Annual Reports on NMR Spectroscopy, Volume 32

ANNUAL REPORTS ON

NMR SPECTROSCOPY

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ANNUAL REPORTS ON

NMR SPECTROSCOPY Edited by

G. A. WEBB Department of Chemistry, University of Surrey, Guildford, Surrey, England

VOLUME 32

ACADEMIC PRESS Harcourt Brace & Company, Publishers

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0 1996 ACADEMIC PRESS LIMITED

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List of Contributors P. S. Belton, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7 U A , UK. David J. Craik, Centre f o r Drug Design and Development, University of Queensland, Brisbane, 4072 Q L D , Australia. A, M . Gil, Department of Chemistry, University of Aveiro, Aveiro 3800, Portugal. Christopher J. Groombridge, Metropolitan Police Forensic Science Laboratory, 109 Lambeth Road, London SE1 7 L P , U K . Kerry A. Higgins, Department of Biochemistry, Monash University, Clayton, 3144 V1C, Australia. B. P. Hills, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7 U A , UK. Katherine J. Nielson, Centre f o r Drug Design and Development, University of Queensland, Brisbane, 4072 Q L D , Australia. William S . Price, Water Research Institute, Sengen 2-1-6, Tsukuba, Ibaraki, 305 Japan.

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Preface It is a veridical fact that NMR has found applications in all areas of modern science and that its appeal continues to expand apace. This year-1995sees the 50th anniversary of the discovery of NMR spectroscopy, and it is almost 30 years since the appearance of the first volume of Annual Reports on N M R Spectroscopy, during which time a large and diverse collection of topics have been covered. The contents of Volume 32 of this series are no exception, and consist of reviews covering four, clearly distinct, areas of science. It is my pleasure to be able to introduce the very interesting accounts on Applications of NMR to Food Science by Dr A. M. Gil, Professor P. S. Belton and Dr B . P. Hills; Gradient NMR by Dr W. S. Price; Pharmaceutical Applications of NMR by Professor D. Craik, Dr K. A. Higgins and Dr K. J. Nielsen; and NMR Specroscopy in Forensic Science by Dr C. J. Groombridge. Expressions of gratitude go to these reporters and to the production staff at Academic Press (London) for their generous cooperation in the creation of this volume. University of Surrey Guildford, Surrey England

G. A. WEBB June 1995

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Contents List of Contributors . . . . . . . . . . . . . . . . . v Preface . . . . . . . . . . . . . . . . . . . . . vii

Applications of NMR to Food Science A . M . GIL. P . S . BELTON and B . P . HILLS

1. 2. 3. 4.

Introduction Water in foods Biopolymers Analysis . References .

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1

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. . 2 . . 3 . . 12 . . 30 . . 43

Gradient NMR WILLIAM S . PRICE

1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction . . . . . . . . . . . Nuclear spins and gradients . . . . . . Diffusion measurements . . . . . . . Non-homogeneous gradients and other problems Pulse sequences for measuring diffusion . Applications to high-resolution NMR . . . Technical aspects of gradient production . . . Specific examples of gradient NMR . . . . Concluding remarks . . . . . . . . . . References . . . . . . . . . . . . .

51

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Pharmaceutical Applications of NMR D . J . CRAIK. K . J . NIELSEN and K . A . HIGGINS 1. 2. 3. 4.

Introduction . . . . . . . . . The role of NMR in drug development NMR techniques in drug design . . . . . . . Selected examples Acknowledgements . . . . . . References . . . . . . . . .

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. 53 . 55 . 58 . 95 . 100 109 121 128 135 135

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X

CONTENTS

215

NMR Spectroscopy in Forensic Science CHRISTOPHER J . GROOMBRIDGE 1. Introduction . . . . . 2. Misused drugs . . . . 3. Toxicology, body fluids . . 4 . Other forensic analysis . . 5. Magnetic resonance imaging Acknowledgements . . . . References . . . . . .

Index

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. 219 . 282 . 284 . 286 . 288 . 288 299

Applications of NMR to Food Science A. M. GIL Department of Chemistry, University of Aveiro, Aveiro 3800, Portugal

P. S. BELTON and B. P. HILLS Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, U K 1. Introduction 2. Water in foods 2.1. Water in solute-biopolymer systems 2.2. Water in heterogeneous food materials 2.3. Water relaxation in cellular food material 2.4. Magnetic resonance imaging of foods 2.4.1. MRI and temperature mapping 2.4.2. One-dimensional imaging protocols 2.4.3. MRI studies of food surfaces 2.4.4. MRI and food quality 3. Biopolymers 3.1. Introduction 3.2. Proteins 3.3. Polysaccharides 3.3.1. Polysaccharides in solution 3.3.2. Solids, gels and heterogeneous systems 3.4. Plant cell walls 3.5. 2 spectroscopy and biopolymers 4. Analysis 4.1. Introduction 4.2. Developments in authentication 4.3. Process applications 4.4. RSC methods 4.5. MAS methods 4.6 Other applications 4.6.1. Tea, coffee and wine 4.6.2. Fruit and vegetables 4.6.3. Lipids 4.6.4. Spices and phytochemicals 4.6.5. Milk 4.6.6. Meat 4.6.7. Other studies References ANNUAL REPORTS ON N M R SPECTROSCOPY VOLlJME 32 ISBN 0-12-505332-0

2 3 3 5 8 9 10 11 11 12 12 12 12 15 15 17 24 29 30 30 31 31 32 34 36 36 37 37 40 41 41 42 43

Copyrighi 01996 Acudemrc Press LImrfed AN rights of reproducrion in any form reserved

2

A. M. GIL, P. S. BELTON AND B. P. HILLS

1. INTRODUCTION

In the first review on applications of nuclear magnetic resonance (NMR)to food science to appear in this series,' it was remarked that the rate of publication in the area was increasing. This trend has continued. The first international conference on the subject was held in 1992, and 1994 saw the second of what is now an established series. Three booksz4 have been published, and in addition several reviews have appeared. Reviews of a more general nature are listed in Table 1; more specific reviews are dealt with under the various subject headings. In this review we attempt to give a fairly comprehensive account of the activity in the area since 1992. This inevitably requires a degree of selectivity since much of food research uses N M R in a routine way and much N M R is relevant to food research. We have tried to focus only on those aspects where N M R plays a major rather than a peripheral role in research and where the main focus is food o r food-relevant systems. A feature of this review is an account of recent work on plant cell walls. This subject is one which is becoming of increasing interest to food scientists and has been benefited by the application of solid state N M R methods. In addition to applications in food science we also consider two new N M R methods which seem likely to prove to be of general usefulness. These are magic angle spinning for improved resolution in proton spectra, and the use of Z spectroscopy in which polarization is transferred from biopolymer protons to water protons to allow mapping of the biopolymer lineshape.

Table 1.

A list of reviews on the applications of NMR in food science.

Subject

Date ~

~

Ref. ~

General General articles on applications of NMR General review General review General review Applications to food and engineering General review

1992 1993 1993 1993 1994 1994

10

Water Protein hydration

1994

11

Imaging General review of imaging Imaging, diffusion and relaxometry

1994 1994

12 13

Biopolymers Protein NMR

1993

14

5 6 7 8 9

APPLICATIONS OF NMR TO FOOD SCIENCE

2. WATER

3

IN FOODS

2. I. Water in solute-biopolymer systems

The amount and dynamic state of water in food systems affects many important properties such as food texture, the rates of enzymatic and non-enzymatic reactions and microbiological activity. NMR water relaxation measurements can provide a powerful probe of the amount and mobility of water in foods, and a number of recent studies have taken advantage of this. Of the three NMR-active nuclei in water (namely protons, deuterium in D 2 0 and oxygen-17 in H2170), proton-decoupled oxygen-17 transverse relaxation measurements provide the most direct probe of water mobility since they are unaffected by the chemical exchange of protons (or deuterons) between solute and water which complicates the interpretation of water proton and deuterium relaxation studies.’ Table 2 summarizes some of the recent oxygen-17 relaxation studies in sugar-biopolymer systems. Most of these studies have attempted to interpret their water oxygen-17 relaxation data in a qualitative way in terms of changing “water mobility” related to various degrees of “water binding” by the solute or biopolymer. Few attempts have been made to interpret the relaxation data with the two-site fast exchange theory developed by Halle et ~ 1 and. extended ~ ~ to sugar solutions by Hills24 and Belton et ~ l . These ~ * theories show that the water at the solute or biopolymer interface reorients anisotropically and needs to be characterized by two reorientational correlation times and a local order parameter. In principle the slow reorientational correlation time can be determined uniquely by combining oxygen-17 relaxation data obtained at two or more spectrometer frequencies. This approach has been used to study the interaction of water and dimethyl sulfoxide (DMSO) with gelatine22 but not, so far, with sugar solutions or solute-starch systems. In addition to the intramolecular quadrupolar relaxation pathway, water deuterium relaxation has potential contributions from the fast chemical Table 2.

”0 relaxation studies of sugar and biopolymer systems.

System Potato starch suspensions Wheat starch-sucrose Starch-based fat replacers Skim milk and caseinate solutions Sugar-water systems Sucrose-containing food system Wheat starch-sugar Gelatine gels

Nucleus

Ref.

1 7 0 , 2 ~ ‘H ,

15

170

16 17

170

170,

IH

18

170

19

170

20 21 22

170

’H, ‘H

4

A. M. GIL, P. S. BELTON AND B. P. HILLS

exchange of deuterons between water ( D 2 0 ) and exchangeable deuterons on the solute. Water proton relaxation in solute-biopolymer systems has contributions from both chemical exchange and intermolecular dipolar interactions between the hydration water and the solute or biopolymer.26 Some progress has been made in quantifying the relative contributions of these pathways, at least in potato starch suspensions by a combined multinuclear relaxation study. l5 The idea behind these experiments is that the ratio of transverse relaxation rates RIR, (where R is the observed relaxation rate and R, is that of pure water) should be independent of the nucleus, whether oxygen-17 or deuterium, provided that only the intramolecular quadrupolar relaxation mechanism contributes. Any difference in this ratio is presumed to arise from chemical exchange of deuterons with the solute. Similarly, in the short pulse spacing limit of the CMPG sequence, the ratio RIR, for protons and deuterons measured in the same system will differ because of the contribution of intermolecular dipole-dipole interactions between water and solute. It was found that both chemical exchange and dipolar interactions make significant contributions to water transverse relaxation in potato starch suspensions, especially at lower water concentrations, above -40% w/w starch. This approach has yet to be applied to other solute-biopolymer systems. observed a doublet splitting in It is interesting to note that Yakubu et ~71.'~ the deuterium spectrum and a triplet splitting of the oxygen-17 spectrum for water inside potato starch granules but that no such splitting has been observed for other cereal starches such as corn, wheat or pea starch. This could arise from the uniquely ordered hexagonal arrangement of amylose double helices in potato starch. The relationship between NMR water relaxation times and glass transitions in food materials remains to be elucidated. Many papers have used non-NMR techniques such as differential scanning calorimetry (DSC) and dynamical mechanical thermal analysis (DMTA) to measure glass transition temperatures in food ingredients such as casein,27 and amylopectin3' and their mixtures with sugars and to show how increasing water content lowers the glass transition temperature. However, very little NMR work has been done on the relationship between water and biopolymer mobility (as monitored by NMR relaxation times) and the glass ~ transi~ transition temperature. Early work by Kalichevsky et ~ 1 on . glass tions in gluten showed that the proton relaxation time characterizing the non-exchanging gluten protons remained constant below the glass transition temperature but increased above it. Corresponding changes in the water relaxation times were not reported. A detailed investigation of water dynamics in 10% maltose glasses3' using proton and deuterium NMR reported a similar increase in the maltose proton relaxation time above the glass transition temperature and, in addition, demonstrated the remarkable mobility of water in the glassy state of the 10% maltose glass. The water

APPLICATIONS OF NMR TO FOOD SCIENCE

5

relaxation data suggested a model of the glassy state in which water molecules undergo rapid rotational and translational motion inside more rigid “cages” or “channels” formed by maltose molecules.

2.2. Water in heterogeneous food materials

An aspect of relaxation theory that has been paid only scant attention in the food literature is the effect of food microstructure (and the microscopic air-water distribution) on water relaxation behaviour. Most work relating water proton relaxation behaviour to microstructure has been carried out on non-food materials such as water-saturated porous rocks, sandstones, chalks and cement pastes where the distribution of water proton relaxation times is used to determine pore size distributions in the porous matrix. This procedure succeeds because water in a pore relaxes by diffusion to the pore surface, which acts as a surface relaxation sink, so that larger pores are associated with longer relaxation times than smaller pores. An overview of the field has been published recently,32 and Table 3 lists a number of recent papers on water relaxation in heterogeneous non-food materials. From the morphological viewpoint there is little fundamental difference between a starch slurry and a cement paste, or between a bread loaf and a porous sandstone, but the more complex chemical composition of most real foods prevents a simple application of the pore size distribution concept to food relaxation behaviour. For this reason most reports on water relaxation in real foods are of a preliminary and empirical nature. The second part of Table 3 lists a number of typical water relaxation studies on food materials Table 3. Water relaxation measurements heterogeneous systems.

System

in

Ref.

Non-food materials Hydrating cement paste Hydrating sandstones Chalk Hydrating zeolite powder plugs Hydrating silica beds Hydrating Sephadex microspheres Glass microsphere beds

33,34 35 36, 37 38 39, 40 41-43 44

Food materials Staling of bread Cooking of cakes Frozen food gels Hydrating flour and starch pastes

45 46 47 48

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A.

M.GIL, P. S . BELTON AND B . P. HILLS

reported at the 2nd International Conference on Applications of Magnetic Resonance in Food Science held at Aveiro, Portugal, in 1994. To try to place these real food studies on a less empirical basis and bridge the gap between the non-food and food literature, a number of systematic water proton relaxation studies have been made on model heterogeneous systems having a simpler morphology. These include randomly packed beds of Sephadex microspheres where both the microsphere radius and the internal dextran chain cross-linking density can be systematically ~ a r i e d , ~ ~ . ~ ~ randomly packed beds of glass microspheres4 and crushed silica of various mean particle diameter^.^',^^ In each of these systems the distribution of water proton relaxation times was measured as the water content was varied between the saturated bed and the almost dry powder. Because of their more clearly defined morphology it was possible to mathematically model the relaxation behaviour by numerically solving the Bloch-Torrey equations.49 When comparing theory and experiment on randomly packed beds of non-porous particles such as glass microspheres and s i l i ~ ait~ ~ , ~ ~ was found that the water proton relaxation behaviour depends not only on the pore size distribution as defined by the radius of the largest sphere that can be accommodated inside a pore but also on the pore geometry. This is an important distinction, because the former definition predicts a continuous distribution of pore sizes and of relaxation times in the glass microsphere beds, whereas the observed relaxation time distribution actually consists of four distinct peaks (Fig. 1). The longest relaxation time peak is thought to arise from water in distorted octahedral pores formed between six microspheres, the next longest relaxation time peak to water in distorted tetrahedral pores and the third longest peak to water in triangular pores or throats formed between three spheres. The assignment of the shortest relaxation time peak is uncertain; one possibility is that it corresponds to higher-order relaxation modes predicted by the Bloch-Torrey equations with restricted d i f f u ~ i o n . ~ 'A . ~ similar ~ four-peak distribution was observed in water-saturated beds of silica particles with mean diameters between 130 and 250 pm. By fitting the changing relaxation time distribution as the overall water content is lowered in the glass microsphere and silica beds from saturation to the almost dry powder it is possible to deduce the changes in the microscopic air-water distribution between the different pore categorie~.~' The results appear to conform reasonably well to the expectations of capillary suction theory, which predicts that air first penetrates the largest pores before entering successively smaller pores. A similar protocol has been followed with randomly packed beds of Sephadex microspheres with the difference that the Sephadex microspheres are porous so that water can diffuse freely between the compartments inside and outside the r n i c r o ~ p h e r e s . In ~~~ this ~ porous system, osmotic forces predict that lowering the water content of the bed should first remove all water outside

APPLICATIONS OF NMR TO FOOD SCIENCE

1.0

5.0 10.0

50.0 100.0

7

500.0

Relaxation time/ms

Fig. 1. The distribution of water proton transverse relaxation times in a water saturated randomly packed bed of monodisperse glass microspheres of radius 200 pm. The shaded peak is the distribution predicted when the pore size is defined as the radius of the largest sphere that can be fitted inside the pore. (Reproduced with permission from Hills and Snaar&).

the Sephadex microspheres, at which point the relaxation is necessarily single exponential with a value equal to the intrinsic relaxation time inside the fully swollen microsphere. Further removal of water causes the relaxation time to shorten as the microsphere shrinks. As with the non-porous silica and glass microsphere beds the concomitant changes in the water proton relaxation time distributions as the air-water distribution and microstructure changes can be modelled, at least semi-quantitatively, using numerical solutions of the Bloch-Torrey equations.4143 Ice has such a short transverse relaxation time (a few tens of microseconds) such that if water in a particular compartment of pore freezes, the relaxation time peak corresponding to that compartment or pore essentially disappears. This idea has been used to study the microscopic distribution of non-freezing water and ice in the packed Sephadex microsphere beds as they are taken through a freeze-thaw cycle.43 By fitting the relaxation time changes with the Bloch-Torrey equations it was then possible to derive a value for the surface relaxation strength of ice. The value for transverse relaxation at -2°C is 1.34 x lop3cm/s, which is sufficiently large to suggest that surface relaxation effects should significantly affect the relaxation time of non-freezing water in frozen foods. It would be interesting to extend these model relaxation studies to other

8

A. M. GIL, P. S . BELTON AND B. P. HILLS

nuclei such as water deuterium and oxygen-17 relaxation. This would help establish the relationship, if any, between the changing microscopic airwater-pore distribution and the oxygen-17 relaxation times that was discussed in the context of unsaturated packed beds of starch granules in the previous review.' It remains to be investigated whether the Bloch-Torrey equations can also be used to rationalize the effects of changing microstructure in more complex food materials such as doughs, breads, crumbles and food mixes. 2.3. Water relaxation in cellular food material

Water in plants is also compartmentalized on a variety of distance scales. On a macroscopic distance scale different types of tissue such as the vascular bundles or parenchyma tissue within the intact plant are distinguished by different cell types and sizes and are expected to be characterized by different water relaxation times or effective water diffusion coefficients. This is the origin of contrast in relaxation or diffusion-weighted magnetic resonance images of intact plant tissue which can be used to monitor the quality of fruit and vegetables. Table 4 lists a number of examples of this use of imaging. Much less work has been done on the more fundamental issue of interpreting the water relaxation and diffusion behaviour of particular tissue types in terms of cell structure and composition. At a microscopic distance scale, water in plant cells is compartmentalized in various membrane-bound subcellular organelles such as vacuoles, starch granules and the cytoplasm, and the water outside the plasmalemma membrane is associated with the plant cell wall. Each of these aqueous subcellular compartments is expected

Table 4.

Water relaxation measurements in plant materials.

Tissue Carrot, onion, apple Apple parenchyma tissue Apple parenchyma tissue Courgette Mango fruit Tomato Red raspberry fruits Bean hypocotyls Potato Carrot Zucchini squash MRI, magnetic resonance imaging.

NMR measurement Water relaxation Water relaxation and diffusion Water relaxation MRI relaxation contrast MRI MRI MRI 'H relaxation of cell walls Water relaxation Water relaxation MRI

Ref. 52

53 54

55 56 57 58

59 60 61 62

APPLICATIONS OF NMR TO FOOD SCIENCE

9

to be characterized by intrinsic water proton relaxation times determined by the chemical composition of solutes and biopolymers comprising the compartment. The observed distribution of water proton relaxation times arising from tissue of a homogeneous cell type therefore largely reflects the distribution of water between these subcellular compartments and, once again, the relaxation time distribution can be interpreted with numerical solutions of the Bloch-Torrey equations provided the compartment morphology, the membrane water permeabilities and the intrinsic compartment relaxation times are known. Cell morphology can be observed with light or electron microscopy, and the intrinsic compartment relaxation times estimated by separating the cell compartments such as starch granules or cell walls. Fitting the observed relaxation time distribution can then give information about the membrane water permeability coefficients. So far this quantitative approach to plant tissue relaxation has been applied only to a but further applications are expected. parenchyma apple Of particular interest to food manufacturers and distributors are the changes in the water/air/ice distribution in fruits and vegetables as they are ripened, dried, rehydrated, frozen and stored. Here again the changes in the distribution of water proton relaxation times during these ripening, processing and storage operations can, in principle, reveal the microscopic (sub-)cellular changes, though the only recent application of this approach concerns the freezing of potato tissue where the changing subcellular distribution of ice and non-freezing water at temperatures down to -20°C were monitored.60 This study established that even at -15°C there is still unfrozen water in the starch granules and cell wall compartments, an observation of potential significance in understanding shelf-lives in food cryopreservation.

2.4. Magnetic resonance imaging of foods

Mention has already been made of magnetic resonance imaging (MRI) studies of fruit and vegetable quality and of the need for a more fundamental understanding of the origin of tissue contrast in these foods. This highlights the recent trend in food imaging away from merely qualitative imaging of “static” food structure to quantitative imaging applications. Indeed, the true potential of MRI in food science undoubtedly lies not in static structure determination but in following, non-invasively, mass and heat transport in foods, in real time during processing and storage. Relevant food-processing operations include drying, rehydration, heating, blanching, frying, microwaving, extrusion, curing, mixing, drainage freezing and freeze drying. This lengthy, but far from exhaustive, list serves to emphasize that MRI has the potential for revolutionizing food-processing science, a point that has been made in a recent book on MRI of foods.’

10

A. M. GIL, P. S . BELTON AND B. P. HILLS

Table 5. MRI studies in foods.

MRI study

System Carrot Potato Model food gels Peach Potato Sephadex Extruded pasta Potato Apple Glass bead beds Cod Peanut butterlbread Milk Barley seeds Alginate gels

Temperature mapping Temperature mapping Temperature mapping Freezing Freeze drying Radial imaging Rehydration, radial imaging Drying, one dimensional Drying, one dimensional Drying, one dimensional Texture Fat transport Cream separation Maltose distribution Spatial heterogeneity

Ref. 63 64 65 66 67 68 69 70 71 72 73 66 74 75 76

Table 5 lists a number of recent quantitative MRI studies of mass and heat transport in foods. For convenience these will be discussed under the following subheadings.

2.4.1. MRI and temperature mapping Temperature mapping is one of the most exciting recent developments in food imaging. It is based on the observation that water proton longitudinal and transverse relaxation times as well as water self-diffusion coefficients in food materials depend on temperature so that an image (or map) of the spatial dependence of any of these parameters can be converted into a temperature image provided a suitable calibration curve exists relating relaxation times or diffusion coefficients to temperature. This calibration curve can be determined separately from non-spatially resolved NMR measurements on uniform samples. Table 5 lists a number of recent publications based on this idea. By modelling the time course of the temperature profiles the thermal diffusivity and surface heat transfer coefficient can be deduced. At subzero temperatures MRI has potentially important contributions to make in the measurement of freezing times and of freeze-drying kinetics. Ice has such a short transverse relaxation time that it appears as a black region in the image. An unfrozen central region in a food therefore appears as a bright region. Whether this can be exploited in an “on-line” non-invasive technique for optimizing a food-freezing operation remains to be seen. An example of a qualitative MRI study of a freezing peach half is given by McCarthy and Kauten.66 Recently, MRI has been used to study the kinetics

APPLICATIONS OF NMR TO FOOD SCIENCE

11

of the freeze drying of potato cylinder^.^' In contrast to the previous application, the central core of unsublimed ice in the freeze drying potato appears as a bright region in the image. This apparent contradiction can be explained by the presence of the unfrozen water in the starch granules and cell walls referred to in the previous section. The time course of the shrinking central ice core permitted the kinetics of the freeze drying process to be m ~ d e l l e d . ~ '

2.4.2. One-dimensional imaging protocols A number of recent papers have pointed out the advantages of simple one-dimensional projection imaging when attempting to follow rapid changes in moisture, temperature or quality factors during food processes such as drying or rehydrati~n.'~.'~ One-dimensional projection imaging assumes that the food can be cut or moulded into cuboids or cylinders and then processed so that mass and heat transport occur only along one of the principal axes of the cuboid or, in the case of the cylinder, either along the axis or in a radial direction. In the latter case the radial profile can be obtained from the projection by an inverse Abel transformation.68 Such one-dimensional imaging is necessarily faster than two-dimensional imaging since no phase-encoding gradients are required. Moreover, since the whole sample cross-section is projected, sufficient signal is usually obtained in just one or two scans. A further advantage of this protocol is the comparative ease of theoretically modelling the profiles in one space variable. Table 5 lists a number of such one-dimensional imaging studies.

2.4.3. M R I studies of food surfaces Real-time MRI moisture and temperature images include the values at or near the food surface. These surface values are particularly significant because the efficiency of processing operations such as drying or baking depends on the magnitude of the heat and moisture surface transfer rates, which also vary during the processing as the chemical state, moisture content and temperature of the surface changes. It is therefore essential to monitor the changing conditions of the food surface during processing and storage. The potential of MRI surface studies has been demonstrated recently during the surface air drying of an initially water-saturated randomly packed bed of glass beads." Here it was found that the surface degree of saturation decreased almost exponentially with drying time, but it is unclear why this should be the case or to what extent this observation depends on the nature of the food matrix and drying conditions. The surface moisture content is also expected to influence the rate of growth of spoilage organisms and surface quality factors such as texture and colour.

12

A. M. GIL, P. S. BELTON AND B. P. HILLS

2.4.4. MRI and food quality

A food-manufacturing operation has to be optimized by maximizing food quality while at the same time minimizing energy expenditure. MRI can assist in this process by providing not only spatial maps of moisture content and temperature but also of certain food quality factors such as lipid and solute concentrations. For example, MRI studies of whole fresh and frozen cod have been used to determine differences in water binding and its effect on the fish texture.73 This is an important storage problem because cod shows marked changes in texture due to protein aggregation during frozen storage, thereby rendering the fish unpalatable. Other recent examples of MRI quality monitoring include the transport of fat into bread from peanut butter66 and the separation of cream from milk over a 9 h period.74 MRI microimaging and localized spectroscopy have been used to image the production and spatial distribution of maltose and other metabolites in germinating barley seeds,75 which affects the quality of beers and spirits. The use of MRI to monitor bruising in fruits has already been discussed in the section on water in cellular food material and in the previous review.' More generally, it is known that water proton relaxation times depend on the degree of protein and polysaccharide aggregation as well as solute concentration, pH and solid-liquid ratios, so MRI should be capable of monitoring any food quality factor dependent on these factors. A recent example involves the T2-weighted imaging of spatial heterogeneity in calcium alginate gels,76 which is important in controlled release applications.

3. BIOPOLYMERS 3.1. Introduction

The area of research into food biopolymers continues to attract considerable interest. A notable feature of work over recent years is the growing activity in the investigation of plant cell walls by NMR. There have also been developments in methodology particularly in the exploitation of polarization transfer methods by the use of Z spectroscopy. 3.2. Proteins One of the most active areas in protein research has been in cereal proteins. This is largely due to the increased availability of pure protein fractions isolated from the cereal seeds or from gluten. Cysteine-rich basic proteins have been isolated from heat,^',^^ barley77 and maize.78 The larger (9 kDa) proteins are lipid transfer protein^^^,^'

APPLICATIONS OF NMR TO FOOD SCIENCE

13

which have a high affinity for lipid and unusual foam stabilization properties.80 High-resolution one-, two- and three-dimensional methods have been used to assign the sequence and characterize secondary The smaller (5 kDa) protein has had its three-dimensional solution structure defined using NOESY data and distance geometry methods followed by dynamical simulated annealing calculation^.^^ The roles of both these proteins in the cereals are unclear, but both may be used for defensive purpose^.'^^^^ There is now a general recognition that the high molecular weight subunits of gluten have a major role in the bread-making characteristics of flour." However, until quite recently these have not been available in sufficient quantities for examination by NMR. They are high molecular weight, insoluble proteins and are thus unsuitable for high-resolution solution state approaches. An interesting hypothesis exists that these proteins are analogous to the mammalian connective tissue protein elastin and that the elastic properties of the high molecular weight subunits derived from the same mechanism as elastin.82 The hypothesized mechanism was that, in analogy to elastin, the high molecular weight subunits had a p spiral structure which on extension behaved rather like a spring. The restoring force in elastin derives from its hydrophobic nature. Good evidence for this hydrophobic interaction has been obtained by pulsed NMR studies of *H20 in contact with e l a ~ t i n . 'When ~ 'H20 is in excess, heating causes contraction of the protein mass and expulsion of water. This is manifested in an increase in intensity of the slowly relaxing component of the 'H transverse relaxation. When the same experiment was carried out using high molecular weight subunits, behaviour the reverse of that in elastin was observed.84 As the temperature rises, the amount of the slowly relaxing component decreases. This is equivalent to the absorption of water by an expanding protein mass. Such behaviour is typical of a hydrophilic system and is therefore not consistent with a hypothesis which requires elastin-like behaviour. Simultaneously with the developments with the high molecular weight subunits, systematic studies of proton relaxation and lineshape in the barley protein C-hordein have been r e p ~ r t e d . ' ~The . ~ ~strategy was to hydrate the protein in 2H20 and then to use the 'H behaviour as an indication of the protein behaviour at different temperatures and levels of hydration. The results showed that the protein, although insoluble, is highly mobile. C-Hordein, like the high molecular weight subunits, has a high density of glutamine and proline residues. Comparison of relaxation when the sidechain amide contained 'H or 'H suggested that rotation of this group was primarily responsible for spin-lattice relaxation in the laboratory and rotating frames. Analysis of these results, as well as transverse relaxation behaviour, led to the idea that the insolubility of C-hordein was the result of the formation of interchain hydrogen bonds by glutamine side-chains.

14

A. M. GIL, P. S. BELTON AND B. P. HILLS

60 40 20 0 -2040-60 6

60 40 20 0 -20-40-60

B

Fig. 2. Static (lower) and magic angle spinning (upper) spectra of hydrated C-hordein containing (A) 27% water and (B) 30% water. (Reproduced with

permission from Belton and Gi1.85)

Hydration resulted in the breaking of these bonds, but because of the high density of glutamine residues it was statistically very unlikely that all bonds could be simultaneously broken. The insoluble hydrated protein mass thus consisted of “loops” of hydrated protein together with “chains” of intermolecular hydrogen bonds.86 This idea has been extended, by analogy, to high molecular weight subunits, and forms a key part of a new hypothesis on the origins of the elasticity of these material^.'^ Not only have these studies contributed to the understanding of the behaviour of cereal proteins, they have also uncovered some rich NMR phenomena. The proton lineshape of hydrated C-hordein consists of a broad line with a narrow line superimposed on it.”@ This observation is fairly common in biopolymers. However, magic angle rotation causes the narrow component to narrow still further and lose fine structure. This is illustrated in Fig. 2. The origins of this effect lie in both chemical shift anisotropy and dipolar effects,85 and represent an interesting and unusual regime of be haviour .

APPLICATIONS OF NMR TO FOOD SCIENCE

15

Milk proteins continue to attract attention. Lamberlet and c o - ~ o r k e r s ~ ~ have evaluated the usefulness of low-resolution NMR in studying thermal effects of milk proteins. They found that the results were affected by ionic strength and the addition of caseinate or casein micelles. They propose that NMR can be used for determining either reversible or irreversible thermal denaturation of whey proteins in model systems. A multinuclear (25Mg, 31P and 43Ca) study of ion binding with p-casein has shown that there are at least two magnesium binding sites, one of which is unexpectedly strong.” Magnesium competes with calcium for binding sites, but sodium does not, under physiological conditions. The data for the dependence of the 43Ca chemical shifts required a five-site model but could not discriminate between the sites being identical and independent or operating with negative cooperativity .

3.3. Polysaceharides

3.3.1. Polysaccharides in solution The amylose content and degree of amylopectin branching were quantified in normal, high-amylose and waxy barley starches.” After enzymatic debranching treatment and gel permeation chromatography, ‘H NMR spectroscopy showed that amylopectin has longer chains in high-amylose starch (containing 40% amylose) than in the other types studied. The interactions of metal salts with amylodextrin were investigated by highresolution 13C NMR, using ethyl, isopropyl and t-butyl alcohols as model compounds.Y2Within the metal salts studied, potassium thiocyanate was an exception since, instead of causing up-field shifts on hydroxyl carbons on amylodextrin and in alcohols, it caused down-field shifts in C I and C4 of amylodextrin. This is similar to the effect observed for amylodextrin triiodide and other amylodextrin helical complexes, which suggests that a similar type of system may be formed in the presence of potassium thi~cyanate.~~ Carrageenans are an important class of sulfated polysaccharides used in food. Structural studies of various carrageenan oligosaccharides, at room temperature, have been carried out making use of high-resolution bidimensional ‘H and 13C NMR methods.93 Carrageenans may contain additional minor structural features that can, however, determine their functional properties. The set of 13C NMR absorptions produced by all diads potentially present in carrageenans have been either calculated or compiled from chemical shift data.94 Structural determination may be carried out by computer-aided matching of experimental data to the data bank reported. High-resolution ‘H NMR has helped to characterize carrageenans from Furcellaria lumbricalis and Eucheurna gelatinae, after degradation to

16

A. M. GIL, P. S . BELTON AND B. P. HILLS

oligosa~charides.~~ P-Carrageenan has been recently obtained from the sun-dried seaweeds E. gelatinae, E. speciosa and E. r n ~ r i c a t u r nCharacter.~~ ized by chemical analysis, optical rotation and NMR, P-carrageenan was shown to be devoid of ester sulfate. Gelling was found to be ionindependent, and the structure of the resulting gel is suggested to be less restrictive relatively to agarose gel.96 13C NMR was also used to help characterize the cell wall polysaccharide of Australian Cutenella nipae, which was found to be a highly sulfated carrageenan type of polysaccharide. The same work established that the dominant component of the extract is i-~arrageenan.~~ ‘H NMR and I3C NMR spectral parameters of eight sulfated uronic acid-containing disaccharides were used to determine the conformational dependencies on the pattern of ~ulfation.’~ These studies help to understand the role of charged groups on the conformations of the major class of sulfated polysaccharides relevant in food science. The role of carboxylate groups and sulfate groups and their interaction with cations on the three-dimensional structure of the disaccharides was extrapolated to the polymeric chondroitin sulfates and related to the resulting rigidity and flexibility of some glycosidic linkages.98 Several gum arabic samples from Uganda were analysed, and structural comparisons carried out by 13C NMR.” Alginic acids are heteropolysaccharides that are composed of varying ratios of p-D-mannuronic (ManA) and a-L-guluronic (GulA) residues. The ability of alginate solutions to form cross-linked matrices upon interaction with calcium ions provides a range of rheological properties that has led to a variety of applications in food products as well as in other areas. In order to investigate the effects of calcium ion binding on the structure of alginic acid, the high-resolution ‘H NMR spectra of poly-GulA (low degree of polymerization (d.p.)), poly-ManA (low d.p.) D-mannuronic acid and D-guluronic acid were fully assigned via two-dimensional methods. loo Conformational changes of the polymers upon calcium titration were examined, and a binding model was proposed. Combined NMR and molecular modelling studies were used to characterize the conformation of methylated pectic disaccharide (4-0-a-Dgalactopyranurosyl-l-~-methy~-cY-D-galactopyranuronic 6,6’-dimethyl diester).”’ The extrapolation of such information to a regular polymer structure shows that different helical structures can result from small changes in conformation, without any drastic variation of the fibre repeat. Algal cellulose may be detected in solution by 13N NMR spectroscopy, using lithium chloride-N-N-dimethylacetamideas a suitable non-degrading mixture for dissolving underivatized cellulose. lo’ A method of rapid analysis of cellulose-like regions in cereal P-glucans has been devised by the use of high-performance anion exchange chromatography, followed by characterization by 13C NMR. lo3

APPLICATIONS OF NMR TO FOOD SCIENCE

17

Wheat bran has been widely used as a source of dietary fibre, having been found to have several desirable functions at the intestinal level. Wheat bran hernicellulose was digested with a commercial Aspergillus japonicus hernicellulase, and some resulting oligosaccharides were purified and characterized by I3C NMR. lo4 Wheat flour oligosaccharides have also been obtained by digestion of alkali-extractable arabinoxylan with Aspergillus awamori. These fractions were identified by 'H NMR, showing chains of (1--+4)-P-~-xylopyranosylsubstituted at 0 3 and/or 0 2 , 3 with arabinofuranosyl groups. 105~106The structure of water-soluble wheat arabinoxylans, as studied by high-resolution NMR, has been correlated with physical properties such as gelling capacity and thermal stability. lo' Some wheat arabinoxylans have also been identified and characterized after acid hydrolysis and methylation

3.3.2. Solids, gels and heterogeneous sjstems Ready access to the wealth of data on the physical properties of complex carbohydrates is of great interest to those involved in the study and manipulation of food carbohydrate structure and functionality. Such information is available in various databases, recently described," including an NMR spectroscopy database. A variety of studies have been carried out on the gelatinization and retrogradation processes of starch. On heating above about 70°C starch absorbs water and gelatinizes. The starch gel so formed is not stable and, on cooling, starch crystallization or retrogradation occurs. This latter process is the main cause for the staling of bread and has, therefore, inspired significant interest in the food science community. 13C cross-polarizationmagic angle spinning (CP-MAS) was used to characterize wheat starch and wheat starch gels. l 2 Separate subspectra for crystalline and amorphous regions were obtained by the "delayed contact" NMR method, which consists of taking combinations of spectra corresponding to moieties with different rotating frame relaxation times. The CP-MAS starch spectrum of the freshly cooled product after gelatinization showed significant changes such as a 65% loss of signal intensity, and a relative increase of the intensity of some peaks in the amorphous region. After a few days, the spectrum showed an increase in the peaks corresponding to the crystalline components of starch. These results suggest that a starch gel consists of at least three types of regions: a portion of liquid-like properties (in which the carbons do not cross-polarize), an amorphous region, practically unaltered by the gelation process, and a crystalline region in which the starch form is strongly dependent on the water content."* The loss of molecular and crystalline order occurring during starch gelatinization was investigated for various types of starch (maize, waxy maize, wheat, potato and tapioca), after defined thermal pretreatment~."~As a short-distance range probe, I3C

'

18

A. M. GIL, P. S. BELTON AND B. P. HILLS

CP-MAS NMR spectra were used to quantify the "double-helix" content, whereas X ray diffraction was used to detect only the double helices that are packed regularly. Both levels of structure were shown to be disrupted during gelatinization. The corresponding gelatinization enthalpic values seem, however, to reflect primarily the loss of short-range order.ll3 The NMR method of Z spectroscopy or cross-relaxation was applied to the study of immobilized polymer in starch ~ a m p 1 e s .Spectra l ~ ~ of starch granules, freshly gelatinized and cooled starch and retrograded starch reflected different amounts and relative rigidities of immobilized starch chains, in each state. The use of wide-line 'H NMR with MAS confirmed the presence of immobilized starch as well as highly mobile starch fractions. The retrogradation process was shown to be accompanied by an increase in fraction of immobilized chains. Through cross-relaxation, the kinetics of ageing were expressed in terms of the changes in the amount and rigidity of solid-like components (see Fig. 4). The relative amounts of immobilized and mobile starch moieties were found to change with the ageing of gelatinized starch.' l 4 Molecular mobility and water organization in starch forms A and B were investigated recently through the application of the two-dimensional 'H-13C heteronuclear wideline separated (WISE) solid state NMR method. 'I5 From WISE spectra measured with a short mixing time it was estimated that 28% of the water present is bound to polysaccharide chains in forms A and B. It was also concluded that water is not preferably bound to particular sites of the glucose rings. Experiments at different mixing times revealed the existence of different water organizations between the starch forms. Spin diffusion studies showed that water mobility in form A is greater than in form B. This fact is related to the lower gelation temperature of the A form of starch. '15 The structural elements of starch gels may, in principle, be built either from macromolecules of single species (amylose or amylopectin) or develop owing to the interaction of different molecules. The roles of both types of polymers on starch structure formation have been investigated by a combination of T2 relaxation measurements, rheology and X ray diffraction,l16 as a function of temperature and relative quantities of the three components: amylose, amylopectin and water. Results showed that aggregates forming during starch gelatinization are composed of amylose and that the polymer amylopectin acts as a precipitating agent. It is suggested that the resulting gel consists of a polymeric network filled by amylopectin macromolecules. 'I6 31P NMR has also been extensively used in the study of starch and its constituents. The phosphate ester groups on potato starch contribute to its clarity and viscosity when it is cooked to a paste. Both the number and the location of phosphate groups determine those properties, and both factors have been studied by 31PNMR. An enzymatic method for the determination

