been rep~rted?'~

7.13 Soils and Related Materials. - Soils in southern Spain are low in organic matter (OM) and nutrients. Understanding the nature and dynamics of OM has potential to improve soil management technologies for sustainable crop production. The objective of this work was to establish the distribution of functional groups in organic-C from these soils using I3CC P MAS NMR spectroscopy and to investigate the influence of vegetation, climatic conditions, soil parameters, parent material, and soil order on these functional groups. No statistically significant variability in the distribution of organic-C groups was found as a result of the influence of either soil order or parent The influence of change in land-use from native vegetation to pasture and subsequent change from pasture to eucalypt plantation on soil organic matter quality was investigated using 13C CP MAS NMR spectroscopy. Main impacts of changed land-use were higher 0-alkyl (carbohydrate) material under pasture than under native vegetation and plantation, and lower aromatic C under pasture than under native vegetation.206In this study, in order to understand the processes involved in soil organic matter (SOM) formation, the chemical structure of OM was characterized using solid-state 13C and 15N C P MAS NMR spectroscopy, along with Proton Spin Relaxation Editing (PSRE) sequences. Four groups of sampling sites were defined based on the NMR spectra. In each group displaying similar NMR characteristics, N content and soil texture could be highly different among sites. Some NMR spectra showed very different N contents.207Chemical interactions between 2-aminobenzothiazole (ABT) and a soil humic acid (HA) extracted from a gleyic planosol were investigated by solid-state 13CC P MAS NMR spectroscopy. The 13CNMR spectra of the HA-ABT adducts indicated that in both noncatalysed and enzyme-catalysed reactions ABT may be bound to humic acid by noncovalent as well as covalent bonds.208The fate of 13C-labelled phenanthrene and fluoranthene in different soil systems during biodegradation was studied. The soil humic acid fraction was isolated followed by structural characterisation 13CC P MAS NMR. It could be demonstrated that especially the ratio between the concentrations of polycyclic aromatic hydrocarbons (PAHs) and soil humus matrix limits the usefulness of this analytical tool. Based on these results a ratio of 13C activity(PAH)/ 13C activity(soi1)approximate to 1.5/1.0in the test material was suggested. The chemical transformation of a PAH and its bound residue formation in a soil system detected by changes of chemical shifts in the 13CNMR spectrum was proven for the first time. Structural information obtained by NMR spectra were verified by alkaline hydrolysis of

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PAH/humus- associations and following identification of cleavage 13CC P MAS NMR with dipolar dephasing (DD) was used to characterize soil organic matter (SOM) in a 2-year-old exotic pine plantation of subtropical Queensland, Australia, under two contrasting harvest residue management regimes. The SOM under no harvest residues displayed an increased extent of decomposition, as determined by the alkyl C/O-alkyl C ratio, and lower potentially mineralizable nitrogen, organic C, total P and total N contents. The CP spectra displayed little evidence of strong aromatic signals derived from lignin or tannin structures. This was confirmed by the DD spectra, which rapidly lost signal in the methoxyl and alkyl C regions, indicating protein and amide structures with little mobility might be dominant in the aromatic spectral region.210 13CC P MAS NMR was used to examine the changes in carbon composition of windrowed harvest residues during the first 3 years of hoop pine plantations in subtropical Australia and assess the impacts of windrowed harvest residues on soil organic matter composition and quality in the 0-10 cm soil layer.21'Accurate quantification of soil organic matter was carried out by spin counting and also a new technique, RESTORE (REstoration of Spectra via TCHand TI,, (TONERho) Editing).212Solid-state double C P MAS 15N and I3C NMR spectroscopy was applied to study the incorporation of TNT transformation products into humifying plant organic matter. For this approach, 13Cenriched plant material (Lolium perenne) was mixed with quartz sand and aerobically incubated for 11 months after addition of "N-2,4,6-trinitrotoluene (TNT). 15N NMR spectroscopy revealed an almost complete reduction of the TNT after 11 months of aerobic incubation. Most of the reduced nitrogen groups underwent further condensation. The corresponding D P MAS NMR spectra allowed the identification of amides that are further substituted by alkyl groups that resist even acid hydrolysk213Sequential extraction and solid state 31PNMR spectroscopy were used to investigate the relative solubility of phosphorus in a series of soils under permanent grassland in New Zealand.21413C CP MAS and DP MAS spin-counting experiments have been carried out on an absolute basis for a specific whole soil and its humin, humic acid, and fulvic acid fractions, as well as a sample of the soil that was treated with 2% HF(aq). The results confirm previous conclusions that a substantial fraction of the carbon content indicated by classic elemental analysis is missed in some samples, especially whole soil and humin, by both CP MAS and D P MAS 13CNAIR methods, and that the problem is more serious for CP MAS than for D P MAS. The relationship between the 'missing carbon' problem and the concentration of paramagnetic centers, especially Fe(II1) centers, is explored in substantial detail.215The influence of different magnetic-field strengths, sampling spinning rates, single and ramped amplitude cross polarization techniques and TOSS pulse sequence were investigated to obtain a more reliable insight into the effect of the chemical shift anisotropy (CSA), especially the first order spinning side bands (SSB) for the quantification of natural organic 7.14 Polymers. - A class of solid state NMR experiments for the investigation of slow dynamic processes in organic solids was developed. These 1D-MAS


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exchange experiments do not require selective isotopic labeling and can be performed with standard solid-state NMR hardware. An example using poly(nbutylmethacrylate) and poly(4-methyl-1-pentene) was given.217.218 The degree of coverage and the structure and thickness of interphases in composite latex particles consisting of a poly(buty1 acrylate) (PBA) core incorporated with poly(methy1 methacrylate) (PMMA) macromonomer of different molecular weights and a PMA shell was analysed by using solid-state NMR spin-diffusion meas~rements.2~~ Silicon and aluminum MAS and multiple quantum MAS were used to characterize the network character of the organicinorganic composite, and spin diffusion measurements were used to determine the nanostructure of the polymer/composite blending.220The microphase structure of ionomers based on an amorphous, maleated ethylene-propylene copolymer was investigated by using small-angle X-ray scattering (SAXS) and solid-state NMR experiments.221 Several 'H solid state NMR techniques were used to obtain structural information on industrial silicone copolymers as thermoplastic elastomers.222Hydrogenbonding interactions, distribution of various hydroxy groups, and surface morphology in organically modified polysiloxane networks were studied by solidstate NMR techniques based on 'H CRAMPS, 'H spin-exchange, double-quantum, and 1H-29Siheteronuclear MAS NMR Fast-MAS 'H NMR was used to probe the structure and dynamics of the adsorbed water and polymer components in polyelectrolyte multilayer (PEM) films and the bulk polyelectrolyte complex (PEC).224Various latex dispersions based on poly(viny1 acetate) stabilised by different surfactants and polyelectrolytes were investigated by 'H, 2H and I3C solid state NMR spectros~opy?~~ The phase structure of a series of ethylene-vinyl acetate copolymers has been investigated by solid-state wide-line 'H NMR and solid-state high-resolution 13C NMR (PBO) fibres was examThe structure of poly(p-phenylene-2,6-benzobisoxazole) ined using solid state NMR.227High-resolution solid-state NMR spectra of cross-linked and high molecular weight polymeric materials by using a combination of MAS and high temperature. The resolution of the spectra can be comparable to that seen in solution NMR spectroscopy.228The morphology of copoly(ether ester) elastomers, based on poly(buty1eneterephthalate) (PBT) hard blocks and poly(eth ylene oxide)-block-poly(eth ylene-stat-but y1ene)-blockpoly(ethy1ene oxide) (PEO-b-PEB-b-PEO) soft blocks, has been investigated by various solid-state NMR A combined study of nuclear magnetic resonance lineshape and T1 and T2relaxation times as functions of temperature and the amount of hydration water in a cross-linked copolymer of sucrose and 1,4-butadienol diglycidyl ether in the hydrogel phase is reported.230The structure of silyl-carborane ( 1,7-CzBloHlo) hybrid diethynylbenzene-silylene polymers with thermal treatment over a wide range of temperatures has been characterized by using "B MQ MAS NMR, and 13Cand 29SiC P MAS NMR Lightinduced aging of lignocellulosic materials has been studied with a new technique involving selectively (a-, p-, and y- 13C-enrichedcell wall-dehydrogenation polymers and solid state 13Cspectroscopy.232Alternating poly(ester amide)s consisting of 1,4-butylene and diol spacing entities (4NTm and 4NTm,p) were studied using solid-state NMR techniques.233The structural changes that occured during

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the accelerated sulfur vulcanization of styrene-butadiene rubbers (SBR) influenced by the curing agent concentration were examined by 13CMAS NMR.234 13CNMR inversion-recovery C P MAS experiments are used to study the phase structure and partitioning of main and side-chain groups in a homogeneous, metallocene-based, ethylene-1-octene copolymer. The results were analysed using a three-phase Side-chain conformation and dynamics for the form I1 polymorph of isotactic poly( 1-butene) were investigated by high-resolution solid-state 13CNMR Solid-state 13CNMR analyses of the structure and dynamics have been performed for liquid crystalline polyurethanes with different spacer methylene lengths. The molecular motion of the mesogen units was examined in detail, 13CCSA spectra were measured at room temperature for the mesogen carbons by 2D SASS 13CNMR spectroscopy and compared with those simulated by using the two-site exchange model for the flip motions of the phenylene group.237Miscibility and phase behavior in blends of poly(sily1ether) (PSE) and poly(methy1 methacrylate) (PMMA) were investigated by thermal analysis and solid state NMR spectroscopy. It was concluded that the PSE and PMMA chains are intimately mixed on the scale of 20-30 Azobenzenemodified cellulose (azocellulose) polymers were synthesized by covalently linking 4-cyanophenylazophenol to commercially available low molecular weight cellulose samples. These azocellulose polymers were characterized by solid-state 13CNMR and FTIR spectroscopic techniques. The analyses of data suggest that the coupling reaction occurs preferentially at the C6 carbon position and the Nanocomposites prepared by in-situ polymerization of 2primary ethynylpyridine (2EPy) in the presence of a layered aluminosilicate such as Ca2+-montmorillonite(MMT) were characterized by solid-state 13CC P MAS NMR spectroscopy. The results of solid-state NMR and X-ray diffraction studies clearly demonstrate a spontaneous polymerization of 2EPY within the galleries of MMTF4' The intercalation of poly(styrene-ethylene oxide) block copolymers (PS-b-PEO) into a smectite clay, hectorite, has been studied by multinuclear solid-state NMR. Polymer intercalation is assessed by two-dimensional 1H-29Siheteronuclear correlation (HETCOR) NMR with spin diffusion and refocused 29Si detection for enhanced sensitivity. Experiments with 'H CRAMPS evolution, with 'H spin diffusion, detectected a sharp -OH proton signal, and after a 'H T2 filter, provided excellent sensitivity for spin diffusion studies with mixing-time series. Two-dimensional 1H-29Sicorrelation NMR without 'H homonuclear decoupling shows that intercalated PEO has a clearly reduced mobility, most prominently for the PEO nearest to the silicate surface.241 Interactions and morphology were investigated by solid-state NMR in macromolecular charge transfer (CT) complexes formed between poly(N-vinylcarbazole) (PVK)and a surfactant consisting of a monomethyl ether PEO oligomer functionalized at one end with a 3,5-dinitrobenzoyl (DNB) group. 13CC P MAS NMR spectra indicated that in the PVK/PEG-DNB complexes containing 33.1 and 50.0 mol % PEG-DNB, the PEG chains demontrated microphase separa t i ~ nA. series ~ ~ ~of poly(ethy1ene oxide) fullerene (C6*)complexes were prepared and the intermolecular interaction and molecular motion in the complex were investigated by solid-state 13C NMR spectroscopy. By measuring the 13C TI


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relaxation times and 'H T2 relaxation times of the complex sample and by comparing the static 13C spectrum of the pure C60 sample with that of the complex sample, it is demonstrated that there exist n-rc interactions between the n-orbitals of the PEO ether oxygen and the n-system of C60.243 13C exchange solid-state NMR methods were used to study two families of siloxane/PEO hybrid materials. The results revealed that the hindrance of the slow molecular motions of the polymer chains were due to the siloxane structures depends on the chain length and the nature of the interaction between the organic and inorganic phases.244Polyelectrolyte multilayers have been prepared by consecutively adsorbing poly(dially1dirnethjrl ammonium chloride) and poly(sodium 4-styrenesulfonate) from aqueous solution onto colloidal silica. The mobility was followed by solid-state NMR spectroscopy. Relaxation measurements show that the mobility of the polyanion is found to remain constant throughout the layer-by-layer assembly, while the mobility of polycation decreases with increasing numbers of l a ~ e r s . 2A~series ~ of poly(3-hydroxybutyrate) (PHB)/PEO blends with different compositions were studied by solid-state high-resolution I3C NMR spectroscopy. The crystallinity, 'H T2and 'H TI, 'H Tl,of PHB and PEO in the blends were measured. It was found that the crystallinity of PHB may keep almost constant over the whole composition range, while that of PEO decreased dramatically with decreasing PEO content.246The structural characterisation of organotin compounds that are grafted onto insoluble crosslinked polymers has necessarily been limited to elemental analysis, infrared spectroscopy, and solidstate NMR spectroscopy. The system was characterised by 13C and '17Sn C P MAS and shows the potential of this technique through the structural characterization of diphenylbut yl- and dichloro butylst annanes, grafted onto divinyl benzene cross-linked polystyrene by means of a suitable linker.247Binary blends formed by two types of ethylene-co-vinyl acetate (EVAc), which have different vinyl acetate contents, and poly(viny1acetate) (PVAc)were prepared. Heterogenity in this system was demonstrated by '29Xeand I3C MAS NMR.248A detailed structural characterization of annealed polyaniline (PANI) powder samples has been carried out by using solid state NMR and FTIR. Comparing annealed PANI with PANI that has been chemically reduced to the leucoemeraldine base (LB) form, the 13C and 15N MAS NMR and FTIR data clearly show the conversion of the quinoid rings to benzenoid rings upon heating at similar to 200°C in vacuum. Measurement of the NMR relaxation parameter also showed that the rate of chain dynamics modified upon c r ~ s s l i n k i n gThe .~~~ extent and types of changes occurring to PANI films cast from solutions containing a gel inhibitor were characterized by MAS NMR. 13Cand "N MAS NMR data show that pyrrolidine adds to the quinoid ring of similar to 50% of the repeat units, converting these units from the emeraldine base form to the leucoemeraldine form. The presence of the pendant groups also disrupts chain packing, increasing the amount of kilohertz regime chain as determined from the magnitude of 'H PANI were synthesized from peroxidase-catalysed polymerization of aniline with and without the presence of a template. The template used was poly(viny1phosphonic acid) (PVP). I3Cand 15NC P MAS NMR techniques were applied to characterize the structure of polyaniline in its self-doped conducting,

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dedoped base, and redoped conducting form of PANI.25'The effect of melamine polyphosphate (MpolyP) on the thermal degradation of both polyamide 66 (PA66) and polyamide 6 (PA6) was studied using a combination of solid-state techniques. The residues were then analysed by X-ray diffraction and both solid-state 13CNMR and "P MAS NMR.252The relative concentrations and 13C T1of the amorphous, intermediate, and crystalline phases of unaged crosslinked polyolefin cable insulation 6oCoy-irradiated, and irradiated + annealed samples were determined by analyses of directly polarized solid-state 13CNMR spectra.253 The miscibility of poly(methacry1icacid) (PMAA) and poly(viny1acetate) (PVAc) blends was investigated by the C P MAS 13CNMR. The 'H T1 showed that the PMAA/PVAc blends are homogeneous on a scale of 20-50 nm, and 'H TI, revealed that the PMAA/PVAc-rich blends are partially inhomogeneous on a scale of 2-5 nm, while the PMAA-rich/PVAc blends, including the PMAA/PVAc = 1/1 blend, are completely miscible on the scale.254 The length scale of heterogeneity in blends of poly(2,6-dimethyl-p-phenyleneoxide) (PPO) with poly(amethyl styrene) (PaMS) was investigated using solid state 13CNMR. 'H T1 and 'H T1, were measured for PPO/(PaMS) blends of various compositions and neat polymer components (PPO, (PcxMS)'H T1, data indicated that P P O and PaMS are mixed at the molecular level for all compositions, showing that the miscibility scale is comparable with that of the classical blend system of PPO and polystyrene (PS).255Changes in the chemical structure of poly(tetrafluoroethy1ene) (PTFE) induced by electron beam irradiation at room temperature in air have been studied by 19FMAS NMR and IR spectroscopy. Chain scission associated with the formation of trifluoromethyl (-CF3),carboxylic acid fluoride (-COF) and carboxylic acid (-COOH) end groups was confirmed to be the predominant process under these c0nditions.2~~ The effect of y-radiation on a perfluoroalkoxy (PFA)resin was examined using solid-state high-speed MAS I9FNMR spectroscopy. New structures were identified including new saturated chain ends, short and long branches, and unsaturated Solid-state 19FNMR has been applied to regiodefective semicrystalline poly(viny1idenefluoride)to investigate, inter alia, the location of the reverse units. The proximity of reverse units to the amorphous and crystalline domains was further investigated by 'H TI, filtered radio frequency driven recoupling (RFDR) and spin-diffusion experiments. The results showed the majority of reverse units to be relatively mobile (ie. amorphous). However, weak RFDR cross-peaks were detected which suggest the presence of some reverse units in relatively rigid domains.258A solid-state NMR investigation of a series of shape-persistent polyphenylene dendrimers of generation 1-4 with different surface functionalization was undertaken. A combination of traditional static and more advanced MAS exchange techniques for the elucidation of slow dynamics as well as fast-MAS recoupling techniques was used for the quantification of dynamic averaging in the MHz range. Fast processes in the MHz regime are shown to be restricted to fast vibrations of terminal phenyl rings with amplitudes of up to 40" at most, with a fraction of rings performing larger-amplitude motions. Slow processes on the time scale of milliseconds to seconds are also restricted to terminal and doubly para-substituted phenyl rings. From the comparison of dendrimers with different surface


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functionalization it was concluded that the molecular dynamics are dominated by intramolecular steric The molecular motions in polyferrocenylsilanes, a novel class of metal-containing polymers with a main chain of alternating ferrocene and organosilane units, have been probed using variable temperature solid-state 2H NMRF6' Solid state 13C NMR experiments on poly(pbiphenylene terephthalate) with long n-dodecyl side chains have been carried out over a wide range of temperatures, in order to elucidate conformational and dynamical behavior of the polyester in the crystalline state and thermotropic liquid crystalline state. From these experimental results, it is found that at temperatures from room temperature to 80°C the n-alkyl side chains take both of the immobile and the mobile regions, and at temperatures above 120°C take only the mobile region.261The use of solid-state MAS and variable temperature (VT) NMR techniques as routine molecular structure characterization tools to analyse insoluble, high molecular weight and/or cross-linked polymers, was demonstrated. Increasing the mobility in the polymers by either swelling in a solvent and/or by elevation of temperature leads to narrow resonances with isotropic chemical shifts in MAS NMR spectra.262

7.15 Glasses and Amorphous Solids. - 'H MAS NMR and 'H T1 and 'H TI,, relaxation times measurements were used to investigate structural aspects of gel derived glasses of composition 10P205.90Si02 and 30P205.70Si02,with the aim of understanding the different humidity sensitive behaviour observed for films of the same composition. MAS in combination with variable temperature static NMR spectra provided detailed information on the network structural features p r e ~ e n t . 2The ~ ~ influence of F content on the formation and stability of Al-rich phlogopite has been investigated. Samples with varying OH/Fratios and nominal gel compositions of K(Mg,-,Al,) (All+,Si3-,0~o)(OH)y(F)2-y (0.0 5 x s 0.8 and 0.5 5 y 41.8) were studied using 29Si, 'H, and 19FMAS NMR spectroscopies. The main phase (phlogopite) and three other impurity phases [corundum (a-A1203),kalsilite (KAlSi04), and potassium aluminum hexafluoride (K3AlF60.5H20)]were clearly identified by NMR, powder X-ray diffraction and electron-probe m i c r ~ a n a l y s i sA . ~new ~ ~ vitreous phase with Na3A12(P04)3 composition has been prepared and crystallised. The 31P, 23Na,27Aland 7Li MAS NMR spectra for these materials provide some insight into the ordered and disordered constitution of the vitreous phases and the low mobility of the alkaline cati0ns.2~~ The objective of this study was to understand the effect of low temperature sol-gel synthesis on the microstructural properties of lithium [ X L ~ ~ O.,Si02; - ( ~ - x = 0.1-0.8 in steps of 0.11 and silver [xAg20-(1,$3i02; x = 0.1-0.8 in steps of 0.11 silicate xerogels via solid state NMR and X-ray absorption near edge structure (XANES)techniques. 7LiNMR shows quadrupolar satellite transitions attributed to LiN03. The silicate network is monitored with 29SiNMR and shows evidence of Li incorporation.266Amorphous B-C-N ceramics were produced by thermolysis of poly(borosesquicarbodiimide) of idealized formula [B2(NCN)3], MAS NMR studies demonstrated that boron is preferentially trigonally bonded to nitrogen.267In several series of lithium, sodium, and potassium borosilicate glasses whose compositions traverse known

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regions of liquid-liquid phase separation, we have applied 3QMAS "B and 1 7 0 NMR to obtain high-resolution information about short-range structure and connections among various network structural units, and their variation with composition and thermal history. 1703QMAS spectra reveal changes in connectivities between silicate and B 0 3and B 0 4units, by quantifying populations of bridging oxygens. "B MAS and 3QMAS at a magnetic field of 14.1 T allow proportions of several borate units to be determined, including B-[3] in boroxol ring and non-ring sites and B-[4] with 3 versus 4 Si neighbors. By combining the "B and 1 7 0 NMR results, detailed information on the sodium borosilicates can be derived, showing, for example, that B-[4] and non-ring B-[3] tend to mix with silicate units, while ring B-[3] is mainly connected to borate groups.268High temperature electrochemical synthesis is a very promising method for the production of refractory boride powders and coatings. The Na2B407-Na3A1F6 binary system is part of the more complex NaC1-Na3A1F6-Na2B407-Ti02 electrolyte proposed for TiB2 electrosynthesis. The changes in 27Al,23Na,"B, and 19F NMR spectra were used and gave evidence for the insertion of aluminum into the glassy network of Na2B407as A104 and diluted AlF6 units as well as formation of BOF2 groups into solidified mixtures.269The sol-gel method was used to prepare two different starting gels containing SiCH3-groupsfor the preparation of SiOC ceramics. Samples calcined at different temperatures up to 1000°C were characterized by 29Siand 13C MAS NMR The local configurations around sodium ions in silicate glasses and melts and their distributions have strong implications for the dynamic and static properties of melts and thus may play important roles in magmatic processes. The quantification of distributions among charge-balancing cations, including Na+ in aluminosilicate glasses and melts, however, remains a difficult problem that is relevant to high-temperature geochemistry as well as glass science. The local environment was explored around Na+ in charge-balanced aluminosilicate glasses (the NaA102-Si02join) and its distribution using 23NaMAS NMR spectroscopy at varying magnetic fields of 9.4, 14.1, and 18.8 T. 3QMAS NMR spectroscopy at 9.4 T, to achieve better understanding of the extent of disorder around this cation. The extent of this disorder in terms of changes in N a - 0 distance (d[Na-01) distributions with composition and present a structural model favoring a somewhat ordered Na distribution, called a 'perturbed' Na distribution model. The peak position in 23NaMAS spectra of aluminosilicate glasses moves toward lower frequencies with increasing Si/Al ratios, implying that the average distance (Na-0) increases with increasing R. The peak width is significantly reduced at higher fields (14.1 and 18.8 T) because of the reduced effect of second-order quadrupolar interaction, and 23Na MAS NMR spectra thus provide relatively directly the Na chemical shift distribution and changes in atomic environment with composit i ~ n . ~New " 27AlNMR data are presented in order to clarify the discrepancies in the interpretation of the previous 27AlMAS spectra from hydrous aluminosilicate glasses. The 27AlMAS data have been collected at much higher magnetic field (14.1 and 17.6 T) than hitherto, and in addition, MQMAS NMR data are presented for dry and hydrous nepheline glasses and NaAlSi7.7017.4 glass that, according to the model of Zeng et al. (Zeng Q., Nekvasil H., and Grey C. P. 2000)


28 1

exhibited Q3-OH species. The NMR did not provide evidence for this Alummosilicate gels with stoichiometric and nonstoichiometric compositions were synthesized by means of colloidal sol-gel method and their mullitization behavior was studied by X-ray diffraction (XRD), 27Aland 29SiMAS NMR. The various coordinated A1 occupancies were clarified by simulating the 27AlMAS NMR spectra with Gaussian lines. The results demonstrate that the so-synthesized aluminosilicate gel is a hybrid gel containing a mixture of a single-phase and a diphasic gel.273Four silica gels were prepared by hydrolysis of tetraethoxysilane (TEOS) in ethanol, using different catalysts. The difference between the xerogels and silica gels were shown by 29SiMAS NMR spectra.274Single crystals of Na2Si205have been synthesized from the hydrothermal recrystallization of a glass. In the 29SiMAS NMR spectrum of Na2Si205 four well-resolved lines of equal intensity are observed at -86.0, -86.3, -87.4, and -88.2 ppm. The narrow range of isotropic chemical shifts reflects the great similarity of the environments of the different Si Two series of CuO-containing sodium phosphate glasses with 40, and 50 mol% P2O5 were prepared. 31PMAS NMR and FTIR spectra were applied to characterize the short-range structure of the glasses. 31PMAS NMR spectra reveal the presence of Q2- and Q'-tetrahedral sites in both glass series. The depolymerisation was also further investigated.276 The structure of (50 - x/2)Na20-x Bi203-(50- x/2)P205glasses was investigated by Bi L-I11 XANES and EXAFS, 31PD Q MAS NMR and 23NaMAS NMR. The sodium environment is not affected by the introduction of bismuth in the glass network. The metaphosphate glass network undergoes gradual and systematic depolymerization by Bi203.277 Solid state 77SeNMR spectra are presented in the AsxSel-,glass family at ambient temperature. They exhibit three different kinds of Se environments. A comparison with the parent crystalline phases permits to assign the lines to Se-Se- Se, Se-Se-As and As-Se-As Se atom neighborhood^.^^^ Some resolved solid state 77SeNMR spectra are presented in the Ge,Sel-, glass family at ambient temperature. Two types of line positions are exhibited related to the presence or absence of Ge atoms as first neighbours around one Se atom.279Local structure of the SnO-B203glasses was investigated using several spectroscopic techniques. IIB MAS NMR spectra suggested that B04 tetrahedral units maximized at around the composition with 50 mol% Sn0.280 7.16 Cements. - The application of the MAS NMR spectroscopy to study the chemical environment of 27Albearing phases in Portland cement-based concrete has been desribed. A specific methodology is described that allows reliable spectra to be determined for combinations of different types of cements and fillers (in this case, Portland cement, fly ash, slag, silica fume, metakaolin and limestone filler).281When crystalline aluminosilicates partially dissolve in a concentrated alkaline medium, an amorphous geopolymeric gel is formed interspersed with undissolved crystalline particles. Some alummosilicates dissolve more readily than others to give an equilibrium ratio of aluminium to silicon in the gel. In this case study, kaolinite and stilbite mixtures were used to investigate the relative reactivity of different minerals when present in different ratios. XRD and 29Siand 27AlMAS NMR were used to determine when a specific mineral was

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completely transferred into the gel phase.282The calcium silicate hydrate (C-S-H) phase resulting from hydration of a white Portland cement (wPc)in water and in a 0.3 M NaA102 solution has been investigated at different hydration times, by 27Aland 29SiMAS NMR spectroscopy. 27AlMAS NMR spectra recorded at 7.05, 9.39, 14.09, and 21.15 T have allowed a determination of the 27Al isotropic chemical shift and quadrupolar product parameter for tetrahedrally coordinated A1 incorporated in the C-S-H phase and for a pentacoordinated A1 29Si CSA data have been determined from 29SiMAS NMR spectra recorded at 14.1 T for a number of synthetic calcium silicates and calcium silicate hydrates. These are p- and y-Ca2Si04, Ca3Si04C12, a-dicalcium silicate hydrate (aCa2(Si030H)OH),rankinite (Ca3Si207),cuspidine (Ca4Si207F2), wollastonite (pCa3-Si309),pseudowollastonite (a-Ca3Si309), scawtite (Ca7(Si6OI8)CO3.2H20), hillebrandite (Ca2Si03(0H)2)), and xonotlite (Ca6Si6017(OH)2)).284

7.17 Microporous Solids and Related Materials. - Solid-state NMR studies of MCM-41 supported with a highly catalytically active cluster were carried An approach for the investigation of layered alkali metal polysilicate hydrous materials is presented by choosing complementary multinuclear NMR methodologies. New insights into the structural and motional features of magadiite, a layered sodium silicate hydrate, have been gained using 'H, 29Siand 23Naoneand two-dimensional NMR experiments. Complementary 'H MAS single- and double-quantum studies have proven to be powerful tools in a clear recognition of the nature of hydrous species involved in hydrogen bonding.286A large panel of solid-state NMR techniques is used to characterize acid sites in steamed H-Beta zeolite. The structural environment of aluminum in hydrated H-Beta zeolite can be classically probed by 27AlMAS NMR.2g7A comprehensive study has been made on the acid properties of the mesoporous H-AlMCM-41 molecular sieves with varied Si/Al ratios using solid-state NMR techniques, namely by 31PMAS NMR of the adsorbed trimethylphosphine and trimethylphosphine oxide probe molecules in conjunction with elemental analysis. Results obtained from 'H and 27AlMAS NMR of the species that were associated with structural frameworks.288 Applying 'H and 27AlCF MAS NMR spectroscopy (CF: continuous flow), the hydration and dehydration of silicoaluminophosphates were studied under in situ conditions in a flow of nitrogen loaded with water vapor or in a flow of dry nitrogen. . The hydration of H-SAPO-34 and H-SAPO-37 is composed of two successive steps, viz. (i) hydration of Bronsted acidic bridging OH groups (SiOHAl) followed by (ii) coordination of water to aluminum atoms.289The dealumination of USY (ultrastable Y) zeolites by nitric acid and oxalic acid treatment was systematically investigated by multinuclear solid-state NMR and M Q MAS NMR experiments. The results show that both acids are very effective in removing non-framework A1 as well as framework A1 but that aluminum is extracted from the lattice at a higher rate by oxalic acid even at low concentrations. Leaching-induced increase in the Bronsted acidity of the USY zeolite was also observed by 'H MAS NMR ~ p e ~ t r ~ The ~ ~thermal ~ p y . ~ ~ ~ stability and dehydroxylation of the microporous silicoalummophosphates HSAPO-11, H-SAPO-18, H-SAPO-31, and H- SAPO-34 were investigated after


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thermal treatment at temperatures of 773-1173 K. While no change of the crystallinity upon thermal treatment at 1173 K was found by X- ray diffraction, 'H MAS NMR spectroscopy indicated a dehydroxylation of 40-50% of the initially existing bridging OH groups. As shown by 27Aland 29SiMAS NMR spectroscopy, this dehydroxylation is not accompanied by a dealumination but rather by a removal of silicon (desilication) in the local structures of the former bridging OH groups?91Using trimethylphosphine (TMP) as a probe molecul.e, acid sites in a series of A1-MCM-41 materials with Si/Al ratios ranging from 16 to 80 were investigated by various solid-state NMR techniques including 27Al,29Si, 31P,'H MAS, and some double- or triple-resonance methods such as 1H/31PCP, 'Hf'A1, and 1H/31P/27Al TRAPDOR (transfer of population in double resonance). By means of the 1H/27AlTRAPDOR technique, which has the ability to establish a correlation between 'H and 27Al,the hydroxyl groups associated with A1 could be discriminated from the silanol groups. Two signals at 3.5 and 1.9 ppm were observed in the 'HP7Al TRAPDOR spectra; they are likely due to the bridging hydroxyl (SiOHAl) and aluminum hydroxyl groups, respectively. 31P MAS NMR strongly supports the formation of zeolite like Bronsted acid sites in the mesoporous material after the incorporation of aluminum.292The first Al-0P analogue of the so-called 4 = 1 Secondary Building Unit (SBU) of zeolites is presented and characterized by single-crystal X ray diffraction (compound 1). Compound I was carefully studied by 31Pand 27Al solid-state MAS NMR, resulting in the precise spectroscopic description of the phosphorus and aluminium sites. Unusual 27Alquadrupolar parameters were measured. Moreover, we also present a high-resolution solid-state 'H NMR study of 1 at 600 MHz and 33 kHz MAS; a remarkable improvement in resolution is observed, allowing definite a~signments.2~~ Cryogenic NMR and NMR T1 (NMRT) measurements of pore-confined water (D20)have been performed using deuterium NMR to probe the pore size distribution (PSD) of, silica materials and porous membranes. NMRT measurements were performed at a temperature slightly below the normal freezing point (277 K) of bulk water (D20)to ensure that all interparticle water was frozen out. PSD derived from cryogenic NMR was in excellent agreement with PSD obtained from N2 adsorption meas~rements?~~ Synchrotron X-ray single-crystal diffraction analyses revealed that as-synthesized and Na-exchanged RUB-29 (Csl-,,Na.x)14Li2~[Si72Li180172].yH20 (x = 0,0.9) displays the lattice symmetry 1222. Using high-field (14.1 T) NMR spectroscopy, at least six separate 6Li resonances could be resolved for the first time by solid-state 6Li MAS NMR spectroscopy and assigned to Li in the framework and non-framework sites of the microporous lithosilicate materials.295The transformation of porous glass spheres into MFI-spheres in a synthesis with low water content was observed by in situ "B MAS NMR spectroscopy. The yield of crystalline material could be determined by means of the intensity of a narrow signal at -3.7 ppm in the "B MAS NMR spectra.296Organic cations, like methylium or ethylium, create chemisorbed covalently bonded alkoxy species in zeolitic structures. Their existence in NaX and NaY was proved by 13CMAS NMR.297The formation of surface alkoxy species on nanosized HZSM-5 and microsized HZSM-5, after exposure to methanol and subsequent conversion to olefins, has

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been investigated by in situ solid state NMR.298The nature of the lowest triplet excited state of acetophenones included in zeolites has been inferred through steady-state and time-resolved emission spectra. Acetophenone shows cationdependent state switching. Static, MAS, and C P MAS spectra of 13Cenriched acetophenone included in MY zeolites confirm the presence of such an interact i ~ n The . ~ dealumination ~ ~ of zeolite H-beta by ammonium hexafluorosilicate (i.e., (NH4)2SiF6,AHFS) treatment was investigated by 27Aland 19Fsolid-state NMR, combined with 27Al/19F double resonance NMR. The NMR results demonstrated that the experimental conditions of AHFS dealurnination, that is, in the presence and absence of ammonium acetate (NH40Ac),strongly affected the amount, state, and nature of extraframework aluminum (EFAl) species. Different aluminum fluoro-complexes after dealumination were detected. 19F and 27AlNMR results revealed that the essential part of the dealumination p r o c e s ~ . ~ the ~ I nwork, the results of 27AlMAS NMR studies carried out for structures of different framework aluminosilicates are presented. The spectra of natural clinoptilolite and its sodium and hydrogen forms were measured. 27Al MAS NMR spectra of all the samples studied were compared with the FTIR spectra measured in the middle infrared region.30127AlMAS and MQ MAS NMR have been used to identify and quantify framework and extra-framework (EFAL) aluminum in Y zeolites stabilized by steaming and EFAL extraction using various leaching agents. It is useful to characterize contributions of different A1 sites in USY. The decomposition of MAS and MQ MAS was carried out with the use of homemade software called MASAI. It takes benefits from the simultaneously analysis of a high resolved 2D MQ MAS and a quantitative MAS 2D M Q MAS NMR has been applied to investigate the coordination of A1 atoms in SAPO-34. The results show that the effects of the template and/or water on the coordination of A1 atoms.303Disordering of Si and A1 in natrolite, scolecite, mesolite, and gonnardite was investigated with 29Siand 27AlMAS NMR spectroscopy. Fits of 29SiMAS NMR spectra and mathematical relations based on A1 avoidance were used to calculate Si and A1 occupancies across the tetrahedral sites in these minerals.3o4Solid-state NMR results on the investigations of the intermediate gel phases formed during the formation of crystalline AlP04-11 molecular sieves. In addition to 31Pand 27AlMAS methods, the 31P/27Aldouble-resonance experiments such as C P are used to obtain valuable information in the gel phase.305Several solid-state NMR techniques in conjunction with powder X-ray diffraction (XRD) and FT-Raman spectroscopy were used to characterize the structures of the intermediate phases of AlP04-11 molecular sieve synthesis. The evolution of the gel phases as a function of crystallization time was followed by 31Pand 27AlMAS NMR to obtain information on the local environments of A1 and P atoms and by powder XRD to detect the long range ordering of the gel samples. 27Al3QMAS was applied to gain additional resolution for monitoring the incorporation of different A1 sites into the framework during the crystallization. 31Pand 27Aldouble resonance techniques such as CP, transfer of populations by doubleresonance, and rotational echo double-resonance have been utilized to select the 31P-O-27Albonding connectivities within the gel phases.306H-mordenites (HMor) with variable


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2/A1203molar ratio (MR) varying from 10 to 206 were incorporated with copper (CuMor). A consistent finding was the disappearance of the NMR line from extra-framework 27Al,whenever Cu is present.307A rapid and convenient route is described for the synthesis of mesoporous MCM-41 and MCM-48 materials and was analysed by 29SiMAS NMR.308Powder and single-crystal X-ray diffraction, combined with MAS NMR measurements, has been used to study the thermal expansion of siliceous zeolite ferrierite as it approaches a second-order displacive phase transition from a low-symmetry (Pnnm) to a high-symmetry (Immm) structure. The results from the single-crystal X-ray diffraction study can be correlated with 29SiMAS NMR chemical shifts for the low-temperature phase.309MCM-41 mesostructured silicas containing molybdenum and tungsten have been prepared in an acidic medium at room temperature. Two procedures were considered: the so-called 0x0-peroxo route with low-nuclearity peroxo metal precursors (route A) and the 0x0-polyoxo pathway, involving or generating polyoxometalates (route B). The native materials have been characterised by chemical analysis, powder X-ray diffraction, Raman spectrometry and 29Si MAS NMR.310The alkali leaching of two aluminosilicates, kaolinite and metakaolinite, with aqueous NaOH has been inestigated, both silicates gave hydroxysodalite (HS) with or without the evanescent zeolite NaA. X-ray diffraction and high-resolution 29Siand 27AlMAS NMR spectroscopy provided information about the reaction sequence of the clay.311The framework of ETS-10 undergoes reversible structural changes upon dehydration and rehydration. Pronounced differences in the 29SiMAS NMR spectra clearly indicate rearrangements in the local environment, while the X-ray powder diffraction patterns provide evidence for a long-range effect of the guest/host i n t e r a ~ t i o n .In ~ 'this ~ work, the molecular dynamics of acetonitrile and acetonitrile-d3 confined in silica pores of nominal diameter 6 and 20 nm are studied by high field 'H and 2HNMR, and the results are discussed with reference to the bulk substances. NMR line-shapes, TI, T2 and diffusivities (D) are reported as a function of temperature. The line-shape and relaxation measurements clearly reveal a two-component system in the solid phase, consisting of a narrow line superimposed on a broad complex resona n ~ e .Perdeuterated ~'~ isobutane-dlo,when it is adsorbed on zeolite ZSM-5, has been shown to exhibit a superposition of two 2H NMR Pake-powder patterns with quadrupole constants CQ= 2.35 kHz and asymmetry parameters q = 0.17 for the CD3 groups and CQ = 23.0 kHz and q = 0.20 for the CD group at 153-373 K. The narrowed Pake-powder patterns have been interpreted in terms of the motional behavior of isobutane molecules located at the channel intersections of the zeolite 7.18 Surface Science and Catalysis. - Observation of a H-agostic bond in a highly active rhenium- alkylidene olefin metathesis heterogeneous catalyst by two-dimensional solid-state NMR spectroscopy was reported.315MAS NMR was used to characterize the substrates of the hydrogenolysis of cyclic alkanes.316 The structures formed by the adsorption of carboxyalkylphosphonic acids on metal oxides were investigated by 'H fast MAS, heteronuclear correlation (HETCOR), and 'H DQ MAS solid-state NMR experiment^.^'^ The adsorption

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and photodecomposition of ethanol that was employed as a model substrate of organic pollutants were investigated in the presence of adsorbed water with T i 0 2 by 'H MAS spectroscopy in order to obtain information about the actual photocatalytic reaction sites of organic pollutants on T i 0 2phot~catalysts.~'~ The fluorination on the y-alumina surface was studied by multinuclear MAS NMR and BET measurements. The fluorine species on the fluorinated y-alumina can be identified by 19FMAS NMR combined with 'H MAS NMR and 27AlMAS and C P MAS NMR. As proven by 'H MAS NMR, fluorination of the alumina preferentially replaced the basic hydroxyl groups. Hydroxyl is substituted by one fluoride without breaking the bridging A1-0-A1 bonds.319Detailed structure of Bronsted acid sites on the surface of S03/A1203catalyst has been proposed based on 'H/27AlTRAPDOR NMR results and the acidity of the catalyst has also been characterized by NMR probe molecules.320'H MAS NMR, 'H spin-echo MAS NMR with A1 irradiation, 29SiMAS NMR, and 'H-29SiC P MAS NMR were used to investigate the deposition of Mo and Ni species on the surface of alumina and ~ilica-alurnina.~~~ 13C MAS NMR analysis of the hydrocarbon products formed from the selectively 13Clabeled n-but-1-ene on zeolite ferrierite (H-FER) in a closed batch reactor revealed the following successive steps of the olefin conversion with temperature increase from 300 to 823 K: a double-bond-shift reaction, scrambling of the selective 13C label in the formed n-but-2-ene, oligomerization (dimerization), conjunct polymerization, formation of condensed aromatics, and formation of the simple Methane dehydroaromatization on Mo/HZSM-5 and W/HZSM-5 catalysts was studied by solid state 13C NMR spectroscopy.323Cumene conversion under an excess of benzene is studied over a series of mordenite (MOR) zeolites (Na-MOR, HNaMOR, H-MOR and dealuminated H-MOR) by in situ 13Cand 'H MAS NMR spectroscopy in the temperature range 298-473 K. By tracing the fate of 13C labeled atoms in the benzene molecule, intermolecular transalkylation, disproportionation and isomerization reactions are highlighted.324Adsorption of nitromethane on oxide surfaces was monitored using 13CC P MAS NMR. Stabilization of the aci-anion formed by reaction of nitromethane on acid-base pairs was apparent. A linear relationship between the 13CNMR isotropic chemical shift of the methylene group of the adsorbed aci-anion of nitromethane and the heat of adsorption of COz measured by microcalorimetry was established.325The environments for oxygen sites in crystalline V205and in layered vanadia gels produced via sol-gel synthesis have been investigated using 1 7 0 MAS and 3QMAS NMR. In addition, by correlating the 1 7 0 MAS and 3QMAS NMR, "V MAS NMR, and thermogravimetric analysis data, the coordination of water sites has been established.32619FMAS NMR of accessible hydroxyl sites on fiberglass surfaces was Tailor-made adsorbents and supports are attracting increasing interest for a wide range of advanced applications in the field of separation techniques and biotechnology. Binding of the silane molecules on surface silanol groups of the crystalline silicic acids was investigated by 29SiMAS NMR spectroscopy.328Vanadium-cerium oxide catalysts, with different V/Ce atomic ratios, were prepared using vanadyl oxalate (V0C204)impregnated on ceria (Ce02)as precursors. These solids have been characterized with different


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techniques: Raman spectroscopy, thermal analysis (TG-DSC), specific area measurements (BET), EPR, and solid-state 51V MAS NMR.329An overview paper of 51VNMR includes both published and original data of the current state of the field in solid-state chemistry. Advantages and shortcomings of different NMR techniques in their applications to vanadium are discussed on the examples of their application to various vanadia based systems (including individual highly crystalline compounds, solid solutions, glasses, catalysts).330

7.19 Inorganic and Other Related Solids. - With increasing magnetic fields and spinning rates, it becomes possible to obtain partly resolved 'H spectra in solid state materials using a simple excitation sequence. A high resolution spectra obtained at high field (14.1 T) and high spinning rates (30 kHz) for two types of hybrid organic/inorganic compounds: silica matrices doped with pH indicators and mesostructured hybrid silicates templated by surfactant molecules was reported.331A mixed cadmium-zirconium-sodium oxalate with an open architecture has been synthesized from precipitation methods at ambient pressure. The dynamic disorder of the system was further investigated by 'H and 23NaMAS NMR.332Solid-state 'H ,23Na,and 29SiNMR spectroscopies, combined with X-ray powder diffraction (XRD) technique, were used to study the hydration process of a layered sodium disilicate SKS-5 (having a crystal structure of a-Na2Si205). NMR and XRD experiments suggest that SKS-5 is very sensitive to absorbed water molecules and the crystal structure of SKS-5 converts gradually from a-Na2Si205 to a new phase during the hydration process.333Two kinds of amorphous aluminum hydroxides, a sample precipitated from admixing AlC13 and NaOH aqueous solutions and the commercial product, were measured by 27Aland 'H MAS NMR spectroscopy. Pentahedral and tetrahedral coordinations, as well as octahedral coordination of oxygen atoms for aluminum, are observed in 27AlMAS spectra of both amorphous samples. In contrast, octahedral coordination is only observed in gibbsite, bayerite, and boehmite. According to 'H MAS NMR spectra under conditions of high spinning rate (35 kHz) and high field (14.09 T), free waters and OH groups coupled with aluminum for amorphous samples are observed at similar to5 and similar to 4.5 ppm, respectively, the latter peak being broader.334The structural properties of aluminasodium-silicate fiberglass (S) and microporous silica-alumina fiberglass supports (CCB18 and CCB20) were investigated using solid state NMR. Deuterium exchange and the thermal dependence of the dehydration of silica-alumina berglass (CCB18 and CCB20) were monitored using 'H and 29SiMAS technique in order to classify surface hydroxyl Apatites in a fluorine-free chemical system have been synthesized hydrothermally at 650°C and 1.5 kbar along the hydroxylapatite-britholite-(Y) join [i.e., Calo(P04)6(0H)2 CaY6(Si04)6(OH)2 join]. 31PMAS NMR spectra could not be fully assigned, the 2.8 ppm resonance that persists even in Si-rich compositions must include the contribution of P atoms involved in P-0-Y bonds. 'H NMR spectroscopy shows that the H content decreases by around 50% from the phosphate to the silicate end-member although all the compounds were synthesized hydrothermally at 650°C.336 A mixed-valence vanadium phosphate, NH4[(V,O3)2(4,4'-

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bpy)2(H2P04)(P04)2].0.5H20, has been synthesized under hydrothermal conditions and structurally characterized by single-crystal X-ray diffraction. The 31P MAS NMR spectrum showed a resonance centered at 80 ppm with a shoulder at ca. 83 ppm in an intensity ratio close to 1:2, which correspond to two distinct P 2HNMR spectra of LuD3were obtained at 8.0 and 8.4 T from 163 to 398 K, revealing only minor changes with temperature. Using partial alignment of the powder particles in the magnetic field and subsequent freezing in wax, the NMR line shape has been studied as a function of field direction, allowing the orientation of the electric-field-gradient tensor to be partly determined.338The decrease in D-A bond lengths, previously reported for some Lewis acid/base complexes, in going from the gas to the solid phases is investigated by obtaining an accurate crystal structure of solid ND3BF3by powder neutron diffraction. The effects of this change in structure and of intermolecular hydrogen-bonding on nuclear magnetic shielding (Le.,chemical shifts) and the nuclear quadrupolar coupling constants (QCC) are investigated by ab initio molecular orbital and density functional theory calculations. These calculations show that the nitrogen ("N and 14N)and boron ("B and log)chemical shifts should be rather insensitive to changes in r(BN) and that the concomitant changes in molecular L i C 0 ~ F e ~ M n ~ ( ~compounds +~,04 (with x = 0.2,0.4, 0.8, and 1, and y = 0.0, 0.2, and 0.4) have been prepared and studied by 6Li MAS NMR.340In situ XRD and 6Li MAS NMR spectroscopy have been carried out during the first charge process for the layered cathode material Li[Li1/9Ni3,9Mn519]02. The 6Li MAS NMR results showed the presence of Li in the Ni2+/Mn4+layers, in addition to the expected sites for Li in the lithium layers.3416Li MAS NMR spectroscopy has been used to study the structure of a series of LiMn204spinel samples prepared using different annealing methods. The NMR spectra of the delithiated samples, LixMn204, exhibited a gradual shift in the position of main spinel resonances (i.e. Li in tetrahedral sites) with decreasing lithium 7LiMAS NMR spectra of 2.5 MeV electron-irradiated LiF crystals have been measured in a field of 9.4 T. Besides the resonance line of the ionic compound, a second well-separated spectrum is observed in the region of the Knight shift value for metallic lithium. The two lines are attributed to different types of metallic Li: one to bulk-like metal, the other to Li present initially under pressure and relaxing to the former under thermal treatment.343A detailed characterization of the structural modifications and redox processes occurring upon lithium deintercalation from the LiXoCol-,MgyO2 materials (x(0) = 1.0 and 1.10; y = 0.0,0.03, 0.05 and 0.06) was performed in order to determine the effect of Mg doping on the cycling properties. Using electrochemical tests, X-ray diffraction (XRD), 7Li MAS NMR and electrical properties measurements, it was shown that the LiXCol,MgyO2system exhibits a solid solution existing in the whole deintercalation range studied (0.30 less than or equal to x less than or equal to 1.0).7LiMAS NMR study shows that the structural defects (0vacancies and intermediate spin Co3+ions) which are present in the starting Mg-doped phases govern the electronic properties upon lithium de i n t e r c a l a t i ~ nOne. ~ ~ and two-dimensional lo9AgNMR demonstrated that silver diffusion in silver iodide/silver phosphate glasses is governed by a very broad, continuous distribution of correlation times. As a consequence, over a


289

wide temperature range, the Io9Ag spectra can be described by a weighted superposition of a Gaussian and a Lorentzian where these line-shape components result from the slow and the fast silver ions.345Xerogels of composition La2B4A12012, treated between 200 and llOO°C, are investigated by NMR and scanning electron microscopy (SEM). The "B MAS NMR results indicate the presence of tetra coordinated boron in all investigated samples.346("B and loB MAS NMR) spectroscopy has been conducted to characterize distorted chemical bonds of wurtzite boron nitride (wBN), in which boron atoms are coordinated by four nitrogen atoms. Although X-ray analysis give the difference from ideal tetrahedral symmetry by simulation, comparison of results of MAS NMR measurements gives proof of anisotropic symmetry. Anisotropy was typically revealed in the broadened linewidth of the loB MAS NMR spectrum.347Hexagonal boron nitride (h-BN) ceramics can be prepared from polymeric precursors by heat treatment under inert atmosphere. The purpose of this paper is to illustrate the strategy that can be followed to characterize by 15Nand "B solid state NMR the polymers as well as their pyrolysis N-(2'-RPhenyl)-9,10-dihydro-9,lO-ethanoanthracene-l1,12-dicarboximides with R = M e (l), OMe (2), OEt (3) and H (4) were investigated. The crystal and molecular structures of 1-3, determined by single crystal methods, show different conformations of the N-(2'-R-phenyl) group. Anti conformations and weak intermolecular CH/n contacts were found in 2 and 3, while anti and syn conformations coexist in 1 with CH/n contacts being absent. Solid-state 13CC P MAS spectra confirm the X-ray molecular structural data.349The structure of silver cyanide has been investigated by solid-state multinuclear NMR spectroscopy. I3Cand 15NNMR spectra of MAS and stationary powder samples of isotopically enriched (AgCN)-13C,(AgCN)-13C-15N, and (AgCN)-15Nhave been acquired at the external applied magnetic field strengths 4.7,7.05, and 9.4T. Axially symmetric carbon and nitrogen chemical shift (CS) tensors provide evidence for linearity of the polymeric (-Ag-CN-), chains. A two-site model is required to successfully simulate the I3C MAS NMR line shape, which is dominated by indirect nuclear spin-spin coupling between lo7Ag/'09Agand I3Cnuclei.35o Short-range order within a novel aluminophosphate-oxalate hybrid material with the unit cell formula CH2(NH3)CH(NH3)CH3A14P6020( OH)4(C204)H20was studied by multinuclear solid-state NMR measurements. The 13C,27Al,31PMAS NMR spectra showed several resonance lines that could not be predicted by the single-crystal X-ray diffraction analysis. 'H-13C C P MAS NNIR spectrum evidenced that the diaminopropane species either occupied two different positions or were present as two different isomers, which also gave rise to two slightly different deformations of one (P304)of the three crystallographically distinct PO4 tetrahedra. Onedimensional 'H and 31PMAS and 2D 1H-31PC P MAS spectra revealed that the water molecule, occupying a well-defined position within the pore.351Solid state NMR spectra of 29Si, are reported from pure and vanadium-doped zircon (V-ZrSi04)samples. The vanadium concentration is varied up to similar tol-mol % V4+by using both conventional-firing and sol-gel routes, and 51VNMR data are also recorded. 1 7 0 NMR of 1 7 0 isotopically enriched samples shows that the initial gel is completely amorphous with the whole range of possible M-0-M-'

290


linkages detected, and that this structure evolves into a fully ordered ZrSi04 structure with calcination. Static 91ZrNMR data is reported from a pure zircon sample. The NMR data are used to quantify the amount of vanadium entering the zircon structure, and to elucidate its site preference within the lattice. Two contact shifted peaks with very different T1relaxation from the main zircon peak but attributable to the zircon lattice are observed in the 29SiNMR spectra for all samples. These spectra are consistent with vanadium substitution on both the tetrahedral and dodecahedra1 sites, with a slight preference for the silicon 19Fand '19Sn MAS NMR spectroscopy have been used to investigate the fluoride ion conductor, BaSnF4,a member of the MSnF4family of fluorite-related anionic conductors containing double layers of Sn2+ and M2+ cations. Two fluorine sublattices were observed by 19FMAS NMR, which could be assigned to specific sites in the lattice. The first sublattice is due to fluorine atoms located in Ba2+ double layers and is rigid on the MAS NMR time scale at room temperature. The second sublattice comprises the fluoride ions between the Ba2+and Sn2+layers, and the few fluorine atoms that inhabit the Sn2+-Sn2+double layers.353 The lI9Sn and 19FNMR chemical shifts in alkali-metal hexafluorostannates are measured. Data of MAS-spectroscopy and ab initio calculations reveal that [SnF6I2-has a distorted structure due to intramolecular correlations in the anion.35423Na MQMAS and 'H-23NaC P MAS NMR techniques have been used to study the variation of the sodium coordination during the hydration processes of the layered sodium disilicates sks-5 and sks-6. Three new types of sodium ions, with different local coordination environments, were identified and correspond to sodium ions coordinated by oxygen atoms of the silicate framework, water molecules and hydrogen-bonded silan01.~~~ The strontium aluminosilicate Sr3Al10Si020 has been synthesised by a spray-drying process and characterised by powder X-ray diffraction. Its structure is built of double A106 octahedra chains interconnected by A104 and Si04tetrahedra forming a three- dimensional channel-like network where the strontium cations are located. The distribution of aluminium and silicon cations in the different tetrahedral and octahedral sites of the structure was probed by 29Si, 27Al and 3Q MAS NMR.356Polymeric aluminosilicate gels with A1203/Si02molar ratios of 1.3/2, 2.5/2, 3/2 and 4.7/2 were prepared by gelling a mixture of tetraethoxysilane and ethyl acetoacetate aluminium diisopropoxide. Mullite, without any other crystalline phase, directly crystallizes from the gel matrix at about 1000°C for all investigated samples. The A1203or Si02crystalline phase can only be detected at relatively high temperatures accompanying the modification of mullite in the lattice structure. 27Aland 29SiMAS NMR studies indicate that segregation of A1 and Si atoms occurs in all samples below 900°C in the amorphous state, regardless of the gel composit i ~ nA. new ~ ~ indium(II1) ~ silicate, K21n(OH)(Si4010), has been synthesized by a high-temperature, high-pressure hydrothermal method. It crystallizes in the monoclinic space group. The 29SiMAS NMR exhibits four resonances at -88.6, -90.1, -97.4, and -98.2 ppm corresponding to four distinct crystallographic Si sites. A 'H- 29SiC P MAS NMR experiment was performed to assign the four resonances.358Solid-state 31PNMR spectra of 12 metal-containing selenophosphates have been examined to distinguish between the [P2Se6I4-,[PSe4I3-,


29 1

[P4Sel0l4',[P2Se7I4,and [P2Se,]" anions. There is a general correlation between the chemical shifts (CS) of anions and the presence of a P-P bond. These were examined using T1 and CSA spectra.359Cadmium hexathiohypodiphosphate (CdPS3) intercalates with potassium and N,N'-dimethylviologen cations have been studied by complementary one- and two-dimensional solid state NMR experiments. The intercalation process results in vacancies in the Cd sublattice, whose detailed distribution is sensitively reflected in both the 31Pand the '13Cd spectra of the host material. Advanced 2-D 31Pdouble quantum and spin echo NMR techniques are used to further investigate the connectivity patterns of the 31Patoms represented by the peaks observed. Simple chemical shift considerations strongly indicate that the vacancy distribution is primarily controlled by electrostatic repulsion effects.360Polycrystalline samples of AlV04 have been prepared by two methods of synthesis and characterized by 27Al and 'lV MAS NMR spectroscopy at 14.1 T. The MAS NMR spectra clearly reveal that essentially pure samples with minor impurities of V2O5 and alumina have been obtained. From these samples 27Alquadrupole coupling parameters and isotropic chemical shifts as well as the magnitudes and relative orientations of the 51Vquadrupole coupling and chemical shift tensors have been determined with high precision for AlV04. These data have been obtained from a combined analysis of MQMAS NMR spectra and MAS NMR spectra of the central and satellite transitions. The 27Aland 51VNMR data show that the asymmetric unit for AlV04contains three isolated vo4 tetrahedra, one pentacoordinated A1 site, and two A106 octahedra?6' LiGaYCol-,O2solid solutions, obtained under a highpressure (3 GPa), were studied by high-frequency electron paramagnetic resonance (EPR) of Ni3+spin probes and 71GaMAS NMR. 71GaMAS NMR experiments exhibit complex spectra that correspond to octahedral coordination and quadrupolar effects.362'I9Sn Mossbauer spectroscopy is used in combination with 7Li MAS NMR spectroscopy and X-ray diffraction to resolve the details of the different Li-Sn and Li-Sb intermetallics formed during the electrochemical reactions of nominal 'SnSb' compounds inside a lithium battery.363HT-LiXoCol-, Mg,02(where x(0) is the Li/(Co + Mg) ratio; x(0) = 0.98, 1.0, 1.10;y = 0.0,0.03, 0.06, and 0.10) materials were synthesized uia a solid-state reaction. These samples were characterized by X-ray diffraction, 7Li MAS NMR spectroscopy, Identification of the local environand electrical properties ments of the ions in a solid-state electrolyte that contribute to the ionic conductivity or remain trapped in the lattice represents a challenge for many experimental probes of structure. A high-resolution 1 7 0 MAS NMR spectra showed that even from the highly disordered, layered materials a-Bi4V2011 and pB&V1.7Ti0.3010.85, different oxide sites in the lattice may be di~tinguished.~,~ The homogeneity range of a fluorite-like solid solution in the NaF-Bi203-BiF3system has been studied by the isothermal annealing method at 6OO0C, X-ray powder diffraction, and 19FNMR.366The temperature and angular dependences of 2H NMR spectra were measured for single crystal of [Mg(H20)6][SiF6].At different temperatures the molecular motion of the D 2 0 was inve~tigated.~,~ The rotational behavior of the interlayer water molecules of deuterated vanadium pentoxide hydrate, V205.nD20,was studied by solid-state 2H NMR for the mono-

292


and double-layer structures of the adsorbed water molecules. The rotational motion and the activation energy were obtained for these sy~tems.~~*Dynamic 2H NMR spectroscopy employing line shape studies and T1 experiments is used to investigate the molecular dynamics of the perdeuterated pyridinium cations in pyridinium tetrafluoroborate in the temperature range between 120 and 290 K. Special interest lies in the two solid-solid phase transitions and the experimental spectra show that the pyridinium cations perform fast rotation around the Two solid-state NMR pseudo C6 axis at high t e m p e r a t ~ r e s . ~ ~ ~complementary techniques - variable contact time (VCT CP MAS) and 2D wide-line separation (2D WISE) spectroscopy have been used to probe the dynamics of methylene chains in an intercalated surfactant bilayer, formed within the galleries of layered cadmium thiophosphate by the insertion of cetyl trimethyl ammonium ions.37o The structure and dynamics of surfactant molecules intercalated in laponite have been characterized by 13C NMR relaxation times and 13C and 29Sidetected proton relaxation times. These results are compared to similar data obtained for a surfactant intercalated in montmorillonite and for different mesoporous mat e r i a l ~The . ~ ~layer ~ silicates are among the most common minerals in the Earth's surface environment, play important roles in many geological processes, and have diverse technological applications. While it has been suggested that 0 isotope exchange and dissolution kinetics in aqueous solutions are controlled by chemical bonding and local atomic structures, the effect of atomic environment around 0 atom sites in clay minerals on their site-specific reactivities with H 2 0 are not well known, mainly because direct experimental evidence is lacking. For the first time detailed high-resolution 1 7 0 NMR data for 170-exchangednatural kaolinite [A12Si205(OH)4]and muscovite [KA12(AlSi3)010(OH)2] using 1 7 0 3QMAS and MAS NMR at high fields have been p r e ~ e n t e d . ~ ~ ~dissolution The mechanisms of forsterite and enstatite were investigated by 29Siand 'H MAS NMR.37329Si,27AlMAS NMR is used to characterize laponite RD and synthetic saponites of variable interlayer charge. The Si/Al ratios are in good agreement with tile calculated charge from chemical analysis except for the lowest-charged saponite. In contrast to the 29SiMAS NMR spectra in which resolved signals are detected, the 27AlMAS NMR spectra show one signal whose linewidth increases with the clay charge. The water content of the clay samples was obtained from 'H MAS NMR. The 2D M Q MAS NMR technique is required to obtain a highresolution spectrum of nuclei with strong quadrupolar interacti01-1.~~~ The first application of variable-temperature 27AlMAS NMR spectroscopy to the satellite transitions in the characterization of a structural phase transition is presented by an investigation of Friedels salt (Ca2A1(OH)6C12H20) over the temperature range from - 121 to 109°C. Accurate values for the 27Alquadrupole coupling parameters (C, and q(Q))and the isotropic chemical shifts are obtained from either the manifold of spinning sidebands for the satellite transitions or the line shape observed for the central transition.


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References 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

29. 30. 31. 32.

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3 74. L. Delevoye, J. L. Robert and J. Grandjean, Clay Miner., 2003,38,63-69. 375. M. D. Andersen, H. J. Jakobsen and J. Skibsted, J . Phys. Chem. A, 2002, 106, 6676-6682.

8 Multiple Pulse NMR BY I. BARSUKOV

1

Introduction

The report covers the progress in the field of multiple pulse NMR over a period of twelve months from June 2002 to May 2003 and is a continuation of the report from last year. Several novel methods or substantial improvement of the existing experiments are worth mentioning here. Two new methods of indirect evolution detection were proposed by Kupce and Freeman’ and Frydman et aL2In the first method direct frequency dimension excitation at Hadamard-encoded preselected frequencies is used instead of chemical shift evolution, while the second method applies gradient-based spatial encoding. Both methods can offer dramatic reduction of the experimental time for multi-dimensional experiments. The ‘ultimate’ reduced dimensionality solution for triple-resonance experiments was proposed by Kim and Szyperski3in the form of G F T NMR spectroscopy. The method allows to combine the evolution of several nuclei in a single dimension and extract individual components through a linear combination of the acquired sub-spectra. Millet et al.4 proposed three additional experiments for measurement of deuterium relaxation in 13CH2Dgroups. Five different relaxation rates for a single deuteron can be evaluated using new and previously proposed experiments, providing a unique possibility for checking self-consistency of relaxation data. A new chemical shift based E.COSY method that uses ‘’N chemical shift instead of a coupling constant to resolve the multiplet components was introduced by Ding and Gronengorn’. This allows to observe the components in the different spectral regions, avoiding the overlap problem of the traditional E.COSY approach. Several publications described sensitivity improvements in triple-resonance experiments through the use of multiple-quantum ‘Ha-13Cacoherence (see in the text), that are particularly effective for fractionally-deuterated proteins. As before the new and improved experiments are summarised in Table 8.1 at the end of the chapter.

2

General methods

Heteronuclear decoupling is usually achieved by decoupling schemes with a periodic phase-modulation such as WALTZ, MLEV, or GARP. van Ingen et a1.6 Nuclear Magnetic Resonance, Volume 33 0 The Royal Society of Chemistry, 2004

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analysed cycling sideband artefacts generated by the sequences. To reduce the artefacts the decoupling in the asynchronous mode is usually applied. The current implementation of this mode by the spectrometer manufactures, however, does not produce complete randomisation and leads to a periodic signal modulation. As the result, horizontal ridges appear in heteronuclear correlation spectra; they are particularly strong in the experiments that select low-intensity equilibrium heteronuclear magnetisation, such as 2D { 'H}-15N NOE sequence. As the artefact originate mainly from the equilibrium proton magnetisation, they can be completely removed through the phase cycle when synchronous decoupling is used. A non-traditional single-scan method was proposed for detection of homonuclear multi-dimensional spectra by Frydman et aL2 The method relies on pulsed filed gradients to induce and decode simultaneous indirect evolution over a range of frequencies. Effectively, the sample is partitioned into a set of subsamples by the application of gradients. In combination with frequency selective pulses, this enables spins throughout the sample to undergo spatially distinct evolution periods. The information about the evolution is decoded by the application of gradients during the acquisition. The intensity of the solute signals needs to be sufficiently high for the successful application of the method. The acquisition scheme was demonstrated for 2D TOCSY and COSY spectra of small molecule, where complete 2D spectra were recorded in a few seconds. The method can be extended to record 3D and 4D spectra.

3

NOE, Chemical Exchange, Relaxation and Diffusion

3.1 NOE and Chemical Exchange. - Solvent accessibility of HN groups can be evaluated by the detection of rapid proton exchange with solvent in earlier proposed SEA-TROSY experiment. The experiment has been successfully applied to deuterated proteins. In case non-deuterated proteins SEA-TROSY experiments may contain contributions from aliphatic protons. Additionally the results may be misleading due to exchange-related NOE contributions from fast exchanging hydroxyl or amine protons and artefacts due to longitudinal relaxation in mixing time. Lin et aL7 proposed a modified version of the SEA element, Clean SEA, that uses spin-echo filter integrated with a double 15N or ''N /l 3 c filter for I5N or 15N/13Clabelled samples, respectively, along with the PhaseModulated CLEAN chemical Exchange (CLEANEX-PM) spin locking sequence during the mixing time. The radiation damping in mixing time is removed by the application of a weak gradient. Additionally, the relaxation artefacts are eliminated through the improved phase cycling. This element can be combined with HSQC or TROSY sequence (Clean SEA-HSQC or Clean SEATROSY) for HN-group detection. 3.2 Relaxation Rate Measurements. - Deuterium relaxation is a powerful method to probe side-chain dynamics in proteins. It can be measured with high sensitivity for CHD and CHZD spin systems in 13C-labelled, fractionally

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deuterated molecules. Such experiments were further developed by Millet et aL4 to include relaxation measurements for deuterium double quantum, quadrupolar order, and transverse anti-phase magnetisation in 13CH2Dmethyl groups. All experiments are based on the same HSQC-type sequence combined with the appropriate relaxation block. The I3CH2Disotopomers are selected with a 90'90" pulse pair introduced during 13C transverse evolution, and the coherence is refocused with one-bond 13C-2Hcoupling. The coherence transfer delays are optimised to ensure that the inner- and outer lines of the I3Cmultiples are 180" out of phase at the end of the refocusing delay. This preparation stage is followed by the specific pulse sequence block that induces the relaxation of the required 2H coherence component. Following the relaxation period, the coherence is transferred back to proton for detection. The relaxation rate is evaluated from the amplitude variation of the 13C-lHcross-peak intensities. The combination of the new and previously proposed experiments allow to measure five different relaxation rate constants for a single deuteron, thus providing a way to check self-consistency of the relaxation data. Experimental results were presented for two small proteins. The measurements of transverse heteronuclear relaxation rates are complicated by cross-correlation between dipole-dipole and chemical shift anisotropy relaxation mechanisms. The cross-correlation effects are usually eliminated by the application of proton 180" pulse trains or decoupling schemes such as WALTZ 16. Korzhnev et a1.* demonstrated experimentally and through the theoretical analysis that these approaches can lead to a significant overestimation of the relaxation rate values when heteronuclear spin-lock method is used to evaluate rotating-frame relaxation rate RIP.The artefacts mainly originate from equal spacing of proton pulses in the decoupling schemes. The authors proposed simple modifications of the decoupling sequences that use variable intervals between proton pulses to eliminate systematic deviations in the transverse relaxation rates. Presented experimental data demonstrate good agreement between the rotating-frame relaxation rates in the modified experiments and rates measured with CPMG sequence.

3.3 Cross-Correlated Relaxation Experiments. - A general scheme for describing cross-correlated relaxation experiments was proposed by Pelupessy et. aL9 The experiment is subdivided into four blocks corresponding to the different steps of the coherence evolution. These blocks can then be conveniently combined to allow the measurement of the particular cross-correlated relaxation. The selection of the relaxation type is achieved by the conversion block that transforms the target multiple-quantum coherence into single-quantum antiphase coherence. conventional sequences use a single conversion period, which can introduce systematic errors due to autorelaxation and pulse errors in the conversion period. These errors are largely eliminated if two conversion periods are used, before and after the relaxation period. The proposed block scheme makes it easy to introduce symmetrical reconversion of the coherences into the pulse sequences. Using the new approach the authors designed four complementary experiments that make the observed relaxation rates largely indepen-


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dent on the experimental parameters and alleviates problems due to violation of the secular approximation. The method has been applied to the measurement of N/NH (CSA/DD) cross-correlated relaxation rates in human ubiquitin. The sensitivity of the new experiments is similar to that in the conventional approaches. Majority of the existing cross-correlated relaxation experiments involve the same nucleus in both correlpted relaxation mechanisms, thus probing localised motions. Pelupessy et al." suggested experiments that can probe correlated motions of sequential amide groups by measuring NHN/NHN(DD/DD) and N/N (CSA/CSA) cross-correlation rates of two-spin coherences involving amide nitrogen nuclei in successive residues. The pulse sequence utilises a cascade of four INEPT-like transfers in order to convert amide transverse coherence into 8Ny(i)Ca,(i)C',(i)Ny(i + 1) coherence, involving two nitrogen spins in successive residues. After a relaxation period this coherence is converted back into observable amide proton magnetisation. The relaxation period can be modified to measure either DD/DD or CSA/CSA cross-correlated relaxation rates associated with two nitrogen nuclei of successive residues by introducing proton and nitrogen refocusing pulses. Two experiments are performed for each type of cross-correlated relaxation - one detecting the decay of the initial coherence and another detecting the coherence resulting from cross-correlated relaxation. The experimental data obtained for ubiquitin suggest the presence of internal motions that are slower than the global correlation time. TROSY method has been incorporated into HNCA-based experiments for measurement of the intra-residue and sequential Ha-Ca/HN-Ndipole/dipole and Ha-Ca/N dipole/CSA cross-correlated relaxation rates by Vogeli and Pervushin." The rates are evaluated from the differential relaxation rates of ZQ and D Q mu1tiplets recorded in ['5N,"C] -DQ/ZQ- [15N,lH] -TROSY and ['5N,"C] DQ/ZQ- ["N,'H]-TROSY-E. COSY experiments. To improve the sensitivity of the experiments all coherence transfer pathways are utilised by recording 16 FIDs for each time increment and recombining them post-acquisition to obtain in-phase components. Two variants of the experiment were presented, one optimised for the use at high B, that utilises full TROSY effect through double inversion of 'HN spins during M Q evolution, and another, suitable for low fields, that omit double inversion pulses. The E.COSY multiplet components in the spectra are displaced by 3,4J(Ha,HN) coupling constants. 3.4 Diffusion Experiments. - The majority of experiments for measurement of protein translational diffusion rates store the information about the localisation of the molecules as proton z magnetisation during the diffusion period. Fast selective longitudinal proton relaxation in large proteins impose limitations on the maximum diffusion time, limiting the application of the experiments to relatively small proteins. Ferrage et a1.'* proposed to use longitudinal magnetisation of slow relaxing 15Nnuclei to increase the range of the available molecular weights. Double INEPT sequence is used to transfer proton z magnetisation to the connected "N. The spatial information is encoded by a pair of gradient pulses in the first INEPT step and decoded in the last back-transfer INEPT step. The

3 10


experiment was successfully applied for the diffusion measurement of a 44 kDa protein/detergent complex. Resonances of different components in mixed solutions can be separated according to their electrophoretic mobilities through the application of the electric gradient in the course of NMR experiment. The electrophoretic interferograms have usually been generated in the flow dimension in two approaches by incrementing either the amplitude or the duration of the electric field. The later method results in severe signal decays due to molecular diffusion and spin-relaxation. Li and He13proposed a modified CT-ENMR experiment based on the constant-time evolution delay that keeps constant the delay between magnetic field gradients and r.f. pulses. As the result molecular diffusion and relaxation processes have constant contribution which improves the resolution in the electrophoretic dimension. The application of the method was demonstrated on a mixture of two small molecules. 4

Coupling Constants Measurements

4.1 Scalar Couplings. - A new quantitative method of 3J(HN,Ha)coupling de'~ termination in uniformly "N-labelled proteins was presented by Petit et ~ 1 . The proposed COSMO-HSQC experiment combines refocusing of the 3J(HN,Ha) coupling through selective inversion of "N-bound amide protons with a BIRD sandwich, and sensitivity enhanced "N-HSQC sequence. The value of the coupling is determined from the ratio of the HSQC peak intensities for the decoupled and reference undecoupled spectra. This ratio has a simple cosine dependence on the duration of J-evolution in the absence of differential relaxation in the modulated and reference experiments. The relaxation can substantially distort the measurements and has to be taken into account by using the derived more complex equations. The experiment is less sensitive to r.f. pulse imperfections than the equivalent experiment based on Ha inversion with selective pulses. Simple HMQC-based experiments were proposed for quantitative measurement of heteronuclear c o ~ p l i n g s .The ' ~ method is based on recording of two HMCQ experiments with and without additional 180" heteronuclear pulse at the end of scalar coupling evolution period. No heteronuclear decoupling in acquisition is applied. Addition and subtraction of the two spectra result in separation of the orthogonal components of the evolution with the coupling constant, which allows to evaluate the value of the coupling from the ratio of their integral absolute intensities. Due to the fact that one component has in-phase, and another - anti-phase line-shape, the coupling constant has to be determined from a least-square fit of the dependence of the intensity ration on the coupling constant evolution time using additional normalisation parameter that reflects partial cancellation of the anti-phase components. In practice the method can only be applied when there is no resonance overlap in 2D spectra, thus suitable only for rare nuclei. The application of the method was demonstrated for determination of 3J(H3'i,Pi+1) couplings in DNA octamer duplex.

4.2

Couplings Across Hydrogen Bond. - Modification of TROSY experiment


311

for measurement of 2hJ" and lhJHNcouplings across hydrogen bonds formed between His ring and protein backbone in large perdeuterated proteins was described by Eletsky et a1.I6 The coupling constants in the proposed 2D 2hJNNq~antitative-['~N,'H]-TROSY and 2D 'hJHN-quantitative-['5N,'H]-TROSY experiments are evaluated from the ratios of cross-peak intensities in the reference spectrum and the spectrum where evolution with the couplings across hydrogen bonds is decoupled. Large difference between backbone, His ring donor and acceptor 15Nchemical shifts makes it posible to use shaped pulses for selective inversion of the corresponding nuclei for selective decoupling. The couplings were measured for a 44 kDa protein with an accuracy of 0.04-0.2 Hz. Vicinal 3 h J ~ 2coupling ~3 in AU base-pairs of uniformly "N-labelled RNA has been measured in a modified 2hJNN-HNN-E. COSY e~periment.'~ The initial INEPT step is tuned for the optimal H2->N1 transfer and followed by coherence transfer across N1 . . , H3-N3 hydrogen bond utilising 2hJNNcoupling. The coupling is generated by E.COSY cross-peak for the measurement of 3hJH2H3 evolving N3 without proton decoupling and then using a selective 90" proton G4-pulse to leave the spin-state of the H3 proton unperturbed during the N1->H2 magnetisation back-transfer. In the resulting spectrum, the H3 proton acts as a passive spin in the E.COSY spectrum. The small 3hJH2H3 coupling can therefore measured accurately from the displacement of multiplet components separated with large 1JH3N3 coupling. Positive values with the magnitude of up to 1.8 Hz were reported for RNA hairpins. 4.3 Residual Dipolar Couplings. - Simultaneous measurement of several coupling constants offers large reduction in the experimental time and reduces variation in conditions due to sample instabilities. This could be particularly critical for residual dipolar couplings as the properties of the alignment medium may change with time. Wienk et al." described J-correlated ["N,'H]-TROSYHNC experiment for evaluation of 'J(N,C') , 'J(N,Ca), and 2J(N,C") coupling constants in proteins. HMQC element is used for simultaneous coherence transfer from N to C' and Ca nuclei and recording chemical shift. Evolution with 13Cu-Ha couplings is refocused with band-selective proton pulse that avoids

perturbation of lHN spin-state. The 15N chemical shift is recorded in nonconstant-time manner to reduce the number of refocusing carbon pulses and to allow long 15Nacquisition times. Slow-relaxing TROSY components are selected with sensitivity-enhanced gradient-based element. The coupling constants are evaluated quantitatively from the ratio of the the corresponding cross-peak to the reference axial peak observed in the same experiment and not modulated with I3Cchemical shift. A novel (G,J)-E.COSY method was proposed for measurement of one-bond 15N-'HN,I5N-l3C'and two-bond 13C'-'HNresidual dipolar couplings in protein^.^ The method is based on simultaneous transfer of 2H,N, and 2H,N, coherences generated during chemical shift evolution of the HSQC experiment into -H, and -2H,N, coherences, respectively. No decoupling is applied in the acquisition time, leading to the observation of two E.COSY multiplet components for each amide groups, separated by single-bond proton-nitrogen coupling in the acquisi-

312


tion dimension and the nitrogen chemical shift in the indirect dimension. By adjusting the spectral width in the indirect dimension and using TPPI method, each E.COSY multiplet component can be located in distinctly different regions of 2D plane. The value of the coupling is measured in the homonuclear dimension, which reduces the effect of proton exchange on the apparent value of the coupling. The couplings involving C' are measured in a slightly modified experiment that does not refocus 'J(N,C') coupling during 15Nevolution and contains N->C' INEPT transfer period prior I5N evolution time. Two (6,J)-E.COSY sub-spectra are acquired with in-phase and anti-phase E.COSY splitting with passive 15N-13C'coupling, and recombined post-acquisition to produce two E.COSY spectra that have multiplet component positions determined by "N1HN, 15N-13C' and 13C'-'HNcouplings and nitrogen chemical shift. All three couplings can be deduced from the experiment. Large separation of the E.COSY multiplet components with the 15Nchemical shift allows to retain the cross-peak separation similar to that of decoupled HSQC spectrum despite the increase in the number of multiplet components. The proposed sequences have smaller number of pulses than those based on the popular IPAP method. Quantitative J experiments normally have higher precision than methods based on the difference in line splitting. However, the accuracy of the method is often reduced by systematic errors, usually caused by r.f. pulses miscalibration or relaxation. Cutting et al.19 proposed a compensation scheme for HSQC-PFG experiment, that encodes coupling information into the phase modulation of resonances. The original experiment uses constant-time evolution with subsequent transfer of both 2N,H, and N, coherences at the end of 15N evolution period into the components of the observable magnetisation. The amplitude of each component depends on the match between the evolution time and the coupling constant, and thus can be used to evaluate coupling constants. Due to the coherence transfer pathway differences for the 2N,H, and N, coherences, the components have different relaxation attenuation, which is particularly strong for large molecules. The authors suggested to make two measurements at different values of the constant-time delay with the difference equal to 0.5/Jnomina1, with Jnominal being the average value of the coupling constants. The effect of the relaxation is opposite for such pairs of the constant time delays, resulting in the higher accuracy for the average value of the coupling constant. The experimental 1 for the modified method. data demonstrated precision of ~ 0 . Hz Long-range residual 'HN-I3Cdipolar couplings can be reliably measured in fully deuterated proteins using quantitative 3D HCN-HSQC experiment.20The pulse sequence consists of two concatenated 'H-13C/'H-15N experiments that allow to detect frequencies of both coupling partners and additionally resolve cross-peaks with 15N chemical shifts. In the first stage of the experiment the coherence is transferred through 'HN-13C coupling evolving over a selected period T. The efficiency of the transfer and hence the cross-peak amplitudes are proportional to Sin2(2n&cT), where KHC is the sum of residual dipolar and scalar couplings. The second stage involves HSQC detection of "N frequency. The coupling constants are evaluated from the ratio of cross and reference peak intensities. The scalar contribution is derived from the isotropic protein sample.


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Homonuclear ‘H-’H residual dipolar couplings provide valuable structural information for proteins with problematic isotope enrichment. Moglich et aL2’ added a TOCSY-based sequence MOCCA-SIAM to the relatively small list of experiments suitable for the unlabelled proteins. The method allows to measure D(HN,Ha)with the accuracy of 0.2Hz, particularly suited for systems with low degree of orientation. In the suggested pulse sequence the element for simultaneous acquisition of in-phase and anti-phase multiplets (SIAM) is followed by TOCSY transfer with MOCCA-XY 16 mixing sequence optimised for dipolar transfer. Two separate FIDs are recorded per each increment, with the addition of the FIDs producing in-phase ZQF-SIAM and the subtraction resulting in the antiphase DQF-SIAM spectrum. The coupling constant is obtained by the Titman-Keeler convolution procedure applied to both spectra simultaneously. The algorithm does not use any assumption on the multiplet lineshape, but relies on the similarity of the lineshapes of the in-phase and anti-phase multiplet components, which is fulfilled in the experiment. The method was tested on BPTI. Rapid rotation of methyl groups around C-C axis makes their ‘H-’H and ‘H-13Cresidual dipolar couplings related to the C-C bond orientation in respect to the alignment tensor, thus providing valuable restraint for structure calculations. Despite the rotation, the values of the ‘H-’H and ‘H-13C residual dipolar couplings are still about 7 and 3 times larger than the corresponding 13C-13C couplings. The couplings are best measured in CH2D groups of partially deuterated proteins. Two new experiments for measurement of the ‘H-’H couplings were proposed by Sibille et aZ.22The 2D DHH-CT-HSQCexperiment is suitable for small proteins with well resolved methyl group cross-peaks. The pulse sequence incorporate CH2Disotopomer selection filter and a/P selection filter, both based on large ‘J(C,H) coupling. The residual dipolar couplings are evaluated from cross-peak displacements between the spectra corresponding to a and P states. Better cross-peak separation, required for larger proteins, can be achieved in 3D DHH-CT-HCCexperiment that utilises additional carbon frequency, measured as constant-time evolution. The filter elements applied for isotopomer and spin-state selections are the same as for the 2D experiment. Resonance overlap is a major problem when measuring H2’-C2’ and H3’-C3’ residual dipolar couplings is sugar moieties of large nucleotides. Vallurupalli and Moore23proposed to use well-resolved Hl’-Cl’ cross-peaks to obtain the information on the coupling constants. Two experiments based on HCCH-COSY method were described: 3D HCcH-COSY for evaluation of the H2’-C2’ coupling and 3D Relay HCcH-COSY for evaluation of the H3’-C3’ coupling. The coherence transfer pathway starts on Hl’, incorporates chemical shift evolution for H1’ and Cl’, and ends on H2’ or H3’, in respective experiments, for the detection. No decoupling in the acquisition time is applied and the proton multiplet components are separated with the IPAP method. The experiments were successfully applied to a 43 nucleotide RNA. HSQC-based experiment for quantitative measurements of residual one-bond ‘H-13C couplings in oligosaccharides at natural abundance was presented by Pham et ~ 1Long-range . ~ ~ proton-carbon and homonuclear proton-proton coup-

314


lings are eliminated with BIRD sequence, refocusing all but ‘J(C,H) couplings. This allows to use long delays for the coupling constant evolution, thus increasing precision of the experiment. Two variants, jch-h and jch-c, of the experiment were proposed, with either proton or carbon evolution in the modulation period. The experiment utilising proton evolution is used for the measurement of ‘J(C,H) couplings in CH2 groups, while the carbon evolution experiment is two times more sensitive. The analysis of the evolution in the modulation period allowed the authors to derive a simple modulation functions that provides a good approximation. For strongly coupled systems the analysis based on the evolution of the density matrix is more appropriate, but in some cases the weak coupling approximation can be used.

5

Inverse Proton Detected Correlation Spectroscopy

5.1 Double-resonance Experiments. - Long-range heteronuclear correlation experiments are widely used for structure determination of small molecules. These experiments require careful selection of coherence transfer delays when applied to complex spin-system, in particular if one-bond couplings have to be suppressed. Systematic analysis of coherence transfer efficiencies in 2D MBOB, broadband HMBC and XLOC long-range experiments was presented by S~hulte-Herbruggen.~~ Theoretical simulations were performed for several combinations of line-widths and coupling constants and the optimal values of the delays were tabulated for each experiment. The use of optimised delays improves sensitivity of the experiments and allows to avoid gaps of zero transfer amplitude. Low chemical shift dispersion of proton resonances in polysaccharides and other complex carbohydrates leads to strong overlap in long-range protoncarbon correlation experiments. Uhrin26 suggested a 3D HSQC-HSQCMBC experiment that additionally resolve long-range correlations with chemical shift of directly bound proton. The proton coherence is initially transferred to the directly bound carbon, and subsequently to the distant proton over the longrange coupling for detection. The signal from the proton attached to 12Ccan be removed with the BIRD sequence incorporated into single-bond coherence transfer step. The experiments utilise only single-quantum evolution which ensures in-phase pure absorption line-shapes of the cross-peaks. 5.2 Isotope Filtered and Edited Experiments. - Isotope filters were used by Phan and Pate127to differentiate between resonances of fully l5N,I3Clabelled and low-level labelled nucleotides. Two HSQC-based experiments were described, both relying on simultaneous presence of neighbouring labelled nuclei, the condition that is true only for the uniform labelling. The first experiment, sCT-HSQC, aimed at the detection of Cl’-Hl’ correlation, incorporates a pair of selective 9W-pulses into CT-HSQC sequence, that acts on the neighbouring and well separated in chemical shift C2’ resonance, to decouple ‘J(Cl’,C2’)coupling in alternative scans. With the constant time delay tuned to induce spin inversion


315

in the 13Cl'-'3C2'group, the subtraction of decoupled and non-decoupled scans results in the selection of the resonances from the fully labelled species, as the the signals of 13C1'-12C2'are unaffected by the decoupling. A different HN(C) scheme was suggested for the selection of resonances when carbon atoms have only neighbouring nitrogens, as in case of purine H2 and H8 protons, with detection of H-N correlations while using 'J(N,C) coupling for the resonance selection. Use of 3D homonuclear TOCSY-TOCSY experiment in combination with 13C and 15Nfilters was proposed by van Ingen et aL2*for assignment of unlabelled ligands in complexes with '3C,'5N-labelled proteins. The experiment includes three independent INEPT-based 13Cfilters tuned to match aromatic, aliphatic and methyl 'J(H,C) couplings, and two l5N-fi1ters.Gradient pulses of opposite polarity are incorporated into INEPT delays to defocus coherence of 13C-bound protons that is not inverted in the INEPT sequence and to refocus the coherence of 12C-boundprotons that is inverted by the sequence. The use of three independent filters ensures high level of suppression. In addition, the experiment has two-fold sensitivity enhancement through the use of both orthogonal magnetisation components transferred by each of the DISPI-2 mixing sequences. Four FIDs are recorded for each increment and recombined to obtain in-phase line-shapes. The authors noted that line-shape distortions due to the exchange between in-phase and anti-phase magnetisations during TOCSY isotropic mixing were absent in the 12.5 kDa peptide/protein complex because of the selfcancelation anti-phase component at the observed line-widths. The mixing times for both TOCSY transfers were restricted to 21 ms to improve sensitivity and reduce number of cross-peaks in the spectrum. Similar isotope-filtering approach utilising several 13C INEPT-based gradient enhanced filters tuned for different ranges of coupling constants was used by Zangger et al.29in 13C-edited NOESY-HSQC experiment. The scheme required three sequential filters for protein samples, which may lead to significant sensitivity loss. 5.3 TROSY. - Systematic analysis of the sensitivity of TROSY-based "N-IH correlationexperiments CRIPT-TROSY, CRINEPT-TROSY and CRINEPTHMQC-['HI-TROSY for deuterated protein of very high molecular weight (100-800 kDa) was presented by Riek et aL3' The performance of the experiments depends critically on the choice of the polarisation transfer times, relaxation delays between subsequent recordings and water-handling routines. During polarisation transfer and frequency labelling, transverse relaxation has a dominant impact on the sensitivity, whereas the longitudinal relaxation rate is the main parameter that effects the choice of the relaxation delay. Saturation of water signal could lead to about 10 fold reduction in the intensity of protein signals through spin-diffusion, thus requiring careful positioning of flip-back pulses in the pulse sequence to keep water magnetisation along +z during the whole experiment. Good agreement between optimal theoretical and experimental values of the acquisition parameters was demonstrated for several proteins with molecular weights of up to 800 kDa. The optimal delays for CRIPT polarisation transfer were found to be 6 ms for a molecular weight of 200 kDa, 2.8 ms for 400 kDa and 1.4 ms for 800 kDa. For the proteins used, TROSY

316


experiment only allowed the observation of cross-peaks corresponding to highly dynamical regions of the protein, while CRIPT-TROSY experiment selected resonances from the structured parts. Both types of resonances were observed in the CRINEPT-based experiments. Sensitivity of TROSY experiments per unit time can be improved through the enhancement of longitudinal relaxation of 'HNprotons by dipole-dipole interactions with other protein and water proton^.^' Modified sequences 2D ["N,'H]LTROSY, 2D ["N,'H]-LHSQC and 3D 3D ["N,'H]-LTROSY-HNCA of the corresponding experiments were presented, that minimise the saturation of 13Cand oxygen-bound protons. These protons serve as a 'thermal bath' in the equilibrium state and accelerate magnetisation transfer to the saturated 'HN protons in the relaxation period. This allows to use short relaxation delays, thus improving the sensitivity per unit measurement time. The minimised saturation of non-observed protons in 2D ["N,'H]-LTROSY was achieved by introduction of carbon inversion pulses and modification of phase cycle. The 2D ["N,'H]LHSQC and 3D ["N,'H]-LTROSY-HNCA experiments require additional application of 'HN band-selective pulses. More than two-fold sensitivity improvement was experimentally observed at 600 MHz. 5.4 Heteronuclear Triple Resonance Experiments. - An alternative to the traditional triple-resonance experiments based on chemical shift evolution for indirect detection was proposed by Kupce and Freeman.' In the described method the time-domain evolution periods are replaced by direct frequencydomain excitation of selected carbon and nitrogen sites using prior knowledge of the relevant chemical shifts. This is achieved with an array of several simultaneous soft r.f. spin-inversion pulses, on or off encoded according to nested Hadamard matrices. As the result, at each heteronuclear site the sign of the product operator term 2I,S, is either reversed or left unchanged, thereby reversing (or not) the sign of the final proton response. The resulted responses are extracted by reference to the same matrices. High definition frequency-domain traces are derived by conventional Fourier transformation of the decoded free induction decays. Like constant-time experiments, they have no intrinsic linewidth in the indirect dimensions. The selectivity in the frequency domain is determined by the length of the direct excitation sequence which is typically 20 ms in triple-resonance experiments. The methods was illustrated on HNCO and HNCA experiments. In the modified sequence the 13Cand 15Nevolution periods were replaced with the Hadamard-encoded selective inversions of 'H and 15N,or 'H and I3Cspins, respectively.The use of the proposed method can produce up to 100 fold time saving relative to the indirect evolution detection. The experimental data were presented for the selective observation of cross-peaks from 8 sequential residues in a 4 kDa protein. The principles of Hadamar NMR spectroscopy were described in by the author^.^^.^^ Simultaneous detection of information on chemical shift evolution of several nuclei types in a single dimension is becoming increasingly popular as a method of decreasing measurement time and number of spectra required for resonance assignments. These methods are based on 4D or 5D sequences and reduce


317

dimensionality by combining evolution in several indirect dimensions. The ‘ultimate’ reduced dimensionality solution was proposed by Kim and Szyperski3 in the form of GFT NMR spectroscopy, utilising encoding of the evolution in the combined indirect dimension through phase modulation defined by a G-matrix. Several indirect dimensions are incremented simultaneously to introduce modulation with all locked frequencies. For each time increment in the combined dimensions several FIDs are recorded with independent 90” phase shifts in each of the combined dimensions. This variation produces combinations of in-phase and anti-phase resonance splitting in the combined dimension, with separate components extracted by a linear combination of the FIDs. Additional FIDs with some of the evolution periods set to zero need to be recorded in case of resonance overlap. (5,2)D HACACONHN experiment based on a straightforward modification of the HACA(C0)NH sequence was described as an illustration of the new method. Evolution of four nuclei is locked in a single dimension and the chemical shifts of Ha, Ca, C’ and N are extracted from the reconstructed 2D spectra. The complete experiment was recorded in 2h 18min. Improvements of the traditional reduced dimensionality approach were also proposed. In the ‘classical’3D method the chemical shift evolution of one nuclei is detected in quadrature and additionally modulated by the chemical shift evolution of the second nuclei. As the result a doublet is observed in the combined dimension centred at the position of the resonance detected in quadrature with the splitting corresponding to the chemical shift of the other nucleus. Due to the resonance splitting the sensitivity of the experiment decreases twofold and the number of cross-peaks in the spectrum increases. Two additional experiments of this kind, HNNCOCA and HNNCACO, were proposed by Xia et In both experiments Ca is detected in quadrature and the signal is modulated by C‘ chemical shift evolution. In the modified method of the dimensionality reduction both nuclei are detected in quadrature. This can be used to improve the sensitivity of the experiment and separate the signals from the two nuclei by careful adjustment of offsets and sweep width in the projected dimension. Two complimentary experiments of this kind, 3D HNNCCAHA] and HNN(CO)[CAHA], are described in the above mentioned p ~ b l i c a t i o n The .~~ experiments are based on the simultaneous use of N,Ca, and N,Ca,Ha, present at the beginning of the evolution in the projected dimension. The second term is initially converted into zz-order, while the fist is evolved with Ca chemical shift. In the second part of the evolution in the projected dimension the second term is converted into double-quantum H-C coherence and the evolution with the Ca chemical shift is decoupled. The use of both coherence terms improves the sensitivity of the experiment and allows to achieve quadrature detection for both nuclei. The proposed experiments are particularly useful for the assignments of Gly resonances. More extreme dimensionality reduction was proposed by Ding and Gronenborn.3sAll backbone chemical shifts can be extracted from four complementing 2D experiments that allow to increase spectral resolution and reduce experimental time. Proposed versions of 2D HN(C0)CACB and HN(C0CA)CACB experiments correlate HNi,Ni, Cai-l,and C’i-1, and HNi,Ni, Cai, and Cpi,respectively.

318


In both experiments the chemical shift information is derived from simultaneous evolution of CA,N,, 2CB,CA,N,, CA,N, and 2CBxCA,Nx terms in a single evolution delay. These coherences are created by 54" "N pulses flanking the evolution delay. The differentiation between sequential and intraresidue correlations is achieved by coherence transfer through C O in the HN(C0jCACB sequence or using a filter scheme based on 'J(Ca,CO) in HN(C0CA)CACB sequence. Six cross-peaks are observed for each residue, positioned at the combined 13C, 15Nchemical shifts in the indirect dimension. Similar approach was used in 2D HN(C0)CAHA and HN(C0CA)CAHA experiments that correlated Ha chemical shifts. The sequences were optimised to reduce the disturbance of water and amide signals. Large carbonyl carbon CSA leads to a rapid increase of the relaxation rate with the increase of the magnetic field. This makes the experiments based on 13C' coherence (such as HN(C0jCA) inherently insensitive when recorded at the magnetic fields above 600 MHz. The HNCA experiment does not have this limitation and can be used to establish sequential connectivities, but often has reduced intensities for the sequential correlation due to small values of 2J(N,Ca). Alternative assignment strategy using a novel 3D multiple-quantum HACACO experiment with direct detection of the 13C'anti-phase coherence was proposed by Pervushin and E l e t ~ k yAn . ~ ~elegant pulse sequence is based on the HMQC double-constant-time coherence transfer scheme with overlapped constant-time delays that are utilised for chemical shift evolution of 'Ha and 13Canuclei and Ca->C' coherence transfer. Favourable relaxation properties of the 'Ha-13CaMQ coherence reduce the signal losses when 13Camagnetisation is transverse. This is partially advantageous for partially deuterated proteins. The signal is detected as C' anti-phase doublet, minimising the time when C' magnetisation is transverse. Additional sensitivity improvements are achieved by returning the 'HNmagnetisation to the + z direction using selective pulses to accelerate the return of 'Ha magnetisation to the equilibrium, and SEDUCE-2 decoupling of Cp nuclei. Anti-phase multiplet splitting of C' resonances is removed through post-acquisition manipulation where multiplet components are first separated by superposition of the spectrum with its absolute value and subsequent recombination following a & 26.5 Hz shifts, corresponding to 'J(C",C'). This procedure reduces the number of cross-peaks in the spectrum and improved signal-to-noise ration by d2. The application of the experiment was demonstrated on a 44 kDa protein with 35% fractional deuteration at 20" and 9°C. The new experiment can be combined with TROSY-HNCA and TROSY-HNCO experiments to establish sequential connectivities in large proteins. The use of 'Ha-13CaMQ coherence to improve the sensitivity of triple-resonance experiments was continued by the above group with 4D TROSY-MQHACANH e ~ p e r i m e n t .Here ~ ~ the HMQC double-constant-time coherence transfer scheme with overlapped constant-time delays was utilised for chemical shift evolution of 'Ha and 13Canuclei and Ca+N coherence transfer. This was followed by INEPT transfer into the observable HNcoherence. Additionally, the authors presented 'out-and-back' 3D TROSY MQ-HN(CA)HA experiment that uses 'Ha-13CaM Q coherence for indirect 'Ha detection. The exchange between


319

TROSY and anti-TROSY components during the excitation of 'Ha-13Ca MQ coherence was prevented by the use of 'HN band-selective 90"pulses. SEDUCE pulse train was applied for C' decoupling. The application of the experiments was demonstrated for a 44 kDa protein with 35% fractional deuteration at 20°C. More traditional, than the above, approach was used by Lohr et aL3' to detect correlations involving Ha protons. In this case partial protonation at Ha position in otherwise fully deuterated protein sample was the result of protein expression in fully deuterated algal lysate medium in 100% H 2 0to overcome the problem of slow back-exchange of amide protons. Three modified experiments H(CA)NH, HCACO and HCACB were presented. All experiments utilised MQ 'Ha-13Ca coherence with selective spin-locking of 'Ha. The use of the MQ coherence removes the dominating 'Ha contribution from 13Carelaxation, while selective spin-lock eliminates dephasing of MQ coherence due to homonuclear 'H-'H couplings. In the case of H(CA)NH experiment that requires indirect 'Ha detection, the duration of the spin-lock is gradually shortened to allow 'Ha chemical shift evolution. As the result, the evolution period is semi-constant time with respect to homonuclear J-coupling and constant time with respect to relaxation. Analysis of the protonation levels for different amino-acid types and the sensitivity of triple-resonance experiments were presented. Development of HNCA-based experiments for sequential assignment in large proteins was continued by Nietlispach et The authors proposed a complimentary pair of 3D intra-HNCA and DQ-HNCA experiments that allow to distinguish between sequential and intraresidue correlations of the HNCA spectrum and improve the sensitivity of the detection of sequential correlations. The intra-HNCA experiment utilises 'J(N,C') coupling for selective observation of the intraresidue correlations. In the DQ-HNCA experiment carbon chemical shift is registered from the evolution of Ca(i)-Ca(i-1) double-quantum coherence created through 4N,Ca(i),Ca(i-l)zdouble-antiphase coherence, generated by simultaneous evolution with one- and two-bond N-Ca couplings. The maximum transfer efficiency for this coherence is higher than for individual antiphase coherence, but requires longer coherence transfer delay. This does not lead to significant decrease of the intensity as TROSY is used for the "N transverse evolution.The double-antiphase coherence is converted into MQ Ca(i)-Ca(i-1) coherence and the evolution of double-quantum component is selected through the phase cycle. The chemical shift in the carbon dimension is equal to sum of chemical shifts of intraresidue and sequential Ca. The cross-peaks are individually rereferenced using the intraresidue chemical shift from the intra-HNCA experiment. Additional experiment for registering intraresidue (Hi,Ni,Cai) correlations (iHNCA) was proposed by Permi.'" The pulse sequences were optimised for deuterated proteins and have similar sensitivity to the normal HNCA experiment when "N transverse relaxation is slow. The experiments are based on the HN(C0)CA sequence and use the evolutions with 'J(N,C') and 'J(Ca,C') couplings for the selection of the intraresidue correlation. Relatively long coherence transfer delays in the new experiment limit its application to small proteins. Selective protonation of valine, leucine and isoleucine Me-group in fully

320


deuterated proteins is often used to detect long-range NOES at high molecular weight. The assignment of the side-chains requires correlation of the side-chain resonances with those of the backbone. The deuteration makes it possible to transfer coherence from the Me-group to the backbone N H for relatively large complexes, as demonstrated by Hilty et aL4'in case of integral membrane protein OmpX embedded into 60 KDa detergent micelles. The authors described HCCH-TOCSY based experiments (H)C(CC)-TOCSY-(C 0 ) -['5N,1H] -TROSY, H(C)(CC)-TOCSY-(CO)-[lsN,lH]-TROSY and (H)C(CC)-TOCSY-['5N,'H]TROSY that improve the sensitivity through the use of TROSY method. In all experiments coherence transfer pathway starts on Me-group, DIPSI-3 sequence is used for the CC-TOCSY transfer, and selective 13C coherence transfer is achieved though a combination of shaped pulses and SEDUCE-1 decoupling. The attenuation of intense detergent signals is achieved with a HN-selectivepulse. Two-fold sensitivity improvement due to the incorporation of TROSY was reported at 500 MHz. A different approach was used by Tugarinov and Kay42for assignments of selectively protonated Ile 61 methyl groups large deuterated proteins. The analysis presented shows significant decrease of the efficiency of C"-CU TOCSY transfer, which is often used for the side-chain-to-backbone correlation, due to Ile side-chain branching. The transfer efficiency is higher when multiple COSY transfer steps are used and 13Cp-13Cy1 coupling is refocused with band-selective pulses. The later is relatively easy to achieve due to the larget3Cchemical shift differences. The method was implemented as 3D (H)C(CA)NH-COSY experiment and applied to a protein with correlation time of 46 ns. On average, the COSY-based experiment was 50% more sensitive than the corresponding TOCSY-based experiment. Additional information can be introduced into 3D triple-resonance experiments by encoding chemical shift information of inactive nuclei through an apparent scalar coupling active during evolution period.43 The method was illustrated on 3D HNCAcodedHA experiment. Coding of chemical shift information of 'Ha through the scalar 'J(H',Cu) coupling is based on the use of a 180" r.f. soft pulse in the middle of C' evolution. The pulse is set on-resonance at the high-field edge of H' chemical shift region. The inversion profile of the pulse ensures complete inversion of longitudinal proton magnetisation at the maximum frequency and the decrease of the inversion to zero at the minimum frequency, resulting in the frequency dependent refocusing of 'J(HU,Cu)coupling. This leads to a variable splitting of Casignal with an apparent coupling value dependent on the chemical shift of the attached proton. The main advantage of the experiment is in incorporation of an additional chemical shift information without extra transfer delays, albeit at the expense of some sensitivity reduction and cross-peak broadening. The method can be incorporated into other tripleresonance experiments that involve evolution of protonated carbons. Additional experiments to facilitate the assignments of exchangeable protons in nucleotide bases were described by Wohnert et al.44 Two new 2D H6/H5(C4N)H and C6/C5(C4N)H pulse sequences simultaneously correlate the chemical shifts of the H6/H5 resonances or the C6/C5 carbon resonances with

Measurement of slow translational diffusion. Constant-time electrophoretic experiment

Detection of solvent accessible HN groups.

3D HCN-HSQC 2D MOCCA-SIAM

2D (6,J)-E.COSY

Residual dipolar couplings 3D J-correlated ["N,'H]-TROSY-HNC

Scalar couplings 2D COSMO-HSQC 2D J-HMQC Couplings across hydrogen bonds 2D 2hJNN-quantitative-[15N,1H]-TRosY 2D 1hJHN-quantitative-['5N,'H]-TROSY 2 h ~ N , - COSY ~ ~ ~ - ~ .

16 17

Measurement of 2hJNN and lhJHN couplings involving His rings in proteins. Measurement of 3 h J ~ 2couplings ~3 in AU base-pairs of uniformly "N-labelled RNA.

Simulations evaluation of 'J(N,C') , J(N,Ca),and 2J(N,Ca)coupling constants 18 in proteins. Measurement of one-bond I'N-IHN, I5N-l3C'and two-bond 13C-'HNresidual 5 dipolar couplings in proteins. Measurement of 'HN-I3Cdipolar couplings in fully deuterated proteins. 20 Measurement of D(HN,Ha)in unlabelled proteins. 21

14 15

10

11

4

12 13

Ref:

Measurement of 'J(HN,Ha)couplings in uniformly "N-labelled proteins. Measurement of heteronuclear couplings for rare nuclei.

Measurements of deuterium double quantum, quadrupolar order, and transverse anti-phase magnetisation relaxation in I3CH2Dmethyl groups. 3D [15N,'3C]-DQ/ZQ-['SN,1H]-TROSY-E. COSY Measurement of the intra-residue and sequential Ha-Ca/HN-Ndipole/dipole 3D ['5N,'3C]-DQ/ZQ- [*'N,'H]-TROSY-E. COSY and Ha-Ca/N dipole/CSA cross-correlated relaxation rates. Measurement of long-range NHN/NHN(DD/DD) and N/N (CSA/CSA) 2D HNCACO-based cross-correlated relaxation rates.

Relaxation experiments 2D 13C-lHHSQC-based

NOE, Chemical exchange and diffusion 2D Clean SEA-HSQC 2D Clean SEA-TROSY 1D double INEPT-based CT-ENMR

Aims

Summary of new or improved experiments and methods

Experimentlmethod

Table 1

!2

w

%

2

'tt

=.

z5

?

(cont.)

22

24

23

Re&

Resolution improvement for long-range correlations in complex carbohydrates. Improvement of sensitivity per unit time through longitudinal relaxation enhancement.

42

3 41

1

26 31

27 Differentiation between resonances of fully "N,I3C labelled and low-level labelled nucleotides. Assignment of unlabelled ligands in complexes with '3C,15N-labelledproteins 28 Improved isotope filtering for large variation in coupling constant values. 29

Measurement of the 'H-IH couplings in CH2Dgroups.

Measurement of H2'-C2' and H3'-C3' residual dipolar couplings in sugar moieties of large nucleotides. Measurements of residual one-bond 'H-I3C couplings in oligosaccharides at natural abundance.

Aims

Heteronuclear triple resonance Selective correlation detection in triple-resonance experiments. Hadamard-encoded 3D HNCO 3D HNCA Reduced dimensionality GFT-based experiment (5,2)D HACACONHN 3D (H)C(CC)-TOCSY-(CO)-[15N,'H]-TROSY, Side-chain assignments in Me-protonated fully deuterated proteins. H(C)(CC)-TOCSY-(CO)-['5N,1H] -TROSY and (H)C(CC)-TOCSY- ['5N,1H] -TROSY Side-chain assignments of Ile 61 methyl groups in fully deuterated proteins. 3D (H)C(CA)NH-COSY

2D ["N,'H]-LTROSY 2D ["N,'H]-LHSQC 3D ['5N,1H]-LTROSY-HNCA

Heteronuclear double resonance 3D HSQC-HSQCMBC

3D X-filtered TOCSY-TOCSY 3D multiple filtered 13C-editedNOESY-HSQC

Isotope filtered and edited experiments 2D sCT-HSQC, 2D HN(C)

2D jch-h 2D j c h - c , 2D DHH-CT-HSQC 3D DHH-CT-HCC

3D HCCH-COSY 3D Relay HCcH-COSY

Experimentlmethod

Table 1

2

Q

4

k

z.

cc,

%

B

3z

h>

h)

w

3D intra-HNCA 3D DQ-HNCA 2D H6/H5(C4N)H 2D C6/C5(C4N)H

4D TROSY-MQ-HACANH 3D TROSY MQ-HN(CA)HA 3D H(CA)NH, 3D HCACO 3D HCACB 3D HNCAcodedHA

313 HNNCOCA 313 HNNCACO 313 HNNCCAHA] 313 HNN(CO)[CAHA] 213 HN(C0) CACB 213 HN(C0CA)CACB 213 HN(C0)CAHA 213 HN(C0CA)CAHA 313 MQ-HACACO 35

36

Reduced dimensionality experiments.

Sequential connectivities in large proteins. Use of 'Ha-13CaMQ coherence to improve sensitivity. Sequential connectivities in large proteins. Use of 'Ha-13CaMQ coherence to improve sensitivity. Correlation in partially deuterated proteins. Use of 'Ha-13CMQ coherence to improve sensitivity.

Encoding of additional chemical shift information through apparent scalar 43 coupling. Sequential intraresidue and sequential correlations for resonance assignments 39 in large proteins. 44 Correlations in pyrimidine bases.

38

37

34

Reduced dimensionality experiments.

w h, w

2

m

2 G-

s

%

??

324


the imino proton and the amino proton chemical shifts in pyrimidine bases. The experiments simultaneously transfer H6/H5 magnetisation by an INEPT step to the C6/C5 nuclei and by homonuclear CC- and heteronuclear CN-TOCSY steps via the intervening C4 nucleus to the N3/N4 nuclei, and then by a reverse INEPT step to the imino/amino protons. High sensitivity was achieved for a 30-nucleotide RNA at loo and 25°C.

References 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

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26. 27. 28. 29. 30.

31. 32. 33. 34. 35. 36. 37. 38.

39. 40. 41. 42. 43. 44.

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J . Magn. Reson., 2002,156,282. D. Uhrin, J . Magn. Reson., 2002,159, 145. A. T. Phan and D. J. Patel, J . Biomol. N M R , 2002,23,257. H. van Ingen, M. Tessari and G. W. Vuister, J . Biomol. N M R , 2002,24, 155. K. Zangger, M. Oberer, W. Keller and H. Sterk, J . Magn. Reson., 2003,160,97. R. Riek, J. Fiaux, E. B. Bertelsen, A. L. Horwich and K. Wuthrich, J . Am. Chem. Soc., 2002, 124, 12144. K. Pervushin, B. Vogeli and A. Eletsky, J . Am. Chem. Soc., 2002,124,12898. E. Kupce and R. Freeman, J . Magn. Reson., 2003,162,300. E. Kupce and R. Freeman, J . Magn. Reson., 2003,162,158. Y. L. Xia, C. H. Arrowsmith and T. Szyperski, J . Biomol. N M R , 2002,24,41. K. Y. Ding and A. M. Gronenborn, J . Magn. Reson., 2002,156,262. K. Pervushin and A. Eletsky, J . Biomol.N M R , 2003,25,147. K. F. Hu, A. Eletsky and K. Pervushin, J . Biomol. N M R , 2003,26,69. F. Lohr, V. Katsemi, J. Hartleib, U. Gunther and H. Ruterjans, J . Biomol. N M R , 2003,25,291. D. Nietlispach, Y. Ito and E. D. Laue, J . Am. Chem. Soc., 2002,124,11199. P. Permi, J . Biomol. N M R , 2002,23,201. C . Hilty, C. Fernandez, G. Wider and K. Wuthrich, J . BiomoE. N M R , 2002,23,289. V. Tugarinov and L. E. Kay, J . Am. Chem. Soc., 2003,125,5701. W. Kwiatkowski and R. Riek, J . Biomol.N M R , 2003,25,281. J. Wohnert, M. Gorlach and H. Schwalbe, J . Biomol. N M R , 2003,26,79.

9 NMR of Proteins and Nucleic Acids BY S. J. MAlTHEWS

1

Introduction

The application of nuclear magnetic resonance (NMR) to proteins and nucleic acids has maintained solid growth, with deuteration, transverse relaxation optimised spectroscopy (TROSY), liquid crystal NMR and studies on membrane bound systems featuring in high impact articles. Recent developments in protein expression technologies, x-ray crystallography and NMR spectroscopy have provided the essential elements for the high-throughput structure determination platforms necessary for structural genomics. A number of articles summarize the role of NMR in this emerging The focus of most hypothesis-driven biological NMR is the detailed study of macromolecular complexes, which is reflected within this chapter.

2

Application and Development of New Methodology

2.1 Automated Analysis. - Current interests in structural genomics, and the associated need for reliable, increased-throughput structure determination methods, offer an new opportunity for NMR methodology development. The time required for structure determination by traditional NMR methods is currently long, but improved hardware, automation of analysis, and new sources of data such as residual dipolar couplings promise to change this. Manual sequential assignment is time-consuming and is a necessary first step in high-resolution NMR studies of proteins. Novel algorithms are being presented for the automated sequential assignment of protein resonances. Hyberts and Wagner has developed a tool for the computer-assisted assignments of protein NMR spectra from triple resonance data. The program is designed to resemble established manual assignment procedures as closely as possible, where the operator has continuous visual and accounting control over the assignment procedure. IBIS achieves complete assignments for those residues that exhibit sequential triple resonance connectivities within a few hours or days.' Andrec and Levy have explored the computational complexity of the sequential assignment problem using only 13Cachemical shift connectivity information, all of which can potentially be obtained from a single three-dimensional NMR spectrum. They showed Nuclear Magnetic Resonance, Volume 33 0 The Royal Society of Chemistry, 2004

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9: N M R of Proteins and Nucleic Acids

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that a straightforward combinatorial search algorithm can be used to find correct and unambiguous sequential assignments in a reasonable amount of CPU time for small proteins? Although the development of such automated approaches for sequential assignment has greatly assisted this process, the performance of these programs is usually less satisfactory for large proteins, especially in the cases of missing connectivity or severe chemical shift degeneracy. Coggins and Zhou have reported the development of a novel computerassisted method for sequential assignment, using an algorithm that conducts an exhaustive search of all spin systems both for establishing sequential connectivities and then for assignment. The efficiency and robustness of this approach was tested with 27 proteins of sizes varying from 76 amino acids to 723 amino acids, and with data of varying qualities." TATAPRO (Tracked Automated Assignments in Proteins) is a another new algorithm for automated NMR assignments in proteins." Complementing these developments is Xia et al. description of a novel suite of projected 4D triple-resonance NMR experiments for efficient sequential assignment.' NMR chemical shifts have been used for decades to predict protein secondary structures. Hung and Samudrala have developed a highly accurate and automated method of assigning secondary structure from NMR data, which is a useful intermediate step in the determination of tertiary structures. The method combines information from chemical shifts and protein sequence using three layers of neural networks. The automation, increased accuracy, absence of gross errors, and robustness make PsiCSI ideal for high-throughput applications, and should improve the effectiveness of hybrid NMR/de novo structure determination metho~is.'~ Modern protein NMR spectroscopy has a rapidly growing need for a local archival system of raw experimental NMR data. SPINS (Standardized ProteIn NMR Storage) is a new object-oriented relational database that provides facilities for high-volume NMR data archival, organization of analyses, and dissemination of result~.'~ Automated assignment of nuclear Overhauser effect (NOESY) spectra is a prerequisite for automated structure determination of biological macromolecules. With the program KNOWNOE Gronwald et al. have presented a novel, knowledge based a p p r ~ a c h . KNOWNOE '~ is devised to work directly with the experimental spectra without interference of an expert. KNOWNOE was tested on NOESY spectra of a medium size protein, the cold shock protein (TmCsp) from Thermotoga maritima. The results show that a high quality structure of this protein can be obtained by automated assignment of NOESY spectra, which is as good as the structure obtained from manual data evaluation. Herrmann et al. have developed novel algorithms for automated NOESY peak picking and NOE signal identification in 2D and 3D NOESY spectra during de novo protein structure determination.I6ATNOS (automated NOESY peak picking) performs multiple cycles of NOE peak identification in concert with automated NOE assignment with the software CANDID and protein structure calculation with the program DYANA. The ATNOS procedure has been validated with experimental NMR data sets of three proteins, for which highquality NMR structures had previously been obtained by interactive interpreta-

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tion of the NOESY spectra. Gutmanas et al. have applied three-way decomposition to the structural analysis of a complete 3D NOESY spectra in MUNIN.17 MUNIN provided an initial characterization of the complete spectrum of azurin by 355 components. This is followed by post-processing yielded a final list of 149 components, 123 of which characterize 1859 NOE peaks from backbone N-H groups. Components from three-way decomposition are defined as direct products of one-dimensional shapes along the three dimensions. Thus, a complete set of distance constraints from this spectrum can be obtained by one-dimensional peak picking of the shapes along the NOE dimen~ion.'~ A general-purpose Monte Carlo assignment program has been developed by Hitchens et al. to aid in the assignment of NMR resonances from proteins.'* The program can also use known assignments from one form of a protein to facilitate the assignment of another form of the protein. This attribute is useful for assigning protein-ligand complexes when the assignments of the unliganded protein are known. The program can be also be used as an interactive research tool to assist in the choice of additional experimental data to facilitate completion of assignments. Determination of very precise and accurate protein structures by NMR generally requires weeks or even months to acquire and interpret all the necessary NMR data. However, even medium-accuracy fold information can often provide clues regarding protein evolution and biochemical function. Zheng et al. demonstrate an automated strategy for rapid determination of medium-accuracy protein backbone structures. Their strategy derives from using deuterated, 13C, "N-enriched protein samples with selective protonation of side-chain methyl groups. Assignments and constraints are determined automatically by the programs AutoAssign and Autostructure. The strategy was illustrated by automatic analysis of a medium accuracy structure of the Z domain of Staphylococcal protein A in a few days.19 In contrast, for homology modelling, structure-based drug design and functional genomics, it is important that the structure is reliable and accurate. Spronk et al. have presented a refinement protocol that improves the quality of protein structures determined by NMRspectroscopy to the level of those determined by high resolution X-ray crystallography in terms of local geometry.20Furthermore, the description of the nonbonded contact terms used in simulated annealing refinement can have a major impact on nucleic acid structures generated from NMR data. Clore & Kuszewski have demonstrated that substantial improvements in coordinate accuracy of NMR structures of RNA can be obtained by making use of two conformational database potentials of mean force. The application of these two potentials is illustrated for the structure refinement of an RNA aptamer/theophylline complex for which extensive NOE and residual dipolar coupling data have been measured in The structure determination of protein-protein complexes is a rather tedious and lengthy process, by both NMR and X-ray crystallography. Furthermore, rapid, accurate structure determination of protein-ligand complexes is an essential component in structure-based drug design. McCoy and Wyss have developed a method that uses NMR protein chemical shift perturbations to spatially localize a ligand when it is complexed with a protein.22Chemical shift perturba-


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tions on the protein arise primarily from the close proximity of electron current density from the ligand. In their approach the location of the center of the electron current density for a ligand aromatic ring was approximated by a point-dipole, and dot densities were used to represent ligand positions that are allowed by the experimental data. The dot density is increased in the region of space that is consistent for the most data. A surface can be formed in regions of the highest dot density that correlates to the center of the ligand aromatic ring, which allows for the rapid evaluation of ligand binding. Dominguez et al. have presented an approach called HADDOCK (High Ambiguity Driven protein-protein Docking) that makes use of biochemical and/or biophysical interaction data such as chemical shift perturbation data or mutagenesis data.23This information is introduced as Ambiguous Interaction Restraints (AIRS) to drive the docking process. An AIR is defined as an ambiguous distance between all residues shown to be involved in the interaction. The accuracy of the approach was demonstrated with three molecular complexes. The best structures generated by HADDOCK were the closest to the published structure of the respective complexes (within 2.0 Angstrom backbone RMSD). Clore and Schwieters have presented another simple and reliable method for docking protein-protein complexes using chemical shift mapping and backbone residual dipolar couplings and have illustrated this with three complexes (EINHPr, IIA(G1c)-HPr, and IIA(Mtl)-HPr).24Drug discovery procedures based on NMR typically require the analysis of thousands of NMR ~pectra.~’For example, in ‘SAR by NMR’, two-dimensional NMR spectra are recorded for a target protein mixed with ligand candidates from a comprehensive library of small molecules and are compared to the corresponding spectrum for the protein alone. Damberg et al. have described an automated procedure for the comparative analysis of large sets of heteronuclear single quantum coherence spectra, which is based on three-way decomposition and implemented as the software package MUNIN. In a single step, spectra with differences in the peak positions (indicating ligand binding) and the affected peaks are identified. By omission of peak picking, ad hoc scoring of the quality of doubtful peaks is avoided?6 2.2 Dipolar Couplings. - Partial alignment of biomolecules in solution has added a new dimension to structural investigation by high-resolution NMR methods, particularly in circumstances where distance information, obtainable from NOE data, is sparse. The resulting residual dipolar interactions are very precise reporters on the average orientation of bonds relative to the molecular alignment frame, and they can be used in a variety of ways to enrich our understanding of protein structure and function. Applications to date have focused primarily on validation of structures, determined by NMR, X-ray crystallography, or homology modeling, and on refinement of structures determined by conventional NMR approaches. Although de nouo structure determination on the basis of dipolar couplings suffers from the degeneracy of dipolar coupling a number of approaches can address this problem and can accelerate the NMR structure determination process.27The recent availability of residual dipolar coupling measurements in a variety of different alignment media raises the

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question to what extent biomolecular structure and dynamics are differentially affected by their presence. A computational method has been presented that allows the sensitive assessment of such changes using dipolar couplings measured in six or more alignment media.28Tolman has presented an approach that allows many of these difficulties to be circumvented when data are acquired in multiple alignment media. The method allows the simultaneous extraction of both structural and dynamic information directly from the residual dipolar coupling (RDC) data even in the complete absence of prior structural knowledge.29 A number of new strategies and methods for utilising RDCs have been presented this year. Mattinen et aE. have described a new principle in constructing molecular complexes from domain structures, NMR data and small-angle x-ray scattering (SAXS) measurement^.^^ Structure of calmodulin in complex with trifluoperazine was built from N- and C-terminal domains oriented based on residual dipolar couplings measured by NMR in a dilute liquid crystal, and the overall shape of the complex was derived from SAXS data. The complex built by this method was found to be consistent with the known crystal structure. Gaponenko et al. have demonstrated a novel methodology to disrupt the symmetry in the NMR spectra of h o m o d i m e r ~ A .~~ paramagnetic probe is introduced sub-stoichiometrically to create an asymmetric system with the paramagnetic probe residing on one monomer within the dimer. This creates sufficient magnetic anisotropy for resolution of symmetry-related overlapped resonances and, consequently, detection of pseudocontact shifts and residual dipolar couplings specific to each monomeric component. These pseudocontact shifts can be readily incorporated into existing structure refinement calculations and enable determination of monomer orientation within the dimeric protein. Furthermore, Bertini et al. has analysed the benefits of using paramagnetism-based constraints for the solution structure determination of metal-containing proteins.32 RDCs from partially aligned molecules provide long-range structural data and are thus particularly well adapted to rapid structure validation or protein fold recognition. Extensive measurements in two alignment media can provide precise de novo structural information from RDCs alone. Beraud et al. have applied a novel combination of these approaches to the study of methionine sulfoxide reductase (MsrA) from Erwinia chtysanthemi. In contrast to the structure of MsrA from E . coli, the reactive side chain of Cys200 is oriented toward the interior of the molecule and therefore closer to the catalytic Cys53, obviating the need for previously proposed conformational reorganization prior to formation of this disulfide intermediate.33Moreover, Giesen et d.have reported the determination of the global fold of human ubiquitin using protein backbone NMR residual dipolar couplings and space long-range NOE data.34Specifically, from three backbone residual dipolar couplings per residue in two tensor frames and backbone HN-HN NOES, a global fold of ubiquitin could be derived with a backbone root-mean-square deviation of 1.4 Angstrom with respect to the crystal structure. Improved measurement methods for dipolar couplings are frequently being reported. Wienk et al. have described a quantitative J-correlation pulse sequence


33 1

that allows simultaneous determination of one-bond and two-bond nitrogencarbon coupling constants for protonated or deuterated proteins. Coupling constants are calculated from volume ratios between cross peaks and reference axial peaks observed in a single 3D spectrum.35Ding and Gronenborn have presented a novel E.COSY-type HSQC experiments for the accurate measurement of one-bond 15N-'H and''N-l3C' and two-bond13C'-'H residual dipolar couplings in protein^?^ Martin-Pastor et al. have proposed 2D-HSQC-carbon selective/proton selective-constant time COSY experiment for the measurement of proton-proton dipolar couplings in crowded 2D spectra of 13C labelled oligosaccharides and oligonucleotides. In addition, they also describe a heteronuclear constant time-COSY experiment for the measurement of one bond carbon-carbon RDC in these m01ecules.~'Sibille and co-workers have described new experiments for the measurement of proton-proton dipolar couplings in CHzDmethyl groups ofl3Clabeled, highly deuterated proteins.38To illustate the accuracy of structural information, the measured RDCs for Valine residues were analysed in terms of torsion angles and stereospecific assignment of the prochiral methyl groups. On the basis of the previously determined backbone solution structure of this protein, the methyl RDC data proved sufficient to determine the chi(1) torsion angles in seven out of nine valines. Methyl RDCs should assist in the determination of side chain conformation in high molecular weight systems. Although reports on new alignment media have dwindled, Riley et al. have presented the magnetic alignment of the Pseudomonas bacteriophage Pfl captured indefinitely in a gel of the aqueous triblock copolymer Pluronic F-127.39In addition to preserving high-resolution NMR spectra for dissolved solutes, the gel prevents the reorientation of the phage allowing mechanical manipulation of the angle between the axis of the phage alignment and the static magnetic field. Furthermore, the compatibility of Pluronic F-127 with NMR studies of aqueous biological macromolecules was demonstrated. RDCs are particular useful in cases where long range information is sparse or problematic to measure, for example in structural studies of complexes, multi domain proteins and partially folded systems. A number of notable examples are listed here. Ma1 et al. have presented a RDC-based approach for characterizing the molecular recognition of Calmodulin (CaM) with five different CaM-regulated kinases.4' Their analyses indicated that CaM kinase I and IV use the same CaM binding mode as the myosin light chain kinase (1-14 motif), which is distinct from those of CaM kinase 11 (1-10 motif) and CaM kinase (1-16 motif). Jain et al. have investigated the ligand-binding properties of a 53 kDa homomultimeric trimer from mannose-binding protein (MBP) using RDCs:l Using a limited set of backbone amide NMR assignments and orientational information, an order tensor for MBP was determined that is consistent with symmetry-based predictions of an axially symmetric ~ystem.'~C-'H couplings for a bound trisaccharide ligand, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (trimannoside) were determined at natural abundance and allowed docking in the binding site of MBP to produce a structural model for MBP-oligosaccharide interactions. RDCs have also played a central role in the solution structures for complexes involved in the E. coli phosphotransferase

332


system. Namely, enzyme IIA(G1ucose) with the cytoplasmic B domain (IIBGlc) of the glucose transporter IICBGlc and the A domain of the mannitol transporter IIMannitol with the histidine-containing phosphocarrier pr0tein.4~3~~ Many important proteins perform their physiological functions under allosteric control, whereby the binding of a ligand at a specific site influences the binding affinity at a different site. Lukin et al. have demonstrated that the quaternary structure of tetrameric human normal adult carbonmonoxy-hemoglobin can readily be determined in solution at near-physiological conditions of pH, ionic strength, and temperature using amide R D C S .The ~ structure is found to be a dynamic intermediate between two previously solved crystal structures, known as the R and R2. Varadan et al. have utlised RDC data to characterise the solution conformations of K48-linked diubiquitin and tetraubiquiti11.4~Their data clearly indicate a switch in the conformation of diubiquitin, from open to closed, with increasing pH. The closed conformation features a well-defined interface that is related to, but distinguishable from, that observed in the diubiquitin crystal structure. This interface is dynamic in solution, such that important hydrophobic residues that are sequestered at the interface in the closed conformation may be accessible for direct interactions with recognition factors. Wiesner et al. have used RDC to accurately define the relative orientation of the WW domains within the pre-mRNA processing protein 40. They revealed that the binding pockets of each domain face opposite sides of the Jacobs et al. have used a similar approach to investigate the influence of target peptides on domain interactions between the WW and catalytic domains in Pin1,a peptidyl-prolyl cis/trans isomerase essential for cell cycle regulat i ~ n . Data ~ ’ indicate that Pin1 can either behave as two independent domains connected by the flexible linker or as a single intact moiety with some hinge bending motion depending on the sequence of the bound peptide. Dvoretsky et al. have calculated the relative spatial orientation of Cardiac troponin C domains bound to cardiac troponin I measured RDCs and long-range distance restraints utilizing a rigid body molecular dynamics protocol. The relative domain orientation reveals that hydrophobic pockets face each other. The structure successfully explains the observed functional consequences of linker region deletion mutants. Together, these studies suggest that, although linker plasticity is important, the ability of Cardiac troponin C to function in muscle contraction can be correlated with a preferred domain orientation and interdomain distance.48 This year, several NMR reports have shown that long-range information provided by RDCs significantly improve the global structure definition of RNAs, DNAs and oligosaccharides. Mauffret et al. have performed simulations aimed at evaluating the impact of RDCs on the structure definition of long oligonucl e o t i d e ~ ?They ~ confirmed the strong impact of RDCs on the structure determination, and found that a small set of RDC constraints detectable at the natural abundance is sufficient to accurately derive the global and local DNA duplex structures when used in conjunction with NOE-distances. McCallum and Pardi have compared several approaches for estimating alignment tensor parameters and incorporating RDCs into RNA structure determinations. Both the local and


333

global structure of the iron-responsive element (IRE) are improved significantly by refinement with RDCs. The RDC refinements also provide insight on the conformational dynamics of the IRE and highlight important issues that need to be addressed when incorporating RDCs.” Vallurupalli and Moore have demonstrated how to use 3D HCCH-COSY and Relay HCCH- COSY to measure one-bond H2’-C2’ and H3’-C3’ couplings for a 42 nucleotide RNA.” Wu et al. have determined the solution structure of d(CGCGAATTCGCG)(2) based on an exceptionally large set of RDCs, including new ‘H-’H and 13P-lH dipolar restraints. High quality DNA structures were obtained solely on the basis of these new restraints, and the structures are in close agreement with those calculated p r e v i ~ u s l yBondensgaard .~~ et al. have demonstrated the use of RDCs in determining the global structure of the hammerhead ribozyme in the absence of Mg2+ and show that it is not consistent with the Y-shaped conformation observed in crystal s t r u c t ~ r e s Vallurupalli .~~ and Moore have obtained the structure for the loop E region of the 5s rRNA from Spinacia oleracia chloroplast ribosomes using residual dipolar coupling data in addition to NOE, J coupling and chemical shift i n f ~ r m a t i o nLawrence .~~ et al. have refined the structure of an engineered form of SL1, a stem-loop from the Psi-RNA packaging signal of the human immunodeficiency virus, using ‘H-13Cresidual dipolar couplings.55Barbic et al. have used RDCs to investigate the disputed origins of DNA curvature. The global bend magnitude and direction revealed by RDCs are in excellent agreement with the junction model parameters used to rationalize gel electrophoretic data and with preliminary results of cyclization Al-Hashimi et al. have demonstrated that RDC-NMR methodology can provide new insights into the effects of Mg2+ on both the conformation and dynamics of RNA.s7 Umemoto et al. have presented a new methodology for analysing the carbohydrate recognition of Galectin-3, a lectin known to bind galactose containing oligosaccharides in mammalian systems.58 The approach is based on the measurement of residual dipolar couplings from NMR spectra in the presence and absence of ligand. The data on the ligand are consistent with the ligand binding geometry found in a crystal structure of the complexed state. However, a significant rearrangement of backbone loops near the binding site appears to occur in the absence of ligand. 2.3 Transverse Relaxation Optimized Spectroscopy (TROSY). - TROSYbased NMR techniques are for solution studies of large molecular and supramolecular structures > 100 kDa, significantly extending the range of macromolecular systems that can be studied by NMR in s o l ~ t i o nAn . ~ alternative ~~~~ approach that does not rely on TROSY methodology is to use standard tripleresonance and isotope-edited-NOE methods together with encapsulation of a protein in a surfactant shell, which is subsequently solubilized in a low viscosity solvent.61 New pulse sequences incorporating TROSY type magnetization transfer have been introduced that assist in making resonance assignments. Most notably, Riek et al. have demonstrated the use of TROSY in combination with either with

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cross-correlated relaxation-induced polarization transfer (CRIPT) or cross-correlated relaxation-enhanced polarization transfer (CRINEPT) to obtain NMR correlation spectra of macromolecules with molecular weights from 110 to 800 kDa.62Pervushin et al. have presented a general method to enhance the sensitivity of the multidimensional NMR experiments performed at high-polarizing magnetic field via the reduction of longitudinal proton relaxation times. The method is based on the use of two vast pools of 'thermal bath' 'H spins residing on proton-bound carbon and oxygen atoms to uniformly enhance longitudinal relaxation of the 'HN spins. The proposed longitudinal relaxation optimization is implemented in the 2 D [I5N-'H]-LTROSY, 2D [15N-'H]-LHSQC and 3D LTROSY-HNCA experiments yielding the factor 2-2.5 increase in signal-tonoise ratio at 600 MHz and more at high field strengthd3The same group have introduced the TROSY principle into an HNCA experiment, which is designed for measurements of the intraresidual and sequential HCa/HN dipole/dipole and HCa/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the 3,4JHaHNcoupling constants measured in an E. COSY manner.64They also have proposed a new method for backbone resonance assignment in large proteins at natural 'H abundance using a combination of the most sensitive TROSY-type triple-resonance e~periments.6~7~~ These techniques include TROSY-HNCO, 13C'-detected3 D multiple-quantum HACACO and the newly developed 3D TROSY multiple-quantum-HN(CA)HA and 4D TROSY multiple-quantum-HACANH experiments. Hilty et al. have devised novel experiments, 3D (H)C(CC)-TOCSY-(CO)-["N'HI- TROSY and 3D H(C)(CC)-TOCSY-(CO)-["N-'H]-TROSY, for the assignment of side chain methyl resonances of Val, Leu and Ile in large, specifically labelled proteins.67They demonstrate these on the outer membrane protein X (OmpX) from E. coli reconstituted in 60 kDa micelles in aqueous solution. Complete assignments of the proton and carbon chemical shifts were obtained for all isopropyl methyl groups of Val and Leu, as well as for the delta(1)-methyls of Ile. The approach is applicable for soluble proteins or micelle-reconstituted membrane proteins in structures with overall molecular weights up to 100 kDa. The sequential assignment of backbone resonances in larger proteins can be achieved by recording two or more complementary triple-resonance NMR spectra of deuterated proteins. For such proteins, higher fields and experiments based on the TROSY method provide the needed resolution and sensitivity. However, increasingly rapid carbonyl relaxation at the high magnetic field strengths renders assignment strategies based on sequential HN(C0)CA-type experiments much less efficient for proteins >40 kDa. Nietlispach et al. have presented two complementary new experiments, which allow backbone assignments with good sensitivity for larger deuterated proteins.6*A 3D intra-HNCA experiment provides uniquely the intraresidue connection, while a 3D DQHNCA experiment, which detects a13C,-'3Ci.lalphadouble-quantum (DQ)coherence, contains the sequential information. The application of TROSY-based techniques to genuine biological questions is becoming increasingly more frequent and the following papers represent a few highlights. McElroy et al. have used TROSY-based NMR experiments to study


335

the mechanism of ligand-mediated allosteric regulation in the 90.6 kDa 11-mer trp RNA-binding attenuation protein Tryptophan binding was found to be temperature-dependent and modulated protein flexibility, indicating a central role in TRAP function by altering RNA-binding affinity. The molecular chaperone Hsp90 sequesters oncogenic mutants of the tumor suppressor p53 that have unstable core domains. It is not known whether p53 is bound in an unfolded, partly folded, or distorted structure. Rudiger et al. have shown by TROSY NMR spectroscopy combined with cross-correlated relaxation-enhanced polarization transfer (CRINEPT-TROSY) that p53 core domain bound in an unfolded state to form an approximately 200-kDa complex with H~p90.~' This mode of binding might be a general feature of substrates of Hsp90. Tugarinov et al. have demonstrated a 4D TROSY-NMR study of Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa). Virtually complete backbone chemical shift assignments of this largely alpha-helical protein were reported.71 The functional interactions between the various components of the respiratory chain are relatively short-lived, thus allowing high turnover numbers but at the same time complicating the structural analysis of the complexes. Chemical shift mapping by NMR spectroscopy is a useful tool to investigate such transient contacts, since it can monitor changes in the electron-shielding properties of a protein as the result of temporary contacts with a reaction partner. Wienk et al. have used TROSY to investigate the molecular interaction between two components of the electron-transfer chain from Paracoccus denitr$cans: the engineered, water-soluble fragment of cytochrome C-552 and the Cu-A domain from the .~~ shift changes for the backbone amide groups cytochrome c ~ x i d a s eChemical were identified in several uncharged residues located around the exposed heme edge in cytochrome C-552. Yuan et al. have used TROSY-CPMG experiments to investigate conformational exchange in the Trp residues of human normal adult haemoglobin and the three mutants.73 The results indicated that the side-chain of beta37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. 3

Macromolecular Structures

In this section I have chosen not to describe the solution structures of free proteins, as such publications have increasingly modest impact. Structures of macromolecular complexes are considerably more informative from a biological perspective and this year has seen many impressive studies. 3.1 Membrane Proteins. - New and refined methods for NMR structural studies on membrane proteins and peptides are become more abundant. The following section summaries the recent developments both in the solid and solution state. The determination of a representative set of protein structures is a chief aim in structural genomics. Solid-state NMR may have a crucial role in

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structural investigations of those proteins that do not easily form crystals or are not accessible to solution NMR, such as amyloid systems or membrane proteins. Castellani et al. have demonstrated a complete structure determination of a micro-crystalline preparation of the alpha-spectrin Src-homology 3 (SH3) domain by solid-state magic-angle-spinning (MAS) NMR.74375 Distance restraints were derived from proton-driven spin diffusion (PDSD) spectra of biosynthetically site-directed, labelled samples obtained from bacteria grown using [1,3l3C] glycerol or [2-"C] glycerol as carbon sources. This allowed the observation of long-range distance correlations up to 7 Angstrom. This MAS NMR procedure should be widely applicable to small membrane proteins that can be expressed in b a ~ t e r i a .Petkova ~ ~ ? ~ ~et al. have presented new backbone and side chain assignment strategies for multiply labeled membrane peptides and proteins in the solid They have demonstrated that the SPECIFIC C P technique can be used to obtain heteronuclear correlation spectra of peptide backbones with greater efficiency than conventional methods. The methods is demonstrated on a 248-residue membrane protein bacteriorhodopsin (bR). Marassi and Opella have described a different solid-state NMR approach for simultaneous resonance assignment and three-dimensional structure determination of a membrane protein in lipid b i l a y e r ~ .A~ ~total of five 2D'H-l5N PISEMA (polarization inversion spin exchange at the magic angle) spectra, from one uniformly and four selectively.'5N-labeledsamples, were sufficient to determine the structure of the membrane-bound form of the major pVIII coat protein of fd filamentous bacteriophage. Pisa (polarity index slat angle) wheels are an essential element in this process, which starts with the simultaneous assignment of resonances and the assembly of isolated polypeptide segments, and culminates in the complete 3D structure of the protein at atomic resolution. Tian et al. have used'H-l5N - dipolar/15Nchemical shift correlation experiments to characterise the tilt angle of a transmembrane helix in M2 reconstituted in DMPC/DMPG liposomes. In addition, the spectra suggest that the tetrameric protein forms a symmetric bundle consistent with literature data.78Wienk et al. have critically investigated the effect of time and spatial averaging on PISEMA spectra of alpha-helical membrane The analysis showed that under certain motional modes the"N chemical shift/'H-''N dipolar pairs for each of the residues do not form patterns that mimic helical wheel patterns. As a result, the unambiguous identification of helix tilt and helix rotation without any resonance assignments or on the basis of a single assignment may be difficult. Lee et al. have demonstrated that three solution NMR experiments on a unif0rm1y'~N labeled helical membrane protein in micelles provide sufficient information to describe its structure, topology, and dynamics.79The backbone amide resonances were assigned with an HMQC-NOESY experiment and the backbone dynamics characterized by the'H-''N heteronuclear NOE, which clearly distinguishes between the structured helical residues and the more mobile residues in the terminal and inter-helical loop regions of the protein. The structure and topology of the helices are described by Dipolar waves and PISA wheels derived from experimental measurements of RDCs and residual chemical shift anisotropies (RCSAs). The results show that the membrane-bound form of


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Pfl coat protein has a 20-residue trans-membrane hydrophobic helix with an orientation that differs by about 90 degrees from that of an 8-residue amphipathic helix. Fernandez et al. have provided a detailed description proteindetergent interactions using NOE contacts between the integral outer membrane protein OmpX and dihexanoylphosphaticlylcholine (DHPC) in aqueous solution.80The NOEs were measured in standard 3D”N and’3C-resolvedNOESY OmpX spectra recorded with selectively methyl-pr0tonated,2H,~~C,~~N-labeled in micelles of DHPC. The OmpX surface area covered with intermolecular NOEs to the DHPC hydrophobic tails forms a continuous cylinder jacket of approximately 28 Angstrom in height, which is centered about the middle of the long axis through the beta-barrel. In addition, some intermolecular NOEs with methyl groups of the DHPC polar head were identified along both boundaries of this cylinder jacket. The experimental data suggest that the hydrophobic surface areas of OmpX are covered with a monolayer of DHPC molecules, which appears to mimic quite faithfully the embedding of the beta-barrel in a doublelayer lipid membrane. Li et d. have characterized a membrane protein containing residues P688-T762 of the integrin beta3 subunit, encompassing its transmembrane and cytoplasmic domains in dodecylphosphocholine micelles.81An amino-terminal helix corresponding to the beta3 transmembrane helix extends into the membrane-proximal region of the cytoplasmic domain. In the presence of membrane-mimicking detergents, the cytoplasmic domain takes on a turnhelix configuration characteristic of the immunoreceptor tyrosine-based activation motif. These structural features of the beta3 subunit may explain its interaction with numerous cytosolic interacting proteins and begin to illuminate the mechanism of integrin activation. RDCs for a ligand that is in fast exchange between a free state and a state where it is bound to a macroscopically ordered membrane protein carry precise information on the structure and orientation of the bound ligand. The couplings originate in the bound state but can be detected on the free ligand using standard high resolution NMR. This ‘transferred-RDC’ approach has been used to study an analog of the C-terminal undecapeptide of the alpha-subunit of the heterotrimeric G protein transducin when bound to photo-activated rhodopsin.82 Rhodopsin is the major constituent of disk-shaped membrane vesicles from rod outer segments of bovine retinas, which align spontaneously in the NMR magnet. Photo-activation of rhodopsin triggers transient binding of the peptide, resulting in measurable dipolar contributions tolJNHand’JCHsplittings. These dipolar couplings report on the time-averaged orientation of bond vectors in the bound peptide relative to the magnetic field, i.e. relative to the membrane normal. Approximate distance restraints of the bound conformation were derived from transferred NOEs, as measured from the difference of NOESY spectra recorded prior to and after photo-activation. The N-terminal eight residues of the bound undecapeptide adopt a near-ideal alpha-helical conformation. The helix is terminated by an alpha(L) type C-cap, with Gly9 at the C’ position in the center of the reverse turn. The angle between the helix axis and the membrane normal is 40°C. Peptide protons that make close contact with the receptor are identified by analysis of the NOESY cross-relaxation pattern and include the

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hydrophobic C terminus of the peptide.82 N-terminal myristoylation of the immunoglobulin-binding domain of protein G (GB1)from group G Streptococcus provides the means to bind the protein to aligned phospholipid bilayers for solid-state NMR structural studies. The myristoylated protein is immobilized by its interactions with bilayers, and the sample alignment enables orientationally dependent15N chemical shifts and 'H-15Ndipolar couplings to be measured. Spectra calculated for the average solution NMR structure of the protein at various orientations with respect to the magnetic field direction were compared to the experimental The best fit identified the orientation of the myristoylated protein on the lipid bilayers, and demonstrated that the protein adopts a similar structure in both its myristoylated and non-myristoylated forms, and that the structure is not grossly distorted by its interaction with the phosholipid bilayer surface or by its location in the restricted aqueous space between bilayer leaflets. The protein is oriented such that its charged sides face the phosphatidylcholine headgroups of the lipids with the single amphiphilic helix running parallel to the bilayer surface. 3.2 Protein Complexes. - Missense mutants that cause the immune disorder Wiskott-Aldrich Syndrome (WAS) map primarily to the Enabled/VASP homology 1 (EVHl) domain of the actin regulatory protein WASP. This domain has been implicated in both peptide and phospholipid binding. Volkman et al. have shown that the N-WASP EVHl domain does not bind phosphatidyl inositol(4,5)-bisphosphate, as previously reported, but does specifically bind a 25 residue motif from the WASP Interacting Protein (WIP).84The NMR structure of this complex reveals a novel mode of recognition of the WIP ligand, which wraps around the domain, contacting a narrow but extended surface. This recognition mechanism provides a basis for understanding the effects of mutations that cause WAS. Monoubiquitination serves as a regulatory signal in a variety of cellular processes. Monoubiquitin signals are transmitted by binding to a small but rapidly expanding class of ubiquitin binding motifs. Several of these motifs, including the CUE domain, also promote intramolecular monoubiquitination. The solution structure of a CUE domain of the yeast Cue2 protein in complex with ubiquitin reveals intermolecular interactions involving conserved hydrophobic surfaces.85The results suggests an occlusion mechanism for inhibiting polyubiquitin chain formation during monoubiquitin signaling. The CUE domain shares a similar overall architecture with the UBA domain, which also contains a conserved hydrophobic patch. Comparative modeling suggests that the UBA domain interacts analogously with ubiquitin. The structure of the CUE-ubiquitin complex may thus serve as a paradigm for ubiquitin recognition and signaling by ubiquitin binding proteins. Intracellular protein interaction domains are essential for eukaryotic signaling. In T cells, the CD2BP2 adaptor binds two membrane-proximal proline-rich motifs in the CD2 cytoplasmic tail via its GYF domain, thereby regulating interleukin-2 production. Freund et al. have presented the structure of the GYF domain in complex with a CD2 tail peptide.86Unlike SH3 domains, which use


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two surface pockets to accommodate proline residues, the GYF domain employs conserved hydrophobic residues to create a single interaction surface. NMR analysis shows that the Fyn, but not the Lck tyrosine kinase SH3 domain, competes with CD2BP2 GYF-domain binding to the same CD2 proline-rich sequence. L M 0 2 and L M 0 4 are members of a small family of nuclear transcriptional regulators that are important for both normal development and disease processes. L M 0 2 is essential for hemopoiesis and angiogenesis, and inappropriate overexpression of this protein leads to T-cell leukemias. L M 0 4 is developmentally regulated in the mammary gland and has been implicated in breast oncogenesis. Both proteins comprise two tandemly repeated LIM domains. L M 0 2 and L M 0 4 interact with the ubiquitous nuclear adaptor protein ldbl/NLI/CLIM2, which associates with the LIM domains of LMO and LIM homeodomain proteins via its LIM interaction domain (ldbl-LID).Deane et al. have reported the solution structures of two LM0:ldbl complexes and show that ldbl-LID binds to the N-terminal LIM domain (LIM1) of L M 0 2 and L M 0 4 in an extended conformation, contributing a third strand to a betahairpin in LIMl A mechanism was proposed by which ldbl can bind a variety of LIM domains that share low sequence homology. Pro-survival Bcl-2-related proteins, critical regulators of apoptosis, contain a hydrophobic groove targeted for binding by the BH3 domain of the proapoptotic BH3-only proteins. The solution structure of the pro-survival protein Bcl-w reveals that the binding groove is not freely accessible as predicted by previous structures of pro-survival Bcl-2-like molecules.88 Unexpectedly, the groove appears to be occluded by the C-terminal residues. Binding and kinetic data suggest that the C-terminal residues of Bcl-w and Bcl-x(L) modulate prosurvival activity by regulating ligand access to the groove. Binding of the BH3-only proteins, critical for cell death initiation, is likely to displace the hydrophobic C-terminal region of Bcl-w and Bcl-x(L). The Alzheimer-linked neural protein SlOOB is a signaling molecule shown to control the assembly of intermediate filament proteins in a calcium-sensitive manner. Upon binding calcium, a conformational change occurs in SlOOB exposing a hydrophobic surface for target protein interactions. The synthetic peptide TRTK- 12 (TRTKIDWNKILS), derived from random bacteriophage fibrary screening, bears sequence similarity to several intermediate filament proteins. McClintock and Shaw have determined the 3D structure of the Ca2+S 100B-TRTK-12 c o m p l e ~ .The ~ ~ ?structure ~~ reveals an extended, contiguous hydrophobic surface is formed on Ca2+-S1OOB for target interaction. The TRTK-12 peptide adopts a coiled structure that fits into a portion of this surface, anchored at Trp(7), and interacts with multiple hydrophobic contacts in helices 111 and IV of Ca2+-S100B.This interaction is strikingly different from the alpha-helical structures found for other SlOO target peptides. Together, these would provide a larger, more complex site to interact with full-length target proteins and would account for the promiscuity observed for SlOOB target protein interactions. Furthermore, a comparison of the S 1OOB-TRTK peptide structure to the structures of apo- and Ca2+-boundSlOOB illustrates that the

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binding site of TRTK-12 is buried in apo-SlOOB, but is exposed in Ca2+-bound ~100~.89,90 The LAP (leucine-rich repeat and PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the pl20-catenins. Skelton et al. have described the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand.” The structure of a phage-optimized peptide in complex with Erbin PDZ contributes to the growing appreciation of the five ligand side chains important in determining PDZ binding and highlight the mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome. Caltractin (centrin) is a member of the calmodulin (CaM) superfamily of EFhand calcium-binding proteins and is an essential component of the centrosomal structures. Caltractin and calmodulin apparently function in distinct calcium signaling pathways despite substantial sequence similarity. Hu and Chazin have elucidated the high-resolution 3D solution structure of the complex between the Ca2+-activated C-terminal domain of Chlamydomonas reinhardtii caltractin (CRC-C) and a 19 residue peptide fragment comprising the putative CDc3lpbinding region of K a r l p (K-19).92Formation of the complex is calcium-dependent and is stabilized by extensive interactions between CRC-C and three key hydrophobic anchors (TrplO, Leu13 and Leul4) in the peptide as well as favorable electrostatic interactions at the protein- peptide interface. Although the overall structures of CRC and CaM domains in their respective complexes are very similar, differences in critical regions in the sequences, of these proteins and their targets lead to clear differences in the complementarity of their respective binding surfaces. These subtle differences reveal the structural basis for the Ca2+-dependent regulation of distinct cellular signaling events by CRC and CaM. Staphylococcus aureus and Streptococcus pyogenes, two important human pathogens, target host fibronectin (Fn) in their adhesion to and invasion of host cells. Fibronectin-binding proteins (FnBPs), anchored in the bacterial cell wall, have multiple Fn-binding repeats in an unfolded region of the protein. The bacterium-binding site in the amino-terminal domain (1-5Fl) of Fn contains five sequential Fn type 1 (Fl) modules. Schwarz-Linek et al. have described the first structure of a streptococcal FnBP peptide (B3) in complex with the fibronectin module pair 1Fl-2Fl.93 B3 forms an additional antiparallel beta-strands on sequential F1 modules - the first example of a tandem beta- zipper. In the process of Fn-mediated invasion of host cells, these bacterial proteins appear to exploit the modular structure of F n by forming extended tandem beta-zippers. The structural proteins of HIV and Ebola display PTAP peptide motifs that recruit the human protein TsglOl to facilitate virus budding. The UEV domain of TsglOl resembles E2 ubiquitin-conjugating enzymes, and the PTAP peptide binds in a bifurcated groove above the vestigial enzyme active site. The structure reveals the molecular basis of HIV PTAP late domain function and


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represents an attractive starting point for the design of novel inhibitors of virus b~dding.9~ Upon phosphorylation of Y221 by Abelson (Abl) kinase, the Crk-I1 adapter protein undergoes an intramolecular reorganization initiated by the binding of its own Src homology 2 (SH2) domain to the pY221 site. Conformational changes induced by phosphotyrosine recognition promote the binding of the Src homology 3 (SH3) domain of the 4 N tyrosine kinase to a proline-rich loop located between the betaD and betaE strands of the SH2 domain (DE loop). Donaldson et al. have determined the NMR solution structure of the ternary complex of the Abl SH3 domain with the Crk SH2 domain bound to a Crk pY221 p h ~ s p h o p e p t i d e .The ~ ~ SH2 domain bridges two ligands that bind at distinct sites. The interaction between the AN SH3 domain and the Crk SH2 domain is localized to a canonical eight-residue site within the DE loop. The structural and dynamic data indicate that these SH2 and SH3 domains do not assume a unique orientation with respect to one another; rather, they appear to be only tethered via the DE loop. Thus, SH2 domain-SH3 domain interactions do not require additional tertiary contacts or restriction of domain orientation when a recognition motif is presented in a mobile loop. The translation initiation factors (IFs) IFl/eIFlA and IF2/eIF5B have been conserved throughout all kingdoms. Although the central roles of the bacterial factors I N and IF2 were established long ago, the importance of their eukaryotic homologs, eukaryotic IFs (elFs) elFlA and elFSB, has only recently become evident. The translation machinery in eukaryotes is more complex and accordingly, eIFlA and eIF5B seem to have acquired a number of new functions while retaining many of the roles of bacterial IF1 and IF2. IF1 and IF2 have been shown to interact on the ribosome but no binding has been detected for the free factors. In contrast, yeast eIFlA and eIF5B have been reported to interact in the absence of ribosomes. Marintchev et al. have identified the binding interface between human elFlA and the C-terminal domain of eIF5B by using solution NMR.96That interaction interface involves the C termini of the two proteins, which are not present in bacterial IF1 and 1F2. It was proposed that eIFlA and elF5B simultaneously interact with the ribosome at two sites that are >50 Angstrom apart: through their C termini and through an interface previously identified in bacterial IF1 and IF2. The essential splicing factors SF1 and U2AF play an important role in the recognition of the pre-mRNA 3’ splice site during early spliceosome assembly. The structure of the C-terminal RRM (RRM3) of human U2AF 65 complexed to an N-terminal peptide of SF1 reveals an extended negatively charged helix A and an additional helix C.97Helix C shields the potential RNA binding surface. SF1 binds to the opposite, helical face of RRM3, inserting a conserved tryptophan into a hydrophobic pocket between helices A and B in a way that strikingly resembles part of the molecular interface in the U2AF heterodimer. This molecular recognition establishes a paradigm for protein binding by a subfamily of noncanonical RRMs.