APPLICATIONS OF NMR TO FOOD SCIENCE

19

of the amount of phosphate bound to glucose C6 atoms in potato starch has been established and applied to a number of potato plants. 31P NMR has been used on samples before and after the enzymatic treatment, showing that the major signal observed represents C6-bound phosphate. '17 The location of phosphates has been studied on a phosphorylated wheat starch, using some mono- and disaccharides as well as potato starch phosphodextrins as model compounds."' High-resolution 31P NMR spectra and 'H NMR spectra (particularly of the anomeric region) of all compounds enabled the assignment of the 31P peaks to mainly glucose C6-bound monophosphate esters and lower levels of C3-bound and CZbound monophosphates. 31P NMR has also been applied to the study of the structure of the starch granule and, particularly, to the location of amylose in the granule.'19 Native maize starch was cross-linked with P0Cl3 and separated in fractions soluble and insoluble in DMSO. The former contained amylose molecules of small size and the latter contained cross-linked amylopectin and larger amylose molecules. 31PNMR peaks at -1.0 and 1.0 ppm, corresponding to phosphate diester, were observed for the insoluble cross-linked fraction whereas peaks at 4.3 and 4.9ppm, registered for the soluble amylose fraction, denoted only a small amount of phosphate monoester. These results were interpreted as indicating that amylose molecules are randomly interspersed in the granule instead of being in bundles. '19 With the increasing interest in starch associated with the food and paper industries, textile manufacture, pharmacology, and biomedical and pharmaceutical research, impressive activities on the modification of natural starch to obtain polymer networks have been carried out. In order to characterize the networks formed, amylose is often used as a model. Amylose networks obtained by complexation of calcium and by covalent cross-linking with epichlorohydrin were studied by solid state 13C NMR and X ray diffraction.'*' The 13C CP-MAS spectral lines of the amylose network prepared by the calcium procedure were very narrow, and fine splitting was observed. The splitting in the C1 region was interpreted as indicating the formation of a short-range helical structure. The spectral lines of the network formed by reaction with epichlorohydrin were broad and almost structureless, indicating a highly disordered and amorphous system. These results were supported by X ray diffractograms.'*' The differences in structure were related to the relatively lower enzymatic degradation rate of the amylose network formed by addition of epichlorohydrin. Phase transitions associated with ordering/disordering processes have been extensively studied, and attention has recently turned to glass transitions. The glass transition of amylopectin was studied by recording the 'H NMR free induction decay (FID) after a 90" p u l ~ e . ~The ' fast decay (rigid) component, with T2R,corresponds to solid polysaccharide protons in the glassy or crystalline states whereas the slow (mobile) component, with

20

A. M.GIL, P. S. BELTON AND B . P. HILLS

T2M,reflects water protons and exchangeable protons. After the rigid lattice limit (RLL), the increase of T2, with temperature is attributed to the onset or increase in frequency of the motion of groups containing hydrogen. The RLL determined by NMR was found to be 20-30°C lower than the Tg measured by DSC. The thermal results also suggest an increase of Tg with crystallinity whereas NMR shows an initial difference between amorphous and 2% crystalline samples, but no difference between 2 and 4% crystalline samples. It is suggested that this is because NMR looks at short-range mobility which may not be detected by X ray diffraction, while DSC and DMTA look at larger-scale effects.30 The CP-MAS spectra of amylopectin were recorded as a function of water content and, at water contents higher than 17.5%, showed the disappearance of the 82 ppm peak, characteristic of amorphous amylopectin, whereas the C1 signal at 100 ppm shows a triplet multiplicity, characteristic of A-amylose. These changes indicate the occurrence of molecular reorganization. The water content at which these changes occur coincides with the one at which the RLL is observed by NMR.30 Potato starch maltodextrins are a special group of reversibly gelling polysaccharides. Pulsed low-resolution NMR, at a field strength of 20 MHz, was applied to follow the gelation process in different thermally reversible rnaltodextrin-water systems. 12' Results were expressed by the ratio of proton populations with high and low relaxation times following a 90" pulse. A linear relation between the solid-liquid ratio from NMR and X ray crystallinity suggested the formation of highly ordered domains as essential constituents of the gel network. The effect of the presence of amylopectin or acetylated maltodextrins on the gelation process was investigated. Part of the economic interest in carrageenan polysaccharides lies in the diversity of their rheological properties, from pure thickening ( A ) , to soft, elastic gels (1) to hard, brittle gels (K). A coil-helix transition is induced by lowering the temperature or adding salt.lz2 The salt effect has been interpreted as stabilization of the polymeric helices by screening out of the electrostatic interactions. In addition to this general salt effect, some ions specifically promote the formation of helices via site binding of the ions. Anion specificity is generally observed to follow the lyotropic series (C1- < Br- d NO3- < 1- < SCN-. However, the K-carrageenan and furcellaran (low-sulfated carrageenans) anion specificity is opposite to the one commonly observed since anions stabilize the helical conformation against the coil conformation. Further, some deviations from the lyotropic series are observed.'** Previous 12'1 NMR studies had shown a dramatic increase in line width with the coil-helix transition, indicating binding of I- to the K-carrageenan helices. The technical difficulties associated with the strong nuclear electric quadrupole of 1271 ( I = "2) have been tackled to some extent by a more recent study.12* The single-pulse excitation was replaced by a composite pulse where the phases of the constituents are cycled

APPLICATIONS OF NMR TO FOOD SCIENCE

21

producing spectra of improved signal-to-noise ratio. The 1271spectra of standard samples in the extreme narrowing regime were recorded and used as signal intensity references. Measurements of transverse relaxation, longitudinal relaxation and spectral signal intensity were registered as functions of temperature. Signal loss caused by motional correlation times outside the extreme narrowing regime made it difficult to analyse line widths. Longitudinal relaxation times, however, proved more suitable for a comprehensive study, enabling a lower limit of lO-'s to be set up for the residence time at the binding site. 122 The role of cations and of water in the gelation of K-carrageenan has been studied by 23Na, 87Rb and 133CsNMR.'23 The NMR intensity of gel-forming cations undergoes significant change in the vicinity of the sol-gel transition whereas the NMR intensities of the non-gel-forming cations, which coexist with the former, d o not change. This was interpreted as being evidence of selective cation interaction with the polysaccharide. Proton T2 relaxation times showed the existence of three populations of water molecules in the gel system: free bulk water, water strongly bound to polysaccharide and water weakly bound to the network.'23 The functional properties of mixed carrageenan gels have been little studied. Such knowledge should enable the detection of small quantities of a distinct carrageenan type in samples supposedly pure in one type of carrageenan. These small amounts may have a great impact on the rheology and apparent ion selectivity of the system. A rheological method has been devised so that as little as 2% of K-carrageenan may be detected in samples of 1-carrageenan.lZ4 Impurity levels measured rheologically were confirmed by measurements of I3C NMR spectra. Decreasing sodium content in processed foods is a primary concern today. A major problem is to make a low-sodium product with similar chemistry, stability, texture and sensory attributes to that of the full-sodium product. Some studies have investigated the effect of the nature of the hydrocolloid (ionic or non-ionic) on the interaction with sodium ions. 125 The ionic hydrocolloids xanthan and K-carrageenan and the non-ionic guar and locust bean gum were studied. After trace metal analysis of the samples, the 23Na transverse relaxation rates (R 2 ) (inverse seconds) were calculated from the line widths at half height, as a function of added sodium. A t low added Na+, R2 decreased rapidly and was larger for the ionic gums, denoting preferential binding to those than to the non-ionic gums (Fig. 3). At high added Na+, R2 values of ionic gums levelled off and approached the non-ionic gum values. A two-state model with fast exchange, between bound and free Na+, was used to interpret the relaxation behaviour in the ionic systems. A study of xanthan at very low Na+ concentrations suggested a possible competition process between Na+ and endogenous concentrations of K + and Ca2f.125 The polysaccharide xanthan is now widely used as a stabilizer of oilin-water emulsions in most salad dressings. Xanthan has also been found

22

A. M. GIL, P. S. BELTON AND B.

100

1

.

~

.

.

.

~

.

I

80 1

v)

. 9. . , , . . . . , . - - -

.

100

I

n

r

,

.

3

80

P. HILLS

-

1

~

.

\

.

.

~

.

.

.

~ I *

.

~

-.-

~ I

----

~

- 8- Kappa-carrageenan 8 - Xanlhan - - o Guar - x - Locust bean

-

'

I

W

rh

~

'

~

I

-

\

K 60

5 z a

z

40

20 0

2 0 0 400 600 8 0 0 l O O O " 3 0 0 0 5000

Added NaCl (mg/lOOml) Fig. 3. 23Na NMR R2 for 100 mg/100 ml wcarrageenan, xanthan, guar and locust bean gums as a function of added NaCI, 20-5000 mg/100 ml (note break in x axis). The insert shows the 20-250mg added NaCl/lOOml range. (Reproduced with permission from Shirley and S~hmidt.''~)

to have the role of suppressing oil peroxidation by the inactivation of metal The property of Fe2+ binding to xanthan has been ions such as Fe2+.126 investigated, and results suggest that the metal binds through a pyruvate residue. This has been supported by the high-resolution 'H NMR spectra of xanthan before and after addition of Fe2+;the selective broadening and shift to low field of the pyruvate peak indicates its proximity to the paragmagnetic metal ion.'26 The gelation of pectins with a high degree of esterification (HDE), in the presence of different co-solutes, was investigated by measuring 'H and 23Na Tl and T2 relaxation times.127 Both proton transverse and longitudinal relaxation times decrease to a constant value, as pectin concentration increases. This has been interpreted as reflecting a progressive decrease in mobility due to the formation of higher number of junction zones, for higher concentrations. The effect of the co-solutes ethanol, t-butanol and dioxane on the relaxation behaviour helped to establish that the formation of junction zones in HDE pectins is largely dependent on hydrophobic interactions between methoxyl groups. Scleroglucan, a highly polar polymer, is soluble in water, and generally yields, at low concentration, highly viscous solutions which are used as

APPLICATIONS OF NMR TO FOOD SCIENCE

23

thickeners in the food industry. This polysaccharide is composed of repeating units of our D-glucopyranosyl residues; one of these is linked as a pendant sugar to the main p(1-3) chain.lZ8 Solid state and solution 13C NMR were used to follow the fixation of water molecules on anhydrous scleroglucan and the evolution of the complex from the solid state to the gel and solution state. Determination of chemical shifts and proton and carbon relaxation times show that hydration to 21% water leads to an increase in solid state order and in chain mobility. The gel state, containing 90% water, was shown to be of an amorphous nature and to be characterized by a triple-helix model of considerable molecular rigidity. 128 Konnyak is a gel of konjac glucomannan (KGM), which is frequently used in traditional Japanese dishes. In order to characterize the structure and behaviour of this gel, measurements of dielectric coefficient, elastic moduli and broad-line NMR were carried out in the temperature range -180 to 150°C. Mechanical and dielectric loss at - 100°C were attributed to rotational motion of hydroxymethyl groups. Decrease of the NMR second moment at 0 to -60°C suggested that such motion is hindered in KGM, to a larger extent than in other polysaccharides such as amylose, dextran or pullulan. Methyl and hydroxylpropylmethyl derivatives of cellulose have the unusual property of forming gels on heating and reverting to the solution state on cooling, with practical industrial applications, including in the food industry. 13" The thermogelation of methylcellulose was studied by 'H NMR, along with other techniques. The proton spectra of the system were obtained at different temperatures in the 3MO"C range. The integrating intensities below 4.2 ppm correspond to most cellulose non-exchangeable protons and to methyl substituents. The changes in proportion of visible NMR signal during heat-induced gelation and dissociation upon cooling were studied. The overall trend is towards higher intensity at lower temperatures, but the slight reduction in the visible NMR signal with decreasing temperature (at the bottom end of the range studied) suggests that in the sol state there is a substantial proportion of the polymer remaining conformationally immobile. 130 The proposed interpretation of these findings is that methylcellulose chains exist in solution as aggregated "bundles", held together by packing of unsubstituted regions of cellulose and by hydrophobic clustering of methyl groups in regions of denser substitution. 130 As the temperature is raised, the bundles come apart, exposing methyl groups to the aqueous environment and causing an increase in volume. At higher temperatures, a hydrophobically cross-linked network is formed. Due to the importance of water retention of dietary fibres in the colon, a variety of dietary fibres have been evaluated according to their hydration ability.131 Pulsed proton NMR was used and the shape of the relaxation curves studied as well as parameters such as the ratio of the different

24

A.

M.GIL, P. S. BELTON AND B. P. HILLS

relaxation components. The behaviours of water-soluble dietary fibres and water-insoluble fibres were compared. Taking into account the distribution and position of polar groups in the molecules, the 6-carboxylate group seemed to determine most of the hydration behaviour of the polysaccharides. Sodium salt forms showed a more pronounced hydration ability than corresponding protonated forms. 13’ The in vitro and in vivo digestion of insoluble dietary fibres was in~estigated.’~~ Proton NMR spectra of the arabinoxylans in digesta of chickens helped to confirm the in vivo studies, which indicated preferential enzymatic degradation of soluble monosubstituted xylose residues, relative to un- and disubstituted residues.’32 3.4. Plant cell walls Plant cell walls are of major importance in foods. They contribute to the texture of plant foods, and as a major source of dietary fibre have an important nutritional role. Much of the literature on plant cell walls has emphasized its phytochemical and botanical importance rather than its relevance to food. However, since much of the methodology developed is equally relevant to food applications the literature is reviewed here. Cell walls can be regarded as a heterogeneous polymer matrix, and one of the classical ways to apply NMR to such a system is to look at proton relaxation processes. Such an approach has been pioneered by Taylor and MacKay and co-workers. In an early paper133 they observed proton relaxation in cellulose, calcium and sodium pectate gels and bean cell walls. The polymers were immersed in 2H20 to avoid interference from water signals. The FIDs of all samples showed two components: a fast relaxing component described as “rigid”, and therefore, supposedly, Gaussian, and a more mobile fraction, presumably exponential or multi-exponential. In cell walls the rigid component represented 60% of the magnetization, and had a second moment intermediate between that of cellulose and pectate. This is consistent with a model in which the rigid component arises from cellulose (30% of cell wall material) and xyloglucans bound to cellulose. The rigid and mobile components also had different T I values. Dipolar relaxation (TD) was two component in the cell walls and was dependent on pD. The results were interpreted in terms of a speculative model in which increasing protonation weakened some intermolecular interactions allowing more motion to occur. A subsequent development to the approach described has been to extend the range of relaxation measurements to include TIP and the use of the 90,-~-90, sequence to measure interpair second moments. These methods have been applied to cellulose’34 and bean cell walls. 135-137 On the basis of these measurements a cell wall model was proposed which incorporated the

APPLICATIONS OF NMR TO FOOD SCIENCE

25

dynamic features of the biopolymers. Rigid cellulose microfibrils have an outer sheath of rigid hemicellulose molecules. Attached to these are pectic and other hemicellulose molecules which are loosely suspended in a large volume of water.13s In a subsequent study, 136 chemical fractionation was used. This showed results consistent with the proposed model but indicated that removal of the pectin resulted in the loosening of the less lightly bound hemicellulose and that removal of this in turn loosened the remaining hemicellulose. The indication of these results is that sequential chemical removal of cell wall polymers does not leave the remaining cell wall unchanged. This conclusion has also been supported by a combination of neutron scattering and magnetic resonance methods. 13* An alternative approach to cell walls is to employ solid state highresolution 13C NMR. The heterogeneity of the cell walls makes the use of the analysis of chemical shift anisotropy difficult, and most work has concentrated on reporting one-dimensional spectra under conditions of rapid rotation. Under the conditions of dipolar decoupling and/or crosspolarization, quantitative information is difficult to obtain. Polarization transfer rates for the uronic acids vary with the age of the tissue,139 and are likely to vary between different polysaccharide types. 139 Aromatic signals from lignified tissue cross-polarize very slowly and tend to have low intensity in the spectrum.14" This problem can be overcome by using a single-pulse excitation method,141 but even here care is required: spin-lattice relaxation in such a system can be very slow and rotation must be sufficiently rapid to spin out side-bands. The slowness of spin-lattice relaxation makes data acquisition very time-consuming; however, by optimizing contact times and using dipolar dephasing to eliminate unwanted carbohydrate signals14' good estimates of the bound phenolic substances in flax have been obtained using cross-polarization methods. The chemical shift of cell wall polysaccharides is dependent not only on chemical constitution but also on the physical state of the material, particularly conformation and packing.I4' Considerable progress has been made in the assignment of resonance^'^^^^^ and the effects of successive removal of polysaccharides in Vigna radiata have been examined14* in order to examine the contribution of the various polysaccharides to the overall spectrum. In order to carry this out, an assumption was made that removal of polymers did not affect the signals from the remaining polymers. This assumption may not be valid in the light of previously reported proton relaxation results,'36 and it would be interesting to carry out a combined proton and 13C study of a system to further investigate this problem. The nature of cellulose in the cell wall is likely to be of importance in the determination of the eating quality of fruits. A detailed CP-MAS study of cellulose in apple cell walls'46 has indicated that most of it is in crystalline form with crystallites containing about 23 polymer chains per crystallite. There are also disordered chains on the crystallite surfaces.

26

A.

Table 6.

M.GIL, P.S. BELTON AND B. P.HILLS I3C chemical shifts and assignments obtained from CP-MAS experiments on plant cell walls.

Chemical shift (PPm)

Origin

Carbon atom

Celery cell walls 175.3 174 171.7 105.1 101.5, 100.2 88.5, 86.9 81.8, 80.0 75.1, 72.2 68.3 64.7 61.6 53.5 21 17.6

Galacturon, non-esterified Acetyl Galacturon esterified Cellulose, P-galactan p-Glucosyl, a-xylopyranose, P-galacturon Cellulose I Morphous cellulose, xyloglucan P-glucosyl Hexoses and galacturonic acid Cellulose I P-Glucosyl, p-galactosyl Amorphous cellulose Acetyl Pectic methoxyl Rhamnose

C6 Carbox yl C6 c1 c1 c4 c4 c2, c3, c 5 C6 C6 C6 Methyl Methyl C6

Oil palm leaf cell walls 172.7 152.9 149.5 147 136 132.8 132.8 116 116 109.5 105.3 88.7 84.2 75.0, 72.8 64.7 64.7 63.3 56.5 32.6, 29.9 21

Acetyl Ether-linked syringyl Ether-linked guaiacyl Guaiacyl and syringyl not ether linked Syringyl Guaiacyl Syringyl Guaiacyl p-Hydroxyphenyl Arabinofuranosyl Cellulose, xylan Crystalline cellulose Amorphous cellulose, xylan Cellulose, xylan Crystalline cellulose Xylan Amorphous cellulose Aromatic Cutin Acetyl

Carboxyl c3, c 5 c3 c3, c 5 c1 c1 c4 c5 c3, c 5 c1 c1 c4 c4 c 2 , c3, c 5 C6 c5 C6 Methoxyl Methylene Methyl

Millet cell walls 170-180 154 140-148 140-148 140-148 130-140 11C130 11C130

Various carboxyl groups Ether-linked syringyl Guaiacyl Syringyl not ether linked Ether-linked syringyl Aromatic rings Guaiacyl p-Hydroxyphenyl

c3, c 5 c3, c 4 c3, c 4 , c 5 c4

c1

C2, C5, C6 c3, c 5

APPLICATIONS OF NMR TO FOOD SCIENCE

27

Table 6.-contd. ~

Chemical shift (PPm) 102-109 102-109 102-109 100-102(?) 89 84 78-80 72-75 72-75 64 64 62 56 29-33 21

Origin Cellulose or xylans Syringyl Fructans Free sugars Crystalline cellulose Amorphous cellulose Xylans Cellulose Xylans Crystalline cellulose Xylans Amorphous cellulose Aromatic Long-chain waxes and alcohols Acetyl

Carbon atom

c1 C2, C6 c2

c1

c4 c4 c4 c 2 , c 3 , c5 c2, c 3 C6 c5 C6 Methoxyl Methylene Methyl

Data compiled from refs 143-145 and 147.

CP-MAS spectra of rind, parenchyma and vascular bundle fractions of pearl millet have been reported and some assignments of resonances made.147 Treatment of the tissues with alkali resulted in the reduction of signals from aromatic residues. A partial list of resonance assignments is given in Table 6. The table needs to be used with some caution. Chemical shifts and lineshapes in polysaccharides can be very dependent on water content and a range of chemical shifts assigned to the same carbon atoms is to be expected. One of the great attractions of solid state carbon NMR is the ability to measure the relaxation parameters of specific chemical activities within the cell wall. This has been very extensively exploited in a study of the suberization of potato cell wall.15" Suberin is a polyester that grows in response to wounds, and its function is thought to be the prevention of tissue invasion by fungi and bacteria. 150 By careful examination of proton T1, as well as 13C T1and T I , using CP-MAS it was possible to show that the effect of suberization was to decrease motion of the cell wall polymers in the megahertz range and enhance them in the kilohertz range. This, in effect, may be regarded as a stiffening process resulting in hampering of local segmental motions. Presumably such a decrease in motion inhibits invasion. Interestingly, the related polyester material cutin enhances motion when in contact with wax chains in lime cuticle.'51 More recently, isolation and spectral characterization of cutin and suberin have been reported. 15'

28

A. M. GIL, P. S. BELTON AND B. P. HILLS

3.5. Z spectroscopy and biopolymers Food mechanical and rheological properties often depend on the flexibility of macromolecular networks. In the food science context, it is important to characterize the structure and dynamics of coexisting structural components but, in many cases, relatively immobile or rigid regions may be present in small quantity, thus making them difficult to detect. A recently developed NMR method, denominated cross-relaxation or Z spectroscopy, enables the selective detection of solid-like domains and is sensitive to the dynamic characteristics of the solid components, over a wide range of mobilities."4 The cross-relaxation experiment probes the magnetic and dynamic properties of the solid components through the observation of the liquid signal. The sample is initially irradiated with a preparation pulse that is offresonance from the liquid signal to be measured. After the preparation pulse, an on-resonance 90" observation pulse measures the effect on the liquid magnetization. The principle of this technique is that due to the cross-relaxation process partial saturation of protons in solid components will transfer to water during the preparation period. The degree of saturation of the solid components depends on the irradiation frequency and on the relaxation rates in the solid moieties. The resonance intensity of the liquid, magnetically coupled to the solid, is plotted as a function of the off-resonance frequency of the preparation pulse producing a spectrum that reflects the solid NMR spectrum: the cross-relaxation spectrum. The cross-relaxation spectrum may be presented in the form of a plot of (1 - MAZ/MAZO)as a function of offset frequency, where MAZ is water intensity with a saturation pulse and MAZo is water intensity without saturation. The lineshape of the spectrum is dependent on the amount and relative rigidity of the solid component: the more solid component the sample contains and/or the more rigid the solid component is, the broader and more intense will be the resulting cross-relaxation spectrum. '14 One advantage of the use of the cross-relaxation NMR method is that it may, in principle, be executed on a low-field (5-20 MHz), low-resolution, singlefrequency ('H) spectrometer of relatively low cost. The Z spectroscopy method was applied to the study of immobilized polymer in starch samples. '14 Comparison of spectra of starch granules, freshly gelatinized and cooled starch and retrograded starch showed that they reflect the amount and relative rigidity of immobilized starch chains (Fig. 4). Spectral intensities for freshly gelatinized starch were significantly dependent on starch concentration, and retrogradation was shown to introduce a broad component in the spectrum. A wide variety of segmental mobilities is suggested, their distribution changing as gelatinized starch ages. The same method of cross-relaxation has been applied to detect and quantify solid components and to follow their changes during ripening of banana.153 The evolution of solid-like phases in banana during ripening was

el

APPLICATIONS OF NMR TO FOOD SCIENCE 1.0

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, 30

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60 -60

-30

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Offset Frequency (kH5) Fig. 4 ‘H cross-relaxation spectra of 10% waxy maize starch: (a) suspension of granules in 0.25% acqueous xanthan solution; (b) freshly gelatinized and cooled ( X ) , and DMSO solution (0); (c) retrograded gel (5°C for 30 days). (Reproduced with permission from wu et al.’ 4,

monitored. Cross-relaxation spectra show that during ripening there is a dramatic decrease in the area of the broad component (from 20 to 2% w/w). This is consistent with the fact that unripe banana is rich in starch and that ripening is accompanied by starch conversion to soluble sugars. Two superimposed spectral components are observed, a broad and a narrow one. The area of the former decreases with ripening whereas the area of the latter increases during the process. The nature of both broad and narrow components in the cross-relaxation spectrum is discussed in terms of the chemical composition of banana. The wide-line proton spectrum of banana was obtained with the solid echo pulse sequence and showed two components, the broader of which arising from starch.ls3 The results of the quantification of soluble sugars by high-resolution ‘H MAS NMR and of solid starch by the cross-relaxation method confirmed the hydrolytic conversion of starch to sugars during banana r i ~ e n i n g . ” ~

4. ANALYSIS

4.1. Introduction

Interest in NMR as an analytical method is continuing to develop, and the area has been reviewed.ls4 One of the interesting trends is the reexamination of the idea that NMR is an insensitive technique. As higher field strengths become available, very high sensitivities may be achieved. Even with the relatively modest (by current trends) proton frequency of 500MHz it has been shown’’5 that for benzene in water the detection limit is 35 ng/ml and for N-nitrosodimethylamine 510 ng/ml. Both these figures represent detection at the sub-parts per million level. Often in food systems straightforward high-resolution solution state methods are not suitable.

30

A. M. GIL, P. S. BELTON AND B. P. HILLS

Lines may be broadened by motional effects, and thus require the use of wide-line or pulse methods, or they may be broadened by susceptibility effects. In the latter case, MAS can restore resolution. For at-line or in-line applications or routine quality control laboratories, cost is often a factor. In order to lower costs, low-field systems are required together with simple electronics. This need continues to drive exploration of low-field pulsed N M R methods and has renewed interest in rapid scan correlation (RSC)NMR. A continuing major application of N M R in food science is the use of isotopic and other methods for the authentication of foods and the detection of adulteration. As the market for foods with specific claims to geographic origin, particular forms of processing or “naturalness” grows, so apparently does the level of fraud. The various topics outlined above are reviewed in the following sections. 4.2. Developments in authentication The importance of authentication and the detection of adulteration by N M R is attested by the continuing output of reviews and reports on the subject. Many of these do not offer any new N M R approaches but reflect applications of standard methods to new areas. One new application is the use of very high-field NMR as a proximate analysis method.’56 The use of I3C N M R combined with discriminant analysis157 has been reported for identifying olive oils. It was shown that it was possible to distinguish different grades of oil by this method. Deuterium N M R is still finding new applications in the detection of adulteration. An example is the distribution of deuterium in the aromatic sites of ben~aldehyde;’~~ this has enabled the detection of synthetic benzaldehyde in bitter almond oil. The remaining publications in this area are listed in Table 7. 4.3. Process applications

N M R does not lend itself very easily to process applications; nevertheless, there continues to be interest in the field and progress is being made. Reade17’ has considered some of the problems involved in applying N M R on-line in the food industry. Among the most important applications of N M R are the determination of oil and water contents171 and the solids contents of fats. For the latter application a new low-field instrument has been devised. 172 The use of N M R to measure oil and moisture contents in seeds is valuable because the non-destructiveness of the technique allows measurement of seeds to be used in breeding programmes as well as in general quality

APPLICATIONS OF NMR TO FOOD SCIENCE

31

Table 7. Recent publications concerned with the authentication of foodstuffs. Subject

Ref.

Comments

Deuterium NMR of wines Preparation of musts Use of deuterium and carbon NMR Isotopic methods SNIF-NMR NMR for authentication and adulteration Authentication of essential oils SNIF-NMR for flavours and fragrances SNIF-NMR for fruit juices SNIF-NMR Authentication of decanolides

159 Refers to Lombardy wines 160 New method proposed 161 Review 162 Review 163 Review 164 Review 165 Use of GC, GC-MS and NMR 166 Review

167 Review 168 Review 169 Uses 2H NMR

GC, gas chromatography; MS, mass spectrometry; SNIF-NMR, site-specific natural isotope fractionation studied by NMR.

Where results are to be shared between laboratories, it is vital to ensure traceability, and certified reference materials need to be available. The Community Bureau of Reference (BCR) has developed these for rapeseed. 177 An unusual and interesting application of N M R is for the control of cake ~ o o k i n g . ' ~ 'The approach was to sample rapidly and slowly relaxing components of transverse relaxation to characterize the liquid-solid ratio in the material. The results were then analysed for an experimental design in which ingredients and cooking times were systematically varied. Transformation of the variables was performed using principal components analysis (PCA). It is clear that much work remains to be done, but this approach seems to suggest a route to the use of relatively simple N M R measurements in the control of complex processes. The non-destructive detection of fruit quality by relaxation time measurements has concentrated on the role of sugars in ripeness'79 and their estimation in intact fruits.'" However, 31P N M R may be used to examine the effects of low oxygen levels and p H changes in fruit ripening.''l 31PN M R has also been used to measure phospholipid contents in peas.'** 4.4. RSC methods

One of the problems of the food industry is that it is typically an industry where profit margins are low. This means that quality control instruments must be of low cost, otherwise they do not pay for themselves. Sophisticated high-field instrumentation is, notoriously, not low cost. If the advantages of N M R are to be brought to the food industries' quality control problems they

32

A. M. GIL, P. S. BELTON AND B. P. HILLS

must come in a suitably low-cost form. One way of doing this is to revert to low-field methods and use the relatively cheap electronics associated with continuous wave methods. Experimentally this brings with it the difficulty of low signal-to-noise ratios and long scan times if distortion of the resonance lines is to be avoided. These problems can to a large extent be ameliorated by the use of RSC NMR.ls3>ls4In this technique the spectral range is scanned rapidly. This results in a spectrum which is strongly distorted by oscillations in intensity following each peak maximum. ls3 This is caused by the convolution of a residual component of transverse magnetization with the swept field. The origins of this are well understood, and the intensity can be described by the simple equation ~ ( t =) exp(-ib?) where b is the sweep rate in radians per second and f is the time after the peak maximum. Deconvolution may be achieved by reverse Fourier transformation of the spectrum, multiplication by the appropriate function in the Fourier domain. Forward transformation then recovers the spectrum. When this method is used for proton spectra on a 60 MHz machine, surprisingly good results can be obtained, an illustration of which is given in Fig. 5 . Ethanol could be

I

1

8.0

1

7.0

I

I

1

I

6.0 5.0 4.0 3.0 CHEMICAL SHIFT ppm

1

1

1

2.0

1.0

0.0

1

Fig. 5. An RSC proton NMR spectrum of a margarine sample. (P. S. Belton and B. J. Goodfellow, unpublished results.)

APPLICATIONS OF NMR TO FOOD SCIENCE

33

measured quantitatively in water to levels as low as 0.01%, and the technique compared favourably with gas chromatography and density methods for the analysis of the ethanol content of wines and beer.'83,184 Similarly, quantitative measurements of glucose and estimates of unsaturation in oil were made.184 In general, the technique seems to hold some promise as a low-cost, non-invasive, minimal preparation method of food analysis.

4.5. MAS methods

Observation of emulsion phases is important in studies of emulsion stability and capacity as well as in quantification. A 'H NMR method has been used to quantify the different phases in the multiple emulsion water in oil in water (W/O/W) whose dispersed oil drops contain even smaller droplets of a dispersed aqueous phase. 18' MAS was applied, showing that it greatly improves resolution by removing broadening due to magnetic susceptibility mismatch among phases. This result will enable more detailed studies of chemical and physical processes in emulsions. Addition of the shift reagent [DyEDTAI- to the external water phase, in combination with MAS, allows separation and quantification of external and internal water phases. 185 Selective observation of internal water was achieved by T2-selective application of the CPMG spin echo sequence, leading to elimination of external water resonance. MAS methods may be applied to study the liquid phase in fruit tissue, 153.186.187 N arrow peaks for water or metabolite nuclei are expected, since the primary line-broadening interactions should be suppressed by fast molecular reorientation. This enables the study of metabolism in intact plant tissue, and its application to foods has been previously reported. 18' High-resolution 13C NMR spectra of intact fruit tissues-grape, peach, persimmon, banana and apple-were obtained while spinning the sample at the magic angle to improve resolution (see Fig. 6). Only low speeds (of the order of a few hundred hertz) were required to reduce susceptibility broadening, which decreased as apple > banana > grape. 18' Fructose, glucose and sucrose resonances appeared with correct chemical shifts, thus permitting their identification and quantification. The most abundant anomers of fructose and glocose were observed. Scalar IH-I3C couplings were observable as multiplets in spectra taken without proton decoupling, thus aiding the assignment. Measured values of the nuclear Overhauser enhancement effect and longitudinal time T1 were consistent with rapid, effectively isotropic tumbling of sugar molecules, at rates very close to those observed in solutions of pure sugars. The method developed is capable of detecting sugar anomers in concentrations at least as low as 0.5%.'*' The relatively low sensitivity of 13C led to long acquisition times, and minor

34

A. M. GIL, P. S. BELTON AND B. P. HILLS

~

110

"

'

l

100

"

'

l

90

"

'

l

m

'

'

70

'

~

'

Bo

Chemical Shift I ppm

Fig. 6. Proton scalar decoupled 50MHz I3C NMR spectra of intact fruit tissue (effect of MAS shown in lower spectra): (A) grape, (B) persimmon, (C)banana and (D) apple. The vertical scaling been adjusted for convenient display. (Reproduced with permission from Ni and Eads.Is6)

APPLICATIONS OF NMR TO FOOD SCIENCE

35

x1

Chemical Shift/ppm

Fig. 7. 'H NMR spectrum (200MHz) of intact banana fruit tissue: (A) non-MAS spectrum obtained in a conventional high-resolution probe with sample axis parallel to magnetic field; (B) MAS spectrum obtained without water peak suppression; (C) vertical expansion of (B); (D) MAS spectrum obtained with water peak suppression. The signal-to-noise ratios in spectra (C) and (D) are 55 and 1137, respectively. The MAS rate was 1.05 kHz. (Reproduced with permission from Ni and Eads.ls7).

components were not visible, which makes routine quantification rather difficult. Another method was advanced, consisting of the recording of the MAS 'H NMR spectra using water peak ~uppression'~'(Fig. 7). As indicated before, MAS reduced susceptibility broadening while water suppression increased the dynamic range, enabling the detection of liquid phase components with concentrations as low as 0.01%. Water, glucose, fructose, sucrose, organic acids (malic, citric, tartaric) and total fatty acyl lipids were quantified. Longitudinal relaxation times of non-exchangeable glucose protons were measured in situ in banana and found to be dominated by

36

A . M. GIL, P. S. BELTON A N D B. P. HILLS

intra- and intermolecular homonuclear dipolar interactions, as found for the in vitro situation. Apparently, rotational motions of the water and sugar molecules within the fruit are relatively ~ n h i n d e r e d . In ' ~ ~a separate study, the increase in soluble carbohydrate during ripening of banana was monitored by high-resolution proton NMR, using low-speed MAS and water peak suppression. 153 Resonances of water, sucrose, fructose, glucose, fatty acyl chains of mobile lipids, and organic acids were easily detected and their changes with ripening quantified.

4.6. Other applications

4.6.1. Tea, coffee and wine

Pu-erh tea is a kind of Chinese tea, manufactured in specific provinces, and which undergoes periods of postfermentation and piling processing longer than those for dark teas. '*' These differences in the processing are believed to have a determining effect on the production of the characteristic aroma and colour components of Pu-erh tea. 13C NMR studies, along with chromatographic techniques, showed that carbohydrates and amino acids as well as catechins are digested during the microbial fermentation process, the main constituents of made-tea being caffeine, gallic acid and 2 - 0 - p - ~ arabinopyranosyl-myo-inositol. The volatile components were identified by gas chromatography and gas chromatography-mass spectrometry. The film of tea scum which forms on the surface of tea brewed in hard water has been characterized by several techniques, including powder diffractometry , electron microscopy, Fourier transform infrared (FTIR) spectroscopy, mass spectrometry and solid state 13C NMR."" The presence of inorganic carbonate was confirmed by FTIR and 13C NMR, and the same techniques pointed to the presence of hydroxyl groups, carbonyl groups and some unsaturated organic linkages.'" Dry tea leaves are known to have a high fluorine content, but its chemical form, and hence its bioavailability, is unknown. "F NMR has been used to study several types of tea infusions showing that F- is the main fluorine form present.lgl To identify forms of accumulated aluminium in tea leaves, 27Al NMR was applied to the study of intact tea leaves and the detection of complexes such as aluminiumcatechin, aluminium-fluorine, aluminium-phenolic acid and aluminiumorganic acid. On the basis of the NMR results and leaf constitution, it was found that most of the aluminium present in intact leaves is in the form of catechin complexes while some portion is bound to phenolic and organic acid complexes."* The in vitro speciation of aluminium in black tea infusion was assessed in order to investigate the quantity of aluminium potentially available for absorption throughout the small bowel. This work included an in vivo study of the breakdown of tea-derived polyphenols to low molecular

APPLICATIONS OF NMR TO FOOD SCIENCE

37

weight phenols, which was measured by analysing ileostomy effluent through high-resolution 'H NMR.'93 At present, the output of coffee amounts to about 40 million tonnes, and almost the same amount of extraction residue of coffee is formed. However, most of the residue is disposed of as waste, and it is therefore of interest to find an effective method of reusing coffee residues. Some recent studies have used NMR to clarify the chemical structure of an unknown antimicrobial substance found in coffee residue in order to use it as an antibacterial substance for foods. 194 Following purification by high-performance liquid chromatography, and characterization using mass spectrometry and 'H and 13C NMR, the substance was found to be 3',4'-dihydroxyacetophenone. Solid state 13C NMR, using CP and MAS, has been applied to analyse insoluble deposits adhering to the inner glass surface of bottled red wine. 19' These deposits were found to be composed of a phenolic polymer of anthocyanins, procyanidins and protein. Some terpene disaccharides of wine have been characterized by a combination of mass spectrometry and high-resolution 'H NMR'96 4.6.2. Fruit and vegetables

Chilling injury (CI) is a disorder associated with a number of fruits and vegetables that occurs when they are stored at low but non-freezing temperatures (S12"C) for periods specific to the species. Brown pits or stains appear on grapefruit peel when the fruit is stored at 5°C for 3 weeks. One of the triterpenone components found to have a direct relationship with CI effects, friedelin, was identified by 'H and 13C NMR.19' A 10MHz 'H NMR technique has been developed to estimate the sugar content of intact fruits.lg8 Proton T2 relaxation times have been calculated for intact grapes and sweet cherries. The results were then interpreted taking into account the chemical exchange process between sugar and water in the fruit. NMR has been used to analyse pesticide residues in field-grown carrots. These were grown in trifluralin-treated soil and sampled regularly. The pesticide residue was measured by 19F NMR and chromatographic methods. The two methods were found to be in agreement, although the latter proved more adequate for the measurement of low concentration^.'^^ 4.6.3. Lipids In contrast to some of the previous sections much of the work on lipids relies on the application of conventional high-resolution methods. These have been used to explore the chemical composition of lipids in foods, and result in experiments which generally give less ambiguous results than those described elsewhere.