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3.3 Nucleic Acid Complexes. - Vertebrate polyadenylation sites are identified by the AAUAAA signal and by GU-rich sequences downstream of the cleavage site. These are recognized by a heterotrimeric protein complex (CstF) through its 64 kDa subunit (CstF-64); the strength of this interaction affects the efficiency of poly(A) site utilization. Canadillas and Varani have presented the structure of the RNA-binding domain of CstF-64 containing an RNA recognition motif (RRM) augmented by N- and C-terminal he lice^.^' The C-terminal helix unfolds upon RNA binding and extends into the hinge domain where interactions with factors responsible for assembly of the polyadenylation complex occur. Consecutive U repeats are required for a strong CstF-GU interaction and is was shown how UU dinucleotides are recognized. Contacts outside the UU pocket fine tune the protein-RNA interaction and provide different affinities for distinct GU-rich elements. The protein-RNA interface remains mobile, most likely a requirement to bind many GU-rich sequences and yet discriminate against other RNAs. The transcriptional regulator RcsB interacts with other coactivators to control the expression of biosynthetic operons in enterobacteria. Pristovsek et al. have reported the solution structure of the C-terminal DNA-binding domain of the RcsB protein from Erwinia a m y l ~ v o r aThe . ~ ~C-terminal domain is composed of four alpha-helices where two central helices form a helix-turn-helix motif similar to the structures of the regulatory proteins GerE, NarL and TraR. Amino acid residues involved in regulator RcsA independent DNA binding were identified by titration studies with a RcsAB box consensus fragment. The RcsAB box is specifically recognized by the RcsAB heterodimer as well as by RcsB alone and role of RcsA is not to alter the DNA binding specificity of RcsB but to stabilize RcsB-DNA complexes. In prokaryotes, the recoding of a UGA stop codon as a selenocysteine codon requires a special elongation factor (EF) SelB and a stem-loop structure within the mRNA called a selenocysteine insertion sequence (SECIS). Fourmy et al. have used NMR spectroscopy to determine the solution structure of the SECIS mRNA hairpin and characterized its interaction with the mRNA-binding domain of SelB.lOO In the free SECIS mRNA structure, conserved nucleotides are strongly exposed for recognition by SelB. Binding of the C-terminal domain of SelB stabilizes the RNA secondary structure. In the protein-RNA complex, a Watson-Crick loop base-pair leaves a GpU sequence accessible for SelB recognition. While most nuclear receptors bind DNA as homo or heterodimers, the human estrogen related receptors (hERRs) are members of a subfamily of orphan receptors that bind DNA as monomers. Gearhart et al. have determined the solution structure of the DNA binding domain (DBD) of hERR2 bound to its cognate DNA."' The structure and base interactions of the core DBD are similar to those of other nuclear receptors. However, high-affinity, sequence-specific DNA binding as a monomer necessitates formation of additional base contacts outside the core DBD. This is accomplished using a modified guanosine-binding 'AT-hook' within the C-terminal extension (CTE) flanking the DBD, which makes base-specific minor groove interactions. The structure of the CTE is stabilized both by interactions with the DNA and by packing against a region of


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the core DBD normally reserved for dimerization. This pseudo-dimer interface provides a basis for the expansion of DNA recognition and implies a mechanism through which dimerization may have evolved from an ancestral monomeric receptor. NHP6A is a non-sequence-specificDNA-binding protein from Saccharomyces cerevisiae which belongs to the HMGB protein family. Masse et al. have presented the refined solution structures of the NHP6A-DNA complex as well as the free 15 bp DNA.lo2Both the free and bound forms of the protein adopt the typical L-shaped HMGB domain fold. The DNA in the complex undergoes significant structural rearrangement from its free form while the protein shows smaller but significant conformational changes in the complex. Structural and mutational analysis as well as comparison of the complex with the free DNA provided insight into the factors that contribute to binding site selection and DNA deformations in the complex. The importance in downstream target regulation of tertiary structure and DNA binding specificity of the protein encoded by the vnd/NK-2 homeobox gene has been analysed. The Y54M mutation inactivates the ability of the vnd/NK-2 homeodomain protein to repress ind and msh. The NMR-determined tertiary structures of the Y54M vnd/NK-2 homeodomain, both free and bound to DNA, were compared with the WT ana10g.I'~The only structural difference observed for the mutant homeodomain is in the complex with DNA and involved closer interaction of the methionine-54 with A2, rather than with C3 of the (-) strand of the DNA. This subtle change in the homeodomain-DNA complex resulted in modifications of binding affinities to DNA and constitute the molecular basis for the phenotypic alterations observed on ectopic expression of the Y54M vnd/NK-2 gene during embryogenesis. The SOS response, a set of cellular phenomena exhibited by eubacteria, is initiated by various causes that include DNA damage-induced replication arrest, and is positively regulated by the co- protease activity of Reca. E . coli DinI, a LexA-regulated SOS gene product, shuts off the initiation of the SOS response when over-expressed in uiuo. Biochemical and genetic studies indicated that DinI physically interacts with RecA to inhibit its co-protease activity. Using NMR spectroscopy it has been shown that DinI tightly binds to the central region of RecA (between the N- and C-terminal domains) and that this interaction is enhanced upon the oligomerisation of RecA.lo4On the other hand, DinI did not inhibit the interaction between 4mer single-stranded (ss)DNA and RecA-ATPgammaS, but had a slight effect on the structure of ssDNA-RecA-ATPgammaS complexes. It was concluded that prevention of repressor binding to RecA protomers by DinI, with a possible conformational change induced in the DinI-bound ssDNA-RecA-ATPgammaS complex, together function to inhibit the co-protease activity of Reca. RNA plays a central role in many biological processes and is therefore an important target for drug development. In recent years an increasing wealth of structural and functional information about RNA-ligand complexes has been obtained using in uitro selected RNAs (aptamers). However, all those studies focused on structure and changes of the nucleic acid and mostly considered the

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ligand as a rigid target. Isotopically labeled ligand in complex with RNA has been analy~ed.'~' Surprisingly asymmetric changes in the13Cchemical shift of the ligand methyl groups indicate that the dye undergoes changes in its conformation and charge distribution upon binding. Results indicated that the uneven charge distribution in the RNA binding pocket provides a major contribution to the driving force of the ligand structural changes. The observation that not only the RNA adapts to the ligand, in what is called adaptive binding, but that the ligand itself also undergoes conformational changes is crucial for the rational design of RNA ligands and for understanding the properties of RNA-ligand complexes. An RNA aptamer containing two binding sites exhibits extremely high affinity to the HIV Tat protein. Matsugami et al. have determined the structure of the aptamer complexed with two argininamide molecules.'06Two adjacent U:A:U base triples were formed, which widens the major groove to make space for the two argininamide molecules. The argininamide molecules bind to the G bases through hydrogen bonds. The binding is stabilized through stacking interactions. The structure of the aptamer complexed with a Tat-derived arginine-rich peptide was also characterized. It was suggested that the aptamer structure is similar for both complexes and that the aptamer interacts with two different arginine residues of the peptide simultaneously at the two binding sites, which could explain the high affinity to Tat. Aminoglycoside antibiotics that bind to 16s ribosomal RNA in the aminoacyl-tRNA site (A site) cause misreading of the genetic code and inhibit translocation. Structures of an A site RNA oligonucleotide free in solution and bound to the aminoglycosides paromomycin or gentamicin C l a have been determined. Recently, the X-ray crystal structure of the entire 30s subunit has been determined, free and bound to paromomycin. The NMR structure of the oligonucleotide-paromomycin complex has been determined with high precision and compared with the X-ray crystal structure of the 30s subunit complex. The comparison shows the validity of both structures in identifying critical interactions that affect ribosome function.

4

Protein Relaxation and Dynamics

Although the high-resolution structure of a protein may provide significant insight into which regions are important for function, it is well-known that proteins undergo significant conformational fluctuations, even under native conditions. This suggests that the static structure alone may not provide sufficient information for elucidation of the thermodynamic determinants of biological function and that an accurate molecular-level description of function requires knowledge of the nature and energetics of the conformational states that constitute the native state ensemble. Information obtained from NMR dynamics experiments provides insight into specific structural changes and the energetics associated with function. This section highlights some specific examples. The temperature dependence of the fast internal dynamics of calcium-


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saturated calmodulin in complex with a peptide corresponding to the calmodulin-binding domain of the smooth muscle myosin light chain kinase has been examined using15Nand2H NMR relaxation method~."~ A significant variation in the temperature dependence of the amplitude of motion of individual side chains is seen. Importantly, analysis of models indicates that changes in generalized order parameters can be robustly related to corresponding changes in residual entropy. A simple cluster model that incorporates features of cooperative or conditional motion reproduces many of the unusual features of the experimentally observed temperature dependence and illustrates that side chain interactions result in a dynamically changing environment that significantly influences the motion of internal side chains. This model also suggests that the intrinsic entropy of interacting clusters of side chains is only modestly reduced from that of independent side chain motion. Finally, estimates of protein heat capacity support the view that the major contribution to the heat capacity of protein solutions largely arises from local bond vibrations and solvent interactions and not from torsional oscillations of side chains. In addition to determining the first solution structure of the unliganded protein, Ferreon et al. have performed 15Nrelaxation and native state hydrogendeuterium exchange experiments on a Src homology domain-3 (C-SH3) from Caenorhabditis elegans .lo* It was observed that the regions of greatest structural variabilility also show low protection and order parameters, suggesting a higher degree of conformational diversity. These flexible regions also coincide with those regions of Sem-5 that have been predicted to be unfolded in many of the most probable conformational states within the native state ensemble. A similar study has been performed on the D/D domain of the cyclic AMP-dependent protein kinase (PKA) holoenzyme, which mediates important protein-protein interactions in the subcellular localization of the enzyme."' The recent solution structures of two D/D AKAP complexes revealed that the AKAP binds to a surface-exposed, hydrophobic groove on the D/D. Analysis of the changes in hydrogen/deuterium exchange and internal motions of the backbone of the D/D when free and bound to the anchoring protein reveal increases in H/D exchange protection factors as well as increases in backbone flexibility. This unusual observation of increased backbone flexibility and H/D exchange protection may be a general effect observed for the stabilization of hydrophobic ligand/hydrophobic pocket interactions. Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound state in solution. 15Nrelaxation rates for the monomeric and dimeric states have been extrapolated from relaxation data acquired at four different protein concentrations."' A qualitative comparison of order parameters in the monomeric and apparent dimeric states shows that loops forming the dimer interface become rigidified upon dimerization. Qualitative information on monomer-dimer exchange and intramolecular conformational exchange was obtained from the concentration dependence of auto- and cross-correlated relaxation rates. The loop containing the catalytically important Asp129 fluctuates between different conformations in both the monomeric and dimeric states. The exchange rate compares rather well with that of the catalysed reaction step,

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supporting existing hypotheses that catalysis and enzyme dynamics may be coupled. The side-chain of Trp49, which is important for substrate specificity, exhibits conformational dynamics in the monomer that are largely quenched upon formation of the dimer, suggesting that binding is associated with the selection of a single side-chain conformer. Ma et al. have characterised the dynamics of the backbone of the electrontransfer protein plastocyanin from the cyanobacterium Anabaena variabilis from 15Nand 13Carelaxation."' The 13Crelaxation studies were performed using I3Cin natural abundance. Overall, it is found that the protein backbone is rigid. However, the regions that are important for the function of the protein show moderate mobility primarily on the microsecond to millisecond time scale. These regions are the 'northern' hydrophobic site close to the metal site, the metal site itself, and the 'eastern' face of the molecule. In particular, the mobility of the latter region is interesting in light of recent findings indicating that residues also on the eastern face of plastocyanins from prokaryotes are important for the function of the protein. The study also demonstrates that relaxation rates and NOESof the 13Canuclei of proteins are valuable supplements to the conventional 15Nrelaxation measurements in studies of protein backbone dynamics. Changes in the dynamic properties in proteins often accompanies ligand binding; several studies have reported these effects in a variety of complexes. The high-affinity inhibition of stromelysin 1 (MMP-3) by tissue inhibitor of metalloproteinases 1 (TIMP-1) helps control tissue remodeling and tumor development. Their unfavourable enthalpy of binding of +6.5 kcal mo1-' is unusual among protein-protein associations, deviates from structure-based prediction, and is compensated by a net entropy increase providing at least 18 kcal mol-' of favorable free energy of binding at a 1 M reference state. Using NMR, bindinginduced changes in the backbone of N-TIMP-1 were checked as one possible source of conformational entropy changes.I12M M P binding increases rigidity in some contact sites in TIMP-1 but increases mobility remotely in the otherwise rigid P-barrel core of N-TIMP-1, increasing 15Nrelaxation evidence of pico- to nanosecond and micro- to millisecond fluctuations of beta-strands A-E. This is a distinctive example of affinity between two well-structured proteins being enhanced by increased conformational entropy in the reservoir of a folding core. Apolipoprotein CII (apoCII), a surface constituent of plasma lipoproteins, is the activator for lipoprotein lipase (LPL) and is therefore central for lipid transport in blood. The 3D structure of human full-length apoCII in complex with sodium dodecyl sulfate (SDS) micelles has been r e ~ 0 r t e d . lIn ' ~ addition to the structure determination, 15Nrelaxation measurements revealed that the Cterminal helix of apoCII, which is responsible for the activation of LPL, has a higher degree of internal motion on the nanosecond time scale that other helices. Bhattacharya et aE. have monitored relaxation rates for the ssDNA-binding domain (RPA70A) in the absence and presence of d-CTTCA.l14Analysis of the data using the Model-Free formalism and spectral density mapping approaches showed that the structural changes in the binding site are accompanied by some significant changes in flexibility of the primary DNA-binding loops on multiple timescales. On the basis of these results and comparisons to related proteins, they

9: N M R of Proteins and NucEeic Acids

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proposed a mechanism of sequence-nonspecific binding of ssDNA involving dynamic remodeling of the binding surface. Subramaniam et al. have used NMR spectroscopy together with limited proteolysis to examine the dynamics of the lambda-Int catalytic domain in the presence and absence of DNA half-site substrates with the goal of characterizing the expected conformational change. Although the C terminus is indeed flexible in the absence of DNA, we find that conformational changes in the tyrosine-containing betahairpin are not coupled to DNA binding.l15 Relaxation experiments have revealed a reduction of order parameters in the flap region of an inactive HIV-1 protease mutant, indicating a small increase in the amplitude of internal motion on the sub-nsec timescale upon binding substrate. In addition, several residues in the flap region are mobile on the conformational exchange timescale, msec-ysec. Flap dynamics of the proteasesubstrate complex have been compared with those of protease-inhibitor complexes.116 Binding of the product inhibitor p-nitrophenol to the monoclonal esterolytic antibody NPN43C9 has been inve~tigated."~Structural information from changes in chemical shift upon binding has been related to the changes in local dynamics in the active site of the catalytic antibody using NMR relaxation measurements. Significant differences in backbone dynamics were observed between the free Fv and the complex with p-nitrophenol. A number of resonances, including almost all of the third hypervariable loop of the light chain (L3), were greatly broadened in the free form of the protein. Other residues in the antigen-binding site showed less broadening of resonances, but still required exchange terms in the model-free analysis, consistent with motion on a slow timescale in the active site region of the free Fv. Binding of p-nitrophenol caused these resonances to sharpen, but some exchange terms are still required in the analysis of the backbone dynamics. It was concluded that the slow timescale motions in the antigen-binding site are very different in the bound and free forms of the Fv, presumably due to the damping of large-amplitude motions by the bound inhibitor. The bond lengths and dynamics of intra- and intermolecular hydrogen bonds in an RNA kissing complex have been characterized by determining the NMR relaxation rates of various double- and triple-quantum coherences that involve an imino proton and two neighbouring I5Nnuclei belonging to opposite base .l18 Then new experiments described allow one to determine the chemical shift anisotropy of the imino protons. The bond lengths derived from dipolar relaxation and the lack of modulations of the nitrogen chemical shifts indicate that the intermolecular hydrogen bonds which hold the kissing complex together are very similar to the intramolecular hydrogen bonds in the double-stranded stem of the RNA. The binding of the HIV tat protein to the TAR (transactivating response region) RNA element activates transcription of the HIV viral genome. The complex of TAR with argininamide serves as a model for the RNA conformation in the tat-TAR complex. The dynamics of the HIV-2 TAR-argininamide complex was investigated by measurements of the relaxation rates of protonated base

348


carbon and nitrogen n ~ c l e i . "Six ~ autocorrelation rates as well as cross-correlation rates were measured to map the frequencies of base motion in the HIV-2 TAR-argininamide complex. These measurements reveal a broad range of dynamic heterogeneity exhibited by the hexanucleotide loop, dinucleotide bulge, and the A-form helical regions. U23 in the bulge undergoes the largest dynamic change on binding argininamide, while U25 remains flexible, reflecting the large conformational change that is triggered upon ligand binding.

5

Protein Folding

NMR continues to play a central role in the structural study of unfolded and partially folded states of proteins. Structural and kinetic information can be revealed under denatured and partially denatured conditions providing insight into the structure of folding intermediates and transition states. Juneja and Udgaonkar have critically examined the relevance of such structural information to protein folding mechanisms.'20 Much of the work covered in this section illustrates the need for integrated biophysical approaches in order to understand the intricate details of the folding reactions of the simplest proteins. A protein in solution is a thermodynamic entity, spanning, in principle, the entire allowed conformational space from the fully folded N to the fully unfolded U. Although some partially folded higher-energy conformers may coexist with N and U, they are seldom detected spectroscopically because their populations are usually quite low under physiological conditions. Akasaka has described a new type of experiment, a combination of multidimensional NMR spectroscopy with pressure, that is capable of analysing the structure and thermodynamic stability of these higher-energy conformers.'21The idea is based on the finding that under physiological conditions the conformational order of a globular protein normally decreases in parallel with its partial molar volume, so that under equilibrium conditions, the population is shifted to a less-and-less- ordered conformer with increasing pressure. In principle, with the high-resolution of multidimensional NMR, the method enables one to explore protein structure and stability in atomic detail in a wide conformational space from N to U with pressure and temperature as variables.121The same group have found that a pressure-stabilized equilibrium conformer of ubiquitin shares a common structural feature with the proline-trapped kinetic intermediate found in a pulse-labelling exchange NMR experiments. The conformer is locally unfolded in the entire segment from residues 33 to 42 and in C-terminal residues 70-76. The close structural identity of an equilibrium intermediate stabilized under pressure with a transiently observed folding intermediate is likely to be general in terms of a folding funnel common to both experiments.'22 Lyon et aE. have developed an NMR technique to probe the structures of disordered, partially folded states of proteins at the level of individual amino acid residues.'23Chemically induced dynamic nuclear polarization (CIDNP) is first generated in exposed aromatic side-chains of the denatured state and then transferred to the high-resolution NMR spectrum of the native state by stimulat-


349

ing rapid refolding of the protein. Application of this method to the low pH molten-globule state of alpha-lactalbumin reveals remarkably native-like environments for the aromatic residues in the primary hydrophobic core of the protein. Canet et al. have reported the combined use of real-time photo-CIDNP NMR and stopped-flow fluorescence techniques to study the kinetic refolding of a set of mutants of a small globular protein, histidine- containing phosphocarrier protein (HPr), in which each of the four phenylalanine residues has in turn been replaced by a tryptophan residue.'24The results indicate that after refolding is initiated, the protein collapses around at least three, and possibly all four, of the side-chains of these residues. The hydrophobic collapse observed for HPr during the early stages of its folding appears to act primarily to bury hydrophobic residues. This process may be important in preventing the protein from aggregating prior to the acquisition of native-like structure in which hydrophobic residues are exposed in order to play their role in the function of the protein. In a complementary study the folding of tryptophan mutants of HPr was investigated by NMR, DSC, CD, intrinsic fluorescence, fluorescence anisotropy, and fluorescence quenching.'2s The heat denaturation of all four mutants can be fitted adequately to a two-state model, in agreement with the observations for the wild-type protein. The guanidinium hydrochloride unfolding transitions of F22W, however, showed the accumulation of an intermediate state at low concentrations of denaturant. F22W also showed two phases in the fluorescence experiments corresponding to the two phases previously observed in ANS binding studies of the wild-type protein. Residue 22 was found from NMR studies to be part of the binding interface on HPr for ANS. These observations indicate that the second slow phase reflects a local, rather than a global, rearrangement from a well-structured highly nativelike intermediate state to the fully folded native state that has less hydrophobic surface exposed to the solvent. Roy and Jennings have reported the real-time NMR kinetics of the denaturant-induced unfolding of interleukin-1-beta and provide direct, global, and residue-specific information on the structural nature of the unfolding reaction.'26 Heterogeneity in the individual amino acid residue kinetics reveals a rugged unfolding landscape. The relative kinetic stability of native-like turns supports low temperature molecular dynamics predictions of turn-controlled unfolding. Mizuguchi et al. have used real-time NMR experiments to monitor folding of apoplastocyanin. Results showed that the local and long-range interactions in the native apoplastocyanin are formed simultaneously, consistent with highly cooperative formation of the native Insights into the earliest events in protein folding can be obtained by analysis of the conformational propensities of unfolded or partly folded states. The structure of the acid-unfolded state of apomyoglobin has been characterized using paramagnetic spin labelling and NMR.l2' Nitroxide side-chains, introduced by coupling to mutant cysteine residues at positions 18,77, and 133, were used as probes of chain compaction and long-range tertiary contacts. Significant interactions are observed within and between the N and C termini while the central region of the polypeptide chain behaves as a random polymer. Even in this highly denatured form, the protein samples transient compact states in

350


which there are native-like contacts between the N and C-terminal regions. Folding studies on proteases by the conventional hydrogen exchange experiments are severely hampered because of interference from the autolytic reaction. Bhavesh et al. have described the NMR identification of the hierarchy of early conformational transitions in HIV-1 p r 0 t e a ~ e . lSecondary ~~ chemical shifts, HN-H, coupling constants, 'H-15N NOES, and 15N transverse relaxation parameters have been used to report on the residual structural propensities, motional restrictions and conformational transitions. The data suggested that even under the strongest denaturing conditions hydrophobic clusters as well as different native and non-native secondary structural elements are transiently formed. On reducing the denaturing conditions, partially folded forms appear. The residues showing high folding propensities were contiguous along the sequence at many locations or in close proximity on the native protein structure, suggesting a certain degree of local cooperativity in the conformational transitions. The dimerization domain, the flaps, and their hinges exhibited the highest folding propensities. Their data suggested that even the early folding events may involve many states near the surface of the folding funnel. The unfolding and refolding of a monomeric mutant of copper-zinc superoxide dismutase was investigated by NMR spectroscopy in the copper-reduced form and by using guanidinium hydr~chloride.'~' It was found that the protein gives rise to a series of intermediates at low guanidinium concentration and to a globular unfolded state at concentrations higher than 3.5 M, which closely resembles a random coil structure, but with a high degree of compactness. The transition into the unfolded structure implies the detachment of the metal ions from native coordination sites, even if non-specific interactions remain. Bartalesi et al. have monitored the reversible unfolding of oxidized Bacillus pasteurii cytochrome c(553) by guanidinium hydrochloride under equilibrium conditions by NMR and optical spectro~copy.'~~ The results indicate that unfolding takes place through a mechanism involving the detachment from heme iron coordination of the Met71 axial ligand and yielding either a high spin (HS) or a low spin (LS1) species, depending on the pH value. Human acidic fibroblast growth factor 1 (hFGF-1) is an all beta-barrel protein, and the secondary structural elements in the protein include 12 antiparallel beta-strands arranged into a beta-trefoil fold. The conformational stability of the human acidic fibroblast growth factor (hFGF-1) has been investigated using amide proton exchange, temperature-dependent chemical shifts and optical s p e c t r o ~ c o p y . ' ~Urea-induced ~~'~~ equilibrium unfolding of hFGF- 1 monitored by fluorescence and C D spectroscopy suggests that the protein unfolds by a two-state (native to denatured) mechanism. In contrast to the equilibrium unfolding events monitored by optical probes, native state hydrogen exchange data show that the beta-trefoil architecture of hFGF-1 does not behave as a single cooperative nit.'^^,'^^ The structure and dynamics of equilibrium intermediate in the unfolding pathway of hFGF-1 has also been presented by Srimathi et ~ 2 l . l ~ ~ Limited proteolytic digestion experiments and hydrogen-deuterium exchange monitored by 'H-15N HSQC spectra show that the conformational flexibility of the protein in the intermediate state is significantly higher than in the native


35 1

conformation. "N relaxation experiments show that many residues located in beta-strands IX, X, and XI exhibit conformational motions in the micro- to millisecond time scale. Furthermore they suggested that residues in the betastrands 11,VIII, and XI1 possibly constitute the stability core of the protein in the near-native intermediate state. The A and B chains of insulin combine to form native disulfide bridges without detectable isomers. The fidelity of chain combination thus recapitulates the folding of proinsulin, a precursor protein in which the two chains are tethered by a disordered connecting peptide. 'H NMR studies of a representative analogue lacking invariant side chains Ile(A2) and Val(A3) have demonstrated local unfolding of the A1-A5 segment in an otherwise native-like structure. This and related partial folds retain efficient disulfide pairing, which suggests that the native N-terminal alpha-helix does not participate in the transition state of the r e a ~ t i 0 n . ITwo ~ ~ insulin isomers containing non-native disulfide bridges ([A7A1 l,A6-B7,A2O-B19] and [A6-A7,All-B7,A20-B19]) are metastable and biologically active. Remarkably, the same two isomers are preferentially formed from native insulin or proinsulin following disulfide reassortment in guanidine hydrochloride. Hua et al. have demonstrated that the isomers contain native-like elements of structure in the B chain, whereas the A chain is largely disordered. This general asymmetry is consistent with a hierarchical disulfide pathway in which nascent structure in the B chain provides a template for folding of the A chain.'36 NMR spectroscopy has been employed to characterize unfolding intermediates and the denatured state of horse ferricytochrome c in guanidine hydrochloride.*37Two equilibrium unfolding intermediates with His-Lys heme axial ligation and two unfolded species with bis-His heme ligation were detected. These results are contrasted with previous results on horse ferricytochrome c denaturation by urea, for which only one unfolding intermediate and one unfolded species were detected by NMR spectroscopy. Urea and guanidine hydrochloride are often used interchangeably in protein denaturation studies, but these results and those of others indicate that unfolded and intermediate states in these two denaturants may have substantially different properties.

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

1

Introduction

A large majority of proteins are the enzymes that cause post-translational modifications of the proteins themselves,production of lipids, glycolipids etc and the whole range of natural products that this review attempts to cover. As in previous years (the last in the series being ref'), in addition to structural and conformational studies, NMR analysis in metabonomics and metabolomics (Section 3) is covered. NMR studies on membranes (Section 4) continue to be on the increase and important not only in understanding how cells are regulated but also in drug discovery, membrane transport and drug activity. Interactions at the outer face of the membrane and in the extracellular matrix (Section 5) reflects my own interests in membrane interactions of viruses and prions and fibril formation and the involvement of proteoglycans. Other protein-to-oligosaccharide interactions are reviewed in Section 6. The diversity of natural products is illustrated with examples from each molecular group (Section 7 ) ending with polysaccharides (Section 8) which leads naturally on to materials science (Section 9).

2

Drug Discovery and Combinatorial Chemistry

Marchioro et aE2 review the part that NMR techniques are increasingly playing in the different steps of candidate drug development from the identification and structural characterisation of a chemical compound to drug screening, structure based design, ex vivo studies on biological fluids and tissues and in vivo studies on organs or animals. The review focuses on combinatorial and screening methods such as solid- and liquid-phase synthesis and drug-screening methods. A pulsed gradient spin-echo NMR diffusion sequence PGSE-WATERGATE has been optimised for the measurement of the diffusion coefficients of small ligands in aqueous solution such as is commonly required in pharmaceutical and combinatorial appro ache^.^ Structure based screening represents an integrated approach for the identification and optimisation of hits for affinities and specificities by the combined use of NMR spectroscopy, homology modelling and X-ray crystallogr a ~ h yA. ~new automated method for rapidly analysing NMR spectra of comNuclear Magnetic Resonance, Volume 33 0 The Royal Society of Chemistry, 2004

357

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pounds synthesised in an 8 X 12 matrix (multiwell plates) has been proposed' using self-organising maps (SOM). Such an unsupervised neural network6 was capable of clustering together NMR spectra containing a common pattern and identifying outliers useful for quality control on combinatorial libraries. Utilisation of a novel microcoil probe allows NMR data to be gathered on low pg/picomolar samples in libraries of natural products being screened in drug dis~overy.~ The approach was demonstrated by the search for anti-cancer activities in components of the bark of Taxus breuifolia. Wolters et a18 add capillary isotachophoresis/NMR to microcoil techniques which allows the highest mass sensitivity nanolitre volume flow cells.

3

Metabonomics, metabolomics and in vivo spectroscopy

Studies which focus on the metabolic profiles of biofluids are referred to as metabonomics. As discussed by Reo9and further in the second half of this section a future challenge is to describe the cellular metabolome for purposes of understanding cellular functions (i.e. metabolomics). Keun et all0 reviewed metabonomic analysis of biofluids and tissues utilising high-resolution NMR spectroscopy and chemometric techniques that has proven valuable in characterising the biochemical response to toxicity for many xenobiotics. The lab-to-lab and day-to-day variability of analysis of urine samples'o showed excellent reproducibility and robustness of metabonomic techniques which makes it competitive to the best proteomic analysis and more valuable than genomic analysis. Collection of normal data by NMR analysis of body samples remains a priority. Other examples from the literature collected by Reo9 demonstrate how 'H NMR and pattern recognition methods are used to obtain the urinary metabolic profile and how this profile is affected by exposure to various toxicants. In order to reduce fluctuations in data acquisition for metabonomic studies, the group of Nicholson et al" have adopted a data filtering method, orthogonal signalling correction (OSC), to biofluid NMR data to minimise the influence of spectrometer variation during acquisition. NMR based metabonomic studies were carried out on the effects of commonly used drug carrier vesicles.12 Human pancreatic juice obtained from patients after partial excision of the pancreas for bile duct cancer was fractionated to isolate glycopeptides which were further purified by ion exchange chromatography and gel filtration and characterised by 'H NMR spectro~copy.'~ The glycopeptides had an unusually high fucose content having novel bi-, tri-and tetra-antennary N-linked oligosaccharide structures with, respectively 3,4 and 5 fucose residues. The capabilities and advantages of multinuclear NMR to provide metabolic information in cells and tissues are now being explored. A review by Govil14 focuses on the application of NMR spectroscopy in elucidating the chemistry of living cells, body fluids and tissues especially when the cells are active. The changes in kinetics of the metabolic processes under the influences of activators or drugs was evaluated and spermatozoa used to illustrate the usefulness of the NMR technique. The extension of 13CNMR techniques to study cellular metab-

10: N M R of Carbohydrates, Lipids and Membranes

359

o h m over recent years has provided valuable data supporting the occurrence, diversity and extent of carbon cycling in the carbohydrate metabolism of microorganism~.~* In mammals the effects of glucose on fructose metabolism in rat hepatocytes was studied by 13C NMR following gluconeogenesis and phosphorylation mechanisms.16''0 assisted 31PNMR has been used to follow creatine kinase phosphotransfer in studies of the synchronisation of membrane metabolic sensors with cell metab~lism.'~ The effect of flutoxetine treatment on carbohydrate metabolism in human blood platelets was studied by 'H NMR.'* Schiller et all9 reviewed the use of 31PNMR (and MALDI-TOF mass spectrometry) in lipid research both with relevance to membranes (see the next Section) and also as applied to crude organic extracts without previous lipid separation. 4

Membranes

A review on the use of NMR for studies of the properties of micelles, discusses2' the structure of self-aggregated micelles, hydration and solubilisation properties as well as interactions between micelles and different kinds of surfactants and biological compounds. Studies on membranes are getting increasingly sophisticated introducing many interacting compounds in types of lipid, protein and low molecular weight compounds such as drugs discussed next. Debouzy et a12' used several techniques to study the effects of mustard gas in membranes: 'H NMR to observe the localisation of sulphur mustard in membranes of small unilamellar vesicles of lecithin; 31PNMR to verify the hypothesis of pore formation in membranes of large unilamellar vesicles; and, pseudosolid-state 31Pand 2H NMR to analyse the dynamic consequences of the presence of sulphur mustard in multilayer dispersions of dimyristoylphosphatidylcholine (DMPC). Dynamic information was obtained by 'H NMR and 2D NOE experiments on the interaction of lidocaine with phosphatidylcholine bilayers in order to assess the location of this anaesthetic in the The interactions of cyclosporines with lipid membranes was studied by solid-state NMR and high-sensitivity titration ~alorimetry.2~ Binding constants were dependent on the concentration of the cholesterol in the membranes. The authors reported that the association of cyclosporin A with the membrane was accompanied by a positive enthalpy change which was over-compensated by positive entropy changes suggesting classical hydrophobic effects, in contrast to other peptide-lipid interactions. Biological membranes contain functional domains (lipid rafts) made up in particular of sphingomyelin, glycolipids, c h o l e ~ t e r oand l ~ ~(g1yco)proteins.These are dealt with in turn in the following: 4.1 Sphingomyelinand Glycolipids. - Mattjus et a125demonstrated that sphingomyelin can dramatically alter the transbilayer distribution of monohexaosylceramides such as galactosylceramide (GalCer) in 1-palmitoyl-2-oleoyl-phosphatidylcholine/sphingomyelin vesicles studied by 13CNMR spectroscopy in the presence of paramagnetic Mn2+.Their results also suggested that sphingolipid-

360


sphingolipid interactions that occur even in the absence of cholesterol play a role in controlling transmembrane distribution of cerebrosides. Several glycosphingolipids less polar than GalCer have been purified from rat brain26and designated fast migrating cerebrosides (FMCs). NMR analysis showed two of them to be 3-0-acetyl-sphingosine-GalCer varying in non-hydroxy and hydroxy fatty N-acylation. FMCs are myelin constituents enriched in both the central nervous system and peripheral nervous system and implicated in dysmyelenating disorders. High resolution MAS natural abundance 13CNMR was used to study, in the presence of phosphatidylcholine and Gd3+, the interaction of a diterpenoid isolated from Podocarpu Spp. which is a potent anti-oxidant and anti-bacterial agent. The latter property was ascribed to the determined orientation in the upper region of the membrane with its hydroxyl group located in the vicinity of the C-3/4 carbon atoms of the phospholipid acyl and nearly perpendicular with respect to the phospholipid acyl chain axis.27The association of GM4 ganglioside with the membrane surrounding lipid droplets was shown by analysis of shark liver glycolipid fraction of six shark species.28The lipid moieties were compared by NMR analysis of GM4 which was the most abundant lipid oligosaccharide in the samples. 4.2 Lipids and Cholesterol. - Nuclear Overhauser enhancement spectroscopy (NOESY) in combination with MAS is introduced as a novel approach to studying the structure and dynamics of the lipid matrix and are highlighting the conformational and spatial disorder of lipids in memb~anes.2~ Proton line narrowing in biomembranes spun at the magic angle for spinning speeds greater than 7kHz was investigated3' by increasing the field strength from 200 to 800MHz and changing the membrane fluidity of liposomes. Solid-state NMR has been used for example to study phospholipid-cholesterol interactions in sphingomyelin-cholesterol in binary systems3'and cholesterol dynamics in membranes of raft c o m p o ~ i t i o n To . ~ ~study cholesterol's ability to modulate the activity of neural membrane proteins, the role of polyunsaturated (docosahexaenoate)phospholipids in controlling membrane cholesterol content was studied by 2H NMR in 1,2-didocosahexaenoylphosphatidylcholine and 1-stearoyl-2-docosahexaenoy1phosphatidylcholine~3 The molecular organisation of cholesterol was studied at different concentrations in the lipid bilayers. Pulsed field gradient NMR has also been used34for direct determination of the lateral diffusion coefficient of cholesterol (labelled at C-6 with fluorine) for an orientated lamellar liquid-crystal phase of dimyristoylphosphatidylcholine (DMPC)/cholesterol water. The phospholipid lateral diffusion coefficient was shown to be approximately four times smaller in the presence of cholesterol. The perturbation induced by second messenger diacylglycerols (DAGs) into bovine brain phosphatidylcholine (BBPC) bilayers in the absence of bovine brain sphingomyelin and/or cholesterol were studied by 2H NMR. The authors35conclude that since both have a tendency to form non-bilayer lipid phases and lateral heterogeneity of the membranes are associated with increased activation of membrane associated enzymes, sphingomyelin and cholesterol enriched regions of biological membrane (rafts) provide an environment with increased sensitivity


361

to the generation of lipid second messengers and modified transmembrane signal transduction properties. Interactions of acylcarnitines with model membranes was studied by I3CNMR and the possible biological effects on the lipid bilayer in vivo discussed.36The affect of amphotericin B on pure and ergosterol- or cholesterol- containing dipalmitoylphosphatidylcholine(DPPC) bilayers was studied by 2H NMR.37 Hauss et aP8 incorporated 10% perdeuterated or protonated squalene into stacked bilayers of dioleoylphosphatidylcholine (DOPC) doped with dioleoylphosphatidyl glycerol (DOPG) to simulate the negative charge found on natural membranes. Squalene was shown to lie predominantly in the bilayer centre parallel to the plane of the membrane. The elastic deformation of membranes has been probed by 2H NMR r e l a ~ a t i o n . ~ ~ 4.3 Proteins and Peptides within Membranes. - The structure determination of membrane proteins in lipid environments is reveiwed by Opella et aE4’using 2D IH-”N polarization inversion spin-exchange and polarity index slant angle methods in a convergence of solution and solid state NMR. Solid-state NMR spectroscopy studies for the determination of the structures of membranes peptide and proteins in lipid bilayers have been reviewed4’including an overview of bacterial expression systems for protein preparation and isotopic labelling. Bechinger et up2 discuss 2H, 15N and 31Psolid-state NMR spectroscopy of polypeptides reconstituted into orientated phospholipid membranes. Gawrisch et a129*43compare high resolution and solid state methods for studying lipidpeptide interactions. The former requires rapid isotropic motions of the peptides achieved by conducting experiments in membrane-mimetic environments. The review goes on to discuss novel NMR techniques utilising rapid magic angle spinning (MAS) or orientated peptide/lipid samples that have dramatically improved resolution of solid-state NMR. The use of systems for orientating molecules including studies on oligosaccharides are further reviewed by Khetrapal et ~ 1 . 4 ~ Van der We1 et al 45 used solid-state 2H NMR spectroscopy and an approach involving geometric analysis of labelled alanines to examine the structure and orientation of a designed synthetic hydrophobic membrane-spanning a-helical peptide in phosphatidylcholine bilayers. The peptide was shown to have an inherently preferred tilted orientation. Analysis of transmembrane helices by solid-state NMR and from X-ray crystallographic data e.g. bacterial rhodopsin and the membrane spanning domain of the M2 protein of influenza virus, also showed a uniform peptide plane tilt as well as backbone torsion angles. Hydrogen bond geometry was also more uniform than expected and the hydrogen bond length was shorter than in water soluble proteins suggesting the dominance of electrostatic versus covalent contributions to these bonds.46Saito et uZ47748 also used bacteriorhodopsin as a typical membrane protein for extensive 13CNMR analysis of dynamic aspects of membrane proteins and membrane associated peptides. Other studies on protein-lipid interactions include NMR solution structure and dynamics of motilin in isotropic bicellar solution49of the neuropeptide APNPY with selectivity for the neuropeptide re~eptor;~’ an antimicrobial pore forming protein, n i ~ i n ZErB; ~ ~ 1 and ErB-2 transmembrane domains

362


(by 2HNMR)." Arshava et a153report the high resolution NMR of seven peptides representing the transmembrane domains of the a-factor receptor from Saccharornyces cerevisiae.The structure and functional characterisation of the periplasmic N-terminal domain of the sugar specific ion channel protein (ScrY) of Salmonella typhirnurium has been carried out including comparative TR-NOENMR spectroscopy on the sugar binding affinity.54 Other strategies include the use of 31PNMR spectroscopy to investigate domain formation in lipid rafts and 19FNMR analysis of "F-labeled tryptophan to monitor the conformation and dynamics of the indole ring in transmembrane peptides, e.g. Trp of gramicidin A (see the next Section for discussion of gramicidin S) in orientated membrane^.^^ Proton inverse detected deuteron (PRIDE) NMR techniques have been used to study gramicidin D in transmembrane ~ r e i n t a t i o nData . ~ ~ from mutagenesis analysis and NMR-based structure calculations of the cytoplasmic tail of the invariant chain Ii associated with class I1 major histocompatibility antigens (MHC class 11)57 showed that Ii molecules on different intracellular vesicles can promote endosome docking and fusion and thereby control endosomal traffic of membrane proteins and endosomal content. 31Pand 2Hsolid state NMR spectroscopy was used to investigate a unique peptide (called CP) of the transmembrane sequence of a T cell antigen receptor.'* The C P peptide, GLRILLKV, is similar to the amphipathic region of many proteins. Unlike antimicrobial peptides however which damage cell membranes C P does not create pores but may act by interfering with signal transduction at the membrane level. The integral outer membrane protein OmpX was studied in dihexanoylphosphatidylcholin~9 to look at protein-detergent interactions using NOE measurements in 3D 15N and 1 3 C - r e ~ ~ l['H, ~ed 'HI-NOESY spectra recorded with selectively methyl-protonated and otherwise uniformly 2H,13C,"N-labeled Ompx micelles of dihexanoylphosphatidylcholine at natural isotope abundance. The global structure and dynamics of human apolipoprotein CII (ApoCII) in complex with SDS micelles was studied by 15N NMR relaxation measurements in isotopically enriched ApoCII and evidence presented for increased mobility of the helix segment in the action of lipoprotein lipase.60Cushley et a161 have reviewed NMR studies of lipoprotein structure. Gottschalk et al 62 reported on transmembrane signal transduction of the aIIP3 integrin and further discuss integrin-cell matrix adhesion which brings us to the next Section.

5

Extracellular Macromolecular Structure

The domain structure and organisation of extra-cellular proteins in the extracellular matrix (ECM) is reviewed for the basic folds of many domains that have been determined by crystallography or NMR s p e c t r o s ~ o p yOtvos . ~ ~ and Cudica consider the role of post-translational modifications in the transformation of PrPCto PrPSC.They agree with our that the glycosylated form has to be arrived at by molecular modelling using conformational data on oligosaccharides and NMR structures of recombinant non-glycosylated prions. The amino


363

acid mutations associated with a predisposition for miss-folding of prions map near the glycosylation s i t e P 5furthering the suggestion that post-translational modifications alone, or in combination with amino acid changes play a dominant role in the pathogenic transformation of PrPC to PrPSC.Important NMR data continue to be acquired on non-glycosylated recombinant prion proteins. The Wuthrich group have published a new review of the NMR structures of wild type and variant human and bovine prion Prions are the only glycoproteins involved in a protein folding disorder that are GPI anchored to the cell membrane which may account for their infectivity. GPI may be cleaved by mammalian phospholipase so the analysis of these enzymes is relevant here. For example, phosphatidylinositol-specific phospholipase C stereoselectivity has been studied by engineering Ca2 binding sites in C-independent enzymes.67 Novel N- and 0-linked diinositols have been synthesised6*and interestingly had anti-galactosidase inhibitory activity. Of the 24 or so non-GPI anchored proteins involved in protein folding disorders the majority of the literature concentrates on amyloid p protein in Alzheimer's disease. There are some data on others, for example, transthyretin in an amyloid fibril studied by Jaroniec et a169 by 13C-MASsolid-state NMR spectroscopy. General insights into the amyloid folding problem from solid-state NMR is reviewed by Ty~ko.~' Ishimura et a?' look at high hydrostatic pressure methods combined with fluorescence, CD and high resolution NMR to study fibril formation, folding intermediates etc. ABPs and their fibrils have been studied by solid state NMR.72373374 The association of ABP with membrane surfaces was also monitored by solid-state NMR.75 Crescenzi et a176 found evidence from NMR in aqueous trifluoroethanol or SDS micelles for the similarity of ABP interactions in apolar environments with virus fusion proteins, e.g. HIV gp41 fusion domain.77 There is also evidence that prions and viruses associate with proteoglycans at the cell surface and in the ECM and several articles on studies of heparin-protein interactions. Islam et aE78studied the effect of periodate oxidation of heparin with 'H NMR characterisation of the products and their interaction with ATIII mediated anti-factor I1 and anti-factorXa activities (reduced). Controlled 2-0desulphation followed by exhaustive periodate oxidation/borohydride reduction and NMR characterisation of the products has also been used to study the interaction of heparin with fibroblast growth factor-2 (FGF2).79As discussed by the authors, heparin activates FGF2 by favouring formation of ternary complexes with its cellular receptors. NMR spin lattice relaxation times and NOES showed that the altered monosaccharide residues act as flexible joints and cause marked distortion of the chain conformation of heparin required for formation of ternary complexes. The resulting heparin has no anticoagulant activity and had a reduced capacity to induce FGF2 dimerisation, but it fully retained FGF2 binding and acquired the ability to inhibit angiogenesis. Heparin oligosaccharides have been isolated from depolymerised porcine intestinal mucosal heparin with the novelty of observations on the role of histamine in modifying deploymerisation and providing competitive binding of oligosaccharides to the heparatinase used.80Dermatan sulphates have been characterised from the skin +

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of the eel, Anguillajaponica." Bacterial cell walls have been studied by cross polarisation MAS and rotational-echo double resonance [13C]and ["N] NMR experiments82performed on intact cells of Staphylococcusaureus labelled with [13C]alanine, ["Nlglycine or with [13C]glycine and [~-"N]lysine. The cells were harvested during stationary or exponential growth conditions, the latter with and without vancomycin. The technique could show that vancomycin reduces the number of petide-stem lysines covalently linked to a pentaglycyl bridge suggesting that it interrupts synthesis in S. aureus by interference with transglycosylation.

6

Ligand-receptor InteractionsInvolving Glycosylated Molecules

The properties of oligosa~charides~~ and polysaccharidess4 as characterised by NMR have been reviewed by Witczak.

6.1 Oligosaccharide-proteininteractions. - Schuman et a?' have probed the conformational and dynamical effects of 0-glycosylation within the immunodominant region of a MUCl glycopeptide, GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue the extra-cellular domain is under glycosylated exposing a highly immunogenic core peptide PDTRP as well as the exposure of normally cryptic core Tn (GalNAc), STn (sialyl(a2-6)GalNAc) and T F (Gal@13)GalNAc) carbohydrates. Grinstead et up6have reported their NMR studies on the same MUCl glycopeptide in interaction with antibody to study the effect of the different glycosylation discussed above on binding. The structural characteristics of a mucin glycopeptide motif from the N-terminal fragment of the cell surface glycoprotein CD43 is describeds7 that has multiple 0-linked chains which serve to extend the protein into the extracellular space and offers multivalent oligosaccharide motifs for recognition e.g. a glycopeptide SSSAVAV triglycosylated with the Ley epitope. The paper confirms the 'profound organisational' effect of a-linked glycosylation on the peptide backbone compared to the (3-link.Sialyl Lex has become a prominent target for biological studies because of its role in inflammation through binding to selectins. Synthesis and molecular tumbling properties of sialyl Lex and derived neoglycolipids was studied in model membranes by NMR spectroscopy.88Siebert et alS9have studied low affinity interactions such as those between small peptide domains and oligosaccharides using NMR in order to establish whether small lectin domains can be of use in therapeutics. NMR reporter screening for the detection of high-affinity ligands has been reviewed." Carbohydrate-to-carbohydrate recognition between Lex antigens is discussed and the role of calcium ions in mediating this reaction .91 One of the areas with a large potential for glycotherapeutics involves the recognition of sialic acids by mammalian sialoadhesins, viral and parasite haemagglutinins and viral neuraminidases. The specificity and affinity of sialic acid binding by the rhesus rotavirus VP8 core haemagglutinin has been explored by NMR spectro~copy.~~ The haemagglutinin required no additional carbohydrate


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moieties for binding and did not distinguish 3' from 6'siallylactose, but had a 10 X lower affinity for N-glycolylneuraminic acid than N-acetylneuraminic acid. In this case the broad specificity coupled with low affinity (Kd determined as 1.2mM) were consistent with this interaction mediating initial cell attachment prior to the interaction that determines host range and Zanetta et up3report their evidence for a lectin activity for human interleukin3 (IL-3), the specificity of the interaction due to GlcA(2S)(f31-3)GalNAc(4S)and modelling of the carbohydrate recognition domain. Emerson et up4 reported the NMR characterisation of interleukin-2 (IL-2) in complexes with the IL-2R receptor component and with low molecular weight compounds that inhibit the IL-2/IL-2R interaction. Schrag et up5 reviewed the recent crystallographic and NMR studies of proteins located in the endoplasmic reticulum (ER), Golgi complex and ER-Golgi intermediate compartment including the lectin domains of calnexin and calreticulin central to mediation of correct glycoprotein folding and secretion. These interactions were further studied by synthesis of the monoglucosylated high mannose type dodecasaccharide and its interaction with calreticulin measured by 'H NMR spectroscopy?6 Binding of the protein cyanovirin-N to oligomannose-8 and oligomannose-9 of gp120 of HIV is crucial to its potent virucidal activity against HIV. Several studies have used NMR to further characterise this r e a c t i ~ n . ~ Suetake ~ l ~ * . ~et~ ~ 1 ' ~ have characterised the interaction of tachycitin, an invertebrate chitin binding protein with an amidated C-terminus and possesses antimicrobial activity against both fungi and bacteria. Lactotetraosylceramide, has been characterised"' as a novel glycosphingolipid receptor for Helicobacter pylori opening up the possibility of an additional target for inhibition of binding of this organism in the gastrointestinal tract where it is associated with human pathology. Gramicidin S analogues have been designedlo2as carbohydrate binding proteins. Asn/Asp/Gln and Trp residues in the peptides were employed for hydrogen binding interactions and the creation units for hydrophobic pocket to promote the interaction. As a result of the 'H NMR study, NOES were observed between aromatic side chains of a peptide [Gln'~",Trp3~']-gramicidinS and mannose. Sperm-egg interactions e.g. in the mollusc bivalve Unio elongutulus where the N-linked glycans have been recently characterised,lo3The structures corresponded to those found on mammalian proteins during biosynthesis i.e. GlclMan9GlcNAc2and Man9GlcNAc2.Their role in the protection of polypeptide chain from proteolytic attack was suggested and discussed in the light of the evolution of the recognition and adhesion properties of oligosaccharide chains in the fertilisation process. 6.2 OligosaccharideConformation and Their Role in Protein Function. - Lommerse et allw have performed extensive conformational analysis of two xylose containing N-glycans of the a-D-haemocyanin of the snail Helix pomatia. Synthesis and NMR characterisation of the six regioisomeric monophosphates of octylGalNAc(f31-4)GlcNAc has been reported.lo5To reveal the function of the carbohydrate portion of glycopeptide and glycoproteins Tagashira et allo6 chemo-enzymatically synthesised artificially N-glycosylated derivatives of eel

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calcitonin and studied their 3D shape and biological activity. The CD and NMR spectra showed that the glycosylation did not change the conformation. Cglycosyl mimetics of the branched core trisaccharide, Man(al-3)[Man(a1-4)] Man, of N-linked oligosaccharides were studied bound to mannose binding lectins by NMR (line broadening, TR-NOE and STD) and surface plasmon resonance (SPR) rnethod~.''~Despite the increased flexibility of the C-glycosyl analog it appeared to be bound in the same conformation as the natural ligand. However the use of the trisaccharide alone may not be ideal to reach any conclusions. Incorporation of a "C label into a carbohydrate ligand, 3,6dideoxy-D-Xyl(a1-3)2-0-methyl Man, permitted the NMR study of its binding to the Fab from a monoclonal antibody. The dissociation rate constants were determined by full line shape analysis of the 13C spectrum."* Asensio et do9 report experimental evidence for the existence of non-em-anomeric conformations in branched oligosaccharides by NMR analysis of the structure and dynamics of aminoglycosides of the neomycin family. Novel methods for conformational analysis include J-modulated 1D-directed COSY for precise measurement of proton-proton residual coupling constants of oligosaccharides."' Two 2D J-modulated HSQC-based experiments were designed" ' for precise determination of small residual dipolar one-bond carbonproton coupling constants in 13Cnatural abundance carbohydrates. A 2D quantum J-correlation NMR experiment for precise measurements of the protoncarbon vicinal coupling constants 3JC2,4-H1t and 3Jc6,8-H1t in 13Cuniformly labelled nucleic acids1I2may have use in conformational studies of oligosaccharides. Similarly detailed studies by solid state NMR in polycrystalline carbohydrates (e.g. of fucose"') may add more conformational data. Conformational studies of blood group A and B oligosaccharides were carried out by NMR measurements of one-bond C-H residual couplings in partially orientated liquid crystal solutions and by the generation of thousands of structures using the Monte Carlo method.'14 The crystal structure of methyl 1,2,3,4-tetra-O-acetyl-b-Dglucopyranuronate has been reported to agree with NMR data.'15 Ferrier et review the characterisation and conformational features of various carbohydrates including acyclic, furanose and pyranose forms, disaccharides and oligosaccharides and 15Nfor analysis of the effects of N-substitution. 7

Natural Products

7.1 Parasites. - Assembly of a protective cell wall by parasites is essential for survival outside the host and for transmission among susceptible hosts. Their structure and biosynthesis obviously presents targets for drug targeting. The structure of the filamentous cell wall of Giardia intestinalis determined by chemical methods, mass spectrometry and 'H NMR spectroscopy''7 showed a 3:2 ratio of carbohydrate to protein (w/w) and the carbohydrate to be composed of a novel homopolymer of (p1-3)-linked GalNAc. Conformational analysis by molecular dynamics and persistence length calculations demonstrated a flexible structure consisting of left and right handed helical elements. It is to be further


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investigated whether in the solid state the polysaccharide forms ordered helices or possibly multiple helical structure having strong interchain interactions. This would lead to the highly insoluble nature of the cyst wall together with a strong association between the carbohydrate and protein. 7.2 Algae. - Polysaccharides of the red seaweed Apophlea lyallii were extracted with hot water, fractionated by DAEA-sephadex ion exchange chromatography and characterised by neutral sugar analysis, linkage analysis and NMR spectroscopy."* A major fraction obtained was composed of a unique (2-0methyl ga1acto)-glucurono-xylo-glucanwith a repeating unit structure [2-0Me-Galp(a 1-4)GlcAp(p1-3)Xylp(a1-4)-1. New dimeric macrolide glycosides have been isolated from the marine sponge Myriastra clauosa, clavosolides A-D.'19

7.3 Fungi. - Lyophilised fungal cells extracted with chloroform/methanol mixtures yields lipid and saccharide components discussed below: 7.3.1 Oligosaccharide. Alkali extractable and water soluble cell wall polysaccharides (PS) have been used as chemotaxanomic markers in the fungal genus Geosmithia:chemical and 'H NMR analysis of the PS revealed similar structures of galactofuranose chains linked to a mannan core of G. namylowskii and G. putterillii with Eupenicillium and G . uiridis shared a glucogalactomannan-type structure with TalaromycesJlauus.120The two main water extractable polysaccharides produced by the basidomycete fungus Pleurotus ostreatoroseus Sing were characterised121using 13C, 'H and 'H, 13C HMQC NMR spectroscopy, methylation analysis and Smith degradation as an (al-6)-linked mannan branched at C-2 with side chains of different lengths containing (al-2)- and (al-3)linked mannose residues and a partially 3-0-rnethylated-(a1-4)-linkedgalactopyran. The former is representative of fungal mannans but the latter has not previously been described. The complex structures of the phosphonogalactomannans of Aspergillus uersicolor mycelia were determined by 31P,'H and 13C NMR with methylation analysis. A similar mannan core as discussed above was substituted at C-2 and C-6 with side chains of (1-5)-linked p-galactofuranose units which became shorter with increasing culture age.'22The structure of the polysaccharides isolated for the cell-wall of three isolates of Verticillium fungolica was investigated by chemical analysis, 1D and 1D 'H NMR and I3CNMR.lZ3 An iso-benzofuranone obtained from the endophytic fungus Pestalotiopsis microspora identified as isopestacin having a substituted benzene ring attached at the C-3 of the furanone ring possesses anti-fungal activity and behaves as an antioxidant scavenging both super oxide and hyroxy free radicals.124The structure of the disaccharide repeating unit of the phenol soluble polysaccharide from Shewanella putrefaciens strain A6 was shown to be composed of two nine-carbon sugar units, 5-acetamido-7-acetamidino-8-O-acetyl-3,5,7,9-tetradeoxy-L-glycero-a-D-galacto-non-2-ulosonic acid (8eLeg) and 2-acetamido-2,6-dideoxy-4C-(3' -caboxamide-2,2'-dihydroxypropyl)-~-D-galactopyranose (proposed as Shewanellose, She).'2s

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7.3.2 Lipid. Approximately 13% of the lipid fraction of the soil filamentous fungi Mortiella alpina were cerebrosides'26characterised by 'H NMR, 13CNMR and MALDI-TOF mass spectrometry as glucopyranosyl sphingadienes having unusual fatty acid composition, 2-hydroytridecanoic (4%), 2-hydroxytetradecanoic (6O%), 2-hydroxypentadecanoic (20%) and 2-hydroxyhexadecanoic (16%) acids. Complex glycoinositolphosphoryl ceramides (GIPCs) were purified from a pathogenic encapsulated wild type strain of Cryptococcus neoformans and from an acapsular mutant.'27 In the mutant as well as 2-hydroxy-tetracosanoic acid the unusual 2,3-dihydroxy-tetracosanoic acid was characterised as the N-acyl group of C18 phytosphingosine. The oligosaccharide moieties were also more varied in the mutant with Manp(al-6), Manp(al-6)Manp(a1-6) and Manp(a12)Manp(a 1-6)Manp(al-6)substituents linked to the non-reducing terminal mannose residue found in the wild type Manp(al-3)[Xylp(fJl-2)]Manp(al4)Galp(Pl-6)Manp(al-2)Ins-l-phosphorylceramide. The definitive structures of 43 sterol molecular species in rat-derived Pneumocystis carinii were elucidated by NMR spectroscopy.'28As reported by Giner et a1,12*mammals cannot alkylate the C-24 position of the sterol side chain nor can they desaturate C-22. Thus the reactions leading to these sterol modifications are particularly attractive targets for the devlopment of drugs against fungal and protozoan pathogens that make them. Ceramide monohexosides (CMHs) were purified from lipidic fractions of the fungus Pseudallescheria boydii which is pathogenic in immunocompromised The biological results suggested that CMHs are involved in fungal differentiation. CMHs have previously been identified as products of Magnaporthe griseus, a fungal pathogen that infects rice leaves and causes rice blast, a devastating crop disease.13' The CMHs were shown to be elicitors of the hypersensitiveresponse. Ceramide dihexosides have now been characterised by chromatographic approaches, MS and NMR.13' A phospholipomannan of Candida albicans has been characterised as a new member of the fungal mannose inositolphosphoceramide family.131

7.4 Mycobacteria. - Mycobacteria contain lipoarabinomannans (LAMS) which act as virulence factors and stimulate the immune system by interacting with the CD14/Toll like receptor system. Mycobacterial LAM structure was compared to the lipoglycan found in Rhodococcus equi a major cause of foal morbidity and m ~ r t a l i t y . 'In ~ ~contrast to mycobacterial LAM there are no extensive arabinan domains but single terminal a-D-Araf residues capping the Man branches C-2 linked to the a-1,6-mannan backbone linked to phosphatidylinositol.

7.5 Bacteria and Vaccines. - Secreted bacterial natural products and polysaccharides (PS) can have various biological activities as exemplified in the next paragraph. The oligosaccharides (OPS) and polysaccharides of their lipolysaccharides (LPS) are studied, for structural information, vaccine development, to define cross reacting epitopes in human autoimmune disease and to differentiate species. A cross section of recent structural studies is given below and structural


369

and conformational studies leading to vaccine development are then discussed. All the information on structure and biosynthesis obtained is useful to define the genome involved in PS and natural product synthesis to help to annotate the genomes not only of bacteria but for fungus and plants with related pathways in biosynthesis. A separate Subsection below is given to bacterial natural products as biosurfactants. Bacterial natural products. A new macroilide antibiotic designated as chalomycin B was isolated from murine Streptomycete. The structure was determined by EI and ESI-MS, 'H, I3C and 2D NMR and by comparison of the data with those of chalcomycin from which it varied by having a COCH2CH3 group at C-3 of one of the two monosaccharides linked to the C17 of ma~roi1ide.l~~ A family of nucleoside-lipoprotein antibiotics also from Streptomyces (sp LLAA896) were characterised by NMR.134Nineteen compounds, the muramycins, were found with core structures containing a glycosylated uronic acid derivative joined by an aminopropane group to a hexahydro-2-imino-4-pyrimidyylglycyl residue containing dipeptide that is further extended by a urea-valine moiety. They exhibited a broad spectrum activity via inhibition of peptidoglycan biosynthesis. The total synthesis of the major catabolite of the anti-tumour agent bleomycin and its various glycosides was achieved to study their respective activities in DNA ~1eitvage.l~~ NMR confirmed the structures. The biosynthesis in Streptomyces species of tunicamycin and an analogue tunicamine (an 11carbon dialdose sugar) has been studied136by identifying isotopically labelled intermediates by 'H, 2H and 13C NMR. Tunicamycin is a useful inhibitor of protein glycosylation and inhibitors with related activities have been used to suppress virus sub-unit assembly. Structural studies. Brisson et provide the structure and conformation by high resolution NMR of the lipid A core oligosaccharide of the LPS of Mannheimia (Paturella) haemolytica serotype Al. The lipid A and the 0-specific polysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn's disease were the subject of a structural The structure of the OPS was determined by chemical analysis and NMR spectroscopy to be a polysaccharide composed of the following repeating unit [-4)Rhap(a1-3)Manp(1-1. Structures of the linkage region between the core and the polysaccharide 0 chain in the LPS of Klebsiella pneumoniae serotypes 01, 02a, 02a,c, 0 3 , 0 4 , 0 5 and 0 1 2 were determined by NMR and chemical methods.139 Pseudomonas aeriginosa immunotype 1 LPS was studied after mild acid and strong alkali degradations by MS and NMR spectros~opy.'~~ The structures of the LPS and lipooligosaccharides of Campylobacter jejuni 8 1116 previously not described have now been reportedI4' and the structure of the OPS of Bordetella h i n ~ i i . ' ~ ~ The structure of the trisaccharide repeating unit of the 0-polysaccharide of Providencia alcalifaciens 0 16 was determined using COSY, TOCSY, NOESY and HSQC experiment^.'^^ The presence of N-acetylmuramic acid was confirmed by solvolysis with trifluoromethane sulfonic acid and its optical rotation determined. The 0-polysaccharides of phytopathogenic bacteria Xanthomonas

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campestris were characterised and shown to resemble those of some Pseudomonas syringae strains,'44whereas the osmoregulated periplasmic glucan (OPG)of X . campestris was shown to have the same core as free living Rhodobactcter sphaeroides: trichloroacetic acid treatment of the latter organism'45revealed a cyclic glucan containing 18 Glc residues that can be substituted by 1-7 succinyl ester residues at C-6 and one or two acetyl groups. Two 0-antigens of strains PCM 1542 and 1544 of Citrobacter gilleni should be classified to different 0-serogroups based on NMR structural ana1~sis.l~~ The lipid A fraction from the LPS of Pseudomonas cochorii has been characterised by NMR and MALDITOF MS and shown to have remarkable heterogeneity with respect to the fatty acid and phosphate components which may have potential biological signifi~ a n c e . 'Production ~~ of tricarboxylic acid anhydrides from fatty acids by a lipase-producing strain of the soil bacterium Pseudornonas cepacia has been studied for its effects of feeding oleic acid on production of lipase inducers.'48The compounds showed moderate antibacterial and antifungal activities. Lutnaes et all4' have reviewed the NMR assignments of selected bacterial carotenoids. The 0-specific polysaccharide of Proteus penneri 103 was studied by 2D COSY, TOCSY, NOESY, 'H-detected 'H, 13C HMQC, 'H, 31PHMQC and HMBC experiments and shown to contain oligosaccharide-ribitol repeating units resembling ribitol teichoic acids of Gram positive ba~teria.'~' This is one of a series of papers by the authors proposing the classification of P . penneri strains based on their OPS. The structure of the OPS of P . vulgaris was characterised with a novel regioisomer of N-acetylneuraminic acid, 2-acetamido-4-O-[(R)-Icarboxy ethyl]2-deo~y-D-Glc.'~'The structure of the OPS of the LPS was ~haracterised'~~ of a prototypical strain of Hafnia alvei that has characteristics of a new species under the genus Escherichia although, except in overall composition, the LPS is not identical to any of the previously investigated LPS 0 chains of E. coli or H . alvei. NMR analysis has been used to characterise the 0-antigen polysaccharide from E . coli F171 shown to be identical to the previously described E.coli 0 2 5 and thus should be ascribed to this subgroup.'53Conformational studies have been performed on a pentasaccharide derived from the 0-polysaccharide from E. coli 0142 and comparison made of the polysaccharide and component repeating units by NMR spectro~copy.'~~ The conformational flexibility of the Group B meningococcal polysaccharide in solution was characterised by high resolution NMR.'" The data were consistent with internal and segmental motions dominating the nuclear magnetic relaxation of the polysaccharides and not molecular tumbling. Statistical distributions of correlation times were found specifically for the pyranose rings, linkage groups and methoxy groups that accounted for the measured relation times and NOE enhancements. Conformational studies by molecular modelling and NMR spectroscopy including NOE and residual dipolar coupling have been performed on the hexasaccharide repeating unit of capsular polysaccharide of Vibrio cholerae 0 3 9 an isolate associated with several outbreaks of cholera in the past decade.'56The structure contains a tetrasaccharide epitope homologous to the human Leb blood group antigen that adopts a relatively well-defined conformation. Saturation transfer difference 1D-TOCSY has been used to map the


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topography of oligosaccharides recognised by a monoclonal antibody directed against the cell wall polysaccharide of Group A Streptoco~cus.'~~ As discussed further by Choi et al,'" in contrast to the conventional dogma that carbohydrates are poorly immunogenic T-cell independent antigens, some polysaccharides can significantly stimulate T-cell proliferation. These authors have studied the type 1 capsular polysaccharide form Streptococcus pneumoniae and PSA2 of Bacteroidesfragilis by comparison of conformation and T-cell activities. Berkin et alls9 report progress towards a synthetic glycoconjugate vaccine against Neisseria meningitides A based on a conjugate of albumin and a complex phosphate containing ManNAc trisaccharide. The conformation of the hexasaccharide repeating subunit from the Vibrio cholerae 0139 capsular polysaccharide was characterised from NMR and molecular modeling'60 of a fragment obtained by a new polysaccharide lyase which cleaves at (1-4)-linked GalA residues. Bacterial genomics. Cloning in Escherichia coli and nucleotide sequence analysis of the Streptococcus sobrinus gtfU gene that produces a highly branched water soluble glucan has been reported. The protein produced was identified by its biological properties and a linkage analysis of the glucans by 13C NMR.16' Studies, involving for example 31PNMR in lipid A biosynthesis,162have been used to follow expression of bacterial enzymes. The lpxH gene is the newly designated name for UDP-2,3-diacylglucosamine hydrolase.162The lbgAB gene cluster of Haemophilus ducreyi encodes a p- 1,4-galactosyltransferase and an a-1,6 DD-heptosyltransferase involved in LPS bio~ynthesis.'~~ Bacterial Surfactants. The structural and conformational changes on micellar solubilisation of surfactants were investigated by 'H and 13C NMR in the presence of different cosurfactant~.'~~ Pulsed field gradient NMR was used to measure the solution equilibria in amino acid and dipeptide terminated micellar and polymeric surfactant ~ ~ l ~ t i oThe n ~flocculent . ' ~ ~ properties of polysaccharides of Klebsiella pneumoniae H12 have been ascribed by NMR and MS methods to a complex polysaccharide which is a linear polymer of a unit of pentasaccharide with a side chain of one D-GlcA to D-Glc moiety.'66Glycolipid surfactants produced by Pseudomonas aeruginosa D2D2 from diesel-contaminated

7.6 Plants. - Liakopoulou-Kyriakides et a116%eviewthe recent advances in the separation and characterisation by MS and NMR of the volatile and nonvolatile components of the dried stigma of Crocus sativus (saffron) and their biological activities. From the flowers of Dregea volubilis three novel polyoxypregnane glycosides named volubilosides A, B and C with glycoside component P9:

D-Glcp(~1-4)6-deoxy-3-0-methyl-D-Allp(~ 1-4)cymaropyranosyl(P 1-4)-Dcymarop yranoside D - G1cp ( p 1-4)6 -d eo x y - 3- 0-met h y 1- D -A 11p ( P 1 -4)cymar opy ran o sy I( P 1-4)-D digitoxopyranoside.

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The corolla of Brassica rapa has an UV absorbing zone in its centre, known as the nectar guide for attracting pollinating insects: the pigment which plays the role as the nectar guide was isolated from the petals and identified to be isorhamnetin 3-0-di-p-D-glycoside on the basis of MS and NMR spectrocopy.'^^ From the ethyl acetate fraction of Conya canadensis Naveen et all7' isolated and characterised by NMR a novel sphingolipid and its p-Dglucopyranoside derivative along with p-sitosterol, stigmasterol, p-sisterol 3-0p-D-glucopyranoside and harmine. From the flowers of Nerium indicum a rhamnogalacturonan and a xyloglucan were isolated and characterised by NMR and other techniques and their structures correlated with activities similar to nerve growth factor on the proliferation and differentiation of pheochromocytoma cells.17* The leaves of Hyparrhenia hirta yielded rare diastereoisomeric flavonolignans and their glycosides that are the first flavonolignan glycosides to be isolated as natural products. A complete set of 'H and I3C NMR chemical shift data has allowed reassessment of the previous literature on related molecule^.'^^ From the aerial parts of Veronica pectinata var. glandulosa three new phenylethanoid glycosides have been isolated and identified and their free radical scavenging activity assessed.'74The leaves of thyme (Thymus vulgaris) yielded natural products which inhibited platelet aggregati~n.'~~ Two new triterpenoid saponins have been isolated from the fruits of Gledistsia dolavayi F r a n ~ h .the ' ~ ~complete 'H and 13Cassignments were achieved on the basis of 2D spectra Including HMQC-TOCSY, TOCSY, 'H-'H COSY, HMBC, ROESY and HMQC of gledistside A and B differing in the methylation of the lipid but both having the same glycoside components p-D-glucopyranosylechincystic acid having D-Xylp(Pl-2)-L-Arap(al-6)-D-Glcp(fil-28-0) and D-Xylp(f313)-D-Xylp(p1-4)[D-Galp( 1-2)]-L-Rha(al-2)motifs. Three acylated iridoid glycosides from the seed pods of Scrophularia nodosa were characterised by 1D and 2D NMR and by comparison with literature data. All three contained a-L-Rha and were able to stimulate the growth of human dermal cells in vivo consistent with the ethnobiological use of mature seed pods in wound healing.'77Weckerle et have characterised by 'H and 13CNMR as well as ESI MS/MS the structures of 3-methylbutanoyl and 3-methylbut-2-enoyl disaccharides from green coffee beans. Five new glycosides were among a large number of phenyphenalenonerelated compounds isolated from Xiphoidium caeruleum of the plant family Haem~doraceae.'~~ The biosynthetic relationships of the characterised structures were explored as the basis to elaborate biogeographic characteristics of this plant family. Thirteen new glycosides (glucosidesand allosides) were isolated from the fern Hymenophyllum barbatum and their structures elucidated by extensive 2D NMR.'" The structures of the aglycones were: 2-methyl-but-2-ene-1,4-diol 2-hydroxymethyl-but-2-ene- 1,4-diol 2-methylene-butane-1,3,4,-trio1 3-hydroxy-5-hexanolide. 0leszak''l has reviewed the chromatographic determination of plant saponins


373

and introduced the use of LC-NMR for online identification. Triterpenoid saponin glycosides of Lupin angustifgolius were isolated from whole seeds and characterised by NMR and for their anti-fungal activity.lS2Antimicrobial terpenoids have been identified from the oleoresin of a Peruvian medicinal plant Copaifera paupera, some with activity against cancer cell lines."3 From the rhizomes of Iris germanica six known isoflavones were isolated, identified by NMR techniques and vsed in studies to elucidate their activity as inhibitors against cytochrome P450 (active) and as anti-tumour agents based on an antiinflammatory mechanism (not active).184From the methanolic extract of the underground parts of Globularia orieiitalis a new anti-oxidant sugar ester was isolated, 6-O-feruloyl-Glc-(P1-6)glucitolcharacterised by 1D and 2D NMR, ESI and FAB-MS and confirmed by chemical means.lS5Three new triterpene saponins were isolated from the roots of Silene vulgaris, gypsogenin 3 - 0 glucuronide, quillaic acid 3-0-glucuronide and glysogenin 3-O-glycoside, characterised by 'D and 2D 'H and 13Cmethods including COSY, HSQC, HMBC and HSQC-TOCSY.lS6From the 95% ethanol extract of the roots of Albizia adianthifolia were isolated and characterised by COSY, NOESY, TOCSY, HSQC, HMBC and FABMS, HRESIMS complex multiply glycosylated oleanine-type triterpene saponins with varying activities in immunomodulation, lymphoproliferation and haemolytic a~tivity."~ A new steroidal saponin from the total plant Tribulus terrestrisl., terretroneoside A containing six monosaccharides was charcterised."' 2D NMR was essential to prove the oligosaccharide linked as a 2,3,4 branched (3-D-Galp having a-L-Rhap at C-2, 26-0-Glcp-22methoxyfuro-stane at C-3 and Xylp((31-3)Xylp((31-2)Glcp at C-4. From the acetone extract of Ginkgo biloba leaves which had strong activity against xanthine oxidase was characterised by 'H and 13CNMRlS9the compounds kaempferol 3-0-a-[6"'-p-coumaroylglucosyl((31-4)rhamnosidel and quercertin 3-0a-[6"'-p-coumaroylglucosyl(~1-4)rhamnosidel. The essential oils of Mentha suaveolens, a plant of the Labiatae family which grows wild in Morocco, were characterised and their anti-microbial activity assessed along with a series of other aromatic molecules.190The main aromatic substituents of M . suaveolens were pulegone, piperitenone oxide and piperitone oxide of which the first amongst all the compounds tested was the most active against 19 bacteria (including Gram +ve and Gram -ve) and three fungi. New pregnane glycosides were isolated from the whole plant Caralluma neogevensis, their structure elucidated by extensive spectroscopic methods and correlated with their cytotoxic and genotoxic activitie~.'~~ From the roots of the African medicinal plant Bulbine frutescens novel phenylanthraquinone glycosides were isolated and charcteri~edl~~ by HMBC, HMQC and ROESY NMR together with CD. From the roots of Muraltia ononidofolia several saponins were characterised with complex Xyl, Ara, Rha and Glc monosaccharide containing oligosa~charides.'~~ From an ethyl acetate fraction of a methanol extract of the root bark Morus alba was ~haracterised'~~ a product called kuwanon G with antibacterial activity against, for example, Streptococcus mutans, S. sobrinus, S. sanguis and Porphyromoans gingivalis which hence might be useful in human oral pathologies. Shim et also report on the

374


action of oleanolic acids from Physalis angulata on P . gingivalis and S. mutans.The complete NMR assignments of seven protopanaxatriol glycosides has been reported from the saponin fraction isolated from mild acid hydrolysis products of crude root isolates of Pana n ~ t o g i n g s e n g . ' ~ ~ From the stems of Kadsura interior two new lignans characterised by MS, CD and NMR and together with 12 other related lignans graded for their potential as anti-tumour promo tor^.'^^ From the bark of Mytragyna inermis two novel 27-nor-triterpenoid glycosides named inermisides I and I1 were isolated and the structures determined by extensive 2D NMR and MS spectral analysis as 6-deoxy-D-Glcp-pyrocincholate and the 6-deoxy-Glcp ester of Glcp(f31-6)Glcppyrocin~holate.'~~ M e r f ~ r thas ' ~ ~reviewed the literature of the last two decades on sesquiterpenes and sesquiterpene lactones including their 13Cand LC-NMR. Lago et a1200characterised 17 sesquiterpenes, one diterpene and four fatty acids form the volatile oils of the stem bark of Guarea macrophylla.The bark extract from the medicinal plant Detarium microcarpum on the other hand gave relatively simple isolates of quinolactone, bornesitol, pinotol, myo-inositol, sucrose, glucose and fructose analysed by NMR of their benzoate derviatives.201 From the heart wood of Punica granatum two new ellagitannins, diellagic acid rhamnosyl(1-4)glucopyranoside and 5-0-galloylpunicacortein D were isolated together with four known tannin metabolites.202A combination of techniques was used to localise the 0-acetyl substituents in xylosyl oligosaccharides present in hydrolysates of hydrothernally treated eucalyptus

8

Polysaccharides

In a series of studies looking at the various components of prickly pear and their natural product make up the group of Vignon et a1204have published about xylans isolated from the pericarp of prickly pear seeds of Opuntaficus-indica.The (p1-4)-Xyl backbone characterised has GlcA residues attached at C-2. The water soluble xylans have one non-reducing terminal residue of 4-0-Me-D-GlcA for every 11-14 Xyl units whereas in the water non-soluble xylans there is only one 4-0-Me-D-GlcA unit for every 18-65 Xyl units. In celery (Apium graveolens) parenchyma cell walls the amount of xylans205was remarked on as being very low (2molOh) with cellulose (43mol%) and pectic polysaccharides ( 5Imol%) making up the larger percentage and smaller amounts of rhamnogalacturonan (28 mol%), arabinan (12 mol%) and galactan (11 mol%). Cross polarisation MAS solid state 13CNMR was used to analyse the proton spin relaxation times of the cellulose (rigid) and rhamnogalacturonan (semi-mobile), whereas the arabinans being the most mobile responded to single-pulse excitation but not cross polarisation MAS 13CNMR. Definitive structural studies have also been performed on the hairy region of pectic polysaccharides from the aerial part of Silene vulgaris containing GalA (43%) arabinose, galactose and rhamnose as the major constituents?06 The arabinofuranose backbone of pectic polysaccharides of olive pomace was chara c t e r i ~ e d .Structural ~~~ studies of the pectic polysaccharide from duckweed


375

Lemna minor L. obtained after partial acid hydrolysis of the 'hairy regions' was shown to contain terminal and P-1,5-linked apiofuranose, terminal and a-1,5linked Araf, terminal and f3-1,3 and f3-1,4 linked Galp, terminal and P-1,4-linked Xylp and p-1,4-linked 2-mono-0-methyl Xylp with some side chains GalA residues.208Further complexity was found in the patterns of methyl and 0-acetyl esterification of GalA and Rha containing spinach pectins.209Studies of the OPS of the plant growth promoting bacterium Azospirillum brasilense Sp245 identified a new rhamman with a pentasaccharide repeating unit.210Exopolysaccharides excreted by thermophilic bacteria from shallow hydrothermal vents were shown by GC-MS and NMR to be a mannan and an EPS with a repeating unit formed by 7 monosaccharides, 6 with a-glucolgalacto configuration and one with P-conformation.21' The structural features of the water-soluble arabinogalactans from Norway Spruce and Scots pine heartwood were determined by 13CNMR and methylation analysis and compared to Siberian larch hardwood.212The affect of acetylation on the material properties of glucuronoxylan from aspen wood was determined by 'H NMR spectros~opy?'~ A HSQC approach via suppression of J-dependence of polarisation of transfer in NMR spectroscopy was applied to wood lignans, a complex polymer where it has been difficult to obtain data on the relative amounts of different structural Lignins have also been studied by 2D and 3D NMR spectroscopy to unravel their complexity.215Cross polarisation MAS solid-state NMR has been used to study archaeological woods.216 New structural features of the high-arabinose, highly acidic heteropolysaccharide from gum ghatti (Anogeissus lati$olia) are The molecular mobility was measured in the xanthan of locust bean gum by solid-state 'H and 2H NMR and 2H high resolution NMR.218The polysaccharide obtained by ethanol precipitation from an aqueous solution of gum tragacanth contained arabinogalactan and tragacanthic acid as well as starch (approx 6%). NMR, GC-MS and ESI-MS showed the structure of the arabinogalactan to be even more complex than previously thought219with core structures containing Arap, P-Arafand a-Galp units as well as the known terminal and 2-0- and 3-0substituted a-Arafunits. Reducing oligosaccharides present in the gum included:

AraA 1-2)Araf(l-4)Arafand Arafi 1-2)AraJT1-5)Araf Galp((3 1-4)Galp(p 1-4)Galp(1-4)Gal, Galp(p 1-4)Galp(p1-4)Galp Glcp(P1-4)Galp(p 1-4)Galp(p 1-4)Galp. The synthesis of arabinofuranogalactan type oligosaccharides has been achieved with characterisation by 'H and 13C NMR and MALDI-TOF mass spectr ometry.220 Relating to food and dietary fibre, the characterisation of the dietary fibre in cocoa shell by solid-state NMR showed that it contained little if any lignan but probably protein-Maillard-tannin complexes.221Other polysaccharide components also identified were pectic polysaccharides, rhamnogalactans fucosylated xyloglucans, glucuronarabinoxylans and cellulose. The glucomannan produced by the plant Amorphophallus konjac was characterised at the component oligosaccharide level isolated from a cellulose digest,222'H and 13CNMR have

376


been used to characterise partially and completely acetylated derivatives of chitin in the intra-lamellar matrix of abalone nacre.223 Shacher-Hill 224 has reviewed the role of NMR in plant metabolic engineering not only to measure metabolite levels and metabolic fluxes (cf. metabolomics studies in Section 3 above), but also to understand plant physiology and to test hypotheses about the effects of engineered traits on plant physiology and function. Leading on to Section 9 one can imagine the importance of this to materials science.

9

Materials Sciences

This section goes full circle to the beginning of the review being primarily concerned with getting compounds to and through membranes. There are many studies on cyclodextrins as drug carriers and their NMR analysis essential for structural and conformational inferences. The stoichiometry, geometry, stability and solubility of the inclusion complexes of melatonin with native cyclomalto oligosaccharides were determined experimentally by high-resolution NMR spectroscopy, calorimetric and solubility measurements and mass Synthesis and characterisation of the inclusion compound of a methyltrioxorhenium(VI1) adduct of 4-ferrocenylpyridene with p-cyclodextrin have been reported.226Highly sulphated cyclodextrins have been studied for use in chiral analysis by capillary electrophoresis the relative position of the C-2 sulphate, C-3 hydroxyl and hydrophobic enclosure of the cyclodextrins provide regio-specific differential interaction with each e n a n t i ~ m e r . ~ ~ ~ New nanoparticles and polymeric vesicles from amphiphiles having a po1y-Llysine backbone and varied levels of hydrophilic methoxypolyethylene glycol and hydrophobic palmitoyl pendent groups have been studied.228The synthesis of glycosylated poly(amido-amines) using novel amino sugar derivatives and the characterisation of the presence of sugar residues in the polymer chains by FT-IR and ‘H and I3C NMR has lead to compounds that are potential soluble drug carriers with specific targetti~~g.”~ The dynamics of polymer layers has been investigated by NMR techniques.230(1-diphenylphosphino-3-benzy1oxy)propan-2-thiol has been prepared as a chiral ligand and studied by NMR as its palladium11 complex.231 Chitosans have proven useful as material in drug targeting. Chitosans with oligosaccharide branches have been prepared by the group of Smidsrod et al.232 The release of model macromolecules controlled by the hydrophobicity of palmitoylglycolchitosan gels characterised by ‘H NMR.233Conjugates of silk fibrin and chiotoligosaccharides characterised by NMR have been investigated as growth retardents of Escheirchia The synthesis and characterisation of a novel glycopolymer with protective activity towards human anti-a-Gal antibodies has been reported.235This has specific application in the clearing of antibodies to pig hearts in xenotransplanation but the principles learned may be appropriate to other filtration mechanisms. The solution structure of two xenoantigens a-Gal-LacNAc and a-Gal-Lex


377

are further described.236 Novel glycolipids which contain 2 and 15 oligomaltose units and a phosphatidylethanolamine were synthesised and characterised by FTIR and 'HNMR.237Conformations of the glycolipids at the air-water interface were proposed based on film balance measurements. The glycolipids demonstrated unique conformations and the ability for sterically stabilising liposomes. Mixed micelle formation by aqueous mixtures of pentaethylene glycol monohexyl ether and diethylene glycol monohexylether was studied by measuring micelle intradiffusion coefficients by a pulsed gradient spin-echo-NMR The experimental evidence suggested that in mixed ethoxylated surfactants the micellar behaviour is mainly determined by the mean number of ethoxy units per surfactant molecule independently of their distribution. Yokohama et review the use of ES-MS and NMR spectroscopy for identification of unknown surfactants. Rasmusson et a1240discuss a dilute surfactant system based on a quaternary mixture of SDS/wa ter/pent anol/dodecane.

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11 Synthetic Macromolecules BY HlROMlCHl KUROSU AND TAKESHI YAMANOBE

1

Introduction

For synthetic macromolecules, NMR has been the most powerful method to characterize and to investigate the relationship between the structure and the physical properties. In the field of synthetic macromolecules, NMR is used not only as the routine analytical method but also as the method that has the infinite possibility. In this chapter, NMR applications for synthetic polymers are reviewed. Cheng and English edited ' NMR Spectroscopy of Polymers in Solution and in the Solid State' including solution and solid state NMR applications to polymers.' Ando et al. reviewed pulse field gradient spin echo NMR and MRI method to investigate the structure and dynamics of polymer g e k 2Kameda and Asakura reviewed dynamic NMR studies of the silk fibroin from silk worms.3 Spiess overviewed the role of NMR in macromolecular science hi~trically.~ Scheler reviewed the topics about the strucure of polymers, polymer dynamics, phase separation of polymers oriented polymers and fluorop~lymers.~ 2D NMR and pulsed gradient diffusion experiments and applications are reviewed by Newmark.6 Reviews about NMR study about the ring-opening metathesis polym e r i z a t i ~ n .the ~ ' ~radiation modification of polymer' and in situ NMR spectroscopy in free radical polymer synthesis."

2

Primary structure

Characterizations of primary structures such as tacticity, regioregularity, end group, sequence distribution, and so on are investigated mainly by solution NMR. Table 1 summarizes the papers in which NMR is used to investigate the primary structure of polymers.

Nuclear Magnetic Resonance, Volume 33 0The Royal Society of Chemistry, 2004 386

16 17 18 19 20 21 22 23

degradation degradation degradation composition end group end group regioregularity, stereoregularity tacticity branch configuration degradation configuration configuration configuration branch branch configuration tacticity end group, molecular weight

25 26 27 28 29 30 31 32 33 34

24

11 12 13 14 15

Re5

block copolymer seqeuence distribution degradation degree of chloromethylation sequence distribution

Nucleus Contents

(star polystyrene)-block-(linearpolydimethylsi1oxane)-block-(starpolystyrene) 6-phenyl-6-deoxy-2,3-di-O-methylcellulose Biodegradable epoxy-polyester resins Chloromethylated polyimide acid, copolyarylenephthalide,ortho-(4’-phenoxybenzoyl)benzoic ortho-(4’-phenylthiobenzoyl)benzoicacid natural rubber Nylon Nylon 6 polyimide,polyimide,2,2-bis(3,4-dicarboxyphenyl)hexafluoro propanedianhydride, 2,3,5,6-tetramethyl-1,Cphenylenediamine, 2,6-diaminotoluene, m-phenylenediamine perfluoroalkoxy resin poly (E-caprolactone), poly (L-lactide) poly((4-(7-octenyloxy)-4’-cyanobiphenyl)-alt-CO), polyketones poly((4-hydroxystyrene)-block-((4-methylstyrene)-co-(4-hydroxystyrene))) poly( 1-(2-hydroxye thoxy)-3,5-bis-(methox ymethy1)-2,4,6-trime t hyl benzene) poly(1,3-butadiene) poly(1,3-cyclohexadiene) poly(1,3-cyclohexadiene) poly(1,3-cyclohexadiene) poly(1,3-dihydroxypropyl methacrylate-est-2,3-dihydroxypropyl methacrylate) poly(1,7-0ctadiyne) poly(1,7-0ctadiyne) poly( 1,9-decadiene) poly( - .1-octadecene) poly(2-(1 H, 1 H,2 H,2 H-pe~uorohexyl)-2-oxazoline)-block-poly (2-methyl-2-oxazoline), H poly(2-nonyl-2-oxazoline)-block-poly(2-methyl-2-oxazoline)

Polymer, monomer

Table 1

cr

8

E

c, rn

2

3

% R

.-

ic;‘c

k

01

(cont.)

poly(2-(1-imidazoly1)ethylmethacrylate-co-tetrahydropyranyl methacrylate) poly(2-(N-carbazolyl)ethyl methacrylate)-block-poly(2-(dimethylamino)ethyl methacrylate) poly(2,2,2-trifluoroethyl a-fluoroacrylate) poly(2,2’-bis(2-thiophene)-l,l’-binaphthlene-co-2,2’-bis(5-bromo-2-thiophene)1,l’binaphthlene) poly(2,2-diphenyl-4,4,6,6-tetramethylsiloxane) poly(2,3,3-trifluoro-2-propen1-01-co-vinylidenefluoride) poly(2,3-difunctionalized 7-oxanorbornene-co-cyclic olefin) poly(2,3-dihydroxy-1,4-napht h ylene) poly(2,6-dihydroxy-1,5-naphth ylene) poly(2-[(2-bromopropionyl)oxy]ethyl acrylate-co-methyl acrylate) poly(2-[(S)-2-(1-pyrrolidinylmethy1)-1-pyrrolidinylmethyl]st yrene), poly(2-[(S)-2-(methoxymethyl)-1-pyrrolidinylmethyl] styrene), poly(2-[(S)-2-(N,N-diet hylaminomethy1)-1-pyrrolidin ylmethyl] styrene) poly(2-ar yl- 1-meth ylenecyclopropanes), poly(2-ethoxycarbonyl-1-met h ylenecyclopropane) poly(2-cyano-3-phenyl-2-propenoates-co-styrene) poly(2-ethy1hexyl acrylate) poly(2-ethynylthiophene) poly(2-hydroxyethyl methacrylate), poly(N-vinyl-2-pyrrolidone) poly(2-hydroxypropyl methacrylate) poly(2-methylbenzyl methacrylate-co-me t hyl methacrylate), poly(4-methylbenzyl methacrylate methyl methacrylate) poly(2-methylene-4-oxa-12-dodecanolide), poly(2-methylene-4-oxa- 16-hexadecanolide), 12-olide), poly(8,9-benzo-2-methylene-4,7,lO-trioxa-8-dodecenpoly(9,10-benzo-2-methylene-4,8,11 -trioxa-9-tetradecen- 14-olide) poly(2-Phenylethyl acrylate-co-methyl acrylate),poly(2-Phenylethyl methacrylate-co-N-vin yl pyrollidone) poly(2-tetrah ydropyranyl methacrylate)-bloc k-pol yfluorene-blockpoly(2-tetrahydropyranyl methacrylate)

Polymer, monomer

Table 1

37 38

39 40 41 42 43 44 45 46 47 48 49 50 51 52

53 54

55

molecular weight molecular weight tactici ty composition sequence distribution molecular weight configuration branch stereoregularity regioregularity composition branch configuration composition degradation composition tacticity composition block copolymer

H H Si F H,C C H,C

H,C

C

35 36

composition composition

Re$

H H

Nucleus Contents

cs 3

5

(/1

=. 2

5

Q

8 %

32 m

00

w

poly(2-vinylnaphthyllithium)-block-poly(tert-Buacrylate) poly(3,3-bis(hydroxymethyl)oxetane-co-3-ethyl-3-(hydroxymethyl)oxetane) poly(3,5-dimethylphenyl acrylate-co-glycidyl methacrylate) poly(3,5-dimethylphenylmethacrylate-co-glycidyl methacrylate) poly(3,5-dimethylphenylmethacrylate-co-methyl methacrylate) poly(3-et hy1-3-hydroxylmethyl oxetane) poly(3-et hy 1-3-h ydroxymeth yloxetane) poly(3-hexylthiophene),3,4-disubstitutedpoly(thiophene) poly(3-hydroxybutyrate) poly(3-hydroxybutyrate-co-3-h ydroxyhexanoate) poly(3-hydroxybut yrate-co-3-hydroxypropionate), poly(3-hydroxybutyrate), poly(3-hydroxypropionate) Poly(3-hydrox yvalerate-co-4-hydroxybut yrate) poly(3-phthalimido-2-hydroxypropylmethacrylate) poly(4-(benzyloxycarbonyl)phenylacrylate-co-methyl methacrylate) poly(4,7-dimeth yl-2-me thylene-1,3-dioxepane-co-methy1 1,3-dioxepane-coacrylate), poly(4,7-dimethyl-2-methylenestyrene), poly(4,7-dimethyl-2-methylene1,3-dioxepane-co-acrylonitrile) poly(4-alk yl triphenylamine) poly(4-bromobenzylmethacrylate-co-ethyl methacrylate) poly(4-ethyn ylbenzoic acid) poly(4-ethynylbenzoic acid) poly(4-hydroxybenzyl alcohol ) poly(4-hydroxybenzyl alcohol) poly(4-methyacryloyloxyphenyl-3’,4’-dimethoxystyryl ketone), poly(4-methyacryloyloxyphenyl-3’,4-dimethoxystyryl ketone-co-glycidyl methacrylate) poly(4-methylpentene-1) poly(4-Vinyl pyridine-methacrylonitrile) poly(4-Vinylpyridine-co-Etacrylate) poly(5-(trimethylsiloxy)isophthaloyldichloride) poly(5,6-Benzo-2-methylene- 1,3-dioxepane-co-Meth yl Met hacrylate)

C H

71 72 73 74 75 76 77 78 79 80 81 82

configuration configuration composition branch composition

67 68 69 70

composition degradation composition composition configuration composition configuration configuration composition composition composition

56 57 58 59 60 61 62 63 64 65 66

block copolymer branch composition composition composition branch branch regioregularity degradation composition degradation

m

W 00

w

0,

a n m = z

3

n

% Q 2

n

Ei.

E5

k

h h

poly(acrylonitri1e-co-methacrylicacid), poly(acrylonitri1e-co-itaconicacid) poly(acrylonitri1e-co-methyl methacrylate) poly(acrylonitri1e-co-styrene-co-glycidylmethacrylate) poly(acrylonitri1e-co-styrene-co-glycidyl methacrylate) poly(alkynylsi1anes) poly(a-Et y,DL-glutamate) poly(a-hydroxyacrylic acid) pol y(a-methylstyrene-co-acrylonitrile) poly(amide ester) pol y(amide-urea) ether) poly(aramid silicone-co-3,4’-diaminodiphenyl poly(ary1ene arylene phosphine oxide) poly(ary1ene arylene phosphine oxide) poly(ary1ene ether phosphine oxide) poly(ary1ene ether sulfone) pol y(asparagine)-graft-poly(capro1actone) poly(aspartic acid-co-lactic acid) poly(P-cyanoethylsilsesquioxane),poly [(P-cyanoethylsi1sesquioxane)-co-(Pmethylsilsesquioxane)] poly(P-hydroxybutyrate-co-e-caprolactone) poly(bispheno1A carbonate) poly(butadiene)

107 108 109

Sequence distribution end group configuration

C H H,C

97

94 95 96

93

88 89 90 91 92

87

98 99 100 101 102 103 104 105 106

83 84 85 86

H,C H,C

poly(5,6-Bis(Chloromethyl)-Norbornene) poly(9-(bicyclo[ 2.2.1.]hept-5-en-2-ylmethyl)-9H-carbazole-co-ethylene) poly(acry1amide-co-methyl methacrylate), poly(acry1amide-co-styrene) poly(acry1icacid) poly(acry1icacid) poly(acry1onitrile-co-but yl acrylate)

configuration stereoregularity composition, block copolymer branch branch, composition H composition, sequence distribution H,C sequence distribution H,C stereoregularity H,C composition H,C sequence distribution H,C,Si,P regioregularity C block copolymer H,C stereoregularity Sequence distribution H Sequence distribution H Branch H Sequence distribution P Branch P Branch Branch H Molecular weight, end group C Graft Branch H,C ladder polymer

Re$

Nucleus Contents

(cont.)

Polymer,monomer

Table 1

2

K

Q

4

33

R‘

2

3

%

r;

(D

z

c

w u 0

end group end group Tacticity composition composition Tacticity configuration Branch tacticity, configuration composition stereoregularity

H,C F H

H P,F C

poly(buty1eneadipate-co-terephthalate) poly(buty1ene succinate-co-ethylene succinate), poly(buty1ene succinate-co-diethylene succinate) poly(buty1eneterephtha1ate)-poly(ethy1eneterephthalate-co-isophthalate-co-sebacate) poly(capro1actone-co-ethylene oxide-co-lactide) poly(carbosi1oxane) poly(chlorotrifluoroethy1ene-co-alkylvinyl ester), poly(chlorotrifluoroethy1ene-co-alkylvinyl ether) poly(cyc1ohexenecarbonate) poly(cyc1ohexeneoxide) poly(cyclohexeny1enecarbonate) pol y(cycloolefin-co-cyanoacrylate ) Poly(D,L-lactic acid)-methoxypoly(ethy1eneglycol) poly(di-but yl i taconates) poly(dicyc1opentadiene) poly(di-ethyl maleate)-graft-pol y(ethy1ene-co-a-olefin) poly(di-ethyl dipropargylmalonate) pol y(dihexy1-fluorene-co-ethylenedioxythiophene) poly(diisopropy1 itaconate)

composition end group block copolymer Sequence distribution, stereoregularity Sequence distribution composition, sequence distribution composition composition branch, stereoregularity Sequence distribution

C

poly(buty1acrylate) poly(buty1acrylate) poly(buty1acrylate-co-glycidyl methacrylate), poly(buty1acrylate-co-methyl methacrylate-co-glycidyl methacrylate) poly(buty1acrylate-co-styrene) poly(buty1a-fluoroacrylate) poly(buty1methacrylate-co-methyl methacrylate) poly(buty1methacrylate-co-styrene)

H C C C H,C H C

Si C

H

C

C

Tacticity composition, sequence distribution Branch Branch Sequence distribution

C C

poly(butadiene-co-isoprene) poly(butadiene-co-styrene)

125 126 127 128 129 130 131 132 133 134 135

120 121 122 123 124

118 119

115 116 117

112 113 114

110 111

pol y(dimethylsi1oxane)-polyamide H poly(dimethylsiloxane-co-hydrogenmethylsiloxane-co-methylphenylsiloxane) Si poly(D1-lactide) grafted gelatins H poly(D1-lactide), poly(1actide-co-glycolide) pol y(E -caprolactone) poly(endo-N-ethyl-7-oxanorbornene-2,3-dicarboxylimide-co-cyclooctene), poly(exo,exo-7-oxanorbornene-2,3-dimet hylester-co-cyclooctene),poly(exo-7oxanorbornene-2,3-dicarboxylic anhydride-co-cyclooctene) poly(epsi1on-caprolactone) H poly(epsi1on-capro1actone)-graft-hydroxypropylcellulose H,C Poly(E-caprolactone) H Poly(E-caprolactone-block-isobutylene)diblock H Copolymers and poly(E-caprolactone-block-isobutylene-block-E-capro1actone) poly(ester-carbonates), poly(5-methyl-5-benzyloxycarbonyl1,3-dioxan-2-one-co-L-1actide) pol y(ethene-co- 1-octene) poly(ethene-co- 1-octene) poly(ethene-co-norbornene) C poly(ether ketone) H poly(et hylene carbonate-co-caprolac t one) poly(ethy1enecarbonate-co-caprolactone) poly(ethy1enecarbonate-co-E-caprolactone-co-L-lactide)

141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158

end group Graft end group block copolymer, end group composition end group end group Tacticity end group Sequence distribution Sequence distribution Sequence distribution

136 137 138 139 140

Ref:

end group Molecular weight, end group block copolymer composition composition, end group, block copolymer block copolymer Sequence distribution branch graft degradation Branch Sequence distribution

H H H H

poly(diisopropy1trimethylene-1,1-dicarboxylate) poly(di-Me vinylbenzylphosphonate) poly(dimethylsi1oxane)-block-poly(2-ethyl-2-oxazoline) poly(dimethylsi1oxane)-block-poly(2-ethyl-2-oxazoline) pol y(dimethylsi1oxane)-block-polystyrene

~

Nucleus Contents

(cont.)

Polymer,monomer

Table 1

n

g.

a’ (9

\o t d

w

pol ~(ethylene-co1,7-octadiene-c0-1,9-decadiene) poly(ethy1ene-co-1-hexene) poly(ethy1ene-co-l-octene)

poly(ethy1ene glycol) methacrylate, poly(propy1ene glycol) methacrylate poly(ethy1ene glycol) methacrylate, poly(propy1ene glycol) methacrylate poly(ethy1ene glycol), poly(ethy1ene terephthalate) poly(ethy1ene glycol)-block-poly(1-lactide) pol y(et hylene glyco1)-block-polyethylenimine pol y(ethylene glyco1)-graft-poly(L-1ysine) poly(ethy1ene oxide) poly(ethy1ene oxide) pol y(ethylene oxide)-block-poly((R)-3-hydroxy but yrate) poly(ethy1ene oxide)-block-poly(4-vinylpyridine) poly(ethy1ene oxide)-block-polylactide-block-poly(ethy1ene oxide) poly(ethy1ene oxide)-block-polylactide-block-poly(ethy1eneoxide) poly(ethy1ene oxide)-b-poly(alky1glycidyl ether) poly(ethy1ene oxide-co-glycidol),poly(ethyleneoxide-co-ethoxy ethyl glycidyl ether) poly(ethy1ene succinate)-block-poly(.vepsiln.-caprolactone) poly(ethy1ene sulfide-co-styrene sulfide), poly(methy1ene sulfide-co-styrene sulfide) poly(ethy1ene terephthalate ionomer)/silicate poly(ethy1ene terephtha1ate)-block-poly(1,4-butylene succinate) poly(ethy1ene terephtha1ate)-co-poly(ethy1ene5-nitro isophtha1ate)-co-poly(ethy1ene nitroterephthalate) pol y(et hylene terephthalate-co-4,4'- bibenzoate) poly(ethy1ene terephthalate-co-butylene terephthalate) poly(ethy1ene terephthalate-isophthalate-tert-Bu isophthalate) poly(ethy1ene-alt-(cis-1,2-cyclopentane)) poly(ethy1ene-butylacrylate-carbon monoxide)

C

C

sequence distribution Sequence distribution Sequence distribution C Tacticity C H, p, F, end group, branch,tacticity Si, Sn. stereoregularity H,C composition C c Sequence distribution

H C F H

H

end group branch, end group end group block copolymer block copolymer composition degradation end group Sequence distribution block copolymer block copolymer degradation, composition composition composition composition, block copolymer composition cross link composition degradation

183 184 185

178 179 180 181 182

159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177

(cont.)

poly(glycidy1methacrylate-co-vinylidene chloride) poly(glycolide-co-caprolactone)-block-poly(ethyleneglycol) -poly(glycolide-co-caprolact one) poly(glyco1ide-co-E-caprolactone) poly(heptadiene) poly(hexa(ethy1eneglycol) methacrylate-co-2-(dimethylamino)ethyl methacrylate-co-methyl methacrylate) poly(hexafluoropropy1eneoxide-co-hexamethylcyclotrisiloxane) poly(iminomethy1enes) poly(indene-co-methyl acrylate), poly(indene-co-ethyl acrylate), poly(indene-co-butyl acrylate) poly(indene-co-methyl methacrylate-co-methacrylonitrile) pol y(indene-co-p-methylstyrene)

199 200 201 202 203 204 205 206 207 208

composition configuration composition composition conformation composition composition Sequence distribution

197 198

190 191 192 193 194 195 196

186 187 188 189

Ref:

composition composition, sequence distribution composition block copolymer

Sequence distribution Tactici ty Tacticity stereo-sequence distributions, chain-ends, tacticity Sequence distribution Sequence distribution Sequence distribution Molecular weight Sequence distribution Tacticit y composition

Nucleus Contents

poly(ethylene-co-5,7-dimethylocta1,6-diene),poly(ethylene-co-3,7-dimethylocta1,6-diene) poly(ethy1ene-co-butene) H,C poly(ethylene-co-butene) poly(ethy1ene-co-n-butylacrylate-co-carbonmonoxide) H,P,F, Si,Sn C pol y(Ethylene-co-norbornene) poly(ethy1ene-co-propylene),poly(ethy1ene-co-1-hexene) C poly(ethy1ene-co-trimethyleneterephthalates) H poly(eth yloxazoline) poly(y-butyrolactone-co-E-caprolactone) H,C poly(glucono1actone) H Poly(glycidy1methacrylate-co- a-methyl styrene ) poly(glycidy1methacrylate-co-methyl methacrylate), poly(glycidy1 methacrylate-co-butyl methacrylate) H,C poly(glycidy1methacrylate-co-styrene) H,C

Polymer,monomer

Table 1

2 m

a

0 3

RJ 2:

n =.

Q m 3

a.

z

B

m

3z

P

a

w

poly(methacry1icacid-co-vinylidenechloride) poly(methacrylonitri1e-co-styrene-co-methylmethacrylate) poly(methoxy hexa(ethy1ene glycol) methacrylate)-block-poly(2-(dimethylamino)ethyl methacrylate)-block-poly(methy1methacrylate) poly(meth yl acrylate) poly(methy1acrylate-co-norbornene) poly(methy1 a-(chloromethy 1)acrylate) poly(methy1methacrylate) poly(methy1methacrylate)

H H H

poly(Me methacrylate) poly(Me methacrylate) poly(Me methacrylate) poly(Me methacrylate) Poly(Me methacrylate)-graft-poly(E-caprolactone) poly(Me methacrylate-co-N,N-dimethylacrylamide) poly(Me methacrylate-graft-polystyrene) poly(methacr ylic acid)-block-poly(2-(dimethy1amino)ethy1 methacrylate)-block-poly(methacry1ic acid) poly(methacry1icacid-co-ethyl acrylate)

H H P

C

H H

H

H,C C H

poly(indene-co-vinyl acetate-co-isopropenyl acetate) poly(iso-Bu vinyl ether), poly(tert-Bu vinyl ether) poly(is0butene-co-4-vinylbenyl N,N-diethyldithiocarbamate) poly(isobuty1ene-block-norbornadiene),poly(norbornadiene-block-isobutylene) poly(isoprene-co-styrene) poly(1actic acid)-block-poly(ethy1eneglycol)-monoamine poly(1actide) poly(L-lactic acid-co-glycolic acid) poly(L-lactide)-block-poly(ethy1ene glycol) pol y(L-lactide-co-E-caprolactone) poly(L-lactide-co-RS-g-malicacid) Pol y(m-chloroaniline) poly(me acrylate-co-4-vinylpyridine)

tacticity sequence distribution end group tacticity, branch regioregularity

composition, sequence distribution composition composition composition, block copolymer

composition stereoregularity composition Molecular weight configuration composition stereoregularity Sequence length block copolymer composition composition configuration composition, sequence distribution Tacticity Tacticity end group Tacticity Graft composition Graft Molecular weight, composition

234 235 236 237 238

23 1 232 233

230

222 223 224 225 226 227 228 229

209 210 21 1 212 213 214 215 216 217 218 219 220 22 1

(cont.)

poly(methy1methacrylate)-block-poly(3-hydroxybutyrate) poly(methy1methacrylate)-block-poly(methylphenylsilane)-block-poly(methyl methacrylate) poly(methy1methacrylate-co-ethyl acrylate) poly(methy1methacrylate-co-ethyl acrylate) poly(methy1methacrylate-co-ethyl acrylate) pol y(met hyl met hacrylate-co-meth yl acryla te) poly(methy1methacrylate-co-methyl acrylate), poly(methy1 methacrylate-co-ethyl acrylate), poly(methy1methacrylate-co-butyl acrylate) poly(met hyl methacr ylate-co-N-(phenyl/p-t olyl) it aconimide) poly(methy1methacrylate-co-vinyl acetate) poly(methy1methacrylate-pyrrolyl methylstyrene)-graft-polypyrrole poly(methy1 n-octadecyl itaconate) poly(methy1sorbate) poly(methy1 sorbate) poly(methylphenylethylsi1ene-co-methylsilyne), pol y(methylphenethylsi1ene-co-phenylsilyne),copolysilanes, poly(m-phenylenevinylene-co-2,5-dioctyloxy-p-phenylenevinylene) poly(N-acryloyl-N-methylpiperazine-co-N-acetyl-a-aminoacrylic acid). poly(n-alkyl propiolate),poly(acetylene ester)

poly(methy1methacrylate) poly(methy1methacrylate) pol y(met hyl methacrylate) poly(methy1methacrylate), polystyrene, poly(buty1 acrylate) poly(met hyl methacry1ate)-block-poly(2-(dimet hy1amino)ethyl methacrylate)-block-poly(hexa(ethy1ene glycol) methacrylate) poly(methy1methacrylate)-block-poly(2,5-bis[(4-methoxyphenyl)oxycarbonyl]styrene)

Polymer,monomer

Table 1

244

sequence distribution, block copolymer block copolymer block copolymer

247 248 249 250 25 1 252 253 254 255 256 257 258 259 260 261

sequence distribution composition, configuration sequence distribution sequence distribution composition, sequence distribution composition regioregularity composition tacticity tacticity tacticity branch configuration composition configuration

245 246

239 240 241 242 243

Ref.

stereoregularity tacticity tactici t y block copolymer composition

Nucleus Contents

K5

R' (P

m

\o

w

poly(N-propargylbenzamides) poly(N-vinyl-2-pyrrolidone-co-butyl acrylate) poly(N-vinyl-2-pyrrolidone-co-glycidyl methacrylate) poly(N-vinyl-2-pyrrolidone-co-methylacrylate) poly(N-vinyl-2-pyrrolidone-co-vinyl acetate) poly(N-vinylcarbazole-co-butylmethacrylate) poly(N-vinylcarbazole-co-methacrylic acid) poly(N-vinylcarbazole-co-methylacrylate) poly(N-vinylcarbazole-co-methylmethacrylate) poly(N-vinylcarbazole-co-vinylacetate) poly(N-vinylpyrrolidinone) poly(N-vinyl-pyrro1idone)-graft-cellulose, polyacrylamide-graft-cellulose poly(octafluorocyc1opentene-co-ethylvinyl ether ) poly(oxybenzoate-p-trimethyleneterephthalate) poly(oxyethy1ene)-block-poly(oxybut ylene) poly(p-(1l(acry1amido)undecanoyloxy)phenyl dimethylsulfonium methylsulfate-co-methylmethacrylate) poly(p-diethyny-benzene-co-propargylalcohol) poly(pheny1-1,3-butadienylene) poly(phosphate ester)

poly(N-butyl acrylate) poly(n-but yl acryla te) Poly(n-bu t yl acrylat e) pol y(N-diphenylmethylitaconimide) poly(n-hexyl isocyanate)-block-polyisoprene poly(N-isopropyl acry1amide.-co-maleic anhydride) hylenedioxy- bis(ethylacrylamide) poly(N-isopropylacrylamide-co-amino-N'-et poly(N-isopropylacrylamide-co-dodecyl met hacrylate ) poly(N-isopropylacrylamide-co-methacrylic acid) poly(norbornene),poly(norb0rnadiene diester)

H H H,P

H

H H

C

H

H,C

H

C C H

composition branch molecular weight, end group

branch branch branch conformation,configuration composition composition composition composition composition tacticity sequence distribution configuration stereoregularity composition sequence distribution composition composition,configuration composition, configuration configuration sequence distribution composition composition end group graft composition sequence distribution composition,block copolymer composition 290 291 292

262 263 264 265 266 267 268 269 270 27 1 272 273 274 275 276 277 278 279 280 28 1 282 283 284 285 286 287 288 289

(cont.)

poly(prory1enecarbonate), poly(cyc1ohexene carbonate) pol y(silphenylenesiloxane) poly(sily1ether) poly(styrene peroxide)

poly(p-phenylene)s,polystyrene, poly(tetrahydrofuran), poly(styrene-alt-tetrahydrofuran) poly(p-phenylene-2,6-benzobisoxazole) poly(p-phenylenephosphaal kene) poly(p-phenylenevin ylene) poly(propene-co-ethene) pol y(propene-co-phenylnorbornene) poly(propy1enecarbonate) poly(propy1enecarbonate) poly(propy1enecarbonate)-block-poly(cyc1ohexen carbonate), poly(p opylene carbonate)-block-poly(6vinyl- 1-cyclohexene carbonate) poly(propy1eneglycol) methacrylate poly(propy1eneglycol), poly(hexamethy1eneadipate), poly(Me methacrylate-co-butyl acrylate) poly(propy1eneimine) poly(propy1eneoxide) poly(propy1eneoxide) poly(propy1eneoxide-co-ethylene oxide ) POlY(ProPYlene) poly(propy1ene-co-ethylene) poly(propy1ene-co-ethylene) poly(propy1ene-co-ethylene) poly(propy1ene-co-ethylene-co-butene-1) poly(propy1ene-co-ethylene-co-butene-1)

Polymer,monomer

Table 1

H,C,Si H,C,Si

C

C C C

C

C

C H

H

H,C

P H C

H,C C

302 303 304 305 306 307 308 309 3 10 31 1 312 313

molecular weight composition end group regioregularity end group sequence distribution tacticit y regioregularity end group sequence distribution composition composition, sequence distribution sequence distribution sequence distribution end group sequence distribution

314 315 316 317

293 294 295 296 297 298 299 300 301

Re$

graft, sequence distribution comosition molecular weight, end group irregularity regioregularity sequence distribution regioregularit y sequence distribution block copolymer

Nucleus Contents

w v,

: m

rn 3

B

h

2 2. c,

3 %

K2

2

00

333 334 335 336 337 338 339 340 341 342 343 344

composition end group tact icity tacticity composition

poly(Styrene-co-tert-Butyl Acrylate-co-Methyl Methacrylate) poly(styry1)lithium poly(tert-Bu acrylate), poly(acry1ic acid)-silica poly(tert-Bu acrylate), poly(tert-Bu methacrylate), poly(n-octadecyl acrylate), poly(n-octadecyl methacrylate) poly(tert-Bu methacrylate-co-2-isopropenyl isocyanate) poly(tert-butyl acrylate) poly(tert-but yl met hacrylate) poly(tert-butyl vinyl ether) poly(tert-butyl-3-isopropenylcumylperoxide-co-sty~ne), poly(tert-butyl-3-isopropenylcumylperoxide-co-sty~n~)-graftpol y(met hyl met hacrylate)

poly(styrene-co-maleicanhydride) poly(styrene-co-p-methylstyrene)

H

H

H

H H,C

H

C H

H

H H

c

H C

composition composition t acticit y composition sequence distribution sequence distribution end group end group composition composition sequence distribution, composition composition composition composition composition, block copolymer end group, configulation branch composition

H H C

322 323 324 325 326 327 328 329 330 331 332

319 320 321

block copolymer composition tacticity, sequence distribution

H

poly(styrene-block-p-nitrophenylmethacrylate) Poly(styrene-co-(Boc-p-aminostyrene)) poly(styrene-co-1-(4-vinylphenyl)-3,5,7,9,11,13,15-heptacyclopentylpentacyclo [9.5.1.13,9.15,15.17,13]octasiloxane) poly(styrene-co-1-(vinylbenzyl)thymine) poly(styrene-co-2-hydr oxyethyl methacrylate) poly(styrene-co-4-methylstyrene) poly(styrene-co- a-methylstyrene) poly(styrene-co- a-methylstyrene-co-acrylonitrile) poly(styrene-co-butadiene) poly(styrene-co-butadiene) poly(styrene-co-butadiene) poly(styrene-co-butadiene-co-acrylicacid) poly(styrene-co-dodecylmethacrylate), poly(styrene-co-octadecylmethacrylate) poly(styrene-co-maleicanhydride)

318

sequence distribution

poly(styrene peroxide)

(cont.)

poly(tetrafluoroethylene) poly(tetrafluoroet hylene) poly(tetrafluoroet hylene) poly(tetrafluoroethy1ene-co-vinylacetate), poly(chlorotrifluoroethy1ene-co-vinylacetate), poly(viny1idenefluoride-co-vinyl acetate) methacrylate), poly(tributylsily1 poly(tributylsily1methacrylate-co-2,2,2-trifluoroethyl methacrylate-co- 1 -trifluoromethyl-2,2,2-trifluoroet hyl methacrylate) poly(urea-imide) poly(urethane-urea) poly(viny1acetate)-graft-polyisobutylene poly(viny1alcohol) poly(viny1alcohol) poly(viny1alcohol-block-st yrene), poly(viny1pivalate-block-styrene) poly(viny1chloride) poly(viny1chloride) poly(viny1chloride) poly(viny1chloride), polypropylene poly(viny1chloride)-graft-polynorbornadiene, poly(viny1chloride)-graft-polyindene, poly(viny1chloride)-graft-poly(cyc1ohexadiene) poly(viny1chloride-co-2-chloropropene) poly(viny1phenol)-block-polystyrene poly(viny1pivalate-block-styrene), poly(viny1alcohol.-block-styrene) poly(viny1pivalate-co-vinyl acetate)

Polymer,monomer

Table 1 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364

sequence distribution end group sequence distribution graft tacticity, irregularity tacticity block tactici t y stereoregularit y tacticity tacticity graft composition block copoloymer block copolymer sequence length, composition

Ref

end group branch branch, cross link composition, sequence distribution

Nucleus Contents

D

R

4

%

z=.

(Q R

3 %

5

2

0

P 0

poly aniline polybutadiene poly bu tadiene

poly(viny1idene fluoride) poly(viny1idene fluoride), poly(viny1idene fluoride-co-hexafluoropropylene) poly(viny1idene fluoride-co-2-benzoyloxypentafluoropropene) poly(viny1idene fluoride-co-hexafluoropropene) poly(viny1methylsiloxane-co-dimethylsiloxane) poly[(2-alkoxy-5-methyl-1,3-phenylenevinylene)-alt-(1,4-phenylenevinylene)] poly[(N-acety1imino)ethylene] poly-[(R)-3-hydroxyoctanoate]-block-poly-[(R)-3-hydroxybutyrate] poly [( S)-1-methylene-2-meth ylprop ylene] poly[3-{ 4-((R)-4-ethyl-2-oxazolin-2-yl)phenyl} thiophene] poly[6-(4-Methoxy-4’-oxy-azobenzene)hexyl methacrylate], poly(ethy1ene oxide) poly [oligo(oxyethylene)oxydimethylsililene] poly [oligo(oxyethylene)oxydimethylsilylene] Polyacetylene polyacrylamide, poly(acry1amide-co-[3-(methacryloylamino) propyl] trimethylammonium chloride), poly(acry1amide-co-acrylicacid), poly(acrylamide-co-[2-acrylamido2-methylpropane sulfonic acid]) pol yacr ylate polyacrylonitrile poly acrylonitrile polyacrylonitrile, polyacrylonitrile-graft-pol yethylene, poly(acrylonitri1e-co-methacrylicacid)-grafte-polyethylene poly amide polyamide-polyester

382 383 384 385 386

end group, branch tact icit y tacticity graft branch composition, sequence distribution configuration configuration configuration

C C H,C

387 388 389 390

366 367 368 369

H,C,Si H,C,Si C C

H

H,C,F Si H H,C H,C C H

F

2F F

370 371 372 373 374 375 376 377 378 379 380 381

365

composition composition, sequence distribution molecular weight block copolymer composition sequence distribution sequence distribution configuration block copolymer,end group composition tacticity regioregularity, conformation block copolymer sequence distribution sequence distribution configuration conformation

poly(vinyl-(5-fluorouracil)ethanoate),poly(viny1-(5-fluorouraci1)ethanoate-co-acrylicacid), poly(vinyl-(5-fluorouracil)ethanoate-co-maleicanhydride) H,C poly(viny1idene chloride-co-methyl methacrylate) H

P

2

2

s.

% n

i$

% 8

E.

c-c

t/l

k

2

M

(cont.)

polybu tadiene, polystyrene polybu tadiene-ol polycarbonate polydimethylsiloxane, polyacrylate polyepichloroh ydrin polyester polyester, 4,4-(1-h ydroxyphen ylidene)diphenol, 4,4’-(9-fluorenylidene)diphenol polyesters, p,p’-bibenzoic acid, dimer acid, alkylene glycol pol yethene polyethene polyethene pol yether polyols polyethylene polyethylene polyeth ylene polyethylene polyethylene polyethylene polyet h ylene polyeth ylene polyet h ylene polyeth ylene polyeth ylenimine-graft-poly(ethylene glycol) polyglycidol polyimide 4,4’-(hexafluoro polyimide,1,3,5-tris(4-aminophenoxy)benzene, isopropylidene) diphthalic anhydride, 4,4’-oxydiphthalic anhydride, 3,3’,4,4‘-benzophenonetetracarboxylic dianhydride

Polymer,monomer

Table 1

C H H

C

C C

C C C C C C C C C

C C H

C H H

tacticity molecular weight branch composition regioregularity branch composition sequence distribution branch branch branch regioregulari t y branch branch branch branch branch branch branch branch branch branch graft branch branch branch

Nucleus Contents 39 1 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 41 1 412 413 414 415 416

Re$

s

Q

4

wzt.

m

5

B

5 ft (b

B

polymethylsiloxane polymet hylsiloxane pol ynorbornadiene polynorbornene pol yperoxide pol yphenylene pol yphosphole polypropene pol ypropene pol ypropene pol ypropiolate polypropylene polypropylene polypropylene polypropylene polypropylene polypropylene polypropylene polypropylene polypropylene polypropylene

polyimide,2,2-bis(3,4-dicarboxylphenyl) hexafluoropropane dianhydride, 2,4,6-triaminopyrimidine polyimide,copolyimide polyisobutylene polyisobutylene-block-poly(2-(dimethylamino)ethylmethacrylate), poly(2-(dimethylamino)ethyl methacrylate)-block-polyisobutyleneblock-poly(2-(dimethylamino)ethyl methacrylate) polyisobutylene-block-polystyrene pol yisophthalester pol yisoprene pol ylactide Polylactide-block-polyisoprene-block-polylactide block copolymers branch configuration stereoregularity block copolymer, molecular weight ladder polymer tacticity, ladderlike polymer branch configuration composition branch regioregularity stereoregularit y tacticity tacticity stereoregularity tacticity irregularity tacticity end group, regioregularity tacticity tacticity tacticit y tacticity tacticity branch

H H H,C H,C H

H,C C C C C C C C

C

H,P C C C

branch sequence distribution end group block copolymer

H H H,C,Si H

445 446

444

425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443

422 423 424

421

420

5

P

E:

z

i0+

8

% Q

6.

(cont.)

pol ypyrrole polyrotaxane, poly(ethy1ene glycol)-block-poly(propy1eneglycol) -block-poly(ethy1ene glycol) Pol ysilane Polysilsesquioxane Polystyrene Polystyrene Polystyrene Polystyrene Polystyrene Polystyrene Polystyrene Polystyrene Polystyrene polystyrene, poly(2,4,6-trimethylstyrene), poly(methacry1ate) polystyrene-block-(poly(tert-butyl acrylate))2 Polyst yrene-block-poly(5,6benzo-2-methylene- 1,3dioxepane),poly(Me methacrylate)-block-poly(5,6-benzo-2methylene-l,3-dioxepane) and poly(Me acry1ate)-blockpoly(5,6-benzo-2-methylene- 1,3-dioxepane) polystyrene-block-polyisoprene-block-poly(2-vinyl pyrid1ne)-block-poly(t-butylmethacry1ate)-blockpoly(ethy1ene oxide) polystyrene-graft-cellulose polystyrene-graft-silicate polytetrahydrofuran-block-polystyrene,polytetrahydrofuran-block-poly(Me acrylate) Polyt hiolene Polythiophene Polyurethane polyurethane,poly (tetramethylene oxide), polyethylene oxide Polyvinyl butyral urea-formaldehyde resin

Polymer,monomer

Table 1

C C

H

H

C

H,CF C H H H

H H H

H,C,Si Si

H,C

464 465 466 467 468 469 470 471 472

463 block copolymer graft graft block copolymer configuration regioregularity composition molecular weight sequence distribution degradation

449 450 451 452 453 454 455 456 457 458 459 460 461 462

447 448

Ref

tacticity ladderlike polymer branch end group end group tacticity end group end group tacticity end group tacticity reactivity end group block copolymer

composition composition

Nucleus Contents

P 0

E

rn

3 c) 3

8ra

%

n

3.

i,?