38

A. M. GIL, P. S. BELTON AND B. P. HILLS

A recent work has reviewed the use of high-resolution 13C methods for the study of lipid structure and composition.200The I3C NMR spectra of Vernoniu galurnensis seed oil and of epoxidized palm super olein, soybean oil and linseed oil have been recorded and interpreted.201 Both natural epoxy oils and epoxidized oils are of commercial interest, and the spectroscopic procedure provides a semi-quantitative method of analysing oils that contain epoxy acids. I3C NMR has also been used to quantify castor oil in various edible oils, such as coconut oil, palm oil, groundnut oil and mustard oil. The C10, C9, C12, C13 and C11 peaks of the main component of castor oil, ricinoleic acid, have been used for quantification down to 2.0 and 3.0%, respectively, for qualitative and quantitative analysis.202 Oil chemical changes during the various stages of plant flowering and fruiting may be studied by making use of 'H and I3C NMR spectroscopic methods, as demonstrated in a recent study of Tugetes minutu Determination of the content of saturated fatty acids in positions 1,3 and 2 of triacylglycerols of olive oil has been carried out by high-resolution 13C NMR methods.204 This work was the basis for the development of an analytical method to detect synthetic esterified oils in mixtures of virgin olive oils. Pure fractions of olive oil highly esterified sucrose polyesters have been produced and subsequently identified by infrared and NMR spectroscopy.205In a separate study, high-resolution I3C NMR spectroscopy was used to characterize the composition of the unsaponifiable matter of 20 olive oils and pomace oils.2o6 Based on the identification of characteristic peaks corresponding to molecular substructures, rather than to individual constituents, it was possible to distinguish between different grades of olive oils. There are some indications that high levels of certain fatty acids of fish oils may be related to a lower incidence of heart disease. Most abundant fish fatty acids contain a double bond in the 0-3 position. The w-3 fatty acid distribution in lipid e ~ t r a c t ~ ' ' ,and ~ ~ ~white muscle208 from the Atlantic salmon (Salmo salur) has been quantified by high-resolution IH and I3C NMR. The effect of storage at below freezing temperatures was discussed.207A structure-specific 'H NMR method for quantification of w-3 fatty acids in fish oils has been ~ u g g e s t e d . ~This " ~ method relies on the different chemical shift observed for the methyl resonance of w-3 fatty acids (6 = 0.95 ppm) relative to that of other fatty acids (6 = 0.86 ppm). Oils from 24 samples of raw, cooked and canned albacore tuna (Thunnus ululunga) were quantified and compared.209 This approach is also useful to obtain structural knowledge about fatty acids and related compounds concerned with by-products or bioreactions of w-3 acid formation. A variety of high-resolution 'H and 13C NMR techniques have enabled the elucidation of the structure of cis-5,8,11,14,17eicosapentaenoic acid ethyl ester (EPA-EE) and cis-4,7,10,13,16,19docosahexaenoic acid ethyl ester (DHA-EE).210The distribution of the two corresponding acids, EPA and DHA, between the a- and P-glycerol chains

APPLICATIONS OF NMR TO FOOD SCIENCE

39

have been determined by high-resolution 13C NMR, for various fish oils. This study showed that DHA is concentrated in the /3 position whereas EPA is randomly distributed between the (Y and /3 positions.211 Oxidative deterioration of vegetable and of salted dried fish oils214,215 was investigated using NMR spectroscopy. The ratios of olefinic protons and divinylmethylene protons to aliphatic protons were determined by NMR and shown to decrease during ~ t o r a g e . ~ *Comparison ~*~'~ with corresponding peroxide values and acid values showed that the ratios of olefinic protons to aliphatic protons may serve as an index of oxidative deterioration. A recent study demonstrated the uses of high-field proton NMR in characterizing the products of the oxidative deterioration of polyunsaturated fatty acids.213 Thermal stressing of culinary oils, rich in those acids, generated high levels of some n-alkanals, trans-2-alkenals, alka-2,4-dienals and 4-hydroxy-rrans-n-alkanals.Yields were compared between different types of cooking oils. The same work discusses the dietary, physiological and toxicological implications of the process. The extent of lipase-catalysed esterification reactions has been studied non-invasively.216 Ratios of ester:alcohol signals were measured and shown to be reproducible and suitable for the study of varying conditions and nature of fatty acid and alcohol substrates on the extent of the reaction. In order to investigate intermediates and products due to chemical transformation of cholesterol during storage and heating of foodstuffs, quantitative analysis of cholesterol oxides in egg powder was carried out by high-resolution 'H NMR.217 Quantified cholesterol derivatives ranged from 4.9 to 9.1 ppm with a detection limit of 0.3 ppm. Pigments responsible for colour formation during blanching and deodorization of canola oils (rapeseed oil) were analysed by 'H and 13C resonance, proving to be trace glycerides. Both the bleaching agent and the deodorization treatment were shown to affect the distribution and concentration of the chromophores.218The essential oil liguloxide, responsible for the tomato ketchup characteristic odorific note, has been characterized by 'H and 13C NMR methods.219 High-resolution I3C spectra of butter, two vegetable fats, four baking fats, seven spreading fats and an infant formula were obtained.220 Peak assignment enabled the identification and semi-quantification of constituents such as butterfat, lauric oils, partially hydrogenated fat, linoleic acid or linolenic acid. The low-resolution pulsed methods to determine solid fat contents have been standardized for animal and vegetable oils and fats, with specification The existing of sample preparation and conditions of experimental techniques for the study of solid-liquid fat ratios, fat structure and polymorphism and fat crystallization have been complemented by the Table 8 summarizes application of imaging and localized the variety of recent NMR studies of fats and oils.

40

A. M. GIL, P. S. BELTON AND B. P. HILLS

Table 8. Recent NMR studies of lipids. System/nucleus

Ref. ~

High-resolution methods Vario~s/'~C and 'H Vegetable epoxidized ~ i l s / ' ~ C Castor 0ii/l3c Minuta oiVl3C and 'H Olive 0ii/l3c Pomace 0i1s/'~C Fish o i l ~ / ' ~and C 'H Oxidative deterioration of oils/'H Cholesterol oxides Canula o i l ~ / ' ~and C 'H

200, 216, 219 201 202 203 204-206 206 207-211, 214, 215 213 217 218

Low-resolution methods Animal and vegetable oils and fats/'H Tripalmatin/2H Cocoa masses/'H Various oilseeds/'H Milkfat ~ h e e s e / ' ~ C / ~ ' P Palm oil/'H Review of methods and sample preparation

221-224 225 226 227, 228 229, 230 231 232

Lipid polymorphism and crystallization have also been studied by 'H solid state NMR.'" The molecular dynamics in three crystalline forms of tripalmitin were investigated, as well as the motional consequences of certain thermally induced structural transitions, in the solid state. Below the melting point, at 20°C, molecular motions were found to be more restricted in the p form than in the a and p' forms. On the other hand, spin-lattice relaxation measurements have shown that the motion of methylene groups is dynamically heterogeneous and faster in the a and p' forms than in the p form. The transition between the a and p forms was followed by lineshape studies of the deuterium spectra, which showed the occurrence of immobilization. Other tripalmitin transitions were studied on the basis of deuterium NMR spectra and relaxation times.225 4.6.4. Spices and phytochemicals Antioxidants are used in processed foods to minimize the undesirable effects of lipid oxidation, and natural compounds have been increasingly needed to replace the conventional agents butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). 'H NMR has helped to characterize six major compounds purified from oleoresin of sage (Salvia oficinalis) .233 They were quantified in sage and in four commercial rosemary extracts and their antioxidative activity measured with an accelerated test. Mushrooms

APPLICATIONS OF NMR TO FOOD SCIENCE

41

contain reducing substances with chemical properties similar to those of ascorbic acid. Four types of these ascorbic acid analogues have been purified from different varieties of mushrooms and their structures characterized by NMR methods.234Capsaicin is a pungent principle of the hot pepper, and its study has more recently focused on its nutritional benefits and physiological functions. However, the use of capsaicin as a food ingredient has been limited because of its strong pungency and low The corresponding glucoside was obtained and characterized completely by 'H NMR; additional tests showed that glucosylation of the phenolic hydroxyl group resulted in loss of pungency and increase in water 4.6.5. Milk

Thermal denaturation, aggregation and gelation of P-lactoglobulin in solutions either with no added salts or with some added mono- or divalent ions were investigated by 'H and '"Cd NMR spectroscopy.236 The aggregatiodgelation kinetics were studied at 70°C, by 'H NMR, showing that the protein folded form unfolds under all salt conditions and is followed by aggregation and gel formation. Binding divalent ions seemed to stabilize the unfolded conformation. "'Cd NMR line widths indicate that Cd2+ ions interact predominantly with carboxylate oxygen sites and are not tightly bound. The physical properties of milk fat and its fractions, blends of milk fat with other fats and mixtures of milk fat with liquid oils have been investigated using methods such as differential scanning calorimetry and NMR.230

4.6.6. Meat Collagen is an important component of meat protein and has, therefore, been in the past a subject of extensive work, including the use of spectroscopic techniques such as NMR. More recently, a proton NMR study of different cross-linked collagens was performed as a function of water and temperature237 in order to obtain information about the different processes of water-collagen interaction. Collagens from the connective tissues of the calf, steer and cow with different numbers of non-reducible cross-links were analysed. Transverse and cross-relaxation times of water protons were accounted by two processes: proton exchange at higher temperatures and dipole-dipole interactions prevailing at lower temperatures. All the relaxation parameters showed specific behaviour for the 0.44 water activity, for every tissue. In addition, the NMR parameters obtained for calf collagen tissue behaved differently from the other tissues. This should relate to the relatively lower number of cross-links and higher solubility of this tissue.237 Ar present, heart muscle is not considered to be a useful by-product of the meat industry, thus justifying the very few studies dedicated to this system.

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However, heart muscle is a potential source of protein and a functional ingredient in the food industry of new products such as beef heart surimi analogues. The behaviour of heart muscle from chicken, pork and beef relative to composition and processing conditions has been studied through 'H NMR relaxation methods.238 The transverse magnetization decay was found to best fit a three exponential component model, which should correspond to three proton populations of average T2 values in the range 2 m s to 0.7s. Heating the system to 6 5 T , during 30min, produced significant increase in the longest T2, in all cases providing a rapid and accurate method to monitor water release and, hence, meat quality.23s Phosphorous NMR has also been found useful to monitor changes in ATP, creatine phosphate and pH in order to help assessing halothane (bromochlorotrifluoroethane) sensitivity and meat quality in pigs.239 Halothane sensitivity is a metabolic defect that produces the well-known condition termed pale soft exudative (PSE) meat, which is of low organoleptic quality and yield. Further research on PSE as well as on dark firm dry meat (DFD) was carried out by 31P NMR.240 Comparison of the phosphorous spectra of several different pig breeds and muscles showed that normal, PSE and DFD meats correspond to clearly distinct compositions in phosphorylated compounds, under 30 min post-mortem conditions.

4.6.7. Other studies The molecular motion of sucrose in water-ethanol-sucrose-casein solutions These was studied by 13C NMR spin-lattice relaxation studied are relevant to the investigation of colloidal stability of cream liqueurs. Results suggest that the sucrose conformation is not affected by concentration, temperature or the presence of alcohol or casein, there being no evidence of interaction between sucrose with either alcohol or protein. 2H NMR has helped the study of baker's yeast fermentation.242 Experiments in deuterated water showed that yeast fermenting on D-mannose, D-glucose or D-fructose yielded trideuterated (R)S-benzylthioglycerate. The extent and stereochemistry of labelling was shown, by 2H NMR studies, to depend on the carbohydrate precursor. The Maillard reaction of model compounds for peptide-bound lysine with reducing sugars was investigated under both stringent and mild condition^.^^' The structure of a new reaction product was determined by a combination of chromatography and NMR methods. One should be cautious of automatically regarding a high dietary fibre level as beneficial. One component of bran, phytic acid, is a potent complexer of divalent cations, being often implicated in calcium and zinc deficiency diseases. Phytate and trace elements of the daily diet of healthy subjects in Taiwan were determined by 31P NMR and by instrumental neutron activation analysis, r e s p e c t i v e ~ y . ~ ~ ~ The volatile components of salak fruit have been isolated, and over 40

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compounds have been identified by a combination of techniques, including 'H NMR.245The conformational preferences of some L-aspartyldipeptide methyl esters to elicit tastes such as sweet and bitter have been investigated by conformational free energy calculations and conformational studies by 'H NMR.246 The presence of the ~-aspartylgroup proved to be a necessary factor for sweet dipeptides whereas the orientation of hydrophobic moiety relative to the structural system AH/B was confirmed to be determinant.

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A. M. GIL, P. S. BELTON AND B. P. HILLS M. A. Murcia and J . Villalain, J . Sci. Food Agric., 1993, 61, 345. H . Barjat, P. S. Belton and B. J. Goodfellow, Analyst, 1993, 118, 73. H. Barjat, P. S. Belton and B. J. Goodfellow, Food Chem., 1993, 48, 307. T. M. Eads, R. K. Weiler and A. G . Gaonkar, J . Coll. Interface Sci., 1991, 145,466. Q. W. Ni and T. M. Eads, J . Agric. Food Chem., 1992, 40, 1507. Q. W. Ni and T. M. Eads, J . Agric. Food Chem., 1993, 41, 1026. T. M. Eads, Frontiers in Carbohydrate Research-2 (ed. R. Chandrasekara). Elsevier, New York, 1992. 2. Gong, Biosci. Biotech. Biochem., 1993, 57, 1745. M. Spiro and D. Jaganyi, Food Chem., 1994, 49, 351. H. Horie, T. Nagata, T. Mukai and T. Goto, Biosci. Biotech. Biochem., 1992, 56, 1474. T. Nagata, M. Hayatsu and N. Kosuge, Phytochemistry, 1992, 31, 1215. J. J. Powell, S. M. Greenfield, J . K. Nicholson and R. P. H. Thompson, Food Chem. Toxicol., 1993, 31, 449. A. Nishina, Biosci. Biotech. Biochem., 1994, 58, 293. E. J. Waters, Z . Peng, K. F. Pocock, G. P. Jones, P. Clarke and P. J . Williams, J . Agric. Food Chem., 1994,42, 1761. V. A. Marinos, M. E. Tate and P. J . Williams, J . Agric. Food Chem., 1994,42, 2486. H. J . Nordby and R. E. McDonald, J . Agric. Food Chem., 1994, 42, 708. S . I. Cho, R. L. Stroshine, I. C . Baianu and G. W. Krutz, Trans. ASAE, 1993, 36, 1217. R. D . Mortimer, D. B. Black and B. A. Dawson, J . Agric. Food Chem., 1994, 42, 1713. F. D. Gunstone, Progr. Lipid Res., 1994, 40, 1215. F. D. Gunstone, J . A m . Oil Chem. Soc., 1993, 70, 1139. H . Sajid, K. Mohd, G. S. R. Sastry and N. P. Raju, J . A m . Oil Chem. SOC.,1993, 70, 1251. R. K. Thappa, S. G . Agarwal, N. K. Kalia and R. Kapoor, J . Essent. Oil Res., 1993, 5, 375. R. Sacchi, F. Addeo, I. Giudicianni and L. Paolillo, Ital. J . Food Sci, 1992, 4, 117. J. J. Rios, M. C. Perez-Carmino, G . Marquez-Rui and M. C. Dobarganes, J . A m . Oil Chem. SOC., 1994, 71, 385. R. Zamora, J. L. Navarro and F. J. Hidalgo, J. Am. Oil Chem. SOC., 1994, 71, 361. M. Aursand, J. R. Rainuzzo and H. Grasdalen, Proceedings of the Qualify Assurance of the Fish Industry, Ministry of Fisheries, Denmark, 1992. M. Aursand, J . R. Rainuzzo and H. Grasdalen, J . Am. Oil Chem. SOC., 1993, 70, 971. R. Sacchi, I. Medina, S. P. Aubourg, F. Addeo and L. Paolillo, J . Am. Oil Chem. Soc., 1993, 70, 225. C. A. H. Hegg, 17th Nordic Lipid Symposium Proceedings, (eds Y. Maikki and G. Lambertsen) Scand. Forum Lipid Res. Technol., Bergen, Norway, 1993, p. 240. F. D. Gunstone and S . Seth, Chem. Phys. Lipids, 1994, 72, 119. U . N. Wanasundara and F. Shahidi, J . Food Lipids, 1993, 1, 15. A. W. D. Claxson, G. E. Hawkes, D. P. Richardson, D. P. Naughton, R. M. Haywood, C. L. Chander, M. Atherton, E. J. Lynch and M. C. Grootveld, FEBS Lett., 1994, 355, 81. H. Saitb and M. Udagawa, J . Am. Oil Chem. SOC.,1992, 69, 1157. H. SaitB and M. Udagawa, Biosci. Biotech. Biochem., 1992, 56, 831. C. J. O’Connor, S. F. Petricevic, J. M. Coddington and R. A. Stanley, J . A m . Oil Chem. SOC.,1992. 69, 295. A. Fontana, F. Antoniazzi, G. Cimino, G. Mazza, E. Trivellone and B . Zanone, J . Food Sci., 1992, 57, 869. D. M. Chapman, E. A. Pfannkoch and R. J. Kupper, J . Am. Oil Chem. SOC., 1994, 71, 401. C. Dragar, V. A. Dragar and R. C. Menary, J . Essent Oil Res., 1993, 5, 507.

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F. D. Gunstone, J . A m . Oil Chem. SOC., 1993, 70, 361. British Standards Institution, British Standard, 1992. BS 684, Section 2.22. M. C. M. Gribnau, Trends Food Sci. Technol., 1992, 3, 186. C. Simoneau, M. J. McCarthy, D. S. Reid and J. B. German, Trends Food Sci. Technol., 1992, 3 , 208. S. Oezilgen, C. Simoneau, J. B. German, M. J. McCarthy and D. S. Reid, J . Sci. Food Agric., 1993, 6 , 101. D. Precht and E. Frede, Fett. Wiss. Technol., 1994, 96, 324. C. V. Hernandez and D. N. Rutledge. Food Chem., 1994, 49, 83. G. Rubel, 1. A m . Oil Chem. SOC., 1994. 71, 1057. P. N. Gambhir, Trends Food Sci. Technol., 1992, 3, 191. L. T. Kakalis. T. F. Kumosinski and H. M. Farrell, J . Dairy Sci., 1994, 7 7 , p. 667. W. L. Ng and C. H. 0 h . J . Am. Oil Chem. SOC., 1994,71, 1135. T. M. Eads, A . E. Blaurock, R. G . Bryant, D. J . Roy and W. R. Croasmun, J . A m . Oil Chem. Soc., 1992, 69, 1057. M. C. M. Gribnau, Trends Food Sci. Technol., 1992, 3, 186. A.-E. Cuvelier, C. Berse and H. Richard, J . Agric. Food Chem., 1994, 42, 665. M. Okamura, J . Nutr. Sci. Vitaminol., 1994, 40, 81. T. Kometani, H . Tanimoto. T. Nishimura, I. Kanbara and S. Okada, Biosci. Biotech. Biochem., 1993, 57, 2192. H. Li, C. C . Hardin and E. A. Foegeding, J . Agric. Food Chem., 1994, 42, 2411. J . P. Renou, M. Bonnet, G . Bielicki, A. Rochdi and P. Gatellier, Biopolymers, 1994, 34, 1615. J . R . Lee, I . C. Baianu and P. J. Bechtel, J . Agric. Food Chem., 1992, 40, 2350. R. Lahucky, J. Mojto, J . Poltasky, A. Miri, J. P. Renou, A . Talmant and G . Monin, Meat Sci., 1993, 33, 373. A. Miri, A. Talmant, J. P. Renou and G. Minin. Meat Sci., 1992, 31, 165. D. G . Cornell, R. L. Dudley, R . F. Joubran and N . Parris, Food Hydrocoll., 1994,8, 19. G. Fronza. C. Fuganti, A. Mele, G. Pedrocchi-Fantoni and S. Servi, Tetrahedron, 1994, 50, 857. R. Tessl, G . Wondrak, R. Kesrten and D . Rewicki, J . Agric. Food Chenz., 1994,42, 2692. C. F. Wang, S. M. Tsay, C. Y. Lee, S. M. Liu and N. K. Aras, J . Agric. Food Chem., 1992, 40, 1030. K . C. Wong and D. Y . Tie, Flavour Fragrance J . , 1993, 8, 321. Y.J. Kim, S. J. Han, S. C. Kim and Y . K. Kang, Biopolymers, 1994, 34, 1037.

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Gradient NMR WILLIAM S. PRICE National Institute of Materials and Chemical Research, Tsukuba, Ibaraki 305, Japan 1. Introduction 2. Nuclear spins and gradients 2.1. Introduction 2.2. BO versus B1 gradients 2.3. BOand B 1 gradient geometries 3. Diffusion measurements 3.1. Introduction 3.2. Free and restricted diffusion 3.3. Correlating the signal attenuation with diffusion 3.3.1. Macroscopic description 3.3.2. GPD approximation 3.3.3. SGP approximation 3.3.4. Numerical methods 3.4. “Diffusive diffraction” and imaging molecular motion 3.5. Simple geometries 3.5.1. Flow 3.5.2. Reflecting boundaries 3.5.3. Absorbing boundaries 3.5.4. Fractals and anomalous diffusion 3.5.5. Anisotropic diffusion 3.6. Validity of the SGP and GPD approximations 3.7. More complicated boundary conditions 3.7.1. Introduction 3.7.2. Polydispersity, polymers and macromolecular systems 3.7.3. Size distributions of the restricting geometry 3.7.4. Obstruction 3.7.5. Exchange and many body effects 3.7.6. Porous media 4. Non-homogeneous gradients and other problems 4.1. Introduction 4.2. Imperfect gradient pulses and sample movement 4.3. Eddy currents and perturbation of Bo 4.4. Internal gradients and relaxation time distributions 5. Pulse sequences for measuring diffusion 5.1. Introduction 5.2. BOsequences 5.2.1. Basic sequences 5.2.2. Reduction of the effects of background gradients 5.2.3. Reduction of eddy current and phase distortion effects ANNUAL REPORTS ON NMR SPECTROSCOPY VOLUME 32 ISBN 0-12-505332-0

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Copyrighl 019% Academic Press Limited All right5 of reproduction in any form reserved

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5.2.4. Steady gradient 5.2.5. Fringe (or stray) field methods 5.2.6. Zero and multiple quantum 5.2.7. Multiple spin echoes 5.2.8. Miscellaneous 5.3. B1 sequences 6. Applications to high-resolution NMR 6.1. Introduction 6.2. Coherence selection and quadrature detection 6.3. Spectral selectivity and editing 6.4. Solvent suppression 6.4.1. Introduction 6.4.2. Coherence selection 6.4.3. Diffusion 6.4.4. Selective excitation 6.4.5. Watergate 6.4.6. B1 solvent suppression methods 6.5. Spectral simplification according to mobility 6.5.1. Introduction 6.5.2. Diffusion-ordered two-dimensional experiments 6.5.3. Electrophoretic mobility 7. Technical aspects of gradient production 7.1. Introduction 7.2. Gradient coil design 7.2.1. Bo gradient coils 7.2.2. B 1 gradient coils 7.3, Power supplies 7.4. Gradient calibration 7.4.1, Bo gradients 7.4.2. B 1 gradients 7.5. Sample shimming and field frequency locking 7.6. Temperature control 8. Specific examples of gradient NMR 8.1. Introduction 8.2. Diffusion-based studies 8.2.1. Diffusion measurements 8.2.2. Restricted diffusion and obstruction 8.2.3. Binding and transport 8.2.4. Liquid crystals and surfactants 8.2.5. Porous media 8.2.6. Polymers and macromolecules 8.3. High-resolution NMR applications 8.3.1. Applications of DOSY and electrophoretic NMR 8.3.2. Coherence selection, phase cycling and solvent suppression 9. Concluding remarks References

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1. INTRODUCTION

In the last decade there has been a tremendous increase in the use of both magnetic (Bo), and to a lesser extent, radiofrequency (rf or B,) field gradients in nuclear magnetic resonance (NMR). Not too long ago a gradient was something to be avoided since it would lead t o poor resolution and complicate the analysis of relaxation experiments in the case of Bo gradients or cause uneven excitation in the case of B1 gradients. However, as is often the case in NMR, phenomena that at first sight seem to be only the cause of artifacts are later found to have practical applications. Gradients now pervade almost all areas of NMR ranging from coherence selection in high-resolution NMR to inputting spatial dependencies into NMR imaging (also known as magnetic resonance imaging or MRI) and NMR microscopy. The increased use has occurred concomitantly with the widespread commercial availability of NMR gradient probes and gradient drivers. In fact, most modern spectrometers now come equipped with gradient probes and amplifiers as standard, sufficient for high-resolution applications. This also provides at least limited scope for performing diffusion measurements. The use of gradient NMR allows diffusion to be added to the standard NMR observables of chemical shifts and relaxation times (i.e. longitudinal or T I ;transverse or T,; and in the rotating frame or Tip). The applications of gradients can be roughly separated into two broad areas; k and q space.' k space involves the spatial spectrum of nuclear spin positions and q space involves the spatial spectrum of nuclear spin displacements. These notions will be further explained below. This chapter is concerned with the theory, applications and technical aspects of diffusion measurements and high-resolution applications of gradients. Since the field is now so large, this chapter will not concern itself with predominantly &-based k space applications such as MR12 and NMR microscopy.= Although, it should be noted that microscopy and imaging are now within the realms of B1 gradient^.^,^ It is not possible to cleanly divide the literature between k space and q space as some studies contain elements of b ~ t h . ~ *Some ~ - ' ~mention will be made regarding homogeneous and inhomogeneous static gradients. A number of reviews on gradient NMR have already appeared in the literature including ones of a general nature'"-16 and also of specific areas of interest such as heterogeneous systemsi7 such as zeolites and porous system^,'^+*^^^ surf act ant^,'^ liquid crystals and membranes,26 and high-resolution application^.^^.'^ The monograph by Callaghan on the closely related field of NMR microscopy covers much of the theory and technical aspects of gradient NMR.4 In this review I have attempted to give a rather general coverage of the field, but with special emphasis on the field of diffusion and transport in restricted geometries and in biological systems. As mentioned above, gradients are now established parts of many areas of

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NMR methodology. Using steady gradients and, more particularly, in the guise of pulsed field gradient (PFG) NMR, gradients afford a powerful tool for not only studying molecular diffusion (under favourable circumstances down to less than m2/s) but also for providing structural information in the range 0.1-10 p m when the diffusion is restricted on the NMR time-scale. In PFG experiments, the ‘tuneable’ parameter is termed q ( = ( 2 r ) - l y S g ; where y is the gyromagnetic ratio, S is the duration of the gradient pulse and g is the gradient magnitude; NB-some authors use k = ySg) and it is analogous to the scattering wave vector in neutron diffraction .29 Neutron scattering, however, is confined to making measurements on the scale of only a few nanometres and thus is usually confined to single scattering events. Hence for observation on the micrometre scale, PFG is a unique method and its measurements may correspond to multiple scattering events. This interesting new area of PFG NMR has given rise to “diffusive diffraction experiments” and q space imaging (see Section 3.4).1,30,31 While in k space imaging the resolution is limited by the signal-to-noise ratio as the voxel size decreases, q space imaging is limited only by the size of the applied gradient and artifacts such as gradient pulse mismatch and sample motion. This is an important distinction. Due to its non-invasive nature, it is especially suited to probing the molecular dynamics and structural details of biological system^.^^,^^ Since PFG NMR does not perturb the system or require labelled probe molecules, it provides an excellent tool for investigating such molecular dynamics by examining the underlying diffusive processes directly. Importantly, it allows measurements to be performed under physical conditions, such as high pressure34 and temperature, where other methods may be precluded. Recently, a report of measuring self-diffusion in the earth’s magnetic field has even appeared.35 Because of the length scale in which PFG NMR is sensitive, it is especially suited to studying the physics of restricted diffusion in materials. Restricted diffusion influences fluid penetration in ceramics and plastics, transport in polymers and biological systems, and sorption and reaction in catalysts. PFG NMR is complementary to NMR relaxation-based molecular dynamic studies in that it studies macroscopic happenings which occur on the time-scale of milliseconds to seconds. Relaxation studies, however, tend to be microscopic in nature since relaxation is sensitive to occurrences on the time-scale of the reorientational correlation time of the species ( T ~ ) Further, . it allows diffusion to be measured directly without the assumptions needed to relate T~ to diffusion and v i s ~ o s i t y . ~ ~ , ~ ~ , ~ However, the diffusion measured on the time-scale of a PFG measurement is normally subject to restriction. To obtain the true diffusion coefficient, D ,as opposed to an “apparent” diffusion coefficient, Dapp,an appropriate model must be used to account for the effects of restricted diffusion. At present anything but the simplest restricted system is beyond the reach of analytical solutions. To study transport and exchange between two domains, the domains must

GRADIENT NMR

55

be able to be distinguished by some measurable property. Traditional NMR methods for studying transport and exchange have relied upon either a difference in chemical shift o r a difference in relaxation rate. However, using PFG NMR a difference in the diffusion coefficient between the domains may be used to separate the two regions even if the chemical shift of the probe species in both domains is the same. Thus, PFG provides the basis of a new method for measuring transport and exchange. There are now numerous gradient applications in high-resolution NMR, including: (1) solvent suppression, (2) coherence selection, ( 3 ) induction of quadrature detection and (4) separation of complicated mixtures in twodimensional experiments based on their diffusion and electrophoretic mobilities. These new methods not only allow multidimensional experiments to be collected in a fraction of the time normally taken, but also allow for significantly improved solvent suppression with the ability of observing resonances which resonate close to the solvent frequency. Bo and B1 gradients have some similarities and yet many differences both theoretically and technically. One of the largest technical problems in pulsed Bo gradient NMR is the effect of eddy currents generated by the rapid rise and fall of the gradient pulses. Much research has been done to overcome these eddy current problems (see Section 4). These problems, however, do not apply when B1 gradients are used. On the theoretical side, B1 gradients are inherently more complicated than Bo gradients since the normally separate steps of coherence transformation and gradient evolution occur simultaneously. This review will first characterize the differences between Bo and B1 gradients and their effects on nuclear spins. It will then discuss the basis of diffusion measurements by gradient NMR including the effects of restriction, exchange, relaxation and problems that confront such measurements. Next, the applications to high-resolution NMR and technical aspects of gradient production and use will be discussed. Finally, the last section will give a brief introduction to a few selected applications. 2. NUCLEAR SPINS AND GRADIENTS 2.1. Introduction

In this section we review relevant theory concerning NMR and Bo and B1 gradients. Since most of the work done so far pertains to Bo gradients, the discussion and theory will be oriented to Bo gradients. Generally the theory for Bo and B , gradients is analogous, and the points at which they differ will be noted. All of the NMR theory pertaining to Bo gradients in the following sections has the Larmor equation as the origin:

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WILLIAM S. PRICE

where w is the Larmor frequency and Bo is the strength of the static magnetic field and we have neglected the small effect of the shielding constant. Since Bo is spatially homogeneous, o is the same throughout the sample. Equation (1) holds for a single quantum coherence (i.e. n = 1). However, if in addition to Bo there is a spatially dependent magnetic field Bo(r), and accounting for the possibility of more than single-quantum coherence, then w becomes spatially dependent,

The important point is that if a homogeneous gradient of known magnitude is imposed through the sample, the Larmor frequency becomes a spatial label. Thus, in PFG measurements the NMR signal is phase encoded according to the molecular displacements over a well-defined time interval, whereas in NMR imaging the signal is phase encoded according to molecular position.38 Equation (2) also shows that successively higher quantum transitions are more sensitive to the effects of the gradient whereas zero-quantum transitions are unaffected by the presence of the gradient. Counsel1 et u I . ~first ~ demonstrated that it is possible to use the natural inhomogeneity of the B1 field produced in a normal NMR probe to mimic Bo gradient pulses. They showed that the sequence (also known as a is equivalent to generatcomposite z pulse) (d2),+,{spin 10~k}~+,/~(7~/2), ing an inhomogeneous rotation about the z axis, which is just the effect of a Bo gradient pulse. The “sign” of the gradient is changed by changing the phase of the spin lock pulse or of either of the d 2 pulses.

2.2. Bo versus B1 gradients

B1 gradients are inherently more complex than Bo gradients. Apart from purely technical considerations, there are three main differences between Bo and B1gradients4’ (1) A Bo field couples only into the spin system along the z axis, thus the effective gradient tensor is always truncated into an effective vector. Rf fields, however, couple into the spin system, from any orientation within the transverse plane. As a result the B1 gradient generally retains its tensor form when it couples into the spin system. (2) When the same rf coil is used for both excitation and detection, any phase variation is cancelled during the measurement. But when an experiment involves two rf fields at the same frequency this cancellation no longer occurs, and phase variations need to be considered. This spatial dependence of the phase difference between the two rf fields presents an additional complication (or opportunity).

GRADIENT NMR

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(3) The third difference is that B1 fields are non-secular and so do not commute with internal Hamiltonians. Thus, unlike a Bo gradient, a B1 gradient cannot be treated additively with respect to internal Hamiltonians. A formalism has recently been introduced for describing the spin dynamics of B1gradient experiment^.^' Importantly, the steps of coherence transformation and gradient evolution are clearly separated in this formalism. In &-based experiments only the gradient strength is varied spatially. In B1 experiments there are two mechanisms that lead to spatially varying spin dynamics: the amplitude variation of the gradient rf field and the phase difference between the gradient and homogeneous rf fields. The amplitude variation is most directly analogous to Bo experiments. The phase variation arises from the symmetry of the rf field and current flow through the coils. A number of technical problems arise in the generation of Bo gradients and also as side-effects of their generation. It is difficult to obtain gradient pulses with very rapid rise times, and the imposition of the gradient pulse generates eddy currents in the surrounding metal parts of the probe. These eddy currents have deleterious effects on the spectral acquisition and resolution. The gradient pulse also perturbs the field-frequency lock system. As will be discussed in Section 4, much effort has gone into finding ways to lessen these problems including special pulse sequences, shielded gradient probes and pre-emphasis of the gradient pulses. At a technical level, B1 gradients have some significant advantages over their Bo counterparts. The main advantages are:42 (1) the switching times are much shorter, (2) the lock channel is unaffected, (3) there is no need for pre-emphasis, (4) the lineshape is not distorted, ( 5 ) no eddy currents are induced and so there is no need for shield coils, etc., and (6) the gradient is frequency selective. However, at present, the absolute strength of B1 gradients (-0.2 T/m) is still far less than that available with Bo gradients (-80T/m). And because B1 gradients generally preserve their tensor form when they couple into a spin system, the design of truly planar rf gradient fields is difficult.

2.3. Bo and B1 gradient geometries The technical aspects of Bo and B1 gradient production will be reviewed in Section 7.2. Here we examine the geometries and consequences of Bo and, in particular, B1 gradients and how B1 gradients can be introduced into pulse sequences so as to behave like Bo gradient pulses. Bo gradients are ideally linear (i.e. planar) along one axis (generally the z axis). Additional gradient coils may be added to provide gradients oriented along other directions. Two types of B1 gradients can be employed depending on the coil geometry: planar or radial (i.e. quadrupolar). The

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amplitude of a planar field (i.e. dBJdx) increases along one axis in the laboratory frame, while the amplitude of a radial field (i.e. dB,/dr, -dB,,ldy) increases along two orthogonal axes. The dephasing due to a planar gradient occurs in a plane perpendicular to the rotating frame axis along which the rf gradient field is applied. For a radial gradient, however, the dephasing, being the result of both an amplitude and phase variation has the effect of scattering the magnetization over the surface of a sphere. Hence, when trying to dephase longitudinal magnetization, a radial gradient is more efficient than a planar gradient. Also because of the radial phase distribution, the gradient phase does not have to be adjusted to the phase of the homogeneous rf coil. A planar gradient can be formed from a radial gradient by inserting T pulses into the gradient evolution. This refocuses the evolution perpendicular to the phase of the T pulses and leaves only gradient evolution along the direction dictated by the phase of the T pulses. An example of such a where g , is a B1 gradient sequence is (gl)~-T,-(gl)8-(gl)e-T~-(gl)~, pulse.42 The effect of this sequence can be evaluated by examining the average H a m i l t ~ n i a n , ~ ~

{ (y) i, (y) i,,} cos

- sin

where u and v are the transverse axes of the laboratory frame. If the phases C$ and 8 of the gradient pulses are the same, then the average Hamiltonian is proportional to i, and represents a planar gradient. An exact analogue of a Bo gradient pulse can be formed if this sequence is sandwiched between two homogeneous ~ / pulses 2 of opposite phase (i.e. composite z pulse); the effect of this sequence is a composite z rotation in which the rotation angle is a function of position in the transverse plane. 3. DIFFUSION MEASUREMENTS 3.1. Introduction

In this section we discuss how the diffusion and flow of spin-bearing species in homogeneous and heterogeneous systems are related to the observable NMR signal. To better understand the mechanism of how diffusion is measured with gradients, we must first review some relevant aspects of diffusion. Next we discuss how diffusion and the boundary conditions are related to the observable NMR signal. An analogy between diffraction and

GRADIENT NMR

59

PFG diffusion studies will be made. We will then present the results for some simple restricting geometries (e.g. cylinders, planes and spheres), followed by more complicated models such as those which include exchange. Finally we will discuss PFG NMR from the viewpoint of being a probe for studying porous media. 3.2. Free and restricted diffusion

For the case of diffusion in an isotropic and homogeneous medium, the conditional probability, P(ro, r, t ) , of finding a particle initially at position ro, at a position r after a time t is equal to

(

‘)

P(ro, r, t) = ( 4 ~ D t ) - ~ ” e x p - (r 4Dt - ro>

(3)

Thus the radial distribution function of the spins in an infinitely large system with regard to an arbitrary reference time is Gaussian. From equation (3) it can be deduced that the root mean square displacement for diffusion in three dimensions is given by

((r - r,J2) = 6Dt

(4)

o r for diffusion along one direction ((x - X”)2) = 2Dt

(5)

Equation (4) provides an alternative definition of the diffusion coefficient and is equivalent to Fick’s first and second laws. Naturally, the probability given in equation (3) is determined by solving the diffusion equation

where D represents the (rank two) diffusion tensor, subject to the initial condition

P(ro,r, 0) = S(ro - r)

(7)

and the boundary condition P-0 as r+m. In the case of isotropic diffusion, which we shall consider first below, the tensor D is replaced by the diffusion coefficient D ,and equation (6) simplifies to

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WILLIAM S. PRICE

7

I

7

I

Acquisition

A

'VVV"' 0 tl

I

71

A

"

1

V

tl + A

71

72

I

Acquisition

B

0'

tl

tl + A

Fig. 1. (A) The Stejskal and Tanner PFG sequence.52This is a Hahn spin echo pulse sequence with a "rectangular" gradient pulse of duration S and magnitude g inserted into each 7 period. The separation between the leading edges of the gradient pulses is denoted by A. The applied gradient is enerally along the L axis (the direction of the static field). (B) The STE ~equence.~"

The solution of this equation becomes much more complicated when the diffusion of the particle is affected by its boundaries, although the solutions of equation (8) for many cases of interest can be found in the l i t e r a t ~ r e . ~ ~ ? ~ ~ Any echo sequence is susceptible to the effects of diffusion. Diffusion measurements are generally performed with some variation of the Hahn spin echo or stimulated echo pulse (STE) sequences incorporating field gradient pulses (i.e. the PFG experiment; Fig. 1). Steady gradient experiments also exist but due to the greater usage and applicability we will concentrate on the PFG approach. In our discussion we will refer to the

GRADIENT NMR

61

simplest possible PFG sequence, namely a Hahn spin echo pulse sequence with a gradient pulse inserted into each T period. The second half of the echo is used as the free induction decay (FID). This is commonly known as the “Stejksal and Tanner” sequence (see Fig. l(A)). The main magnetic field is taken to be in the z direction, and the gradient pulses are also applied along the z direction. Normally, the experiment is started with the spins being at thermal equilibrium (i.e. equilibrium magnetization along the z axis). In the absence of magnetic field gradient pulses, the application of the d 2 pulse rotates this magnetization into the x-y plane. The spins then dephase during the first 7 period. At a time 7,a T pulse is applied which reverses the dephasing process so that an echo is formed at time 27. The T pulse also has the effect of refocusing chemical shift effects and frequency dispersion due to residual Bo inhomogeneity. The first applied magnetic field gradient pulse spatially ‘‘labels’’ the spins with respect to their position along the z axis. The spins then continue to diffuse during the time A, at which point they are subject to a second gradient pulse of equal but negative magnitude (NB-the T pulse has the effect of changing the sign of the gradient). If the spins have not moved with respect to the z axis the effect of the two applied gradient pulses cancel, but if the spins have moved, the degree of dephasing due to the applied gradient is proportional to the displacement in the direction of the gradient (i.e. the z direction) moved in the period A. Any dephasing will result in a diminished echo signal, M ( q , t ) , at time 27, and thus the effect of diffusion is monitored by the attenuation of the echo signal. In correlating the signal attenuation to diffusion, the spin echo signal is generally normalized by dividing the echo signal obtained in the absence of field gradients, M ( q = 0, t). Thus we define the attenuation, E ( q , t ) (or E ) , of the echo signal as

The experiment is performed by varying one of the experimental variables (i.e. 6, A or g). 7 is generally kept constant, and thus by using the normalized signal attenuation and not the echo, bulk relaxation effects are factored This “factorization ansatz” will be further considered in Section 3.7.6. The degree of dephasing is determined by the “area” of the gradient pulses (i.e. 6g) and the displacement of the spin along the z-axis. We need to equate the attenuation ( E ) of the echo signal to the experimental variables, the diffusion coefficient and the available diffusion space. At this point it is appropriate to introduce the concept of restricted diffusion. We define R as being the characteristic distance of a restricting geometry (e.g. the radius of a sphere or cylinder, or half the separation between planes), we also define the dimensionless variable, 6 = DA/R2, which is useful in characterizing restricted diffusion. Consider a case where we have two particles diffusing at the same rate, one is freely diffusing (i.e.