R cp

z

B

m"

2

h

11: Synthetic Macromolecules

3

405

Liquid Crystalline Polymers

The molecular ordering in a nematogenic liquid with dispersed polymer networks was simulated by Monte Carlo method. From obtained parameters, 2H NMR spectra were d i c ~ s s e d .Pulse ~ ~ ~Field ~ ~ Gradient ~~, Spin Echo method was applied for polymer liquid crystals. The self-diffusion coefficients were measure and discussed about the dynamics of liquid crystals for a-helical poly(n-ocpoly(ciiethylsi10xane)~~~~ 478 The combination of pulse tadecyl L-gl~tarnate),''~~ field gradient NMR and MAS NMR is applied to anti-cancer drug Carboplatin. The influence of polyethylene glycol chains on the drug mobility is For the main-chain polyester liquid crystals, 13C NMR measurements were applied and discussed the local ordering, morphology and dynamics of aromatic cores and flexible alkyl spacers.478The odd-even effect of flexible space length of poly((phenylsulfony1)-p-phenylene alkylene bis (4-oxybenzoate)) was investigated using solid-state 13CNMR spectroscopy. NMR results indicate that the odd-even fluctuations of the activation energy of local polymer chain motion and trans-gauche exchange arises from the differences in molecular packing.479To describe the structure and dynamics in a lyotropic xanthan liquid crystal, 23Na and 14NNMR measurements were applied. Conventional spin-lock experiments revealed the existence of an extremely low frequency, submegahertz dispersion. The corresponding correlation times are on the order of tens of microseconds.480 Solid state I3Cand 15NNMR was applied to liquid crystalline polyurethanes. It has been suggested that the flip angles of fluctuations around the phenylene axis is limited to less htan about 20 degree without any 180 degree flip . Selective deuteration of liquid cyrstalline polymers can provide the information about the dynamics and orientational order of liquid crystal at molecular level. 2H NMR study were carried out for polymethacrylates containing phenylbenzoate,482poly(ethylenepropylene)-co-poly(ethy1eneo~ide)~~~ polyacrylamide-stabilized Pfl phage liquid poly~iloxane,4~~ BL038-5CB liquid crystal mixture in a holographic polymer!86 H-decoupled deuterium one and two dimensional NMR spectroscopy at natural abundance level was developed. The spectroscopic separations and the S/N ratio from the spectra are large enough to measure the enantiometric e ~ c e s s . 4 ~ ~

4

Imaging

Photopolymerization of a commercial dental resin has been investigated by 'H stray-field(STRAF1) magnetic resonance. The influence of the curing protocol was observed by this method!88 NMR microimaging was used to reveal the heterogeneous changes in the aged nitrile rubber elastomers. TI imaging shows that there is no spatial distribution of Proton NMR imaging techniques were used to characterize a silicone rubber and silicone rubber networks unfilled, filled with silica and in the presence of a guest polymer.490,491 The fracture mechanism of two types of extruded polyamide 6/maleic anhydride grafted ethyl propylene blends was studied by MRI. A clear relation is observed between the

406


toughness and the dimensions of the plastic zone.492A newly designed NMR cell was developed to study the shrinkage process of a hydro-poly(methacry1ic acid) gel under the application of an electric field. The 3D images of the shrinking of gel on the application of a DC electiric field were observed in real time.493Single point ramped imaging with T1 enhancement (SPRITE) sequence was used in conjunction with raising the sample temperature to obtain images of rigid polymers. It was demonstrated that this approach provides a straightforward and reliable method for imaging a rigid polymers.494Multipoint k-space mapping is ahybrid between constant-time and spin-warp imaging. This method was implemented for NMR imaging of semi-solid materials. The method's potential is illustrated with 3D proton images of synthetic polymers.495

5

Characterization of the Synthetic Macromolecules

The heterogeneity in segmental chain order of a poly(dimethylsi1oxane)(PDMS) layer grafted onto a silica surface was investigated by 'H multiple-quantum NMR. These grafted PDMS layers consist of partially immobilized chain segments at the PDMS-silica interface and mobile chain portions outside the interface which is supported by measurements of homonuclear double-and triple-quantum buildup curves. These curves reveal a bimodal distribution of the residual dipolar couplings along the PDMS chain. The segmental orientations detected in the interface and mobile regions are related to the average chain length and reflect the competing effects of the surface-induced orientation and chain conformations. The different behavior of the two chain fragments is evident from temperature-dependent The structure and rheological behavior of atactic poly(viny1 alcohol) (a-PVA) hydrogels prepared by freeze/thaw cycles were investigated as a function of polymer concentration and number of freeze/thaw cycles. The presence of phases with different mobilities was observed using 13Ccross polarization(CP)/Magic Angle Spinning(MAS) and direct polarization(DP)/MAS NMR experiments. Results obtained showed that an increase in the number of freeze/thaw cycles induces an increase in the degree of crystallinity in the polymer-rich phase together with an increase in the storage modulus. Moreover, both the percentage of protons in a rigid environment measured by 'H NMR and the storage modulus values tended to a limiting value after six freeze/thaw cycles. These results show that the first five or six freeze/thaw cycles play a very important role in determining the hydrogel structure and rheological properties. A more detailed comparison of NMR and rheological data led to the conclusion that the storage modulus is mainly controlled by the a-PVA crystallinity while the hydrogen bond interactions have a much smaller ~ o n t r i b u t i o n . 'H, 4 ~ ~13C['HI-, and 2D HETCOR NMR spectroscopy have been employed to characterize acrylonitrile (AN) copolymers of methacrylic acid (MAA) or itaconic acid (IA) prepared by DMF-water suspension polymerization. The chemical composition of AN-MAA copolymers was determined using 'H NMR and that of AN-IA copolymers using quantitative 13C['H]-NMR spectra. AN-IA copolymers show two separated signals due to


407

two carboxylic groups in different magnetic environments of IA, whereas ANMAA copolymers show only one in the region of 6 6.32-7.60 ppm in 'H NMR and 6 171.0-176.0ppm in '3C['H]-NMR spectra. Evidence of acrylamide formation by the partial hydrolysis of AN groups is obtained by the small broad signal at 6 7.9 ppm in 'H NMR spectra. No carboxylic proton signal was observed in copolymers containing 12 mol% MAA or IA, which indicates the formation of anhydride. The microstructure was obtained in terms of triad sequences from 13C['H]-and ''H NMR spectra of the copolymers, which showed configurational and compositional sequences. This is confirmed by 2D HETCOR NMR spectra also. The distribution of AN sequences is found to be random (no nm N 2.0). The tacticity values are not affected by the comonomer content.498High-resolution solid-state NMR spectra can be obtained of crosslinked and high molecular wt. polymeric materials by using a combination of magic-angle spinning and high temperature. The resolution of the spectra can be comparable to that seen in solution NMR spectroscopy. This enables standard solution 2D NMR pulse sequences, such as COSY, TOCSY, and HMQC, to be used to assign resonances in these systems. It has been found that short duration pulse sequences work better than long sequences. For observing heteronuclear correlations, carbondetected 2D pulse sequences appear to have less stringent instrumental setup requirements than proton-detected 2D sequence^!^' The structure of silyl-carborane (1,7-C2B1OH10) hybrid diethynylbenzene-silylene polymers with thermal treatment over a wide range of temperatures has been characterized by using "B MQ/MAS NMR, and 13C and 29SiCP/MAS NMR methods. From the experimental results, the following was found: (1) naphthalene rings form by diene reaction between the P h - C d group and C=C group at temperatures from 250 to 500" ;(2) three-dimensional network forms by addition reaction between the vinyl group and C d group at curing temperatures from 250 to 350°C and addition reaction between m-carborane group and the vinyl group and/or the C=C group at curing temperatures from 250 to 350°C; (3) the hybrid polymers are decomposed by oxidation at curing temperature of 500°C; and (4) structural change of the m-carborane hybrid polymer leads to desirable modification of the oxygen index, heat deflection temperature, and flexural ~ t r e n g t hA. ~structural ~ analysis of copolymers of syndiotactic polypropylene with small amounts of 13C-enrichedethylene, in the range 0.4-2.6 mol %, is reported. X-ray diffraction and solid-state 13CNMR CP/MAS data indicate that the as-prepared copolymer samples are crystallized in the conformationally disordered modification of form I1 of sPP containing kink bands5" The role of reactivity ratio of monomers on the chain structure of N-vinylcarbazole-vinyl acetate copolymers (V/A) of different components prepared by solution polymerization using AIBN initiator was studied. The copolymer composition was determined from quantitative 13Cand 'H NMR spectroscopy. The reactivity ratio of monomers was calculated using the Kelen-Tudos (KT) and non-linear error in variable (EVM) methods. Twodimensional heteronuclear single quantum correlation (two-dimensionalHSQC) and total correlation spectroscopy (TOCSY) spectra were used to resolve the complex 'H NMR spectra of the copolymers.502CP dynamics between several abundant spin baths has some inherent complications that lead to

-

408


misinterpretation of the C P rates and relaxation parameters if not properly accounted for. A general thermodynamics description of C P dynamics involving up to six spin baths is developed and implemented with MATLAB. The CP dynamics for a fluorinated polymer, (2-perfluorohexylethyl acry1ate)-styrene co-polymer, was analysed as a system with five spin baths-four 19Fand one ‘H. The C P behavior was successfully simulated above the glass-transition temperature. The C P rates and Tlp’s are discussed in terms of the structure and dynamic behavior of the polymer.503 The noncrystalline structures of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymers were studied by variable temperature solid-state wide-line ‘H NMR and solid-state high-resolution I3CNMR spectroscopy. It is found that at room temperature there exists a rich and rigid component in the noncrystalline region of PHB and PHBV. The content of this component decreases with the increase in 3-hydroxyvalerate content in PHBV and with the increase in temperature. The brittleness of PHB may be partly attributed to the rigidness of the noncrystalline region at room temperature, while the improvement of the properties of PHBV may come from the enhanced mobility of the noncrystalline region.’04 The smaller 129Xeshift indicates the presence of larger sorption sites in high permeability polymers relative to conventional polymers. The temperature dependence of solubility in these polymers is drastically different from the behavior in conventional polymers. There is a rapid exponential increase in solubility in high permeability polymers as temperature decreases corresponding to a large negative enthalpy change on sorption and it is this increase in solubility which leads to a large increase in shift with decreasing temperat~re.’~’ Solid polymer electrolytes based on polyurethane/poly(dimethylsiloxane)segmented copolymers (PS55) have been characterized by differential scanning calorimetry (DSC), ionic conductivity, and multinuclear solid-state NMR measurements. Solid-state 13C NMR results from cross-polarization time constant (TCH) measurements along with two-dimensional (2D) WISE NMR suggest that a significant decrease in the mobility of the soft segment as the salt is added. Polysiloxane backbone is not affected until at a higher salt concentration, as observed by the line width change in the 29SiNMR spectrum. The onset temperature of 7Li motional line narrowing is correlated with the soft segment glasstransition temperature (Tg).The activation energies obtained from ionic conductivity, 7Liline width, and spin-lattice relaxation time in the laboratory frame (T1) measurements indicate that there is a strong correlation between the ionic conductivity of the solid polymer electrolyte and the mobile lithium Interaction between a cationic copolymer (acrylamide-trimethylaminoethyl acrylate) P(AAm-TMA) and an anionic perfluorinated surfactant (lithium perfluorooctanoate) (LiPFO) was studied using ‘H and 19FNMR. To clarify the effect of the charge density, copolymers having a different composition of comonomers have been used. In each copolymer-surfactant system the presence of the polyelectrolyte in solution strongly modifies the 19F chemical shifts. Consequently, a strong polymer-surfactant interaction is evidenced. The chemical shifts and line widths observed have been interpreted as dependent on two main factors: the aggregation state of the system and the rate of exchange among

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different sites. 'H spectra, which run at a constant concentration of polymer and different concentrations of surfactant, appear rather broad and poorly defined. The spectral broadening becomes extremely marked at certain value C of the surfactant concentration, where a strong super-Lorentzian line appears, surmounted by weak sharp lines in which the chemical shift is still visible. Increasing the charge density the critical concentration C increases. A further increase in the surfactant concentration causes a sharp precipitation which is accompanied by the disappearance of the super-Lorentzian resonance. A model of interaction has been proposed on the basis of the results The chemical composition and the structure of poly(p-phenylene-2,6-benzobisoxazole) (PBO) fibers is investigated by solid state NMR. CP-DD-MAS 13CNMR including dephasing delay experiments permits to determine the chemical composition of PBO. Studying molecular dynamic of PBO (wide line 'H NMR), the structure of PBO is examined. According to the model of Kitagawa et al., the structure of PBO is then e~plained.~" The effect of y-radiation on a perfluoroalkoxy (PFA)resin was examined using solid-state high-speed magic angle spinning (MAS) 19FNMR spectroscopy. Samples were prepared by subjecting them to y-radiation in the dose range 0.5-3 MGy at either 303, 473, or 573 K. New structures identified include new saturated chain ends, short and long branches, and unsaturated groups. The formation of branched structures was found to increase with increasing irradiation temperature; however, at all temperatures the radiation chemical yield (G value) of new chain ends was greater than the G value of long branch points, suggesting that chain scission is the net process.509N-vinyl-2pyrrolidone/methyl acrylate copolymer composition of these copolymers was calculated from 'H NMR spectra. The radical reactivity ratios for N-vinyl-2pyrrolidone and methyl acrylate were 0.09 and 0.44, respectively. These reactivity ratios were determined using the Kelen-Tudos and nonlinear least-squares error-in-variable methods. The 13C{ 'H } and 'H NMR spectra of the copolymers overlapped and were complex. The complete spectral assignment of the 13Cand 'H NMR spectra were done with distortionless enhancement by polarization transfer and two dimensional 13C-'H heteronuclear single quantum correlation spectroscopic experiments The two-dimensional 'H-'H homonuclear total correlation spectroscopic NMR spectrum showed the various bond interactions, thus inferring the possible structure of the cop01ymers.~'~ Poly(viny1 alcohol) (PVA) samples with 55% and 61% syndiotacticity, and their related dry and hydrated gels obtained by two different freeze-thawing cycles have been studied using the solid-state 13CCP/MAS NMR technique. From a comparative analysis of the spectra, evidence was obtained that the gelation process largely disrupts the intramolecular hydrogen-bonded network of the PVA. The addition of water to the dry gels favors their swelling, destroying intra-chain hydrogen bonds between hydroxyl groups as a function of the degree of tacticity and the gelation procedure, and promotes the formation of new networks of interchain hydrogen bonds. Information on the dynamics of the polymeric domains in the kilohertz range has been obtained from the analysis of the'H and I3C spin-lattice relaxation times in the rotating frame (TlpHand TIPC),indicating that homogeneous arrangements of the amorphous or swollen polymeric chains exist, independent

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of the preparation method or the tacticity of the PVA chains.511The molecular structure and morphology grown from isotactic polypropylene (iPP)/odichlorobenzene solution were studied by thermal analysis and high-resolution solid-state 13CNMR. The morphology varied depending on incubated temperature. A gelation occurred below 70"C, while the crystallization took place over 80°C. The crystal obtained was identified with the a-form, which is the most stable crystalline modification of iPP. In CP/MAS 13C NMR spectrum, the chemical shift of methylene, methine, and methyl carbons provided almost the same shift for both the gel and a-form, but the characteristic doublet for methylene and methyl signals of the gel was different in intensity ratio with the a-form. These results indicate that the gel-forming crystal is identified with a 1-form, being the disordered a-form. Indeed, the lower melting temperature and smaller melting enthalpy than the crystal were observed by thermal analysis. This result connects to the incompleteness of crystal formed in the gel.512 Characterization of clarithromycin polymorph was performed by solid-state cross polarization and magic angle spinning (CP/MAS) I3CNMR spectroscopy. Two polymorphs, form I1 and form I, of clarithromycins indicated characteristic resonances of C1 carbonyl carbon at 176.2 and 175.2 ppm, respectively. Since each peak of C1 carbon was well separated in the spectrum of the two polymorphs, quantitative analysis of the polymorphic fraction from the peak area of these peaks was performed. The peak area of form I was found to linearly increase with an increase of its content, with a correlation coefficient of above 0.99. Solid-state NMR was found to be a useful technique to determine the characteristics of the polymorphic forms.513The copolymer composition of N-vinyl-2-pyrrolidone/butyl acrylate (V/B)copolymers was determined from the quantitative 13C{ 'H } NMR spectra. The monomer reactivity ratios for N-vinyl2-pyrrolidone (V) and Bu acrylate (B)were found to be rv = 0.11 0.07, rB= 0.54 - 0.19, using the Kelen-Tudos and non-linear least-square error-in-variable methods. The 13C{'H } and 'H NMR spectra of these copolymers are overlapping and complex. The complete spectral assignment of the carbon and proton NMR spectra were done by employing distortionless enhancement by polarization transfer (DEPT) and two-dimensional (2D) 13C-'H heteronuclear single quantum correlation spectroscopy experiments The 2D total correlation spectroscopy (TOCSY) ('H-'H homonuclear TOCSY) NMR spectrum was used to ascertain the various geminal and vicinal couplings in the copolymer.514The phase structure of ethylene-vinyl acetate copolymers was investigated by solidstate wide-line 'H NMR and solid-state high-resolution 13CNMR. Not only the degree of crystallinity, but the relative contents of the monoclinic and orthorhombic crystals within the crystalline region varied with the vinyl acetate (VA) content. Biexponential I3CNMR spin-lattice relaxation behavior was observed for the Crystalline region of all samples. The component with longer 13CNMR spin-lattice relaxation time was attributed to the internal part of the crystalline region, whereas the component with shorter spin-lattice relaxation time to the mobile crystalline component was located between the noncrystalline region and the internal part of the crystalline region. The content of the mobile crystalline component relative to the internal part of the crystalline region increased with

+

1 1 :Synthetic Macromolecules

41 1

the VA content, showing that the 13CNMR spin-lattice relaxation behavior is closely related to the crystalline structure of the copolymer^.^'^ A series of main-chain hydrogen-bonded supramolecular complexes have been prepared by self-assembly of proton donor (diacid) and acceptor (bipyridyl ethylene) through the formation of intermolecular hydrogen bonds. Solid-state CP/MAS I3CNMR spectroscopy experiments revealed that an induced upfield shift ( A6 2 4 ppm) of carbonyl carbon in solid state was observed as a result of the formation of intermolecular hydrogen bond. The natural abundance CP/MAS "N NMR spectroscopy also supported that the molecular complexes with hydrogen bond characteristics rather than zwitterionic complexes were formed on the basis of the change in 15Nchemical shift.516Poly(methy1acrylate) (PMA) was characterized by 2-dimensional NMR spectroscopy. The intensity ratio of the methylene proton signals showed that 50% of the PMA is in isotactic and 50% is in syndiotactic configuration. The CH2-and CH-carbon resonances were assigned to various configurational sequences. These assignments were justified with the help of 2D heteronuclear single quantum correction (HSQC) and total correlation spectroscopy (TOCSY). 517 A detailed structural characterization of annealed polyaniline (PANI) powder samples has been carried out by using solid state NMR and FT-IR. Comparing annealed PANI with PANI that has been chemical reduced to the leucoemeraldine base (LB) form, the 13Cand 15NNMR and FT-IR data clearly show the conversion of the quinoid rings to benzenoid rings upon heating at 200°C in vacuum. Combining interrupted-proton decoupling with "N NMR reveals the presence of tertiary amine nitrogens generated from the crosslinking of the annealed polymer. The tertiary amine sites occur upon new bond formation between quinoid rings of one chain with the imine site of an adjacent chain, yielding a N,N'-diphenylphenazine structure at the site of crosslinking. The NMR data also shows that upon heating crosslinking is the predominant reaction occurring in PANI powder and that it follows a single mechanism. Measurement of the NMR relaxation parameter also shows that the rate of chain dynamics of the polymer is modified upon cros~linking.~'~. Solid-state 19FNMR has been applied to poly(viny1idene fluoride)(PVDF) to investigate, inter alia, the location of the reverse units. The application of relaxation filters in pulse sequences revealed fundamental differences relating to the domain structure of PVDF. A TlpFspin-lock experiment gave a spectrum of the crystalline phase along with some intensity from signals associated with reverse units. The proximity of reverse units to the amorphous and crystalline domains was further investigated by TlpF-filteredradio frequency driven recoupling (RFDR) and spin-diffusion experiments The results showed the majority of reverse units to be relatively mobile (i.e. amorphous). However, weak RFDR cross-peaks were detected which suggest the presence of some reverse units in relatively rigid domains. A signal arising from a highly mobile site was detected at 6 F = -115 ppm by a delayed acquisition experiment and is tentatively ~ ' ~ microphase structure of polyisoassigned to - CH2CF2He n d - g r o u p ~ . The prene-block-poly (methyl methacrylate) diblock copolymers was studied using solid-state NMR techniques. Wideline separation spectroscopy reveals a narrow interphase between the two polymers. The domain sizes of a lamellar sample and

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a sample with hexagonally ordered cylinders were determined using 'H spin diffusion. The lamellar sample shows a domain size of 16 f 2 nm for the minor polyisoprene phase and a long period of 33 & 4 nm. The cylindrical structure has a long period of 38 f 7 nm, the diameter of the poly(methy1 methacrylate) (PMMA) cylinders is 21 f 4 nm. These results are about 20% below the estimates obtained from theoretical calculations according to Helfand and Wasserman.s20

6

Dynamics of the Synthetic Macromolecules

The noncrystalline 13C spin-lattice relaxation times of atactic and isotactic polypropylene and those of an ethylene- 1-octene copolymer of low crystallinity have been measured by classical inversion and saturation recovery methods as well as by a CP/MAS-based pulse sequence. The latter is a saturation recoverytype sequence that involves cross-polarization. It samples preferentially the soft non-crystalline regions of semicrystalline polymers. The method is found to be useful in determining TICof the amorphous regions of semicrystalline iPP at room temperature. It is found that the atactic PP molecule and the non-crystalline iPP regions have the same average segmental relaxation rate. The TICof some of the carbons investigated was less than that of TIHand the experimental recovery curves showed complex exponential behavior from the contribution of a transient nuclear Overhauser effect (NOE) to the 13Cmagnetization. Moreover, the experimental data were fitted with a double exponential function obtained from solving the Solomon equations. The fitting leads to TICin very good agreement with the values obtained by classical inversion or saturation recovery sequences. The same TICvalue was obtained with the CP-based sequence when transient NOES were eliminated by saturation of the proton magnetization during the delay period. The hexyl branches of the ethylene copolymer lead to an increased average backbone C-C intermolecular distance in the non-crystalline regions compared to those of the linear polyethylene chain and, thus, to a higher backbone methylene segmental mobility.s21 High-resolution solid-state 13C NMR spectroscopy and MM3 molecular mechanics calculations have been applied to investigate side-chain conformations, side-and main-chain dynamics, and phase transformations of the form I11 polymorph of isotactic-poly(1-butene) (I-PB). MM3 calculations indicate that the t-g' side-chain conformation is much more stable than the g'-g and g-t side-chain conformations in the crystal lattices of form 111. The I3CCP/MAS NMR spectrum at 199 K shows that the methyl group in the side chain adopts only the t-g' conformation. Upfield shift for the 13C methyl signal above 338 K indicates dynamic disorder in the side-chain conformations. The 13C two-dimensional exchange NMR reveals that the polymer chains in form I11 consist of two components. The 74% polymer chains execute the 90" helicaljump motion with an activation energy of 79.3 & 5.6 kJ/mol, while the residual chains are immobile. It is found that the 90" helical jump motion begins to occur at the temperature very close to the Tg. Upfield shifts for the 13C main-chain signals at 338 K indicate the motional mode change from the 90"

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helical jump motion to ‘more or less’ rotational or jump motion with different angle displacements. Above 338 K, two phase transformations from form I11 to forms I1 and I’ are confirmed in the 13CCP/MAS spectra. The ‘H spin-lattice relaxation times in the rotating frame show that the transformation from form I11 to form I’ proceeds within the form I11 phase via the crystal-to-crystal process, whereas the line-shape analyses of the I3CDP/MAS NMR spectra indicate that the transformation from form I11 to form I1 proceeds via the quasimelting/recrystallization process. The frozen chain conformations in the amorphous phase below Tg are also investigated on the basis of the I3Cchemical shifts. It is suggested that most of the main chains adopt the helical (tg’)n conformation, Different forms of solid and the residual part adopts the (tt) polyaniline (Pani) and polyaniline intercalated into montmorillonite clay layers were characterized by solid-state deuteron quadrupole echo and magic angle spinning NMR experiments. Quadrupole echo spectra reveal different fractions of fast flipping phenyl rings for conductive emeraldine salt and nonconductive emeraldine base samples. Deuteron MAS spectra of emeraldine salt show two partially resolved sets of sidebands, whose centers are separated by 6.0 1.5 ppm. For conductive polyaniline, the temperature dependence of line width and intensity is consistent with the existence of metallic domains embedded in a disordered insulating matrix, such that approx. 40% (T = 223 K) to 60% (T = 373 K) of the aromatic deuterons experience a Knight shift. This implies that polarons play an important role in the conduction mechanism.523A combination of molecular modeling, DFT calculations, and advanced solid-state NMR experiments is used to elucidate the supramolecular structure of a series of benzoxazine oligomers. Intramolecular hydrogen bonds are characterized and identified as the driving forces for ring-shape and helical conformations of trimeric and tetrameric units. In fast MAS ‘H NMR spectra, the resonances of the protons forming the hydrogen bonds can be assigned and used for validating and refining the structure by means of DFT-based geometry optimizations and ‘H chemical shift calculations. Also supporting these proposed structures are homonuclear ‘H-’H double-quantum NMR spectra, which identify the local proton-proton proximities in each material. Quantitative 15N-’H distance measurements obtained by analysis of dipolar spinning sideband patterns confirm the optimized geometry of the tetramer. These results clearly support the predicted helical geometry of the benzoxazine polymer. This geometry, in which the N-H . - 0 and 0-H 0 hydrogen bonds are protected on the inside of the helix, can account for many of the exemplary chemical properties of the polybenzoxazine materials. The combination of advanced experimental solid-state NMR spectroscopy with computational geometry optimizations, total energy, and NMR spectra calculations is a powerful tool for structural analysis. Its results provide significantly more confidence than the individual measurements or calculations alone, in particular, because the microscopic structure of many disordered systems cannot be elucidated by means of conventional methods due to lack of long-range order.524The molecular dynamics of the different molecule subunits in the amorphous polymer poly(n-hexyl methacrylate), PnHMA, was investigated by recently developed solid-state 1D-MAS exchange NMR *

*

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methods at temperatures close to the glass transition temperature. The molecular mobility of main-chain, side-chain, and carboxyl carbons could be assigned to different relaxation processes as identified by dielectric and mechanical relaxation spectroscopy as well as caloric methods. Information about both the time constants as well as the geometry of the processes could be obtained. It was found that the side chain contributes in the dynamic window of NMR exchange experiments (T c 1 ms to 0.1 s) mainly to the P process while the main chain and carboxyl group are found to contribute to both the P and a processes. The data suggest that the contributions of side and main chain to the p process are different. All dynamic processes exhibit a wide distribution of correlation times.525I3CNMR chemical shifts have been measured and assigned for epimeric methyl 3a/~-hydroxy-5~-cholan-24-oates (methyl lithocholate [3a-OH epimer] and methyl iso-lithocholate [3P-OH epimer]). Their molecular dynamics simulations suggest that for both epimers there exists two predominant gas phase conformations, which have been further forwarded for ab initio/HF optimizations and DFT/GIAO based I3C NMR chemical shift calculations. Excellent linear relationships have been observed between experimental and calculated 13C NMR chemical shifts for both epimers. For methyl lithocholate (MeLC), the other min. energy conformation equates very well with the single crystal X-ray structure (orthorhombic, space group P212121,unit cell a = 7.14710(10) A, b = 11.9912(2) A,c=26.4368(5) A).The crystal packing of MeLC consists of continuous parallel intermolecular hydrogen bonded [3a-OH - - 0 = C241 head-to-tail polymeric chains, which are further cross-linked by many simultaSelf-diffusion coefficients of neous weak C(sp3)H 0-type of linear and cyclic alkanes in melt, in blends with equivalent linear alkanes, and dissolved in linear polyethylene, were measured by pulsed-gradient spin-echo NMR at various temperatures. The results indicate the following: (i) at the same carbon number, cyclic alkanes diffuse more slowly than linear alkanes in their respective melts, but h e a r s and cyclics share a similar rapid rate of decrease with increasing carbon number; (ii) in blends of linear and cyclic alkanes at the same carbon number the single average diffusion coefficient observed varies monotonically as a function of composition; and (iii) two distinct diffusion coefficients are observed in the cycloalkane/linear polyethylene blends, with the extrapolated trace cycloalkane diffusion consistent with Rouse behavior. The results are compared with recent numerical simulations and with experiments in other polymer systems, forming a consistent picture of the effects of diffusant mass, molecular shape and flexibility, and the dynamic attributes of the host mat e ~ i a l . Pulsed-field ’~~ gradient NMR spectroscopy was used to study the diffusion of three different pol y(pr opyleneimine) dendrimers with h ydr ophilic t riethylene glycol methyl ether terminal groups (generations 2, 4, and 5) in poly(viny1 alcohol) aqueous solutions and gels. The effects of the diffusant size, polymer concentration (from 0 to 0.26 g/mL), and temperature on the self-diffusion coefficients have been studied, and the model of Petit et al. [Macromolecules 1996, 29 , 60311 was used to fit the experimental data. The Stokes-Einstein hard-sphere radii were also calculated in the zero concentration limit and were compared with those of the linear poly(ethy1ene glyco1)s under the same condiCJ

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tions. The proton NMR relaxation times (T1and T2)were measured to study the mobility of the dendrimer core part and terminal group as a function of the dendrimer size.528 Dynamics, structure, and domain size in polyimidepoly(dimethylsi1oxane)networks (PI-PSX) with various compositions were investigated by solid-state NMR spectroscopy. 29Siand 13CMAS NMR spectroscopy revealed the presence of PSX chains in 2 motional states, and their relative content was estimated. The overall mobility as well as local motion was studied by 1D and 2D 'H wide-line spectroscopy. 2D WISE spectra proved large differences in molecular mobility of PSX and PI chains. High mobility of PSX is not imparted to rigid PI, and the interfacial region is located in the PSX phase. This region corresponds to the immobilized PSX chains at the surface of PSX domains, which link PI chains. The spherical morphology and domain sizes at the nanometer scale were determined from analysis of spin diffusion processes. Molecular mechanics and dynamics calculations were performed to design PSX domains at the atomic level. Correlation of the structure and morphology with mechanical and thermal properties was Two sets of independent NMR measurements of self-diffusion and proton transverse relaxation in molten cis 1,4-polybutadiene (PB) performed to study chain dynamics properties were reported. Self-diffusion coefficients were measured as a function of temperature and of molecular weight (M) over the range lo4 to 6.7 x lo4 g/mol. The crossover from the Rouse-type behavior (D M-') to the reptation one was found to occur for Mcross 3 x lo4g/mol; for M > Mcrossthe data were consistent with the scaling dependence: D M-2.4f0.05, in agreement with the data analysis recently reported in the literature. The thorough analysis of the transverse relaxation of protons attached to highly entangled PB chains (6.7 x lo45 M 5 43 x lo4 g/mol) gave evidence for the dynamics partition of one chain into two end-submolecules and one inner part clearly discriminated from one another. The number NEndof monomeric units in one end-submolecular, independent of M, is closely related to the monomeric friction coefficient h measured from short chain diffusion over the temperature range 25 to 85°C.The interpretation both of diffusion results and of proton relaxation of inner monomeric units lead to the associated with definition of an effective friction coefficient hEff &4M/NEnd)0'4 the curvilinear diffusion of one chain in its tube. The friction coefficient c L o c associated with local monomeric rotations is discriminated from from its weaker temperature dependence. This approach was applied to polyethyleneoxide chains in solution (DMF, 0.18 5 c 5 1, w/w) where the segmental size of end-submolecules was found to vary as l/c. Experimental results are well matched by this specific NMR approach which accounts for the novel properties of the proton relaxation function.530The 2H spin-lattice relaxation time, TI, of poly(ethy1ene-alt-propylene)(EP)soft segments above their glass transition temperature was measured by solid-state 2H NMR spectroscopy. It was found that the block copolymers had a fast and a slow TI component whereas E P copolymer had only a fast component. The fast T1components for poly(styreneb-EP) diblock copolymer (SEP) and poly(styrene-b-EP-b-styrene) triblock copolymer (SEPS) are similar to the T1 value of EP above ca 20". The slow T1 component for SEP and SEPS exhibited a minimum at 60" and approached the

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value of the fast component near the Tg of polystyrene. The motional behavior of the E P units for SEP is similar to that of SEPS over the entire range of t e m p e r a t ~ r e . 13C ~ ~ ’NMR TI, NOE, and Tlp were measured for two entangled polyethylene melts (M = 16K and 37K) at 5 and 75 MHz 13CLarmor frequencies from the m.p. up to 530 K. Two models for entanglement dynamics are tested against the experimental data. This comparison shows that entanglements not only stretch out those relaxation times longer than the entanglement time ze but also have a spatial effect in restricting the motion of entangled points on the time scale of ze. For polyethylene, the amplitude of residual orientation on the time scale of ze is lower than the theoretical prediction for a Gaussian chain of the entanglement length with both ends fixed. The opposite conclusion was drawn from recent double quantum NMR experiments on polybutadiene. The magnetization decay curve for the Tlp experiment is nonexponential, indicating that the loss of orientation on long time scales depends on position along the chain backbone.532Solid-echo ‘H NMR relaxation was analysed for ultrahigh molecular weight polyethylene reactor powders. Their high crystallinities reveal the characteristics of crystalline free induction decay (FID). A simple but appropriate fitting function for the crystalline component: combination of Weibullian and sine functions were proposed. The applicability of this function was tested for the series of samples having much higher crystallinity of over 90%, which was achieved by fuming nitric acid etching at room temperature over a period of 1 year. The typical beat at 20-30 ps on the observed FID was further emphasized by prolongation of etching time, which corresponds to ‘crystalline’ relaxation. An introduction of the simple Weibullian/sine function for FID fitting reproduces well this beat profile. On the other hand, the FID relaxation of the amorphous chains could be represented by the single Weibullian function. However, they were classified into two categories: intermediate and mobile, depending on their relaxation characteristics. Among these three components (crystalline, intermediate, and mobile ones) exhibiting different chain motions, the intermediate amorphous relaxation was an effective guide for characterization of phase arrangements in the powder samples. The component percentage of this phase decreased with prolongation of the etching time, which was coincident with the crystallinity development detected by thermal analysis. Also, the spin-spin relaxation time of intermediate amorphous chains gradually decreased with the progress of the etching treatment. These results indicate that the molecular motion of the surviving intermediate amorphous phase is restricted. A comparison of morphological changes and these FID characteristic transformations obtained during etching suggested that such an intermediate amorphous phase is located in the region between crystalline domains within the powder

7

Polymer Blend of the Synthetic Macromolecules

Miscible blends of perdeuterated poly(ethy1ene oxide) monomethyl ether (d4PEO)and poly(methy1 methacrylate) (PMMA) were studied using deuterium


417

NMR over the concentration range of 0.5-30% d4PE0 using 2-4 Larmor frequencies ranging from 31 to 76 MHz. Spin-lattice relaxation times and line widths were measured from 300 to 475 K. Over this range PEO is liquid-like or rubbery in terms of its dynamics even though many of the measurements are below the blend glass transition temperature. There is no indication of the DSC glass transition in terms of a jump in either the spin-lattice relaxation times or the line widths. A model suitable for a rubber solid was used to interpret the spin-lattice relaxation times in terms of segmental motion and backbone libration. Segmental correlation times for d4PE0fall in the nanosecond range with a broad distribution of correlation times described by a KWWP of about 0.27. The segmental dynamics of d4PE0 are 12 orders of magnitude faster than PMMA segmental dynamics for a 3% d4PE0blend near the blend Tg. Over the temperature range studied, d4PE0 segmental dynamics are nearly independent of composition for blends from 0.5% to 30% d4PEO. At the lowest concentration studied, d4PEO is in the dilute solution range; this eliminates intermolecular concentration fluctuations as an explanation of the rapid &PEO dynamics. These observations are unusual for miscible polymer blends and cannot be described by current models.534.The length scale of heterogeneity in blends of poly(2,6-dimethyl-p-phenyleneoxide) (PPO) with poly( a-methyl styrene) (PaMS) was investigated using solid state 13CNMR. The PPO/P a MS blend system was homogeneous at ambient, but phase separation at or above the lower critical solution temperature (LCST) was confirmed by polarized-light microscopy (PLM) and DSC. TIHand TlpHwere measured for PPO/P a MS blends of various compositions and neat polymer components (PPO, P a MS). TlpHdata indicated that PPO and P a MS are mixed at the molecular level for all compositions, showing that the miscibility scale is comparable with that of the classical blend system of PPO and polystyrene (PS).535 The effect of the repeated unit length on the substantially increasing molecular motion and entropy change (-T A Sm) of polymer blends was investigated with solid-state 13CNMR and DSC within a miscible window. The hydrogen-bonding strength, from the formation of the phenolic-polyester interaction, was not high enough to overcome the breaking-off of the self-association of the phenolic. With respect to the increasing repeated unit length, the polyester resonance intensity of the solid-state 13C NMR spectra was weakened because of the reduction in the CP efficiency in highly mobile samples. The glass-transition temperature of the blend and the proton spin-lattice relaxation time from NMR experiments were also reduced. The effect of the reduced hydrogen-bonding strength on blending brought about a tendency of higher entropy (-TASm) and higher molecular mobility of the blend. Accordingly, poly(decamethy1ene adipate) possessed the longest repeated unit length and exhibited the most mobile one in this phenolic/polyester blend family. The molecular segmental motion and entropy progressively increased while the repeated unit length of the guest polymers increased within a miscible Poly(methylt hiomet hyl met hacrylate) (PMTMA) is miscible with PVA over the whole composition range as shown by the existence of a single glass transition temperature in each blend. The interaction between PMTMA and PVA was examined by Fourier transform IR spectroscopy, solid-state NMR

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spectroscopy, and XPS. The interactions mainly involve the hydroxyl groups of PVA and the thioether sulfur atoms of PMTMA, and the involvement of the carbonyl groups of PMTMA in interactions is not significant. The measurements of proton spin-lattice relaxation time reveal that PMTMA and PVA do not mix intimately on a scale of 1-3 nm, but are miscible on a scale of 20-30 nm. In comparison, the authors have previously found that PMTMA is miscible with poly(p-vinylphenol) and the 2 polymers mix intimately on a scale of 1-3 nm.537 The blends of poly(hydroxyether of bisphenol A) (phenoxy) with poly(4-vinyl pyridine) (P4VPy) were investigated by DSC, FTIR and high-resolution solidstate NMR spectroscopy. To examine the miscibility of the system at the molecular level, the high resolution 13CCP/MAS together with the high-power dipolar decoupling (DD) NMR technique was employed. Upon adding P4VPy to the system, the chemical shift of the hydroxyl-substituted methylene carbon resonance of phenoxy was observed to shift downfield in the 13C CP/MAS spectra. The TIHand the TlpHwere measured as a function of the blend composition In light of the proton spin-lattice relaxation parameters, it is concluded that the phenoxy and P4VPy chains are intimately mixed on the scale of 20-30 The I3C and 129Xehigh-pressure (HP) MAS NMR methods were used to study the effects of dissolved Xe on segmental motions of polystyrene and poly(viny1 methylether) (PS/PVME) = 50/50 blends under Xe pressure of 0.0-4.0 MPa. The chemical shift of 129Xedissolved in the blend moves to the high-frequency side and the line becomes narrow with increasing Xe gas pressure. The 129Xehigherfrequency shift is explained in terms of an increase of the Xe concentration in the blend. The line narrowing suggests that the Xe atomic diffusion is enhanced with increasing Xe concentration in the blend. On the other hand, the 13Cline widths under MAS and 'H dipolar decoupling (DD) show that the dissolved Xe atoms enhance the segmental motions of the individual component polymers (plasticization effect) at Xe gas pressures higher than 1.0 MPa. The observed pressure and temperature dependence of the line width demonstrates that the motion frequency difference between PS and PVME at high pressures is less than that observed at high temperatures. The dissolved Xe atoms enhance segmental motions of PS and PVME and lower the motion heterogeneity in the blend.539 The miscibility of phenolic resin/poly(vinyl acetate) (PVA) blends was investigated using DSC, FTIR and solid state 13CNMR. The blends display a single Tg over the entire range of composition indicating miscibility in the amorphous phase due to H-bonding between the OH groups of the phenolic resin and carbonyl groups of PVA. Quantitative measurement of the fraction of hydrogenbonded carbonyl group using both 13CNMR and FTIR analyses result in gave good agreement between these two spectroscopic techniques. According to proton spin-lattice relaxation time in the rotating frame, the blend is intimately mixed on a scale less than 2-3 nm.540Poly(p-vinylphenol) (PVPh) and poly(Nacryloylmorpholine) (PAcM) form interpolymer complexes in ethanol/water (1:l)solution. However, only ordinary blends are obtained from DMF solution. Each of the complexes and ordinary blends shows one composition-dependent glass transition temperature, indicating its single-phase nature. Fourier transform IR spectroscopy and 13Csolid-state NMR spectroscopy reveal the existence

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of hydrogen-bonding interactions between the hydroxyl groups of PVPh and the carbonyl groups as well as the ether oxygen of PAcM in the blends and complexes. Measurements of TlpH,reveal that each of the complexes and ordinary miscible blends has one composition-dependent TlpH, indicating an intimate mixing on a scale of about 1.5 nm. The blends show a higher degree of surface enrichment of PVPh than the cornplexe~?~' The thermal stability of a poly(2hydroxyethyl methacrylate) (PHEMA)/poly(methacrylicacid) (PMAA) blend at the same unit mole ratio was interpreted by the formation of the intermolecular bond and its mobility. The mobility change between the intermolecular bond,COOCH2CH200C-,which is formed between the side-chains of PHEMA and PMAA, and the side-chain of PHEMA was investigated by 2D WISE NMR.542 Binary blends formed by poly(vinylpyrro1idone)(PVP)and poly(ethy1ene oxide) (PEO) were studied by solution and solid-state NMR to determine their physical interaction, homogeneity, and compatibility for use as membranes to separate water/alcohol. From the NMR solution it was possible to evaluate the microstructure: PVP presented a preferential syndiotactic distribution sequence and PEO presented two regions, one crystalline and the other amorphous. Considering the solid-state NMR results, it was possible to evaluate molecular dynamics and all binary blends, showing that PEO behaves as a plasticizer; some intermolecular interaction was also observed. An important point was to evaluate the microstructure of the carbonyl PVP that was not observed before.543The miscibility of poly(methacry1ic acid) (PMAA) and poly(viny1 acetate) (PVAc) blends was studied by the CPMAS 13CNMR. The TIHshowed that the PMAA/PVAc blends are homogeneous on a scale of 20-50 nm, and theTlpHrevealed that the PMAA/PVAc-rich blends are partially inhomogeneous on a scale of 2-5 nm, while the PMAA-rich/PVAc blends, including the PM AA/PVAc = 1/1 blend, are completely miscible on the scale. The domain size of the PMAA/PVAc = 1/1 blend was also estimated from the Goldman-Shen experiment. The obtained maximum domain size of 2.5 nm is consistent with the results of TlpH The decomposition of 13C NMR spectra for the carboxyl (COOH) and carbonyl (COO) regions and the TICsuggested the existence of an interpolymer interaction between PMAA and PVAc: the interaction is most likely a hydrogen bond and acts an important role for a great miscibility. The dependence of TICon the PMAA molar ratio indicates that the molecular motion of PVAc is largely affected by PMAA.544Miscibility and phase behavior in the blends of phenolphthalein poly(ether sulfone) (PES-C) with poly(hydroxyether of bisphenol A) (PH) were investigated by means of DSC, high resolution solid state NMR and FTIR. It was found that the homogeneity of the as-prepared blends depended on the solvents used; N,N-dimethylformamide (DMF) provided the segmental mixing for PH and PES-C, which is confirmed by the behavior of single, composition-dependent T,s. To examine the homogeneity of the blends at the molecular level, the TlpHwere measured via 13CCP/MAS NMR spectroscopy as a function of blend composition. In view of the TlpHvalues, it is concluded that the PH and PES-C chains are intimately mixed on the scale of 20-30A.545

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441. M. Mitani, R. Furuyama, J. Mohri, J. Saito, S. Ishii, H. Terao, T. Nakano, H. Tanaka, T. Fujita, J . Am. Chem. Soc. 2003,125 (14), 4293-4305 442. R. A. Ozzetti, A. Pedro de Oliveira Filho, U. Schuchardt, D. Mandelli, J . Appl. Polym. Sci. 2002,85 (4), 734-745 443. Y. -W. Shin, H. Nakatani, T. Uozumi, B. Liu, T. Sano, K. Nitta, M. Terano, Japan Advanced Institute of Science and Technology, School of Materials Science, Ishikawa, Japan. Polymer International 2003,52 (l), 29-34 444. R. Thomann, Y. Thomann, R. Mulhaupt, J. Kressler, K. Busse, D. Lilge, J. C . W. Chien, J . Macromol. Sci., Phys. 2002, B41 (4-6), 1079-1090 445. J. C. Yoder, J. E. Bercaw, J . Am. Chem. Soc. 2002,124 (ll), 2548-2555 446. W. Weng, W. Hu, A. H. Dekmezian, C. J. Ruff, Macromolecules 2002,35 (lo), 38383843 447. G. J. Lee, S. H. Lee, K. S. Ahn,K. H. Kim, J . Appl. Polym. Sci. 2002, 84 (14), 2583-2590 448. H. D. Park, W. K. Lee, T. Ooya, K. D. Park, Y. H. Kim, N. Yui, Journal of Biomedical Materials Research 2002,60 (l), 186-190 449. R. Shankar, A. Saxena, A. S. Brar, Journal of Organometallic Chemistry 2002, 650 (1-2), 223-230 450. C. -L. Chiang, C. -C. M. Ma, J . Polym. Sci., Part A: Polym. Chem. 2003, 41 (9), 1371-1379 451. A. Hirao, N. Haraguchi, Macromolecules 2002,35 (19), 7224-7231 452. P. Li, K. -Y. Qiu, Polymer 2002,43 (lo), 3019-3024 453. J. R. Lizotte, T. E. Long, Macromol. Chem. Phys. 2003,204 (4), 570-576 454. Y. Luo, Y. Yao, Q. Shen, Macromolecules 2002,35 (23), 8670-8671 455. J. -F. Lutz, K. Matyjaszewski, Polym. Prep. (Am. Chem. Soc., Div. Polym. Chem.) 2002,43 (2), 229-230 456. A. J. Maliakal, N. J. Turro, Abstracts of Papers, 224th ACS National Meeting, Boston, M A , United States, August 18-22,2002 2002, PMSE-005 457. J. Rong, Z. Yang, Macromol. Muter. Eng. 2002,287 (l), 11-15 458. S. -I. Yusa, T. Yamamoto, A. Hashidzume, Y. Morishima, Polym. J . (Tokyo, Japan) 2002,34 (3), 117-124 459. C. -T. Zhao, M. Do Rosario Ribeiro, M. F. Portela, Macromol. Chem. Phys. 2002, 203 (13), 1983-1987 460. K. Hatada, T. Kitayama, T. Nishiura, W. Shibuya, J . Polym. Sci., Part A: Polym. Chem. 2002,40 (13), 2134-2147 461. R. Francis, B. Lepoittevin, D. Taton, Y. Gnanou, Macromolecules 2002, 35 (24), 9001-9008 462. J. -Y. Yuan, C. -Y. Pan, Eur. Polym. J . 2002,38 (8), 1565-1571 463. N. Ekizoglou, N. Hadjichristidis, J . Polym. Sci., Part A: Polym. Chem. 2002,40 (13), 2 166-2170 464. R. A. Cruz, A. M. Mendoza, T. Heinze, International Journal ofPolymeric Materials 2002,51 (7), 661-674 465. R. Advincula, Q. Zhou, J. Mays, Materials Research Society Symposium Proceedings 2002,676 (Synthesis, Functional Properties and Applications of Nanostructures), Y3.44. 1-Y3.44. 6 466. I. Cianga, Y. Hepuzer, E. Serhatli, Y. Yagci, J . Polym. Sci., Part A: Polym. Chem. 2002,40 (13), 2199-2208 467. H. T. A. Wilderbeek, J. G. P. Goossens, C. W. M. Bastiaansen, D. J. Broer, Macromolecules 2002,35 (24), 8962-8968 468. L. Zhai, D. D. Laird, R.D. McCullough, Polymeric Materials Science and Engineer-

43 6


ing 2002,87 288-289 469. N. Ma, A. Petit, L. Yahia, 0.L. Huk, M. Tabrizian,Journal ofBiomaterials Science, Polymer Edition 2002,13 (3), 257-272. 470. X. Duan C. M. Griffith, M. A. Dube H. Sheardown, JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 2002,13 (6), 667-89 471. A. K. Dhaliwal, J. N. Hay, Thermochimica Acta 2002,391 (1-2), 245-255 472. P. Christjanson, K. Siimer, T. Pehk, I. Lasn, Holz als Roh-und Werkstoff 2002,60 (6), 379-384 473. C. Chioli, Y. Lansac, P. Pasini, J. Stelzer, C. Zannoni, Molecular Crystals and Liquid Crystals Science and Technology, Section A:MoEecular Crystal and Liquid Crystals, 2002,372, 157-162 474. C. Chiccoli, P. Pasini, G. Skacej, C. Zannoni, S. Zumer, Physical Reuiew E:Statistical, Nonlinear, and Soft Matter Physics, 2002,65., 051703/1-051703/7 475. Y. Yin, C. Zhao, A. Sasaki, H. Kimura, S. Kuroki and I. Ando, Macromolecules, 2002,35,5910-5915 476. S. Kanesaka, H. Kimura, S. Kuroki, I. Ando and S. Fujishige, Polym. Prepr., 2003, 44,357-358 476 . S. Kanesaka, H. Kimura, S. Kuroki, S. Fujishige and I. Ando, Abstracts of Papers, 225th ACS National Meeting, New Orleans, LA, United States March 23-27,2003, 2003, POLY-565 477 . A. Pampel, D. Michel, R. Reszka, Chemical Physics Letters, 2002,357, 131-136 478. D. McElheny, J. Grinshtein, V. Frydman, L. Frydman, Macromolecules, 2002, 35, 3544-3552 479. M. Mizuno, A. Hirai, H. Matsuzawa, K. Endo, M. Suhara, M. Kenmotsu, C. D. Han, Macromolecules, 2002,35,2595-2601 480. J. R. van derMaarel, D. E. Woessner, M. E. Merritt, J . Physical Chemistry B, 2002, 106,3864-3871 481. H. Ishida, F. Horii, Macromolecules, 2002,35,5550-5558 482. E. B. Barmatov, J . Polym. Sci., Part A: Polym. Chem., 2002,40,2044-2048 483. C. Stubenrauch, C. Frank, R. Strey, D. Burgemeister, C. Schmidt, Langmuir, 2002, 18,5027-5030 484. J. -F. Trempe, F. G. Morin, Z. Xia, R. H. Marchessault, K. Gehring, J . Biomolecular NMR, 2002,22,83-87 485. H. Sibert, P. Becker, I. Quijada-Garrido, D. A. Grabowski, C. Schmidt, Solid State Nuc. Magn. Reson., 2002,22, 31 1-326 486. M. Vilfan, B. Zalar, A. K. Fontecchio, M. Vilfan, M. J. Escuti, G. P. Crawford, S. Zumer, Phys. Rev. E:Stat. Nonlin. Soft Matter Phys., 2002,66,021710 487. P. Lesot, M. Sarfati, J. Courtieu, Chemistry-A Eur. Journal, 2003,9, 1724-1745 488. T. G. Nunes, G. Guillot, S. G. Pereira, R. Pires, J . Phys. D:Appl. Phys., 2002,35, 1251-1257 489. M. Garbarczyk, W. Kuhn, J. Lkinowski, S. Jurga, Polymer, 2002,43,3169-3172 490. M. Gussoni, F. Greco, M. Mapelli, A. Vezzoli, E. Ranucci, P. Ferruti, L. Zetta, Macromolecules, 2002,35,1714-1721 491. M. Gussoni, F. Greco, M. Mapelli, A. Vezzoli, E. Ranucci, P. Ferruti, L. Zetta, Macromolecules, 2002,35, 1722-1729 492. P. Adriaensens, L. Storme, R. Carleer, J. D’Haen, J. Gelan, V. M. Litvinov, R. Marisse, J. crevecoeur, Macromolecules, 2002,35, 135-140 493. Y. Hotta, I. Ando, J . Mol. Struct., 2002,602-603,165-170 494. B. MacMillan, M. Halse, M. Schneider, L. Fardy, Y. H. Chui, B. J. Balcom, Appl. Magn. Reson., 2002,22,247-256


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495. M. A. fernandez-Seara, S. L. Sehrli, F. W. Wehrli, J. Magn. Reson., 2003, 160, 144-150 496. Wang, Mingfei; Bertmer, M. ; Demco, D. E. ; Bluemich, B. ; Litvinov, V. M. ; Barthel, H., Macromolecules (2003), 36 (12), 441 1-4413. 497. Ricciardi, Rosa; Gaillet, Christine; Ducouret, Guylaine; Lafuma, Francoise; Laupretre, Francoise., Polymer (2003),44 (1l), 3375-3380. 498. Bajaj, Pushpa; Sreekumar, T. V. ; Sen, Kushal; Kumar, Rajeev; Brar, Ajaib Singh., Journal of Applied Polymer Science (2003),88 (9,1211-1217. 499. Thakur, Khalid A. M. ;Newmark, Richard A. ;Kuehn, Nicole T. ;Gregar, Travis Q, Macromolecules (2003),36 (3), 719-723. 500. Kimura, Hideaki; Okita, Koichi; Ichitani, Motokuni; Sugimoto, Toshiya; Kuroki, Shigeki; Ando, Isao., Chemistry of Materials (2003), 15 (l),355-362. 501. De Rosa, Claudio; Auriemma, Finizia; Talarico, Giovanni; Busico, Vincenzo; Caporaso, Lucia; Capitani, Donatella., Macromolecules (2002), 35 (4), 1314-1318. 502. Brar, A. S. ; Kaur, Manpreet., Journal of Molecular Structure (2002), 606 (1-3), 23 1-240. 503. Hazendonk, Paul; Harris, Robin K. ; Galli, Giancarlo; Pizzanelli, Silvia., Physical Chemistry Chemical Physics (2002), 4 (3), 507-513. 504. Chen, Yan; Yang, Guang; Chen, Qun., Polymer (2002),43 (7), 2095-2099. 505. Wang, Yingzi; Inglefield, Paul T. ;Jones, Alan A., Polymer (2002),43 (6), 1867-1872. 506. Lin, Chia-Liang; Kao, Hsien-Ming; Wu, Ru-Rong; Kuo, Ping-Lin., Macromolecules (2002),35 (8), 3083-3096. 507. Proietti, N. ; Amato, M. E. ; Masci, G. ;Segre, A. L., Macromolecules (2002),35 (1l), 4365 -4372. 508. Bourbigot, Serge; Flambard, Xavier; Revel, Bertrand., European Polymer Journal (2002), 38 (8), 1645-1651. 509. Dargaville, T. R. ; George, G. A. ; Hill, D. J. T. ; Scheler, U. ; Whittaker, A. K., Macromolecules (2002),35 (14), 5544-5549. 510. Brar, A. S. ; Kumar, Rajeev., Journal of Polymer Science, Part A: Polymer Chemistry (2002), 40 (14), 2225-2236. 511. Lai, Simona; Casu, Mariano; Saba, Giuseppe; Lai, Adolfo; Husu, Ivan; Masci, Giancarlo; Crescenzi, Vittorio., Solid State Nuclear Magnetic Resonance (2002), 21 (3/4), 187-196. 512. Nakaoki, Takahiko; Inaji, Yoritsugu., Polymer Journal (Tokyo, Japan) (2002), 34 (7), 539-543. 513. Tozuka, Yuichi; Ito, Atsutoshi; Seki, Hiroko; Oguchi, Toshio; Yamamoto, Keiji., Chemical & Pharmaceutical Bulletin (2002), 50 (8), 1128-1130. 514. Brar, A. S. ; Kumar, Rajeev., Journal of Molecular Structure (2002),616 (1-3),37-47. 515. Zhang, Qiujin; Lin, Weixin; Yang, Guang; Chen, Qun., Journal of Polymer Science, Part B: Polymer Physics (2002),40 (19), 2199-2207. 516. Xu, Jianwei; He, Chaobin; Toh, Kee Chua; Lu, Xuehong., Macromolecules (2002), 35 (23), 8846-8851. 517. Brar, Ajaib S. ; Kumar, Rajeev; Kaur, Manpreet., Applied Spectroscopy (2002), 56 (lo), 1380-1382. 518. Mathew, Raji; Mattes, Benjamin R. ; Espe, Matthew P., Synthetic Metals (2002), 131 (1-3), 141-147. 519. Wormald, Philip; Apperley, David C. ; Beaume, Francois; Harris, Robin K., Polymer (2002), Volume Date 2003,44 (3), 643-651. 520. Werkhoven, Thekla M. ; Mulder, Fokko M. ; Zune, Catherine; Jerome, Robert; de Groot, Huub J. M., Macromolecular Chemistry and Physics (2003),204 (l),46-51.

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521. Alamo, R.G. ;Blanco, J. A. ;Carrilero, I. ;Fu, R., Polymer (2002),43 (6), 1857-1865. 522. Miyoshi, Toshikazu; Hayashi, Shigenobu; Imashiro, Fumio; Kaito, Akira., Macromolecules (2002), 35 (7), 2624-2632. 523. Goddard, Yanina A. ;Vold, Robert L. ;Hoatson, Gina L., Macromolecules (2003), 36 (4), 1162-1169. 524. Goward, Gillian R. ;Sebastiani, Daniel; Schnell, Ingo; Spiess, Hans Wolfgang; Kim, Ho-Dong; Ishida, Hatsuo., Journal of the American Chemical Society (2003), 125 (19), 5792-5800. 525. Pascui, Ovidiu; Beiner, Mario; Reichert, Detlef., Macromolecules (2003), 36 (1l), 3992-4003. 526. Virtanen, E. ; Nissinen, M. ; Suontamo, R. ; Tamminen, J. ; Kolehmainen, E. , Journal of Molecular Structure (2003), 649 (3), 207-218. 527. von Meerwall, E. ; Ozisik, R. ; Mattice, W. L. ; Pfister, P. M., Journal of Chemical Physics (2003), 118 (S), 3867-3873. 528. Baille, W. E. ; Malveau, C. ; Zhu, X. X. ; Kim, Y. H. ; Ford, W. T., Macromolecules (2003), 36 (3), 839-847. 529. Brus, J. ; Dybal, J. ; Sysel, P. ; Hobzova, R., Macromolecules (2002), 35 (4), 12531261. 530. Guillermo, Armel; Cohen Addad, Jean-Pierre., Journal of Chemical Physics (2002), 116 (7), 3141-3151. 531. Nakajima, Takeshi; Akiyama, Tsutomu; Furuya, Hidemine., Magnetic Resonance in Chemistry (2002),40 (2), 161-164. 532. Qiu, XiaoHua; Ediger, M. D., Macromolecules (2002), 35 (5), 1691-1698. 533. Uehara, Hiroki; Aoike, Taku; Yamanobe, Takeshi; Komoto, Tadashi., Macromolecules (2002), 35 (7), 2640-2647. 534. Lutz, T. R. ;He, Yiyong; Ediger, M. D. ; Cao, Haihui; Lin, Guoxing; Jones, Alan A., Macromolecules (2003),36 (5), 1724-1730. 535. Woo, Ea Mor; Chou, I-Chi; Chang, Li Ling; Kao, Hsien-Ming., Polymer Journal (Tokyo, Japan) (2003), 35 (4), 372-378. 536. Hung, Albert Y. C., Journal of Polymer Science, Part B: Polymer Physics (2003),41 (7), 679-686. 537. Yi, J. Z . ; Goh, S. H, Polymer (2003),44 (6), 1973-1978. 538. Zheng, Sixun; Mi, Yongli., Polymer (2003),44 (4), 1067-1074. 539. Miyoshi, Toshikazu; Takegoshi, K. ; Terao, Takehiko., Macromolecules (2002), 35 (l), 151-154. 540. Huang, Mei-Wen; Kuo, Shiao-Wei; Wu, Hew-Der; Chang, Feng-Chih; Fang, SuYun. National Chiao Tung University, Institute of Applied Chemistry, Hsinchu, Taiwan. Polymer (2002),43 (S), 2479-2487. 541. Yi, J. Z . ; Goh, S. H., Polymer (2002), 43 (16), 4515-4522. 542. Asano, Atsushi; Kurotu, Takuzo., Polymer Degradation and Stability (2002),78 (l), 137-141. 543. Diniz, Teresinha M. F. F. ; Tavares, Maria I. B., Journal of Applied Polymer Science (2002), 85 (14), 2820-2823. 544. Asano, Atsushi; Eguchi, Masaru; Shimizu, Miho; Kurotsu, Takuzo., Macromolecules (2002),35 (23), 8819-8824. 545. Zheng, Sixun; Guo, Qipeng; Mi, Yongli., Polymer (2002),Volume Date 2003,44 (3), 867-876.

12 NMR in Living Systems BY M.J.W. PRIOR

1

General Applications and New Methodologies

The magnitude of signal errors in 31PNMR ISIS estimations of metabolite concentrations has been investigated using simulations and measurements in phantoms. At low ratios of TR/Tl the contamination of signals was found to increase rapidly especially in small ( ~ 2 cm') 7 volumes of interest. When TR/T1 = 1, the signal from a 27 cm3VOI was 20% too high and from an 8 cm3VOI was 150% too high. Although the signal in an ISIS experiment was found to vary linearly with concentration, measurements from a low concentration VOI within a larger phantom of higher concentration were too high.' The ratios of Nacetylaspartate/(total creatine) (NAA/tCr), (choline-containing compounds)/tCr (Cho/tCr) and myo-inositol/tCr (mI/tCr) have been obtained from 2 x 2 ~ 2cm voxels located in the frontal lobe of healthy, young male and female volunteers using a semi-automated system with the aim of producing reference data.2 Spectra from the human brain have been obtained at 7 Tesla using 'H NMR following the adjustment of shims assisted by use of FASTMAP. Ultra-short echo-time (6 ms) STEAM was used to reduce J-modulation effects and signal attenuation due to the T2 of metabolites. It was possible to detect glucose, at 5.23 ppm, in single shot spectra obtained from an 8 cm3~ o x e lA. ~new method for the estimation of cerebral glucose utilization (CMRGLC)in the rat has been r e p ~ r t e d . ~ After a 24 h fast, rats were injected with a bolus, and then infused with, [1-"C] deoxyglucose for 50 min. After 30 min, rats were then given a bolus of 500 mg kg-' 2-[6-'3C]deoxyg1ucose and data were collected for another 20 minutes. During this period the value of CMRGLC was estimated to be 0.6 0.4 pmol min-' g-'. The longitudinal and transverse relaxivities of Gd(DTPA)2-,Gd(D0TA)- and Gd(DTPA-BMA) have been determined in solutions containing choline, creatine, N-acetylaspartate. The maximum potential influence of the agents on in vivo metabolite signals was calculated using the data acquired in s ~ l u t i o nThe .~ effects of Gd-DTPA on the intensity of the peak representing choline containing compounds (Cho) observed in 'H NMR spectra have been investigated in patients with gliomas. Although there was broadening of the Cho signal by Gd-DTPA in T1-weighted spectra, there was no increase in signal intensity that has been observed in prior studies implying that previous effects could be attributed to T2*/susceptibilityeffects! The apparent transverse relaxation times Nuclear Magnetic Resonance, Volume 33 0 The Royal Society of Chemistry, 2004

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for water, N-acetylaspartate (NAA) and creatine plus phosphocreatine (tCr) have been measured at 4 T and 7T in the human brain. A significantly reduced value . ~ possible sources of errors in for T2*was observed at 7, compared to 4, T e ~ l aThe the measurements of NAA in the human brain have been assessed. Software was written to minimize systematic errors and the concentration of NAA in was determined in two subjects.8

2

Cells

Microorganisms. - A review of the metabolism of lactic acid bacteria studied by NMR methods has been produced? The effects of different levels of NADH oxidase on glucose metabolism has been investigated with 13Cand 31PNMR in three strains of Lactococcus lactis: the parent strain of L. Zactis MG1363, a NOX- strain with a deletion of the gene for NADH oxidase, and a NOX+ strain with a hundred-fold increase in NADH oxidase activity. In all strains under aerobic conditions the level of fructoes-1,6bisphosphate (F16P) was lower than under anaerobic conditions, and the rate of F16P depletion was very high. The levels of 3-phosphoglycerate and phosphoenolpyruvate (PEP) were enhanced under aerobic conditions and significantly lower in the NOX- strain. Glycolytic flux was decreased in the presence of saturating levels of oxygen, but was not altered by NADH oxidase levels." In a study of the effects of selenium on the growth of cyanobacterium (Spirulina plantensis (Nordst.) Geitl. IPPAS B-256), 31PNMR has been used to measure phosphorous metabolites and intracellular pH (pHi) of cells in suspension. There was a significant shift in pHi, from 7.17 to 6.18, and a decrease in sugar phosphate concentrations when growth was inhibited by exposure to larger amounts of selenium." The dynamics of the build up and breakdown of polyphosphate in Corynebacteriurn glutarnicurn has been followed using 31PNMR with a time resolution of 50 seconds. Under conditions of 60-80% saturation with 02,the rapid accumulation of high levels of polyphosphate were detected when Pi and, glucose or acetate were added to cell suspensions. Furthermore, there was a similarly rapid decrease in polyphosphate, and an increase in sugar phosphates when the O2 supply was switched off.12 The metabolism of [3-13C]propionate under anaerobic and aerobic conditions has been followed with 13CNMR whilst phosphorous metabolites were detected by 31PNMR in activated sludge obtained from a wastewater treatment plant. During an initial anaerobic phase, the conversion of [3-13C]propionate to l3C-labe1led polyhydroxyalkanoates was observed in vivo and the composition of the peak was confirmed by NMR analysis of extracts. In a subsequent aerobic phase, cells were incubated without a carbon source, but in the presence of phosphate. During this period, the consumption of 13C-labelledpolyhydroxyalkanoates was observed in vivo along with the accumulation of glycogen. 31PNMR detected the accumulation of intracellular Pi, although no polyphosphate formation was detected. During the final anaerobic incubation with unlabelled propionate, the consumption of l3C-labe1ledglycogen was observed. There was a small increase in the amount of

2.1

12: N M R in Living Systems

441

13C-labelledpolyhydroxyalkanoates, which indicated the conversion of glycogen to this group of compound^.'^ The effects of dissolved oxygen on the pH value of the mycelium of Aspergillus niger have been measured with 31PNMR. The obtained values were then used in vitro to examine the effects of pH on the kinetics of key enzymes.14Intracellular pH homeostasis in the filamentous fungus Aspergillus Niger, immobilised in Ca2+-alginatebeads, has been investigated with 31PNMR. The fungus maintained constant cytosolic pH (pH,,,) and vacuolar pH (pH,,,) values of 7.6 and 6.2, respectively, when the external pH was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a ApH across the cytoplasmic membrane of 6.6-6.7 was reached. Perfusion with various hexoses and pentoses revealed that the magnitude of ApH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the source. Furthermore, larger ApH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Exposure of A . niger to 1,2 or 10 pM carbonyl cyanide m-chlorophenylhadrazone led to decreased ATP levels and increased respiration, and to a partial, transient or permanent collapse of ApH of the vacuolar membrane, respectively. Non-lethal levels of azide caused a transient decrease in pH,,, that was closely paralleled by a transient vacuolar a~idification.'~

2.2 Blood. - The diffusional water permeability (Pd) of dog and cat red blood cells (RBC) membranes has been investigated using a doped-water NMR method. The values of Pd for cat RBC were 3.0,3.5,4.2,4.4 and 5.9 xlOV3cm s-' at 15,20,25,30 and 38 "C, respectively. The values of Pd for dog RBC were 3.8, 4.6, 5.0, 5.9 and 7.9 xlOP3 cm s-' at 15, 20, 25, 30 and 38 "C, respectively. Inhibition of Pd by p-chloromercuribenzene sulfonate was maximal in dog RBC in 15-30min and in cat RBC in 60 min. The maximal inhibition of Pd in dog RBC was higher at all temperatures s t ~ d i e d .The ' ~ effects of p-chloromercuribenzene sulfonate (PCMBS) on the water permeability of marsupial RBC membranes have been measured with an Mn2+-doping'H NMR r n e t h ~ d .In ' ~ koala (Phascolarctos cinereus) RBC at 37", the maximum inhibition was reached in 60 min with 1 mM PCMBS and in 15-30 min with 2 mM PCMBS. In RBC from red kangaroo (Macropus rufus) and the swamp wallaby (Wallabia bicolor) the maximum inhibition was reached after 90 min of incubation at 37" with 2 mM PCMBS. The maximal inhibition of Pd in RBC of all species studied was 60-70% at 37". 2.3 Cultured Mammalian and Tumour. - A combination of MAS high resolution NMR and pattern recognition methods have been used to examine batches of liver cell spheroids incubated with or without D-galactosamine. Principle component analysis revealed separation of control and treated cells based on the concentration of triglycerides and cholesterol.'* The effects of 2,Sanhydromannitol on ATP content, oxygen consumption and substrate oxidation have been investigated in isolated hepatocytes from rats fed on a high fat-low carbohydrate (HFLC) diet or a low fat-high carbohydrate (LFHC) diet. Cells from

442


HFLC fed rats had similar ATP content, but lower oxygen consumption, decreased fructose oxidation and increased palmitate oxidation compared to cells from LFHC fed rats. Fructose and palmitate oxidation were affected by exposure to 2,5-anhydro-mannitol only in cells from LFHC fed rats, and there was a larger decrease in ATP content in cells from LFHC fed rats, compared to cells from HFLC fed rats exposed to 2,5-anhydro-mannito1.l9 The metabolism of A549 cells grown on microcarrier beads has been monitored with 31Pand 13CNMR. Quantitative spectra of phosphorous metabolites were collected that demonstrated that the increase in total phosphorous metabolites reflected the increased cells mass. Furthermore, the metabolism of [l-t3C]g1ucose was monitored in these cells and peaks for glucose and lactate were detected. The rate of glucose consumption was measured to be 0.25 pmol min-' lop8cells.2o

3

Plant Studies

A review has been produced on the use of magnetic resonance spectroscopy and imaging in plant metabolic engineering.2t The metabolism of glycerophosphocholine in perfused carrot (Daucus carota) cells has been investigated using 31PNMR. When glycerophosphocholine was added to the perfusion medium a decrease in its 31PNMR peak was observed in the first hours of the experiment. This decrease was concomitant with the appearance of another peak at 3.2 ppm representing phosphocholine. Further analysis of the isolated perfusion medium and cell extracts revealed that the medium contained glycerol-3-phosphate, Pi and glycerophosphocholine, but phosphocholine was present only in cells.22The effects of overproduction of animal cell death suppressor proteins, Bcl-xL or Ced-9, in transgenic tobacco cells have been investigated using 31PNMR. Seeds of those plants over expressing Bcl-xL or Ced-9 protein were able to germinate, and seedlings were able to grow, in the presence of 0.2M NaC1. Furthermore, transgenic seedlings were more tolerant to cold compared to wildtypes. 31PNMR was used to investigate intracellular changes in cell suspension cultures exposed to high [NaCl]. Cytoplasmic acidification and vacuolar alkalinisation were found in wildtype cells immediately after 0.3M NaCl treatment; pHcytwas 0.3 and 0.9 at 20 and 80 min, respectively. However, pHcytin transgenic cells was constant for 80 min whilst pHvac decreased transiently at 20 min. In transgenic cells exposed to 0.5M NaCl, pHCytdecreased from 7.3 to 6.1 within 15 min, but remained constant for a further 85 min, while pHvac was maintained at 4.9 for 50 min, rapidly increased to 5.6, and then this was maintained for a further 50

4

Tissues

Brain. - A review of 13C NMR studies of the compartmentalisation of cerebral glucose metabolism has been produ~ed.2~ 4.1


443

Microdialysis has been combined with in vivo 13CNMR to investigate the uptake of glutamate from the extracellular space of the rat brain. Brain Glu C5 was enriched by an i.v. infusion of [2,5-'3C]glucose, which was chased by ["C] glucose to remove 13C-labelfrom the glial pool. Glial uptake of extracellular neurotransmitter [5-13C]Glu was monitored in vivo through the formation of [5-13C, "NIGln during the infusion of '5NH4A~.25 The labelling time coarse of glutamate C4 and C3 has been monitored in vivo in the rat brain after treatment with 3-nitropropionic acid. 'H-observe 13C-editedspectra were collected and the data were fitted to a mathematical model to yield the TCA cycle rate (VTCA) and the exchange rate of a-ketoglutarate and glutamate ( V K G ) . Treatment with 3NP induced a decrease in V T C A from 0.71 & 0.02 to 0.58 & 0.02 pmol 8 - I min-' and an increase in V K G from 0.88 0.08 to 1.33 0.24 pmol g-' min-'.26 The metabolism of [1,6-'3C]gl~c~~e has been followed in the rat brain using 'Hobserved l3C-edited NMR. The labelling of [4-'3C]gl~tamate and [4-"C] glutamine was detected in a 0.1 cm3 volume. The formation of 13C-labelled [2-' 3C]aspartate, [I3-13C]aspartate, [3-13C]lactateand [3-13C]alaninewas also observed.2713CNMR in the rat brain has been used to assess the concentration of glycogen, and its turnover.28Animals were fed [l-'3C]glucose for 48 hours before experimentation and the labelling patterns were assessed. Labelling of glycogen, NAA, Glu, Gln, Asp, and glucose was observed, but the amount of labelling was variable. The turnover of brain glycogen was estimated to be half that of NAA and the total concentration of glycogen was calculated to be 3.3 f 0.8 pmol g-'. The effects of lactate as an energy source in ischaemic-preconditioned rat brain slices has been investigated with 31PNMR. Slices were obtained from the left-brain hemisphere of rats where the contralateral middle cerebral artery had been occluded 48 h previously, or they were obtained from the left hemisphere of normal rats. All slices were pre-treated with iodoacetate and fluorocitrate and perfused with artificial cerebrospinal fluid containing lactate. Stimulation with high-K+ buffer caused the disappearance of PCr and ATP in spectra of preconditioned and normal slices. However, PCr recovered only in the preconditioned ~lices.2~ The effects of aging on Cho, tCr and NAA in the rat brain have been studied with 'H NMR in vivo and in tissue extracts. The ratios of Cho/NAA and Cho/tCr increased in the aged hippocampus, whereas the ratio of tCr/NAA was similar in the aged and adult hippocampus. The ratios of Cho/NAA, Cho/tCr and tCr/NAA remained similar in the cortex of adult and aged rats. In brain slices it was found that the activity of the low-affinity choline uptake system increased in the aged cortex and hippocampus. However, incorporation of choline into phospholipids exhibited no change in high- and low-affinity kinetic^.^' The effects of propofol on lactate accumulation and oedema formation has been investigated in the rat brain under hyperglycaemic conditions. Propofol(l2 mg kg-' or 60 mg kg-') was given 30 min prior to 2 g kg-' glucose i.p. After a further 30 min, the middle cerebral was occluded for 60 min and was followed by a 120 min period of reperfusion. Lactate accumulation increased continuously during ischaemia and reperfusion in the 12 mg kg-' propofol group, this accumulation was attenuated in the 60 mg kg-' propofol group, which ended the reperfusion

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period with near baseline levels of lactate. Similarly, the accumulation of oedema, measured with diffusion weighted MRI, occurred continuously in the low dose group, whereas the high dose group had little remaining oedema at the end of reperf~sion.~' In an investigation of the role of leukocytes in ethanolinduced microvascular damage in the rat brain, 31P NMR has been used to measure phosphorous metabolites. Administration of alcohol produced dose dependent venular vasospasm, rolling and adherence of leukocytes to venular walls, and a decrease in leukocyte velocity. Intermediate to high doses of ethanol resulted in the infiltration of leukocytes and macrophages across venular walls, concentration dependent increases myeloperoxidase staining in parenchyma, and rupture of post-capillary venules with focal haemorrhages. Administration of 4 g kg-' or 6 g kg-' ethanol resulted in whole brain losses of ATP and PCr, increased Pi and a decreased pHi. Vinblastine-depletion of circulating leukocytes prevented or ameliorated alcohol-induced microvascular damage and depletion of high-energy phosphate^.^^ 4.2 Heart. - The NMR in the study of the mouse heart has been reviewed.33 The effects of nicorandil on cardiac blood flow and high energy phosphates (HEP) have been studied in the pig heart. Myocardial infarction was caused by proximal left circumflex coronary artery ligation, 31PNMR was used to measure phosphorous metabolites and radioactive microspheres were used to measure changes in blood flow. The ratio of PCr/ATP in infarcted hearts was 2.01 f 0.11, 1.85 0.10 and 1.59 & 0.11 in the subepicardium, midwall and subendocar0.09, dium, respectively, compared to 2.22 f 0.11, 2.01 f 0.15 and 1.80 respectively, in the normal heart. Nicorandil had no effect on HEP of the normal heart but increased the ratio of PCr/ATP to 1.87 & 0.10 in the subendocardium of infarcted hearts. However, there was only a small redistribution of blood flow away from the subendocardium of the infarcted heart.34 The effects of hypoosmotic shock on the rate of Rb+ efflux, pHi and cellular energetics have been investigated in Langendorff-perfused rat hearts using 87Rb and 31PNMR. Two models of hypoosmotic shock were compared: 1) normal hearts perfused with 70 mM NaCl buffer 2) hyperosmotic hearts, equilibrated with additional Methyl a-d-glucopyranoside or urea, perfused with normosmotic buffer. Four minutes after hypoosmotic shock, the Rb+ efflux rate constant transiently increased twofold, while pH decreased slightly, without any affect on ATP or PCr. Dimethylamiloride abolished activation of the Rb+ efflux in the second model, but pHi was unaffected. There was a 120% increase in the rate of Rb+ efflux after 12 or 20 min of global ischaemia. Treatment with glibenclamide partially decreased the rate constant, but reperfusion with hyperosmotic buffer had no effect on its value.35The role of cardiac ATP-sensitive K + channels induced by the HMG-CoA reductase inhibitor, pravastatin, in myocardial metabolism during ischaemia has been investigated in the isolated rabbit heart. Hearts were untreated (control group), or treated with pravastatin, pravastatin plus glibenclamide or pravastatin plus L-NAME 60 min prior to 45 min of global, normothermic ischaemia. Compared to the control group during ischaemia, increases in Pi, and decreases in ATP and PHi, were significantly


445

inhibited by pravastatin, but were not affected by pravastatin plus glibenclamide or pravastatin plus L-NAME.36The roles of coronary flow and metabolism in the uptake of Rb+ have been studied in the isolated pig heart with 87RbMRI and 31PNMR. Following loading with Rb+, perfusion to the left anterior descending artery bed was stopped (no flow), reduced (low flow) or replaced with deoxygenated buffer (hyp) for 2 hours; perfusion was then restored. The signal intensities of 87Rbin the left anterior wall were higher, and the fluxes greater, in the hyp and low flow hearts compared to the no flow hearts. Furthermore, PCr and ATP decreased less in the hyp and low flow hearts.37The effects of P-1075, a potent opener of ATP-sensitive K + channels, have been investigated in the perfused rat heart using 31Pand 87RbNMR. A 20 min infusion of P-1075 depleted PCr and ATP by about 40% and 33%, respectively; Pi increased by over 100%. There was an almost complete recovery of PCr 10 min after the infusion ceased, but ATP did not recover. Co-administration of glibenclamide with P-1075 prevented the fall in PCr and ATP, but a combination of 5-hydroxy decanoic acid with P-1075 did not. Treatment with 2,4-dinitrophenol had a similar effect to that of P-1075, but its action was not affected by glibenclamide. The efflux of Rb+ from pre-loaded hearts was increased by the presence of P-1075 or 2,4dinitrophenol, and reduced to baseline when glibenclamide was used in combination with P- 1075 or 2,4-dinitrophen01.~~ The role of phospholamban in ischaemic injury has been investigated in the perfused mouse heart using "P NMR. Wild type and phospholamban-knockout hearts were subjected to 20 min of ischaemia followed by 40 min of reperfusion. In knockout hearts, ATP and pH values were lower during ischaemia, and post-ischaemic contractility was less compared to wildtype hearts. After shorter ischaemia, the recovery of ATP and pH was greater in female knockout hearts compared to those in male knockout hearts. Recoveries of function, ATP and pH were less in female knockout hearts treated with L-NAME, and greater in male knockout hearts treated with the NO donor S-nitroso-N-acetylpenicillamine?9 Isolated perfused hearts from transgenic mice with no sarcomeric mitochondria1 creatine kinase have been studied with 31PNMR. Left ventricular performance was similar in transgenic and wildtype hearts, but the ratio of PCr/ATP was higher in the wild-type hearts. Furthermore, free [ATP] was higher and the free energy release from ATP was lower in the transgenic hearts.40The effects of over expression of P-adrenergic receptors on cardiac function during 20 min ischaemia and 40 min reperfusion has been investigated in Langendorff-perfused from male (MTG4) and female (FTG4) mice. Hearts were pre-treated with a non-specific N O synthase inhibitor (L-NAME) or a specific inducible N O synthase inhibitor (1400W), whereas controls received no pretreatment. During ischaemia, ATP and pH fell lower in MTG4 hearts, and there was a lower recovery of post-ischaemic contractile function, PCr and ATP, compared to FTG4 and wildtype hearts. In FTG4 hearts treated with L-NAME, ATP and pH fell as low during ischaemia as those in untreated MTG4 hearts and the recovery of post-ischaemic contractile function, PCr and ATP where comparable to that in untreated MTG4 hearts. In contrast, 1400W had no affect on FTG4 hearts, and L-NAME and 1400W had no affects on MTG4 hearts.41The effects of