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WILLIAM S. PRICE

m

Fig. 2. The effects of restricted diffusion are schematically presented for the three relevant A time-scales for a particle diffusing within a sphere of radius R : (1) 6 (=DAlR2)< 1 (the short time limit); the particle does not diffuse far enough in the time A to feel the effects of restriction. Measurements performed within this time-scale lead to the true diffusion coefficient (i.e. 0). (2) 6-1; some of the particles feel the effects of restriction, and the diffusion coefficient measured with this time-scale will be apparent (i.e. D,,,) and be a function of A. (3) 6> 1 (the long time limit); all particles feel the effects of restriction. In this time-scale the displacement of the particle is independent of A and depends only on R .

an isotropic homogeneous system) while the other is confined to a reflecting sphere (Fig. 2). Assume that we measure the motion of a particle by taking a measurement at time t = 0 and a second measurement at time t = A. In the case of freely diffusing particles the diffusion coefficient determined will be independent of A for any value of A. However, for particles confined to the sphere the situation is entirely different. For short values of A such that the diffusing particle does not “feel” the effect of the boundary (i.e. [< 1)

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63

the measured diffusion coefficient (i.e. D,,,) will be the same as that observed for the freely diffusing species. As A becomes larger such that 6 - 1, the particle will begin to feel the effects of the boundary, and the diffusion coefficient determined will be dependent on A. At very long A, the maximum distance that the confined particle can travel is limited by the boundaries, and thus the measured diffusion coefficient again becomes independent of A. A further complication arises if the restricting geometry is not symmetric with respect to the gradient (e.g. a cylinder). In this case the diffusion becomes anisotropic, and the measured diffusion coefficient will depend on the direction of measurement. In free solution, averaging makes the measurement isotropic (i.e. independent of the field direction).

3.3. Correlating the signal attenuation with diffusion We will now discuss the mathematical formulations necessary to relate the signal attenuation to the diffusion coefficient and boundary conditions in the PFG experiment. We will not pursue the analysis for steady gradient diffusion experiment^.^^ Both macroscopic and quantum mechanical (i.e. density matrix) formulations exist. The quantum mechanical basis of the spin echo experiment is well known’3’15,49and will not be presented here. The theory used to interpret B1 gradient experiments is essentially the same as that for Bo gradient^.^" We will first give an outline of the macroscopic approach, which is necessary to understand later discussions on gradient pulse shapes. The macroscopic approach allows for the finite length of the gradient pulse. However, in the case of restricted diffusion the macroscopic and density matrix approaches become mathematically intractable. Thus, in the general case one is forced to use different approximations to find formulae relating E to the diffusion coefficient, boundary and experimental conditions. There are two common approximations, the first is the Gaussian phase distribution (GPD) approximation and the second is known as the short gradient pulse approximation (SGP). It is found that even using these approximations, analytic solutions are generally not possible and numerical methods must be used. Some solutions using the GPD and SGP approximations are presented in Section 3.5, and the validity of the SGP and GPD approximations is discussed in Section 3.6. Finally, some solutions and discussion are made for more complicated systems, such as porous media and those including transport.

3.3.1. Macroscopic description The macroscopic treatment is based upon the “magnetization fluid” approach of Torrey.’l In the following discussion we consider only the case

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WILLIAM S. PRICE

of isotropic diffusion. The more general case where the diffusion coefficient scalar D is replaced by the tensor D has been considered elsewhere52 and in the presence of gradients in more than one direction.53754The magnetization, M(r, t) is considered to be both a time- and space-dependent function. By combining the Bloch equations with Fick's second law, we obtain, for isotropic diffusion,

where Mo is the equilibrium magnetization, Mx,y,z are the components of local magnetization along the directions of the respective unit vectors ex,y,z. Further, it is assumed that the static magnetic field is oriented along the z axis such that B = (0, 0, BO).Thus, only the z component of the time dependent external magnetic field must be considered,

.

BZ(r,t) = Bo

+g. r

(11)

where g is defined by g=-

a& e y + -e, aB* dBz e x + aY az ax

The transverse magnetization can now be defined as

V

= (Mx

+ iMy)exp( -iwot + t/T2)

Inserting equation (13) into equation (10) we get

Setting

(1')

"(r, t ) = T(t)exp - iyr

and inserting this into equation (14) leads to

which has the solution

g(t')dt'

(13)

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65

This can then be rewritten as

As noted above, the effect of a 72 pulse is to change the sign of the gradient. Further, the effective gradient is zero for longitudinal magnetization. Replacing g with the effective gradient, g e f f , allows the attenuation due to diffusion to be calculated for any pulse sequence (including steady gradients). In the simplest PFG sequence as shown in Fig. l(A), including a background gradient, go, equation (18) becomes”

+ r ’ g * g o D 8 [ ~ + ~ + 8 ( t , + t 2+2/382-2?] ) \

v

g. gocross-terms

The direction in which the diffusion is measured is the same as the direction of the gradient. Generally the condition g > > g o holds, and thus the g-go cross-terms can be neglected in equation (19). The (A - 813) factor in the go = 0 term reduces to A when A > > 6 , which corresponds to the solution found using the SGP approximation. It must be noted, however, that equation (19) only holds for free diffusion. In fact, analytical solutions only exist for free diffusion and for free diffusion superimposed upon flow.4

3.3.2. GPD approximation The Gaussian phase approximation results from the method of phase accumulation, which was originally suggested by Douglass and McCa1LS6In this scheme the phase of the ith spin at the end of the PFG sequence is given by

1,

ti+A+ 8

flf8

4ii(q,A) = yg(

zi(t)dt -

Zi(t) /,+A

1

dt

(20)

where zi(t) is the spin position in the direction of the field gradient and the time c1 is defined in Fig. 1. The echo intensity is proportional to the resultant magnetic moment, which is given by ?j

(21)

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WILLIAM S. PRICE

where P(4, A) is the relative phase distribution accumulation function. If, as in the case of free diffusion, it is assumed that the phases have a Gaussian distribution then the echo attenuation is given by57

3.3.3. SGP approximation In this approximation, motion during the gradient pulse is ignored (rigorously, one assumes that 6+ 0 and lg(+ while their product remains finite). Operationally this condition is approximated by keeping S << A. The PFG spin echo amplitude is given by52,58

where p(ro, 0) is the starting spin density, which is usually taken as being equal to the equilibrium spin density, p(ro). This assumption requires that insignificant relaxation occur between the first rf pulse (i.e. excitation) and the first gradient pulse. In practice this is normally the case. P(ro, r , A) is the Green's function or diffusion propagator,52959the conditional probability that a spin starting at position ro will migrate to r in a time A. The q (ro - r ) vector product is the change in phase of the spin by moving from ro to r ; this also indicates that the diffusion is measured in the direction parallel to the applied gradient. Thus the total signal is a superposition of signals (transverse magnetizations), in which each phase term is weighted by the probability for a spin to begin at r o and move to r. The double integration results from the need to average the result over all the sample (i.e. all possible starting and finishing positions). It is because the method only measures diffusion in one direction that it provides such a direct means of measuring diffusion anisotropy . 0

3.3.4. Numerical methods

There have been numerous attempts at numerically simulating both steady and PFG experiments. Numerical simulations have been based upon finite differencing schemes and lattice methods,6G62 a Fourier a p p r ~ a c h , and ~~.~ Brownian dynamic and Monte Carlo sir nu la ti on^.^^,^^,^^-^^ The Monte Carlo method has been used in combination with neural networks to simulate the results for a particle diffusing in a biconcave It should be noted, however, that while it is possible to analyse more complicated geometries and systems using numerical methods, little insight is gained into the physical problem at hand.

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3.4. “Diffusive diffraction” and imaging molecular motion

We define the probability that a molecule at any starting position will displace by R = r - ro during the period A as15.59 P(R, A) =

i

drop(ro) P(ro, ro + R, A)

(24)

F(R, A) has also been termed the “average propagator”. Using equation (24) we can rewrite equation (23) as

E ( q , A) =

I

dR P(R, A) e x p ( i 2 ~ q*R)

Physical insight can be gained by noting from equation (25) that there is a Fourier relationship between E(q, A) and P(R, A).59 Thus, Fourier transform of E ( q , A) with respect to q returns an image of F(R, A). Conventional (i.e. k space) imaging returns an image of p(ro). Thus, equation (23) in Section 3.3.3 is analogous to the scattering function which applies in neutron scattering, and q corresponds to the scattering wave vector. However, there are major differences in the temporal and spatial time-scales of each type of experiment. Further, E ( q , A) is measured in the time domain of A in PFG experiments and in the frequency domain for neutron-scattering experiments. A direct analogy can be made between PFG NMR of a spin undergoing restricted diffusion in an enclosed pore and optical diffraction by a single s1it.30,66,73,74 In the long-time limit (i.e. A+.) all species lose memory of their starting positions, and so P(r0, r, 00) = p(r)

(26)

and thus, in the long-time limit, the average propagator becomes P(R, 03) =

I

dro p(ro + R ) p ( r ~ )

(27)

Thus, P(R, a)is the autocorrelation function of the molecular density p(ro). By the Wiener-Kintchine t h e ~ r e m , ’ E(q, ~ m ) is the power spectrum of p(ro). Hence, from equation (23)

where p(q) is the Fourier transform of p(r). p(q) is analogous to the signal measured in conventional imaging.2 However, unlike conventional imaging which returns the phase sensitive spatial spectrum of the restricting pore,

68

WILLIAM S . PRICE

\\

;ri

1E-5

-

1E-6

-

1E-7

-

1E-8

0

20000

40000

60000

80000

4 (m-'1 Fig. 3. A plot of E(q, a) versus q for two values of the slit width (2R), 2R = 26 pm (---) and 3 0 p m (-). It can be clearly seen that the diffractive minima and maxima

are R-dependent.

E(q, 03) measures the power spectrum, lp(q)I2. Cory and G a r r ~ w a y ~ ~ remarked that a rectangular pore reproduces exactly the diffraction pattern of a single slit of width 2R, and in this case equation (28) can be rewritten as E(q, w) = 1 sinc(2~qR)

=

2[1- C O S ( ~ T ~ R ) ] (4.rrqR)*

(29)

Since Fourier inversion does not yield p(r) but instead the autocorrelation function of p(r), and in consideration of the diffraction analogy, structural information can be obtained in q space by using the characteristic features of the diffraction pattern such as the position of the nodes. In Fig. 3, E(q, A = a) is plotted against q for two values of R . Clearly, by having more than two gradient pulses with the pulses having different directions, PFG can be turned into a multiple wave vector experiment and thereby gain more information.7G78 The simplest case would be to have two independent wave vectors (either orthogonal or collinear) .76,77 Mitra78has considered the case where the relative orientation

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69

is arbitrary. H e showed that the dependence of M ( q , t) on the angle between the two wave vectors contains information that is absent in conventional PFG measurements. 3.5. Simple geometries Here we summarize some of the solutions given in the literature describing the attenuation in PFG experiments. In agreement with the discussion concerning free and restricted diffusion (see Section 3.2), the solutions for the restricting geometries reduce to that of free diffusion in the short time-scale limit (i.e. & 1) the solutions become dependent only upon the restricting geometry.

3.5.I . Flow The attenuation due to plug flow in the Stejskal and Tanner sequence, in the absence of background gradients, is given by55

(30) where v is the flow velocity. Thus, the signal is phase shifted by the effect of the plug flow and exponentially damped by the diffusion. A theoretical description of the signal attenuation from a turbulent flowing liquid in a PFG experiment has also been developed.79380

3.5.2. Reflecting boundaries (i) Reflecting planes. For the case of spins confined between two infinitely large perfectly reflecting parallel planes (separation 2R) with the direction of the field gradients perpendicular to the planes, using the SGP appr~xirnation,~~

m

+ 4(47rq~)'C exp n=l

In the long time limit this reduces to

1- (-l)"cos(47rqR) [(47rqR)'- (n7r)2]2

(31)

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WILLIAM S . PRICE

which is, of course, identical to equation (29). Similarly, using the GPD approximation,68

2 - 2L(S) (2s-

+ L(A - S ) (2n

+

2L(A) + L(A + 6) 1)’dDi(2R)’ -

)]

(33)

where L(r) = exp[ - (2n + 1)2?Dt/(2R)2].

(ii) Reflecting cylinder. For a gradient applied along the polar axis direction (i.e. across a diameter), the GPD approximation solution isx1

c

n=l

2 a i D 6 - 2 + L(6) t L ( A - 8) - 2L(A) -2L(A cw;(R’a: - 1)

+ 6)

)

(34)

where L(t) = exp(-aiDt). The an are the roots of the equation J;(a,R) = 0, where J is the Bessel function of the first kind. (iii) Reflecting sphere. For the case of spins confined to a perfectly reflecting sphere, the SGP solution is given by6’

m

n=O

rn

where an,, is the mth root of the equation j;(a,,) = 0 and j is the spherical Bessel function of the first kind. In the long-time limit, equation (35) reduces to58

E(q,A) =

9[(2.rrqR)cos(2rqR) - sin(2.rrqR)I2 (2.rrqW

(36)

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71

Similarly, using the GPD approximation,82

c

2a:DS - 2

+ 2L(S)

n=1

+

L ( A - S) 2L(A) - L ( A a: (R2 a: - 2 ) -

+ 6)

)

(37)

where L ( t ) = exp(-a:Dt) and the a, are the roots of ( C Y , R ) J & ~ ( ~ , R ) 1/2J3/2(an R ) = 0. The corresponding equation for the Karlicek-Lowe alternating PFG experiment has also been d e r i ~ e d . ’ In ~ the limit (>> 1 and S << A, equation (37) becomes5’

E ( q , A) = exp[ - (2.rrqR)’/S]

(38)

3.5.3. Absorbing boundaries Enhanced spin relaxation at boundaries has recently received much attention since it is closer to reality than a completely reflecting boundary. This corresponds to the partially absorbing boundary condition,

where r = (rl and H (m/s) is the permeability coefficient. A schematic diagram of spins diffusing inside a spherical interface having the partially absorbing boundary condition is given in Fig. 4. The same condition is fulfilled if the interface itself is covered by a layer of active relaxation centres.84 H would then stand for the surface relaxivity or the density of such centres. Another situation leading t o equation (39) is where the external spin diffusivity significantly exceeds the internal.” A t this point it is convenient to define a reduced (i.e. dimensionless) permeability coefficient by h = HR/D. The microscopic first-order rate constant, ktr, for transport through the interface can be related to the permeability. For example, in the case of diffusion through a sphere,86 k,, = 3HlR = 3Dh/R2. The solutions given in Section 3.5.2 are merely special cases (i.e. H = 0) of the corresponding solutions given below.

(i) Absorbing planes. Tanner*7 solved the closely related problem of diffusion in an infinite system of parallel semipermeable planes. For the case of a pair of absorbing planes, a solution using the SGP approximation was first published by Mitra and Sen,84 and later by Snaar and Van As,47 and

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WILLIAM S. PRICE

Fig. 4. The partially absorbing wall model. R denotes the radius of the sphere and H denotes the permeability (m/s) through the interface. In this model, when a spin reaches the external medium, its signal is instantly annihilated. (Adapted from Barzykin et ~ 1 . ~ ~ )

Coy and Callaghan.69.88Here we present the result derived by Coy and Callaghan, with the gradient being applied perpendicular to the planes,

[ ( Z T ~ R ) sin(27rq~)cos a, - a, cos(27rq~)sin anJ2 [(2TrqR)2 - a;]2

where the eigenvalues a, and

pmare given by a, tan(an) = h

and

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73

Some experimental data and simulations using equation (40) are given in Fig. 5. Coy and Callaghad9 noted that wall relaxation effects do not significantly perturb the diffraction pattern, and the major effect is to shift the position of the diffraction minima, resulting in a reduced value of R . They found that small variations in R had a much larger effect. A n extension of equation (40) which includes relaxation before and after the gradient pulse has also been presented.69 (ii) Absorbing cylinder. The relaxing boundary is at a radial distance R from the cylinder centre and, as before, the gradient is applied across the polar direction. The SGP solution iss8

where the eigenvalues are given by

(iii) Absorbing spheres. For the case of spheres with relaxing walls, the SGP solution iS46,8436,88

where an, are the positive roots of

In a typical experiment the condition [> 1 holds, and only the lowest

74

WILLIAM S. PRICE

0

5000

10000

15000

0

5000

10000

15000

1

0.1 r?

Q CY

G 0.01

0.001

I

Fig. 5. Plot of E ( q , A) versus q for a PFG experiment of pentane diffusing in a capillary stack (2R = 100pm) for different values of A ( 0 , 200ms; a, 300ms; 0 , 700 ms; m, 900 ms). The theoretical lines represent regressions of equation (40) on to the experimental data. In the upper graph H was fixed according to the known relaxation time, and R and D allowed to float. In the lower graph a distribution of R was allowed. (Reproduced with permission from Coy and C a l l a g h a r ~ . ~ ~ )

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75

eigenvalue a = aoo is important. Thus, from equation (43), a satisfies the following equation:

In this long-time limit the normalized echo amplitude is independent of and is given by

(

a2 (hh a’- (27rqR)’

sin (2rrqR) 2rrqR

6,

+ cos(2rrqR)

In the limit of weak permeability (i.e. h-0, reflecting boundary), the lowest positive solution of equation (44) is a2 = 3h, and equation (45) transforms into

which is, of course, the same as equation (38). In the limit of infinite permeability (i.e. h+w, perfectly absorbing boundary), we have

The approximate equalities in equations (46) and (47) are obtained for 2rrqR << 1 . The amplitude of the echo signal for a typical set of experimental parameters is plotted in Figs 6 and 7 as a function of (qR)’ for a range of surface permeability values. Only a weak dependence on permeability is noted. Further, there is an interesting effect in that there is a weak decrease in the apparent diffusion coefficient as given by the initial slope, i.e.

as h is increased. The reason for the apparent slowing of diffusion is that as the permeability increases, more spins leave the sphere and no longer contribute to the signal. At high permeability values, only those spins survive which do not reach the surface by time A. It has been noted8’ that, providing h 7 1, the relaxation due to the absorbing surface is single mode with relaxation time R/3H while the PFG data depends on both R and h. D can be found from low q short A (i.e. [<< 1) PFG measurements leaving H and R as the only unknowns. Thus by combining relaxation and PFG measurements, R can be found iteratively.

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WILLIAM S. PRICE

h 0 3 10

Fig. 6. A plot of the normalized echo amplitude versus (qR)* for various values of

the permeability coefficient h. The calculations were performed using 6 = 0.765 which, for instance, corresponds to D = 1 x lo-'' m2/s, A = 60 ms and R = 2.8 x m, according to the values used in an experimental study of bicarbonate diffusion in red blood cells.'18 The solid lines are calculated from equation (42) whereas the dashed lines were plotted using the long-time limit formula of equation (45). The plot clearly shows that the positions of the minima are h-dependent. In reality the values of h are always small. However, we have included a large range of h values so that the effects may be easily visualized. (Adapted from Barzykin el

3.5.4. Fractals and anomalous diffusion Diffusion in fractal networks and some polymer systems gives rise to anomalous diffusion. In order to understand this phenomenon and its consequences for gradient measurements, we must introduce the randomwalk dimension d,.

For normal unrestricted motion the time-independent proportionality constant a = 6 0 ,and d , = 2, and equation (49) reduces to equation (4), but in the case of fractal networks d , > 2. This has the result that the diffusion in a fractal network is not invariant against time translation and the mean square displacement increases less than linearly with the observation time.

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77

0.

(@I2 Fig. 7 . An expansion of Fig. 6 for small values of qR. The solid lines are calculated from equation (42), and from Ieft to right the values of h used are 0, 3, 10 and m , whereas the dotted lines were plotted using the long-time limit formula for 0 (left) and m (right) permeability and the short-pulse approximation (equations (46) and (47)). The parameters used in the calculations are given in the caption to Fig. 6. The plot shows that the a parent diffusion coefficient decreases as h increases. (Adapted from Barzykin et al.8F)

Using the GPD approximation, the PFG spin echo attenuation for diffusion in a fractal network is given by89

YYff

E(q’ A) = exp{ 3 ( +~ 1 ) (+~2)

- Vi(A where

K =

+ AK+2- %(A + 6)K+2]j

2/dw.Using the SGP a p p r o ~ i m a t i o n , ~ ~ , ~ ~ E ( q , A) = exp( -

A solution for the steady field gradient case has also been derived.89

3.5.5. Anisotropic diflusion Isotropic diffusion is really a special case, and generally the self-diffusion coefficient is described by a Cartesian tensor (see equation (6))’ for example

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WILLIAM S. PRICE

diffusion in a liquid crystal. In the case of the equation describing the echo attenuation due to free diffusion (i.e. equation (19)), the 8 D term must be replaced by g.D.g. Thus, the direction that the diffusion is measured is determined by the gradient and it is actually a diagonal element of D’, the diffusion tensor in the gradient frame that is measured. The diffusion tensor in the gradient frame can be linked to the tensor in the molecular frame by using rotation matrices,

where R is the relevant rotation matrix. Thus the off-diagonal elements of D’ will vanish only when the director of the restriction and laboratory frames of reference coincide. Thus, in the general case both diagonal and off-diagonal elements of D will effect the measured echo attenuation.s3354 The dependence of D on the orientation with respect to the gradient thus provides an additional structural probe. Ideally, it is possible to determine the dimensions of the restricting geometry from the restricted displacement. But this requires prior knowledge of the sample orientation so that it is possible to align the gradient in the relevant direction. This is usually not possible, and the measurement returns a mixture of the different elements of the diffusion tensor. A solution is to determine the diffusion coefficients in six different directions so as to define D. Since diffusion measurements are not without error, this method provides a poor estimate of D, and a better approach is to perform a much larger number of trials and to determine D s t a t i s t i ~ a l l yHowever, .~~ as a further complication, the restricting geometries may not all be aligned in the same direction (e.g. a suspension of red blood cells). The effective D’ is then an average of the different orientations. Van Gelderen et a1.81 have proposed measuring diffusion in three orthogonal directions so as to determine the trace of the diffusion tensor, ?hTr(D’) =

l/3(0i1

+ D$2 + 05,) = %Tr(D) = D,,

(53)

As the trace is invariant under rotations, the orientational dependence is removed. Generally, diffusion is only measured in one direction, and if an anisotropic system is not oriented in one direction, it is necessary to perform a “powder average”. It is mathematically equivalent to consider that there is only a single domain with a defined direction and that it is the field gradient randomly oriented, thus equation (23) become^^^,^^

4?r

d8dc#JdVodVp(ro,O)P(ro, r, A) cos[2?rq-(r - ro)]sin 8

GRADIENT NMR

79

where 0 and 4 are the polar angles and 1/(4r) sin 0 do d 4 is the probability of q being in the direction defined by 0 and 4. The situation is much more complicated, though, if in the time-scale of A the diffusing molecules change from one domain, specified by a unique local director orientation into a n ~ t h e r . ~ Equation ~?'~ (54), for polycrystalline systems, may be further simplified to

For one-dimensional (or capillary) diffusion, we have91s93 m

E ( q , A) = 1+ n=l

1 (-1)" n!(2n l)! (2r q AD)"

+

(56)

and for two-dimensional (or lamellar) diffusion,

The three-dimensional case is, of course, free diffusion (see equation (19)). Callaghan4 has given a detailed discussion on how one-, two- and threedimensional diffusion each gives characteristic echo attenuation curves. As is often the case for more complicated systems, exact analytical solutions are not available, and equation (54) must be evaluated numerically (see Section 3.3.4),67391

3.6. VaIidity of the SGP and GPD approximations

There has been much discussion recently about the validity of the GPD approximation (e.g. see refs 84 and 94-96). The GPD approximation is exact in the limit of free diffusion (i.e. R+m), i.e. where the phase distribution is Gaussian. However, the SGP equation is only strictly valid for infinitely small 6. Balinov er aL6* used computer simulations of Brownian motion to test the validity of the GPD and SGP approximations. They found that the Murday and Cotts equation (see equation (37)), which is based on the GPD approximation, simulated the data very well in the limit of [< 1, fairly well for 5- 1, and well for &>1, in agreement with the predictions by N e ~ m a n . ~The ' SGP equation described the data well for large values of 5 and small gradient strengths. The results showed that at 5- 1 the long-time limit of the SGP equation (i.e. the attenuation has become independent of A) is already applicable. In the GPD approximation the problem of finite gradient pulses is treated by writing an expression for the phase of the

80

WILLIAM S. PRICE

magnetization in terms of an integral over particle displacements. However, a severe approximation must be made when this approach is adapted to treat restricted diffusion, and the Gaussian phase condition is enforced and the phase factor is calculated from the mean squared phase deviation. This approach therefore does not yield interference effects.69 Blees6' extended the finite difference approach of Zientara and Freed6' to obtain solutions for diffusion between two planes using gradient pulses of finite duration, and compared this to the SGP result (see equation (31)). Blees focused on the effects of the gradient pulse duration on the diffraction peaks that occur at high q values. He found that as the gradient pulse duration increased, the diffraction peaks shifted toward higher values of q (i.e. making the pore size appear smaller than the actual size); further, the higher-order minima were more greatly affected (see Fig. 8). Recently, it has been shown that at finite gradient pulse lengths, the echo amplitude is the spatial Fourier transform of a "centre of mass" p r ~ p a g a t o r .As ~~ expected, as b 0 , the centre of mass propagator reduces to the usual diffusion propagator.

3.7. More complicated boundary conditions

3.7.1. Introduction Above we have considered only simple systems, where species diffusing within a restricting geometry do not interact with other restricting geometries. In real systems (e.g. biological cells or porous systems) though, it may be necessary to consider the effects of (a combination of) exchange, obstruction and polydispersity in addition to surface and bulk relaxation. The case of exchange rapidly becomes very complicated since the spins in each domain may have different diffusion coefficients and relaxation rates as well as being subject to restricted diffusion. 3.7.2. Polydispersity, polymers and macromolecular systems Since a number of reviews (e.g. see refs 13, 19 and 98) have been concerned with the application of gradient NMR for determining the diffusion coefficients and other related physical parameters of polymers, we will only touch upon this topic briefly. A major problem in using PFG to study polymers is their p ~ l y d i s p e r s i t y . ~Apart ~ " ~ from the distribution of diffusion coefficients, the different molecular weights and molecular mobility of polydisperse species lead to different relaxation rates (see Section 4.4), and, hence, the observed echo signal is not weighted by the respective concentrations alone. STEbased experiments are less influenced by this relaxation problem. Analysis

GRADIENT NMR

81

0

-1

-2

-3

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Fig. 8. Failure of the SGP approximation. The effect of the finite duration of the gradient pulse on the spin echo attenuation is shown. The numbers given in the key are the ratio 6/A. The calculations were performed setting ( 2 R ) * / 2 0 A= 1. (Reproduced with permission from Blees.61)

of the data from polydisperse samples is considerably simplified if one is able to assume a particular diffusion coefficient di~tribution.''~Recently a new, gradient based method, called DOSY (diffusion ordered spectroscopy) (see Section 6.5.2) provides another alternative for tackling polydisperse systems. By directly measuring the polymer diffusion coefficient at high dilution, it is possible to obtain the effective hydrodynamic (or Stokes) radius, r,, of the polymer using the Stokes-Einstein equation,

where T is temperature and 7 is the solvent viscosity. However, as the polymer concentration increases, the interaction between polymer chains causes the diffusion to become concentration dependent. Thus, the concentration dependence of the polymer and solvent diffusion coefficients provide information about the shape of the polymer. In polymer melts and in

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WILLIAM S. PRICE

solutions with high polymer concentrations, the dependence of the diffusion coefficient on concentration and molar mass provides information about entanglement of the polymer chains. In some polymer gel systems the diffusion is described by the particles diffusing in an isotropic medium while being harmonically bound to an attractive For small values of A, the observed displacement may be smaller than the end-to-end distance of the polymer chain. Hence, a polymer system can be considered as being heterogeneous at short A values. The diffusion of the polymer molecules is restricted by instantaneous tubes formed by the bulk phase of the surrounding molecules. The reptation time, tr, is defined as the time required for a polymer molecule to cover a curvilinear diffusion path of the order of its contour length. For A > & , the confining tube will be completely uncorrelated with the previous tube and the observed diffusion coefficient will appear to be constant (i.e. it will not scale with time; see Section 3.5.4). Conversely, for A < tr, the motion of the polymer segments is subject to a correlated confinement. Accordingly, the apparent diffusion coefficient will decrease with increasing A. This model of molecular "reptation" in polymers has been confirmed with PFG NMR for both polymer melts'05-107 and solutions. 108~109 Polymeric microgels are quite distinct from polymers. They are semi-rigid and are unable to penetrate each other. While, similar to polymers, their diffusivity is determined by mutual interactions, their transport properties are quite different from polymer chains. In a study with cross-linked polystyrene beads, it was noted that with increasing concentration a large decrease in mobility is observed and the deviation from ordinary diffusion increased.'" This indicated that the individual microgels are in a cage formed by their neighbours. However, as the cages are not perfectly confining at large A, the motion of the microgels appears to occur via normal diffusion.

3.7.3. Size distributions of the restricting geometry A related problem to polydispersity is where spins diffuse inside a polydisperse set of restricting geometries. With an appropriate model, PFG data can be analysed to give the size distribution. As an example, we consider the attenuation of the NMR signal resulting from spins diffusing inside a polydisperse set of spheres (e.g. an emulsion). The possibility of exchange between the two domains is neglected. Taking into account the distribution of the sphere radii, P(R), the attenuation is given by"' io

[

dR R3P(R)E(q, A)

GRADIENT NMR

83

where E ( q , A) is given by equation (37). Often P ( R ) is taken to be a log-normal function. Callaghan et a1.'l2 developed a similar method using another size distribution. An improvement based on the recognition that the echo attenuation data obtained in the long-time limit contains all the necessary information on the radii distribution has been presented.' l 3 More recently, Fourel et al. 'I4 supplemented equation (59) by adding an unrestricted diffusion component. The unrestricted component is either to account for the case where the distribution of radii is not perfectly log-normal or to account for a freely diffusing component.

3.7.4. Obstruction

Obstruction occurs when a small molecule, for example in a colloidal system, is excluded from a fraction of the total volume by the colloidal particles. This causes a lengthening of the diffusion paths which results in an effective diffusion coefficient, Deff.The description of self-diffusion in such systems is mathematically very difficult since the diffusion path of the molecule can be very complicated; also, the colloidal particles are not distributed in space in a totally ordered way nor in a totally random way. Using a cell model, Jonsson et ul."' derived the obstruction effect for a system containing spherical monodisperse particles; neglecting binding effects, the effective diffusion coefficient is given by

Hence, it can be seen that in the case of obstruction by spheres the limiting value of Deff is 2D/3. They also considered the cases of obstruction by spheroidal particles and by polydisperse systems. Since the degree of obstruction is dependent on the shapes of the obstructing particles, it provides an additional source of structural information. Recently, Blees and Leyte'I6 derived a model for the effective translational diffusion coefficient of particles diffusing within colloidal crystals modelled as a lattice of immobile spheres. The centres of the spheres are located on the lattice points of a cubic array (either simple cubic, body-centred cubic or face-centred cubic lattices). In their model, the diffusing particle has dimensions much smaller than those of the colloidal particles and is able to move freely inside both the colloidal particles and the continuous phase. The diffusing molecule is characterized by both a diffusion coefficient and a concentration in the continuous phase, and a diffusion coefficient and concentration inside the colloidal particles. They found that for low volume fractions of the obstructing particle ( 4 < 0.3), the calculated self-diffusion coefficient is independent of the lattice type and is in agreement with the simple cell model of Jonsson et ~ l . ~ ' '

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WILLIAM S. PRICE

3.7.5. Exchange and many body effects The simplest case of exchange that we consider is that between two freely diffusing regions, with each region being characterized by a different diffusion coefficient. Ignoring relaxation time differences between the two compartments, the echo attenuation is given by a superposition of exponentia~s,’~J~’

E ( q , A) = P1 exp[ - ( 2 ~ q ) ~A]D +, P2 exp[ - ( 2 ~ q ) ~ D ~ A l(61) where D 1and D2 are the apparent self-diffusion coefficients defined below, and P1 and P2 are the population fractions (relative signal intensities) given by

D1(2)=

‘i 2

D,+Di+

1 ~

(2Tq)2(

-+ie

ii

)

and

where D, and Di are the diffusion coefficients in the two domains. Similarly P, and Pi are the relative populations, and T, and T~ are the mean residence lifetime in each domain. The above equation is based on the assumptions that the exchange rates are much faster than the transverse relaxation rates of the species in question. This approach involves a serious approximation, transport between different subregions is introduced through the mean residence times and conditional hopping probabilities, by combining Fick’s second law with the Chapman-Kolmogorov equations.” Thus, the space coordinate is applied in a macroscopic sense, leaving the space unit much larger than the diameters of the individual subregions. However, this approximation considerably simplifies the solution of the underlying diffusion problem. If the exchange is in the slow or fast exchange limit, equation (61) can be further ~imp1ified.l~ In biological systems such as red blood cell suspensions, transport occurs between a restricted and an unrestricted (or perhaps obstructed) domain. In the present discussion we will assume that the restricted domain is constituted by a sphere. In this case the solution to equation (8) becomes very complicated. A means of making the system more mathematically tractable is to achieve the “partially absorbing wall” condition (see Section

GRADIENT NMR

85

3.5.3 and Fig. 4). In this case a two-site system is modelled by a one-site system, implying infinitely fast relaxation in the other domain (see equation ( 4 2 ) ) . In cell or vesicle systems the partially absorbing wall experiment is, at least in principle, possible as the relaxation properties of the exterior medium may be manipulated by addition of relaxation agents. However, in practice it is experimentally difficult to attain the instant quenching condition,86~"8and the spins in the exterior medium contribute considerably to the observed echo signal. Further, even if the true partially absorbing boundary condition is achieved, this model is only weakly sensitive to t r a n ~ p o r t . ~An ~ , ' extension ~ of the partially absorbing wall condition for the experimentally more realizable model where the spins that transport through the interface are rapidly relaxed but not instantly quenched has been developed.86 In this formulation, the finite lifetime of the spins in both domains is included. Accordingly, the diffusion equation (i.e. equation (8)) must be complemented with a decay term and is given by

T2 and D are different in each domain and are denoted below by the subscripts i and e, corresponding to the interior and the exterior, respectively. The boundary conditions for the Green's function are given by Di--/ a Pi ar

r=R

De$( r=R

and

Equation (65) describes the flux continuity over the interface, while equation (66) states that the flux is proportional to the concentration difference between the domains weighted by the appropriate permeability coefficients. At equilibrium, there is no flux across the interface, and [Pi/P,leq = He/Hi. The contribution to the echo signal from the spins found at time A inside the sphere is found to be r

E i ( q , ~= ) 6 C (2n + l)exp(-tiaL)fn(anm) n,m

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WILLIAM S. PRICE

where anmare the positive real roots of the equation a n m j n + 1 ( a n m ) [PnmKn+3/2(Pnm)- ( n - he) Kn+l/~(Pnm)l

= jn(anm)[(n+ hi)PnmKn+3/2(Pnm)- n ( n + h i - he)Kn+l/2(Pnm)] (68)

Pnm = (&-I with E = DeT2,IR2 and 5 = DiiD,; hi = H i R / D i , he = H e R / D e , K is the modified Bessel function of the third kind, and fn(anm) is a constant and is further considered below. It is assumed that at least one root exists; this requires small Tze and sufficiently small permeability. The latter condition is normally fulfilled experimentally for most systems of interest. As the lifetime in the outer domain is decreased (i.e. T Z e + 0 ) , Pam tends to infinity, and the inner component of the echo reduces to equation (42) as expected. The contribution to the echo signal from the spins found at time A outside the sphere iss6 m

Ee(q, A) = 6hil

C (2n + 1)exp(-tia;m) n,m

f

The roots are defined as above. For n = 0, the functions f and are related via

The outer component vanishes as T2, tends to zero, as expected. In the long-time limit, only the lowest eigenvalue a = am is important, which, from equation (68), satisfies the following equation:

where P = Poo. The corresponding constant fo(a) in equation (67) is given by

GRADIENT NMR

87

The overall normalized long-time echo amplitude is again independent of A. The components are given by

and

[&p2 +

a

(2~q)~R~ (74)

where h = hi/[l + hJ(1 + p)]. The amplitude of the echo signal for a typical set of experimental pararneters1l8 is plotted in Fig. 9 as a function of (qR)2 for a range of permeability values. It is assumed that the permeability coefficient and hence the equilibrium spin concentrations are equal in both domains. In contrast to the partially absorbing wall case, finite relaxation rates in the external medium lead to an increase in the apparent diffusion coefficient given by the initial slope as the permeability increases. This is what was experimentally observed in a study of bicarbonate ions diffusing through red blood cell membranes into an Mn2+-doped extracellular medium. Even though the external relaxation rate is very fast (but not infinite), a small population of spins survive at the end of the PFG experiment and result in a considerable enhancement in the observed apparent diffusion coefficient. Typically, two stages are observed, a rapid initial attenuation due to the external component followed by a slower attenuation due to the internal component. When the ratio of the internal and external diffusion coefficients is very small (i.e. [<< l ) , the initial stage is too fast and may not be detected. When there is no restriction on the lifetimes in either phase the mathematics become more cumbersome. A schematic diagram of such a system is given in Fig. 10, and some experimental data for hypophosphite (H2P02-) transport in human red blood cells is presented in Figs 11 and 12, and for bicarbonate transport in human red blood cells in Fig. 13. The theory has now been extended for the SGP limit to the case where there is no restriction on the lifetimes in either domain, and this will be presented

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WILLIAM S . PRICE

h

I

0.0

0.2

0.5 0.7

0.2 -

0.0 0.00

0.02

0.04

0.06

Fig. 9 . A plot of the normalized echo amplitude calculated versus (qR)2 using equations (73) and (74) of spins diffusing through a spherical interface into a rapidly (but not instantly) relaxing external medium. The calculations were performed with 6 = 0.765, A = 60ms, R = 2.8 x 10-6m, Tze = 26 ms, Di= 1 X 10-"m2/s and m2/s. In contrast to the case of a perfectly absorbing external medium D, = 1 x (see Figs 6 and 7), an increase in hi is accompanied by an increase in the apparent diffusion coefficient. Smaller values of hi were considered than in Figs 6 and 7, so that p in equation (71) was real. For comparison the corresponding plots of the contribution to the echo signal from inside the interface (equation (73)) are also included as dashed lines. For hi = 0 the lines are coincident.

elsewhere. The theoretical amplitude of the echo signal as a function of S is plotted for a range of surface permeability values in Fig. 14. 3.7.6. Porous media Many aspects of PFG and diffusion that are relevant to porous media have been discussed above as isolated cases. Here I wish to dwell specifically on PFG of porous media such as rocks. Porous media constitute the most general case of restricted diffusion. As mentioned in Section 3.2 it is normally assumed that the relaxation can be factored out (i.e. equation (9)). However, this assumption only holds when H is sufficiently small such that DIH is much larger than V,IS and S and V , are the surface area and volume of the pore, respectively. Vp equals $V, where 4, is the porosity and V is the total volume.'*'

m.

GRADIENT NMR

89

Fig. 10. A schematic representation of diffusive transport through a spherical interface. The subscripts i and e refer to the internal and external medium, respectively. Hi refers to the outward permeability and He to the inward permeability. When either D, >>Dior Tzi >> Tze this model becomes equivalent to the partially absorbing wall condition (see Fig. 3). (Adapted from Price et ~ 1 . ” ’ )

In a porous structure a diffusing spin can migrate between pores, and this has the effect of changing the single-slit “diffraction” into a “diffusion grating”.30 For the purpose of our discussion we will imagine that we have numerous pores of size R and spacing L . For describing E ( q , A) in a porous medium there are three relevant diffusion coefficients, D , D e f fand Dapp.D is the true unrestricted molecular diffusion coefficient, Deffis the effective long-range diffusion coefficient across the permeable structure, and the apparent diffusion coefficient Dappis determined from the time dependence of E ( q , A) at any value of q (see equation (48)). Information about the pore spacing and connectivity is contained in the ratio DapdDeff. For long time and low q , Dappapproaches Deffand, as expected, when t<1 Dappreduces to D (see equation (48)). The elements of the “diffraction grating” are weighted according to a diffusion envelope C(2, A) (a Gaussian, see equation (3)), which describes the probability that a spin will move to another pore a displacement 2 parallel to q from the starting pore after a time A. Hence, provided R2/D<< L2/Deff(i.e. the pore equilibration assumption), P(ro, r, A) becomes a product of the pore density and probability of jumps between pores.

where ISO(q)l2 is the average pore structure factor, L ( Z ) is the “lattice correlation function” describing the relative position of the pore centres, F

tDNDS

-DNDS 60

40

20

0

-20

-40

6 (103 x

S)

-60

(Hz)

Fig. 11. 31P PFG NMR of H2P02- transport in a suspension of human red blood cells at 310 K. The intra- and extracellular species give separate resonances, and the transport can be blocked by the addition of dinitrostilbene (DNDS). The experimental parameters were A = 100 ms and g = 0.28 T/m. The haematocrit of the cells was -0.7 and the cell volume was about 70 fl, which, assuming the cells to be spherical, gives an effective radius of about 2.5 p m . AS denotes the difference in signal attenuation of the intracellular species due to transport. The signals of the extracellular species, which have little obstruction, attenuate very rapidly. (Adapted from Price et u/."*)

GRADIENT N M R

91

0.00 -0.25 -0.50

-0.75 -1.oo -1.25 -1-50 -1-75

0

2

4 2

6 (A-M3)

8

6 (10

5

3

X S

10

)

Fig. 12. Plot of the H2P02- attenuation data shown in Fig. 11 versus S2(A - 6/3). The intra- and extracellular signals from the DNDS-treated cells are denoted by 0 and W , respectively. Similarly the intra- and extracellular signals from the untreated cells are denoted by and 0 , respectively. The effect of transport is clearly visible on the intracellular signal but negligible on the extracellular signal. The solid line through the extracellular data of the DNDS treated cells is the result of linear regression of equation (19) on to the data (NB-with go = 0), which gave an apparent diffusion coefficient of Dapp= 5.9 X lo-"' m2/s. The true diffusion coefficient, as measured in cell-free supernatant, was 1.60 X lop9m2/s. This indicates the presence of obstruction effects for the extracellular species. The dashed line represents a simulation using equation (37) where the cells are assumed to be spherical with a radius of 2.5 prn, giving the intracellular diffusion coefficient 1.5 X lo-'" m2/s. The solid line through the intracellular data of the untreated cells is a visual interpolation. The dot-dash line is a simulation where it was assumed that 59% of the NMR signal arose from the intracellular species diffusin within spheres with a radius of 2.5 p m with a diffusion coefficient of 1.5 X lo-'' m2/s with the remainder of the signal resultin8 from diffusion in spheres of 8 p m radius with a diffusion coefficient of 1.6 x 10- m2/s. (Adapted from Price et d.'")

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WILLIAM S . PRICE

2

Fig. 13. Plot of the attenuation data resulting from 13C PFG NMR of Hi3C03- in a suspension of human red blood cells at 310 K versus @(A - S/3)12, where I is the current used to generate the gradient pulses. Cells were prepared with ( 0 ) and without (4) the transport inhibitor DNDS. The results of a diffusion experiment performed on the bicarbonate ion in cell-free supernatant are also shown (m). The experimental parameters for the cell experiments were A = 140 ms and g = 0.45 T/ rn, and for the supernatant sample were A = 100 ms and g = 0.2 T/m. The Z2 term is included in the abscissa to allow comparison of the data since they were acquired with two different gradient strengths. The solid line through the supernatant data is the result of linear regression of equation (19) on to the data (NB-with go = 0), which gave a diffusion coefficient of D = 1.26 x 10-'0m2/s. The data from the DNDS-treated cells result from two-component diffusion with the signal from the rapidly diffusing extracellular bicarbonate being superimposed upon the slowly attenuating signal from the intracellular species. The solid line through data from the DNDS-treated cells is a visual interpolation. The solid line through the untreated cells is from linear regression, giving an apparent diffusion coefficient of 9.4 x lo-'' m2/s. (Adapted from Price et

0.0

-0.2

-0.4 n n

Q-

m

-0.6

W

-

Y v fi

-0.8

-1.0

-1.2

0

5

10

15

20

25

30

Fig. 14. Example attenuation curves of the intracellular signal versus 6 for a variety of values of the permeability when there is no restriction on the lifetime in either domain. The parameters used in the simulation were A = looms, g = 0.3T/m, R = 2.5pm, D, = 1.5 x m2/s and D , = 5.9 x lo-'' m2/s. The concentration and volume ratios (i.e. [internal] : [external]) were 41 : 66 and 1 :0.6, respectively. (Adapted from Price et

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WILLIAM S. PRICE

denotes Fourier transform and 63 represents convolution. F{L(Z)} is the reciprocal lattice which is broadened by F{C ( Z , A)}. This broadening arises because the number of “scattering centres” in the lattice increases with time as the spins diffuse to more distant pores. If the pore spacing L is irregular, the maximum E ( q , A) will occur at 141 = L-’. Thus, using small values of q (
where u is the standard deviation of the pore spacing. This model predicts coherence peaks in E(q, A) when qL is an integer. It was noted that DeBis already effective at times when a single spin would have oniy diffused across one pore width, while pore structure effects are not visible until a spin has had time to visit a neighbouring pore.38 By performing inverse Fourier transforms of the experimental data, Callaghan et aL3’ were able to construct an “image” of the spherically averaged autocorrelation function. In a later work, diffraction effects were studied in a more polydisperse set of polystyrene spheres.121It was found that the coherence peaks were strongly influenced by the sphere size and polydispersity. In a related work to that of Callaghan et aZ.38 Sen and c o - w o r k e r ~ ~ ~ ~ ~ ~ have proposed a simple ansatz to link PFG experiments and pore geometry via the two-point pore space correlation function

P(ro, r, A)

=

ro - r

S(r, - r)

where S(r, - r) is the pair correlation function and C(A) is a normalization constant. These parameters are determined by the condition^^'.'^^ I d . P(ro, r, A) = 1

(78)

VP and 1

r

dro dr P(ro,r, A) I ro - r 1

=

6ADapp

(79)

where the integrals in equations (78) and (79) are over all space. At small q E ( q , A) is Gaussian function but deviates with increasing q.45 In porous

GRADIENT NMR

95

media this deviation occurs at q = (Dt)-1'2.45As noted above, small q measures D; however, at longer times Dappstarts to decrease from D as the spins start to interact with the interface. The decrease depends on the ratio S/Vp.45 For a smooth pore surface with wedge-shaped regions, exact asymptotic results were obtained.46 These results were used by Latour et ~ 1 . to ' ~ determine ~ S/V, in a number of porous systems. By combining measurement of the short-time behaviour of Dappand E ( g , A), the surface relaxivity H can be obtained. The long-term effective diffusion constant, DeH,derived from PFG gives information on the tortuosity of the connected space. The diffusion coefficient measured by PFG equals that derived from electrical conductivity only when H = 0. Deffcan yield the geometrical information factor F.'22,'24 F is defined as

F=

D Deff 4 p

where +p is the porosity. In fact for large q, E ( q , A) has been shown to be proportional to

where r(A) is a time-dependent function characteristic of the geometry, in analogy to the Debye-Porod law. 120*1253126 Mitra7* has demonstrated that multiple wave vector extensions of the PFG experiment can be used to distinguish between diffraction-like behaviour resulting from restricted diffusion and multicompartment diffusion.

4. NON-HOMOGENEOUS GRADIENTS AND OTHER PROBLEMS

4.1. Introduction

The theory presented in Section 3 does not consider the myriad of experimental and technical problems that can occur. Nor were multiple l ~ ~ the interaction interactions considered. For example, Zhang et ~ 1 . studied between non-ionic micelles and a non-ionic polymer using PFG. They found that the micelle diffusion is influenced by the polymer in two ways (i.e. obstruction by the polymer network and association of the micelles to the polymer chain). There are a number of other problems, some of them technical and some of them inherent to the experimental system, that must be considered. For example, in coupled spin systems it is desirable to keep A constant to avoid problems imposed by J coupling. Most of the technical problems that will be

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s. PRICE

discussed are relevant only to Bo experiments. The problems that we will consider here are: (1) not perfectly reproducible gradient pulses or pulses of different than intended shape due to technical limitations; (2) eddy currents induced in the surrounding conductors by the gradient pulses; (3) static magnetic field disturbances due to perturbations of the magnet and lock system by the gradient pulses; and (4) internal gradients in the sample due to non-homogeneous magnetic susceptibility. These problems generally lead to increased signal attenuation and, therefore, overestimates of the diffusion coefficient and misinterpretation of the experimental data. Here we consider the effect of these problems and some methods to alleviate them.

4.2. Imperfect gradient pulses and sample movement

Ideally the gradient pulses would be of a specified shape and magnitude and perfectly reproducible. Mismatched gradient pulses cause a “phase twist” which results in large signal attenuations which are unrelated to the effects of diffusion. In practice the gradient driver (amplifier) may be incapable of delivering perfectly reproducible gradient pulses. A further complication can arise from heating of the gradient coils and therefore changes in coil resistance. The most obvious solution is to use a better (normally constant current) power supply and to provide temperature control of the gradient coil (i.e. cooling). Additional appropriately spaced gradient pulses prior to the “start” of the rf pulse sequence may also help to alleviate motional disturbances during the encoding and decoding gradient pulses (see Section 5.2.3).128C a l l a g h a ~ ~ has ’ ~ proposed ~ a method named MASSEY (modulus addition using spatially separated echo spectroscopy), which incorporates the static spatial resolution properties of read gradients (k space imaging), to resolve the phase twist (see Section 5.2.3). Eddy currents can also result in mismatched gradient pulses (see the following section). Nearly all PFG sequences prescribe rectangular^' gradient pulses. However, due to finite rise and fall times (typically of the order of 50 /AS) and the attempts to reduce the generation of eddy currents, the gradient pulses may be non-rectangular. In fact, trapezoidal pulses are commonly Here we examine the effect of such pulses in the standard Stejskal and Tanner PFG pulse sequence in the absence of background gradients. For the case of rectangular or nearly rectangular gradient pulses (Fig. 15(A)), the solutions can be determined by using equation (18), and are found to be of the form130 E ( q , A)

=

exp{ - + 2 D [ i j 2 ( A - 6/3) + B ] }

where the B term is defined in Table 1. Some further variations have also been considered by Merril.131 The results show that, so long as the pulses

GRADIENT NMR

z

I

z

97

I

ti + A

z

I

I

‘z

A

I

I

P

L

Fig. 15. The Stejskal and Tanner pulse sequence with imperfect pulses. (A) PFG sequence with approximately rectangular gradient pulses; E represents the rise and fall times of the gradient pulses. (B) PFG sequence based on the pulses being a sine or sine’ function of time. (Adapted from Price and K~che1.I~’)

are nearly rectangular, the precise shape is unimportant as long as the “area” (i.e. Sg) of each pulse is equal to that of the ideal rectangular pulse. In the case of sine or sine’ gradient pulses (Fig. 15(B)) the following relations can be deri~ed:’~’

-92D6’[cos236/2+ sin‘(4A - 6)/2](N7r)} Esinz= exp{-?806’[A/4 + 6/12 + 5 6 / ( 4 N ~ ) ~ ] }

Esin= exp{

183)

(84)

where 2N denotes N full oscillations of the gradient pulse. Sample movement has a similar effect to mismatched gradient pulse except that all spins in the sample receive an equal phase shift (as in the case

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WILLIAM S. PRICE

Table 1. The B term in equation (82) for various Bo gradient pulse shapes in the Stejksal and Tanner sequence. Gradient pulse shape

B

Identical

0

Exponential rise and fall

) X (6 + l/k) -2/kz(l/k - 6) + 4/(keek)(&S- l/kz- ~ ~ / 22/(k2ez"k)

Exponential rise and fall with overshoot and undershoot

[-86/k' - 12/k' + e-&(2.s2 - 4 ~ ' 6+ 6 ~ ' / k- 86 Elk + 12~lk'- 861 k2 + 12/k3)]/g 4e-"k(~2- ES- S/k)/(g2k)+ e-2"k(2~26 + 6~'/ k + 4 ~ 6 / k+ 6dkZ + 26/k2 + 3/P)/g' + (26 - 3/k)/(g2kz)

Sine rise and fall

~ ~ E ' ( E- 36)/d - 6 4 ~ ~ 1 6 ~ E ~-(2 F6 ) / +

Ramped rise and fall

~'/30- 6 216

Mismatched

E2(2T- t,

+

+

-A -

6 - 2813)

From Price and Kuchel. I3O

of flow; see Section 3.5.1), instead of a phase twist. Sample movement can be reduced by more securely fastening the sample in the probe. 4.3. Eddy currents and perturbation of Bo

The eddy currents induced by the gradient pulses cause significant problems, and the solution is by no means trivial. If, for example, in the Stejskal and Tanner sequence the eddy current tail from the first gradient pulse extends into the second transverse evolution period, then the total field gradient during the second evolution period will not equal that in the first, and thus both the echo time and amplitude may be affected.'32 This will cause additional attenuation unrelated to diffusion; however, the effect will be indistinguishable from an increased diffusion coefficient. To determine if eddy current effects are significant, a measurement can be performed on a sample with a diffusion coefficient lower than that which can be detected with the experimental system in question (i.e. 8 , A and g). If signal attenuation is observed, then the presence of eddy current effects is implied. If the eddy currents extend into the acquisition time the signal may be distorted. 132 There has been some recent interest in finding ways of mapping the eddy currents (e.g. see ref. 133). Several methods exist for handling the eddy current problems. The most effective solution is to use shielded gradient coils (see Section 7.2.1) whereby the gradient coils are designed to produce the desired gradient within the active sample volume but negligible gradient outside the active volume. This method is particularly convenient since no experimental adjustments are necessary. Another method is termed "pre-emphasis". The method is based on the Lenz's law requirement that the sign of the fields generated by the eddy currents will be opposed to the change which induced them. Thus, the

GRADIENT NMR

99

current at the leading and trailing edges of the gradient pulses is overdriven, and in this way the coils self-compensate for the induced eddy currents. This is generally performed by adding numerous small exponential corrections of different amplitude and time constants to the desired current waveIn practice, pre-emphasis is experimentally complicated and the method is not perfect since the spatial distribution of the fields produced by the eddy currents in the surrounding metal and those produced by the gradient coils are not identi~al.'~' If after applying pre-emphasis and shielded gradient coils the eddy current problems still persist, the gradient pulses may be individually adjusted so that the total area of the gradients in the two transverse evolution periods is the same. However, this method is an empirical approach and is dependent on the experimental times and gradient strengths used and does not account for distortion during the spectral acquisition. Sequences and postprocessing for removing phase instability such as MASSEY129 can also be used to remove these distortions. The imposition of field gradient pulses can have serious effects on the stability of the main magnetic field. The rapid rise and fall of the gradient pulse can directly affect the stability of the main magnetic field by inducing additional currents into the solenoids producing the main magnetic field or indirectly by affecting the lock feedback system. The final result is that the main field may be caused to oscillate or at least shift from its normal value. Thus, in addition to shielded gradient coils it may be necessary to use some ~ ~provide dynamic ~himming.'~'Crozier et al. 139 form of p ~ s t p r o c e s s i n g 'or have developed a method for minimizing eddy current induced Bo shifts based on direct frequency modulation of the spectrometer and receiver reference frequencies.

4.4. Internal gradients and relaxation time distributions Ideally the only magnetic gradients present would be those purposely applied. In practice non-homogeneous internal gradients are common within many samples (e.g. red blood cells, metal hydrides, colloids and porous media) due to differences in magnetic susceptibility. For example, it is estimated that red blood cells have gradients up to 2 x lop2T/m. 140,141 Even minute air bubbles lead to large background gradient^.'^^ These inhomogeneous background gradients result in a decrease in the observed T2 through the effects of translational diffusion of nuclear spins.95,96*143-149 In fact, there is no exact theory for treating the effects of diffusion in a general non-uniform gradient.9s Also, cross-terms between the applied gradients and background gradients greatly complicate interpretation of the attenuation data (see equation (19)) since the attenuation due to the background gradient may be indistinguishable from the attenuation due to the applied

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gradient. It has been suggested that background gradients can cause effects that can be mistaken for restricted diffusion.147 Further, measured anisotropic diffusion could result from anisotropic background gradients. 150~151B Y looking at the form of equation (19), it can be seen that one solution to the problem is to use an applied gradient much larger than the background gradients. lS2 When this is not possible, more sophisticated pulse sequences must be used (see Section 5.2.2). Another related problem is that of a distribution of relaxation times in a heterogeneous system. Heink et al.153 have suggested replacing the initial 7r/2 pulse in the Stejskal and Tanner sequence or STE sequences (see Fig. 1) with either a primary or STE sequence. The durations in the preparatory sequence may be varied while leaving the diffusion part of the sequence unchanged. If the signal attenuation due to the gradients is independent of the durations in the preparatory sequence, artifacts due to a distribution of relaxation times can be excluded.

5. PULSE SEQUENCES FOR MEASURING DIFFUSION 5.1. Introduction All Bo-based pulsed field gradient sequences for measuring diffusion have the Stejskal and Tanner (i.e. Hahn spin echo) and STE sequences (see Fig. 1) as their common ancestor. At present, steady gradient methods are not widely used and, accordingly, I will give them only brief coverage. The recently developed B1 gradient sequences will also be discussed.

5.2. Bo sequences

5.2.1. Basic sequences The Stejskal and Tanner pulse sequence is suitable for general diffusion measurement, but for particular sample/experimental conditions, modified versions lend specific advantages. The STE sequence is more appropriate when Tl >> T2 because it causes the information on spin phases to be stored along the applied field direction for a time interval between the gradient pulses. Thus, in the STE sequence A is limited by T I ,whereas in the Stejskal and Tanner sequence it is limited by T2.

5.2.2. Reduction of the effects of background gradients The basis of most sequences for the removal of background gradients is to add additional 7r pulses to the PFG sequence to refocus the dephasing

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effects of go in a way analogous to the Carr-Purcell-Meiboom-Gill sequence. In 1980, Karlicek and L ~ w e proposed ' ~ ~ the use of alternating (bipolar) pulsed field gradients in a modified Carr-Purcell sequence (Fig. 16(A)) to eliminate the contribution of the g.go cross-terms. In the case of the Karlicek and Low sequence, the echo attenuation can be shown to be

E ( g , 2n.r)

= e~p{-~/3?Dd[n&

+ ( n - 1)3g2]}

(85)

where the integer n is defined in Fig. 16(A). Systematic errors due to the cross-term can also be eliminated in a Carr-Purcell sequence that only uses pulses of one polarity,155 but this sequence is not as efficient as that of Karlicek and Lowe, especially when T2< T l . The Karlicek and Lowe sequence154is limited by T2, and thus it is desirable to have STE-based pulse sequences. Cotts and c o - ~ o r k e r s presented '~~ three modified STE sequences incorporating alternating pulsed field gradients (Fig. 16(B)) which greatly reduce the effects of the background gradients. Latour et al.148have recently proposed a pulse sequence that combines features of the Karlicek and Lowe and Cotts pulse sequences in which the gradient pulses in the normal STE echo pulse sequence are replaced by a series of short gradient pulses of alternating sign (Fig. 16(C)). Lian and c o - w o r k e r ~have ' ~ ~ demonstrated that an image of D or (Dgz) can be obtained without the corrupting g-goterms by appending a standard imaging sequence to an alternating pulsed field gradient sequence154or a Carr-Purcell sequence.

5.2.3. Reduction of eddy current and phase distortion effects Griffiths et a1.15' proposed an experiment in which a train of 7~ rf pulses is used to refocus the stimulated echo so as to delay the acquisition until after the eddy currents have subsided. However, since the magnetization is transverse during this period it is susceptible to transverse relaxation, J modulation and phase distortions from the eddy currents. Gibbs and co-workers have proposed the LED (longitudinal eddy current delay) pulse sequence (Fig. 17(A)).158This is a modified STE experiment, and is useful when the Tl values of the species in question are longer than the lifetime of the eddy current transients. However, the LED sequence does not solve the problem of the eddy current tail extending from the first gradient pulse into the second transverse evolution period. A partial solution is to precede the sequence by a train of identical gradient pulses with the same separation as Another problem common to both the that used in the LED LED sequence and to the STE sequences is that extensive phase cycling is required. For the LED sequence at least 64 steps are required. The phase cycling requirements of both sequences can be greatly reduced if a homospoil pulse is included after the second rf pulse.

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n/2 n

.. , . . ...

A

0

n/2

z

(8n+ 1 ) ~

32 n

...-

0

C

2

(A

4It...... kl.. ....

Fig. 16. Sequences for removal of background gradients. (A) The Karlicek and and (C) the Lowe sequence,'54 (B) the nine-pulse sequence of Cotts et improved stimulated echo sequence of Latour et ~ 1 . ' ~ '

Wider et al. have recently proposed self-compensating magnetic field gradient pulses (Fig. 17(B)). In the method, a gradient pulse is replaced by two pulses of half the duration with a n- rf pulse in between the two gradient pulses. The two gradient pulses are of opposite sign. In this way the two gradient pulses in quick succession but opposite sign attempt to cancel out imperfections of the individual pulses. Using this sequence the signal

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A

x/2

x

B

Fig. 17. Sequences for removal of eddy current effects. (A) The LED pulse ~equence.'~'Ideally the delay T, is of a duration sufficient for the eddy currents to have dissipated before acquisition begins. A common addition to the sequence is to prefix a series of gradient pulses separated by a duration A, to allow the eddy current effects to reach a steady state. Homospoil pulses are commonly employed in the period 7, to reduce the phase c cling requirements. (B) The self-compensating gradient sequence of Wider et al. I X

attenuation due to diffusion is related by the following equation (the time periods are defined in Fig. 17(B)):

The MASSEY sequence has been developed by Cal1aghanlz9for minimizing phase instability in very high-gradient NMR spectroscopy (Fig. 18). This method incorporates a read gradient (i.e. k space), G, into the standard Stejskal and Tanner sequence. The addition of G allows for the restoration of spatially dependent phase shifts such as caused by a mismatch in the ( q

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I

z

z

I

Fig. 18. The sequence used in the MASSEY technique for removing phase in~tability.”~ The sequence is a combination of the Stejskal and Tanner sequence with a read gradient, G.

space) gradient pulses. The restoration of the phase occurs at t = -A27rq/ yG with respect to the centre of the echo. The phase twist caused by the pulse mismatch is resolved by Fourier transformation of the whole echo with respect to 27rq. Finally, the effects of the phase twist together with any net phase twist due to sample movement is removed by subsequent modulus calculation. 5.2.4. Steady gradient

Steady gradient methods have a number of inherent problems: (1) because the rf pulses are imposed in the presence of the gradient, it may be difficult or even impossible to evenly excite the whole spectrum; (2) measurements are normally limited to samples containing a single component since the resonances are greatly broadened by the gradient; (3) it can be difficult to separate relaxation and diffusion effects; and (4) the time over which diffusion is measured is not so well defined. However, they do have one large advantage compared to the pulsed gradient sequences-no eddy current problems. Norwood and Quilter16* have developed a constant time, pulse and gradient amplitude diffusion experiment (CTPG) which circumvents a number of problems associated with steady gradient experiments. Subsequently, N o r ~ o o d ’has ~ ~considered several new sequences for measuring diffusion, some of which can be used to measure restricted diffusion and species having coupled spins and short T2.

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5.2.5. Fringe (or stray) field methods Kimmich et al.i64 have recently considered the use of steady gradient methods in the fringe field of superconducting magnets. The enormous gradient so provided (10-180 T/m)'65 has the potential for measuring very small diffusion coefficients. However, the inherent properties of using the fringe field are a major drawback. Because of the large field inhomogeneity, only a thin layer of the sample is on-resonance, and the signal-to-noise ratio is dramatically reduced. Further, multicomponent and spatially resolved diffusion coefficients are not possible. The large gradient also means that the echo will become very sharp, and so it may be difficult to digitize the echo properly. Later studies have extended the fringe field approach to allow the production of relaxation-independent diffusion decays, multislice experiments, experiments using shaped rf pulses and two-dimensional variants. More recently, Demco et al. '61 have developed constant relaxation methods using a pulse sequence based on a stimulated echo and Carr-Purcell mixed echo pulse sequences. 5.2.6. Zero and multiple quantum It is often desirable to work with heteronuclei, especially when measuring the diffusion coefficient of nuclei in a complex mixture such as a biological fluid. However, heteronuclei generally have a sensitivity far beneath that of protons. Further, because of the low magnetogyric ratios of heteronuclei, larger gradients must be used. The most straightforward means of alleviating the signal-to-noise problem is through the use of specifically labelled/ enriched probe molecules. Large gains in sensitivity can be made through using pulse sequences to generate polarization transfer from protons to the heteronuclei. This approach has the advantage of generating multiplequantum transitions. If the attenuation of the multiple-quantum coherence can be studied (instead of the single-quantum coherence), the same degree of attenuation can be achieved but with smaller gradients and therefore smaller eddy current problems. In multiple quantum experiments, it is the effective sum of the y values of the nuclei involved in the coherence which is relevant to the attenuation. Thus, for the Stejskal and Tanner pulse sequence in the case of the free diffusion and neglecting the effects of background gradients, the formula relating echo signal attenuation to diffusion can be written as

For the normal (i.e. single-quantum) experiment f(y) = -$. For homonuclear multiple-quantum experiments f ( y ) = For heteronuclear multiple-quantum experiments the definition of f(y) is not SO straight-

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I

'I:

I

4

,

'I:

4

Fig. 19. A pulse sequence for detecting homonuclear zero-quantum coherence in an

inhomogeneous magnetic field (phase cycling not shown). The first 7 delay corresponds to the preparation period, the evolution period is denoted by tl and the second r delay corresponds to the detection period. forward. For Z spin-detected heteronuclear double-quantum experiments with an I-S spin system, f(y) = [(y, + y ~ ) / y ~ ] * y , . ~ ~ ~ The use of heteronuclear inverse distortionless enhancement by polarization transfer (DEPT)-based sequences (i.e. inverse detection) and inverse heteronuclear correlation spectroscopy (1HETCOR)-based sequThe DEPT-based sequence has significant ences has been investigated, 168~16y advantages for working with low y nuclei due to the polarization transfer from the I spin (usually 'H) to the S spin. However, the IHETCOR pulse sequence is more suited to the observation of protons as the unfavourable polarization transfer from the less abundant heteronuclear population to the proton population. Coherence order selection may be incorporated into the diffusion experiment to provide solvent suppression. Multiple-quantum steady gradient experiments have also been devised, and Norwood has recently presented a multiple-quantum version of his CTPG e~perirnent.'~' Hall and N o r ~ o o d ' ~ have ' pioneered the use of zero-quantum spectra for measuring diffusion. Zero-quantum spectra are unaffected by magnetic field gradients. Since zero-quantum coherences are "spin forbidden", they can only be excited and observed indirectly. A zero-quantum coherence can only be formed in a spin system consisting of at least two coupled spins, and its precession frequency will be the difference in chemical shifts of the contributing spins. A typical pulse sequence used for observing zeroquantum coherences is shown in Fig. 19. Diffusion will be encoded with respect to t,. The signal attenuation is given by14,52

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107

where TFQc is the transverse relaxation time of the zero-quantum coherence which evolves during t l . Hence the decay of the tl FID signal due to relaxation and diffusion is given by

Thus the amplitude and linewidths of zero-quantum coherences are diffusion dependent. Assuming a Lorentzian lineshape, the linewidth of a zeroquantum coherence at half peak height due to relaxation and diffusion will be

The diffusion-dependent part can be separated by acquiring a zero-quantum coherence using a sequence not containing an echo, for example by adding a n- pulse to the middle of each 7 period of the sequence given in Fig. 19. The difference in the measured linewidths will be determined solely by diffusion. There are two major problems with this method: first, high F1 resolution is required and, second, the signal-to-noise ratio decreases as g increases. Although this is a steady gradient experiment, it does allow separate measurements of individual components.

5.2.7. Multiple spin echoes There have now been a number of reports of multiple spin echoes (MSE) for spin I = Y2 nuclei occurring after a sequence of only two pulses. It has been shown that these MSE are generated by the dipolar demagnetizing field. 172~173The stimulated echo sequence, 9W1-t1-9OoX-t2-9Vx, is particularly favourable for generating MSE. If t2 = ntl for integer n , it is likely that one of the MSE will interfere with the stimulated echo. The amplitude of the MSE has been related to the pulse separations, magnetic field gradient and the diffusion coefficient.

5.2.8. Miscellaneous A “single-shot” method based on a Carr-Purcell-like sequence with incremented gradient pulses in each 7 period has been proposed.’74 While the method is susceptible to the effects of imperfect n- pulses, it does, under ideal conditions, offer a means of determining the diffusion coefficient in the one experiment. It was noted that two possible applications of this method are for measuring macroscopic incoherent motions and also, if gradient pulses are applied alternatively along three orthogonal directions,

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anisotropic diffusion effects. Van Gelderen and c o - ~ o r k e r s ’have ~ ~ proposed another single-shot diffusion method based on a series of gradientrecalled echoes. The method offers improved time resolution and reduced sensitivity to bulk motion. The method consists of a spin echo experiment during which a series of echoes are created by alternating pulsed gradients. Since subsequent echoes are attenuated by additional gradient pairs, the total diffusion curve can be acquired within a single experiment. In the presence of a static field gradient (i.e. Bo inhomogeneity), neglecting go2 terms, the signal attenuation for the nth echo is given by

En = exp{ - 2 ? L l [ 2 r ~ 6 ~ / 3- g o * g ( d 2- ns3)]} for n odd

(91)

En = exp[ - 2 y D ( g 2 n S 3 / 3 - go-gnS3)] for n even

(92)

and

Thus, the difference in dependence of the even and odd echoes on the go-g terms may be used to estimate static local gradients. The phase induced by bulk motion is

Thus, if the motion can be considered as constant within a time interval 46, the even echoes will be motion compensated. Diffusion can be determined from the experiment in two ways. In the first method the experiment is performed twice, changing only the gradient strength. The ratios of the corresponding echoes can then be compared to extract the diffusion constant. In the second approach, just one experimental data set is acquired, and the ratio of the peaks symmetric in time to the echo maximum are recorded. However the second method does not account for T2 effects. 17’ Li and S ~ t a k have ’ ~ ~ described a pulse sequence that combines both a stimulated echo and a spin echo component. It is noted that this sequence has potential for measuring anisotropic and restricted diffusion since the two echoes can be used to observe diffusion in different directions and over different time-scales. It should be mentioned that “multiple wave vector” sequences are also now being developed which contain more than two gradient pulses but in different directions.”

5.3. B1 sequences B1 gradients can also be used for diffusion measurement^,^^ combined with water upp press ion'^^ or a sequence for measuring the longitudinal relaxation

GRADIENT N M R

109

t, + A

A Fig. 20. B1 Gradient pulse sequences for diff~sion.~'This sequence is the B1 analogue of the Stejskal and Tanner sequence. The B1 gradient pulses are represented as grey rectangles.

along the gradient axis.'78 The B1 analogue of the Stejskal and Tanner sequence is given in Fig. 20. As noted by Canet and co-workers, this experiment is merely a transposition in the z-y plane of the rotating frame of the conventional Stejskal and Tanner sequence which is performed in the x-y plane. 6. APPLICATIONS TO HIGH-RESOLUTION NMR

6.1. Introduction In this section, we briefly review the recent applications of both Bo and B1 gradients to high-resolution NMR. The effects of diffusion as discussed in the preceding sections may play a part in the mechanism of a particular technique, but determination of the diffusion coefficient is not the aim. Gradients have long been used in high-resolution NMR as a homospoil, and here we consider applications such as coherence selection, quadrature detection, spectral selectivity and solvent suppression. Also included in this section are diffusion-ordered experiments and electrophoretic NMR. These latter two techniques are difficult to classify into a particular experimental category. For example, the diffusion-ordered experiment can be thought of as a method for measuring diffusion but, and certainly more importantly, it can be thought of as a means of spectral simplification of complex mixtures. A similar argument can be made for electrophoretic NMR. Caution should be used when comparing the efficiency of gradient and non-gradient methods, and also when comparing different gradient methods, since in most cases, inherent effects such as relaxation have not been

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considered. Some recent reviews have also considered gradients in highresolution NMR. 28*179 6.2. Coherence selection and quadrature detection Often a single scan gives a sufficient signal-to-noise ratio, and thus phase cycling results in an inefficient use of spectrometer time. Field gradients can be used instead of phase cycling for selecting a particular order coherence'" and also for inducing quadrature detection. 181-183 Through the incorporation of field gradients at appropriate times into the pulse sequences, desired coherences can be selectively rephased, while dephasing those that do not follow the desired coherence pathway.180y181 We can imagine that during a gradient pulse of duration 6, a spin of coherence order n will acquire a complex phase factor (see equation (2))

If an rf pulse transfers the coherence from n to n' and then a second gradient pulse of equal duration and magnitude but opposite sign is applied, the spin will acquire an additional phase of

Clearly, if n6 = n'6, then the dephasing effects of the two pulses cancel and the resonance is refocused. Coherences which do not fulfil this requirement will acquire spatially dependent net phases, and thus the effects of the two gradient pulses will not cancel. Thus, in the general case only those coherences are observed for which the cumulative phase factor is zero. In the case of heteronuclear coherences, a composite coherence order is defined, thereby allowing the inclusion of the gyromagnetic ratios. Thus, a particular coherence transfer pathway can be selected according to the ratios of the gradient pulses used. Assuming a sufficiently strong sample, multidimensional experiments can be acquired with one scan per increment. Further since the coherence selection does not rely on subtraction, there is a large reduction in f 1 noise. An example of a phase-cycled COSY (correlation spectroscopy) sequence and its gradient counterpart are given in Fig. 21. The gradients in the COSY sequence in Fig. 21(B) can perform either N- or P-type selection in a single acquisition depending on the sign of the gradient. Gradients of the same sign result in N-type selection and produce quadrature detection in w l . If we assume an AX spin system, the observable signal, in terms of product operator^,'^^'^^ is lS3 */2 USycos (dfl) exp ( - iws tl)

GRADIENT NMR

+;

-1

111

;

+;

-1

._\__I___

Fig. 21. Schematic diagrams of (A) phase-cycled and (B) gradient COSY sequences. The pulses P I P2 must be cycled in the phase-cycled sequence in an appropriate manner to effect N- or P-type selection.

where US, is the initial coherence of the X spin and w, is the Larmor frequency of the X spin. A disadvantage in using gradients for coherence selection in this sequence is that the gradient pulses can only refocus one of the two possible coherence pathways (see Fig. 21(B)). An absorption mode spectrum can be obtained by recording separate P- and N-type data sets. An example of a double-quantum COSY sequence which uses gradients for coherence selection is depicted in Fig. 22.18’ A formalism for representing coherence transfer by pulsed field gradients has recently been presented. lg6 Since gradients select only one of two possible coherence pathways, there is at least a V? loss in sensitivity compared to the equivalent phase-cycled

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w2

:

0 -1 -2

Fig. 22. Schematic diagram of a gradient-selected double-quantum filtered COSY

sequence. experiment. The sensitivity of experiments which use gradient pulses for coherence pathway selection has recently been investigated. lX7 However, if gradients are used simply to purge unwanted coherences rather than to select coherences, there is no loss of signal intensity compared to nongradient-based methods. To minimize intensity losses due to diffusion effects, the strength and duration of the gradient pulses should be kept as small as possible. Similarly, the duration between gradient pulse pairs should be kept as short as possible. B1 gradients as well as Bo gradients can be used for coherence selection and phase cycling. In the case of B1 gradients, if the geometry of the rf coil is such that the amplitude and/or the phase of the B1 field is a function of space, then this heterogeneity can be used to distinguish between different coherence p a t h ~ a y s An . ~example ~ ~ ~of ~a heteronuclear ~ ~ ~ ~ ~ single~ ~ ~ ~ ~ quantum correlation (HSQC) experiment that incorporates B1 gradients is given in Fig. 23. 6.3. Spectral selectivity and editing Spectral editing is normally based on subtraction, but such methods are limited by dynamic range difficulties and the precision of the subtraction. It

GRADIENT NMR

113

0

0

9

9 0

65

70

75

3 30

15

I0

0

I PP.