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increasing sarcoplasmic reticulum Ca2+-ATPase activity by gene transfer has been studied in the rat heart following aortic banding. Following heart failure, rats were infected with an adenovirus (SRCATP) carrying the gene for sarcoplasmic reticulum Ca2+-ATPase, or a control virus @Gal-GFP)carrying the genes for P-galactosidase and a green fluorescent protein under the control of different promoters. Control rats were sham operated and either uninfected or infected with one of the two viruses. Four weeks after transfer the survival rate of rats with heart failure was 9 % in rats infected with PGal-GFP and was 63% in rats infected with SRCATP. 31PNMR analysis of perfused hearts showed that the ratio of PCr/ATP was lower in failing hearts infected with PGal-GFP compared to sham operated hearts infected with PGal-GFP. Infection with SRCATP in the failing heart improved the PCr/ATP ratio, but infection with SRCATP in the sham heart decreased the PCr/ATP ratio.42 The effects of myosin isoenzyme composition on the cyclical changes in myocardial ATP, PCr, Pi and pHi have been investigated in the perfused normal, hypothyroid and hyperthyroid rat heart using the pacing-gated 31PNMR method. Maximal decrease in ATP and PCr, and maximal increase in Pi at the peak of systole was observed in normal hearts. However, the magnitude of fluctuations was smaller in the hypothyroid hearts and larger in the hyperthyroid hearts. The cardiac myosin isoenzyme patterns were also different between groups.43The effects of inhibition of tumour necrosis factor-a (TNF-a) on post-ischaemic recovery of hypertrophied hearts have been investigated in neonatal rabbit hearts subjected to aortic banding. Myocardial TNF-a protein expression increased progressively with left ventricular hypertrophy, but serum TNF-a was only detected after the onset of heart failure. Before the onset of heart failure hypertrophied hearts were perfused in the Langendorff mode and subjected to 45 min ischaemia and 30 min of reperfusion. Post-ischaemic recovery was impaired in hypertrophied hearts, but treatment with anti-rabbit TNF-a antibody restored post-ischaemic function. Inhibition of TNF-a was also associated with faster post-ischaemic recovery of PCr, ATP and pH assessed by 31PNMR.44 The effects of chronic oestrogen status on the susceptibility of hearts to ischaemia-reperfusion injury have been investigated with 31PNMR. Hearts from rats that had been ovariectomised, not ovariectomised or ovariectomised and treated with S.C.oestrogen pellets were perfused isovolumetrically and subjected to 15 min of global ischaemia at 37"followed by 30 min of reperfusion. Preischaemic mechanical function was similar in all groups except for a small, significant decrease in (left ventricular developed pressure) LVDP in the oestrogen treated group. This group had improved recovery of LVDP following reperfusion compared to the untreated ovariectomised rats. However, 31PNMR did not detect any differences between groups before or after ischaemia and reperfu~ion.~~ The accumulation of 2-deoxy-D-glusoce-6-phosphate has been monitored in the perfused rat heart in an investigation of the effects of lactate on the translocation of GLUT1 and GLUT4? 13C NMR at 7 T has been used to appraise myocardial metabolism in vivo in Sprague-Dawley rats, fasted overnight, and infused with l3C-labe1ledglucose, 3-hydroxybutarate and acetate. At the end of


447

the perfusion, tissue extracts were prepared in order to confirm information observed in v i m Acetate and 3-hydroxybutyrate were readily taken up by the myocardium and supplied 53 & 9 and 42 6% of the acetyl CoA in the TCA cycle, respectively. Glucose was also readily taken up by the myocardium, but did not contribute to myocardial oxidative metabolism!’ In an investigation of the impact of ischaemic glycolysis on the integrity of the sarcolemmal membrane and myocardial viability, 31PNMR has been used to measure phosphorous metabolites and ‘H and 59C0NMR, in conjunction with cobolticyanide, have measured cellular volumes; glycolytic activity was assessed from lactate production. Isolated hearts were subjected to 30 rnin of ischaemia followed by 30 rnin of reperfusion. Increased lactate production resulted in delayed onset of ischaemic contracture, cytosolic acidification and cellular swelling during ischaemia. Furthermore, there was reduced diastolic tone, improved recovery of HEP and increased contractile force during reperfusion. Inhibition of ischaemic glycolysis by exposure to iodoacetate, and depletion of glycogen by exposure to 2-deoxyglucose, resulted in early onset of ischaemic contracture, elevated post-ischaemic diastolic pressure and reduced coronary flow rates.48 The effects of pretreatment with the combination of an angiotensin converting enzyme inhibitor (TEM), an angiotensin I1 type 1 receptor antagonist (CV), and/or a NO synthase inhibitor (L-NAME) on myocardial metabolism and contraction during ischaemia and reperfusion has been studied with 31PNMR in the Langendorff-perfused rabbit heart. Hearts were subjected to 20 rnin of normothermic global ischaemia before 30 min of reperfusion. During ischaemia the groups treated with TEM + CV or TEM + CV + L-NAME showed significant inhibition of the decrease in ATP and showed a significant inhibition of the increase in the left ventricular end-diastolic pressure (LVEDP) compared to the control group. Furthermore, the two test groups had significant improvement of the level of ATP and a significant decrease in the LVEDP compared to the control group during reperfusion. No differences were seen between the groups treated with TEM + CV or TEM + CV + L-NAME.49Ischaemiareperfusion injury has been investigated in male and female heart from 1295mice pre-treated with 3 mM Ca2+or isoproterenol L-NAME prior to 20 rnin of ischaemia and 40 min of reperfusion. Basal contractility increased equivalently in female and males hearts with isoproterenol or Ca2+treatment. A lower postischaemic contractile function, ATP and PCr was observed in male hearts, compared to female hearts, treated with Ca2+or isoproterenol. Endothelial NO synthase expression, and ischaemic N O production, was higher in female hearts and L-NAME increased injury in isoproterenol-treated female hearts.” The effects of L-NAME (a nitric oxide inhibitor) on the action of the cardioprotective agent, JTV-5 19, has been investigated in the Langendorff-perfused rabbit heart. JTV-519 was administered 40 min prior to 20 rnin of normothermic global ischaemia and 30 min of reperfusion. During ischaemia, the JTV-519 group showed significant inhibition of the decrease in ATP compared to the control and JTV-519 plus L-NAME groups. However, the JTV-519 and the JTV-519 plus L-NAME groups showed significant inhibition of increased LVEDP compared to controls. After reperfusion, the JTV-5 19 group had significant improve-

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ment of ATP levels.’’ The effects of increased workload, hypoxia or low-flow ischaemia on AMPactivated protein kinase (AMPK) and its affect on creatine kinase activity have been investigated in the isolated, and in situ, rat heart. Under normoxic conditions, where [AMP] and the velocity of AMPK increased, there was a positive relationship between the rates of creatine kinase and AMPK. Under conditions of hypoxia or low-flow ischaemia there was modest increase in AMPK activity and small decrease in creatine kinase velo~ity.’~ Magnetisation transfer 31PNMR has been used to discriminate between three different CK fluxes in the isolated beating heart. The analysis of four different protocols of magnetisation transfer and in uitro biochemical knowledge, e.g. oxygen consumption and size of the mitochondrial ATP and PCr compartments, allowed the discrimination of three different CK activities. These were for: 1) cytosolic CK functioning at equilibrium, 2) CK localized in the vicinity of ATPases, 3) mitochondrial CK displaced towards PCr ~ynthesis.’~ 4.3 Kidney. - The effects of RSR13, a synthetic allosteric modifier of haemoglobin oxygenation-binding affinity, on renal function in a model of chronic renal failure has used 31PNMR to assess ATP levels. Treatment with RSRl3 exacerbated the effects of renal failure causing a further increase in serum creatine and a 45% decrease in ATP levels. Treatment with furosemide prevented the effects of RSRl3 on this model of renal fai1u1-e.’~ 4.4 Liver. - The effects of ethanol on ATP content of perfused livers isolated from fed rats has been monitored with 31PNMR whilst O2consumption has been measured with Clark electrodes. The mitochondrial ATP synthesis in the whole organ was estimated by subtraction of the glycolytic ATP supply from the total ATP production. An increase in ATP production in the presence of ethanol was observed along with an enhanced net ATP consumption, which resulted in a decrease in the level of ATP. However, the mitochondrial respiration remained unchanged and the in situ mitochondrial ATP/O ratio increased from 0.33 0.035 to 0.42 & 0.015 in the presence of ethan01.’~The effects of 2,5-anhydromannitol on feeding and hepatic phosphorous metabolism have been investigated in rats fed on a high fat-low carbohydrate (HFLC) diet or a low fat-high carbohydrate (LFHC) diet. 31PNMR showed that 2,5-anhydro-mannit01(300 mg kg-’ i.p.) caused a decrease in liver ATP in LFHC fed rats, whereas this response was attenuated in HFLC fed rats. Furthermore, the administration of 2,5-anhydro-mannitol increased food intake in rats given the LFHC diet.56‘H NMR has been used to assess the triacylglycerol content of the liver in an investigation of the effects fructans on the metabolism of obese Zucker fa/fa rats. When fructans were added to the diet, rats had a lower energy intake, lower body weight and less triacylglycerol accumulation in the liver compared to control rats, or those supplemented with cellulose. The results were confirmed by ex uiuo biochemical and histochemical analy~is.’~ The role of F1,6P in the regulation of hepatic carbohydrate metabolism has been investigated with 13CNMR in ADM mice over expressing a double mutant bi-functional enzyme, 6-phosphofructo-2-


449

kinase/fructose- 1,6-biphosphatase. The rate of label incorporation into glycogen C1 was not different between the control and ADM mice. However, the rate of label incorporation into glycogen C6 was 5.6 0.5 pmol g-' h-' in the ADM group and 3.7 f 0.5 pmol g-' h-' in the control group. The rates of net glycogen synthesis, determined from the group of signals from glycogen C2, C3, C4 and C5 signals,were twofold higher in the ADM The effects of acute liver failure, induced by cc14, on hepatic TCA cycle and gluconeogenic fluxes have been investigated with 13CNMR following an infusion of [U-'3C]pr~pionate.Liver injury caused an increase in gluconeogenic flux relative to citrate synthase flux from 0.80 & 0.10 to 1.34 f 0.24, and from 1.36 f 0.16 to 1.80 0.22 in fed and fasted animals, respectively. However, there was also a 47% reduction in citrate synthase flux. Recycling of PEP via pyruvate and oxaloacetate was not significantly altered by CCl, injury, but there was a 26% decrease in hepatic glucose output? The quantification of glycogen signals during synthesis has been investigated with 13CNMR in the perfused liver. Livers were perfused with [1-'3C]glucose and this was chased by perfusion with [12C]glucose.Signals were monitored in the liver and perfusate was collected to account for any glucose released or metabolised. A decrease in the glycogen signal could not be accounted for by the metabolism of glycogen and was probably caused by changes in line shape that occurred during continued synthesis.6027AlNMR has been used to detect aluminium(II1)chelates of triazacyclononane polycarboxylate ligands in the liver of rats following an infusion. Samples of blood and urine were also examined which indicated that the chelates remained stable under physiological conditions.61A triple-tuned, surgically implanted NMR coil has been used to monitor 31Pand 'H-decoupled 13Csignals from the rat liver in vivo. Following the administration of [2-13C]glycine peaks for glycine, glutathione and serine were observed. Hepatic HEP and pH were also monitored.62 The effects of mitochondria1 and cytosolic creatine kinase on free ADP levels in the livers of transgenic mice have been investigated using 31PNMR and saturation transfer. The velocity of CK was related to the total enzyme activity of either enzyme expressed, and was found to increase with increasing concentrations of creatine. The fluxes for both enzymes in both directions were equal. Hepatic free ADP concentrations, calculated by assuming equilibration of the CK reaction in vivo, decreased from 84 & 9 to 38 4 nmol (g wet weight)-'.63 The effects of magnetisation transfer on the lactate signal in the perfused liver have been investigated. The size of the motionally restricted lactate pool, determined using a two-pool model fit, was 1% in the perfused liver and about 1.8-2.5% in ischaemic liver.64 4.5 Muscle. - The kinetics of phosphocreatine at the onset of muscle contraction has been investigated with 31PNMR in the MM creatine kinase knockout mouse. Depletion of PCr after 2 s of stimulation at 5 Hz was 2.2 f 0.6% of initial PCr in knockout mice compared to 9.7 f 1.6% in wildtype mice. Initial PCr/ATP and pHi were not significantly different between groups, and there was no detectable change in pHi or ATP. The initial difference in ATP depletion was

450


maintained for the first minute of stimulation at 5 Hz, though there was no difference in the quasi-steady-state PCr level after 80 s of ~timulation.6~ The role of muscle metabolites in muscle fatigue has been measured in an in uiuo rat muscle model. Two electrical stimulation protocols were used, high intensity stimulation followed by medium intensity stimulation (high group) and low intensity stimulation followed by medium intensity stimulation (low group). Metabolic fatigue was based on [H2P04-] measured by 31PNMR and excitation-contraction coupling (ECC) fatigue was measured as the fatigue in excess of metabolic fatigue, and as the relative decline of force at low compared to high stimulation frequencies. During the initial stimulation period, the high group had greater metabolic fatigue and greater ECC fatigue. During the second stimulation period and recovery, the high group had no difference in metabolic fatigue and greater ECC fatigue.66The effects of salinity on the pseudo-first order unidirectional rate constants and flux rates for arginine kinase in the forward and reverse directions have been measured in the levator muscle of the blue crab (Callinectes sapidus) using 31PNMR saturation transfer. Animals were acclimated for seven days to a salinity of 5, 17 or 35%, which corresponds to a haemolymph osmolarity of 640, 720 and 960 mosmol- ', respectively. During kinetic measurements, isolated dark levator muscles were superfused with saline corresponding to the acclimated osmolarity and also with saline that was hyperosmotic or hypoosmotic compared to the acclimated conditions. There were no differences in the rate constants or flux rates of arginine kinase under any acclimated conditions and a 1.7-fold range of variation under hypo- or hyperosmotic conditions. However, this variation was reduced even further when changes in cell volume were taken into account. Arginine kinase flux was reduced by hyperosmotic treatments and enhanced by hypoosmotic t r e a t r n e n t ~The .~~ diffusion coefficients for the 31Presonance of phosphoarginine (PA) and the 'H resonances of betaine, arginine plus phosphoarginine, and CH2plus CH groups have been measured in giant muscle fibres of the spiny lobster (Panulirus argus) radially and axially to the fibre direction. Axial diffusion coefficients were always higher than radial diffusion coefficients, and the latter decreased over time in a manner that was consistent with previous results from fish and mammalian tissue.68 The mechanism of the therapeutic action of rosiglitazone has been investigated in the Zucker rat under insulin-stimulated glucose disposal. Zucker fa/fa rats treated with rosiglitazone (FRSG) were compared to untreated obese litter mates (FC) or untreated lean litter mates (LC). Rates of glycolysis and glycogen synthesis in skeletal muscle were assessed after treatment by monitoring [1,613C]glucoselabel incorporation into [l-13C]glycogen, [3-'3C]lactate and [3-"C] alanine during a euglycaemic hyperinsulinaemic clamp. Treatment with rosiglitazone caused increased insulin sensitivity; whole body glucose disposal rates were: 24.4 f 1.9,17.6 & 1.4 and 33.2 & 2.0 mg kg-' min-' in FRSG, FC and LC, respectively. Treatment with rosiglitazone also caused normalisation of glycolytic flux to 52.9 & 9.1 nmol g-' min-' compared to 56.2 f 16.6 and 18.8 i8.6 nmol g-' min-' in LC and FC, respectively. Furthermore, glycogen synthesis flux was 56.3 & 11.5 nmol g-' min-' in FRSG compared to 75.2 f 15.3 and 16.6


45 1

+

12.8 nmol gg' min-' in LC and FC, respectively. 31PNMR revealed that during insulin clamp in FRSG and LC groups glucose-6-phosphte (G6P) increased by 13.0 k 11.1% and 16.9 f 5.8% of baseline values, respectively, whereas G6P declined by 23.3 f 13.4% in the FC g r o ~ p . 6 ~

4.6 Tumour. - The effects of hyperglycaemia on oxygenation, radiosensitivity and bioenergetics status has been investigated in subcutaneous RIF- 1 tumours. A 0.3 M solution of glucose was administered to maintain blood glucose levels at 17 f 1 mM and 31PNMR was used to record spectra during the infusion. There were no significant changes observed in the 31PNMR spectra and there was no effect on tumour radio~ensitivity.~' In an investigation of the synergistic effects of a combination of cPrGHC1 and Epirubicin on a human breast cancer cell line (MDA-MB-231), 31PNMR has been used to measure a drug-induced acidification of PHi in the tumour grown in nude mice.71 4.7 Whole Organisms. - The effects of exposure to pentachlorophenol in veliger larvae of the red abalone (Haliotis rufescens) has been assessed with 31P NMR. Changes phosphoarginine, nucleotide triphosphates (NTP), sugar phosphates, Pi and pHi were monitor during 1 hour exposure to clean seawater followed by 2 hours exposure to 1.2 mg dm-3 PCP and during 2 hours of recovery in clean seawater. During PCP-exposure, Pi rose to 302 f 63%, PHi declined from 7.12 0.03 to 7.04 f 0.04, and PA and NTP declined to 38 f 10% and 61 16% of control values, respectively. On re-exposure to clean seawater, pHi recovered to 7.08 0.11, whilst Pi, P A and NTP recovered to 196 - 53%, 89 & 12% and 89 13%, re~pectively.~~ Phosphorous metabolite concentrations and the forward rate constant for arginine kinase have been measured in red abalone during an eight-hour exposure to 66 pg dmg3copper in seawater. A decrease in PA, with a corresponding increase in Pi was observed while ATP remained constant. Furthermore, there was a significant elevation in the forward rate constant for arginine kinase compared to controls. In the same study, the changes in cellular metabolites of medeka embryos (Orysias Zatipes) during development from fertilization through to hatching have been recorded. In medeka, a decrease of phosphomonoesters (PME) concurrent with an increase in PA and ATP was observed as the eggs approached hatching.73The uptake and metabolism of trifluoromethylaniline in the earthworm (Eisenia veneta) has been followed using I9FNMR.74Metabolism of trifluoromethylaniline was observed when worms were exposed to filter papers containing 10 pg cmg2,and to a lesser extent at 1 pg cmg2.

+

5

Clinical Studies

The use of 'H NMR in the characterisation of breast lesions has been reviewed.75

5.1 Brain. - The uses of 'H NMR in the study of dementia and Parkinson's disease:6 in clinical investigations of the brain77and in the study of hepatic

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encephalopathy have been reviewed.78 Quantitative multi-slice 'H NMR spectroscopic imaging (TE = 280 ms) has been used to determine the regional metabolite levels in the adult posterior fossa. Concentrations were determined by the phantom replacement method. The concentrations (mM) of Cho, tCr and NAA, respectively, were found to be: 2.3 +_ 0.4,8.8 k 1.7 and 7.6 f 1.0 in the cerebellar vermis, 2.2 f 0.6,8.9 f 2.1 and 7.5 k 0.8 in the cerebellar hemisphere, 2.2 k 0.5,4.3 & 1.1 and 8.3 f 0.9 in the pons, 1.8f0.5,7.8*2.0and8.0+ l.lintheinsularcortex,l.3f0.3,5.7+ 1.1,7.2+ 0.9 in the parieto-occipital grey matter, and 14 f 0.3, 5.3 f 1.3 and 7.5 k 0.8 in the occipital white matter.79 The levels of metabolites in the human brain, expressed as their ratio to tCr, have been measured with 'H NMR. The distribution of metabolite ratios was analysed by principle component analysis to determine possible metabolic links between different regions of the brain.80 Localised 'H NMR at 4 T has been used to measure the level of BOLD effects on the water signal and the methyl resonances of tCr and NAA in the visual cortex. Changes in peak height and spectral linewidth were observed in the water peak as well as in the peaks of NAA and tCr, which are mostly found in the intracellular space.*' 'H NMR has been used to record spectra from the grey matter of the posterior singulate gyrus and the precuneus, and from the parieto-occipital white matter in nine Alzheimer's disease patients and twelve controls subjects. In patients, NAA/tCr ratios were reduced in the grey and white matter whereas (glutamate - glutamine)/tCr (Glx/tCr) was reduced in the grey mater only; there was a correlation between Glx/tCr and NAA/tCr in the grey matter.82'H NMR has been used to assess mI/tCr and lactate/tCr in the first week of term infants with neonatal encephalopathy. Infants were assessed by MRI for anomalies in the basal ganglia, and by Griffith's developmental scale to determine outcome at one year. The ratios of mI/tCr and lactate/tCr were higher in infants with abnormal MRI and outcome. Furthermore, mI/tCr and lactate/tCr were correlated and both were correlated to Griffith's development ~ c a l e s . ~ ~ Ttemporal he evolution and spatial distribution of macromolecules and small molecules has been investigated in stroke patients. Single-voxel spectra were obtained with or without inversion recovery suppression of small molecules; subtraction of the suppressed from the unsuppressed spectra resulted in spectra without overlap of macromolecules. Signals from macromolecules were elevated in lesions relative to contralateral brain and tended to increase in the subacute period, even as lactate declined. Regions of increased lactate, increased macromolecules and decreased NAA were closely correlated in 2D spectroscopic images.84'H NMR has been used to measure changes in Cho peak intensity in the corpus colosum and adjacent periventricular white matter of multiple sclerosis (MS) patients before and after plaque appearance. The Cho peak is known to increase in acute MS plaques, though any pre-lesion changes had not been previously investigated. MRI was used to determine the presence of lesions or normal appearing white matter (NAWM). It was found that there was a significantly higher baseline Cho/tCr ratio in NAWM voxels that displayed MRI visible lesions 6 months later compared to NAWM that remained unchanged. There was, also, a similar

+


453

pre-lesional elevated Cho/tCr 12 months before plaque appearance. Furthermore, voxels that contained a lesion at baseline that increase in lesion volume at 6 months also showed a significantly higher ratio of Cho/tCr compared to lesions that did not change.85Changes in NAA levels in the brains of patients with Alzheimer's disease and motor neurone disease have been detected by 'H NMR. A significant reduction of the NAA/tCr ratio was observed in the motor cortex of patients with motor neurone disease and a significant reduction of NAA/tCr was observed in medial temporal lobe of patients with Alzheimer's Metabolite changes in the brains of patients with dementia with Lewy bodies have been investigated with 'H NMR. In comparison to age matched healthy controls, patients had significantly lower NAA/tCr, Glx/tCr and Cho/tCr in the white matter. There were, however, no differences observed in the grey matter of patients compared to The use of 'H NMR in the detection of upper motor neurone loss or dysfunction in amyotrophic lateral sclerosis (ALS) has been compared to the use of transcranial magnetic stimulation (TMS). Abnormal NAA/Cho ratios were detected in 53% of ALS patients and TMS detected abnormalities in 65% of patients. 'H NMR detected more patients with upper motor neurone involvement in the suspected El Escorial subgroup, where TMS detected more patients with upper motor neurone involvement in the possible, probable and definite El Escorial subgroups.88I n vivo 'H NMR has been carried out on 11 patients with neuroepithelial tumours prior to surgery and the collection of biopsy samples. Analysis of biopsy samples using MAS NMR revealed that glycerophosphocholine, phosphocholine and choline contributed to the Cho peak observed by 'H NMR in vivo. Metabolite ratios from in vivo spectra correlated with ratios in ex vivo The relaxation times of metabolites in gliomas and brain have been measured and used to correct absolute concentration values obtained using 'H NMR. In low-grade glioma, the T2 of NAA was shorter, and the T2 of water was longer, when compared to values obtained from normal subjects. In high-grade glioma, the T2 of NAA and tCr were shorter, and the T2 of water was longer compared to those in normal subjects. In gliomas, [NAA] and [tcr] were decreased, and [Cho] was increased, compared to normal subjects. High-grade gliomas had significantly lower tCr and Cho than low-grade gliomas and low [tcr] was the most reliable indicator of malignancy?' The effects of 1-month creatine supplementation on the 'H NMR-visible metabolites of the brain have been investigated in 15 patients with sporadic amyotrophic lateral sclerosis and normal volunteers. Before supplementation, patients had a lower NAA/tCr ratio than the control group. Following supplementation the control group showed a decline in the NAA/tCr, NAA/Cho, Glx/tCr and Glx/Cho ratios. However, in patients the NAA/tCr and NAA/Cho ratios remained unchanged whilst the Glx/tCr and Glx/Cho ratios decreased following s~pplementation.~' In an investigation of the effects of recombinant insulin-like growth factor-I in multiple sclerosis 'H NMR spectroscopic imaging has been used to determine regional metabolite ratios.92Kinetic investigations of 15 patients with classic phenylketonuria have been performed using stimulated echo 'H NMR before and after oral phenylalanine loading (100 mg kg-' body

454


weight). The brain phenylalanine concentrations of patients were found to be quite different despite similar blood phenylalanine levels. Individual variations of the apparent transport Michaelis constant covered a wide range from 0.1 to 1.03 mM and there was a variation between 2.61 and 14.0 in the ratio of the maximal transport velocity over the intracerebral consumption rate. These parameters, and the brain preload phenylalanine levels correlated significantly with the degree of cerebral white matter abnormalities in MRI s ~ a n s . 9 ~ The 1~~ influence of pyridoxal 5' -phosphate on the effects of vigabratin have been measured with 'H NMR in healthy subjects. Pyridoxal 5'-phosphate alone did not influence the ratio of GABA/tCr in the occipital lobes, whereas vigabratin alone increased the ratio of GABA/tCr in both hemispheres. However, the combination of pyridoxal5'-phosphate and 1000-2000 mg day-' vigabratin did not cause an increase in the ratio of GABA/tCr, and the combination of pyridoxal 5'-phosphate and 3000-4000 mg day-' vigabratin had different effects depending on the sequence of the drugs and dose of ~igabratin.9~ The effects of lorazepam on 'H NMR spectra of the brain have been investigated in 10 healthy volunteers. Spectra were recorded from the left dorsolateral prefrontal cortex prior to and one hour after administration of 2 mg of lorazepam. No effects on the levels of metabolites or their ratios were observed?6 The effects of the administration of citicholine on the phospholipids of the brain have been investigated with 31PNMR. Subjects, with a mean age of 70.3 f 5.7 years, were given 500 mg citicholine orally each day for 6 weeks followed by either a further 6 weeks of citicholine or a placebo. Treatment with citicholine for 6 weeks was associated with a 7.3% increase of phosphodiesters from baseline levels. There were no significant effects observed in subjects who had a further 6 weeks of citicholine treatment?' Serial metabolic changes in frontal lobes of patients with deep intracerebral haemorrhage have been performed with multi-voxel 'H NMR. The ratio of NAA/tCr in the white matter of the primary motor and premotor areas were measured bilaterally within 48 hours, at 2 weeks and at 1 month after onset. In the primary motor area on the affected side, where the haematoma did not extend, the NAA/tCr ratio decreased sequentially. At 48 h and at 2 weeks after onset, a negative correlation was found between NAA/tCr and haematoma volume, but there was no correlation 1 month later. At 2 weeks, NAA/tCr correlated with motor impairment, and there was a significant correlation with clinical outcome as early as 2 week after onset.98The effects of severe carotid stenosis and carotid endarterectomy on brain metabolites have been assessed using quantitative 'H NMR measurements of metabolite levels; single proton emission computed tomography was used to measure blood flow. There was a significant correlation between the degree of internal carotid artery stenosis and [NAA], and a significant correlation between cerebral blood flow and [NAA] in the basal ganglia.99The metabolism of [1-13C]acetate in the human brain has been followed with I3C NMR. There was rapid incorporation of 13C-labelinto cerebral bicarbonate, and C5 of glutamate and glutamine. The rate of acetate oxidation, calculated from steady state enrichment of bicarbonate, was 0.13 0.03 mmol g-' min-' in fasted subjects. Furthermore, when the effects of val-


455

proate on acetate metabolism were observed in a single subject the accumulation of 13C-labelin C5 of glutamine was increased and the production of labelled bicarbonate was decreased.'@' A comparison has been made between 13CNMR measurements of the rates of glutamine synthesis and TCA cycle obtained from experiments using oral or intravenous administration of [l-13C]glucose.101 In an investigation of the use of DL sodium P-hydroxybutarate (PHB)in two 6-monthold infants with persistent hyperinsulinaemic hypoglycaemia, 'H NMR has been used to detect PHB in the brain.lo2 The effects of post-traumatic stress disorder on brain metabolites in the hippocampus have been studied with 'H NMR. Compared to age matched control subjects, there was no change in hippocampal volume measured by 'H NMR, but there was a 23% bilateral reduction of NAA in the hippocampus, and a 26% reduction of tCr in the right hippocampus of post-traumatic stress subjects.'03 Fluctuation in y-aminobutyric acid (GABA) levels in the occipital cortex across the menstrual cycle in 14 healthy women and 9 women diagnosed as having premenstrual dysphoric disorder (PMDD) have been measured using 'H NMR. Levels of gonadal hormones and neurosteroids were determined from blood samples on each scan day. Cortical GABA levels declined across the menstrual cycle in healthy women, whereas women with PMDD had an increase in cortical GABA levels from the follicular phase to the mid and late luteal phases. Significant between group differences in the relationship between hormones and GABA were observed for oestrodiol, progesterone and allopregnanolone.'@' The metabolites of the medial prefrontal and perietal lobes have been measured with 'H NMR in adults with Asperger syndrome and control subjects. Frontal metabolite concentrations were correlated with scores on the Yale-Brown Obsessive Compulsive Scale and the Autism Diagnostic Interview. Increased pre-frontal [NAA] was significantly correlated with obsessional behaviour, and increased prefrontal [Cho] were significantly correlated with social function. There were no differences observed in perietal lobe metabolite concent r a t i o n ~An . ~ investigation ~~ into possible links between mood and choline levels in the bilateral frontal and occipito-perietal white matter has been carried out with 'H NMR in subjects with no neurological or psychiatric disorders; subjects were graded according to the Positive and Negative Affect Scale. In the left frontal lobe, choline was found to be inversely correlated with Positive Affect.lo6 The concentrations of NAA, tCr, Cho and mI have been measured in the anterior superior cerebellar vermis and frontal lobe white matter in 31 alcoholics and 12 normal subjects. Subjects were examined within 3 to 5 days of their last drink and then after 3 weeks and 3 months using an online repositioning technique to localize the measurements to the same region. At 3 to 5 days of abstinence, frontal white matter metabolite concentrations were not significantly different compared to controls, but brain tissue in the area of interest was reduced. However, cerebellar [NAA], [Cho] and cerebellar volume was decreased, though [tcr] and [mI] were not significantly different. Upon re-examination, [NAA] was reduced in patients who relapsed before the three-week examination. After three months of abstinence, cerebellar [NAA], and brain and cerebellar volumes increased, whereas cerebellar [Cho], [tCr] and [mI] and

456


brain tissue in the area of interest were not significantly different. Cerebellar [NAA] increased with abstinence, but reduced [Gho] persisted beyond three months.lo7The effects of methamphetamine use on 'H NMR-detected metabolites in the basal ganglia has been investigated in 13 abstinent methamphetamine users compared to 11 healthy control subjects. The methamphetamine users showed a significant reduction in the ratio of tCr/Cho in the bilateral basal ganglia compared to controls. The reduction in the ratio of tCr/Cho was significantly correlated with the duration of methamphetamine use and with the severity of residual psychiatric symptoms. However, the ratio of NAA/Cho in the bilateral basal ganglia did not significantly differ between the two groups.'O* 5.2 Heart. - The relationship between cardiac energetic and myocardial hypertrophy has been investigated in patients with Friedreich's ataxia compared to healthy controls. A significant reduction of PCr/ATP, and a significant increase in Pi/PCr was observed in spectra from the hearts of patients compared to controls. There was a significant correlation between septa1 wall thickness and PCr/ATP or Pi/PCr. Furthermore, patients with left ventricular hypertrophy showed a significant decrease in PCr/ATP and a significant increase in Pi/PCr. However, in those patients without left ventricular hypertrophy the changes in metabolite ratios were smaller and the change in PCr/ATP was insignificant. The ratio of PME/PCr remained unchanged in all patients.'"

Joints. - The Na content of the human wrist has been measured in six healthy volunteers with no known history of arthritis or pain. The Na content was 115 to 150 mM in non-cartilaginous regions and from 20 to 210 mM in cartilaginous regions."'

5.3

Liver. - Hepatic ATP levels have been measured in a study of ATP reserves and replenishment in obese compared to non-obese subjects. After obtaining baseline measurements, 0.5 cm3kg-' of a 50% fructose solution was injected to deplete hepatic ATP levels and spectra were then recorded every 5 min. The baseline ATP content was found to be inversely related to the BMI of the subjects, and fructose caused ATP depletion in all subjects. However, there was no correlation between post-fructose ATP, or the extent of recovery, with BMI."' Serial changes in liver metabolism in patients treated for obstructive jaundice has been investigated with image guided 31PNMR. Conventional liver function test were also performed. Compared with controls, liver spectra from jaundice patients contained an excess of PME metabolites. Biliary decompression was achieved in all cases and plasma biochemistry improved consequently; this was accompanied by an increase in the ratio of ATP/Pi measured in the liver. Furthermore, a decrease in the PME peak of liver spectra was observed after treatment, which was probably due to a reduction in bile content as a strong PME peak was observed in bile samples examined by 31PNMR in uitro.'12 5.4

5.5

Muscle. - A review of the use of NMR in the study of cellular mechanisms


457

of insulin resistance in human has been produced.'13The pathogenesis of skeletal muscle insulin resistance in type-2 diabetes has, also, been reviewed.' l4 The 'H NMR chemical shift of water and imidazoles protons relative to cholines (-N+CH3)and creatines (-CH3)have been used to estimate temperature and pH in muscles of five normal volunteers. The temperatures in gastrocnemius and soleus mhscles under ambient temperature of 21-25 "C were 33.6 f 0.4 and 35.3 k 0.4 "C, respectively. The pH of these muscles was found to be 6.97 k 0.01 and 6.96 f 0.02, respectively. When the surface temperature was 40 or 10 "C the temperature of the gastrocnemius muscle was 35.8 f 0.4 and 26.2 k 1.2 "C, respectively, whilst the pH was 6.95 f 0.01 and 7.01 f 0.01, respectively. However, the pH and temperature of soleus muscle was unaffected by these external temperature^."^ 'H NMR has measured the intramyocellular lipid (IMCL) content in the vastus lateralis muscle of physically fit, non-diabetic men after 12 and 84 hours of fasting. The ratios of IMCL/water and IMCL/tCr increased from 0.00623 f 0.00065 to 0.0142 f 0.0015 and 6.82 f 0.87 to 14.96 f 1.73. Analysis of blood samples revealed that FFA, serum triglyceride and 3-hydroxybutarate concentrations increased, whereas plasma glucose and serum insulin levels decreased during fasting.' l6 A two-pedal ergometer, equipped with position and force transducers, has been designed and tested using a series of steady-state exercises of increasing intensity. The results showed that mechanical power of subjects ranged from 1.5 0.2 W to 11.0 f 1.6 W. Oxygen consumption increased from 0.28 f 0.04 dm3min-' at rest to 0.48 k 0.1 dm3min-' at the highest load whilst decreases were observed in the PCr/(PCr k Pi) ratio from 0.94 k 0.01 at rest to 0.73 f 0.04 at the highest 10ad.l'~The effects of muscle activation on fatigue and metabolism have been studied in the human medial gastrocnemius muscle. It was found that stimulation at 100 Hz and 80 Hz produced similar rates and amounts of fatigue. Furthermore, 31PNMR spectra collected during 2 ischaemic, 8-train stimulations at 80 or 100 Hz showed similar changes in [Pi], [PCr], pH and the turnover of ATP."* The relationships between in vivo 31PNMR measurements of skeletal muscle oxidative function and in vitro measurements of mitochondrial function have been studied in 27 women with varying levels of fitness. Following 90 s of isometric plantar flexion exercises, the PCr recovery time constant, the ADP recovery time constant, the rate of change of PCr during the first 14 s of recovery and the apparent maximal rate of ATP synthesis were measured with 31PNMR. These in vivo markers of mitochondrial function correlated moderately with the percentage of type IIa oxidative fibres and citrate synthase activity, but correlated weakly with cytochrome c oxidase activity."' The metabolic cost of isometric force generation and maintenance has been investigated in the human gastrocnemius muscle. Muscles were stimulated at 200r 80 Hz for 300, 600, 1200 or 1800 ms under ischaemic conditions. The cost of force generation was determined from the ATP turnover during the shorter pulse trains that did not attain a steady force level. Estimates of the cost of force maintenance at each frequency were determined by subtracting the ATP turnover during the shorter duration trains from that in the longer trains. The force generation phase of contraction was more metabolically costly than the force maintenance phase during 20 and 80 Hz stimulation. Further-

458


more, the mean rate of ATP hydrolysis appeared to decline as contraction duration increased.12' 31PNMR has been used to investigate changes in PCr in the tibialis anterior muscle during sustained isometric exercise carried out at 30% of maximal voluntary contraction until fatigue. Three phases of PCr hydrolysis were measured from regression lines fitted to the slopes of the PCr data. An initial high rate of PCr hydrolysis was observed during which there was a transient increase in pH. A moderate rate of PCr hydrolysis in the second phase was accompanied by a splitting of the Pi peak such that two pH values could be assigned to the muscle. Finally, a slow rate of hydrolysis was observed in the third phase and the Pi peak remained split until the end of the exercise period. The mean rates of PCr hydrolysis were -0.44 0.19% s-', -0.07 & 0.04% s-' and -0.29 f 0.10% s-' for the fast, moderate and slow phases, respective1y.l2l The effects on post-exercise muscle glycogen replenishment of a supplement containing carbohydrate plus protein (CHO-Pro) have been compared with replenishment from supplements containing equal carbohydrate (LCHO) or the equal calorific content in of the CHO-Pro supplement in carbohydrate form (HCHO).Natural abundance 13CNMR was used to measure glycogen content in the vastus lateralis in seven age and weight matched subjects. Before exercise the glycogen content was: 141.8 k 15.4 mmol dm-3, 150.8 & 9.5 mmol dm-3 and 148 f 9.8 mmol dmP3for the CHO-Pro, HCHO and LCHO groups, respectively. Immediately following exercise the glycogen contents were: 40.9 f 5.9 mmol dmP3,41.9 f 5.7 mmol dmp3,and 40.7 k 5.0 mmol dmP3for the CHO-Pro, HCHO and LCHO groups, respectively. After 240 min of recovery, the muscle glycogen content was significantly greater at 88.8 & 4.4 mmol dmp3 for the CHO-Pro group, compared to 75.5 h 2.8 mmol dmP3and 70.0 f 4.0 mmol dm-3 for the HCHO and LCHO groups, respectively.'22 In an investigation of the effects of three months treatment with rosiglitazone on whole body insulin sensitivity and peripheral adipocyte insulin sensitivity, 1H NMR was used to assess the effects on liver and muscle triglyceride content. Rosiglitazone treatment resulted in a 68% and a 20% improvement in insulin stimulated glucose metabolism during high dose and low dose insulin clamps, respectively. These changes were associated with a 39% increase in extramyocellular lipid (EMCL) content and a 52% increase in the sensitivity of peripheral adipocytes to the inhibitory effects of insulin on l i p ~ l y s i s .The ' ~ ~ effects of short term plasma amino acid elevation on whole-body glucose disposal and cellular insulin action in skeletal muscle has been investiga.ted in 7 healthy men during 5.5 h euglycaemic, hyperinsulinaemic, fasting glucagon, growth hormone and somatostatin clamp in the presence of low or increased plasma amino acids. Glucose turnover was measured with D-[6,6-2H2]gllucose and i.m. glycogen and G6P concentrations were determined by 13C and 31PNMR, respectively. A 2.1-fold increase in plasma amino acids reduced whole-body glucose disposal by 25% and reduced glycogen synthesis in the muscle by 64%; this was accompanied by a reduction in G6P starting at 130 minutes.124An investigation has been carried out to determine if IMCL and EMCL contents are increased early in the development of juvenile obesity and to explore the relationship of IMCL and EMCL levels to insulin resistance. Eight non-obese and 14 obese adolescents


459

were investigated with 'H NMR to determine IMCL and EMCL content of the soleus muscle, with a 2 h euglycaemic hyperinsulinaemic clamp to determine insulin sensitivity, with dual energy x ray absorptiometry to measure total body fat, and with MRI to determine the distribution of abdominal fat. The IMCL and EMCL content of the soleus muscle were greater in obese subjects and a strong inverse correlation was observed between IMCL and insulin sensitivity even when allowing for percentage total body fat and abdominal S.C.fat mass. In obese adolescence, higher IMCL and EMCL stores accompanied increases in total body fat and central a d i ~ 0 s i t y .The l ~ ~ effects of insulin treatment on the intracellular lipid content of the liver and muscle of diabetic patients have been studied with 'H NMR. At the start of the study, non-diabetic subjects had a two-fold higher insulin sensitivity and lower IMCL and intrahepatic cellular lipid content compared to diabetic subjects. In diabetic subjects, the IMCL lipid content correlated negatively with insulin sensitivity. After 67 h of insulin infusion, with near normal blood glucose, in diabetic patients IMCL content of the tibialis anterior muscle correlated positively with insulin sensitivity, whereas fasting glucose production decreased by The relationship between IMCL content, lipid oxidation and insulin action has been investigated in fourteen, young non-obese women with body fat ~ 3 0 %or >30% and fourteen, young non-obese men with body fat ~ 2 5 %or >25% with no family history of diabetes. Subjects with higher body fat had normal insulin-stimulated glucose disposal, IMCL content in the soleus and tibialis anterior muscles, and plasma FFA levels. This was associated with increased lipid oxidation, resting energy expenditure, resting oxygen consumption and plasma leptin levels in the post-absorptive condit i ~ n .'H ' ~NMR ~ has been used to measure the IMCL content of the soleus and tibialis anterior muscles in women with previous gestational diabetes (pGDM), compared to women with normal glucose tolerance (NGT), at 4-6 months after delivery. Body fat mass was assessed from bioimpedance analysis; insulin sensitivity and glucose effectiveness were measured using a glucose tolerance test. In women with pGDM, there was a 45% increased body fat mass, 35% reduced insulin sensitivity and glucose tolerance, and 40% and 55% higher IMCL in the soleus and tibialis anterior muscles, respectively. The IMCL of the tibialis anterior correlated with insulin sensitivity and glucose tolerance index independent of the body mass index. Insulin-resistant pGDM subjects had higher IMCL than NGT and insulin-sensitive P G D M . ' ~The ~ IMCL content and whole body lipid oxidation has been investigated in HIV patients with lipodystrophy syndrome of peripheral fat wasting and central adiposity induced by antiretroviral therapy. Patients had impaired insulin sensitivity, increased plasma triglyceride and cholesterol concentrations compared to controls. Furthermore, 'H NMR demonstrated that muscular triglyceride content was higher in the tibialis anterior and soleus muscles of patients, even in patients where the basal metabolic rate was comparable to that in control subjects, post absorptive lipid oxidation was significantly impaired.'29


460

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32(12), 2797-2802. 85. Tartaglia, M.C.; Narayanan, S.; De Stefano, N.; Arnaoutelis, R.; Antel, S.B.; Fran-

86. 87. 88.

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10.3.

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13 Nuclear Magnetic Resonance Imaging BY TOKUKO WATANABE

1

Introduction

This review is a continuation of the last seven years’ and covers the progress in the field of NMR imaging (NMRI), NMR microimaging, NMR microscopy or MR (micro) tomography as well as MRI (Magnetic Resonance Imaging). Papers mainly recorded in CA Selects on Proton Magnetic Resonance and on Carbon & Heteroatom NMR over a period from June 2002 to May 2003 are collected. The topics were limited to mainly basic, technical and applicable aspects in the non-clinical research field, such as physicochemical, biological, botanical, physiological, pharmaceutical, geological, environmental, food science and technology, and various industrial applications. Recently, the term ‘MRI’ instead of the term ‘NMR imaging’ has become much more popular, even in the nonclinical field of research. In this review, the author has quoted the term used by the original author(s). Review articles are separately included as reviews in the first section of each topic. A lot of papers on clinically oriented MRI using a whole body MRI machine have been published in the period, but almost all such papers have not been picked up because of their very professionally-orientated topics in clinical or medical interest. In some cases the technical development for imaging methods or pulse sequences proceed in the clinical MRI machine rather than in the high-resolution NMR machine or MRI machine for animal use because of the very severe, specific demands for using with patients. Such technical developments have been exploited in the high-resolution NMR machine. Journals, that concentrate on the development and applications within the topics, include Journal of Magnetic Resonance, Magnetic Resonance of Medicine, Journal of Magnetic Resonance Imaging, Magnetic Resonance Imaging, N M R in Biomedicine, Magnetic Resonance in Chemistry, Journal of Computer Assisted of Tomography, as well as more clinical publications such as 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 appearance in the Review of Scientific Instruments. Concepts in Magnetic Resonance which present fundamental aspects of the technique and Magnetic Resonance Quarterly which contains review articles of various topics of the field are recommended from the Nuclear Magnetic Resonance, Volume 33 0 The Royal Society of Chemistry, 2004

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educational view point. The abstract from the Annual Meeting of International Society for Magnetic Resonance in Medicine (ISMlRM) is strongly recommended as a means of deriving concise, up to date information on developments within the subjects.

2

Overview and Physicochemical Applications

In chemistry, or materials science, NMR is a standard method for structural analysis as a spectroscopic method. NMR also has successes as NMR imaging which can be exploited to visualize structures in hetlerogeneous objects comprising product and process control in particular in soft materials like gels, elastomers, polymer melts and food, which cannot be resolved by many other methods. In this period, the pulse sequences for recording signals from such samples containing nuclei with very short spin relaxation times has been developed from previous years. NMR imaging is beirtg used more and more to characterize the heterogeneity and the distribution of relevant properties in materials like gels, polymers, elastomers, and catalysts, as reviewed in sections 7 and 8. As an inevitable consequence, in situ reaction processes are demanded targets for visualization. Such applications have appeared in this term and reviewed as a new trend in section 2. The use of NMR imaging or MRI in dynamic phenomena, such as diffusion, flow velocity, and mass transportation, is conspicuous, especially in the industrial applications, which is reviewed in sections 6 and 8. It is expected in future that various applications in dynamics will be done in various fields by employing fast imaging techniques. Also, spatially resolved NMR can be used for the studies of particle dynamics in granular media and parameters of mass transport, a topic of interest in chemical engineering. Research into the structures induced by flow in various colloidal or suspension systems has started to focus in the past few years on large-scale structures and inhomogeneous flow phenomena. Gas-phase NMR has also great potential as a probe for a variety of interesting physical, biomedical, and industrial problems that are not amenable to study by water or similar liquid, as reviewed in section 5 and included in sections, 6 to 8. The optical pumping techniques for enhancing the polarization of the noble gases, 3He and '29Xe,has developed and recently we can get the gases at lower cost and more easily in the laboratory. Therefore, a variety of NMR experiments of laser-polarized noble gases have been reported in molecules, materials, and organisms in addition to clinical applications. Non-clinical applications have been increasingly exploited in connection with velocity imaging of convective flow, diffusion in heterogeneous porous media, porosity distribution, xenon chemical shifts in different physicochemical environments and so on. Diffusion tensor magnetic resonance imaging (DT-MRI) has recently gained popularity in axonography of human brain, because of its potential in providing crucial information about intercommunication between different regions of brain (section 11). This technique exploits the sensitivity of MRI to random water diffusion in tissues in the presence of diffusion gradient pulses incorpor-

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ated into the imaging sequence. DT-MRI is interesting and useful in material science as well. Molecular imaging is an emerging field of study that deals with imaging of disease on a cellular or genetic level rather than on a gross level. Recent advances in this field show promise, particularly in the imaging of gene expression. These topics will be reviewed in section 11. 2.1 Review. - Typical reviews concerning the principles or basic technologies of NMR imaging will be picked up in this section and majority of reviews will be presented as review in each section. A review with 232 references concerning modern applications of NMR tomography in physical chemistry.*The scope and limitations of the method, its capabilities, and some of the most widely used applications in physical chemistry are discussed. The technique is able to provide a vast variety of information about the state of objects under study and on the physicochemical processes occurring in them, which can be gained owing to the specific features of the technique. This review predominantly covers the studies of the structure and properties of various liquid-containing objects. 2.2 Catalysed Reaction. - The first application of NMR imaging to study the progress of a multiphase heterogeneous catalytic reaction in situ was r e p ~ r t e d . ~ , ~ Various stationary regimes of a-methylstyrene (AMS) hydrogenation on a single Pt/y-A1203catalyst pellet have been investigated. The two-dimensional maps of the liquid-phase distribution within the pellet have been obtained in the course of the catalytic reaction. It has been demonstrated that despite a substantial broadening of the NMR lines of liquids permeating porous solids the quantification of the relative amounts of AMS and the major reaction product cumene with spatial resolution across the pellet is feasible. In situ magnetic resonance techniques were also applied to explore the spatial variation in chemical conversion of a catalysed reaction occurring within a fixed-bed reactor, the liquid-phase esterification of methanol and acetic acid, catalysed by a proton exchange (Amberlyts 15 ion exchange resin) c a t a l y ~ tThe .~ paper highlights the application of one-dimensional chemical shift imaging and volume selective magnetic resonance spectroscopy techniques to measure directly and quantitatively the spatial distribution of chemical conversion within a fixed-bed of catalyst particles. The authors suggest that magnetic resonance visualization techniques are now able to provide a quantitative tool to aid in the integrated design of catalyst and reactor. The enzymically catalysed esterification reaction ( i e . , propionic acid + 1butanol += Bu propionate + water) inside an immobilizing alginate hydrogel environment has been investigated by means of spectroscopically resolved NMR imaging6 Both the gel-forming water and the solvent (cyclohexane) were fully deuterated to simplify the identification of small amounts of reactants. The self-diffusion process of the separate reactants (propionic acid and butanol) and the concentration buildup inside the gel bead is shown. The reaction itself has been observed with chemical resolution in a time series of up to 40 h, clearly demonstrating the reduction of 1-butanol and production of water inside the gel bead.


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2.3 Mesophase Formation. - The developing interfacial region between a soap bar and water was studied using a suite of spatially resolved Stray field imaging (STRAFI) m e t h ~ dThe . ~ dynamics of water ingress into the soap was followed. The T2 contrast employed in the STRAFI method is not sufficient to resolve detail of the mesophase formation at the interface. However, double-quantum filtered 2H spectroscopy at different positions in the interfacial region allowed water concentration and mesophase distribution to be mapped over the first 120 h of dissolution. 3

Instruments and Materials

3.1 Reviews. - Scaling down imaging was reviewed on molecular mapping of cancer in mice.' The development of miniaturized imaging equipment and reporter probes has improved our ability to study animal models of disease, such as transgenic and knockout mice. These technologies can now be used to continuously monitor in vivo tumor development, the effects of therapeutics on individual populations of cells, or even specific molecules. An application of rare earth magnets and the factors that limit wider utilization was reviewed from recent advancements in permanent magnet materials.' The development of high-energy product rare earth-Fe-B has enabled the application of permanent magnets to MRI. As permanent magnet materials have developed, attempts have been made to take advantage of their improved properties in medical applications.'0 The overview, based on an extensive review of the literature, chronicles the development of magnets in medicine and summarizes areas where future research will be beneficial. Uses of magnets in medical applications range from their simple use for retention through orthopedics and fracture healing, to magnetomotive artificial hearts and pioneering brain surgery to guide catheters. Diffusion tensor magnetic resonance imaging (DT-MRI) has recently gained popularity in axonography of human brain. Large diffusion weighting that is necessary for the generation of axonography with high SNR is achieved by increasing the magnitude of diffusion pulses. However, strong eddy currents in the metallic structure of the cryostat are induced by large diffusion gradients, resulting in distortion of magnetic resonance images. Therefore, characterization of eddy current effects is essential for optimizing the scanning parameters and improving image quality. The effect of eddy currentis on images obtained using the DT-MRI of human brain were characterized on 1.5-T GE scanner, using single shot diffusion weighed echo planar imaging sequence." The current status of development of the niobium based intermetallics with special attention to Nb3(A11-,,Ge,)was highlighted as a review.12 Discussion is focused on the materials science aspects of conductor manufacture, such as P-phase (A15) formation, with particular emphasis on the maximization of the superconducting parameters. Many successful manufacturing techniques of the potential niobium-aluminide intermetallic superconducting conductors are described, compared and assessed. This new technique has potential to revolution-


469

ize the existing superconducting industry enabling reduction of cost orders of magnitude. 3.2 Imaging System. - A scanning Hall probe microscope for large area magnetic imaging down to cryogenic temperatures (35 K) was designed and con~tructed.'~ The authors describe the performance of the system and illustrate its potential with images of the local magnetic induction at the surface of various magnetic recording media at room temperature as well as an array of superconducting YBa2Cu3O7-b2at 40 K. Using a low-field magnetic resonance scanner, both images of gaseous polarized '29Xeand water cells at room temperature was ~ b t a i n e d .This ' ~ potentially low-cost imaging technique offers the possibility of high-resolution imaging using both polarized noble gas and proton MRI of tissues in the same scanner. While NMR for analysis of chemical structures requires highly homogeneous magnetic fields and thus expensive technology, many issues in materials science and process control can be tackled by NMR in such inhomogeneous fields as generated by simple permanent magnets. Inexpensive sensors was realized with an open geometry.15The fields of flow NMR and unilateral NMR are addressed in particular in view of development and applications of the NMR MOUSE (mobile universal surface explorer). The main application area of such unilateral NMR concepts is in nondestructive analysis of large objects. In addition to static objects, the stream lines in flowing liquids, granular media, and complex mixtures can be followed by NMR (flow NMR).

3.3 Magnets. - A two-stage pulse tube cryo-cooled MRI magnet was presented.16 The compact, cryogen-free 0.5 T superconducting magnetic resonance imager magnet was conductively-cooled using a two-stage pulse tube cryorefrigerator. With the absence of cold moving parts and seals, pulse tube cryorefrigerators offer lower transmitted vibrations to a MRI magnet and potentially longer MTBF rates. The pulse tube system provided an estimated 1.3 W of cooling power at 4.2 K. The fabrication and the test results of the superconducting magnet for a whole body MRI are pre~ented.'~ According to the testing result, the central field of the magnet is 1.5 T with a field homogeneity of 9.3 ppm on 40 cm DSV (the diameter of spherical volume). Some magnetic resonance images for human body have been achieved by employing this magnet. Based on a widely adopted effective model Hamiltonian with competing antiferromagnetic (AF) and d-wave superconductivity (DSC) interactions, the vortex charge in high Tc superconductors is investigated by solving self-consistently the Bogoliubov-de Gennes equations. New imaging experiments should be able to probe the vortex charge directly.'* 3.4 Probe, Resonator, Coil and Transceiver.- A new, large probe was developed for the stray field NMR imaging of soil water and preliminary results were presented.'' The authors introduced a new, unique, large 5 cm diameter STRAFI probe, and it was used for three preliminary test cases. Development, character-

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ization, and preliminary results of a recent technique capable of local measurements of pore-size distribution by a spatially resolved low resolution NMR technique are also described. Superconducting (Bi,Pb)2Sr2Ca2C~3010 tapes fabricated by the oxide-powderin-tube method have been employed to assemble L-C resonating circuits operating in the radio frequency domain2' The quality factor of these superconducting devices has been evaluated in liquid nitrogen bath for resonating frequencies. Bi-based resonators, conceived for working frequencies in the range between 5 and 27 MHz and temperature, presented an improvement of the quality factor by more than an order of magnitude. The present results have been evaluated to take into account a possible application of such a device as an MRI detector. Picoliter 'H NMR Spectroscopy and chemical shift imaging (CSI) experiments were performed with a 267 pm-diameter solenoid transceiver to acquire localized 'H NMR spectra.21The measured signal-to-noise ratio (SNR) at 500 MHz is within 20-30% of theoretical limits formulated by considering only its resistive losses. In CSI experiments performed using a few thousand total scans, the choline Me line has an acceptable SNR in resolved volume elements containing only 50 pL of sample, and localized spectra are resolved from just 5 pL in the Xenopus globule. Two types of micromolding were used to realize high quality factor microcoils operating at room temerature for NMR application.22The mold is a positive thick resist (AZ 4562) and the electrodeposition process is optimized for Cu structures. Classical Cu micromolding was used to obtain NMR imagery antennas in the range of 500 kHz -1 GHz. First measurements at ambient temperature showed resonant frequencies of 174 and 102 MHz, and Q factors of 74 and 60, respectiely, for the five turns coils realized on each side of glass and Kapton substrates.

3.5 Optical Pumping System for Polarized Gases. - A Rb spin exchange optical pumping system was designed for high capacity production of >65% spin polarized 129Xe gas23and for 3Heat pressures near 1 bar24.The system for '29Xeis based on a fiber coupled multiple laser diode array and capable of producing an unprecedented 210 W of circularly polarized light at the pumping cell with a laser line width of 1.6 nm.16The maximum 129Xe nuclear polarization of 67% was achieved using a 0.6% Xe mixture. The spin exchange optical pumping of 'He at pressure of near 1 bar was explored by using broadband laser diode arrays (LDA), and the authors have obtained 55% 'He nuclear polarization in the glass cells (TI = 840 h).17These results are particularly relevant to the application of 3He-basedneutron spin filters to neutron scattering and magnetometry as well as polarized gas magnetic resonance imaging. 3.6 Cryogenic Systems. - A new compressor system was developed for cooling MRI magnets which has more displacement than the present compressor. By increasing the displacement from 2.9-5.2 L/s at 60 Hz it is possible to provide more refrigeration at lower temperatures with both G M and pulse-tube expand e r ~This . ~ ~paper describes essential design features and the extensive testing


47 1

that was done to characterize the compressor over a wide range of operating conditions including extremes that it might experience. The steps taken towards the ultimate goal of a superconducting magnet being as simple as a household fridge were described.26 Pulse tube refrigeration systems were developed by using unique multiple stage phase shift mechanism and novel compressor technology to produce refrigeration at 4, 40, and 80 K.27The developments in IGC-APD on the twostage pulse tube cryocooler systems have focused primarily on refrigeration capacity, temperature stability, performance repeatability, and long life of whole refrigeration system. This paper describes the two-stage pulse tube cryocoolers designed for MRI cooling and their performance characteristics. 3.7 Artifact via Implant Biomaterials. - To quantify the susceptibility artifacts produced from various biomaterials for neurosurgical implants in 0.5,1.5 and 3.0 T MR scanner, MR imaging of six kinds of ceramics, two kinds of Co-based alloys with different combination, pure titanium, titanium alloy and stainless steel were performed.28On all biomaterials, susceptibility artifacts developed parallel to the direction of the main magnetic field at both ends. The sketch of the carbon fiber reinforced, multifunctional puncture needle is presented.29 A hollow multichannel needle made of carbon fiber reinforced plastic with an outer diameter of only pm was developed for MRI-supported surgical operations. This puncture needle enables the surgeon to realize a complete, highly precise and artifact-free supervision of the operation area in tumors of the abdomen and the spinal cord, including the microinvasive treatment of prolapsed intervertebral disks.

3.8 Temperature Measurement.- Based on the fact that the electric impedance of biological tissues is very sensitive to temperature, a method to monitor local temperature was pr~posed.~'Using an analytic model and a finite element method model, the effect of the local temperature change on the phase image obtained by the magnetic resonance current density imaging technique (0.3 T) was analysed. 4

Pulse Sequences and Data Processing

4.1 Pulse Sequences. - Improved strategies for acoustic ringing effect31 and reduction of the experimental time, or temporal r e s ~ l u t i o n have ~ ~ - ~been ~ reported. A new pulse sequence is introduced for compensation of acoustic ringing effects, which occur in rotating-frame images obtained with the rapid imaging pulse trains (RIPT). The new sequence (RIDE'n RIPT) combines features of ring down elimination (RIDE), the most common difference-spectroscopy sequence for acoustic-ringing compensation, with the advantages of RIPT for fast acquisition of magnetization profiles in B1 field gradient^.^^ RIDE'n RIPT was used to record 1-dimensional profiles of the proton magnetization in supercritical fluid samples of methane in carbon dioxide, To withstand the high pressures required

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for the supercritical carbon dioxide mixtures, a toroid cavity autoclave (TCA) was used as the NMR resonator and pressure vessel. ‘Spectroscopic RARE’ sequence for fast spectroscopic imaging with high signal-to-noise ratio is presented, which is based on spatial localization by the fast MRI method of rapid acquisition with relaxation enhancement (RARE) and encoding of the chemical shift information by shifting the position of a refocusing n pulse in a series of m e a s u r e m e n t ~ In . ~ ~contrast to spectroscopic ultrafast low-angle RARE (U-FLARE), the formation of 2 echo families (odd and even) is suppressed by using a train of n R F pulses with an internal 4-step phase cycle. The pulse sequence was implemented on a 4.7 T imaging system, tested on phantoms, and applied to the healthy rat brain in vivo. The NMR measurement of molecular diffusion with magnetization-grating rotating-frame imaging (MAGROFI) was improved by utilizing the tightest magnetization grating (Nyquist grating).33If applied to toroid cavity NMR detectors, phase-cycled fast two-transient NYGROFI (Nyquist-grating rotatingframe imaging) reduces the experimental time to minutes or only seconds depending mainly on relaxation times. The feasibility of echo planar proton detected I3Cimaging using CYCLCROP based upon the PRAWN module was demonstrated in some initial experim e n t ~By . ~the ~ combination of Cyclic J cross polarization (CYCLCROP), which is a sensitive method for the noninvasive monitoring of I3Cdistributions and flux, with echo planar imaging (EPI) for spatial encoding of the proton detected carbon signal, efficient use of the available signal to be made is allowed and therefore, a significant improvement in the temporal resolution of any study was permitted. Two methods that can be used to eliminate the effect of the J-splitting in the EP images are presented. In addition, a fast, image-based B1 field-mapping method which may be used to quantitative map the low frequency RF field in a dual resonant (13C/’H)probe is presented. 36 The electronic Overhauser effect was employed to enhance NMR Overhauser imaging was obtained in a magnetic field of only 16 mT.35Despite of the weak magnetic field, the technique can reveal, in some systems, various aspects of ionic transport and kinetics apparently not observed in conventional high-field magnetic resonance imaging. The spatial and temporal evolution of the gelation of sodium alginate by calcium ions was monitored. The sensitivity of the Overhauser enhancement to small changes in gel component permits a stringent test of the underlying mechanisms of transport and chemical kinetics. Overhauser enhanced magnetic resonance imaging (OMRI) for tumor oximetry was described by using a low-field magnetic resonance scanner and a paramagnetic contrast agent Ox063 (a highly derivatized triarylmethyl radical).36Spatially resolved pO2 (oxygen concentration) images from OMRI experiments of tumor-bearing mice exhibited heterogeneous oxygenation profiles and revealed regions of hypoxia in tumors (c10 mmHg; 1 mmHg = 133 Pa). This work illustrates that anatomically coregistered p 0 2 maps of tumors can be readily obtained by combining the good anatomical resolution of water proton-based MRI, and the superior pOz sensitivity of EPR. A new method for selectively detecting sodium ions in anisotropic environ-


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ments is p r e ~ e n t e dA . ~spin-lock ~ (SL) sequence, followed by a coherence transfer pulse, generates rank-two zero-quantum coherences, and converts them into observable transverse magnetization. The quadrupolar polarization is only generated when there are residual quadrupolar couplings in the sample. This filter has proved to be more efficient than a double-quantum magic-angle (DQ-MA) filter in generating observable signal from ions in anisotropic media.

4.2 Data Processing. - In the tilted rotating frame (TRF), the transverse relaxation time TZp depends strongly on the orientation of TRF with respect to the usual rotating frame. Since the orientation of the two reference frames and, to some extent, the role of the spin Hamiltonians in TRF are controllable experimentally, the TZprelaxation can be made sensitive to molecular mechanisms related to the selected spin interaction. The realization of a contrast Hamiltonian-dependent in solid-state NMR Imaging was proved.38The solid-state imaging approach is based on the magic angle in the rotating frame. Some results on simple solid polymers are presented. An original scheme based on synchrotron radiation computed micro-tomography was presented to assess the efficiency of segmentation algorithms requested to quantify bone architecture from three-dimensional (3-D) micro-magnetic resonance imaging (yMRI) technique~.~~ The Fourier-based formalism to analyse structural measurements obtained using the long-range liquid dipolar field in NMR was presented in the case of a two-spin system and in the case of cylindrically symmetric systems.40In the latter cases the measured data are related to the Meijer transform (or K transform) of the radial distribution function of the spin density. Experiments were performed on structured phantoms exhibiting cylindrical symmetry over the length scale probed, and were in excellent agreement with the theory. Finally, changes in image contrast with the strength of the modulation gradient can be expressed in terms of the action of a dipolar filter. Software, which can use digital three-dimensional images to determine structural parameters such as strut length distribution, connectivity, and cell size, is being developed?' Analysis of a reticulated polyurethane foam sample using these methods yielded a reasonable approximation of the structural skeleton of the sample. This skeleton was used to compute strut length, interior angle, and window size and shape distributions for a small sample comprised of about 20 cells. 5

Hyperpolarized Noble Gases, Gas Phase Imaging and Other Nuclei

5.1 Review. - Two reviews42'43 describe the principles and magnetic resonance applications of laser-polarized noble gases. The enormous sensitivity enhancement afforded by optical pumping can be exploited to permit a variety of novel NMR experiments across numerous disciplines. Many such experiments are reviewed, including the void-space imaging of organisms and materials, NMR and MRI of living tissues, probing structure and dynamics of molecules in

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solution and on surfaces, NMR sensitivity enhancement via polarization transfer, and low-field NMR and MRI?2 Pulse sequences for optimum use of the nonrenewable magnetization require a different imaging strategy than traditional proton MRI. Examples are discussed for static lung images, diffusion images for characterization of the local alveolar structure, and high temporal resolution images.43 Novel, non-medical applications of laser-polarized noble gases for both NMR imaging and spectroscopy were reviewed and highlight progress with examples from the authors' laboratory including high-resolution imaging at low mT of the applied field strength and velocity imaging of convective flow was presented.44 Porous media microstructure was probed with both thermal and laser-polarized Xe, as Xe is an ideal probe due to low surface interaction with the grains of the porous media.

5.2 Hyperporalized Nobel Gases. - The use of a new carrier agent, lipid-based helium microbubbles, for intravascular laser-polarized 3He imaging was investigated.45 The average diameter of 3 pm, which is smaller than that of the capillaries, makes it possible to conduct in vivo studies. In vivo images of encapsulated 3He were acquired with signal-to-noise ratios (SNRs) larger than 30. The first images of 3Hemicrobubble distributions in the lungs were obtained. Initial NMR studies of xenon gas diffusion in model heterogeneous porous media and continuous flow laser-polarized xenon gas were presented with a brief overview of this area.46Both areas utilize the pulsed gradient spin-echo (PGSE) techniques in the gas phase, with the aim of obtaining more sophisticated information than just translational self-diffusioncoefficients. Gas velocity imaging is, however, found to be limited to a resolution of about 1 mm s-' owing to the high diffusivity of gases compared with liquids. The authors also presented the first gas-phase NMR scattering, or diffusive-diffraction, data, namely flowenhanced structural features in the echo attenuation data from laser-polarized xenon flowing through a 2 mm glass bead pack. Laser-polarized 129Xeand a high-Tc superconducting quantum interference device (SQUID) were used to obtain magnetic resonance images in porous materials at a magnetic field of 2.3 mT, corresponding to a Larmor frequency of 27 ~ H z !Image ~ resolution of 1 mm was obtained with gradients of only 1 mT/m. The resolution of Xe chemical shifts in different physicochemical environments at ultralow fields is also demonstrated. Details of the circulating flow optical pumping apparatus and the SQUID spectrometer are presented. 5.3 Imaging via Other Gases. - Deuterium (2H)microscopic images (SE and FLASH pulse sequences) with resolution of tens of microns and localized 2H spectra with volumes in the micro liter range have been obtained on a 400 MHz (9.4T) wide bore spectrometer equipped with a SOG/cm gradient system?* In order to realize 2H experiments at 61.4 MHz, a simple RF coil was built. A phantom consists of a 2 x 2 x 2 mm3 cube with C D 3 0 Dimmersed in D20.As an example for a biological application, the absorption process of D20 in a plant fragment (Mikania Cordifolia) has been monitored by acquiring consecutive SE


475

microimages along various hours. Application of MRI to pressurized gas was described.49In a specially-designed nonmagnetic, cylindrical sample chamber (12 mm internal diameter and 114 mm in length), methane gas or hydrogen gas was supplied up to 10.0 MPa to study its feasibility of gas MR imaging. It is shown that signal intensity on MR images of hydrogen was much less than that of methane due to high diffusivity of hydrogen molecules, which was also confirmed by numerical calculation. It is demonstrated that NMR flow imaging on the basis of phase encoding of flow velocities as well as NMR flow tagging can be successfully employed to visualize laminar, but not necessarily fully developed flow of thermally polarized hydrocarbon gases at atmospheric pressure. Gas flow in the nonconsolidated bed composed of solid glass beads and porous silica gel grains was characterized by the displacement NMR spectroscopy on the basis of PFGSTE NMR techniq~e.~' '29XeNMR of adsorbed xenon used as a probe and 'H NMR imaging have been used to study the diffusion of hydrocarbons (benzene, n-hexane, paraxylene) during their adsorption or desorption in a fixed bed of zeolite ~rystallites.~' The 'H NMR imaging allows one to visualize the progression of the diffusing molecules in the zeolite bed and to determine their intracrystal diffusion coefficients in the simplest cases. By the simulation of experimental '29Xespectra using the nonuniform or the shrinking core models, the concentration profiles of hydrocarbons and intracrystal diffusion coefficients were given, in the bed and in the zeolite crystallites during their adsorption. Direct visualization of gaseous xenon and methane in the void spaces of aerogels, offering unique information and insights into the pore structure and molecular diffusivities of occluded sorbates were done by NMR pulsed-field gradient (PFG) technique^.^^ Exchange and diffusive motion of sorbed xenon gas at equiliblium was characterized. Spatially resolved adsorption isotherms of thermally polarized perfluorinated gases (C4F8,octafluorocyclobutane) in Y-TZP (yttria-stabilized tetragonal-zirconia polycrystal) ceramic materials was investigated with NMR imaging of nuclear spin density.53NMR images of as a function of gas pressure permits spatially resolved measurements that are analogous to conventional bulk Brunauer-Emmett-Teller adsorption isotherm measurements.

Other Nuclei. - A spatial distribution of the magnetic domain structure in the U2D2phase of solid 3He was examined by a special MRI technique.54There were three kinds of magnetic domains in a single seed crystal of a few mm3 size and each domain size was as large as the seed crystal size. The distribution and growth process of domains was studied. The first magnetic resonance imaging profiles of chloride content in low- and high-permeability Portland cement mortar have been obtained using a novel material science imaging technique. The penetration of water ('H) as well as chloride (35Cl)and sodium (23Na)ions, into mortar specimens was monitored for a period of 72 h.55These preliminary experiments show significant differences

5.4

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between the extent and nature in the penetration of chlorides compared to water and sodium.

6

Dynamics: Diffusion, Flow and Velocity Imaging

The mobility of the species under investigation is determined not only by its chemical structure and size, but also by the physical environment in which it is located. Therefore, the ability to visualize and quantify the mobility of particular components in multi-component solutions by NMR imaging has had a profound impact on various fields of science and technology. In this session, NMR imaging for dynamics, such as diffusion, flow, dispersion and mass transfer in gases, liquids and solids are introduced.

6.1 Reviews. - Measurement and analysis of self-diffusion coefficients using the pulsed-gradient spin-echo NMR technique (PGSE-NMR, sometimes referred to as PFG-NMR) permits a non-perturbing in-situ probing of complex structure. This is especially true for colloid science since the characteristic structural dimensions are comparable to the displacements on the NMR timescale. NMR self-diffusion studies of polymeric surfactants were reviewed.56 Modern physicochemical applications of NMR tomography to the investigation of mass transfer processes were described.57The mass transfer involved in the sorption of gases, fluids and solutes, swelling of polymers and coal and drying of various materials is considered. Various methods of studying isotropic, anisotropic and restricted diffusion in porous media are discussed. The approaches to the investigation of filtration and the flow of fluids, gases and granulated materials are presented. Visualization of fluid flow and transport in porous media were reviewed, and illustrated with experiments showing applications of velocity imaging, NMRI measurements of multiphase flow.58The examples show the capacity of NMRI to give structural information both of the medium and the fluid distributions as well as their temporal development. NMR imaging to describe flow in heterogeneous porous media was demonstrated on sandstone samples. Porosity distributions, the spin density, the intrinsic magnetization intensity (proportional to the amount of saturating fluid), permeability distributions and the velocity field within the samples are reviewed.59 Contribution of magnetic resonance techniques in chemical engineering is described as a review.6oTopics were the principles and applications of NMR in the fields of fluid flow characterization, process applications, gas-phase imaging, fast imaging techniques and future perspectives. 6.2 Data Processing. - Flow, diffusion, dispersion, and thermal convection in percolation clusters was investigated by NMR experiments and numerical simulations with finite-element method (FEM) or finite-volume method (FVM).61 Random-site, semi-continuous Swiss cheese, and semi-continuous inverse Swisscheese percolation models above the percolation threshold were considered. The


477

water-filled pore space was investigated by using NMR imaging and in a pressure gradient by NMR velocity mapping. 6.3 Diffusion.- The self-diffusion coefficients (D)of a-helical poly (n-octadecyl L-glutamate) (POLG) and chloroform (CHC13)as solvent in the isotropic, biphasic, and liquid crystal phases were measured as a function of POLG concentration at 30°C by the PFGSE 'H NMR method and by the diffusion NMR imaging method to study diffusional behavior of the polypeptide and solvent.62 Through-plane diffusion of moisture in paper samples was measured by MRI.63The mechanism of moisture transport through the plane of paper sheets, and the moisture distribution as a function of time for a thick two-ply paper was discussed by using two different experimental arrangements. The data clearly support a model in which moisture is transported in two stages: as vapor through the void spaces of the paper and subsequent sorption by the fibers on a longer time scale. The anisotropy, Dpara/Dperp, of water diffusion in fully hydrated bilayers of dimyristoyl-phosphatidylcholineat 29°C has been measured by PFG-NMR and NMR imaging.64In this experiment, magnetic field gradients in an arbitrary direction with respect to a stack of macroscopically aligned lipid bilayers is used by employing the special hardware system, and then the translational diffusion of water was measured as a function of the angle between the direction of the magnetic field gradient and the normal of the lipid membrane. The obsd. diffusion coefficient is found to depend strongly on this angle. The results are discussed in terms of the relatively low permeability of water across the lipid bilayer, instrumental limitations, and/or possible defects in the lamellae. 6.4 Flow in Suspensions and Colloids. - An increasing number of industrially relevant suspensions, e.g., stabilized flows, backfill, and codisposal systems, differ from conventional transport systems. Analysis of these complex hybrid suspension flows is difficult due to their non-Newtonian behavior and increased particle/particle interaction. Results by NMR imaging were analysed s t a t i ~ a l l y . ~ ~ - ~ * Particle concentration and fluid velocity maps in a 100 mm diam. pipe test loop were presented for a number of flow conditions, which show that laminar flow of these suspensions is stratified rather than hom0geneous.6~Shear-induced migration of particles was studied during the slow flow of suspensions of neutrally buoyant spheres, at 50% particle volume fraction, in an inelastic but shearthinning, suspending fluid.66 The suspension is flowing in between a rotating inner cylinder and a stationary outer cylinder. NMR imaging demonstrates that the movement of particles away from the high shear rate region is more pronounced than for a Newtonian suspending liquid. A continuum constitutive model for the evolution of particle concentration in a flowing suspension proposed by Phillips et al. was tested. Vertical profiles of the fluid fraction in batch flotation experiments, measured by one-dimensional NMR imaging, were input into a new method for extracting the hindrance function, G.67This simple, explicit calculation was tested on concentrated monodisperse and polydisperse samples.