0

15 IDn

I

-

"

'

I

-

5.0

'

.

'

41s

1

4 .O

"

"

I

"

"

3.5

I

Fig. 23. A 400 MHz 'H-13C HSQC experiment of sucrose in 'HzO observed using an rf sequence incorporating planar B1 gradient pulses. (Reproduced with permission from Maas and Cory.30')

can be shown that both zero-quantum and two-spin order (correlated z order) are unaffected by a Bo gradient whereas single- and double-quantum transitions are dephased.'" This can be used as the basis for editing procedure^.'^^ However, with zero-quantum filtering, since only part of the coherence is converted into observable signal, there is a factor of 4 loss of signal. The situation is better with z-order filters.''' Selection of a particular frequency domain can be achieved by either (1) frequency, phase and amplitude modulated soft rf or (2) DANTE (delays alternating with nutations for tailored excitation)-like sequences which employ a train of hard pulses of appropriate duration and phase. Canet et UZ.~'have ~ developed sequences relying on trains of B1 gradient pulses to

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achieve such selectivity. Due to the greater technical problems involved, a Bo gradient equivalent is not possible.

6.4. Solvent suppression

6.4.1. Introduction

Large solvent resonances (e .g. the water resonance in biological samples) present formidable challenges to proper acquisition of NMR spectra. They prevent optimal use of the available dynamic range of the analogue-todigital converter of the spectrometer and, perhaps more seriously, obscure peaks near the solvent resonance. Additional problems arise depending on the method used to suppress the solvent resonance. Methods of solvent suppression have recently been reviewed. Here we will review the gradient-based methods of solvent suppression and contrast them with the more traditional NMR methods. Solvent suppression methods fall into two general categories: (1) magnetization destruction prior to excitation of the remaining spectrum and (2) non-excitation. Traditionally, selective irradiation of the solvent peak (i.e. a magnetization destruction method) has been the most common method since it is both simple and easily incorporated into multidimensional experiments. However, it suffers from the disadvantages that signals close to the solvent signal are also saturated, species that are in exchange with the solvent are difficult to observe, and in the case of water as the solvent, it is impossible to observe hydration water. Selective excitation (i.e. a nonexcitation method) has the advantage that exchangeable protons can be observed. Its disadvantages are that it does not provide such a large degree of water suppression and the resulting spectrum suffers from intensity, phase and baseline distortions. Gradients offer far superior methods of water suppression. There are three ways in which gradients may be used to effect solvent suppression: (1) through coherence selection, (2) by differential diffusion between the solvent and solute or (3) by selective excitation of the water and subsequent destruction of the magnetization. 1937194

6.4.2. Coherence selection

In this method, gradients are used to select multiple-quantum coherence and thereby suppress the water resonance. However, problems arise since either large gradients or small gradients but with longer durations must be used. Thus, there are solute signal losses due to diffusion and, especially when using longer gradient pulses, due to relaxation. Hurd and co-workers have shown that coherence selection alone is insufficient to fully remove the H 2 0 signal from a multiple-quantum filtered COSY.19’

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115

6.4.3. Diffusion In the differential diffusion m e t h ~ d ' ~ (DRYCLEAN ~.'~~ (diffusion-reduced water signals in spectroscopy of molecules slower than water); i.e. magnetization destruction) the faster diffusion of the solvent compared to the solute forms the basis of the water suppression. For example, at 298K, water has a diffusion coefficient of about 2.3 x m2/s and a large protein has a diffusion coefficient nearly two orders of magnitude smaller (see Table 3). Thus, the solvent resonance will be greatly attenuated compared to the protein resonance if a pulse sequence is used that incorporates some form of gradient spin echo sequence (e.g. see Fig. 1). Clearly this method requires there to be a large difference between the diffusion coefficient of the solute and solvent molecules to work efficiently, and it also entails the use of large gradients. 6.4.4. Selective excitation In this method, depicted schematically in Fig. 24(A), a selective pulse is used to excite the solvent signal to become a transverse coherence. This transverse coherence is then dephased by a gradient pulse. A hard 7d2 pulse may then be used as the final excitation pulse, resulting in uniform Thus, this technique is excitation with minimal phase distortion. both frequency and lineshape independent. Conversely, excellent lineshape is not a prerequisite to obtaining good solvent suppression. A one rf pulse/gradient pulse combination is extremely sensitive to any mis-setting of the ad2 pulse. The solvent suppression can be improved by using further applications of the selective rf pulse/gradient combination before the excitation pulse, although the subsequent gradients should be, ideally, in a different direction to avoid creating echoes. The negative aspects of this method are that a highly selective rr/2 pulse is required and that if the solvent TI is very short and on the order of the time for the dephasing procedure then some (unwanted) z-magnetization will be re-established prior to the excitation pulse. One method of circumventing the problems imposed by a short solvent T I is to follow the dephasing gradient by a weak gradient to broaden the solvent signal to approx. 180Hz. A noise pulse is then applied and thus, following gradient collapse, the remaining solvent magnetization is randomized and no coherence is read by the final excitation pulse. 197 191,1953197

6.4.5. Watergate Watergate198*199o r water suppression by gradient-tailored excitation is a combination of selective excitation with pulsed field gradients. The basis of the method, schematically represented in Fig. 24(B), consists of a gradient

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selective TG

B

XI2

selective TC

Fig. 24. Solvent suppression by Bo gradients. (A) Selective excitation and then dephasing. (B) the Watergate sequence. (C) An example of a two-dimensional sequence incorporating a Watergate unit (i.e. a Watergate TOCSY (total correlation spectroscopy)).

pulse, a selective 7~ pulse and then a second gradient pulse. All coherences that are dephased by the first gradient pulse can only be rephased if they are subject to a 7~ pulse. Ideally, the coherences of interest are rotated by 180" while the net rotation of the water in near 0". Providing the echo interval is kept short to minimize J modulation, T2 relaxation and molecular diffusion effects, the desired resonances are retained in the spectrum with full intensity while the water peak should be suppressed by a factor of at least lo4. In one-dimensional usage a 90" non-selective excitation pulse can be applied before the Watergate sequence. In cases where it is desired to observe exchangeable protons, a selective 90"-, pulse (selective of the water

GRADIENT NMR

117

resonance) can precede the non-selective 90". Watergate can be incorporated into two- and higher-dimensional experiments (Fig. 24(C)).

6.4.6. BI solvent suppression methods The residual inhomogeneity of (supposedly) homogeneous rf coils have been used to suppress water. 2oo,201 In these experiments the spins of interest were spin locked while the water magnetization, which was previously placed perpendicular to the spin-locking direction, was dephased. Maas and Cory202 have developed a water suppression technique based on the formation of B1 gradient spin echoes or B1 gradient recalled echoes (i.e. rotary echoes). The method is in effect the B1 gradient analogue of the Watergate sequence. The B1 gradient sequence can be used to selectively suppress a resonance by preventing the formation of an echo for a specific resonance while allowing the other resonances to refocus. Maas and Cory202 note that a radial (i.e. quadrupolar) B 1 gradient is more efficient for dephasing longitudinal magnetization than a planar gradient since it has a spatially dependent phase variation in addition to the amplitude variation. Their El solvent suppression technique is reasonably insensitive to artifacts such as J modulation, off-resonance excitation, radiation damping and, chemical shift evolution, and will allow the observation of exchangeable protons. The B1 gradient suppression sequences of Maas and Cory differ from the Bo gradient Watergate sequence in that the magnetization to be observed rotates in a plane perpendicular to the applied rf field and the relaxation is given by an effective TI defined by 1

Tleff

zjl+k) 1 1

=

whereas in the Watergate sequence the relaxation of the observed spins depends on T2. In a preliminary study, Canet and c o - ~ o r k e r s ~have ' ~ shown that a DANTE train of B1 gradient pulses may be used to selectively suppress water. The DEBOG (DANTE elimination by B-one gradient) sequence may be written as

where (gl)xis an rf gradient pulse of duration 7,7' is the precession interval which governs the selectivity process and (d2),, is a homogeneous read pulse. They remark that while it is desirable to have the B1 gradient as strong as possible, the natural B1 gradient in normal saddle coils may be sufficient for most cases. An example of a COSY incorporating E l gradient water suppression is given in Fig. 25.

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Fig. 25. A B , gradient COSY experiment incorporating water suppression of a sample of 0.9 M glucose in H 2 0 recorded at 200 MHz using a sequence involving the DEBOG suppression scheme203and the equivalent of a double-quantum filter. The B , gradient stren th was about 0.03T/m. (Reproduced with permission from Mutzenhardt et al. 8 4)

6.5. Spectral simplification according to mobility 6.5.1. Introduction

Diffusion and electrophoretic mobility provide a criterion by which to separate mixtures of species according to their size and charge, respectively, Schulze and Stilbs204 have described a method which uses the complete spectral bandshape to extract the individual component contributions. Below we will discuss two, more elaborated, methods: diffusion-ordered spectroscopy and electrophoretic NMR.

GRADIENT NMR

119

6.5.2. Diffusion-ordered two-dimensional experiments

Recently a two-dimensional diffusion experiment (or DOSY), has been proposed for separating species according to their diffusion coefficient^.^^^-^^^ In the SGP limit, the acquired FID is transformed with respect to t2 (the acquisition time) to obtain an NMR spectrum of the form N.

An(v) is the one-dimensional spectrum of the nth species ( q = 0), including the effects of transverse and longitudinal relaxation, Dn is the diffusion coefficient, and NA is the number of components. Here q is incremented to obtain data for the second dimension. The analysis consists of inverting the q dimension to obtain the spectrum of diffusion coefficients. Generally the inversion is mathematically ill-conditioned, and thus additional information must be supplied. For example, whether the distribution of diffusion coefficients is discrete or continuous, which corresponds to whether the components are monodisperse or polydisperse. When there is a discrete number of components the inversion programs DISCRETE209 and SPLMOD210 can be used. Alternatively, if there is a distribution the program CONTIN211 can be used. An example of a DOSY spectrum is given in Fig. 26. Recently, Johnson207 has investigated the effects of chemical exchange in DOSY spectra.

6.5.3. Electrophoretic mobility Electrophoretic NMR (ENMR) resolves the NMR spectra according to the electrophoretic mobility of ionic species. The NMR method has a number of advantages over traditional methods, including needing only a relatively short drift period and not requiring labelled ions. Johnson and He212 have recently reviewed the theory and applications of ENMR. Early ENMR experiments were based on the Stejskal and Tanner sequence and used a U-tube electrophoresis chamber.2123213The ionic drift velocities were determined from the co-sinusoidal dependence of signal intensity on the electric field in the sample. The method was subsequently extended to two dimensions through Fourier transformation of the NMR intensities with respect to either the duration or the amplitude of the electric field Morris and Johnson217 have reported an improvement called mobility ordered two-dimensional NMR (or MOSY: mobility ordered spectroscopy) which achieves resolutions of approximately 1 part per 100 in the mobility dimensions for ions in mixtures and permits the display of both positive and negative mobility. In this method, the LED pulse sequence is used and the

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lo-*

1

D (m2s-')

8.0

7.0 6.0

5.0

4.0

3.0

2.0

1.0

0.0

lo-''

6 (PPm) Fig. 26. DOSY contour plot for a solution containing BSA (bovine serum albumin) (2 g/dl), SDS (sodium dodecyl sulfate) (2 g/dl) and P-mercaptoethanol (0.01 M) in phosphate buffer. The unlabelled line represents the reaction p o d u c t , HOCH2CH2SSCH2CH20H.(Reproduced with permission from Chen ef af. 84)

U-tube electrophoresis chamber is replaced with a cylindrical cell allowing observation of unidirectional flow. Agarose gels are used to stabilize the samples when higher currents are needed. For ENMR collected with the LED sequence (see Fig. 17(A)) the complex data set is given by k

~

x

=y

2 ~xPc~(P~(oI./~(T~, 727 7,)

j=O

where

and

(97)

GRADIENT NMR

121

k is the number of species, A = T] + T~ is the electrophoretic drift time, I is the current, A is the cross-sectional area of the electrophoresis cell and K is the conductivity of the solution. Dl and pJ are the diffusion coefficient and the electrophoretic mobility of the jth species, respectively. M,j is the equilibrium magnetization, and T< and T2I are the spin-lattice and transverse relaxation times, respectively, of the jth species. The drift velocity of the jth species is given by uJ = p,Edc, where Edc = IIAK is the electric field in solution. The real and imaginary parts must be acquired in separate experiments. The mobility spectra (i.e. the second dimension) are obtained by transforming with respect to A or I (depending on what was incremented in the experiment). Incrementing I is preferable since it excludes diffusional broadening. Transformation with respect to I gives, for ions with mobility pl,peaks at u, = qpJA/AK in the mobility dimension. If a cylindrical electrophoresis chamber is used and two FIDs are acquired using the LED sequence at each current value (i.e. one FID for each polarity), the resulting phase-modulated NMR spectra, S , is given by k

where the subscripts "+" and "-"denote the polarity, and uJ(o)and dj(w) are the absorption and dispersion lineshapes for the jth species. From appropriate linear combinations of these spectra, two data sets of absorption mode spectra, one modulated by cos(2~quA)and the second modulated by sin(2rquA), are obtained. These data sets can then be analysed by means of Fourier transformation or linear prediction to yield a MOSY spectrum with pure absorption mode peaks in both dimensions.

7. TECHNICAL ASPECTS OF GRADIENT PRODUCTION 7.1. Introduction

The technical aspects of pulsed field gradient NMR have been discussed by a number of authors (e.g. see refs 4, 14, 15, 104 and 218). Most of the following discussion will be relevant to Bo gradients. The design of a Bo gradient probe is essentially similar to that of a microscopy probe (e.g. see refs 4 and 219) except that the gradients used for the BOgradient probe are larger and normally in one direction only. To perform gradient NMR, it is necessary to have a coil to produce the gradient, a power supply to drive the coil and to have the switching (and perhaps the magnitude) of the gradient under software control (i.e. in appropriate synchronization with the rf pulse sequence).

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7.2. Gradient coil design 7.2.1. Bo gradient coils

Ideally the gradient coils should produce a perfectly linear gradient, but it has been found in practice that a reasonable deviation from perfect linearity is allowable for many experiments.220Many other types of gradient coil are possible (see ref. 218), and typically quadrupolar (for g, and gz) and planar array (for g y ) coils are used in electromagnet-based systems, whilst saddle coils (for g, and g y ) or Maxwell pair coils (i.e. anti-Helmholtz) for g , are used in superconducting geometries. In fact, a design suitable for use in a magic angle spinning probe has recently been presented.221 Since most recent experiments have been performed in superconducting magnets using a shielded Maxwell pair, our discussion will be based upon on this geometry. The magnetic field strength at any point can be estimated from the Biot-Savart law ,2227223

where

u=

J

4pr2

(p

+ r2)2 + z2

K and E are the elliptic integrals of the first and second kinds, respectively. r2 is the radius of the point at which the gradient is calculated, p is the radius of the gradient coil and z is the displacement along the z axis from the coil. Equation (100) may be used as a starting point for determining how many turns are necessary in the primary gradient coil. However, shield gradient coils need to be introduced into the design such that the desired gradient is imposed over the sample volume but a greatly reduced (ideally nothing) gradient is generated outside the coils (see Fig. 27). In this way no (or at least greatly reduced) eddy currents are generated. The idea of shielded gradient coils was originally proposed by Mansfield, Chapman and B o ~ l e y The . ~ theoretical ~ ~ ~ ~ ~aspects of shielded gradient coils have recently been summarized by Callaghar~,~ and will not be discussed here. Numerical optimization procedures for designing coils have been It should be noted that the shield coils decrease the strength and linearity of the gradient that would be produced if the primary et al.232 discussed techniques to gradient coil were u n ~ h i e l d e d . ’ Carlson ~~ design shielded gradient coil systems; specifically, they considered the design compromises between gradient homogeneity, construction complexity, accessible bore and coil efficiency. Although PFG experiments are generally performed with a gradient in

GRADIENT NMR

123

Insert glass

rf coil Shield Coil

1

I-.-

.. .. .. ..

..

.. ...... .. ......

Coil

.... .. ........ .. .... .. .. ..

..... ...... ..... ...... ..... ..... ..... .. .. .. .. .. ...... . .. .. .. .. .....

Thermocouple

Fig. 27. Shielded gradient coils. Schematic diagram of a probe head containing shielded gradient coils.

one dimension only, it is now increasingly common, especially with the advent of imaging and microscopy probes, to perform diffusion experiments in three dimensions so as to obtain the diffusion tensor. Basser and co-workersS3 have proposed that measured diffusion anisotropy in anisotropic media can be used as a basis for calibrating and aligning magnetic field gradients. It is important to account for “cross-terms’’ between gradients, otherwise they will lead to incorrect estimates of the diffusion coefficient .54,233

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The gradient coils can have a strong mutual inductance with the rf coils. This has two deleterious effects, first the Q of the rf coil(s) is diminished, and secondly the gradient coils can have the effect of coupling in external radio sources since the current leads act as antennae.

7.2.2. B, gradient coils Various B1 gradient coil geometries have been proposed. Counsel1 et aZ.39 used the residual inhomogeneity of a conventional transmitter coil to produce a planar gradient. As only the one rf coil is used, both the gradient and homogeneous field are exactly in phase, and thus only the amplitude variation of the fields needs to be considered. The disadvantage of this design is that the gradient is very weak. Canet and c o - w ~ r k e r sused ~~ a remote single-turn coil to create an approximately planar B1 gradient. This design delivers a modest gradient of 0.02-0.03 T/m.234 More recently, Maas and have used an inverted Helmholtz coil which creates a radial gradient. This configuration gives a much higher gradient strength of about 0.1-0.2Tim. In their NMR probe design, the gradient coil is placed on the outside of a conventional homogeneous inner coil. Both coils are driven by the same transmitter, and therefore the response of a spin system to rf from either coil is coherent. This gradient coil design is useful in that it generates two orthogonal gradients and the resulting rf field has cylindrical symmetry. This symmetry obviates the need to know the phase angle between the homogeneous and the gradient rf coils, which would otherwise need to be known for a planar gradient. In using a radial gradient to form a planar gradient (see Section 2.3) this phase angle is variable through the phases of the gradient pulses. This setup, however, requires an odd sample geometry (i.e. an annulus).

7.3. Power supplies Two factors limit the maximum current switching speed; the first is that the power supply voltage must equal RI L dlldt, where L and R are the load (i.e. gradient coils + leads) inductance and resistance, respectively, and the second is the “slew rate” of the power supply. Since the current through a gradient coil induces heating, this causes the coil resistance to change; a constant current power supply is generally preferred so that the gradient pulses are more reproducible. A number of power supply designs have recently been discussed. 128,236 The design by Boerner and Woodward’28 is interesting in that the driver regulates the total charge delivered to the gradient coil independent of the coil inductance or resistance. The design by Saarinen and W ~ o d w a r dincludes ~ ~ ~ provision for electrophoresis pulse generation.

+

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125

7.4. Gradient calibration 7.4.I . Bo gradients The different Bo gradient calibration methods have recently been reviewed by Holz and Weingartner.237 In theory the applied gradient could be calculated from the known dimensions, geometry and the number of turns of wire in the coil and the current applied. In practice, this method should give an estimate with an error of S10%, the major reason being interaction with nearby metal in the probe. Thus, other methods are needed to get accurate gradient calibrations. The simplest way of calibrating a gradient is to use a "standard sample" of known diffusion coefficient. Ideally, a reference compound should have a diffusion coefficient and T2 that are not strongly temperature-dependent. Further, caution should be used with standards containing coupled spins to avoid artifacts arising from J modulation. Some suitable standard samples and their diffusion coefficients are listed in Table 2. Apart from sample-dependent problems, the effects of eddy currents and/or mechanical vibrations will result in this method giving only an apparent calibration. Further, because eddy current effects increase with gradient strength, a calibration at one current value cannot be used to determine the gradient strength at another value of the applied current. This method of gradient calibration is further limited by the need to have a compound containing a nucleus that can be observed with the probe at hand and with a similar diffusion coefficient. For lower diffusion coefficients, suitable reference compounds become more scarce. Glycerol has often been used as a reference, but its diffusion coefficient is greatly affected by water

Table 2. Some selected reference compounds and their diffusion coefficients at 298 K useful for calibrating PFG experiments. A more comprehensive listing can be found in the paper by Holz and W e i r ~ g a r t n e r . ~ ~ ~ Observed nucleus

'H 2H ' ~ i 13C 'YF *'Ne 23Na 31P 129~e 133cs

Compound H20

'HzO LiCI (0.25 M) in H 2 0 C6H6 C6H6F

Ne (4 MPa) in 2H20 NaCl (2 M) in H 2 0 (C6HM' (3 M) in C6D6 Xe (3 MPa) in H 2 0 CsCl(2 M) in H 2 0

Diffusion coefficient (m2/s) 2.30 x 1.87 x 9.60 x 2.21 x 2.40 x 4.18 x 1.14 x 3.65 X 1.90 x 1.90 x

10-9 10-9 lo-'' 10-9 10-9 10-9 10-9 lo-'" 10-9 10-9

Ref. 302 303 237 304 237 245 305 237 244 237

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content as well as having a highly temperature-dependent diffusion coefficient and T2.14,237 Hrovat and Wade132,160*238 have suggested using the time displacement of the echo maximum caused by the intentional mismatch of gradient pulses. It is possible to calculate the gradient strength using the echo shape from a sample of known geometry, such as a ~ y l i n d e ? or ~ ~other geometries.’60 However, this method is prone to a number of systematic errors. Another possibility, although limited by the spectrometer bandwidth, is a onedimensional image.2”2.240One procedure for calibrating the gradient with a one-dimensional image is to use a sample of known length in the direction of the gradient (a bulb is typically used). The FID is recorded in the presence of the gradient for a number of different applied currents, and then by plotting the width of the spectrum versus the current the gradient strength can be calibrated.222 This method requires that the length of the samplecontaining cell be known accurately, and the final calibration will have an error of around 5 % . The virtue of this method is that the calibration can be performed without any knowledge of the sample diffusion coefficient. If confronted with an uncalibrated coil, a realistic calibration procedure is to first perform a theoretical calculation of what gradient the coil should produce for a given current. A one-dimensional image should then be used for experimental verification. Finally, if a suitable reference compound exists, this should be used for “fine tuning” of the calibration.

7.4.2. BI gradients As for Bo gradients, B1 gradients can be calibrated using reference compounds. However, B1 gradients can also be calibrated, for example, by determining the 360” pulse for a substance inside a capillary at different locations in the sample. One complication, though, is that since gl is directly proportional to the rf field strength, the gradient is proportional to the pulsewidth (and therefore probe tuning). Canet et ~ 1 . ~have ’ shown that after calibration of a B , gradient coil, on changing the sample (and therefore the tuning) the gradient can be determined for the new sample by comparing the 360” pulse width at the centre of the sample relative to that found in the calibration.

7.5. Sample shimming and field frequency locking The sample should be firmly held inside the linear region of the gradient coils, and thus the sample is normally contained in a volume not more than 1 cm high or 1cm in diameter. Such a sample, though, has large changes in magnetic susceptibility close to the rf coils. Accordingly, it is very difficult to achieve good resolution. A solution is depicted in Fig. 28, This method,

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127

Sample

I

Solvent

2/

Fig. 28. A possible sample configuration for gradient NMR measurements in a 10 mm NMR probe. The sample (0.5 ml) is placed in the 8 mm i.d. flat-bottomed NMR tube which is coaxially supported in a 10mm tube. The space beneath the flat-bottomed tube is filled with water, and the sample capped with a Teflon vortex plug to minimize susceptibility differences between the sample and its adjacent

environment, and thus allow the sample to be more readily shimmed. (Adapted from Price .222) compared to just coaxially inserting a bulb into an NMR tube, has the advantage in that it is easy to clean the sample tube or to work with viscous substances. With the use of increasingly higher gradients and the desire to measure ever smaller diffusion coefficients, the rigidity of the sample is of great concern. A sample spinner suitable for use in PFG experiments has been developed that allows for the spinning to be arrested during the motion-sensitive part of the experiment and yet spun, to achieve higher resolution, during acquisition.241 The normal 2H lock is coupled to the Z shim coil to counteract the natural drift of the magnet. A Bo gradient pulse will obviously affect this mechanism. The simptest solution, in the case of low-resolution experiments, is simply to turn the lock off. A better solution is just to gate the lock off for the duration of the gradient pulse.

7.6. Temperature control

Since the temperature regulation in NMR probes is by heated (or cooled) air (or gas, e.g. nitrogen) through the base of the probe, it is possible for

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temperature gradients to be produced along the long axis of the sample. If the temperature gradient is large enough, convective flow may be induced, resulting in an overestimate of the diffusion coefficient. Goux et al.242 studied the effects of thermal convection in PFG experiments. They concluded that, while the best solution is improved temperature control, the effects of convective flow can be minimized by using narrower sample tubes, decreasing the sample height, or increasing the sample viscosity.

8. SPECIFIC EXAMPLES OF GRADIENT NMR 8.1. Introduction

It is beyond the scope of this chapter to provide a comprehensive survey of the literature related to gradient NMR. Instead, this section is intended to give an introduction to the types of systems that have been studied, and the information obtained, and also to discuss some of the novel applications of gradient NMR. 8.2. Diffusion-based studies

8.2.1. Diffusion measurements

A list of diffusion coefficients for interesting species is given in Table 3. Recently, measurements of the self-diffusion coefficients of H2,243the noble gases Xe244 and Ne245 in water, and of Cm in b e n ~ e n e - dhave ~ ~ ~been ~ reported. 23Na PFG NMR has been used in conjunction with TI measurements to study the relationship between the diffusion coefficient, conductivity and T~ of sodium ions in water-glycerol solutions.33 Gibbs and Johnson have studied polyammonium cation diffusion in aqueous solutions of DNA.247 Even the diffusion behaviour of sodium monofluorophosphate (MFP2-), Na+, H20 have been studied in toothpaste-like pastes based on aqueous silica dispersions.248 B1 gradients have been used to measure the self-diffusion coefficient of hydroxyquinoline in aqueous solutions of micellized sodium dodecyl sulfate, and also to determine its partition coefficient as a function of

8.2.2. Restricted diffusion and obstruction have used 13C PFG NMR to study the diffusion coefficient of Price et glycine inside human red blood cells. Glycine transports across the red cell membrane slowly on the NMR time-scale. It was found that the intracellular glycine diffusion coefficient was one-third that of the free solution value.

GRADIENT NMR

129

Table 3. Selected diffusion coefficients of species of biological and chemical significance. The species in parentheses denote the solvent, where applicable.

Observed nucleus 'H

l3C 31P

Species Ovalbumin 2H (H20) c 6 0 (l mM) (c6D6) [2-13C]Glycine(H20) H 13C03- (H20) H2P02-(H20)

Diffusion coefficient (m2/s)

Temperature (K)

Ref.

7.92 X lo-'' 4 x 10-9 8.3 x lo-'" 1.18 x 10-9 1.26 x lo-]" 1.60 X lo-'

Ambient 296

276 243

298

246 32 118 118

310 310 310

PFG NMR has been used to measure water diffusion and pore volume in wood pulp cellulose fibre^.^^',^'^ Two components were observed: one with a self-diffusion coefficient independent of time and the other with a time-dependent "apparent" diffusion coefficient. The two components were attributed to the bulk water between the cellulose fibres and water within the fibres, respectively. Van Gelderen and co-workersX1studied the diffusion of phosphocreatine in the cylindrically shaped fibres of rabbit skeletal muscle. By using diffusion measurements in three orthogonal directions, they were able to determine the trace of the diffusion tensor with time. This allowed them to evaluate the diameter of the cylindrical cells and the unrestricted diffusion coefficient. They found the radius of the cells to be in the range of 8-9pm, and the phosphocreatine diffusion coefficient to be in the range 7 x 10-l' to 9 x 10-" m2/s. Obstruction effects are quite common in both chemical and biochemical systems. Kuchel et measured the diffusion coefficients of H2 and H 2 0 in H 2 0 and in an H 2 0 solution of bovine serum albumin. They found that in H 2 0 the diffusion coefficients of H2 and H 2 0 were 4.0X and 2.1 x lo-' m2/s, respectively, at 296 K. However in the protein solution the diffusion coefficients were reduced to 1.0 x lo-' and 1.8 x m2/s, respectively. The larger reduction in the diffusion coefficient of H2 than H 2 0 was attributed to the greater obstruction felt by the faster diffusing H2. Latour et ~ 1 studied . ~the ~time-dependent ~ water diffusion coefficient in packed erythrocytes. They found that the long-time diffusion coefficient, Deff, was very sensitive to the extracellular volume fraction. Using an effective medium formula, they were able to estimate the erythrocyte membrane permeability. From the short-time behaviour of the diffusion coefficient, they estimated the surface-to-volume ratio of the cells to be approximately (0.72 p n - ' . Blees and L e ~ t e studied ' ~ ~ the self-diffusion of organic solvents in water and in a colloidal dispersion of poly(butylacry1ate) in water. They found

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WILLIAM S. PRICE

that, for the colloidal systems with A = 45ms, the self-diffusion was dominated by exchange effects amongst the colloidal particles. The diffusion coefficient was also found to depend strongly on the volume fraction of the colloidal particles. They noted that the partition of the organic solvent between the water and the colloid particle could be determined from the experiment. PFG has found great application in the measurement of droplet sizes and distributions. Systems that have been studied include emulsions stabilized by anionic, cationic and non-ionic surf act ant^."^^"^^^^^ In a study of water-inoil emulsions, it was noted that as the temperature changed the standard deviation of the droplet size remained constant, but there was a large decrease in the mean diameter of the dist~ibution.''~It was surmised that the most likely explanation of this effect was water interdroplet diffusion. Thus, there must be a significant probability for water molecules to move from droplet to droplet in aggregates as observed in oil-in-water emulsions255or by random coalescence and break-up providing additional distances for motion.256 Diffraction-like effects have been observed in a highly concentrated water-in-oil emulsion.257From the experiment the mean droplet size of the emulsion was able to be determined.

8.2.3. Binding and transport Hydration numbers may be determined from the concentration dependence of the water self-diffusion coefficients.2589259 31P PFG NMR has been used to measure the diffusion coefficient of 2,3-bisphosphoglycerate (DPG) in haemoglobin solutions in both free solution and in intact erythrocytes.260 The dependence of the measured diffusion coefficients on the amount of DPG bound to haemoglobin was used to estimate the dissociation constants for DPG complexed to carbon-monoxygenated, oxygenated and deoxygenated haemoglobin. An example of using PFG NMR to study the transport of hypophosphite and bicarbonate ions in human red blood cells"* has already been discussed in Section 3.7.5. More recently, Waldeck et aZ.261used PFG NMR to study the ionophore-mediated transmembrane exchange of Li+ in liposomes. They applied the two-region approximation approach of Karger (i.e. equation (61)) and found good agreement between the transmembrane exchange rates obtained from the PFG measurements and those using traditional NMR methods.

8.2.4. Liquid crystals and surfactants Extensive reviews on the application of gradient NMR to liquid crystal and result surfactant systems have recently been p r e ~ e n t e d .As ~ ~a, ~ ~ we will not spend any time reviewing this area apart from mentioning that gradient

GRADIENT NMR

131

NMR provides a superb tool for such systems and has produced useful results in systems such as water self-diffusion in polycrystalline lamellar systems,262 hexagonal mesophases," smectic liquid crystals,263 and surfactan t systems. 264-267 As would be expected for restricting geometries, strong diffusional anisotropy effects have been noted in studies of biological cells. 150,151 In fact, the observation of the anisotropy has been noted as being a useful clinical probe of demyelinating disorders, white matter infarcts, neoplasms and neonatal brain and spinal cord development.1so Schoeniger et a1,268 studied water diffusion in neurons. They noted that water in the nucleus has different diffusion properties than that in the cytoplasm. Similarly, LeBihan and c o - w o r k e r ~have ~ ~ ~proposed that measurements of diffusion coefficients have clinical applications including functional assessment, tissue characterization and treatment monitoring.

8.2.5. Porous media Zeolites and rocks are well-known examples of porous media that have been studied by gradient NMR. PFG measurements can unambiguously discriminate between the limiting cases of (1) intracrystalline diffusion, (2) restricted self-diffusion and (33 long-range self-diffusion. The information that can be obtained about zeolite beds via PFG NMR has recently been summarized by K a r g e ~ - Consequently, .~~ we will just review some of the more recent applications. 'H NMR imaging has been combined with PFG NMR to study sorption in zeolites.270 Figure 29(A) contains an example of 'H NMR spectra of n-hexane in a bed of zeolite NaX after the onset of adsorption through the upper right face (right-hand side of the spectra) of the zeolite bed in the NMR sample. The data show that the observed diffusivities are a function of the local sorbate concentration (Fig. 29(B)). PFG NMR has also been used to study catalytic reactions271and the molecular diffusion of CH4, CO, C 0 2 , n-hexane and Xe in zeolite^.^^".^^^,^^' It has also been noted that the geometrical information factor F , which can be determined from PFG measurements, is important for transport phenomena in porous systems such as fluid flow, electrical conductivity, and fourth sound.'22 PFG NMR has also been used to study the pore size and to determine the surface-areato-volume ratio and surface relaxivity in 8.2.6. Polymers and macromolecules

Gradient NMR provides a convenient means for studying the physical chemistry of proteins. Gibbs and co-workers have used 'H PFG NMR to study ovalbumin intradiffusion coefficients as a function of protein concentration in free solution .276 They noted that PFG measurements in

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WILLIAM S. PRICE

m-0 m-2 In-4

m-2

m-6

m=9

L

a

B

\ 0

50

100

150

200

Fig. 29. Applications of PFG N M R to zeolite systems. (A) ‘H N M R resonances of n-hexane in a bed of zeolite NaX, (a) 50min and (b) 150min after the onset of adsorption with restricted adsorbate supply for different field gradient pulse widths 6 = 0.4 v m ms. The curve with m = 0 represents the concentration profiles. (B) Apparent self-diffusion coefficients of intracrystalline n-hexane observed by timeand space-resolved ‘H PFG N M R in zeolite NaX with restricted (a) and unrestricted ( 0 ) sorbate supply. The open symbols represent the true diffusivities. The solid line with error bars indicates the range of intracrystalline diffusivities as observed in previous PFG N M R studies. (Reproduced with permission from Karger et al.270)

GRADIENT NMR

133

combination with the macroscopic boundary relaxation technique and independent measures of the protein activity coefficient offer a means of comparing the magnitudes of the frictional coefficients for mutual diffusion and intradiffusion. Later, using both 'H and 19FNMR, they extended their studies to the diffusion of ovalbumin in porous gel filtration chromatography media.277 PFG can provide an enormous amount of information about polymers both in solution and in polymer melts. For example, PFG NMR has recently been used to measure the critical overlap concentration for polystyrene in t e t r a c h l ~ r o m e t h a n e .Callaghan ~~~ and Coy,1o8 using very large values of q(27rq (13.0 nrn)-'), have obtained evidence for reptational motion of high molar mass polystyrene in semidilute solution using 'H PFG measurements. In a related study, Appel et dio7 used the fringe field of a superconducting magnet to measure the self-diffusion of poly(dimethylsi1oxane) , polybutadiene and polyisoprene for times smaller than the reptation time. A time-dependent apparent self-diffusion coefficient was observed. Solutions of block copolymers are interesting systems for the study of self-organization since, due to hydrophobic and hydrophilic moieties, the polymer molecules may form micelles over a certain temperature interval. As a result, the diffusion of some polymers can have strange temperature dependencies, such as has been observed with the triblock copolymer PEO-PPO-PEO in 2H20.279Interestingly, it was observed that even for the case of A = 3 ms the attenuation of the echo signal was single exponential. Thus, the exchange time of the polymer molecules between the micelles and unimers must be much shorter than the observation time. PFG also provides a means for studying ion motions in conducting polymers. For example, 7Li PFG NMR has been used to study lithium diffusion in polymer electrolytes. 280

-

8.3. High-resolution NMR applications

8.3.1 Applications of DOSY and electrophoretic N M R The DOSY experiment has opened up new possibilities for analysing spectra from samples containing more than one component. Apart from the examples presented in Section 6.5.2, it has been applied to the analysis of polydisperse systems,2o8 and for studying equilibria involving binding, absorption and partitioning in surfactant systems.281 The DOSY experiment has been used to study the non-Newtonian to Newtonian transition in a hexadecyltrimethylammonium bromide (CTAB)sodium salicylate-water viscoelastic micellar system induced by the addition of a soluble yet slightly hydrophobic polymer.282 It is shown that the diffusion coefficient of the different species provides information on the

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WILLIAM S. PRICE

binding and aggregation processes in the system. Hinton and have used sucrose trapped inside vesicles as a marker to measure the diffusion coefficients of vesicles in a DOSY experiment. Since the size of the vesicles is much larger than the solvent, the Stokes-Einstein equation (equation (58)) can be used to relate the diffusion coefficient to the size of the vesicles. The method also allowed the determination of the trapped and free fractions of sucrose. In a later study, the DOSY experiment (see Fig. 26) was used to determine the binding isotherm and size of the bovine serum albumin-sodium dodecyl sulfate complex.284 Electrophoretic NMR has been used to study the tetramethylammonium ion, N , N , N ’,N’-tetramethylenediamine and tetrahexylammonium ion in polyacrylamide gels285and surfactant 8.3.2. Coherence selection, phase cycling and solvent suppression Since this area has only just been reviewed28we will not dwell on it greatly, but it is of note that gradient-enhanced multidimensional experiments have now become common place. A big advantage of gradient-enhanced multidimensional NMR experiments is that the gradients can also be used for water signal suppression. Thus, the one protein sample dissolved in H 2 0 , can be used for experiments which detect the amide chemical shifts during acquisition and experiments where the alpha protons are recorded (e.g. see ref. 288). Further, gradient-enhanced versions of many sequences are now in common use, for example: the selective one-dimensional COSY experiment,289 heteronuclear multiple-quantum correlation (HMQC),290 I5N-’H HSQC,291 triple-resonance three-dimensional NMR experim e n t ~and ~ three-dimensional ~ ~ , ~ ~ ~ homonuclear J COSY experiments.294A PFG-based method for determining the excitation profile of a shaped rf pulse has been presented.295 Kriwacki and c o - ~ o r k e r shave ~ ~ ~ studied water molecule binding to macromolecules. They employed modified nuclear Overhauser effect spectroscopy (N0ESY)-HMQC, rotating frame Overhauser enhancement spectroscopy (R0ESY)-HMQC and total correlation spectroscopy (T0CSY)HMQC sequences which incorporated a PFG “diffusion filter”. These modified sequences allowed them to select only those peaks arising from slowly diffusing species. Absorption mode phase-sensitive zero-quantum coherence has been developed by N o r ~ o o d This . ~ ~work ~ is interesting in the light of allowing acquisition of high-resolution spectra from samples containing strong internal magnetic gradients. B1 gradients have been used to improve the resolution and sensitivity in NOE and ROE experiments with water.298The B1 gradients were used to suppress radiation damping effects.

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9. CONCLUDING REMARKS In this chapter, I have endeavoured to summarize the main features of gradients in NMR. I have tried to emphasize that gradients in NMR can be a problem if unaccounted for, a probe of porous media if understood, and panacea for the high-resolution spectroscopist. Gradient NMR allows a very convenient means of non-invasively measuring diffusion and transport in biological and chemical systems. q space imaging opens up the possibility of being able to image porous materials at higher resolution than that available with conventional k space imaging. With improving technology (i.e. pulse sequences, shielded gradient coils and fringe fields) larger values of q become possible. Although diffraction effects only become visible at large attenuations, with increasingly higher static magnetic fields and probe technology the use of diffraction effects may become a generally useful method for many systems and not just specific model systems. Further extensions to “multiple wave vector” experiments have potential for providing increased information. Gradient methods that separate species according to their mobility and diffusion coefficient such as electrophoretic NMR and the DOSY experiment have enormous potential in the analysis of complex mixtures containing many species such as biological In high-resolution NMR, gradients allow many experiments to be achieved in a fraction of the time taken by their non-gradient counterparts. This has made the higherdimensional experiments much more practicable. Although Bo gradients are currently more popular than B1 gradients, it is expected that the application of B 1 gradients to high resolution NMR will increase due to their technical simplicity.

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Pharmaceutical Applications of NMR D. J. CRAIK and K. J. NIELSEN Centre for Drug Design and Development, University of Queensland, Brkbane, 4072, Q L D , Australia

K. A. HIGGINS Department of Biochemistry, Monash University, Clayton, 3144, VZC,Australia

1. Introduction 1.1. Instrumentation 1.2. Methodology 2. The role of NMR in drug development 3. NMR techniques in drug design 3.1. Drug conformations 3.2. Protein structure determination 3.3. Protein-ligand complexes 3.3.1. NMR titrations of complexes 3.3.2. NOESY analysis of complexes 3.3.3. Isotope editing 3.3.4. Transferred NOES 4. Selected examples 4.1. Endothelins 4.1.1. 3D structure of ET-1 and related peptides 4.1.2. Comparison of NMR and X ray structures of ET-1 4.1.3. 3D structure of cyclic pentapeptide ET antagonists 4.2. Conotoxins 4.2.1. 3D structure of w-conotoxin GVIA 4.2.2. Structure-activity relationships 4.2.3. Structurally related peptides 4.3. Insulin 4.3.1. 'H NMR studies 4.3.2. I3C NMR studies 4.3.3. Summary Acknowledgements References

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1. INTRODUCTION Since its discovery, nuclear magnetic resonance (NMR) spectroscopy has played an important role in the pharmaceutical sciences. An early application of NMR was to assist in the identification and characterization of A N N U A L REPORTS O N NMR SPECTROSCOPY VOLUME 32 ISBN 0-12-505332-0

Copyright 01996 Academic Press Limifed A // rights of reproduction in any form reserved

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chemically synthesized drugs or biologically active molecules derived from a variety of natural sources which formed the basis of pharmaceutical products. The role of NMR as essentially an analytical tool has now been supplemented by a more fundamental application-providing information to be used in the design of new drugs. This application has become possible because of the significant advances in NMR instrumentation and methods that have occurred in recent years. This review will focus primarily on applications in drug design, rather than analytical or other applications of NMR in the pharmaceutical industry. The importance of NMR in drug design is demonstrated by the recent publication of several books and reviews on the topic,14 and related articles on protein-ligand interaction^.^.^ Applications of NMR to drug design had their origins in the 1960s and 1970s, with the determination of structures and conformations of biologically important organic molecules, typically with molecular masses of up to 1000 Da. These studies were done initially on 60-100 MHz spectrometers, where the conformational information was derived from chemical shifts, coupling constants, and nuclear Overhauser enhancement (NOE) measurements. Such studies proved useful in defining solution conformations; however, these did not necessarily reflect the biologically active form at the receptor site. Nevertheless, for cases where the molecules of interest are relatively rigid, a determination of their solution conformations is valuable. The increasing availability of higher-field (200-500 MHz) spectrometers in the late 1970s and early 1980s dramatically increased the complexity of molecules that could be studied. In parallel with these developments, the availability of wide-bore magnets made it possible to examine intact organs and even whole animals using surface coil technology. This allowed, for example, the study of the effects of drugs on metabolism.’ Further advances in high-resolution NMR have occurred since the mid-l980s, with the increasing availability of 500, 600 and now 750 MHz spectrometers, and new NMR methods. In particular, the development of methodology for assigning and determining the structures of peptides and proteins by two-dimensional (2D) NMR’ has enabled a wide range of new applications in the field of drug design. These arise because of the importance of biologically active peptides as potential targets in analoguebased drug design, and proteins in receptor-based drug design. Initially, the new technology provided the capability to extend the molecular weight range and allow the study of small proteins (<10kDa), but with the development of 3D and 4D NMR methods and high-field spectrometers this has been extended to more than 30 kDa.”12 In addition, methods have been developed which allow the nature of protein-ligand complexes to be characterized in detail. NMR is distinguished from many other forms of spectroscopy in that there are numerous techniques that can be used to address different problems. These techniques are based on a vast array of pulse sequences for

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extracting different types of information from the sample, as well as examining a range of nuclei. Thus, the development of new applications in the field of drug design has resulted not only from major advances in instrumentation, but also from improvements in NMR techniques.” These two areas of development are discussed below.

1.1. Instrumentation

The basic requirement of a spectrometer to be used in drug design research is sufficient sensitivity and resolution to detect signals from drugs and/or their receptors when they are dissolved in aqueous solution at millimolar concentrations. The minimum requirement for these studies is usually a 500 MHz instrument, and most major pharmaceutical companies currently utilize one or more 500 or 600MHz spectrometers in their drug development programmes. Although some studies can be done on lower-field instruments, the number of NOES detected is usually substantially less than for high-field instruments. Currently, 750 MHz systems are being installed in a number of laboratories around the world, including in those of pharmaceutical companies. These offer the opportunity of significantly increased sensitivity and dispersion, which leads to higher-quality data sets in the same measurement time, and ultimately higher-quality structures. Alternatively, the increased sensitivity available on 750 MHz systems can be used to obtain similar quality data, but in a shorter time, to that obtained with 600MHz systems. This is important in cases where the molecule under study is unstable over the course of the required 2D or multidimensional NMR experiments. Another advantage of the greater sensitivity is that more dilute solutions may be examined in the same measurement time. This is important for proteins which tend to aggregate, an occurrence which is not uncommon at millimolar concentrations. In many cases, dilution from 1 to 0.5 mM can make a substantial difference to the degree of aggregation and hence spectral linewidths. Another approach to the study of dilute solutions has been the trend over recent years towards larger volume sample tubes. Dissolution of the same amount of sample in a larger volume results in a more dilute solution, but as the number of spins detected by the receiver coil is the same there should be minimal difference in sensitivity. The associated reduction in aggregation and consequent improved linewidths leads to a net improvement in the spectral quality. A difficulty with this approach in the past has been the generally poorer homogeneity of both Bo and B1 fields over the larger sample volume, but improvements in shim and rf systems mean that similar homogeneity can be achieved in 8 or 10 mm tubes to that in 5 mm tubes. At the other end of the scale there have also been significant developments in microvolume probes over the last few years. These are useful

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where the amount of sample available is limited, for example where only small amounts of a natural product lead compound can be extracted, or where submilligram quantities of a protein are available. The latter situation is becoming less common with the increasing availability of recombinant proteins, where production of tens of milligrams of purified protein is often feasible. Nevertheless, in the early stages of protein studies by NMR, a number of preliminary experiments to establish the optimal conditions of temperature, pH and other solution conditions are performed, and in some cases these lead to destruction of the sample. If these preliminary tests can be done on small sample quantities in a microprobe, then the remainder of the sample is preserved for the extended data acquisition experiments. Another situation where only limited supplies of protein may be available is when specific isotope labelling is used. This may be achieved using molecular biology (where all residues of a particular amino acid type are labelled) or solid phase peptide synthesis for smaller proteins (where a specific amino acid may be labelled). In either case moderately expensive labelled amino acid precursors are required, and the amounts of available sample may be limited. The microprobes currently in use require 40-100 PI of solution, compared with 450-600~1 in conventional 5mm tubes. Thus the reduction in the amount of sample required is more than five-fold. The decrease in sensitivity associated with the smaller amount of sample is offset by the higher sensitivity per unit volume of these probes. The use of susceptibilitymatched sample tubes and spacers assist in maintaining good lineshapes for small volume probes. As most studies are performed in aqueous solution, a mechanism for suppression of the large solvent signal ( - 1 0 0 ~ in protons, compared to M for the sample of interest) is an essential requirement. Until recently, presaturation of the water resonance was the preferred method, but with the advent of gradient-based methods a greater degree of water suppression can be achieved readily.I3 Associated with gradient methods are other advantages, including reduction in the loss of amide proton intensity due to saturation transfer with the solvent peak and reduction in the loss of Ha intensity due to the bleaching effects of presaturation. 1.2. Methodology

In addition to the instrumental advances described above, improvements in methodology have contributed significantly to applications of NMR in drug design and development. In some cases, such as gradient technology, these have been closely associated with advances in hardware technology, but in other cases they have been associated with developments in biochemical methodology or with sophisticated new pulse sequences. An example of the

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latter is the use of heteronuclear NMR methods for structure determination of larger protein^.^-" This has been driven to a large extent by the increasing ease with which uniform labelling of proteins with 15N or 13C isotopes may be achieved. Other examples of advances in methodology will be apparent from the applications described later in this chapter.

2. THE ROLE OF NMR IN DRUG DEVELOPMENT

It is apparent from the above discussion that NMR instruments are now sufficiently powerful to examine samples ranging in complexity from solutions of drug molecules, to their receptor proteins, to intact organs and animals (albeit with a more limited range of experiments possible). To illustrate how these capabilities fit into a drug design and development programme it is first necessary to provide a simplified description of such a programme. Figure 1 schematically illustrates the drug development p r o ~ e s s . ’As ~ indicated, this starts with a design or discovery phase. Discovery refers to the process of identifying a lead compound from a random screening programme, or from chance observation. A large proportion of existing drugs were indeed initially discovered by these routes. Design implies a more directed and rational approach to the production of a lead compound. There are several possible design strategies, but two of the most common may be categorized as either “analogue-based” design, where knowledge of the structure or conformation of a known drug or bioactive molecule is used to design either antagonists or more potent analogues, and “receptor-based’’ design, where knowledge of the structure of a macromolecular target is used to design compounds to interact with that target.

Fig. 1. Schematic illustration of the drug development process, showing the contribution of NMR. (Reproduced with permission from NMR in Drug Design, copyright CRC Press, Boca Raton, Florida.)

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In the case of either design or discovery, once an initial drug candidate is identified the next step is chemical synthesis, followed by biological testing. This may take the form of a simple in vitro assay (i.e. receptor binding or enzyme inhibition). At this point, depending on the results of the initial assay, the design hypothesis may need to be modified and there could be several cycles (indicated by the left-hand loop in Fig. 1) before a compound is submitted for more extensive in vivo testing. Further chemical input may then be required to protect the active compound from enzymatic breakdown and metabolism, and so there are likely to be several cycles of the right-hand loop before a drug candidate emerges. This is a simplified description, but it allows the possible NMR approaches at each stage to be identified. Three classes of NMR experiments can be identified-small-molecule NMR (studies of molecules of less than a few thousand daltons in molecular mass), macromolecular NMR (studies of molecules of tens of kilodaltons in mass) and in vivo NMR (whole organ or whole animal studies). The ways in which these contribute to the drug development process are indicated in Fig. 1. Small-molecule NMR contributes both as an analytical tool for the verification of the structure of synthesized molecules and at the drug design stage, i.e. by providing conformational information it is a valuable tool in analogue-based drug design. Macromolecular NMR clearly has the potential to assist in receptor-based drug design by providing the structures of key protein targets, and is also extremely valuable at the in vitro testing stage. The latter application arises because of the utility of NMR for the study of ligandprotein interactions. Once it has been confirmed that a drug candidate binds to a particular receptor macromolecule, it is important to establish the mode of binding so that further development of lead compounds is based on correct information about the intermolecular interactions. Finally, in vivo NMR is valuable for investigating the effects of drugs on target organs. This is an area that will not specifically be addressed, apart from the example described below which illustrates the type of applications possible. The example chosen is the use of 31PNMR to study the effects on cardiac metabolism of drugs designed to protect the heart during periods of ischaemia (reduced blood flow). With a simple set-up it is possible to perfuse a rat heart inside the magnet bore and obtain signals from abundant phosphorus-containing species in the beating heart. These include the high-energy phosphate species adenosine triphosphate (ATP), phosphocreatine (PCr) and inorganic phosphate (Pi). With a few minutes of data acquisition it is possible to obtain spectra (Fig. 2) in which the relative abundance of ATP, PCr and Pi may be readily measured. These provide an important indication of the bioenergetic status of the heart. In addition, by measuring the chemical shift of the inorganic phosphate peak the intracellular pH, another important marker of cardiac viability, can be determined. By monitoring the spectrum as a function of time following an ischaemic

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insult to the perfused heart, the levels of these various metabolites can be followed (together with pH). The profile for a 24min period of ischaemia (induced by turning off the flow of perfusate to the heart) followed by reperfusion is given in Fig. 3 . Here it is seen that within 3 min of global ischaemia the PCr levels have dropped to zero, but the ATP levels decline at a slower rate. Concomitant with these decreases is a rise in Pi, as well as a drop in pH (not shown). The modulating effects of drugs on the rate of decline of the high-energy phosphorous metabolites can readily be determined by comparing the above profiles for control hearts and animal hearts which have received drug pretreatment. Figure 4, for example, shows the reduced rate of decline of PCr levels in hearts pretreated with anipamil. By comparing different drugs and treatment protocols, improved cardiac protective drugs can be developed. The remainder of this chapter will focus on the applications of N M R to drug design (i.e. those fitting into the small-molecule N M R and macromolecular N M R categories described above). Section 3 describes some of the N M R methods used in these studies, while Section 4 describes selected applications to specific classes of bioactive molecules.

3. NMR TECHNIQUES IN DRUG DESIGN The basic questions relevant to drug design which N M R can answer include: What is the structure of the drug? What is its bound conformation and what charge state does it bind in? Is there more than one bound conformation

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and what are the dynamics at the receptor site? Where is the binding site and what functional group interactions are involved in ligand recognition and binding? Some of these questions can be answered by studies of the drugs themselves, but most require examination of the target macromolecule and its complex with the drug. 3.1. Drug conformations

Determination of the chemical structure and conformation of a drug is an essential starting point in understanding its action and thereby attempting to improve upon it. Structure determination of organic molecules (which make up the majority of drugs) by NMR is a well-established application which has been fully described in many NMR texts and will not be addressed in detail here. It suffices to say that a careful analysis of chemical shifts, coupling constants and NOES can lead to a detailed understanding of conformational phenomena. This is illustrated by recent studies on the antipsychotic compound butaclamol (1). 15-17 Despite the apparently rigid appearance of this molecule, molecular mechanics calculations suggested the possibility of four low-energy conformations for the protonated form of butaclamol.16 These had a chair conformation of the E ring, but either a trans (torsion angle HNCH4a close to 180") or cis (torsion angle HNCH4a of 50") junction of the D and E rings, and either of two possible conformations of the ethane bridge connecting the A and C rings, differing in the torsion angle about the C8-C9 bond; one of the trans conformers was found in the crystal. NMR studies15 showed that the hydrochloride exists as a single conformer in chloroform, with NOE data and the magnitude of the couplings to H4a

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D. J. CRAIK, K.J. NIELSEN AND K. A. HIGGINS

consistent with a trans junction of the D and E rings. By contrast, spectra in dimethyl sulfoxide (DMSO) showed the presence of two conformers in slow exchange, as indicated by two distinct NH resonances in a ratio of 4:1.l 6 * I 7 Analysis of couplings revealed that the major conformer was identical to that observed in chloroform, whereas the minor conformer had a cis junction between the D and E rings. l6 Variable-temperature spectra and saturation transfer studies were used to determine the interconversion barriers between conformers. In general, these studies illustrate the value of combining computational approaches (to define a range of likely low-energy conformers) with NMR (to determine the actual conformers present and interconversion barriers between them). X ray methods tend to yield information only on the lowest energy form in the crystal, whereas NMR studies illustrate that even apparently rigid molecules may have multiple conformers, and that protonation can have a significant effect on conformational equilibria. Studies of isolated drug molecules in solution may not necessarily define the receptor-bound conformation; however, by studying them under a range of different solvent conditions and protonation states, an indication of the range of conformers which may be adopted at the receptor binding site can be obtained.18 When conformational exchange occurs on a faster time-scale than seen above (where conformers were in slow exchange on the chemical shift time-scale) a quantitative understanding of the processes involved can be obtained from NMR relaxation times. In particular, 13C T1 measurements provide information on the overall and internal molecular flexibility, as illustrated in a series of studies on the drugs imipramine (2), amitriptyline (3), and related tricyclic antidepressants. lSz3

13

/”

PHARMACEUTICAL APPLICATIONS OF NMR

Table 1.

I3C NTI (s) values for irniprarnine (2) and arnitriptyline (3). ~~

Compound

153

P

C10, C11

~~

C12

C13

C14

C15

1.18 1.91 1.89 2.54

1.30 2.25

1.31 2.60

1.38 3.12

3.72 4.22

1.09 1.3111.27 1.80 1.9411.90

1.09 1.79

1.38 2.78

1.38 3.11

3.40 4.20

(2)

Hydrochloride Free base

(3)

Hydrochloride Free base

“Aromatic carbons.

Table 1 shows NT, values for the carbon atoms in these molecules and illustrates the varying degree of flexibility at different sites. An unexpected finding from the T1 values for imipramine was the significant degree of flexibility of the ethane bridge (i.e. C10, C l l ) in the central sevenmembered ring. This flexibility is reduced in amitriptyline. Segmental motion of the propylamino side-chains of these molecules was also detected by these measurements and found to depend markedly on the solution conditions and protonation state of the molecules. These studies were extended to include investigations of the interaction of these drugs with model r n e m b r a n e ~ . ~ ~ . ~ ~ The organic molecules described above are rather simple. As noted earlier, perhaps the most significant advance over the last few years in the application of NMR to the “small-molecule” category noted in Fig. 1 has been the increasing complexity of the molecules which can be studied. These include complex metabolites derived from plant, fungal and microbial sources as well as newly discovered endogenous compounds in animals. In particular, bioactive peptides represent a rich source of new drug leads, and NMR has been at the forefront of structure determination of these specie^.^^.^^ Examples of the structural and conformational information which can be obtained are given in Section 4.

3.2. Protein structure determination

NMR spectroscopy is now widely recognized as an invaluable tool for the structural characterization of biological macromolecules with molecular masses up to approximately 30 kDa. The quality of structures obtained using NMR methods is comparable with those derived from X ray crystallography but, in addition, NMR offers the possibility of obtaining quantitative information on molecular flexibility.

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Procedures for determining protein structures by NMR have been the subject of numerous reviews.2c3" In summary, it is generally found that traditional homonuclear 2D methods,' based on a combination of COSY (correlation spectroscopy) and TOCSY (total correlation spectroscopy) for assignments of residue type, and NOESY (NOE spectroscopy) for making sequential connections, is sufficient to assign proteins of up to about 100 amino acids. Beyond this, resonance overlap and poor magnetization transfer for larger proteins mean that heteronuclear methods are required. The increased resolution afforded by '5N-dispersed 3D spectra of uniformly "N-labelled proteins extends the size limit to about 150 amino acids, but larger systems generally require double (13C and "N) labelling. Modern heteronuclear 3D and 4D NMR experiments make use of coupling via the heteronuclei to unambiguously make sequence specific assignments. Examples of proteins of pharmaceutical interest whose solution structures have recently been determined using heteronuclear and/or multidimensional NMR techniques include the calmodulin target peptide the HIV-1 matrix protein,32 the trimethoprim-dihydrofolate reductase complex,33 the DNA-binding Hu protein34 and apokedarcidin, a newly discovered antibiotic c h r ~ m o p r o t e i n . ~ ~ 3.3. Protein-ligand complexes

The study of biologically active molecules bound to their enzyme targets or receptor molecules is one of the most exciting areas to which NMR has been applied. Knowledge of the bound conformation of a ligand, and particularly of the key functionalities involved in binding, are crucial to the rational design of more potent and specific drugs. With the preparation of NMR-active isotopically labelled ligands or, alternatively, deuteration of the target molecule, the signals of one species may be distinguished from the other. This, and the use of multidimensional NMR methods applied to labelled proteins, has made it possible, in some instances, to assign the signals arising from bound ligands, and to use intramolecular NOEs to determine bound conformations. In addition, NOEs from the ligand to the target molecule may be used to determine the regions of the two species which are directly involved in the intermolecular interaction. The results of such studies have provided some unexpected insights into the nature of binding processes. For example, NMR studies of the immunosuppressant drug cyclosporin A bound to its target protein cyclophilin showed that the bound conformation of this cyclic undecapeptide is completely rearranged compared to the free conformation .36,37 Likewise, NMR studies of an FK-506 analogue, ascomycin, bound to FK-506 binding protein do not correlate with the ascomycin structure derived in ~ o l u t i o n . ~ ' This may be, in part, due to the non-aqueous environment in which the

PHARMACEUTICAL APPLICATIONS OF NMR

155

10

coo-

Chorismate

Prephenate

Fig. 5. Structures of chorismate and prephenate showing the CM-catalysed reaction pathway.

structures of the free drugs were determined; but the potential of the bound conformation of a molecule to deviate from its free form is clearly evident. The studies mentioned above represent the state-of-the-art in terms of determining the complete geometry of a protein-ligand complex, and because of their importance they are described in more detail later. These types of studies are generally only feasible for tightly bound complexes. Different approaches can be adopted for weakly bound complexes or for cases where extensive isotope labelling of either the ligand or receptor is not possible. The types of experiments possible in a given system depend on the exchange regime (i.e. slow, intermediate or fast) on the time-scale of the NMR parameters being monitored. Methods for establishing these regimes have recently been reviewed in a book on practical aspects of NMR of macromo~ecules.~~ If the slow exchange condition applies (as occurs for tightly bound complexes) then separate signals are detectable for free and bound forms of both the ligand and the macromolecule, while for fast exchange (weaker affinity binding) the observed resonances from the complex are an average of those from free and bound forms. The following examples illustrate some of the techniques that are useful in the study of protein-ligand complexes. 3.3.1. N M R titrations of complexes The condition of slow exchange on the chemical shift time-scale can be conveniently illustrated by a study of the interaction between the enzyme Bacillus subtilis chorismate mutase (CM) and its substrate, chorismate, using I3C NMR spectros~opy.~~) The reaction catalysed by this enzyme involves rearrangement of chorismate to prephenate, which is one of the first steps in the biosynthesis of tyrosine and phenylalanine (Fig. 5). In the complex

156

D. J. CRAIK, K. J . NIELSEN AND K. A. HIGGINS

formed from the addition of chorismate to CM, the product, prephenate, exists in a stable form bound at the active site. Thus, upon titrating either uniformly or partially labelled [13C]chorismate with the enzyme, the corresponding labelled prephenate could be observed and distinguished from the protein signals. The CM-prephenate complex (M,44 kDa) gave rise to signal linewidths of -15Hz. As a consequence, chemical shifts could be accurately determined, although only coupling constants greater than 5 Hz were able to be measured. The assignment of the prephenate 13Csignals was carried out in a solution of CM to which substoichiometric amounts of uniformly 13Clabelled chorismate were added. Under these conditions it could be presumed that all the prephenate produced was in its bound form, Kd = 7 0 p ~ and , that the chemical shifts reflected the structure and chemical environment of the product within the active site of CM. Assignments were made on the basis of the chemical shifts expected for the various functional groups, and the relative sharpness of the carbon signals lacking directly attached protons, compared with methine or methylene 13C nuclei. The assignments were confirmed with specifically labelled [2,6,913C]- and [1,3,5,8-13C]chorismate added to CM, which allowed clear differentiation between the two sets of labelled sites. The exchange regime of the enzyme-product complex was determined by adding an excess of uniformly labelled [13C]prephenate to the enzyme. Figure 6 shows that at 25°C and 75 MHz, the signals from free and bound forms of the product are in intermediate exchange. Decreasing the temperature or increasing the frequency resulted in slow-exchange spectra in which both free and bound product signals were visible. The fact that slow exchange conditions were achieved, even with a rather moderate affinity, illustrates the value of 13C NMR. The large chemical shift range of 13C means that the resonances of free and bound states are sufficiently separated to result in slow exchange spectra. In the case of the CM-prephenate complex, the exchange rate for the complex was estimated to have a maximum value of 270/s, based on the chemical shift differences. This value is in good agreement with those derived by biochemical methods. The shifts of bound prephenate were compared with free prephenate to determine the sites at which the binding interaction caused the greatest changes. This provided information on the chemical nature of the binding site of the enzyme, and on the chemical state of the bound substrate molecule. Table 2 shows that shift changes of several parts per million were observed. Both upfield and downfield shifts were measured for the 13C signals of bound prephenate, presumably due to either general effects of the altered dielectric environment, specific protein-substrate interactions, an altered substrate conformation, or the changed protonation state of the pyruvoyl or carboxylic groups. To determine the cause of the chemical shift changes, prephenate models were examined in different solvent environ-

PHARMACEUTICAL APPLICATIONSOF NMR

200.0

180.0 PPm

157

60.0 PPm

Fig. 6. Exchange of the free (f) and bound (b) uniformly labelled [13C]prephenate. I3C spectra of the B. subtilis CM-['3C]prephenate complex were obtained under the following conditions: (A) 75MHz, 25°C (3600 scans); (B) 75 MHz, 8°C (10000 scans); (C) 150MHz, 25°C (2500 scans). The sample used in the NMR exchange studies contained 5.8 mM B.subtilis CM and 15 mM [13C]prephenate in a 5 mm NMR tube. The spectra were processed with 15Hz line broadening. (Reprinted with permission from Rajagopalan et ~ 1 . ~ )

ments and with different protonation states. This indicated that the observed shift deviations for sites on the pyruvoyl side-chain were consistent with the partial protonation of this group. Likewise, it appeared that the shift change observed for C10 could be explained by partial protonation of this group. Some coupling constant evidence also supported this hypothesis. lJclo-cll was found to increase by 5 Hz on binding, consistent with partial protonation. The largest chemical shift change occurred at C5 and C6, suggesting that the binding interaction causes a particularly strong perturbation of the C5-C6 bond. Also of interest was the non-degeneracy of the C2,C6 and C3,C5 shifts in the bound prephenate, suggesting that the environment in the vicinity of the product was asymmetrical, or conformationally restricting, resulting in non-averaged signals. In summary, this study was possible due to the purity of the enzyme-

158

D. J. CRAIK, K. J. NIELSEN AND K. A . HIGGINS

Table 2.

I3C chemical shifts of bound and free uniformly labelled ['3C]prephenate."

Carbon atom

Free

c1 c2 c3 c4 c5 C6

48.6 131.7 127.9 61.6 127.9 131.7 170.1 204.3 48.1 180.0

c7 C8 c9 c10

Bound 49.0 129.4d 129.4d 59.8 133Sd 125.1d 166.5 202.9 46.2 177.9

AJJHz'

A 4 0.4 -2.3 1.5 -1.8 5.6 -6.6 -3.6 -1.5 -1.9 -2.1

'JC7-,-8

= O+ 1

'JcScs = 4.5

'JclWl

1.5

= 5 rt 1

"Ref. 50. bChange in chemical shift. 'Change in coupling constant values between the free and bound states. dAssignment not certain.

product complex, and its stability over the time required for the NMR experiments, as well as its relatively low molecular mass (44 kDa) and slow-exchange binding kinetics. By indicating which functional groups were most affected in the bound state, it provided several clues as to the nature of the enzymatic mechanism. It was proposed that the binding site was probably asymmetrical and that the similarity of this binding site to those of other types of CM enzymes could be judged on the basis of analogous studies with labelled prephenate.

3.3.2. NOESY analysis of complexes When slow exchange conditions exist it is sometimes possible to assign NOESY spectra of macromolecule-ligand complexes and to determine the geometry of the complex via intermolecular NOEs. This approach is usually only possible for smaller complexes, and one situation where it is commonly used is in the study of drug-DNA complexes where oligonucleotides are used as models for DNA. This can be illustrated by the binding of the ligand Hoechst 33258 (4) to the oligonucleotide d(GGTAA'ITACC)2.41 An NMR study of the 1:l complex41showed that the system was in slow exchange and it was possible to obtain complete chemical shift assignments for bound ligand and DNA signals. From the pattern of chemical shift perturbations on the DNA, the drug-binding site was located in the minor groove of the central tract of four AT basepairs. Intermolecular NOEs were used to further refine the location of the ligand when bound, and to determine its orientation. NOEs between protons on the concave edge of the ligand (the curvature is apparent from the structure (4) and the DNA confirmed the close contact between this curved surface and the com-

PHARMACEUTICAL APPLICATIONS OF NMR

159

plementary shape of the DNA minor groove. Similar findings had earlier been reported42 for the same ligand with another oligonucleotide, d(GTTTGCAAAAG)*, but in this case a 2: 1 stoichiometry, corresponding to two binding sites, was observed. Several other NMR and X ray crystallographic studies of related complexes have been 3.3.3. Isotope editing One of the difficulties in the application of NOESY techniques to the structure determination of larger macromolecule-ligand complexes is that spectral assignments become extremely difficult and the ability to discriminate between the intra- and intermolecular NOEs is lost. Recently developed isotope editingK~ltering~'.~' techniques overcome both these limitations, i.e. assignments are simplified and cross-peaks from various types of protons (intraligand, intraprotein and protein-ligand) can be selectively observed. Isotope filtering makes use of heteronuclei labels in one part of the system to select 'H magnetization from adjacent protons. If uniform I3C or 15N labels are incorporated into the ligand, 'H NMR studies of the bound ligand can be done without interference from signals of the unlabelled protein. The same sorts of experiments which have traditionally been applied in 'H NMR of free ligands can be used in the case when they are bound. If the structure of the protein binding site is already known, then measurement of chemical shift changes, intermolecular NOEs, and other parameters such as slowexchange amide protons allow the orientation of the ligand to be determined. The technique may be illustrated by the binding of cyclosporin A (CsA) to cyclophilin. CsA is a hydrophobic undecapeptide which exhibits potent immunosuppressive activity and has important applications in the prevention of organ and bone marrow transplant rejection. Its activity is thought to involve the interaction of CsA with the proline isomerase protein cyclophilin (CyP), a 17.7 kDa cytosolic protein, via the inhibition of isomerase activity.

160

D. J. CRAIK, K. J. NIELSEN AND K. A. HIGGINS

Different approaches have been employed for NMR studies of the CsA-CyP complex. Weber et al.37 prepared both uniformly 13C-labelled CsA bound to CyP in D 2 0 and uniformly labelled I5N-labelled CsA bound to CyP in H20. Homonuclear 2D 'H NMR spectra of the 13C-labelled preparation were recorded with a '3C(01, 02) double half-filter,51enabling all intramolecular signals arising from I3C-bonded protons of CsA to be observed. CyP signals were thus not present in these spectra, which could be interpreted in the same manner as standard 2D 'H NMR spectra. I3C(w1, w2) doubly selected COSY and TOCSY experiments allowed many of the spin systems to be recognized. A set of '3C(w1, 0 2 ) double half-filter NOESY spectra was then used to obtain all of the intramolecular NOEs between the I3C-bonded protons of CsA in its bound state. The amide protons could be connected to their respective side-chain protons using a TOCSY-relayed [15N,'H]COSY experiment of the "Nlabelled preparation. In this experiment, 15N nuclei are correlated with their directly bonded protons. Also using the 15N-labelled preparation, a ['H, 'HINOESY experiment with a I5N(w1) half filter was recorded. This gave rise to spectra which contained only the CsA amide proton lines along wz (one of the frequency axes in the 2D spectrum) and all proton lines of CsA and CyP along w1 (the other frequency axis), so that all intra- and intermolecular NOE cross-peaks involving the "N-bound CsA amide protons were observed. These and other experiments allowed the CsA signals to be fully assigned, intramolecular NOE connectivities to be measured and 3&H.Ha coupling constants to be obtained. As well as facilitating structure calculations using distance geometry calculations, the NOEs observed between CsA and CyP were used to determine which regions of CsA were involved directly in the intermolecular interaction. The solution conformation of CsA bound to CyP was found to contain no regular secondary structure and no intramolecular hydrogen bonds. The structure was different from the crystal structure of free C S A , ~which ~ contained hydrogen bonds. The crystal structure has a cis amide bond between residues 9 and 10, whereas all of the amide bonds in CsA bound to CyP are trans. In the crystal structure of CsA all but one of the NCH3 groups are oriented towards the molecular surface, whereas in the bound CsA structure only two NCH3 groups are exposed, including one that is buried in the crystal structure. The NMR solution structure of free C S A is~ in ~ agreement with the crystal structure of free CsA, confirming that quite a drastic structural rearrangement occurs in solution upon binding to CyP. Concurrent work was performed by Fesik et al.36 using uniformly I3C-labelled CsA bound to CyP. NMR signals were assigned from an analysis of 2D 'H/I3C and 13C/13C correlation experiments, again taking advantage of the differentiation between CsA and CyP signals possible due to the I3C labelling. NOE data were measured from a '3C-resolved 3D

PHARMACEUTICAL APPLICATIONS OF NMR

161

experiments4 in which a series of 'WIH NOEs was obtained in spectral planes corresponding to directly correlated 13C signal shifts. NOEs arising from intramolecular CsA proton signals could be identified, since these NOEs appeared in two of the "C-separated planes. The remaining NOEs, therefore, resulted from intermolecular interactions between CsA and CyP, and could be used to determine the functionalities of CsA involved in binding. In a third study, by Hsu and Armitage," an alternative strategy was used to examine the CsA-CyP complex. They prepared completely deuterated CyP, and were subsequently able to run normal 'H NMR spectra of the CsA-CyP complex without interference from the non-exchangeable CyP protons. Although NH connectivities and intermolecular interactions cannot be identified using this method, a distinct advantage is the relative inexpense of preparing deuterated expressed proteins, and the more efficient data collection possible for direct 'H detection. Another advantage is that this approach is well suited to the study of a series of analogues bound to the target molecule, since specific labelling of each analogue is not required. All of these studies give rise to almost identical structures for bound CsA-demonstrating the effectiveness of each approach. Such methods can be extended to examine the structure of not only the bound ligand, but also of the entire complex, provided it is less than -30 kDa. This is illustrated by an elegant study from Fesik's l a b ~ r a t o r yon ~ ~the structure of the CsA-CyP complex. The structure, one of the largest to be determined by NMR, was solved using heteronuclear 3D NMR methods applied to uniformly labelled CSA-['~C,''N]C~P, CsA-["N]CyP and ['3C]CsA-CyP preparations. It was found that the structure of the bound CsA was essentially the same as that determined by previous methods. In addition to determining the bound conformation of ligand and receptor, the orientation of the ligand in the binding site was determined. At the same time, the X ray structure of a CsA-CyP crystal complex, solved to 2.8 A, was reporteds7 and found to be in agreement with the NMR data. Such complementary studies are extremely valuable for the complete characterization of drug-target complexes. An advantage of the X ray method is that it is applicable to larger complexes; a disadvantage is that protein packing within the crystal lattice may result in a slightly altered structure to that obtained in solution. Thus, even if an X ray structure is available, it may be important to obtain the NMR structure. 3.3.4. Transferred NOEs

In cases of lower affinity binding or where it may not be feasible to determine the complete structure of a protein-ligand complex, it is often possible to derive information about the bound conformation of a ligand using the transferred NOE (TRNOE) t e ~ h n i q u e . ~ ~ This " ' is suitable when

162

D. J. CRAIK, K. J. NIELSEN AND K. A. HIGGINS

exchange is fast on the relaxation time-scale, and it makes use of the fact that NOES build up faster in the bound ligand than in its free counterpart. Exchange of the “bound” NOE information back to the free ligand signals occurs under certain limiting kinetic conditions, allowing the readily detected free ligand signals (which can be in large excess relative to the protein) to convey information about the bound state. The technique is applicable to situations in either slow or fast exchange on the chemical shift time-scale, as long as exchange is fast on the relaxation time-scale, although applications are more common in the latter case. The appropriate theory has been described p r e v i o u ~ l y ~and ~ ’ the applicability of the technique may be illustrated by such examples as the substrates to enzyme^,"^^^ peptides binding of nucleotides to to a n t i b o d i e ~ , and ~ ~ .hormones ~~ to carrier proteins.69 4. SELECTED EXAMPLES

In the examples described above, the focus has been on illustrating how a range of new NMR techniques can be used to derive information about drugs, their receptor proteins and their complexes. To conclude this review we describe in more detail some studies on particular classes of biologically active molecules. The emphasis is on peptides, as it is widely believed that a significant proportion of new drugs developed in the future will be based on peptides or their analogues. This belief stems from the vastly increased knowledge that has accumulated over the last few years on the structures and activities of peptides discovered in living systems. While peptides are extremely potent, their application as drugs themselves is limited by their poor bioavailability when administerd orally. In addition, their flexibility means that they are capable of interacting at multiple receptor sites, potentially leading to side-effects. Thus, there is much interest in the design of novel conformationally restricted peptide analogues and peptidomimetics which resist catabolism. The starting point in the design of such analogues is the following types of structural studies. 4.1. Endothelins

Members of the endothelin (ET) family of endogenous peptide hormones are potent vasoconstrictors, originally discovered in mammalian aortic endothelial cells. They have similar biological activities and are associated with the impairment of renal and cardiovascular functions. The ET peptides exist in four isoforms (ET-1, ET-2, ET-3 and endothelin p) consisting of 21 amino acid residues with two disulfide bridges, between residues 1 and 15 and residues 3 and 11, respectively. They share high sequence identity,

PHARMACEUTICALAPPLICATIONS OF NMR

163

especially in the C-terminal region, with most substitutions elsewhere being conservative in nature (Fig. 7(A-D)). The sarafatoxins (SRTs), a group of four cardiotoxic peptides isolated from the venom of the Egyptian burrowing asp, are similar in sequence to the E T peptides (Fig. 7(E-H)) and also have potent vasoactivities, suggesting an evolutionary relationship. An extensive review on the ET peptides is given by Cheng et ~ 1 . ~ ' The genes for a number of different E T receptors have been cloned, isolated and identified. These receptors fall into three subtypes denoted ETA, ETB and ETc. ETA, localized in vascular smooth muscle, is responsible for the mediation of vasoconstrictor activity and has higher affinity for ET-1, ET-2, SRT-6211 and SRT-6b, than for ET-3, SRT-6c and SRT-6d. ETB, showing equal affinity for ET-1, ET-2 and ET-3 and for the SRT peptides, is more widely distributed than ETA, and modulates vasodilation and vasoconstriction, simultaneously. The third receptor subtype, ETc, is specific to ET-3.7' The ET receptors are G-coupled with seven transmembrane domains and belong to the rhodopsin-like receptor superfamily. The E T peptides have attracted considerable attention from the pharmaceutical industry, with a primary goal being the design of an effective ET antagonist for the treatment of angina, hypertension and acute renal failure. Cyclic pentapeptdies BE18257A and BE18257B were found to be weak but selective ET-1 antagonists, acting at the ETA receptor in millimolar c o n c c n t r a t i o n ~ . ~ Structure-activity *-~~ studies utilizing these peptides as lead compounds have led to the discovery of more potent analogues, BQ123 and BQ153, both of which are active in nanomolar concentrations and have improved solubility over the original lead^.^"'^ In the following section, the solution conformations of two of these cyclic ET-1 antagonists, BE18257B and BQ123 (Fig. S), are described. Both compounds are highly selective for the ETA receptor, but display different binding affinities (ICso = 0.47 and 0 . 0 2 2 p ~ ,respectively). Knowledge of the 3D structures of these constrained anatgonists provides a better understanding of the structural requirements of the ETA receptor, which may, in turn, aid in the future design of novel peptidic and non-peptidic E T antagonists. There have been numerous structure-activity relationship studies on the E T and SRT peptides (for a review, see ref. 70) which have shown that residues important for ETA agonism include Asp8, GlulO, Phel4, Hisl6, Leul7, Asp18 and Trp21. Aside from Leul7, these residues are conserved throughout the ET and SRT families. The greatest hypervariability in primary sequence occurs in the regions incorporated by residues 2-7. As the ET and SRT peptides with low ETA affinities have threonine at position 2 instead of serine, this site is considered to be important for ETA ~ p e c i f i c i t y Formation .~~ of a linear ET-1 analogue, by replacing all cysteine residues with alanine, significantly reduces ETA affinity, suggesting that the 3D structure of the head group region in ET-1 is important for ETA

(E) SRT

(B)ET-

Q SRT

@)Endothelin-b

O I ) SRT

Fig. 7. The primary sequences of the (A-D) ET and the (E-H) SRT peptides showing regions of sequence variability with respect to ET-1 (shaded circles) and disulfide connectivities (thick lines).