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Mixing in a multicomponent batch system, a 10% carbon black powder suspended in aqueous solution, was evaluated by MRI.68The combination of the surfactant type and level, and the mixing mode and speed were changed in the sample preparation. Different results obtained were statistically discussed in terms of mixing effectiveness. Coupled, controlled velocity, magnetic resonance imaging (MR1)-rheometry experiments were carried out with colloidal su~pensions.6~ The velocity profiles between coaxial cylinders are analysed for not tool high relative velocity of the tools (< 70 rpm). The thixotropic and yielding behavior of suspensions was directly determined by NMR method. It is suggested the apparent rheological behavior, i.e., as deduced from usual rheometric,al tests without taking into account this discontinuity in shear rate, does not represent the effective behavior of the material. The steady-rheological properties concerning polydimethylsiloxane (PDMS) colloids was investigated by NMR spectra and NMR imaging.70These experiments render the colloids the potential application as contrast agent for gastrointestinal NMR imaging.

Flow in Tubes. - A procedure, based on Tikhonov regularization, is developed to solve the problem of extracting shear rate profiles of steady laminar tube flow of Newtonian and non-Newtonian fluids from experimentally measured velocity profiles. These velocity profiles was taken from recent literature, and are representative of what is currently achievable with NMR imaging and laser Doppler techniq~es.~' The spatially resolved determination of velocities and concentration of single components in two-phase systems consisting of water and oil by means of snapshot MRI techniques was described and applied to steady tube flows with regard to the total flow rate.72The insight into the inner micro flow processes and microstructure allows to characterise fluid mixtures or emulsions. A dynamic MRI technique for the visualization of the two-phase flow in ceramic monolith (43 mm in diameter and 0.15 m in length, 400 channels per square inch of 1.2 mm width) was d e ~ e l o p e d .Compressed ~~,~~ air and water were pressed through this ceramic monolith with flow rates of 1 and 6 L/min respectively. The visualized flow characteristics allowed to tailor 2-phase flows for chemical engineering. MRI techniques were used to visualize flow phenomena within packed beds in a tube with one cylinder and a bundle of ten cylinders. The three-dimensional (3-D) porosity and velocity distribution within the packing is determined.75Axial and radial dispersion coefficients, porosity and velocity distributions averaged in angular and axial direction was derived from the NMR data. This information is necessary for modeling the structure and the flow within a packed bed and for verifying theoretical predictions with regard to the distribution of the porosity and the velocity. NMR velocity imaging was used to study the flow behavior of three types of hyaluronan solutions pumped through Teflon The shape of the velocity profiles varied significantly with Mw, concentration and apparent shear rate. Transitions were observed from near Newtonian fllow to shear thinning to wall

6.5


479

slip with increasing Mw and concentration.

6.6 Gas Flow. - Particle motion in gas-fluidized granular systems was monitored by NMR k-space and q-space imaging methods for the first time.77Thetime-averaged density variations and the random motion of granular particles in the presence of gas flow of variable velocity in a model gas-fluidized bed reactor. The transitions toward fluidization, bubbling phase, and slugging phase are visible as sudden changes in the density and the root mean-squared displacement. The effective diffusion coefficients are measured and the distributions of particle displacements, or propagators, are presented. In gas flow experiment with the beds composed of porous silica gel grains, strong adsorption of hydrocarbon gases was observed by the displacement NMR spectroscopy on the basis of PFGSTE NMR technique.” 6.7 ConcentrationProfiles in Columns. - MRI was applied as an in situ imaging technique to study transient adsorption/desorption and chromatographic processes in microporous ads or bent^.^^ Transient concentration profiles were measured for several different types of systems: water vapor into a bed of 4A zeolite, hydrocarbons through a chromatographic column of 5A or 13X zeolite, and the transient adsorption/desorption of propane or water vapor in a single extrudate of 13X zeolite. The possibility of distinguishing between the concentration profiles in the gaseous and adsorbed phases by MRI methods is examined. Water flow in the porous media was investigated by a fast low angle shot (FLASH) ~equence’~ and microimagingsO.The transport of Ni(I1) in a column, filled with porous media, was observed in three- dimensions and time by using a fast low angle shot (FLASH) MRI sequence in a clinical MRI scanner (isotropic spatial resolution of 1.5 mm).79The porous media was glass beads or quartz sand in a saturated continuous flow mode. The Ni(I1) concentration-dependent effect to TI of water proton was used to obtain the water flow in the porous medium. The fluid flow velocity field was calculated by a front-tracking method and the statistical properties of the velocities were investigated. The flow of water in a column (14 mm in diameter) packed with glass beads was imaged at a spatial resolution of 0.15 x 0.15 x 2 mm3.80Water was pumped through the column using a peristaltic pump, at flow rates of 125 and 250 mL/h, corresponding to mean velocities of 0.5 and 1 mm/s, given a porosity of 0.46 m3/m3.The convection-dispersion equation was fitted to the breakthrough curve. A one-dimensional model of mechanical deformation of compressible chromatographic columns, which is based on linear elasticity and continuum mechanics, was presented and compared to a more complete two-dimensional model and one-dimensional porosity profiles measured by NMR imaging methods.81The model provides a quantitative description of compression and the effects of wall support during scale-up, but it is suggested that a more complete model may be valuable for anticipating the effects of scale and aspect ratio on pressure-flow behavior of compressible columns. 6.8

Porous Media. - Solute transport in saturated artificial porous media was

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observed in a series of laboratory experiments usirig NMR imaging to study a situation of density-dependent flow in three dimensions both qualitatively and quantitatively.82The time-dependent measurements visualized inflow from below of dense salt water into a freshwater reservoir, internal density-driven flow and the behavior of a salt water layer below freshwater flow including plume development by dispersion. Flow (at velocities 0.15-6.67 mm/s) and diffusion of water in natural porous media, quartz sand, and calcareous gravel were measured using a 1.5-T clinical MR t ~ m o g r a p hThe . ~ ~spatial resolution of the dynamic measurements was 1.32 x 1.32 x 5 mm3, and the time between two cross-sectional measurements was approximetly 10 s. The pore space was imaged with a spatial resolution of 0.5 x 0.5 x 0.5 mm3.The porosity of pores that are >0.2 rrim can be measured directly, for smaller pores a calibration is necessary. The use of a pulsed gradient spin-echo imaging sequence for the three-dimensional (3D) imaging of water transport properties in two porous media (2 mm glass-beads and 0.15 mm quartz-sand mixed with 2 mm glass-beads) was reported.84In contrast to tracer methods, which monitor the tracer motion by its effect on the signal relaxation of 'H, this sequence measures the echo signal intensity I. without and with applied diffusion gradient, respectively. For the fine porous medium, frequent contacts of the water molecules with the pore boundaries lead to a significant decrease of I. by increased T2 relaxation, which is caused by the restricted diffusion in the fine pore system.

6.9 Miscellaneous. - To analyse the correlation between the increase in the shear stress and the porosity, agglomeration processes and the type of flow, oxide ceramic masses were tested in a shear device especially developed for pastes and analysed by rheometric experiments, NMR imaging and particle size analysis.*5 Structural changes (hardening, increase in the mean porosity) of the material during the peptization, the fraction of bound (more:generally, the immobilized) water, and crushing of primary particles was discussed. On a macroscopic scale these phenomena cause hardening. NMR imaging visualized flow-induced agglomerates, which form owing to the shear flow and increase the porosity averaged over the whole sample. Coupled flow and dissolution processes are milch more complex and unpredictable than commonly assumed, even under simplified conditions. NMRI is applied to noninvasively measure flow and dissolution patterns in natural, rough-walled, water-saturated halite fractures.86Quantification of the developing fracture morphology and flow patterns was presented based on threedimensional images of water density and flow velocity acquired with NMRI. The correlations of the dissolution patterns to the fracture morphology, flow patterns, and mineralogical component of the rock matrix were a function of the overall dimensionless Damkohler number (DN). Extrusion behavior of pastes with a piston extruder for the determination of the local solid and fluid concentration, the local porosity, and saturation and displacement profiles was investigated by means of 'H- and 19F-NMR imagi n g ~The . ~ ~19F-MRIprovides the local solid concentration.


48 1

Aerosol separation from flue gases of future power stations was fundamentally studied by NMR imaging. The space- and time-resolved liquid distribution in a packed-bed aerosol filter was studied by NMR tomography and a mathematical model was developed.88Randomly packed glass spheres and silicone oil sprays were used for experiments. Measurements of the bed porosity showed a radial course which can be described by a function similar to a damped oscillation function (with the highest value immediately on the wall and periodical changes dying out towards the bed center). Two- and three-dimensional waves in falling film flow in the nonlaminar flow regime were studied by NMR.89The NMR velocity-encoded imaging results as well as propagators are presented for the nonlaminar flow regime of falling films. The film is generated by a continuous flow of silicon oil along a vertical poly(methylmethacry1ate) plate. The in situ saturation development for reservoir core plugs was monitored during spontaneous imbibition using MRI.” The images monitored the movement of the imbibition front and the saturation change across the front as a function of initial water saturation. The oil saturation uniformly decreased everywhere in the core during imbibition. Results suggest two concurrent mechanisms of water influx: (1) plug flow water displacement of oil and (2) a longrange process, possibly a thickening of pre-existing capillary water films on the surface. The immunomagnetic labeling and MRI were applied for the noninvasive visualization of changes in bacterial density distributions as a function of time in a water-saturated porous medium.” Magnetite particles (50-60 nm diam.) were attached via a monoclonal antibody to the surface of Escherichia coEi K12 NR50 cells. Diffusive migration for both motile and nonmotile E. coli through a porous medium with a particle-diam. distribution of 250-300 pm was compared. Noninvasively visualizing bacterial concentrations within an opaque porous medium in real time provides researchers with a powerful tool for studying bacterial transport in porous media, which is important for understanding the impact of bacterial transport on remediation strategies for environmental cleanup of polluted groundwater.

7

Polymer

7.1 Reviews. - NMR imaging used to interrogate structure in a variety of natural and synthetic polymers, polymer blends, and polymer composites was The relevance of chemical, functional, and dynamical information at the micron to millimeter length scale was discussed. Many different experimental strategies range from imaging imbibed solvent spins in a swollen polymer, spins of the swollen polymer, solid polymers, the surface of a solid polymer, polymers under stress/shear, to selectively imaging only one polymer component in a multi-component blend. Particular attention was given to explaining the available contrast mechanisms used to generate images, and the relevance of the imaging data to performance properties of the polymer. Standard ‘H spin-echo

482


imaging methods for low T, polymers such as synthetic elastomers, and recently advanced multiple-pulse methods and single-point,/constant-time imaging techniques for rigid, high T, polymers were routinely used. The structure and dynamics of polymer gels by solid-state NMR, diffusion process of polymer gels by pulse-field-gradient spin-echo NMR, and spatial information response of hydropolymer gels under stimulus 'H-NMR imaging method were described.93 Dynamic NMR studies of the silk fibroin from silkworms were reviewed with references. The chain dynamics of B. mori and S.C.ricini silk fibroins in aqueous solution, in the silk gland of living silkworms, and in fiber were studied various type of NMR spectroscopy. The chain dynamics of silk fibroin absorbed solvent were detected by 'H and 13Csolid-state NMR methods, and also using an NMR imaging method.94

7.2 Characterization.- Gravimetric Flory-Rehner experiments are combined with MRI experiments to study the spatially dependent degree of crosslinking in unfilled, 1,6-hexamethylenediamine-cured PIB (poly-(isobutylene-p-methylstyrene-p-bromomethylstyrene)) t e r - p ~ l y m e r sM . ~RI ~ relaxometry revealed two proton T2 relaxation decay times in swollen specimens: the constrained chain segments near crosslinks and entanglements as fast decaying component and less constrained, remote chains as slow decaying component. The local crosslink density in inhomogeneously cross-linked specimens was determined based on the spatially dependent T2 decay times. The ingress kinetics of cyclohexane in PIB terpolymers was revealed to be Fickian. Characterization of aged nitrile rubber elastomers was revealed by NMR spectroscopy and m i ~ r o i m a g i n g . ~ ~ Aging proceeds mainly via additional chain crosslinking, and scission of the polymer chains does not appear to contribute. TI imaging shows that there is no spatial distribution of spin-lattice relaxation times. The values of equivalent cross-relaxation rate (ECR) were measured by using an off-resonance saturation pulse under conventional field-echo imaging at frequency within +/- 75 ppm apart from the water resonance freq~ency.9~ Based on the ECR-7 (the value at the frequency offset of 7-ppm) the sample gels were divided two classes corresponding to hydrophilic and hydrophobic ones. The synthetic copolymer gels, which were composed of any two or three monomers among 2-hydroxyethyl methacrylate (HEMA), glycidyl methacrylate (GMA), N-vinyl-2-pyrrolidinone (N-VP), methyl methacrylate (MMA) and benzyl methacrylate (BMA), were used. The values of (ECR)l correlated well with water conditions in various copolymer gels and nature of malignant cells. It is suggested that the ECR values could be a new parameter for malignancy and cell proliferative activity of the breast carcinomas with non-invasive modalities by MRI. Magnetic resonance imaging has rarely been applied to rigid polymeric materials, due primarily to the strong dipolar coupling and short signal lifetimes inherent in these materials. SPRITE (single point ramped imaging with TI enhancement) is particularly well suited to imaging solid materials. We have to improve the minimum phase encoding time limited (usually tens of microseconds) with refinements in technology. The SPRITE sequence is used in


483

conjunction with raising the sample temperature to obtain images of rigid polymers that have largely frustrated conventional imaging methods?* This approach provides a straightforward and reliable method for imaging a class of samples that, up until now, were very difficult to image. 7.3 Process Analysis. - Magnetic resonance images of the formation of an alginate gel was performed by employing the electronic Overhauser effect in an imaging mode to enhance the proton signal at a field of only 16 mT.99The high sensitivity of the Overhauser effect to small changes in mobility permits to monitor spatial inhomogeneity in the very early stages of the gelling process. The proton NMR image patterns of ethylene-vinyl alcohol copolymer (EVOH)/dimethyl sulfoxide (DMSO) gels with various ethylene contents were measured to elucidate the process of solvent exchange between DMSO and water in the gels soaked in water.lm The results indicate that the rate of solvent exchange between DMSO and water in the gels increased with an increase in the ethylene content of EVOH copolymers. Composition and phase changes during non-solvent induced coagulation of cellulose were studied by the stray field magnetic resonance imaging (STRAFI).''' Changes in composition and diffusion were readily observable at different depths within the cellulose gel. Large increases in diffusion coefficient and relaxation times were attributable to the phase separation and the progressive development of pores within the gel. The application of NMR microimaging (STRAFI) to the visible light curing of dental restorative resins revealed differences in composite shade and cure time produced changes in the cure profiles."* A one-dimensional probe was used to measure the magnetization in 40 pm thick slices at 100 pm intervals along the length of the specimen. A quadrature pulse sequence was applied and the magnetization decay recorded in a train of eight echoes. A value for T2 could be obtained only for the polymer (59 +/- 16 ps). This first investigation has demonstrated that STRAFI is well suited to polymerization studies. 7.4 Polymer Gel Dosimetry. - Polymer gels whose NMR and optical properties change when irradiated offer unique advantages for measuring radiation dose distributions. For Fricke-agarose gello3,acrylic polymer and N-vinylpyrr~lidone-Ar-(VIPAR-)gel'~~, radiation dose distribution and stability as the dosimetry was examined by MRI. Fricke-agarose gels have elicited much interest in the field of radiation dosimetry, as tissue-equivalent dosimeter~.'~~ Magnetic resonance relaxation rates are measured for dose reconstruction. A major problem of Fricke-agarose gels is the diffusion of the ferric ions formed after irradiation. Two-dimensional Ferric ion distribution, the corresponding images and the doses at different times after irradiation were reconstructed taking into account the calculated diffusion coefficients. A new formulation of acrylic dosimeter gel, named MAGIC (Methacrylic and Ascorbic acid in Gelatin Initiated by Copper), has been developed that responds well in normal atmosphere.'" At room air the water proton spin relaxation rate R2in MAGIC gels is proportional to absorbed dose though the precise relationship depends on the

484


composition of the gel and the initiating complex. The sensitivity to detect small dose changes, i.e., the slope-to-intercept ratio of the dose-response curve varied from 0.08 to 0.17, comparable to hypoxic gels described earlier. MAGIC gels can be imaged by MRI or optical scanning. Three-dimensional (3D)MRI techniques were employed in N-VinylPyrrolidone-Ar-(VIPAR-) based polymer gel d~simetry.''~ The 3D dose distributions obtained with the 3D MRI method were in good agreement with the corresponding Monte Carlo calculations, for brachytherapy and intravascular irradiations. The method allows the reconstruction of isodose contours over any plane, with increased spatial resolution and accuracy following a single MR acquisition.

7.5 Tablet Disintegration, Swelling, Drug Release. - Four papers concerning tablet swelling and its kinetics have been published with a view of better understanding the process of drug release.'06~'09 The disintegration process of paracetamol tablets was visualized in real time arid in situ by MRI using the Snapshot FLASH method. lo6 The total time of the single experiment is 425 ms. The study was carried out in vitro under acidic gastric pH conditions to predict the behavior of the tablets in the stomach after oral administration. The hydration of hydroxypropylmethylcellulose (HPMC) samples in water was monitored by two-dimensional mapping of properties such as spin density and relaxation times at pH = 2 and 6, and T = 25 and 37"C.lo71twas shown that transport behavior of water into HPMC changes from being almost completely relaxation controlled (case 11)at pH = 2 to Fickian behavior for pH = 6. Carbohydrate polymers are widely used for pharmaceutical applications such as the controlled release of drugs. The swelling and water mobility in highamylose starch tablets were studied at different time intervals after the immersion of the samples in water by NMR imaging.lo8NMRI shows clearly the anisotropy of the water penetration and the swelling along the radial and axial dimensions of the tablets. Results show that water uptake and tablet swelling strongly depend on the size of the tablets. Layers of hydrogel represent a diffusional barrier that retards the process of drug release. For better prediction of drug release, a method for evaluating the polymer concentration profile was developed by quantification of the MR images of hydrogels based on the NMR relaxation measurement^.''^ 8

Chemical Engineering and Industrial Application

The application of NMR imaging to industrial materials is one of the main topics in recent years. Four reviews and various applications for liquid, soft and rigid phases have been reported. 8.1 Reviews. - A review titled 'Magnetic resonance: ongoing and future role in chemical engineering research' was described?' Topics were the principles of magnetic resonance techniques, and applications in the fields of fluid flow characterization, process applications, gas-phase imaging, fast imaging tech-


485

niques and future perspectives. Another review described the theory, features, performance, and experimental tips of NMR imaging and its application to nondestructive analyses of inhomogeneous industrial materials such as refractories, cokes, and The functional properties of optical glass fibers are largely determined by molecular structure and network density of UV cured primary (rubbery) coating and secondary (glassy) one. Possibilities of solid state NMR for characterization of primary and secondary coatings on optical fibers as a whole were explored."' Two different NMR methods are used in this study: volume averaged 'H NMR T2 relaxation experiments and NMR microscopy or imaging. By the second method, heterogeneity in the crosslink density in the radial direction was determined. It is shown that some fibers reveal large network heterogeneity that is apparently caused by non-uniform light intensity distribution in the radial direction in draw tower(s). The principles in MRI of soft condensed matter and the application of MRI to the drying of layers of polymer colloids, or, less prosaically, to drying paint, are reviewed and discussed.'12MRI has already yielded valuable insight into phase morphology, flocculation, particle coalescence, and drying phenomena. Finding the missing oil can lead to significant economic windfalls because the infrastructure for additional oil recovery is already in place and the cost of production is likely to be minimal. All existing monitoring techniques, including 4D seismic and downhole seismic sensors, were re~iewed."~ This was followed by a comprehensive review of emerging technologies in subsurface monitoring, such as multi-well seismic, electric resistivity tomography, electromagnetic and ultrasonic imaging, acoustic and fiber-optic imaging, as well as laser/IR or MRI/NMR visualization near the wellbore region. Finally, IR/laser or MRI/NMR imaging of a wellbore is still in its nascent state of development, but holds great promises for the future applications of real-time monitoring and eventual dynamic reservoir management.

8.2 Process Analysis. - Strong competition in the process industry forces the operator in this area to search for solutions which improve and maximize the potentiality of the plant. The online magnetic resonance analyser was developed for improvement of plant performance, which can offer to the user tangible economic improvement and solid support to control and manage the process to which it is a~p1ied.l'~ 8.3 Drying Processes. - A stray-field 'H imaging, and solid-state NMR ('HCRAMPS and 27Al- M(3 and 5)QMAS) were performed to study of the drying process of precasting materials used in a steelmaking converter and the change in chemical structure by the drying The drying of a real refractory mortar was done by using stray-field imaging. The free-water loss rate was relatively independent of water content. However the bound-water loss rate is more affected. With this data, the drying process and time in steel plants can be adjusted and optimized. The governing equations of mass transfer and static equilibrium are presented,

486


based on the assumptions related to porous media. The mechanical stresses generated by the drying strains are expressed according to the linear-elastic model. The von Mises cracking criterion is introduced in order to locate the area where risk of cracking occurs. The model was applied to the drying of Kaolin clay.'16 Moisture evolution was measured by NMR imaging, during the drying of a piece of Kaolin clay. The time-dependent liquid water distribution in cement mortar mixtures during water absorption, i.e., determination of isothermal unsaturated capillary flow in high performance cement mortars, was determined using a 'H NMR imaging technique.' l7 The variation of the material water diffusion coefficient with the water content was established. In addition, the effect of drying treatments on the water transport properties was also investigated. Test results finally show that a reduction of the mortar water/binder ratio and the use of silica fume tend to significantly decrease the absorption of water.

8.4 Coating Process. - A new method of applying hydrophobic coatings to cement-based materials based on exposure to the treatment vapor is proposed. The method is demonstrated for the application of monomeric alkyl (isobutyl) alkoxy silane to cement paste and mortar samples manufactured and cured under a variety of conditions. The efficacy of the treatment is compared to conventional flood coating by using MRI (specifically SPRITE) to monitor the time dependent uptake of water into treated samples."* The mobile NMR (NMR mouse) surface imaging sensor was used to investigate the curing process in a polyurethane adhesive join in a glass-metal compound (such as an automobile windscreen) by measuring relaxation times."' The method proved useful for temperature-cure and 2-component PU adhesives and allows online quality control in automobile construction 8.5 Softening and Melting Process - Coal and Coke. - Both the mechanism of softening and melting process for coals and the coal mapping for the softening and melting properties using in-situ imaging experiments were proposed between 25 and 600°C with newly developed high-temperature microimaging probe and systems.'20In this paper, 3D-SPRITE was demonstrated at first, and then the three-dimensional distribution of mobile components and T1 and T2* mappings based on SPRITE methods were presented in order to clarify the chemical heterogeneity of coals. The location of coke deposits in industrial HZ,SM-5 pellets contained in a fixed-bed reactor was revealed in two different ways by 'H NMR imaging (MRI).121In the first way, coke depositions in the reactor were detected directly by using the SPRITE technique, a special MRI sequence for detecting materials with short T2 relaxation times. In the second, indirect way, carbonaceous residues were visualized by applying spin-echo sequences to a coked catalyst sample loaded with propane. It was possible to distinguish between fresh and coked zeolitic areas within the bed.


9

487

Plants, Trees and Woods

9.1 Reviews. - NMR can be used to measure metabolite levels and metabolic fluxes, to probe the intracellular environment, and to follow transport and energetics nondestructively. A review was aimed to highlight recent studies in which NMR and NMR imaging has contributed to metabolic engineering of plants and to illustrate the unique characteristics of NMR measurements that give it the potential to make greater contributions in the future.'22

wood fiber124and surface layer'25 9.2 Trees. - Moisture content in were investigated by different NMR imaging techniques. MRI experiments were performed on sapwood (SW) and hardwood (HW) samples of Scots pine wood (Pinus sylvestris L.) having different moisture contents: SW 124% and HW Simulating characteristics of wood tissue were done just after cutting of the tree. SPRITE (Single Point Ramped Imaging with T1 Enhancement) permits visualization of water content in previously inaccessible wood fiber systems. The superiority of SPRITE met hods, in comparison to conventional MRI methods, for studying fluid content in low water content wood materials (4 species) was demon~trated.'~~ Both methods were also used to examine relative moisture content during forced drying of a white ash wood sample. The moisture content profile in the surface layer of Scotch pine (Pinus sylvestris) sapwood was measured using the MRI technique.125A novel high-gradient permanent magnet was used for the measurement of the moisture profile in the wood during the drying process, giving a depth resolution of >20 pm. The possibility of measuring the moisture content depth profile in the surface layer from raw state down to 4% moisture content without removing the wood sample from the apparatus was demonstrated for the first time.

10

Food

10.1 Review. - The use of NMR and MRI to study moisture migration, proton mobility, and glass transition in bread and other baked products was discussed in a review on the basic NMR and MRI methodologies.'26 10.2 Moisture Migration. -- Moisture migration and proton mobility of baked products,'26 d a ~ g h , ' ~beef,129 ~ - ' ~ ~orange juice,'29 strawberry slices,'3o and plant material such as seeds: has been investigated by NMR and NMR imaging. The fermentation process for frozen dough using freeze-sensitive yeasts and freezetolerant yeasts was traced by NMR imaging.'27.12* Grain network structures of baked breads were also visualized by MRI. The first fermentation before freezing and the second fermentation with punching or resheeting after thawing are useful for obtaining good quality breads from frozen dough using freeze-tolerant yeast. The spatial distribution of the non-frozen water in foods during freezing and frozen storage was investigated for samples such as beef, orange juice, and The content of liquid-like components, such as unfrozen water, was

488


measured in the temperature range between 20 and -40°C. One- and twodimensional images of the proton distributions in the frozen samples were obtained by SPRITE. To develop improved models for predicting water loss during osmotic dehydration and/or air-drying, one-dimensional MRI protocols were used to follow temporal and spatial changes in water mobility via T2profiles, water content via Mo profiles, and structural shrinkage of strawberry slices during osmotic dehydrati~n.'~' Modeling of the air-drying of osmotically pretreated slices would be complicated by the variable amounts of sucrose solution remaining after osmotic dehydration. Drying and rehydration of plant material such as seed is an important industrial operation and requires knowledge of the moisture diffusion throughout the material. Partial rehydration by soaking in water, prior to milling, is being applied to green coffee beans especially of the Robusta type to improve the organoleptic properties of the coffee beverage. To evaluate changes in the physico-chemical properties of water molecules in green coffee beans after washing or steaming performed during the industrial processing at Pacorini Silocaf (Trieste), NMR microscopy was applied to probe water distribution and mobility in Vietnam Robusta green coffee beans (Coflea ~ a n e p h o r a ) . ' ~ ~ 10.3 Solid State Fermentations. - NMR imaging was used as an easily automated, reliable technique to investigate axial mixing within rotating drums as a model for solid state fermentation^.'^^ Moist bran can be clearly differentiated from dry bran using MRI allowing a non-segregating tracer for axial mixing. Gradients in moisture and glucose content during cultivation of Aspergillus orysae on membrane-covered wheat-dough slices were calculated from 'H-NMR images.'33 These gradients can have a strong effect on the physiology of the microorganisms but have hitherto received little attention in experimental studies. The quantitative analysis of water and glucose gradients at the particle level during solid-state fermentation is needed to improve our understanding of the response of fungi to this nonconventional fermentation environment.

10.4 Fat Contents, Distribution and Migration. - New methods of meat-quality evaluation are always required. Recent advances in the area of computer and video processing have created new ways to monitor quality in the food industry. Fat in beef meat'34,loins'35and were investigated. An image-processing technique by NMR has been used to determine fat content and distribution in beef meat.134The various structures in meat, such as muscle, fat, and connective tissue, were visualized and the volumetric content of fat was measured. A novel methodology to non-invasively and objectively predict sensory traits and intramuscular fat content of dry-cured Iberian loins was Texture analysis of magnetic resonance images was performed on 47 dry-cured Iberian loins. The results are encouraging for the application of this NMR imaging, allowing in situ, automatic, objective and non-destructive classification of loins. The effect of fat migration on the quality of filled chocolates was briefly reviewed and mechanism and kinetics of fat migration were The


489

determination of triglycerides, of distribution of fatty acids, of the fat content, differential scanning calorimetry, pulsed NMR spectroscopy, magnetic resonance imaging, and analysis of texture are described as measuring methods. 10.5 Fruits and Vegetables. - Non-invasive localization of aromatic monoterpene thymol bccumulation in Curum cupticum (upiaceae)fruits has been done by chemical shift selective magnetic resonance imaging.137 The imaging showed that thymol accumulation was localized to the secretory structures (canals) situated in the fruit wall. Differentiation of texture parameters of NMR images and dynamics of T2 relaxation times during storage of apples has been in~estigated.'~~ Two apple varieties, titratable acids and firmness of fruits, were analysed during storage (March- June) by texture analysis (TA)of images obtained by NMR techniques. 10.6 Tubers. - The human ability to detect, interpret, assess and report visual stimuli is employed for qualitative and quantitative image analysis of 60 tubers of intact potato (2 varieties x 2 storage x 15 replicate tubers). Descriptive sensory image analysis and conventional computer-assisted gray tone intensity histogram descriptors for cross-sectional T1 weighted MR images were c ~ m p a r e d . ' ~ ~ Both analyses were able to detect differences between varieties as well as storage times. The sensory image analysis gave better discrimination between varieties than the computer-assisted image analysis presently employed, and was easier to interpret. 11

In vivo Application

11.1 Reviews. - Molecular imaging and gene therapy was reviewed from the view of the use of nuclear medicine, magnetic resonance, and optic imaging to visualize gene expre~sion.]~~ The review presents current in vitro assays for protein and gene expression and the translation of these methods into the radiological sciences. The merging fields of molecular biology, molecular medicine, and imaging modalities may provide the means to screen active drugs in vivo, image molecular processes, and diagnose disease at a pre-symptomatic stage. In vivo detection of nitric oxide by combining EPR and NMR was reviewed, in which the ability of localized ESR to monitor nitric oxide (NO) production in vivo is The feasibility of using MRI strategies combined with spin trapping to map NO distributions in tissues and organs at higher spatial resolution is demonstrated. 11.2 Gene Expression. - Depending on the reporter molecule, gene expression can be detected by the addition of an enzyme substrate, visualized with an UV light, or analysed with sophisticated instrumentation for flow cytometry, magnetic resonance imaging, and positron emission tomography. The materials and procedures used to detect the expression of three reporter molecules, including

490


P-galactosidase, chloroamphenicol 0-acetyl transkrase (CAT),green fluorescent protein, were d e ~ c r i b e d . ' ~ ~ Current methods of studying angiogenesis are limited in their ability to serially evaluate in vivo function throughout a target tissue. Microarray gene expression analysis of murine tumor heterogeneity was defined by dynamic contrastenhanced MRI (DCE-MRI) and pharmacokinetic modeling provide a useful method for evaluating tissue vasculature based on contrast accumulation and washout.'44 11.3 Migration, Flow and Drug Release. - The applicability of MRI for noninvasive in vitro studies of parenteral vehicles without use of marker substances was i n ~ e s t i g a t e d .A' ~wide ~ range of extended release (ER) formulations such as oils, a lipid emulsion, and water solutions of a cyclodextrin and block copolymers were visualized in vitro and in vivo by a 'H-MRI technique.

11.4 Functional MRI. - APP23 transgenic mice overexpressing amyloid precursor protein (APP75 1) reproduce neuropathological changes associated with Alzheimer's disease such as high levels of amyloid plaques, cerebral amyloid angiopathy, and associated vascular pathologies. Functional magnetic resonance imaging (fMRI) was applied to characterize brain functionality in these mice through global pharmacological stimulation. '46 Focal brain lesions can be associated with proton magnetic resonance spectra ('H-MRS)-detectable mobile lipids, reflecting severe tissue degradation and necrosis. However, advanced fitting procedures, such as the LCModel, fail to adequately fit spectra in the presence of lipid resonances. A simple way to efficiently include lipid resonances in the LCModel, allowing a better fit of in vivo short-TE 'H-MRS, was proposed and the authors demonstrated the diagnostic potential of quantitative assessment of mobile lipids in brain tumors.'47 MRI measurement of blood-brain barrier (BBB) permeability following spontaneous reperfusion in the starch microsphere model of ischemia in the rat was r e p ~ r t e d . 'Recently, ~~ improvements in MRI tracer kinetic models and in MR hardware design mean that an estimation of Permeability in vivo can now be obtained with acceptable accuracy and precision. Authors reported the use of such methods to study acute changes following spontaneous reperfusion in an animal model of ischemia. The mechanism of action of the contrast media on the LDH (lactate dehydrogenase) activity was studied in rat blood and it was found that changes in the enzyme activity are correlated with the toxicity (LD 50) of diagnostic Roentgen contrast media (RCM).'49The groups of risk from the standpoint of side reactions to the intravenous RCM introduction are determined. An in vivo MRI study employing a high field (7T) magnet and a TI- and T2-weighted imaging sequence with subsequent histopathological evaluations was undertaken to develop and evaluate MRI-based volumetric measurements in the kainic acid (KA) treated rats.'" Volumetric: analysis of the hippocampi demonstrated a reliable and valid method for the measurement of the rat hippocampus in vivo using MRI with a high field magnet, thereby providing a

13: Nuclear Magnetic Resonarice Imaging

49 1

useful tool for future studies of rodent models of neurodegenerative diseases.

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83. Baumann, T., Petsch, R., Fesl, G., Niessner, R., J. Environ. Qual. 2002,31,470-476. 84. Herrmann, K.-H., Pohlmeier, A., Gembris, D., Vereecken, H., J. Hydrology (Amsterdam) 2002,267,244-257. 85. Gotz, J., Zimehl, R., Coll. Polym. Sci. 2002,280, 389-397. 86. Dijk, P.E., Berkowitz, B., Yechieli, Y. Water Resources Res. 2002,38,5/1-5/12. 87. Gotz, J., Kreibich, W., Peciar, M., Rheol. Acta 2002,41, 134-143. 88. Van Buren, V., Reimert, R., Chem. Ingen. Tech. 2003,75,236-240. 89. Heine, C., Stapf, S., Bluemich, B., Appl. Magn. Reson. 2002,22,223-233. 90. Baldwin, B.A., Spinler, E.A. J. Petrol. Sci. Engin. 2002,35,23-32. 91. Sherwood, J.L., Sung, J.C., Ford, R.M., Fernandez, E.J., Maneval, J.E., Smith, J.A., Envir. Sci. Tech. 2003,37,781-785. 92. White, J.L., Polym. Mater. Sci. Eng. 2002,87, 189-190. 93. Ando, I., Kuroki, S., Kobayashi, M., Zhao, C., Matsukawa, S., Polym. Gel. Networks 2002,235-307. Eds. Osada, Y., Khokhlov, A.R., Marcel Dekker, N.Y. 94. Kameda, T., Asakura, T., Ann. Rep. NMR Spectrosc. 2002,46,101-149. 95. Adriaensens, P., Pollaris, A., Kelchtermans, M., Gelan, J., Macromol. 2003, 36, 706-711. 96. Garbarczyk, M., Kuhn, W., Klinowski, J., Jurga, S., Polymer 2002,43,3169-3172. 97. Matsushita, S., Takasu, A., Inai, Y., Hirabayashi, T., Era, S., Sogami, M., Sasaki, F., Ohsaki, H., Kinosada Y., Magn. Reson. Imaging, 2002,20,285-293. 98. MacMillan, B., Halse, M., Schneider, M., Fardy, L., Chui, Y.H., Balcom, B.J., Appl. Magn. Reson. 2002,22,247-256. 99. De Souza, R.E., Engelsberg, M., Barros, W., Jr. Carvalho, L.B.Mo1. Cryst. Liq. Cryst. Sci. Tech., A, 2002,374,249-254. 100. Kanekiyo, M., Kobayashi, M., Ando, I., Kurosu, H., Amiya, S., J.App1. Polym. Sci. 2002,86,504-508. 101. Laity, P.R., Glover, P.M., Hay, J.N., Polymer 2002,43, 5827-5837. 102. Lloyd, C. H., Scrimgeour, S. N., Lane, D. M., Hunter, G., McDonald, P.J., Dental Mater. 2001,17,381-387. 103. de Pasquale, F., Luciani, A. M., Pacilio, M., Guidoni, L., Viti, V., d’Errico, F., Barone, P., Sebastiani, G., Radi. Protec. Dosimetry 2002,99( 1-4, Microdosimetry), 363-364. 104. Fong, P.M., Keil, D.C., Does, M,D., Gore, J.C. Phys. Med. Biol. 2001,46, 31053113. 105. Baras, P., Seimenis, I., Kipouros, P., Papagiannis, P., Angelopoulos, A., Sakelliou, L., Pappas, E., Baltas, D., Karaiskos, P., Sandilos, P., Vlachos, L., Med. Phys. 2002, 29,2506-25 16. 106. Tritt-Goc, J., Kowalczuk, J., Eur. J. Pharm. Sci. 2002, 15, 341-346. 107. Tritt-Goc, J., Pislewski, N., J. Control. Release 2002, 80, 79-86. 108. Malveau, C., Baille, W.E., Zhu, X.-X., Marchessault, R.H., Biomacromol., 2002, 3, 1249-1254. 109. Sepe, A., Baumgartner, S., Kristl, J., Lahajnar, G., Jarh, O., Cell. Mol. Biol. Lett. 2002,7, 154-156. 110. Saito, K., Materia 2002,41, 878-879. 111. Litvinov, V.M., Dias, A.A., Proc. Int. Wire Cable Slymp. 2001,50,267-273. 112. McDonald, P.J., Keddie, J.L., Europhys. News 2002,33,48-51. 113. Islam, M.R. Tech. 2000: Your Competitive Advantage, Annual Technical Meeting of the Petroleum Society, 2000,51, 56-60. Pub.: Petrol. SOC.CIM, Alberta. 114. Bozzini, C., Masiello, A., Chim. Indust. (Milan, Italy) 2002,84,61-64. 115. Saito, K., Kanehashi, K., Saito, Y., Godward, J., Appl. Magn. Reson. 2002, 22,


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

1

Introduction

The report on the oriented molecules is published in alternate volumes of this series and the present one covers the available literature during the biennial period ending May 2003. The complete literature on the subject can be traced back by referring to earlier volumes of this series. During the current period, the developments in various areas have followed the same trend that was pointed out in the previous report.' Thus use of weakly ordered media for orienting biological macromolecules continues the rapid growth witnessed in recent years with the literature coverin,ga range of topics from developments of new orienting media and novel experimental techniques and pulse schemes to applications to proteins, peptides, nucleic acids and carbohydrates. New and improved procedures for structure calculation using residual dipolar couplings (RDC) and other anisotropic parameters have been dealt with. Several reviews on such applications have also berm published. It is interesting to compare the development of the use of NMR spectroscopy of oriented molecules for structural studies starting from small and symmetrical molecules to biological macromolecules during the past four decades. For a while, applications of NMR spectroscopy of oriented molecules for structural studies had significantly declined but with the possibility of achieving weak ordering this has become one of the hottest areas of research. Consequently, a large section of this report is devoted to this area. Developments in other areas have continued at a moderate pace as in previous years. In some areas such as the study of small molecules oriented in thermotropic liquid crystals, attempts to simplify the spectra using various experimental techniques have been continuing. The use of organic chiral liquid crystals for enantiomeric excess analysis has been extended further. The network of the strongly coupled spin systern, provided by the liquid crystalline environment, is being exploited for quan tum information processing. Similar developments in other areas to accelerate the progress of research have continued and hold promise of increased future activities. The classification of the topics is essentially the same as that followed in earlier volumes and the available literature is presented under the following headings: Reviews, Theory and General Studies; New Techniques; Dynamic NMR Studies; Chiral, Smectic, Lyotropic, and Polymeric Systems; Relaxation Studies; Orientational Order in liquid crystals; MemNuclear Magnetic Resonance, Volume 33 0The Royal Society of Chemistry, 2004 491

498


branes and Molecules Oriented therein; Structure and Shielding Tensor Studies of Small Molecules; Quantum Computing; Weak Ordering and Biomolecular Studies.

2

Reviews, Theory and General Studies

Volume 9 of the Encyclopedia of NMR has been published. It contains several reviews on some of the recent developments pertaining to the NMR of oriented systems.*A book on ‘NMR of ordered liquids’ withi 19 chapters and 455 pages has been published by Kluwer Academic publish ere^.^ The chapters in this book are classified under four parts. Part I covers basics with 5 chapters: (1) Basics of NMR of molecules in uniaxial anisotropic environments; (2) Density matrix methods in NMR; (3) Coherent averaging and correlation of anisotropic spin interactions in oriented molecules; (4) Multiple quantum NMR spectroscopy in orientationally ordered fluids and ( 5 )Spectral analysis of orientationally ordered molecules. Part I1 covers NMR of solute atoms and molecules and it consists of 4 chapters (1) NMR of noble gases dissolved in liquid crystals; (2) NMR of partially ordered solutes with emphasis on structure determination; (3) Observation and interpretation of residual dipolar couplings in biomolecules and (4) The search for high resolution NMR methods for membrane peptide structure. Part I11 covers theory, models and simulations consisting,of 6 chapters: (1) Solutes as probes of simplified models of orientational order; (2) Molecular models of orientational order; (3) Molecular theory of orientational order; (4) Very Flexible solutes: alkyl chains and derivatives; ( 5 )NMR studies of solutes in liquid crystals: small flexible molecules and (6) Simulations of orientational order of solutes in liquid crystals. Part IV covers Dynamic aspects and relaxation with 4 chapters: ( 1) Spin relaxation in orientationally ordered molecules; (2) Low-frequency NMR relaxometry of spatially constrained liquid crystals; (3) NMR on macroscopically oriented lyotropic systems and (4) Dynamic NMR in liquid crystals and liquid crystalline solutions. A review with 77 references on the NMR study of rigid molecules in liquid-crystalline phases has also been published! It describes how the orientational order parameter and the conformational distribution of molecules in liquid crystalline phases can be determined from NMR data. A review of relaxation and the dynamics of molecules in the liquid crystalline phases is a ~ a i l a b l eCarbon-13 .~ NMR studies of liquid crystals have been reviewed.6Studies of solute molecules and bulk liquid crystals using one, two and three dimensional NMR techniques and use of liquid crystals in NMR quantum computing have been dealt with. Use of separated local field NMR technique for the study of order in liquid crystals in which dipolar oscillations observed during cross polarisation constitutes the evolution in the indirect dimension has been pre~ented.~ Pulse schemes including the one in which local proton dipolar fields can be estimated have been discussed. Several reviews on the use of RDCs for biomolecular structure determination have been published. These have been covered under the section on weak ordering and biomolecular structural studies. A new formulation of the short-range anisotropic orientating potential experi-

14: Oriented Molecules

499

enced by a biaxial apolar particle dissolved in a uniaxial mesophase has been given, where the interactions between the solute and the single molecules constituting the solvent are explicitly taken into account. The model has been tested by calculating the order parameters of naphthalene* and 1,4-difluorobenzene, 1,4-dichlorobenzene, and 1,4-dibromobenzene molecules9 by using the Monte Carlo-Metropolis method and the results compared with the experimental 'H NMR data. It is observed that in the 'magic' nematic mixture of 55 wt% ZLI 1132 (a Merck eutectic mixture of alkylcyclohexylcyanobenzenes and alkylcyclohexylcyanobiphenyl) and EBBA [N-(4-ethoxybenzylidene)-4-n-butylaniline] in which solute molecules experience zero average electric field gradient, lo the orientations predicted by the model match the experimental Saupe matrices. Implications of this result are discussed in terms of the absence of long-range orientational effects in the magic mixture and the nature of the interactions in the different nematic phases. This model has further been applied to apolar molecules namely biphenylene, l ,4-dicyanobenzene, 1,4-dinitrobenzene, and p-benzoquinone and on so-called magic solutes cyclohexane, 1,4-trans-dimethylcyclohexane, and trans-decalin (whose electronic structure is expected to preclude significant anisotropic long-range interactions) providing further evidence of the validity of the approach". The possibility of the occurrence of a biaxial nematic phase in thermotropic materials has been discussed.'2. The reasons why this phase proves to be elusike are explored using molecular field theory and the design of thermotropic riesogens, which are likely to form this phase, is discussed. Structural and optical anisotropy of biaxial nematics has been considered.13It has been suggested that biaxial thermotropic nematics have anomalously small parameters of biaxial orientational order, which are one or two orders of magnitude lower than the limit of their detection by NMR leading to the dramatic discrepancy between the optical and NMR data on biaxiality of these compounds. The superposition of scalar and residual dipolar couplings giving rise to the cylindrical mixing Hamiltonians has been considered and analytical polarization and coherence transfer functions obtained for three cylindrically coupled spins under energy-matched ~0nditions.l~ Also, the transfer efficiency has been analysed as a function of the relative coupling constants for characteristic coupling topologies. The orientational order parameters of four aromatic solutes in nematic solvents, derived from proton dipolar couplings, which show a correlation with the dielectric anisotropy of the solvents has been considered and the experimental behavior explained by a molecular theory based on the reaction field appro~imation.'~ A procedure to test the relation connecting the order of probe molecules dissolved in binary nematic mixtures with that exhibited in each nematic component at the same reduced temperatures has been proposed.'6 'H studies at different temperatures were carried out using as solutes 1,4-difluorobenzene, 1,4-dichlorobenzene, 1,4-dibromobenzene and naphthalene in 152, EBBA and in mixtures of I52/EBBA. The results suggest that the orientations of the solutes in the mixtures can be predicted in a satisfactory way as a function of the mixing rat:ios, independently of the solutes and temperatures. A method is presented for the reconstruction of interaction vector orientations of tensor interactions of rank 2, such as dipolar coupling and chemical shielding


500

anisotropy interactions, in solids and partially aligned system~.'~ The method is based on a principal component analysis of the covariance matrix of the NMR parameters collected for multiple alignments. The method is demonstrated for an isotropic distribution of sample orientations as well as for finite sets of orientations and internuclear vectors encountered in protein systems. A model for the ab-initio calculation of the shielding tensors for molecules in liquid crystal solvents has been proposed." The electrostatic interactions between the solute and the anisotropic solvent are described within the integral equation formalism framework and the shielding tensors have been calculated for the nuclei of CH3CN and C6H5N02in different solvents. The use of heteronuclear multiple quantum (MQ) spectra as an aid in the automatic analysis of NMR spectra of both rigid and flexible molecules dissolved in liquid crystalline phases is presented.19The applicability of a procedure for use with the decoupled heteronuclear M Q spectrum that significantly reduces the computing requirements of the automated spectral analysis is also discussed. The interaction between nuclear spins with I = 3/2 and an anisotropic organized environment has been considered.20A model based on a solution to the Poisson-Boltzmann equation for the estimate of quadrupolar couplings in filamentous bacteriophage Pfl solutions has been developed and compared with experiment.

3

New Techniques

Methodological developments aimed at simplifying and accelarating the analysis of the spectra of oriented molecules as well as extracting new information have been reported. A three-dimensional multiple-quantum NMR experiment that produces individual spectra of all quantum orders has been described.21The separation of different quantum orders is accomplished via Fourier transformation with respect to the phase of the first two pulses of a generic three-pulse multiple-quantum sequence. This dramatically reduces the time required to obtain several selectively detected spectra and enhances the sensitivity and digital resolution from that obtained using the original two-dimensional technique. The experiment has been demonstrated for protons of para-chlorotoluene dissolved in the nematic liquid crystal ZLI-1132. Multiple-quantum proton NMR spectra of the 8-spin system of indene partially oriented in a nematic mesophase have been acquired using a magnetic field gradient and from the obtained dipolar couplings the molecular shape has been determined.22A novel method to estimate the Saupe order parameters from the observed spectral widths of the high-order multiple-quantum spectra has also been proposed. The application of switched angle spinning (SAS) to correlate the first-order dipolar spectrum of a sample in the anisotropic phase with the isotropic magic angle spinning (MAS) spectrum in a two-dimensional experiment has been reported.23 In this experiment the degree of dipolar couplings introduced is selected via mechanical manipulations of the liquid crystal director in an oriented sample. The I9FSAS-COSY correlation of iodotrifluoroethylene, an AMX spin system, dissolved in the nematic liquid crystal 4-octylphenyl-2-chloro-4-(4-heptylben-


50 1

zoy1oxy)-benzoate provides assignment of both the J and dipolar couplings in a single experiment. The method has been extended to the case where the couplings are scaled to a degree such that the spectrum approaches strong coupling.24 Additionally by using a more complex SAS method which involves magnetization evolution along three angles, the spinning side band patterns have been correlated with the isotropic chemical shifts. A proton detected local field technique has been reported wherein homonuclear decoupling has been achieved with low r.f. power to obtain heteronuclear couplings in fluorinated compounds.25 The polarization transfer dynamics for the deuterium- carbon correlation method has been studied and a technique, which exploits heteronuclear multiplequantum coherences, has been proposed for measuring and assigning 2H quadrupolar couplings in ordered media.26A two-dimensional (2D) isotropic-anisotropic chemical shift correlation experiment to accomplish 13C NMR spectral assignments for nematic liquid crystals has been prop0sed.2~In the obtained 2D spectrum the anisotropic shift parameters are represented as sharp lines by gamma-encoding. The experiment has been demonstrated on p-methoxybenzilidene-p-n-butylaniline (MBBA). The 2D HSQC NMR experiment has been applied to molecules containing rare spins such as 13C in natural abundance dissolved in a liquid crystalline solvent to obtain spectra free from the strong lines of the abundant isotopomer.28 The technique also gives the individual chemical shifts of the rare spins, and, for a molecule containing another abundant nucleus, such as fluorine, the rare spin - 19Fanisotropic couplings are also obtained. It has been suggested that cross-polarisation from the dipolar reservoir under the mismatched Hartmann-Hahn condition can provide an estimate of the local dipolar fields and thus provide a handle on the order parameters of liquid crystalline molecules.2gA new method for selectively detecting sodium ions in anisotropic environments has been r e p ~ r t e d . ~A' spin-lock sequence, followed by a coherence transfer pulse, is used to generate rank-two zero-quantum coherences and convert them into observable transverse magnetization. It is reported that this filter is more efficient than a double-quantum magic-angle filter in generating observable signal from ions in anisotropic media in both a nasal bovine cartilage sample and a liquid crystalline DNA sample. The possibility of achieving selective excitation in coupled multispin systems has been theoretically studied and preliminary experimental results for the case of 1,2-dichloro-4fluoro-5-nitrobenzene dissolved in ZLI-1132 pre~ented.~' 4

Dynamic NMR Studies

The order and dynamics of two aromatic polyamides in their lyotropic phases have been investigated using variable-director studies in which macroscopicallyaligned liquid crystalline solutions were rotated away from equilibrium for brief periods of time, and their natural abundance 13C-NMR spectra recorded as a function of angle between the director and the magnetic field. From the experimental line shapes a detailed description of the macromolecular order and

502


dynamics in these fluids has been obtained.32A study on the NMR spectra of mixed liquid crystals of opposite diamagnetic susceptibility anisotropies near critical point under variable angle sample spinning, reported in a previous volume of Specialist Periodical Reports,’ has been published.33Deuterium NMR studies to probe the dependence of the relaxation time for the director alignment on a specific group in a liquid crystalline molecule have been undertaken using different electric field strengths and angles between the magnetic and electric fields and at different temperature^.^^ For all the experiments on perdeuterated 4-pentyl-4’-cyanobiphenyl,the director relaxation times are found to be independent of the group used to determine the director orientation during the alignment process, in contrast to observations made using vibrational spectroscopy, but in accordance with the known concepts of hydrodynamic theory of nematics. The static director distribution in thin nematic liquid crystal cells with different film thicknesses and different surface anchoring strengths have been studied using a combination of 2H-NMR spectroscopy and continuum theory.35 2H NMR spectroscopy has been employed to investigate the director dynamics in the nematic liquid crystal, 4-pentyl-4’-cyanobipheny17confined between two glass plates and subject to orthogonal magnetic and pulsed electric fields.36When the pulsed electric field, whose intensity is strong enough to make the director align normal to the magnetic field, is switched off, the director relaxes back to being parallel to the magnetic field. Deuterium NMR spectra have been used to study the degenerate pathways of the director in the realignment process which could also be predicted by an analysis based on continuum theory. Experiments have also been performed so that the electric field is applied at 45O to the magnetic field, thus avoiding the degeneracy of the director realignment pathThe process of reorientation of the director of nematic liquid crystals by the application of an electric field has been s t ~ d i e d . ~Using ’ , ~ ~ the electric field to set the desired initial angle of the nematic director with respect to the magnetic field, the reorientation processes in the presence of both electric and magnetic fields has been studied for various initial angles. The reorientation time measured as a function of the electric field has been be used to calculate the rotational viscosity at different temperatures.The measurement of residual dipolar and quadrupolar coupling constants in the liquid phase by using an electric field to destroy the isotropic nature of molecular tumbling has been discussedPO Molecular self-diffusion coefficients for nematic and smectic-A thermotropic liquid c r y s t a l ~and ~ ~ ,a~columnar ~ phase of a discotic liquid crystal43have been obtained using stimulated-echo-type 2H and ‘H pulse-field-gradient spin-echo (PGSE) nuclear magnetic resonance. The orientation-dependent molecular diffusion in the nematic liquid crystal 4-pentyl-4-cyanobiphenyl has been measured and the results described in terms of the affine transformation model of diffusion in hard-ellipsoid nernatics.# The anisotropy of the solvent self-diffusion coefficients within suspensions of nanoparticles has been measured by ‘H NMR with pulsed field gradient and used as a new procedure to detect nematic 0rdering.4~The potentiality of this method has been illustrated using aqueous clay dispersions exhibiting such ordering. Anisotropic self-diffusion measurements in an antiferroelectric smectic A liquid crystal, studied using PGSE


503

method combined with quadrupole coil rotation, have been *H NMR spectra have been monitored as a function of the shear rate using a cone-and-plate rheometer in a nematic dimer. The orientation of the director relative to the magnetic field has been obtained and the analysis of the experiment has lead to information on the viscoelastic properties of the liquid crystal system.'" Rheology and deuterium NMR measurements performed on the nematic lyotropic system sodium dodecyl sulfate, decanol, and water has been reported and the studies indicate that the calamitic and discotic phases of the system are textured nematics of the flow-aligning type!'

5

Chiral, Smectic, Lyotropic and Polymeric Systems

The use of polymeric chiral liquid crystalline solvents based on homopolypeptides to discriminate between enantiomeric pairs of chiral hydrocarbons using proton-decoupled deuterium one- and two-dimensional NMR spectroscopy at natural abundance has been discussed.49Accuracy of the estimates of the enantiomeric excess, the range of applications and the advantages of the technique have also been presented. The potential of the method for routine analysis of the enantiomeric composition of organic chiral building blocks involved in the syntheses of natural and synthetic bioactive products has also been described." The use of chiral liquid crystalline solvents in the investigation of interconverting chiral conformers has been illustrated by a deuterium one- and two-dimensional NMR study of cis-decalin in various chiral and achiral polypeptide oriented solvent system^.^' Organic solutions of poly-y-benzyl-L-glutamate (PBLG) and poly-E-carbobenzyloxy-L-lysine(PCBLL) in dichloromethane or chloroform allowed the chiral invertomers of cis-decalin to be differentiated at low temperatures while at high temperatures it was possible to distinguish between deuterium nuclei of the molecule which become enantiotopic under rapid kinetic averaging. Enantiomeric and enantiotopic analysis of the NMR spectra of compounds derived from the functionalized cone-shaped core, cyclotriveratrylenes dissolved in organic solutions of PBLG have been de~cribed.'~ Using 2H and 13CNMR studies, it has been shown that enantiomeric discrimination of chiral molecules with a 3-fold symmetry axis is possible in this system. However, measurements on compounds with flexible side groups having on the average C3"-symmetry revealed no spectral discrimination of the enantiotopic sites which could be understood in terms of available theoretical models. Experimental evidence of the differentiation of 'H-lH, 13C-lH,and 13C-2H enantiotopic directions in prochiral molecules with C,- and C2"-symmetry dissolved in a chiral liquid-crystalline phase using 13C and ZH -{lH} NMR spectroscopy at the natural abundance has been p r e ~ e n t e d Studies .~~ on endo-bicyclo[2.2.2]oct-5ene-2,3-dicarboxylic anhydride, bicyclo[2.2.l]hepta-2,5-diene,and ethyl alcohol oriented in organic solutions of PBLG or PCBLL have been presented and discussed. Using malononitrile as a model, the first NMR differentiation of enantiotopic directions in a molecule with CZvsymmetry and with no prostereogenic carbon has also been presented. The possibility of monitoring the

504


differential ordering of enantiomers included into cyclodextrins through deuterium NMR studies in lyotropic liquid crystals has been discus~ed.~~Enantiomeric discrimination of fluoroalkanes using 'H- NMR has been d e ~ c r i b e dA. ~ ~ two-dimensional NMR strategy that allows determination of the diastereoisomeric and the enantiomeric composition of a mixtures6and another 2D experiment for simplifying the analysis of proton spectra in chiral media5' have been reported. Orientational order, molecular structure and phase transitions of the chiral smectic liquid crystal (S)-[4-(2-methylbutyl)phenyl]-4'-octylbiphenyl carboxylate (8BEF5), partially deuterated in both the chiral chain and the phenyl ~ ~ additive moiety have been investigated by using 2H NMR s p e c t r o s c ~ p y .The potential method has been applied in the Smectic A phase and the order parameter of the average conformer of the whole molecule has been obtained. 31P and 13CNMR studies are reported foi. a smectic A liquid crystal of hexakisC4dodecyl(4-biphenoxy)]cyclotriphosphazene.S9 Orientational order parameters for the cyclotriphosphazene fragment and the biphenylene fragment have been determined from the "P and 13C NMR spectra respectively and have been observed to be the same. The orientational order of two liquid crystals, namely, N-[6-[4-(4-nitrophenylazo) phenyloxy]hexyl]diethanolamine (C6) and l-bro-

20

0 Hz

20

Figure 1

Proton selective refocusing 2D spectrum selective on the methyl of 3-methyl4,4,4-trichlorobutyric-~-lactone 21.5 % enriched in the S enantiomer and dissolved in PBLG/CHC13 at 295 K . (Reproduced with permissionfrom reJ57)


505

mo-l0-[4-(nitrophenylazo) phenyloxyldecane (B10) has been studied by 2H NMR spectroscopy with hexamethylbenzene-d18as the probe molecule.6' The director in the smectic A phase of C6 and the nematic phase of B10 aligned parallel to the magnetic field. A relatively higher orientational order parameter of the solute in C6 has been attributed to the formation of intermolecular hydrogen bonds. Lamellar systems containing salts of n-hexadecylpyridinium and sodium, 1hexanol, and D20 have been investigatede61'H, "F and 23Naspectra of the systems reveal quadrupolar splittings for 2H and 23Naand dipolar splitting for 19Fand the results have been correlated with the phase behavior of the lyotropic systems as a function of temperature. The lyotropic liquid crystalline system, cetyltrimethylammonium bromide(CTAB)/ DzOhas been studied by 'H, I4Nand I7O NMR.62 The residual quadrupolar splittings of these nuclei have been obtained and the results discussed in terms of various dynamics processes and order parameter of the system. A method for the alignment of lyotropic La phases in a magnetic field has been suggested and the alignment process studied by 2H NMR rneas~rements.~~ The phase diagram of the nematic mesophases present in the tetradecyltrimethylammonuim bromide/n-decanol/water system has been determinedP4 Two distinct biaxial nematic mesophases with opposite diamagnetic and optical anisotropies have been identified using 'H NMR spectroscopy and optical microscopy. NMR studies of conformation of two homologous series of bi-swallow tailed compounds exhibiting transitions between lamellar, columnar and three-dimensional phases have been reported.65 The utility of proton coupled 13CNMR spectra has been shown for the determination of both sign and magnitude of the orientational order parameter induced in a uniaxially compress poly(dimethylsiloxane) network.66The flow behavior of nematic liquid crystalline polysiloxanes of the side-chain type has been studied by in situ 2H NMR spectroscopy on samples under shear in a cone-and-plate cell and the director orientation as a function of applied shear rate determined from the quadrupole splitting of the spectra.67A deuterium NMR study of molecular ordering in a holographic-polymer-dispersed liquid crystal has been made.@A series of N-acetyl-L-glutamic acid oligopeptide benzyl esters have been synthesized and the behavior of the liquid crystalline phases examined by the use of 'H NMR.69 6

Relaxation Studies

The Redfield high-temperature theory of spin-lattice relaxation has been applied to the calculation of the dipolar order relaxation time TIDof para-azoxyanisoled6 in the context of discrepancies noticed between experimental data and standard theory based on two spin model.70It is observed that this theory gives the same expressions as the standard theory for the simple case of an ensemble of isolated spin pairs and even when larger number of spins are considered it fails to explain the discrepancies. The measurement of cross-correlations between the relaxation processes originating from the chemical shift anisotropy of 13Cand its

506


dipolar coupling with the attached proton for various carbons in a liquid crystal have been reported.71The motional parameters have been extracted from the measured self-relaxation, cross-relaxation and the cross-correlation rates. The potential of the method is demonstrated by the determination of the rotational (MBBA) at 297 diffusion coefficients of N-4-methoxybenzylidene-4-butylaniline K undergoing magic angle sample spinning. 14N quadrupolar splitting and spin-lattice relaxation times Tlz and TIQhave been measured in the hexagonal phase of a binary mixture of dodecyltrimethylammonium chloride and Based on a model of classical aggregates, it has been shown that the surface self-diffusion around the cylindrical axis of the aggregate together with fast local motion about the surface normal explains the experimental results. Deuterium spin-lattice relaxation and the quadrupolar splittings have been measured for a deuterated decyloxy chain in the chiral liquid crystal, 1-methylheptyl 4'-(4-ndecyloxy-benzoyloxy)biphenyl-4-carboxyate in the uniaxial smectic-A phase.73 From the measurements, the orienting potential of mean torque, chain dynamics and the overall rotational diffusion constants have been obtained. 2H NMR studies have also been carried out on a chiral polymorphic liquid crystal smectogen74and a discotic ~ m e c t o g e nand ~ ~the relaxation behaviour has been analysed using the availble motional models. Proton dipolar order relaxation time, TID, has been measured for partially deuterated para-azoxyanisole (PAA-d6) as a function of both temperature and concentration in perdeuterated PAA.76From the studies it is concluded that the intermolecular contribution to the relaxation is negligble in this compound. The existence of intra-pair and inter-pair proton magnetic quasi-invariants in chain deuterated and normal 4-n-pentyl-4'cyonobiphenyl (5CB) has been experimentally verified and their relaxation behaviour ~tudied.~' Experimental evidence for director fluctuations in a micellar lyotropic liquid crystal, studied by 'H spin-lattice relaxation rate, has been reported.78 The experiments have been performed on potassium laurate/l-decanol/water system over a broad range of Larmor frequencies using fast field-cycling NMR relaxometry. The NMR spin-lattice proton relaxation dispersion of the liquid crystal butylcyano-phenylcyclohexaneis studied over several decades of Larmor frequencies at different temperatures in the nematic phase.79The results show that the order fluctuation of the local nematic director contribution to spinlattice relaxation undergoes a transition between two power regimes on going from low to high Larmor frequencies. An enhancement of order director fluctuations have been observed on sonication studies on 5CB and the results analysed in terms of a coupling between acoustic waves and nematic order director fluctuations." The Zeeman and quadrupolar spin-lattice relaxation times of the aromatic deuterons of a smectic liquid crystal have been measured throughout the smectic A phase at two different frequencies.'l The obtained spectral densities have been analysed by a global target fitting procedure using diffusional models for the overall molecular reorientations and the internal motions. By combining *HNMR order parameter measurements with relaxation studies, a comprehensive picture of lipids in the liquid-crystalline state has been obtained.*2'H and 2H NMR studies in the mesomorphic phases of the micellar lyotropic mixture


507

potassium laurate/l-decanol/heavy water is reported.83The TI dispersion studies indicate that in the nematic phases the slow reorientational dynamics of water is closely related to the slow reorientation of the micellar aggregates, while the quadrupolar splittings indicate that in the nematic phases the water reorientations do not average out the deuterium quadrupolar interaction. A proton NMR study of molecular dynamics in the liquid crystal 4-octylphenyl 2-chloro-4-(4cyanobenzoy1oxy)benzoate has been carried out in all its mesophases over a broad frequency range by using conventional as well as field cycling technique.84

7

Orientational Order in Liquid Crystals

The orientational ordering in the nematic phase of 4-alkyl-4-cyanobicyclohexanes has been studied using two-dimensional I3C NMR.85Semi-empirical linear relations between the order parameters obtained from the I3C-lH dipolar coupling constants and anisotropic 13C chemical shifts have been established. 'H-13C splittings in the I3C spectra of monodeuterated compounds have been used to measure the long range dipolar coupling constants in three ferroelectric liquid crystals in their racemic forms and the order parameters obtained.86Long range 'H-I3C dipolar coupling constants in 4'-( l-deuteropentyloxy)-4-biphenylcarbonitrile have been measured from the proton-decoupled I3C NMR spect r ~ m . In ' ~ addition 'H -I3C dipolar coupling constants have also been obtained by using I3C twofrom non-deuterated 4-n-pentyloxy-4'-cyanobiphenyl(50CB) dimensional separated local field spectroscopy. Long range I3C -I3C dipolar couplings including couplings to carboxyl carbons have been obtained in the liquid crystal, 4-cyano-4-biphenyl hexanoate-l-I3C labeled with a single I3C nucleus, from the proton-decoupled I3C spectrum of this compound.8' Short terminal polyoxyethylene chains have been introduced into mesogens containing lateral s u b s t i t ~ e n t sSuch . ~ ~ systems are found to exhibit a large nematic range near room temperature. Lithium and boron ions of LiBF4 are oriented by these liquid crystalline media and the ordering of the ions have been monitored by the corresponding quadrupolar splittings. These results as well as the thermal evolution of I3C chemical shift in the oxyethylene units as a function of the salt concentration indicate that the interaction between the ions and the mesogens is weak. Ordering in symmetrical nematogens with a lateral crown ether and shortoxyethylene units in the terminal chains has been studied by I3C NMR spectroscopy?' The I3Cchemical shift anisotropy and the local C-H bond order parameters have been obtained for the nematic phase for the crown ether fragment and the terminal chains. The study indicates that the lateral crown ether protrudes markedly from the core with the consequence that the molecular shape is far from rod-like in geometry. Ordering in nematic liquid crystals from NMR cross-polarisation studies has been considered and several pulse schemes presented with illustrative examples?l Thin molecular depositions of 4-pentyl-4-cyonobiphenyl (5CB) mesogenic molecules have been investigated by deuterium nuclear magnetic resonance spectroscopy?2 The surface orientational order parameter, the surface biaxiality,

508


and the diffusion coefficient have been determined from the spectral patterns obtained at different sample orientations in the external magnetic field. The order and dynamics of the mesogenic groups in a series of side chain liquid crystal copolymers have been investigated in their nematic phase by 'H NMR.93 The main chain is built with a variable content of randomly distributed acrylic and methacrylic units, while the mesogenic groups are based on a phenyl benzoate moiety. The analysis of the orientational order and the spectral densities have been carried out and the processes contributing to relaxation have been identified. Orientational order and thermodynamic properties of main chain trimer liquid crystals of the type 4,4-bis[o-(4-cyanobiphenyl-4'yloxy)alkoxy]biphenyls) have been studied by a combined use of 2H NMR, PVT and high-pressure differential thermal analysis.94The structure, magnetic alignment, and orientational order of polysiloxane liquid crystal copolymers have been studied by x-ray diffraction and 2HNMR s p e c t r o s ~ o p yThe . ~ ~ orientational order of nematic liquid crystal droplets confined in a polymer matrix has been investigated by 13C-NMR.96 Proton decoupled 13Cspectrum of 2,2'-difluor0-4"hexoxy-4-penty1-[1,1':4',1"]-terpheny1in the nematic phase has been analysed to determine the relative conformation of the two fluorinated ri11gs.9~ 8

Membranes and Molecules Oriented Therein

Interaction with membranes of mastoparan, a 14-residue peptide from vasp venom, has been studied by'H -NMR spectroscopy in detergent micelles and by solid-state *H -NMR and "N-NMR spectroscopies in oriented lipid b i l a y e r ~ . ~ ~ Two distinct orientational states have been observed for the mastoparan helix, namely an in-plane and a trans-membrane alignment with a population of 90:lO. Solid-state NMR spectroscopic characterization of a long chain phospholipid bilayer system which spontaneously aligns in a static magnetic field has been reported.99The oriented membrane system is composed of a mixture of phospholipid palmitoylstearoylphosphatidylcholine (PSPC) and phospholipid dihexanoylphosphatidylcholine (DHPC) which form bicelles that are highly hydrated. Using 'H and 31PNMR spectroscopy, an alignment of the bilayer normal perpendicular to the static magnetic field was observed with a PSPC/DHPC molar ratio between 1.6 and 2.0 at 50". Paramagnetic lanthanide ions (Yb3+)were observed to flip the bilayer disks such that the bilayer normal was parallel with the static magnetic field. The topologies of zervamicin I1 and alamethicin oriented in lipid bilayers have been investigated by "N solid-state NMR spectroscopy."' The alignment and ordering of these molecules on factors such as the lengths of the fatty acyl chains and lipid concentrations have been studied. The observed differences in orientation between zervamicin and alamethicin have been discussed with reference to the differences in their lengths, helical conformations, distribution of hydroxyproline residues, and hydrophobic moments. The application of the proton inverse detected deuteron (PRIDE) NMR technique to the measurement of the orientation of membrane-bound peptides has been demonstrated with Gramicidin D, a transmembrane peptide,


509

and ovispirin, a surface-bound peptide, being used as model systems."' Numerical simulations to analyse the prospects for high-resolution solid-state NMR of large isotope-labeled membrane proteins macroscopically oriented in phospholipid bilayers has been considered using the known X-ray structures of rhodopsin and porin as models.'02On the basis of the results obtained several new high-resolution experiments for sequential protein backbone assignment and structure determination have been proposed. The binding of doxorubicin, a potent anthracycline cancer drug to magnetically oriented phospholipid bilayers containing cardiolipin has been studied using 31Pand 2H NMR spectros~opy.'~~ High resolution 13C NMR spectra on a DMPC-cholesterol sample, oriented between glass plates have been obtained.lMSources of line broadening quantified and possibility of performing chemical shift- dipolar coupling correlation 2D experiments demonstrated.

9

Structure and Shielding Tensor Studies of Small Molecules

NMR spectra of partially oriented propargyl iodide and iodoallene yield the complete structures of these molecules. lo5 Temperature and solvent effects on these structures have also been analysed and discussed. Molecular structures of tolan and trans-azobenzene dissolved in the nematic liquid crystal ZLI 1132 have been determined from the 'H NMR spectra and with the aid of theoretical calculations.lo6To aid the spectral analysis of such a molecule with 10 protons, the initial spectral parameters that allow spectral assignment have been obtained by analysing integral curves by a genetic algorithm. Structural analyses have been carried out and it is observed that the determined rotational barriers at the dihedral angle of 90° in the liquid crystal are larger than those in the gas phase, indicating that the liquid crystal solvent stabilizes the planar or nearly planar structures of solutes. The molecular structure of N-methylpyrrole obtained by gas phase electron diffraction has been reinvestigated using the rotational constants of 13Cand 15N-substituted specie^.'^' Inter-proton distances of the compound dissolved in MBBA, EBBA and ZLI 1167 have been earlier obtained."' In the present study,lo7the proton spectrum of the compound in ZLI 1167 including 13Csatellites has been obtained and the determined structure agrees with that in the gas phase within experimental uncertainties. Ordering of apolar and polar solutes in nematic solvents has been ~ t u d i e d . ' ~ ~ ' ~ ' ~ The order parameter profiles of two apolar and polar solutes namely para- and ortho-dichlorobenzene are found to be very different both in the apolar and polar nematic solvents namely hexyl- and pentyloxy-substituted diphenyl diacetylene but show little variation from one solvent to the other. The results have been analysed and interpreted in terms of the underlying molecular interactions and it is concluded that local atomic charge densities rather than the overall electrostatic character of the solute is responsible for solute ordering. The magnetic shielding tensors for sp-hybridized carbons in methylethynylketone, deuteroethynylbenzyne and its p-dimethylamino and p-nitro derivatives have been determined using liquid crystal NMR studies and the results compared

510


with theoretical calculations."' It is observed that the substituent strongly differentiates the shielding tensors of substituted and unsubstituted acetylenic carbons. The nuclear shielding tensor parameters and the self-diffusion coeffcient of xenon-129 dissolved in the critical mixture of two nematic liquid crystals with opposite diamagnetic anisotropy112 have been determined from '"Xe NMR s p e c t r o ~ c o p yIn . ~the ~ ~ critical mixture, the director reorientation offers a means of determining the self-diffusion coefficients in the parallel and perpendicular directions relative to the liquid crystal director which has been utilized. The deuterium decoupled proton NMR spectrum of a sample of diphenylmethane-d3 dissolved in a nematic liquid crystal solvent has been analysed to yield a set of dipolar coupling^."^ These have been used directly without going through the intermediate step of characterizing the potential energy surface and a conformational distribution centered on 56.5' 0.5 O has been obtained for rotation about the two ring-CH2 bonds. Deuterium NMR spectroscopy has been applied to acenaphthene which has a point group symmetry of CZv,but behaves as though the symmetry is C2when dissolved in chiral nematic liquid crystalline solvent^."^ To quantify this effect the quadrupolar splittings of the perdeuterated compound were obtained and used to determine the orientational order parameters when dissolved in PBLG/CHC13 chiral liquid crystalline system. The proton and the deuterium spectra in a non-chiral liquid crystal have also been used to aid the analysis. The lowering of the symmetry of the orientational distribution function has been quantified by the angle by which the principal axes of the orientational order matrix of acenaphthene dissolved in PBLG/CHC13 are rotated out of the plane defined by the aromatic ring. The orientational order parameters of six solutes with different shapes and sizes dissolved in two nematic solvents have been measured as a function of temperature.'16 The obtained parameters have been correlated with the molecular geometry and the anisotropy in the polarizability as well as with the results of molecular dynamics simulations. The effects of the nature and concentration of the solutes 1,3,5-trichlorobenzene, benzene, chlorobenzene, o-dichlorobenzene, m-dichlorobenzene, and p-dichlorobenzene on orientational order in the nematic liquid crystal N-(4-ethoxybenzylidene)-2,6dideutero-4-n-butylaniline have been investigated."' Seven separate samples containing differing concentration of all solutes have been investigated and various schemes for comparison of orientational order among the samples examined.

10

Quantum Computing

Quantum computing has become an important area of research in recent years. Use of oriented molecules offers several advantages for quantum information processing by NMR. These include a larger network of coupled spins, shorter computation time and the possibility of exploitation of spins I z 1/2. The disadvantages are the strong coupling among the spins and larger linewidths. The literature in this area covers some of these aspects as well as presents some innovative new developments. Use of dipolar and quadrupolar couplings for


511

quantum information processing by NMR of oriented systems has been deUse of CH3CN, 13CH3CN,7Li (I = 3/2, 2 qubit system) and 133Cs, (I = 7/2, 3 qubit system) oriented in liquid crystals has been made. Creation of pseudo-pure states and the implementation of logic gates and arithmetic operations such as a half-adder and subtractor have been presented. Quantum information processing (QIP) using strongly coupled spin systems has been described.121Orienting spin systems in a liquid crystal matrix increases mutual coupling between spins, which is an advantage for QIP. However, homonuclear spin systems become strongly coupled and spins loose their individual identity as qubits. The use of even such systems for QIP has been demonstrated experimen-

(4 Pseudopure preparation

"GHZ

(+)" ($)x

Hz

1

i

10000

15000

1 8000

Figure 2

6000 4000

2000

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(a) Pulse sequence f o r creation and tomography oflGHZ>O, and u/alcl) for oil-in-water (o/w) L1 phases (normal micellar solutions), I1 (discrete cubic L.C.) and HI (normal hexagonal L.C.). Negative curvatures (HcO, and u/al>O) occur for waterin-oil (w/o) Lzphases (reverse micellar solutions), Iz (reverse discrete cubic L.C.) and HZ(reverse hexagonal L.C.) phases. The lamellar La L.C. phases have zero curvature and u/al = 1. Similarly, also bicontinuous cubic (V, and V,) phases have average H PI 0, and v/al= 1. Several types of phases of cubic symmetry with Gaussian curvature varying continuously over the surface have been characterized by SAXS for the space group and by NMR self-diffusion for bicontinuity conditions. In these phases amphiphilic domains are based on bilayers where the bilayers midplane surface can be often described by an Infinite Periodical Minimal Surface (IPMS). Figure 1 shows the three most popular IPMS found in L.C. phases, e.g. Primitive (space group Im3m), Diamond (space group Pn3m), and Gyroid (space group Ia3d). L, and L20r L.C. phases can solubilise oil or water and form swollen L1 and Lz or other L.C. phases. With further solvent addition micellar systems Nuclear Magnetic Resonance, Volume 33 0 The Royal Society of Chemistry, 2004

53 1

532


Primitive (Qp) lm3m space group

Diamond (QD) Pn3m space group

Gyroid (QG) la3d space group

Figure 1

I P M S used to describe bicontinuous cubic L.C. phases

15: N M R of Liquid Crystals and Micellar Solutions

533

can evolve to oil-in-water (o/w) or water-in-oil (w/o) microemulsions or emulsions. Microemulsions (and sometimes emulsions) are thermodynamically stable. Emulsions are usually kinetically stable.

2

General Articles: Reviews, Methods, Models

Interesting review articles have appeared. A review was reported on the use of I3C NMR techniques to investigate the structural aspects of dissolved molecules either in thermotropic or lyotropic L.C., including bicelles.2 The author addressed also to studies of bulk L.C. using lD, 2D, and 3D I3CNMR spectroscopy and to the use of L.C. media for NMR quantum computing. Multinuclear relaxation and self-diffusion NMR studies, based on 'H, 'H, I3C, 14N,23Na,35Cl, 79Br,and '29Xenuclei, were reviewed with highlight on the modeling of relaxation data to obtain microstructural and dynamic information on L.C. phases of both thermotropic and lyotropic type.3In this review models for molecular processes such as order director fluctuations, molecular rotation, self-diffusion, and internal motions were also presented. Another article covered progresses on NMR techniques to study physical properties of micellar systems? In this paper, micellization, structure of micelles, hydration and solubilization properties along with interactions with different types of surfactants, biological compounds and polymers were discussed. Micelle size and shape determinations via NMR relaxation and self-diffusion measurements were presented in a review article where complementary information from other techniques such as fluorescence quenching and scattering (DLS, SAXS, SANS) were i n c l ~ d e d Similar .~ approach was adopted to present methods used to characterize the microstructure of various types of microemulsions.6 A review on the NMR techniques, particularly wideline 2H and 31PNMR, used for the study of drugs solubilized in phospholipids (PL) bilayers was reported.' Another review article was reported on the various NMR approaches to study structure and dynamics of biomembranes.' Of particular interest is the review on self-diffusion studies of polymeric surfactants' where the ability of pulsed gradient spin-echo (PGSE) NMR techniques to give detailed information on the microstructure of complex colloidal systems was outlined. As to NMR self-diffusion technique it should be mentioned that, due to the improvement of technical performances, the number of scientific papers based on this technique has increased enormously in the recent years. The journal 'Magnetic Resonance in Chemistry' has dedicated a special issue in 2002 (volume 40) to NMR self-diffusion articles among which the top review article" describes methods and advices to measure anisotropic self-diffusion in oriented L.C. phases. Anisotropic diffusion was shown to reveal new structural features in L.C. phases and in biological tissues. Another article, dealing with anisotropic diffusion in nematic and smectic thermotropic L.C., demostrated that stimulated-echo-type 'H and 'H pulse-field-gradient spin-echo (PGSTE) NMR combined with multiple-pulse dipolar decoupling and slice selection is particularly informative on temperature-dependent diffusion processes." The multicomponent diffusion and the non-Gaussian diffusion or restricted diffusion in colloidal

534


systems of different complexity was discussed in an interesting article12 where models to obtain the real molecular self-diffusion coefficients (D) were also proposed. Modeling of data from PGSE NMR experiments performed in lyotropic mesophases was shown to produce a reliable structural fingerprint of the L.C. phase, provided that correct algorithms are found.I3Among methodological approaches, it is worth mentioning the application of switched angle spinning to correlate the 1st order dipolar spectrum of a L.C. with the isotropic magic angle spinning (MAS) spectrum in a two-dimensional e~periment.’~ New ways to align La and hexagonal (Hex) L.C. in the magnetic field were presented in some methodological article^.'^“' The hydrolysis reaction of methylformate added to a L3 phase or to a highly concentrated alkyldimethylamine oxide solution, in the presence of a high magnetic field, brought to the formation of macroscopically aligned La and Hex L.C..”. l6 Alternatively, sublimable solid naphthalene was added to a lipid bilayers. Sublimation process was shown to enhance alignment in the magnetic field significantly.” In a general review on the preparation of functional colloids with reactive surfactants via emulsion polymerization, several NMR methods useful for the characterization of the interactions and of the steps of the reactions were outlined.” Finally a new Brownian dynamics simulation method of translational diffusion on curved surfaces to rationalize NMR relaxation and self-diffusion data should be mentionedIg in this paragraph. In this paper, bicontinuous cubic phases, commonly found in lyotropic L.C. systems, were considered. These cubic phase are described by triply periodic minimal surfaces (TPMS) (Figure 1 shows some of them). It was demonstrated that particularly spin relaxation data of quadrupolar nuclei can provide quantitative information on the bicontinuous cubic structure and are sensitive to the topology of the surface. 3

Liquid Crystals

In this paragraph studies on L.C. phases of both thermotropic and lyotropic type along with their use as solubilizing media are reported. Perdeuterated or selectively deuterated mesogens and amphiphiles are frequently used to profit by the peculiar NMR parameters of the ’H quadrupolar nucleus such as Avq splittings and lineshape analysis. The solubilization of drugs, proteins, peptides and other molecules into an anisotropic L.C. phase which aligns in a magnetic field has become routine strategy for structural investigations. Indeed parameters related to the degree of long-range order and also to steric and anisotropic molecular interactions can be particularly informative to investigate microstructural features. 3.1 Thermotropic Liquid Crystals. - Organic molecules, based on aromatic rings or having suitably located unsaturations to produce elongated shapes (which are due to molecular entanglements and sterical hindrance to free rotations) are sometimes responsible of thermotropic mesomorphism. Besides the most typical nematic, smectic, and cholesteric thermotropic phases, some


535

mesogens, due to their peculiar shape, give rise to columnar, discotic and rod-disk mesophases. 3.1.1 Structural Characterization. A novel method to accomplish 13C NMR

spectral assignments for nematic L.C. using 2D chemical shift y-encoding NMR was proposed.” Substantially, the I3C NMR spectral assignments were made with the aid of the 13Cisotropic shifts obtained for the same compound in the isotropic liquid state. Monte Carlo simulations were reported as another strong aid to interpret structural configurations in nematic droplets.21 Many investigations deal with the characterization of the structure and the dynamic features of several types of newly synthesized mesogens. Often the thermotropic L.C. phase behavior is determined by the presence in the elongated molecule of aromatic ring. This is the case of substituted phenylisoxazoles 22 for which nematic and smectic phases were observed. Also, 4‘-methoxybiphenyl-4oxy-alkane hydrochlorides derivatives were shown to form two different types of smectic rne~ophases.2~ In other cases, specific intra- and intermolecular interactions are responsible of the self-assembly shapes. For instance some triazine derivatives were shown to form disk-shaped aggregates due to strong intramolecular H - b ~ n d i n gColumnar .~~ discotic and other long-range ordered phases were suggested for several alkyl-substituted hexabenzocoronene mesogens due to n-n interactions as ascertained by a variety of advanced solid-state NMR methods.25The effect of the nature and concentration of chlorine-based solutes on orientational order in the nematic L.C. formed by a deuterated butylaniline derivative was reported?6 Some metal complexes are also reported for their thermotropic phase behavior. Smectic A phases were identified for lanthanidecontaining Schiff s base complexes with chloride counterions from ‘H NMR correlation spectroscopy?’ Columnar discotic phases were demonstrated for the Zn, Ni, Cu, and Co complexes of octa(n-decy1thio)porphyrazinefrom 13CNMR2* Another important topic is to investigate linewidth and quadrupolar splitting line shape variations due to the confinement of mesogens in restricted geometries. In a case it was shown through ’H NMR that the presence of cylindrical surfaces induces a long range order in various mesogens above the smectic A to ’H NMR was used also to evaluate reorientation isotropic phase tran~ition.’~ processes for cyanobiphenyl (CB) derivatives confined between two glass plates in the presence of magnetic and electric fields.3013CNMR was used to ascertain the intermolecular interactions responsible of an increase of nematic order due to the inclusion of CB derivatives in a polymer matrix.31Thin molecular depositions of 5CB mesogenic molecules at the alumina-air interface, investigated through quadrupole-perturbed 2H NMR, showed two different configurations: a bulklike one with parallel axial arrangement, and a surface one with planar radial arrangement.32The partition between these two arrangements depends on the total 5CB concentration. In the recent years new types of polymer-based mesogens have been considered and their characterization has been performed through NMR techniques mainly. Studies on the structural characterization and phase transitions of cyano- and azobenzene substituted poly-methacrylate, poly-ethyleneoxide, and

536


poly-ethylenglycol was 34 A discotic columnar shape was proposed, on the basis of solid-echo 'H NMR data, for a poly-methacrylate derivative, deuterated in the benzoic ring.35Smectic A and cholesteric phases were identified for chiral side-chain poly-siloxane through 'H NMR.36A nematic order was suggested for a 5CB derivative in a holographic polymer dispersed L.C. used for switchable diffractive optical element^.^' An aliphatic poly(acety1ene ester), due to presence of the triple bonds, exhibited thermotropic L.C. behavior. Particularly, a columnar assembly was suggested from 'H and 13CNMR, x-ray diffraction and AFM studies3*Smectic L.C. were identified for polyesters based on 1,6hexanediol, di-Me 4,4'-biphenyLdicarboxylate, and di-Me-5-(sodiosulfo)isophtalate mixtures from 'H NMR.39 The kinetics of transesterification in poly(ethy1ene terephtalate) L.C. polyesters containing bis(2-oxazoline) as chain extender was studied using 'H NMR.4' Rate constants and activation energy were determined. Interesting information on structure, order and dynamics can be obtained from NMR relaxation experiments. The use of specifically deuterated molecules and 'H NMR spectroscopy, particularly relaxation experiments, was shown to provide precious details on the molecular dynamics involved in different types of mesogens as a consequnce of different substituted functional groups. Several articles based on this approach specifically applied to polysiloxane derivatives:' to copolymer-based nematic phases4' and to 4'-octylbiphenylcarboxylate-based chiral smectic A phases43were reported. The analysis of 'H NMR relaxation data for mesogens having laterally substitued aromatic groups (one of which is specifically deuterated), taken at two different frequencies, and rationalized using diffusional models, allowed to estimate a diffusion coefficient for the lateral phenyl ring three orders of magnitude higher than that for the whole molecular t ~ m b l i n g . 4 ~In3 ~another ~ article the effect of sonication on the order and on the NMR relaxation behavior was investigated for a nematic 4-pentyl-4'-CB mesophase.46The effect of different methylene-based spacer lengths on the structure and dynamics of L.C. polyurethanes was investigated using solid-state I3C and "N NMR.47 Limited flip angles of fluctuations around the phenylene axis were ascribed to the presence of intermolecular H-bonding between N H and C O groups. Few articles deal with the use of NMR self-diffusion techniques to evaluate the anisotropic diffusion in a smectic A phase formed by an antiferroelectric chiral L C 4 *and a discotic L.C. phase formed by a triphenylene-based c0mpound.4~In the latter case a self-diffusion coefficient around m2/s, three orders of magnitude lower than in the isotropic phase, was measured. This suggested that the diffusion is dominated by solid-like jump processes. 3.1.2 Solubilization in Thermotropic L.C. Thermotropic L.C. are sometimes used to dissolve more or less rigid solutes and to investigate the effects of orientational constraints on the molecular conformations of the solute. Multiple-quantum 'H NMR spectra of indene partially oriented in a nematic phase were performed using a magnetic field gradient method.50The obtained intramolecular dipolar constants were used to determine molecular shape while Saupe order parameters


531

were obtained from spectral width. The 'H NMR dipolar coupling constants observed for some aromatic solutes oriented in nematic solvents, showed a correlation with the dielectric anisotropy of the ~olvent.~'

3.2 Lyotropic Liquid Crystals. - Heavy water is often used to examine lyotropic L.C. phase behavior. 2H NMR quadrupolar splittings Avq appear in the case of anisotropic L.C. phases. Alternatively, deuterated molecules, particularly in the case of lipids and PLs, can be used. For structural investigations of PLs 31P NMR is also used. The analysis of the shape of the 2H NMR signals can give additional information on the orientational order and dynamics. In general, a full characterization of phase behavior of amphiphile molecules in binary or multicomponent systems is often based on NMR experiments, and accompanied by SAXS, and SANS, or DLS measurements. As mentioned above, self-diffusion measurements can give detailed information on the dimensions of the organized microdomains, on the obstruction effects (due to concentration, shape and soft boundaries), and on the spatial connectivity of the microdomains, i.e. the topology of the long-range ordered structure. Here the L.C. phase behavior is presented according to the chemical nature of the amphiphiles, e.g. ionic, nonionic, polymeric, lipidic and phospholipidic types and their use as solubilizing matrices. Vesicles and other dispersed L.C.phases are presented as section apart. 3.2.1 Ionic Amphiphiles. A variety of ionic amphiphiles, mostly cationic, are investigated for their phase behavior in water (W). The main focus is on the effect of different alkyl chain lengths or the effect of different counterions in homologous series. A microstructural investigation through 'H NMR self-diffusion on the quaternary system comprising sodium dodecyl sulfate (SDS)/water/pentanol/dodecane suggested the presence of a L3 phase, made of connected perforated bilayers or possibly a connected three-dimensional network of reverse cylinder^.'^ In addition, the presence of flow birefringence was interpreted as a shear-induced phase transition to an La phase. A 2H NMR relaxation study of the micellar phase of the potassium laurate/l-decanol/heavy water showed that in the nematic phase water reorientation was not enough to average the deuterons quadrupolar l i n e ~ h a p e Some . ~ ~ articles deal with alkylammonium chlorides and bromides. The water self-diffusion coefficients measured in the lamellar, hexagonal and cubic L.C. phases formed by Cl0-, CI2-,and C14-NH3C1 surfactants in water showed correlations with the microstructures which were interpreted through Mitra The use of 'H, 'H, and 13CNMR showed lamellar polymorphism in multicomponent systems based on alkyltrimethylammonium bromide deterg e n t ~The . ~ ~sequence of phase changes, hexagonal, calamitic No biaxial NBx+, biaxial NBX-, discotic ND,and lamellar mesophases was observed by 2H NMR in the tetradecyltrimethylammonium bromide/n-decanol/ water system with increasing decanol ~ o n t e n t . 'The ~ lateral self-diffusion coefficient of the partially fluorinated surfactant 1,1,2,2-terahydroperfluorodecylpyridiniumchloride was measured in a defective lamellar phase through an innovative I9F NMR technique which involves the combination of a PGSE sequence with homonuclear

538


decoupling and slice selection.57The diffusion coefficient was obtained from a two-step experiment based on the measure of the exponential decay constant with increasing diffusion times. Then the variation of this constant with increasing the gradient strength was analysed. In another article the careful analysis of I9F NMR chemical shift allowed to state the formation of lamellar L.C. of ammonium perfluorooctanoate adsorbed on porous alumina.5sThe use of solid state 'H and 13C NMR in the characterization of the phase behavior of silicate/cetyltrimethyammonium bromide (CTAB) mesophases, used to prepare MCM-41 catalysts, gave evidence of the role of different cosurfactants in setting the pore size of the final porous solid.59It was also observed that high cosurfactant/CTAB ratios induce a transition from hexagonal to La phase. The lecithin(LCT)/di-dodecyldimethylammoniumbromide (DDAB) mixed system in water, investigated through 'H NMR, SAXS, cryo-TEM, and optical microscopy, showed peculiar L.C. microstructures.60 As found for DDAB/water systems, two La phases, one collapsed at low water content, and the other swollen at high water content were observed. A bicontinuous cubic phase with Ia3d space group, not present in DDABjW binary system, was ascertained in the waterpoor part of the phase diagram. 3.2.2 Nonionic Amphiphiles. The most popular nonionic amphiphiles are typically formed by a linear alkyl chain bound to a number of ethylenoxide groups for short C,EO,. with the general formula CH3-(CH2),,.1-(O-CH2-CH2),OH, Several techniques including 13CNMR spectroscopy were used to characterize the phase diagram vs. temperature of C12EO6. HI, Laand bicontinuous cubic V1 L.C. phases were identified.61 Moreover, from the comparison with the homologous surfactants having EO, and EOs polar groups it was shown that the La phase expands to higher temperatures whereas the H I region shrinks toward lower temperatures with decreasing the number of E O units. H1, Laand H2 L.C. phases were identified through 'H NMR with increasing the temperature for a multicomponent sample containing equal amounts of water and n-decane oil stabilized by the surfactant mixture C12E06/polymer(poly-EO and poly-PO units)!* The hexagonal phase formed by Triton X-100 in water was used for the preparation of silver n a n ~ p a r t i c l e sThe . ~ ~ Ag particles grew from an initial size of 1-7 nm up 30 nm. However the hexagonal microstructure was not altered as demonstrated by 'H NMR and x-ray diffraction. Ag nanoparticles were shown to form also in reverse H2 L.C. templates based on nonionic amphiphile where the EO groups reduce the Ag ions while being oxidized to aldehydes as demonstrated by 'H NMR.64 3.2.3 Polymer Amphiphiles. Polymer amphiphiles are tipically based on blocks of groups having different hydrophilicity, e.g., the hydrophilic ethylenoxide E O and the more hydrophobic propylenoxide P O units, often referred as A and B blocks respectively. The phase behavior of the ABA triblock copolymer poly(ethy1en glycol) distearate, with an average MW of 930, doped with alkali metal salts was investigated by 'H, 7Li, 13C,and 23Nasolid-state NMR.65 Orderdisorder phase transitions were deduced from line shape analysis in comparison


539

with DSC and SAXS results. The phase behavior in water of an amphiphile made of a rigid aromatic core with two terminal poly-EO chains of various lengths and laterally attached to a polysiloxane backbone was investigated using 2H NMR.66 Due to the peculiar shape, lyotropic smectic phases which align spontaneously parallel to an external magnetic field were mostly identified. A characterization of the aggregation behavior of block copolymers bearing blocks of lipid-mimetic aliphatic double chain units was performed using 'H NMR along with other te~hniques.6~ Mostly nanoparticles were found with poly-ethylene glycol-water domains randomly distributed in the cores. Two new polymer surfactants consisting of a polysiloxane backbone with EO groups as side chains were characterized for the phase behavior in water by polarizing microscopy and 2H NMR.68 Large L, L.C. regions were identified. The study of steady shear flow in situ by 2H NMR in cone-and-plate geometry indicated the formation of multilamellar vesicles (onion-like) at intermediate shear rates. Studies on shear orientation of the L, L.C. phase formed by a diblock copolymer AleBlowith formula CH3(OCH2CH2)18[OCH2CH(C2H5)]loOH revealed from SAXS measurements deviations from ideal swelling and from 2H NMR data the formation of pore defects in the L, phase (so called mesh phase).69The effects of addition of neutral salts to the cholesteric mesophase structure of aqueous solutions (2H20)of hydroxypropyl cellulose were investigated by 'H NMR.70The cholesteric helical pitch was shown to increase upon addition of various ions whose effectiveness was in the order for anions C1- c Br- c NO3- c I- c SCN- and for cations Cs+ c K + c Na+ c Ca2+c Lit c Mg2+c A13+ 3.2.4 Lipids. Natural lipids, particularly mono-acyl-glycerides and PLs, show a very rich polymorphism which is always a topic of great interest. Many investigations deal with the peculiar structures and/or the phase transitions observed for either single lipids or mixtures of them in water and in the presence of other additives (salts, drugs, peptides, etc). Recently great attention was also given to study the molecular conformational properties and the interactions with additives leading to the formation of defective L.C. phases. An interesting general article has appeared where Brownian and molecular dynamics simulations of a lipid bilayer were described and the calculated frequency-dependent 13CNMR spin-lattice relaxation times were compared with experimental values.71 The effects of cholesterol (CHOL) and its precursor lanosterol on molecular conformational order and phase equilibria of lipid-bilayer membrane were investigated using 'H NMR on multilamellar lipid-sterol systems in combination with Monte Carlo ~ i m u l a t i o n s .Solid-state ~~ 13Cand 'H NMR along with other techniques were used to investigate the effect of pH on the phase behavior of CHOL/palmitic acid/water mixtures.73The pH affects the acid ionization and modulates the interactions. Particularly high pH's values favor the participation of both CHOL and palmitic acid to form stable La phases. A number of investigations fucuses on the 1-monoglycerol-oleate, also called monoolein (MO), and several results are reported. The existence of two types of bicontinuous cubic phases, namely the gyroid QG (Ia3d space group) and the diamond QD(Pn3m space group), is well known and demonstrated for the

540


MO/water system. M O in the presence of small amounts of l-palmitoyl-2diastearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-E0)]additive and of the anti-cancer drug Carboplatin in 'HZ0forms another type of bicontinuous cubic phase, e.g a primitive Q' with Im3m space (cfr. Figure 1 for the corresponding IPMS). In this system the self-diffusion coefficients of all components were measured combining 'H P G and MAS NMR. It was also shown that the Poly-EO chains interact with Carboplatin. A study of the self-assembly of M O in mixtures of water and the polar aprotic solvent 1methyl-2-pyrrolidinone (NMP) was performed using SAXS, PGSE NMR and rheological m e a ~ u r e m e n t sTwo . ~ ~ liquid phases were found along with L3,La and cubic Vz L.C. regions in the phase diagram at 20°C. Both the typical Ia3d and Pn3m (at high water content) space groups were ascertained for the Vz phases, but it is worth mentioning that at high N M P contents the Pn3m symmetry transforms into a Im3m space group, as observed in the case of Carboplatin addition.74The phase behavior and dynamics of Melaleuca alternifolia essential oil dispersed in MO/water L.C. phases was investigated through 'H NMR quadrupolar splittings for the identification of the monophasic regions and through 13C T1 relaxation and PGSE NMR measurements for the dynamic aspect^.'^ O n the basis of the NMR results it was suggested that the main component of the essential oil, namely the terpinen-4-01, is partitioned between the apolar domain and the interface and induces important modifications of the phase behavior. Acyl migration and natural hydrolysis effects in MO-based systems were monitored using 13CNMR.77These phenomena were shown to be strongly influenced by the amount of water and by storage conditions. Indeed they can become of crucial importance since a certain amount of free oleic acid always promotes negative curvatures of M O bilayers, either in the La or in the bicontinuous cubic L.C. phases, thus inducing a transition towards Hz L.C. phases. 3.2.5 Phospholipids and Solubilization in P L Bilayers. Many scientific NMR investigations comprise PL based systems among which PL bilayers used as membrane models, PL dispersions and unilamellar (so called bicelles) and multilamellar vesicles are particularly considered. Important studies based on PLs mixtures and PLs/lipids of different nature (particularly CHOL and polyunsaturated species) mixtures, and also PLs/additives of biological interest have been reported. The phosphatidylcholine (PC) polar head is the most abundant PL moiety in the naturally available sources, e.g. lecithin (LCT) from soybean seeds and egg yolk. 31Pnuclei, from the lipid polar head, and 'H nuclei, from perdeuterated or selectively deuterated carbons of the alkyl chains, are the usually investigated NMR nuclei. In addition, many studies have used PL bilayers to investigate peptide interactions and orientation. Defined mixtures of phosphoglycerolipids (PG) and sphingolipids were investigated using 'H NMR.78It was ascertained that the sphingolipid chain is always more ordered than that of P G lipids. In addition, the presence of sphingolipids in the PL mixture induced higher ordered chains also in the PG lipids. A new class of PLs 1,2-dipalmitoylsn-glycero-phosphatidyl-oligoglycerols was studied through FT-IR, and 'H


54 1

NMR and also solid-state 31Pand 13CNMR.79The use of specifically deuterated methylenes in the chains allowed to obtain information on the gauche-transgauche, double gauche, and end-gauche conformers in relation with headgroup structure and hydrophilicity, sample composition, and temperature. The molecular order and lateral diffusion in fluid cationic bilayers with perdeuterated chain 1,2-dipalmitoyl-&2-trimethylammoniumpropane (DPTAP)on a spherical solid support were obtained from 'H NMR relaxation and compared to the results obtained for the zwitterionk 1,2-dipalmitoyl-&2-sn-glycero-3-phosphocholine (DPPC-d62).8u Despite the cationic charge, the supported DPTAP bilayers showed a tighter molecular packing than the supported DPPC. The elastic deformation of membrane PL-based deuterated bilayers was investigated using 'H NMR relaxation." In this article, NMR relaxation data showed that the viscoelastic properties of membrane, at MHz frequencies, are modulated by the bilayer thickness, the interfacial area of the polar group, and also by the inclusion of a nonionic surfactant such as ClzE80sor by the presence of CHOL leading to various bilayer softness. Substantially, viscoelastic properties are modulated by the effective packing v/al. Some articles are related to the dynamics of water layers in the PL L, phases. The anisotropy of water diffusion, expressed as parallel and perpendicular self-diffusioncoefficients,in fully hydrated bilayers of dimyristoylphosphatidylcholine (DMPC) was determined through PG NMR imaging:' The results were related to the low permeability of water across the lipid bilayer and/or possible defects in the lamellae. An estimation of differently bound water molecules for the gel phase of the DMphosphatidylethanolamine(DMPE)/W system was obtained from DSC and 'H NMR r e l a ~ a t i o n Freezable .~~ interlamellar water and bulk water components of TI values were identified. Structure and dynamics of interfacial water in the L, phase of D P P C p H 2 0 system was investigated using 'H NMR quadrupolar splitting and lineshape along with molecular dynamics simulation^.^^ The water reorientation correlation function revealed a mixture of fast and slow decaying components. The light-induced unloading of small molecules from novel photolabile PLs was investigated using 'H and "F PFG NMR.85 Several articles deal with the effect of addition of CHOL, protein and peptides to PL bilayers. The interest is justified by the fact the CHOL and some proteins or peptides are natural consituents of biological membranes. The sphingomyelin (SM)/CHOL binary system was investigated using 'H, 31Pand 13CMAS NMR.@ It was suggested that the higher proportion of saturated fatty acid (FA) chains in SM compared to other PLs may be the most significant factor influencing interactions with CHOL. This was proven also by order parameters obtained from 'H NMR measurements. Some lipid mixtures based on SM, CHOL and palmitoyloleyl-PC (POPC) were investigated through 'H and "P solid-state NMRs7 in fully hydrated liposomes of POPCjCHOL and SM/CHOL. The measured axis of anisotropic rotation of CHOL indicated that the molecule is quasiperpendicular to the membrane plane. CHOL order parameters greater than 0.8 indicated that the sterol resides in a very motionally restricted environment. The mixture POPC/SM/CHOL showed an intermediate ordering situation between those of SM/CHOL and POPC/CHOL, thus suggesting that

542


CHOL could be in fast exchange on the NMR time scale between different microdomains or 'rafts'. A different behavior was found for CHOL in mixtures with POPC or POPS (PS = phosphatidylserine) as proven by 13Ccross polarization MAS NMR." It was shown that adding 0.5 mol fraction of CHOL to POPS, about 80% of CHOL is present as crystallites which undergo structural rearrangements after many hours of incubation. The addition of CHOL to DMPC bilayers was investigated under osmotic stress" using 'H NMR and isothermal microcalorimetry. One important conclusion was that the rippled P, gel phase can dissolve CHOL more readily than the L, phase. In addition, CHOL favors the P, phase formation. The lateral diffusion coefficient of CHOL in DMPC bilayers was determined using I9FPGSE NMR techniques on fluorine labeled at the 6-position CHOL.90 The polyunsaturated didocosahexaenoylphosphatidylcholine (DHAPC, the most unsaturated PL found in vivo) was shown to control membrane CHOL content using X-ray diffraction and 'H solid-state NMR.91A steric incompatibility between the rigid steroid CHOL molecule and the highly disordered DHA chain was suggested. The most important conclusion is that the solubility of CHOL in DHA-rich membranes decreases significantly. The effect of solubilizing the adrenocorticotropic hormone was shown to induce polymorphic transitions in PC-based membranes9' as demonstrated through EPR, 'H and 31PNMR. The dynamics aspects of solubilizing the adrecorticotropin (1-24) peptide in solvated dodecylPC micelles and DMPC bilayers was investigatedP3 A different binding of the more hydrophobic 1-10 residue compared to the more polar 11-24 part was demonstrated. In addition, molecular dynamics simulations were in excellent agreement with NMR results. The influence of Gramicidin D (GD) polypeptide incorporation on the structure and phase behavior of fully hydrated neutral D P P C bilayers was investigated as a function of temperature and pressure using 'H NMR.94Important modifications of the PL bilayer order were found depending on G D concentration. In another article Gramicidin A was incorporated in DMPC-ds7 bilayers and a structural investigation was carried out using I5N, 'H heteronuclear correlation NMR experiments based on cross-p~larization.~~ The incorporation of various membrane proteins or peptides in PL bilayers generally alters the original protein secondary structures. To investigate these modifications through NMR techniques specifically labeled proteins and peptides can be used. Protein conformations were investigated in several articles, in particular rhodopsin and p ~ r i n , ~ ~ P-purothionin,9' the outer membrane protein X (OmpX) from Escherichia coli,98 and other various peptides and The interactions of cyclosporins with PL -based membranes were examined using 'H NMR.'02 Binding constants of cyclosporins to liposomal membranes were found to depend on the CHOL content. It was also shown that the drug is essentially located in the interior of the bilayer membrane. "P NMR was used to investigate the partition of SDS and Li-DS between the water medium and PL bilayers.'03 It was found that complete membrane solubilization requires a ratio of 2.2 mol SDS bound per mol of total lipid at 56°C. Perfluorooctyl bromide was shown to have a limited membrane solubility and to be located within the carbon core of the POPC bilayer as proven by 'H and 31PNMR and by 19F NMR relaxation performed in the

1 5 : N M R of Liquid Crystals and Micellar Solutions

543

presence of the paramagnetic relaxation agents Gd3+and 02.'04 Location at the membrane interface of ( + )-totarol, a diterpenoid molecule having potent antioxidant and antibacterial activity, in P L model membranes was established using 13CNMR relaxation in the presence of the paramagnetic Gd3+ 3.2.6 Vesicles, Bicelles and L.C. Dispersions. The crucial driving force to form vescicles is an effective packing parameter v/al= 1 of the amphiphilic constituents. Possible different conformations of the polar head, which arranges with opposite interfacial curvature in the inner and in the outer layers, are needed. Opposite interfacial curvatures can be achieved mixing different amphiphiles with different polar heads so to obtain an average v/al= 1. Vesicles prefer to form in dilute solutions. Moreover some energy such as sonication is often necessary, e.g. they do not form spontaneously. Hence, rigorously speaking, vesicles are not thermodynamically stable systems. D M P C and DMPC/CHOL vesicles were investigated for the viscoelastic properties using 'H NMR transverse relaxation experiments.'06 Two different relaxation processes, i.e. vesicle shape fluctuations and molecular translational diffusion, were suggested from the analysis of dispersion profiles based on a slow-motional model. Characterization of newly synthesized oligomaltose-grafted lipids having applications in the preparation of sterically stabilized liposome dispersions was carried out using FTIR and 'H NMR.'" Enumeration of DPPC-based liposomes, dispersed in glucose solution, was performed using 31PNMR to assess the unilamellarity of the vesicle, and "0 NMR or 'H NMR relaxometry to evaluate the total internal water content."' A similar approach was used also for D P P C liposomes containing a paramagnetic Gd-derivative used as contrast agent in magnetic resonance imaging (MRI). NMR data resulted in agreement with photon correlation spectroscopy. Egg Yolk P C liposomes were used to assess the properties of the newly synthesized acyclic tetrabutylamides having H+/Cl- transport activity across bilayer membrane.'" The study was performed by pH-stat assay and 35ClNMR experiments. A potential use of these molecules for diseases involving C1- transport malfunction was suggested. Liposomes based on D M P C and D M P G were investigated as carriers of amphiphilic Gd-chelates using 'H NMR relaxation.'" Highly lipophilic Gd-chelates were required to ensure complete liposome incorporation. The amounts of Gd-chelate and of CHOL in the small-sized DMPC/DMPG liposomes were found to be the crucial factors to optimize, in vitro, relaxivity. 31P NMR was used to investigate the efficacy of Triton in promoting domain formation in lipid rafts (functional domains) mixtures based on sphingomyelin (SM), CHOL, glycolipids and some proteins."' Unilamellar bilayered micelles (vesicles)which consist of a mixture of long and short chain PLs are now commonly termed bicelles and are popular membrane model systems. Bicelles may form an isotropic phase or an aligned phase in the magnetic field, depending on PL types, concentration and temperature. Bicelles formed by the newly synthesized PL l-dodecanoyl-2-(4-(4-biphenyl)butanoyl)sn-glycero-3-phosphocholine (DBBPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) in a ratio between 5.1/1 and 6.5/1 in water were investigated using 31P,*H, and 23NaNMR.'12 Bicelles aligned with their normals parallel to

544


the magnetic field. Investigation of the phase behavior of a DBBPCiDHPC mixture with 6/1 molar ratio showed that at low temperatures (10-54°C) the system is dominated by bicelles, whereas at higher temperatures (54-75°C) isotropic micelles form and coexist with bicelles. The structure of bicelles formed by DHPC/DMPC mixtures were investigated by NMR and fluorescence probe.l13 To justify several inconsistencies among the experimental data, the authors suggested that the structure of bicelles could be an interconnected network of D M P C bilayers interrupted by DHPC rimmed pores at high temperatures. The influence of salts, namely KCl, NaCl, CaC12 and MgC12, on bicelles formed by a mixture of D M P C (molar fraction 0.78) and dicaproylPC was studied using solid-state "P and 'H NMR.'14 In the presence of K + and N a + , bicelles had diameters about 300 A, whereas in the presence of divalent cations diameters increased to about 500 A. The structure and dynamics of the gastrointestinal peptide hormone motilin, consisting of 22 amino acid residues was studied in the presence of P L bicelles in acidic solution, using 13CNMR techniques and specifically 13C-labeledm ~ t i l i n . "It~ was shown that motilin interacts with bicelles and becomes less structured and more flexible in neutral solution compared with acid media. A novel 13CNMR method to assess effects of free FA on molecular interactions of CHOL within, and transfer between, model membranes1l6was proposed. Large unilamellar PL vesicles (LUV) with PC/CHOL = 4/1 ratio served as CHOL donors, while small unilamellar PC vesicles (SUV) were the acceptors. The I3C4-CHOL peak, which is narrow in SUV and broad in LUV, was used to monitor the interactions. Oleic acid and other long chain free FAs dose-dipendently increased mobility of lipids in LUV and CHOL transfer rates, whereas short (C8-ClO) and very long (C24) chain free FAs did not. 31P NMR spectra of PL-based SUVs often give two peaks, assigned to outer and inner leaflets of the membrane. This effect was observed also for SUVs formed by both phytanyl phosphate and phytanyl phosphate/phytanol-5 mol%."' The asymmetry of the membrane was ascribed to a different ionization state of the phosphate groups. This, in turn, implies the presence of vectorial properties induced by the electrochemical gradient. Nanoparticles and polymeric vesicles from new poly-L-lysine based amphiphiles were prepared and characterized for their potential use as drug delivery systems."' Hydrophilic methoxypolyethylene glycol and hydrphobic palmitoyl pendant groups were attached to the lysine backbone and vesicle formation was possible only over a narrow spectrum of polymer architecture and was favored by a low MW and a low level of palmitoyl substitution. 19FNMR was used to determine the partition coefficients of some fluorinated psycotropic drugs (trifluoperazine, flunitrazepam, and flurazepam) between PC bilayers of SUVs and water buffer.'I9 Vesicles constituted by mixtures of polyE0400-dimethacrylate (PEG.DM) and cholesteryl methacrylate (CHMA) were investigated for their stability and efficacy as drug carrier using 'H NMR and other techniques."O From drug release experiments where vincristine sulfate, a water soluble anticancer drug, was incorporated in the liposomes it was found that the more stable PEG.DM/CHMA mixed liposomes represent also better carriers for drug delivery.


545

-

31PNMR and DLS were used to prove the self-assembly of deoxyguanosine 5'-monophosphate in water.121A slow diffusion with D m2/s was assigned to translational motions of large globular aggregates. Close to the isotropiccholesteric phase transition a faster motion, related to translational and rotational diffusion of the columnar stacks of guanosine molecules, was suggested from the appearance of a secondary resonance in the 31PNMR spectra. The critical concentrations pertaining the L.C. formation in supercoiled DNA saline solutions were obtained from 31PNMR, polarized light microscopy and phase equilibria experiments.'22The isotropic-cholesteric transition was interpreted in terms of L.C. theory including the effects of charge, orientation entropy, and excluded volume effects. A pitch of about 4 pm was found in the cholesteric phase. In addition the molecular free energy associated with the topology of the superhelix was found to be of paramount importance in controlling the width of the isotropic-cholesteric biphasic region. 4

Micellar solutions

NMR parameters are very sensible to the strong intermolecular interactions occurring as a result of self-association phenomena. NMR data become relatively easy to deal with in the case of isotropic liquid systems. Hence, information on critical micelle concentration (cmc), molecular conformations and interactions, counterion binding, hydration can be obtained from chemical shifts, relaxation, and self-diffusion NMR measurements.

4.1 Micelles in Amphiphile-Solvent Systems. - In this paragraph studies on micellar aggregates formed by single and mixed amphiphiles in a monophasic solvent (often water) are reviewed according to the amphiphile type, e.g. ionic, nonionic, polymer, and mixed amphiphiles. 4.1.1 Ionic Amphiphiles. Ionic surfactants have been well known for many technological applications from detergency to drug delivery systems. Nowadays the trend is to move from the traditional soaps towards more sophisticated surfactants characterized by the presence of specific functional groups. Another important factor that has addressed research towards new types of surfactants is the request of bio-compatibility. Ionic surfactants are often aggressive towards biological membranes (anionic surfactants interact directly with the bilayers, whereas cationic surfactants often act as immunosuppressive agents) and are not fully biodegradable also because of electrostatic reasons. Few ionic amphiphile possess bio-compatibility. An example is sodium taurodeoxycholate (NaTDC) which is the simplest bile salt mimetic.'H PGSE NMR and rheological measurements were performed to investigate the microstructure of the large micellar region formed by NaTDCpH20.123Particularly, water self-diffusion coefficients indicated that spherical micelles undergo a transition to cylindrical aggregates at 12 wt% NaTDC. In the range 12-25 wt% NaTDC, the Ll solution behaves as a Newtonian viscous fluid and at 37 wt% a hexagonal L.C. phase forms. In other

546


investigations new amphiphiles were synthesized with the double aim of improving bio-compatibility and functional activity through the addition of specific chemical groups in specific positions. For ionic species, also counterions and added salts can play a specific role. The potassium and triethanolamine salts of N-lauroyl-L-glutamic acid at different degrees of neutralization were investigated for the phase behavior in water.'24'H NMR self-diffusion measurements demonstrated that the micellar aggregates occur either in the isotropic L1 phase or in the discrete cubic phase whose stability increased with increasing the degree of neutralization and where a Pm3n space group was identified by SAXS experiments. 'H and 31PNMR were used to characterize the micellar phases formed by two series of P,P'-dialkyl pyrophosphate gemini surfactants having diimidazolium and disodium counter ion^.'^^ Fiberlike and rod/tubule shapes of aggregates were suggested on the basis of phase-contrast optical microscopy also. Ellipsoidal micelles were found from SANS and 'H PGSE NMR measurements for an unusual gemini double-chain surfactant where two different polar heads occur, namely [S04Na] and [O(CH2CHz0)lzCH3].'2h Another unusual stilbene-containing gemini double chain surfactant with two ammonium bromide polar groups was shown to form large vesicle-like aggregates when the stilbene moiety of the just synthesized surfactant is in trans conf~rmation.'~' Upon UV-irradiation a dimeric surfactant forms and small (radius about 20 A) spherical charged micelles were suggested on the basis of 'H NMR and SANS experiments. The effect of different organic counterions on the surface activity, micellar formation and dye solubilization behavior of decyl- and dodecylammonium carboxylates in water was investigated by 'H NMR and other techniques.I2*Particularly, cmc values decreased with increasing the chain length of the organic counterion. The micellar system formed by a fluorocarbon-hydrocarbon hybrid anionic surfactant was investigated through 'H PGSE and 19F NMR.129PGSE experiments showed that small micelles with a hydrodynamic radius of 0.6 nm form and an unusual very slow monomer-micelle exchange of the hybrid surfactant occurs so that two NMR signals of the terminal -CF3group can be seen in the I9F NMR spectrum. Reversible pH-induced formation and disruption of an unimolecular micelle formed by a random copolymer of sodium 2-(acrylamido)-2-methyl-propanesulfonate and 11-acrylamidoundecanoic acid were investigated using 'H NMR relaxation and other technique^.'^' Results indicated that the unimer micelle is disrupted into an open chain conformation at basic pH values. A sort of reverse micelles formed by mixtures of barium nonylphenolate/barium carbonate were investigated through 'H and solid-state I3C NMR.I3' It was suggested that the nonylphenolate rings were oriented towards the solid core of barium carbonate while the highly branched aliphatic chains pointed outward to the hydrocarbon solvent. "0 and 'H NMR experiments were used to investigate the parameters influencing proton relaxivity for a acid (DOTA) series of Gd3+ 1,4,7,1O-tetraazacyclododecane-1,4,7,1O-tetraacetic derivatives as potential liver-specific MRI contrast agents.'32Micellization processes and cmc were evaluated. The rate of water exchange was not sensitive either to micellization or to differences in the aliphatic chain. Two articles reported characterizations of the micellar solutions formed by alkyammonium


547

surfactants. In the former the analysis of the relaxation dispersion curves at different frequencies, gave information on local and collective molecular dynamics of the alkyl chain of decylammonium chloride in water.'33 In the latter the effect of the addition of NaBr on cmc, type of aggregate and degree of ionization in the aqueous solutions of n-hexyltrimethylammonium bromide was investigated through the analysis of 'H NMR chemical shifts.'34 4.1.2 Nonionic Amphiphiles. When the ionic polar head of an aliphatic amphiphile is substituted by a number of EO groups biological aggressiveness decreases significantly, although the global bio-compatibility cannot be ensured. Indeed, also the typical nonionic EO-based surfactants do not undergo easy biodegradation. Nevertheless, they have interesting features and unique applications. The micellar self-assembly and structure, including aggregation number, hydrophobicity, micelle size and shape were studied by DLS, SANS and 'H NMR for four C12E07 having different EO distribution^.'^^ Depending on the EO chain polydispersity, changes from spherical to oblate and prolate shape of the micellar aggregates were suggested. Micellar formation of crown ether surfactants, e.g. decyl 15-crown-5 and decyl 18-crown-6, was investigated using fluorescence probe and NMR methods.'36 Chemical shifts and TI measurements allowed the determination of reverse micelles formation (L2 phases) in different organic solvents. Because of their peculiarities, different classes of sucroesters are becoming important types of nonionic amphiphiles. First of all they are generally fully bio-compatible species and their hydrophilic-lipophilic balance (HLB) can be easily modulated by changing the hydrophobic moiety, particularly the chain length, and the nature of the polar sugar head, from a monosaccharide to polysaccharides. This confers all the characteristics observed for typical C,EO,type amphiphiles, but with the advange of a full bio-compatibility. A detailed characterization of maltose FA (stearic, palmitic, myristic, and oleic chains) monoesters as interesting biosurfactants was performed using surface tension and 'H NMR mea~urernents.'~~ As expected, with increasing the FA chain length cmc values decreased significantly. It is worth mentioning that these amphiphiles, unlike sucrose derivatives, possess an anomeric carbohydrate carbon position thus retaining their reducing properties. In addition they were newly synthesized from renewable sources. 4.1.3 Polymer Amphiphiles. Several newly synthesized polymer-based amphiphiles having specific functional groups have been recently reported and most work concerned with the characterization of self-assembly in aqueous and apolar solvents in the perspective of selective applications. Amino acid residues and sugar groups have become popular substituents to modify the HLB of polymer-based amphphiles. The formation of amphiphilic complexes of poly(ally1ammonium chloride) carrying pendant maltose residues with sodium alkylsulfonates (alky = octane, dodecane, and hexadecane) were investigated in aqueous solution using 'H NMR and other technique^.'^^ Some complexes formed micelle-like aggregates, with a hydrophobic core constituted by the sulfonate surfactants bound to the polymer and a hydrophilic outer shell con-


548

stituted by maltose residues. The effect of alkyl core structure on micellar properties of block copolymers based on FA esters of poly-EO-blockpoly(hydroxy1 alkyl L-aspartamide) was investigated using 'H NMR.13' An increase of micellar size and reduction of cmc values were observed when the level of FA attachment to the polymeric backbone was raised. In turn, the level of side chain attachment was found to be easily controlled by the length of spacer groups. Micelle size and shape as well as drug solubilization was investigated for a diblock-EO/oxybutylene(B) methylated (Me) copolymer E011BsMe.'40Compact micelles were found to be stable at low temperatures, whereas large, probably wormlike, micelles were suggested upon heating the L1 solutions to temperatures within 10°C of the clouding boundary. Micelle formation was observed for a nonamphiphilic diblock copolymer consisting of only solventphilic blocks, e.g. poly(vyni1 phenol)-block-polystyrene in the presence of 1,4b~tanediamine.'~' On the basis of 'H NMR experiments micellization occurred as a result of H-bond crosslinking between the two species. Polystyrene bearing a perfluoroalky group at one polymer chain, characterized by 'H and 13CNMR, was shown to form micelles in benzene as a result of a strong self-association of ~ ~ of the perfluoroalkyl chains, demonstrated by I9F NMR r e 1 a ~ a t i o n . lEffect polaxamer polydispersity on micellization in water was investigated by 'H NMR.143It was shown that polydispersity affects the characteristic temperatures of micellization and enlarges sphere-to-rod and phase separation transition regions. Terpolymers of quaternized N-(N',N'-dimethylaminopropy1)-acrylamide, N-dodecylmethacrylamide, and a methacrylate substituted with poly(dimethylsi1oxane) chain were investigated for self-association behavior in water using 'H 2D, NOE, and T1 NMR.'# The dodecyl- and siloxane chains associated together to form mixed microdomains with partial incorporation of the trimethylammonium residues of the quaternized unit, thus demonstrating a strong interchain van der Waals attraction. The determination of the selfdiffusion coefficients of poly(allylcarbosi1ane) dendrimers in water, through 'H PGSE NMR as a function of the polymer concentration, allowed to point out a concentration dependence of self-diffusion coefficients similar to that found for globular proteins.'45'H PGSE NMR was also used to study the size of aggregates formed by three different poly(propy1eneimine) dendrimers with hydrophilic triethylene glycol methyl ether terminal groups in aqueous solutions and gels.'46 Stokes-Einstein hard-sphere radii, according to the equation: R

=

kT/6~r;yD

(1)

were calculated and mobility of the dendrimer cores as a function of size was determined from NMR relaxation data. The assembly of long chain FAs onto the surface of amphiphilic poly(propy1eneimine) (PPI) dendrimers by electrostatic interaction were shown to form reverse micelles in different solvent^.'^' Self-assembly was proven through 'H and I3C NMR. I3CNMR relaxation was used to investigate the microstructure. In non-polar solvent, perfect reverse micelles formed with the ion pairs wrapped inside and long aliphatic chains on periphery, whereas in polar solvents the ion pairs showed the tendency to be exposed out with entangled aliphatic chains.


549

4.1.4 Mixed Amphiphiles Systems. - Mixed micellar systems have attracted wide scientific attention, particularly in view of industrial applications. Indeed, the use of different amphiphiles such as homologous series with different chain lengths, oppositely charged, ionic and nonionic or polymeric pairs, has become a common practice nowdays. Particularly in the case of ionic nonionic pairs, controversial results are often reported by different authors. This is probably due to the delicate self-assembly equilibria imposed by the interplay of different intermolecular forces. Thus purity of the chemicals, molecular and aggregate polydispersity, composition, and temperature (which affects especially nonionic surfactant phase behavior) are expected to play crucial roles. The 'H NMR chemical shifts, relaxation, self-diffusion, and 2D NOESY measurements were used to investigate the microstructure of mixed micelles formed by the nonionic polyethylene glycol lauryl ether (PEGC12) with CTAB and SDS.'48 CTAB molecules were suggested to be located close to the first EO groups next to the hydrophobic chain; these E O groups gradually moved outward from the hydrophobic core with increasing CTABjPEGC12 ratio. On the contrary, no conformational variations of PEGC12 chains were found in the presence of SDS mixed micelles. In another investigation on SDS/PEG mixed systems it was demonstrated that cmc of SDS is 3.3 mM, that is well below the normal value (8.6 mM).'49 This was ascribed to solubilization of PEG molecules inside SDS hydrophobic micellar cores. Substantially, in the presence of the anionic SDS surfactant, an increased oil-like behavior of PEG can be suggested. The C6EO&E02 mixed systems in water were investigated using 'H NMR selfd i f f ~ s i o n .Both ' ~ ~ surfactants co-aggregated and the micellar behavior was mainly determined by the mean number of EO units per surfactant molecule, independently of their distribution. In another similar investigation the CsEO~/octyltrimethylammonium bromide (OTAB) mixed system was con~idered.'~' It was shown from self-diffusion coefficients that the nonionic amphiphile had a larger tendency to self-associate than OTAB. 'H multifrequency NMR relaxation, used to study mixed systems formed by methoxy poly-EO macromonomer micelles in the absence and in the presence of SDS, indicated that SDS micelle formation is not favored while that of the macromonomer is dominant in the mixed system.'52 The inclusion of C12E04in P-cyclodextrin was studied in aqueous solution by 'H NMR.'53The competition between micellization and adsorption was quantified and further supported by semi-empirical M O and molecular mechanics calculations. The effect of benzyl alcohol on the micellar microstructure of CTAB/KBr aqueous system was investigated by 'H NMR and other technique^.'^^ The alcohol molecules were suggested to be located preferentially at the interface thus promoting the formation of much larger micelles. These evolve towards rodlike and then to oblate spheroid shapes with increasing alcohol content. These shape transitions increase also viscosity. The microstructures of the micelles formed by dimeric and gemini surfactants with a (CH2)"spacer (where n = 2,3,4,6,8,10,12) or an aromatic spacer (0-,m-, or p-phenylenedimethylene) were investigated for their conformational changes through 13C NMR s p e c t r ~ s c o p y . ''H ~ ~ PGSE NMR was used to investigate the association of toluene, chlorobenzene and benzyl alcohol with amino acid and dipeptide terminated polymerized surfac-

5 50


tants (PS).156A preferential association of the organic solvents with unpolymerized surfactant with respect to PS was found from the analysis of self-diffusion coefficients. Two articles dealing with alkylpolyglucosides (APG) based mixed systems were reported. In the former mixtures of n-decyl p-D-glucopyranoside and n-nonyl P-D-gluco-pyranoside were shown to form a miscibility gap in water.15' Self-diffusion NMR experiments proved the formation of bicontinuous structures of interconnected rods on both sides of the miscibility gap. In the latter, a 13CNMR study, using 13C-enriched FA probes, was performed to investigate the effects of headgroup size, chain length, and concentration on FA pK, at the nonionic micellar interface of APGs.lSs An increase of about 2 pK, units for the probe inside the micellar systems compared to the value of acetic acid in water was determined. Some articles based on fluorocarbon-hydrocarbon mixed surfactants systems were also reported. The interaction between the cationic copolymer (acrylamide-trimethylaminoethyl)acrylate and the anionic lithium perfluorooctanoate was studied by 'H and 19FNMR.'59Strong polymersurfactant interactions were suggested. Critical aggregation concentrations were determined as a function of surfactant/polymer ratio. The occurrence of superLorentzian lines, surmounted by weak sharp lines in both 'H and 19F NMR spectra suggested an incoming gelification of the mixture. Another binary mixture of fluorocarbon-hydrocarbon nonionic surfactants derived from tris(hydroxymethy1)acrylamidomethane was studied through 19FNMRl6' at different fluorocarbon/hydrocarbon ratios. As a result of the incompatibility of the two surfactants two types of micelles, fluorocarbon-rich and hydrocarbon-rich, were found to coexist. The experimental results could not be adequately described by current models on demixing micellar systems probably because of the interactions between the polyhydroxylated headgroups of the surfactants. The formation of two different types of micelles was observed also for the cationic surfactant mixture formed by cetyltrimethylammonium chloride and N-(1,1,2,2tetrahydro-perfluorodecany1)pyridinium chloride.161From 19F NMR spectra and self-diffusion it was suggested that the two surfactant species constituting the demixed micelles were 'microphase-separated'. 4.2 Solubilisation, Microemulsions and Emulsions. - Micellar systems are widely used as solubilizing media for both hydrophilic and hydrophobic species. A variety of articles have been reported, e.g. foundamental research interaction studies, structural investigations of the immobilized solubilizate (a protein for instance), uses as drug delivery systems, and also as reaction media. Both normal (L1) and reverse micelles (L2) can be swollen by oil and water to obtain water/oil/amphiphile ternary or pseudo-ternary systems. Ternary systems based on water, oil, and surfactant mixtures can form a variety of L1 and Lz monophasic regions. When these systems form isotropic solutions spontaneously, they are termed microemulsions. The formation of a microemulsion is related mainly to a substantial decrease of the interfacial tension (yolw) at the oil-water interface, in agreement with the typical equation:


551

where AG is the Gibbs free energy, AA is the interfacial area variation, and AS is the configurational entropy associated to the dispersion process, at the defined temperature T. Both AA and AS are positive values. The increase of AA is generally higher than that of AS, thus, in order to produce negative values of AG, the interfacial tension yoiw must decrease several order of magnitude (typically from 50-60 to 0.01 mNs-'). In such a case, the emulsification process leads to a microemulsion which is a thermodynamically stable system characterized by small aggregates and low polydispersity. If the added surfactant does not decrease yoIwsufficiently, AG > 0, and emulsification needs additional work. Consequently, emulsions, which are, rigorously speaking, non-thermodynamically but only kinetically stable systems, can be obtained. Hydration of the polar head group of the surfactant and oil penetration are crucial factors to determine the spontaneous curvature, H,, of the surfactant film for both microemulsions and emulsions. In the latter case, a long-term kinetical stability arises from the interplay of several additional parameters among which the relative densities and viscosities of the dispersed phase and of the dispersing medium are certainly determinant to prevent or at least to slow down destabilizing processes such as creaming, sedimentation, flocculation, coalescence and Ostwald ripening. 4.2.1 Solubilization Media. Many investigations have been reported on the interaction between a solubilizate and a micellar system. The solubilization kinetics of triglycerides in micellar solutions formed by nonionic surfactant and triblock copolymer was investigated.'62 Evidence of inhibition of the solubilization process in the presence of anionic surfactant was given, due to enhanced electrostatic interactions. The entrapment and condensation of plasmid DNA in the small inner space of reverse micelles formed by nonionic surfactant was investigated by NMR and other technique^.'^^ DNA was suggested to assume a ring-like structure, probably toroids. The micellar solutions formed by various nonionic commercial surfactants were shown to be good solubilization media for the general anesthetic 2,6-diisopropyl phen01.l~~ Through various 'H NMR experiments (relaxation, self-diffusion, NOE) it was demonstrated that enhanced solubilization generally occurs since the drug is partitioned between the swollen micelles and the aqueous medium. The solubilization of cytochrome c oxidase in lauryl maltoside neutral micelles was investigated by accurate 2D 'H, and 13C NMR measurernent~.'~~ From NMR data analysis, particularly paramagnetic relaxation contributions (due to Cu and Fe presence in cytochrome c) a model where micelles interact with the protein via the polar groups was suggested. The conformation of the secondary structures of integral membrane protein OmpX solubilized in dihexanoylPC micelles was investigated using a variety of NMR techniques (3D 15Nand 13C-resolved ['H, 'HI-NOESY spectra).'66 The conformation of other peptides extracted from horseshoe crab was investigated in the presence of dodecylPC mi~elles.'~' An increased exposure of specific hydrophobic side chains was suggested from 'H NMR data. A tryptophan-rich anticoccidial peptide in the presence of SDS micelles was found to modify its structure.'68 From 'H NMR chemical shift analysis specific interactions between the Trp-7, Trp-9, and Arg-9 residues either with SDS micelles or with Eimeria acervulina

552


living sporozoites was deduced. Conformational changes of the CTAB chains, in micellar solution, were ascertained through ‘H self-diffusion and 13Cchemical shifts analysis as a consequence of the addition of amphiphilic additives Different solubilization interactions dependent on C,Hz,+ lX(X = OH, NH2).169 ‘n’ values were suggested. Often micellar solutions represent suitable reaction media because of their selective solubilization ability. The enzymatic synthesis of conducting polyaniline was performed in micellar solution of sodium dodecylbenzenesulfonate (SDBS), CTAB, and Triton X-lOO.’io SDBS micelles were shown to have the best performance for peroxidase-catalysed template-guided polymerization of anyline. In a study related to the synthesis of chitosan-stabilized polymer dispersions, capsules, and chitosan grafting products via miniemulsion, NMR analysis showed that the efficiency of the reaction medium increased in the presence of a highly flexible costabilizer which crosslinks to the chitosan molecules.’” Micelles formed by hexadecyltrimethylammonium 0,O’-diethylphosphoro-dithioate surfactant were reported to react with 2-chloroethyl phenyl sulfide, a mustard ~ simulant, and to form a stable complex in a pH 9 borate b ~ f f e r . ”Copolymerization of methyl methacrylate with acrylonitrile was reported in microemulsions formed by SDS and 1-octanol as c ~ s u r f a c t a n t . ’Polymeric ~~ nanogels having controlled size, morphology, and composition, as demonstrated by ‘H NMR and other techniques, were produced via inverse mi~roemulsions.”~ Synthesis of gold nanoparticles in a w/o-type microemulsion formed by ethylether/AOT/W (where AOT is the used acronymus for bis-ethylhexyl sodium sulfosuccinate surfactant) in the presence of dodecanethiol was r e p ~ r t e d . ” ~ 4.2.2 Microemulsions - NMR techniques have given huge contributions to the understanding of microstructural features and transitions in microemulsion systems. Now it is well accepted that not only o/w or w/o droplets but a variety of microstructures from discrete to bicontinuous aggregates, exactly as in the L.C. state, occurs in L1 and Lz microemulsion phases. The driving force is always the effective packing parameter ‘vial'. The v/al parameter depends on the shape of the amphiphile and, more importantly, depends on the van der Waals and electrostatic interactions among the amphiphile, the apolar and the polar domains and between the aggregates. The presence of ionic species in the systems induces great sensibility also to specific ion effects. The transition of the dispersed phase from a disconnected to an interconnected domain is generally referred as percolative threshold. Perolative behavior is often observed in w/o microemulsions upon increasing the surfactant/oil ratio. The percolation threshold depends on several parameters such as type of surfactant, counterions, oil penetration and water content. So far, with increasing the volume fraction of the dispersed phase @d, discrepancies with respect to the hard-sphere model (cfr. eq. 1) have been discussed in terms of strong interparticle interactions, microstructural transitions and percolation behavior. The short range attractive interactions can produce clusters of droplets which, in turn, generate water networks throughout the liquid phase. Important changes of the transport properties occur. Within the percolation theory, a mobility parameter (z) - usually from conductivity experi-


553

ments - along an oil dilution line (i.e. constant water/surfactant ratio, that is constant polar/apolar interface) can be reproduced by two separate asymptotic scaling power laws having different exponents below and above the percolation threshold: z

-

($dc

~

z = ($c

$d)+

at $6$dC (below percolation threshold)

(3a)

$dc)‘

at $d > $dC (above percolation threshold)

(3b)

Percolation occurs when the volume fraction of the dispersed phase $d reaches a Critical value $dC at constant temperature. The critical exponent ‘t’ generally ranges between 1.5 and 2, whereas the exponent ‘s’ allows to assign the time dependent percolation regime. Thus, ‘s’ < 1 (generally around 0.6) identifies a ‘static percolation’ regime, and ‘s’z 1 (generally around 1.3)identifies a ‘dynamic percolation’ regime. The static percolation is related to the appearance of bicontinuous microemulsions. The dynamic percolation is related to rapid processes of fusion-fission among the droplets. The interpretation of these interactions in terms of static or dynamic percolation, is obviously strictly dependent on the time scale of the experimental technique. NMR can offer a suitable time scale, the unique at a molecular level, to monitor percolation behavior. In the case of ternary microemulsions formed by DDAB with the n-alkane oils n-C10 and n-C12 and the aromatic oil toluene (TOL) the interpretation of the 14NNMR relaxation and slow correlation times in terms of the percolation theory gave critical exponents in agreement with static and dynamic regime depending either on the volume fraction of the dispersed phase or on the oil type.176The percolation threshold was easily identified from the 14N NMR relaxation rates as shown in Figure 2. In the case of n-C10 and n-C12 oils, a ‘static percolation’, characterized by large aggregates having slow reorientational correlation times s was found. In the case of the microemulsion formed with close (zcsl,,) to TOL oil smaller aggregates formed and three different regimes of interaction were observed, from a ‘static percolation’ at low oil content to molecular dispersion close to the oil corner. It was demonstrated that 14N NMR relaxation represents a very sensitive probe to monitor delicate microstructural transitions such as percolative phenomena in microemulsions. As often remarked in this chapter, and previously demonstrated 19, also selfdiffusion NMR experiments are very informative about size and shape of the microdomains over which molecules are allowed to move. Mixtures of an amphiphilic graft copolymer in W/AOT/cyclohexane oil-continuous microemulsions were investigated using ‘H PGSE NMR.’77Polydisperse, but mostly finite, polymer-droplets aggregates were shown to coexist with a large fraction of free w/o droplets. Solubilization of vinylic and acrylic monomers in SDS micelles were shown to produce o/w microemulsions.’78From NMR data it was demonstrated that different domains of the SDS micelles were involved depending on the polarity of the solubilized monomer. The microstructure of the quaternary w/o microemulsions formed by CTAB/W/n-pentanol/n-hexane was investigated using ‘H PGSE NMR over a wide range of c o m p ~ s i t i o n . NMR ‘ ~ ~ self-diffusion data, analysed in terms of suitable models, allowed the representation of a


554

,

1500

14NNMR Relaxation and percolation thresholds

R2.R1 S.l

1000

-

7a b

TOL

~

t

0

0,2

0.4

0.6

0,8

1 @d

Figure 2

Percolative behavior of DDABI Wloil (n-decane, n-dodecane, toluene) microemulsions

'master plot' of the microstructure, e.g. hydrodynamic radii were plotted as a function of pentanol content for both oil and water dilution lines. The water in supercitical COz microemulsions stabilized by a perfluoropolyether ammonium carboxylate amphiphile were investigated for the dynamics features using selfdiffusion and relaxation NMR experiments.'" By decreasing the density of the COzmedium, the droplet clusters were suggested to form channels that permit water molecules to diffuse freely over distances on the order of microns. In other words, bicontinuity occurs. The amount of water in w/o microemulsions formed by the nonionic tetraethylene glycol monododecylether surfactant was shown to be the crucial factor to allow DNA condensation inside the reverse micelles as proven by NMR spectroscopy.181A 'H PGSE NMR study of a ternary homopolymer -poly(dimethylsiloxane) (PDMS),poly(ethylethy1ene) (PEE)- and nearly symmetric PDMS/PEE diblock copolymer demostrated the formation of bicontinuous microemulsions.***Various types of motion were identified and rationalized, particularly one associated to a disordered state and other two diffusional processes related to the microemulsion aggregates. Food-grade microemulsions based on ethoxylated sorbitan esters and other emulsifiers were investigated by self-diffusion NMR.'83 Structural aspects were found in agreement with the solubilization capacity of lycopene, a natural lipophilic antioxidant moiety from tomato. 4.2.3 Emulsions. The preparation of stable emulsions is often more dependent on a skilled artist than on rigorous scientific rules. Liquid-liquid dispersions are widely used in numerous technological applications such as agrochemicals, cosmetics, pesticides, lubricants, detergents, bitumen in road works, food, and

15: N M R ofLiquid Crystals and Micellar Solutions

555

pharmaceutical and oil industries. Emulsions systems have minimal thermodynamic stability when they form, and tend to phase separate. The primary driving force for phase separation is droplet interfacial free energy. Inclusion of a surfactant which concentrates at the oil/water interface imparts stability by lowering interfacial tension. In addition, reduction of interfacial tension facilitates emulsion formation and prevents immediate droplet recoalescence during preparation. Stability of emulsions is dependent on the interdroplet interactions. These interactions can be related to the hydrodynamic forces, i.e. Brownian motions, H-bonds, London attractive forces, electrostatic repulsive forces, and steric barriers due either to adsorbed surfactant molecules or fine solids. The stability of emulsions with respect to creaming is of great industrial and theoretical interest. A number of additives having different stabilizing roles are commonly used to prolong kinetic stability. Many investigations related to emulsion performances are concerned with natural and processed foods. Modern food industry relies increasingly on large-scale production operations. The development of new foods, and the efficient running of food processing require a more systematic and rigorous approach than was used previously. In addition, microstructural characterizations have become of paramount importance to improve reproducibility of the productive processes. For instance, o/w emulsions stabilized by whey protein and P-lactoglobulin were shown to be extremely stable to coalescence, provided that a saturated adsorbed protein layer forms at the droplet surfaces.184As demonstrated by NMR, if such a film layer is present, the amount of water can be highly reduced thus obtaining very concentrated emulsions. A series of siloxane polymers with various amounts of simple nonionic hydrophilic groups were shown to give self-emulsification and to emulsify silicone. A 13CNMR characterization was re~0rted.l’~ Standard PGSE and stimulated echo pulsed field gradient (PGSTE) ‘H NMR were used to investigate the translational diffusion of the polymeric oil PDMS in o/w emulsions stabilized by two different nonionic surfactants, e.g. polyE04-lauryl ether, and polyEOZ3-lauryletherlg6A detailed analysis of the free Gaussian diffusion of PDMS, of its restricted diffusion inside the droplets, and of the Brownian diffusion of the droplets themselves was reported. The correct consideration of all these different diffusional processes allowed to determine the size distribution of the emulsion droplets. In very diluted systems, by increasing the diffusion time, the Brownian contribution dominates and the Stokes-Einstein model can be applied to obtain the size distribution. Another article dealing with the theoretical aspects related to the modeling of restricted NMR self-diffusion in highly concentrated w/o emulsions was reported.lS7It was demonstrated that standard two pulse spin-echo sequence fails to investigate the short time-scale for diffusion inside a single emulsion droplet with radius 0.7 ym. The investigation of restricted diffusion from PGSE NMR experiments was reported as an effective tool to investigate food emulsions.lS8Droplet size distribution and self-diffusion coefficients of the dispersed phase were obtained on the basis of a suitable modeling of the experimental data.”


556

References 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12.

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