PHARMACEUTICAL APPLICATIONS OF NMR

165

t

D-allo-Ile3

H

Leu4

H a \

r.

"/H

D-Glul .*I

Fig. 8. The primary structure of the ET-1 antagonists (A) BE18257B and (B) BQ123.

166

D. .I.CRAIK, K . J. NIELSEN AND K. A . HIGGINS

s p e c i f i ~ i t y . The ~ ~ highly conserved C-terminal or tail region may be important for ETB specificity, since the ET and SRT peptides have equal affinities for this receptor subtype. Furthermore, ET analogues lacking disulfide bridges or the entire 1-8 region have affinity for ETB. Saeki et have reported that only residues 10-21 are important for effective binding to the ETB receptor. The structural requirements of the ETc receptor have not yet been identified. To elucidate structural features important in receptor binding, and sub-type discrimination, it is important to determine the 3D structures of the ET peptides. 4.1. l . 3 0 structure of ET-1 and related peptides

(i) ET-1. The solution structures of ET-1 and its various isoforms have been determined by several groups using 'H NMR spectroscopy combined with 3D structure calculations.8~9'Because of aggregation and solubility problems the structures of the ET peptides were determined under a range of solvent conditions, resulting in some minor discrepancies amongst the conclusions drawn from these studies. Despite this, the results are in general accordance, and the secondary structure of the ET peptides can be described in terms of four regions of structure which are summarized as follows. While the N-terminal region is predominantly random coil, residues 5-8 form a /3 turn, which is often found to be stabilized by a 1,4 hydrogen bond. Following this is a region of a helix stretching from residue 9 to residues 14-17, depending upon the solvent. The C-terminal region is of interest due to the differences in conformation of the terminal hexapeptides of ET-1 and ET-3. Studies on ET-3 have indicated that the C-terminal region folds back on to the bicyclic core.84,85With one exceptions2 this has not been observed for ET-1. Most studies reported an undefined conformation in this region.80T81 A recent 'H NMR study on a disulfide-deficient analogue of ET-1 in a 10%/1.S%/88.5% acetonitrile/acetic acid/H20 co-solvent system9' shows that the absence of the 1-15 disulfide bridge has little effect on the 3D structure, apart from greater conformational variability in the N-terminal region, even though this peptide has reduced affinity for the ETA receptor but equal affinity for the ETB receptor compared to ET-1. An example NOESY spectrum (Fig. 9) shows the sequential assignment and illustrates the good spectral dispersion, indicative of defined regions of secondary structure. Coles et suggest that the position of the N-terminal charge is important for ETA selectivity. In monocyclic ET-1, the p turn and a helix are well defined in the set of 3D structures. The /3 turn and a helix are evident in the representative structure shown in Fig. 10. While the C-terminal tail has no elements of classical secondary structure, some structural restraint is implied by the presence of slowly exchanging NH

PHARMACEUTICAL APPLICATIONS OF NMR

167

13 14 I1

1

4@ 18 21

I

8.6

8.4

,

8.2 F2

8.0

8

7. 8

‘6

(ppm)

Fig. 9. The NH-Ha region of the NOESY spectrum (mix = 200 ms) for monocyclic [1,15 AbaIET-1 recorded at 600 MHz ( T = 293 K) showing sequential connectivities. Intraresidue cross-peaks are indicated by residue number. (Reproduced with permission from Coles et al. ,92 copyright American Chemical Society.)

protons at Ile19 and He20 and several NOEs involving these residues. These residues appear to exist in a predominantly extended conformation despite the fact that the C-terminal region, as a whole, undergoes conformational averaging. For residue 16, low values of the angular order parameter, which measures the degree of spread for the 4-$ dihedral angles, indicates that this site may act as a hinge for the segmental motion of the C terminus. The fact that a number of NOE restraints involving Ile19 and Ile20 were violated suggests that this region of the molecule undergoes motional averaging, similar to what may be expected in native ET-1. The conclusion drawn from this was that the NOEs observed in this region could not be interpreted in terms of a single predominant c~ n fo rmer.~The ’ results of this study and others on disulfide bond replacement in ET-1 have indicated that the position of the N-terminal charge relative to the rest of the peptide as well

168

D. J. CRAIK, K. J. NIELSEN AND K. A. HIGGINS

3

Fig. 10. Stereoview of the lowest energy structure for [1,15 AbaIET-1 showing backbone atoms and the heavy atoms of the disulfide bridge. (Reproduced with permission from Coles et al. ,92 copyright American Chemical Society.)

as the nature of the residues in the region 2-7 are important for agonist activity at the ETAreceptor, but not for the ETB receptor, which tolerates a wide range of substitutions at these sites.

(ii) Sarafatoxins. Although SRT-6b aggregates at concentrations greater than 2 . 2 m ~in H20,93 it is more soluble than the ET peptides, and its solution structure in this solvent has been determined.89,94In H20, SRT-6b adopts a p turn from residues 3 to 6, followed by a region of a helix from residues 8 to 16. The identification of characteristic NOES between the side-chain protons of GlulO and amide proton of Thr7, and between the y CH3 protons of Thr7 and the amide of Lys9, suggests that Thr7 acts as the N-terminal stop signal for the (Y helix.94 A superimposition of the 43 refined structures for SRT-6b is given in Fig. 11. Residues 2-6 and 8-16 are well defined, but are connected by a region of conformational variation. The orientation of the C-terminal region with respect to the bicyclic core is not defined due to conformational variation around residues 17-18. In summary, SRT-6b has similar secondary structural features and adopts a similar 3D

PHARMACEUTICAL APPLICATIONS OF NMR

L

terminus

\

169

terminus

Fig. 11. Stereoview of the backbone atoms of the 43 final structures of SRT-6b, overlaid over residues 1-3 and 8-16. (Reproduced with permission from Atkins el al. ,94 copyright American Chemical Society).

structure to ET-1. The main difference is in the positioning of the @ turn, which is shifted two residues towards the N terminus in SRT-6b. It is not known whether this is due to solvent effects, since the ET peptides have not been studied in solely aqueous solutions. Considering the high degree of sequence identity and the conservation of the positions of the disulfide bridges, it is not surprising that the SRT peptides adopt similar 3D structures to the ET peptides.

4.1.2. Comparison of N M R and X ray structures of ET-1 Recently, the structure of ET-1 was solved by X ray crystallography to 2.1 A r e s ~ l u t i o n providing ,~~ the first example of the successful use of a partial NMR-derived model for the initial phase determination of the X ray structure. The overall structure of ET-1, as determined by X ray crystallography, consists of an N-terminal extended @ strand with a @ bulge between residues 5 and 7, followed by a hydrogen-bonded loop between residues 7 and 11, and an irregular a helix that extends from residue 9 to the C terminus. Although the overall fold of ET-1 is similar, there are significant differences in the secondary structure determined using the two different techniques. Notably, the region of greatest sequence variability (residues 5-7) and the C-terminal “tail”, which are important for the binding specificity and vasoactivity of the peptide, respectively, are the regions which show the greatest differences in secondary structure.

170

D. J . CRAIK, K . J . NIELSEN AND K . A. HIGGINS

Lee et u I . have ~ ~ made several observations which indicate that the solid state conformation is not the dominant form in the conformational equilibrium presented by monomeric ET-1 in solution. They point out that since ET-1 was crystallized from an aqueous medium, under conditions in which it would be aggregated, the resulting dimeric structure may have intermolecular hydrophobic forces that override secondary structural prop. that ~ ~ ensities in the monomeric state. To counter this, Wallace et ~ 1 claim the most extensive crystal contact interactions occur around residues 11-14, which is the part of the peptide most similar in the NMR and X ray structures. It is of significant irlterest that the C-terminal tail (residues 16-21) forms an ordered a helix in the crystalline form. Furthermore, the tail is found to be neither more flexible nor more disordered than the globular core. This represents a significant difference to what is observed by ‘H NMR spectroscopy for ET-1 and its analogues, where the C-terminal tail is thought to adopt local extended structure around Ile19-Ile20, but to undergo motion in its relative position to the bicyclic core. [Pen3,15,Nle7]ET-l, a potent agonist of ET-1, shows increased helicity, but even in this comparatively rigid analogue, the a helix is extended only to residue 17.97 This is supported by an analysis of H a chemical shifts which are highly sensitive to secondary structure, with upfield shifts reflecting a helix and downfield shifts reflecting 2, strands.‘* A comparison of the deviation of Ha chemical shifts from random coil values for ET-1 and various analogues” is given in Fig. 12. The consistent change from upfield to downfield shifts at residue 15 shows the a helix is interrupted at this site. From this it is clear that the structure of ET-1 is different in solution and in the crystalline state. These differences may be attributed to environmental effects (i.e. solvent) and to crystal-packing forces. Solvents such as H20 and DMSO, in which some of the ET peptides and analogues have been examined, are disruptive to helix formation, since they have the potential to interfere with intramolecular hydrogen-bonding interactions. Other solvents which have been used, such as ethylene glycol and acetonitrile, may be less disruptive to peptide hydrogen bonds but may not be conducive to helix stabilization. The effects of solvent may be more dramatic on the conformations of the exposed terminal regions of these peptides than on the more compact bicyclic core region. To investigate further the extent of a helix possible in the ET peptides, it would be of interest to use a solvent such as 2,2,2-trifluoroethanoI, which provides an environment that is conducive to a-helix stabilization. Interestingly, ET-1 is not predicted to adopt an a helix beyond residue 17 in aqueous or in hydrophobic environments. Figure 13 shows the results of secondary structure predictions (unpublished results) using the program ALB,99 which bases its predictions on the physical properties of the amino acids. In both environments examined, ET-1 is

PHARMACEUTICAL APPLICATIONS OF NMR

Oa60

T

171

HFlP Pen3.15 Nle7 glycol PcnY.1S Nle7

0.40

ET-1 0.20

Ala3.11.17 Nle7

0.00

-0.20

-0.40

-0.60

-0.80

1

Fig. 12. Histograms for the deviations from H a random coil values versus residue number from residues 3 through 18 for ET-1 and various analogues. The C-terminal residues were excluded from the plot since these deviations were insignificant for all of the peptides analysed. Analogues represented are, left to right at each residue number, [Pen3,15,Nle7]ET-l in 14%/43%/43% hexafluoroisopropanol/ethylene glycol/H20 (black bars); [Pen3,15,Nle7]ET-l in 50%/50% ethylene glycol&120 (hatched bars); native ET-1 in 60%/40% ethylene glycol/H20 (black bars); and [Ala3,11,17,Nle7]ET-l in 65%/35% ethylene glycol/H20 (unshaded bars). (Reproduced with permission from Lee ef ~ 1 . ~ ~ )

Sequence

Environment

1 10 20 C S C S S L M D K E C V Y F C H L D I I W

aqueous

T T T

hydrophobic

T T T T

h h h h h h h h h h h h B B B B H H H H H n n n r i n h

B B B

T-turn; h=helix probable; Hahelix definite; B=P-strand probable.

Fig. 13. The results of secondary structure predictions on ET-1 simulated in aqueous and hydrophobic environments.

172

D. J. CRAIK, K. J. NIELSEN AND K. A . HIGGINS

predicted to adopt the (Y helix from approximately residue 7 to 17, and to adopt an extended structure in the C-terminal region. The secondary structure predictions correlate more closely to the structures derived by NMR than that of the crystalline state. O n the other hand, the results of a recent alanine scan study,"' where residues 13, 14, 17 and 21 were found to be important for ET binding and activity, have been reported to be most comprehensible if the entire C terminus was helical in its biologically active form, thereby placing these residues on the same surface. While there are valid arguments for both cases, the biologically active conformation of the C terminus remains speculative. These studies do, however, provide an example of a case where the 3D structure of the solution and crystalline forms are different, thus highlighting the importance of both techniques in determining possible bioactive conformations.

4.1.3. 3 0 structure of cyclic pentapeptide ET antagonists

(i) BE18257B. The structure of the weak but highly selective ETA antagonist BE18257B has been determined recently in DMSO by 'H NMR spectroscopy. Information derived from studies of the temperature dependence of the NH chemical shifts, NOE experiments and coupling constant measurements was used to calculate a set of structures for this molecule using molecular dynamics and simulated annealing calculations. Of the 40 structures calculated, 36 satisfied the experimental restraints with a backbone RMSD (root mean square deviation) of 0.12 A. Two views of these structures are shown in Fig. 14. Two hydrogen bonds -0C-Ile3 and Ile3-NH. * -0C-Glul) stabilizing two (Glul-NH. turns, a type I1 p turn centred on Leu4-Trp5 and an inverse y turn about Ala2, were observed for this compound. To ensure that adequate conformational space was sampled, two further sets of structures were calculated, one in which no NOE or dihedral restraints were used and another in which only NOE restraints were used. An analysis of the +I) dihedral angle conformations for all of the structures in this study (Fig. 15) show that the experimental restraints have a significant impact on the quality of the final structures. This is an important consideration for small, cyclic peptides where the range of conformational space is limited by the nature of the closed backbone. The structures calculated with no experimental restraints show a wide spread of torsion angles, indicating a good sampling of conformational space. The addition of NOES results in a set of structures with +-I)dihedral angles that are clustered tightly in discrete groups for each residue. The conformational variability is reduced and the quality of the structures is further improved by the addition of the dihedral restraints.

-

PHARMACEUTICAL APPLICATIONSOF NMR

173

A

Leu 4 u.32

Na 2 Ala 2

B

I I-Glu 1

0-allo.

Leu 4

DaUo

0-Trp 5

Leu 4

D-Trp5

Fig. 14. Stereoview of the 36 final structures of BE18257B superimposed over the backbone atoms. Two views are shown (A) from the side and (B) from above. (Reproduced with permission from Coles et al. ,lol copyright American Chemical Society.)

(ii) BQ123. The 3D structure of the highly potent ETA antagonist BQ123 in DMSO and in mixed aqueous solvent systems has been determined by several groups using 'H NMR spectroscopy. 102-104 These results showed that BQ123 adopts a well-defined conformation, but there was significant variability in the side-chain orientations amongst the studies. This may be attributed either to mixed solvent effects or to true flexibility in solution.lo2 To examine the effects of solvent on the conformations of this peptide, the structures of BQ123 and its sodium salt (BQ123-h) in DMSO and in H 2 0 , respectively, have been determined recently using 'H NMR spectroscopic techniques. lo5 Figure 16 shows regions of the ROESY (rotating frame Overhauser enhancement spectroscopy) spectrum (mix = 300ms) of BQ123 in H20. The 3D structures of this peptide in both solvents were generated from NOE-derived distance restraints and restrained MD calculations using the program XPLOR.lo6 For the H 2 0 data, 66 NOE restraints were used in the calculation of the structures, while for DMSO, 73 NOE restraints were

PHARMACEUTICAL APPLICATIONS OF NMR

175

0 W N

4H71'

4HN

@ 4HO

I

1

I

8.5

8. 0

7. 5

(PPm)

I

rHt3

ina I

I

I

8.5

8. 0

7.5

Fig. 16. Regions of the ROESY spectrum of BQ123-h in H 2 0 (mix = 300111s) showing some of the key NOES responsible for fixing the backbone and sidechain conformations. (Reproduced with permission from Gonnella ef al. ,Io5 copyright 1994 Munksgaard International Publications Ltd, Copenhagen, Denmark.)

176

D. J. CRAIK, K. J. NIELSEN A N D K. A. HIGGINS

A

B

Fig. 17. Stereoviews of the backbone superimpostions of five representative structures of (A) BQ123 in DMSO and (B) BQ123-h in HzO. The positions of the side-chains are shown. (Reproduced with permission from Gonnella et a/. ,lo5 copyright 1994 Munksgaard International Publishers Ltd, Copenhagen, Denmark.)

used. Four restraints were also used to define two hydrogen bonds between sOC-D( ~ - A s p 2 - " H - * aOC-~-Va14) and between (~-Va14-"H. Asp2). The resultant structures satisfied the experimental restraints with no NOE violations greater than 0.5 A. Comparison of the DMSO and H 2 0 structures showed excellent agreement in the backbone conformations. A superimposition of the structures for BQ123 in both solvents (Fig. 17) highlights the similarity in the backbone conformation, which can be described as follows. A type I1 p turn is formed

-

PHARMACEUTICAL APPLICATIONS OF NMR

177

Fig. 18. Stereoviews of the backbone superimposition of BQ123 in DMSO and BQ123-h in H20. The backbone superimposed with a root mean s uare deviation of 0.18 A. (Reproduced with permission from Gonnella et d.," copyright 1994

Munksgaard International Publishers Ltd, Copenhagen, Denmark.) by the residues -D-Val-Leu-D-TrpD-Asp and a y turn is formed by residues -D-ASp-Prc+D-Val. Also reported is a hydrophobic cluster between the tryptophan ring and the leucine side-chain. This is consistent with previous reports of the structure of BQ123. 102-104 Figure 18 shows a backbone superimposition of the representative structures for BQ123 in DMSO and BQ123-h in H20. The low RMS deviation of 0.18 A shows that the backbone conformations are almost identical. The side-chains, however, adopt distinctly different orientations in the two solvents. This is most pronounced for the tryptophan ring, which undergoes a 180" flip in DMSO compared to that observed for BQ123-h in H20. The difference in side-chain orientation can be accounted for by differences in the observed NOES. Similarly, the side-chain orientation of the leucine residue differs in the two solvents, resulting in a closer association of the y-protons of the leucine residue to the D-Trp aromatic ring in DMSO. Based on the NOE, J coupling constant and chemical shift data, the stereospecific assignments for the &methyl groups of Leu5 could be made, indicating a well-defined side-chain conformation. In contrast, the side-chain of this residue for BQ123-h in H 2 0 is not well defined. Two sets of Leu/D-Trp conformers were obtained from the H 2 0 data, the first in which the Leu5 &methyl protons are oriented towards the tryptophan aromatic ring and the y-protons of the same residue are solvent exposed, and the second where only one of the Leu5 &methyl groups is in close proximity to the tryptophan ring.

178

D. J. CRAIK, K.J. NIELSEN AND K. A. HIGGINS

Differences in the orientation of the valine side-chain were also apparent from the NOE and coupling constant information. In H20, the 3JH,_Hp coupling constants and NOE data were consistent with a low-energy trans conformation of the Ha-HP protons. In DMSO, no preferred conformation for the valine methyl groups could be derived from the NOE data. This was supported by the 3JH,-Hpcoupling constants, which were indicative of freely rotating conformers. Molecular modelling calculations using two different solvation methods were performed to determine whether the backbone and side-chain conformations of BQ123 could be predicted. lo5While the observed backbone structure was predicted, neither method was able to reproduce precise side-chain placements. Furthermore, neither modelling study could predict the backbone conformation based on lowest energy as the sole criterion. The large number of different low-energy conformers generated from the conformational search during molecular modelling demonstrates the difficulty of predicting solution conformations a priori. The studies of the structures of BQ1231°5 and BE18257B''' demonstrated the importance of NMR spectroscopy in determining preferred solution structures and, as a consequence, reducing the number of conformations to be mimicked in the rational design of drugs. Although BE18257B and BQ123 are both ETA antagonists with high sequence identity, they have different binding affinities for the ETA receptor. It is therefore important to compare their 3D structures to make an appraisal of their structural similarities and differences. Table 3 gives a comparison of the average 4 and $ dihedral angles for the BE18257B'" and BQ1231"5 structures, both calculated from the NMR data in DMSO. The extremely low RMSD (root mean square deviation) values reported for these structures suggests that an analysis based on averaged dihedral angles is valid. Both peptides have remarkably similar backbone conformations in DMSO, although there are some local +-$ differences which occur, unexpectedly, in the region of sequence homology (i.e. at the Leu-Trp residues). The similarity in the backbone structures of these ETA antagonists combined with available activity data which varies with amino acid substitution make it possible to evaluate the relationship between the structure of these cyclic pentapeptides and their activity as ETA antagonists. There is some disagreement over which region of ET-1 these cyclic pentapeptides mimic as ETA antagonists. Satoh and Bar10w'~' proposed that, as ETA antagonists, these peptides adopt a conformation which mimics the P turn 5-8 in ET-1. The residues of this turn are hypervariable amongst the ET and SRT peptides, so that the Leu ( E T - l ) - + T r p (ET-2) and Met (ET-1) -+ Leu (ET-2)+ Trp (cyclic pentapeptides) substitutions ought to be tolerated in an antagonist, corresponding to residues 6 and 7 in ET-1 and ET-2. This is not the case, however, since substitutions of D-Phe or D-Leu

PHARMACEUTICAL APPLICATIONS OF NMR

179

dihedral angles for BQ123 (DMSO) and BE18257B (DMSO).

Table 3. A comparison of the 4J and

BQ123 Residue

4J

D-Trpl D-Asp2 Pro3

74 138

D-Val4

Leu5

-58

83 -61

BE18257B

* 5 - 108

89 - 144

143

Residue ~-Trp5 D-GM Ala2

D-allo-Ile3 Leu4

4 109 148 -74 91 -61

v 6 -97

82 - 148

103

for D-Trpl in BQ123 result in a dramatic loss of binding affinity.'"8 Substitution of D-Trpl for an alanine residue results in an inactive analogue. Another argument which would suggest that the cyclic pentapeptides do not mimic the 5-8 turn in ET-1 is that in SRT-6b, which also has a high affinity for the ETA receptor, the position of this turn is shifted by two residues compared to the ET peptides. Structure-activity studies on the cyclic pentapeptides and ET-1 support the proposal that the cyclic pentapeptides, in fact, mimic the C-terminal region. In particular, the profile of activity for the Trpl substitutions in BQ123 correlates with that of the Trp21 substitutions in the ET-1. For example, similar to what is observed for the T r p l replacements in BQ123, Trp21- Tyr and Trp21- Phe substitutions in ET-1 yield analogues with reduced ETA affinities, while the T r p 2 1 4 Ala replacement yields an inactive analogue."' If the cyclic pentapeptide antagonists of ETA mimic the C-terminal region of the ET peptides, the question remains as to why they are not also antagonists at the ETB receptor. The specificity may result from differences in the recognition sites of the receptors, which, in the case of the ETB subtype, do not allow the receptor to recognize these cyclic pentapeptides as antagonists. The structure of the antagonists BQ123 and BE18275B provides an insight into the possible conformation of the C-terminal region of the ET peptides in the membrane-bound state. While this region of the ET and SRT peptides is not well defined in solution, X ray analysis shows that the C terminus adopts the a helix in the crystalline form. If the cyclic antagonists do indeed mimic the C terminus, they are likely to mimic a folded as opposed to an extended state. The type of studies described above illustrate that solvent effects have a significant effect on the 3D structures of peptides, whether determined by NMR or X ray crystallography. In turn, this is important in rational drug design since knowledge of the range of possible binding conformations is essential in the design of conformationally restricted peptides and nonpeptidic analogues.

180

D. J . CRAIK, K. J. NIELSEN AND K. A . HIGGINS

4.2. Conotoxins

Reef-dwelling snails of the genus Conus have developed a remarkable array of peptide toxins, termed conotoxins o r conopeptides, which are major components of the venom that they inject to capture prey. Many of the conotoxins identified to date are characterized by their small size (approximately 10-31 amino acids) and high cysteine content (4-6 cysteine residues). The venoms of the different Conus species (approximately 500) are described as conotoxin "cocktails", with each constituent toxin having a specific biological activity. llo The presence of complex cocktails of bioactive peptides in Conus venoms appears to be a strategy for synergistic action.'" Conotoxins have become important tools in neurological investigations since they have high specificities for a diversity of receptor targets. They are also attracting considerable interest for their potential as sources of lead compounds in drug design applications. The conotoxins can be classified into four major classes, namely the a-, p-, 6- and w-conotoxins, on the basis of their receptor specificity, amino acid sequence, and disulfide-bonding patterns (for a review, see ref. 112). However, the continued discovery, isolation and identification of new conotoxins may lead to the definition of further classes. The a-conotoxins selectively block nicotinic acetylcholine receptors, in skeletal muscle and neuronal tissue, and are generally composed of 13-19 residues with two disulfide bridges. The p-conotoxins are 22 amino acids in length, have three disulfide bridges, and block the voltage-activated Na+ channel, which plays a key role in signal transmission along nerve and muscle membranes. The w-conotoxins are composed of approximately 24-27 amino acid residues, have three disulfide bridges and are Ca2+ channel blockers. A more recently identified class is the 6-conotoxins, which have a similar size and disulfide connectivity to the w-conotoxins but are selective towards Na+ channels, acting at a site different to that of the p-conotoxins. The conantokins are another class of peptides found in the venom of cone snails. These peptides are composed of 17-21 residues, have no cysteine residues but have a high concentration of y-carboxyglutamate (Gla) residues. The conantokins target a subset of glutamate receptors called NMDA receptors (so named for their activation by the agonist N-methyl-Daspartate, a glutamate analogue). Preliminary evidence suggests that the conantokins adopt a-helical conformations in the presence of calcium, presumably by chelation of Gla to Ca2+.'13 The disulfide connectivity patterns of the four major classes of conotoxins are given in Fig. 19. The relatively large number of disulfide linkages limit the flexibility and stabilize the 3D structure of the conotoxins, making them ideal for study by NMR spectroscopy. While there are regions of sequence hypervariability amongst the peptides of each class of conotoxins, the cysteine residues, which may play a role in

-

181

PHARMACEUTICAL APPLICATIONSOF NMR Conotoxin class

Disulfide bonding pattern

a

2-100P

- c c - - - c - - -

6

4-loop

c - - - - - - c - - - - - - c c - - - c - - - c

-

I

I

-

C

,

-

c

I

the minimum number of residues detected between cysteine residues.

Fig. 19. The disulfide connectivity patterns for the four major classes of conotoxins.

maintaining the 3D structure, are highly conserved, suggesting that the 3D structure of each toxin within a class is similar. The pattern of selectivity in receptor specificity that is observed for each of the peptides within a class may be due to the variability of the amino acid side-chains which project from a structurally conserved backbone. The concept that the backbone structure of these peptides provides the framework upon which active side-chains extend makes the conotoxins ideal prototypes in the design of novel drugs. The small size of these peptides makes them amenable to synthesis, further enhancing their application to drug research. In particular, the pharmaceutical industry has developed an interest in w-conotoxins for their potential use as therapeutic agents in the treatment of several conditions. w-Conotoxins, administered to rats subjected to temporary forebrain ischaemia, give some degree of protection to neurons from subsequent neuronal degradation. These peptides thus have potential application in the treatment of stroke victims, where there is a need to selectively block Ca2+ channels. A further potential application is in the treatment of traumatic head injury since brain cell death results, in part, from the influx of calcium through N-type voltage-gated calcium ~hanne1s.l'~ Similarly, pain-relievers such as opioids act by blocking these channels in spinal cord neurons, so there is also some scope for the use of o-conotoxins as analgesics. Currently, w-conotoxin MVIIC is being used in the immunoassay for diagnosing Lambert-Eaton myasthenic syndrome, a disorder which predominantly affects small-cell lung cancer patients, causing profound skeletal muscle weakness. Several w-conotoxins have been isolated and characterized (Fig. 20). Apart from the cysteine and Gly5 conservation, there is considerable hypervariability in the primary sequences of the w-conotoxins. However, there are some consistent sequence trends: all of the w-conotoxins are

182

D. J. CRAIK, K . J. NIELSEN AND K. A . HIGGINS

PEPTIDE

SEQUENCE

conus geographus GVIA GVIB GVIC

SVIA SVIB

conus Kame MVIIA MVIIB m11c MVIID 0-hydroxyproline

Fig. 20. The amino acid sequences of the w-conotoxins characterized from three different species: Conus geographus, C. striatus and C . magus.

amidated at the C terminus, have a net positive charge and have the disulfide-bonding pattern (1-4, 2-5, 3-6). In general, these peptides have a high percentage of hydroxylated amino acids; for example, these constitute 50% of the amino acids, including three hydroxyproline residues, in GVIA. While all w-conotoxins are effective Ca2+ blockers, their pharmacological profiles are not the same, due to their abilitiy to differentially bind Ca2+ channel receptor subtypes. Several Ca2+ channel subtypes exist, and a description of these is presented in an extensive review on Ca2+ channel diversity.'" The ability of the w-conotoxins to interact with different Ca2+ channel subtypes may be exploited in the design of Ca2+ channel blockers with highly specific effects. So far there have been few structural studies of the w-conotoxins. The isolation of sufficient quantities for structural studies is generally not feasible and the synthesis is complicated by the presence of multiple disulfide bridges. These can rearrange during oxidation to form incorrectly disulfidebonded isomers (i.e. there are 15 possible arrangements for three disulfide bridges). Peptides of this size are notoriously difficult to crystallize, rendering X ray methods unsuitable. However, the w-conotoxins are ideally suited to structure determination by NMR, and with increasing interest in these peptides and continued improvements in synthetic procedures many of their NMR structures are likely to be determined in the future. The only w-conotoxin structure available so far is for GVIA, which is described below. 4.2.1. 3 0 structure of w-conotoxin GVIA

The 3D structure of w-conotoxin GVIA has been studied by several groups using 'H NMR spectroscopy.'16-12' This peptide has a highly defined 3D

PHARMACEUTICAL APPLICATIONS OF NMR

183

,o TI I h 3

I

R17 Iv

3

r25

I I

?I i

Y

iI6

0

I 0

ui 7.0 Fig. 21. An example of the TOCSY spectrum of GVIA showing the amide proton to

aliphatic proton connectivities. The vertical lines connect the spin systems, which are shown for all residues that contain amide protons, aside from C1. (Reproduced with permission from Skalicky et a,."') structure, as demonstrated by the large range in NH and H a chemical shifts and the good peak dispersion within this range (Fig. 21). The NOE, coupling constant and slow exchange NH data show that the dominant structural feature of w-conotoxin GVIA is a short, triple-stranded p sheet comprising residues 5-8 and 17-21 as the peripheral p strands and residues 24-27 as the central p strand. The NOE data used to identify the triple-stranded p sheet are summarized in Fig. 22.lI8 Residues 17-27 are linked by a five-residue turn to form a p hairpin structure. Other secondary structural features include a type I1 p turn (residues 3 4 , a type I p turn (residues 9-12), and a chain reversal involving residues 15-18 which did not

184

D. J. CRAIK, K. J. NIELSEN AND K. A. HIGGINS

Fig. 22. Schematic representation of the triple-stranded p sheet in GVIA showing interstrand backbone NOES (double-headed arrows) and slow-exchange amide protons (circled). (Reproduced with permission from Skalicky et ~ 1 . ” ~ )

match any of the established turn types in the study by Pallaghy el al. 12” but was found by Davis et ul.“’ to adopt a type I p turn in 9/21 structures and the more unusual type VIII p turdz1 in the remainder of their structures. A set of structures for GVIA is given in Fig. 23, which shows a stereoview of ~ full relaxation the 21 refined structures calculated by Davis et ~ 1 . l ’where matrix analysis was used to generate distance restraints from NOESY cross-peak intensities. Examination of the structures shows that some regions of the peptide are more defined than others. In particular, the type I p turn 9-12 and the p hairpin turn are less defined than other regions of the peptide. The well-defined p sheet is somewhat distorted, with many of the dihedral angles deviating from those of a classic p sheet. This has been rationalized to arise from strain caused by the presence of a half-cysteine on each of the three strands of /3 sheet.”’ The presence of structural strain is not surprising considering the small size and relatively large number of disulfide bridges in this peptide. The six cysteine residues and Gly5 that are conserved in the w-conotoxins are clearly insufficient to specify function, especially since the 6-conotoxins, which target Na+ as opposed to Ca2+ channels, also exhibit these features. The role of the invariant Gly5 appears to be structural, as it is involved in a well-defined p turn. Since it has a positive 4 angle, which is easily

+I)

PHARMACEUTICAL APPLICATIONS OF NMR

c1

185

Cl

'

Y22

'

-Y22

Fig, 23. Stereoview of the backbone superimposition of the 21 refined structures for GVIA calculated by Davis et al."' The structure coordinates were obtained from the

Brookhaven Protein Data Bank under accession code lomc. accommodated by glycine residues, this conformation may be difficult to reproduce with any other residue, in this instance. Two of the disulfide bridges (1-16 and 8-19) are surface exposed and are poorly defined at the local level, while the third disulfide bridge (15-26) is at the core of the peptide and is well defined with a type I conformation'22 around the S-S dihedral angle. 12" An interesting feature of w-conotoxin GVIA is the disulfide knot, consisting of a ring formed by the two poorly defined disulfide bridges and interconnecting backbone, through which the third disulfide bridge passes. 4.2.2. Structure-activity relationships

Structure-activity studies on w-conotoxin GVIA have shown that Lys2 and Tyrl3 are important for activity and may interact with the specific amino acid residues of the N-type Ca2+ channel through hydrogen bonding and ionic interactions, respectively. 123 Tyrl3 in GVIA is not fully conserved within the w-conotoxin class, so this residue may be important for Ca2+ channel receptor subtype specificity. Prior to the identification of MVIID, it was thought that a positively charged residue at position 2 was an important feature for Ca2+ channel receptor recognition. However, since MVIID has a glutamine residue at position 2, it is clear that some sequence variation is permissible at this position'24 and, furthermore, since MVIID is not highly specific for N-type Ca2+ channels, a positively charged residue at position 2 may be important for N-type specificity.

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D . J . CRAIK, K . J. NIELSEN AND K. A . HIGGINS

The role of the positively charged arginine residues in the w-conotoxins is not fully understood. Substitution of Argl7 and Arg2.5 with alanine results in analogues with no significant change in activity. This is in contrast to p-conotoxin GIIIA, where the guanidino group of Argl3 is a key residue for binding to the Na+ channel. The major subunits of the Na+ channel have high sequence identity to those of the Ca2+ channel, apart from some residues which are essential for receptor selectivity and sensitivity. Sitedirected mutagenesis studies have shown that two glutamate residues, Glu387 and Glu945, are essential for the sensitivity of tetrodotoxin, a Nat channel blocker. These residues may also interact with the positive charges of Argl3 in GIIIA. The corresponding amino acids in N-type Ca2+ channels are threonine and glutamine, so they do not carry a negative charge. From this, and the structure-activity relationships, it has been suggested that the guanidino groups in GVIA are not crucial for binding to N-type calcium channels. 126 The role of the three ammonium groups in GVIA has been investigated using selective acetylation. 127 The positively charged N terminus is considered to be important for GVIA activity, since acetylation at this position caused the greatest decrease in potency. Acetylation of the &-amino group on Lys2 also resulted in reduced potency, but to a lesser extent, while there was little change in activity upon acetylation of Lys24. For MVIID, biotinylation of the a-amino group resulted in a 10-fold increase in the ICs0, whereas biotinylation of the sole &-aminogroup increased the ICs0 500-fold. This indicates that the &-amino group of MVIID is critical for the Ca2+ channel interaction. 128 Studies on a linear analogue of GVIA (with no disulfide bridges) have shown that this peptide assumes a random conformation and does not have biological activity.'23 This indicates that the 3D structure of GVIA is essential for the activity of w-conotoxin GVIA, and, presumably, this can be extrapolated to all of the conotoxins. As mentioned above, GVIA is the first w-conotoxin to have had its 3D structure determined experimentally. As more NMR structures of this class are solved, knowledge of the 3D structures of the different members of the w-conotoxin family will aid in the interpretation of the plethora of structure-activity data that is currently being accumulated. The availability of the coordinates (Brookhaven Protein enables a Data Bank) for some of the published structures of GVIA"9~L20 preliminary analysis relating 3D structure to function. The 3D structure of w-conotoxin GVIA shows an asymmetric distribution of polar and non-polar side-chains. The basic residues form a distinct hydrophilic, positively charged face consisting of the N terminus, Lys2, Argl7, Lys24 and Arg2.5 (Fig. 24(A)). While only the N terminus and Lys2 are considered crucial for the specificity of the peptide to N-type Ca2+ channels, the other positive charges may aid in increasing the solubility of the peptide and in presenting a positively charged, hydrophilic surface for

PHARMACEUTICAL APPLICATIONS OF NMR

A

187

B R17

Y13

K2

R25

Fig. 24. Ribbon views of one of the 21 structures of GVIA,"9 showing the positions of the (A) positively charged side-chains and the N terminus, and (B) the side-chains of the hydroxyl-bearing residues.

binding. Most of the hydroxyl-bearing residues are solvent exposed, being distributed over the surface of the peptide (Fig. 24(B)). These residues may be important for the solubility of the peptide and for forming hydrogenbonding contacts with the N-type Ca2+ channel, in vivo. On the basis of the conservation of the cysteine position and disulfide pairings, it may be expected that other w-conotoxin peptides have similar 3D structures to GVIA. However, given the sequence deletions and high sequence variability between the w-conotoxins, it would be of interest to determine the 3D structures of further members of this class. This may reveal whether there are subtle structural changes and differences in side-chain positions amongst these peptides which account for their ability to interact with different Ca2+ channel receptor subtypes. According t o Pallaghy et al.,120 an accurate picture of the receptor-binding surface of w-conotoxin GVIA must come from a systematic analysis of synthetic analogues assayed on a set of defined Ca2+ channel subtypes. Information derived from structure-activity studies may aid in the development of Ca2+ channel subtype discriminating peptidomimetic ligands.

4.2.3. Structurally related peptides One exciting aspect that has arisen from structural studies of w-conotoxin GVIA is that this peptide adopts a fold similar to other peptides that are apparently unrelated with respect to biology or functionality. lZ9 In

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D. J . CRAIK, K. J. NIELSEN AND K. A . HIGGINS

particular, GIVA has a topology similar to members of a family of plant polypeptide serine proteinase inhibitors which are found in the seeds of “squash” plants. In addition, an unusual cyclic polypeptide with uterotonic contracting properties called kalata B1, found in the leaves of the African plant Oldenlundia uffinis DC, forms a 3D structure similar to w-conotoxin GVIA.13’ While there is little sequence identity amongst these peptides, they share several features of primary structure. They are all of similar size, being of 27-29 residues in length, and have six cysteine residues which form three disulfide bridges. Most importantly, the pattern of disulfide connectivity is the same for each peptide, namely, 1-4, 2-5 and 3-6. The 3D structures in aqueous solution of kalata Bl13’ and the squash peptides, CMTI-1131-133and EETI-II,’347135have been determined from NMR data, enabling a comprehensive comparison of their structures with that of o-conotoxin GVIA.129 It is apparent from this study that these peptides display similarities at both the secondary and tertiary levels. In each peptide, a cysteine knot is formed, where the third disulfide bridge passes through a closed ring consisting of the first two disulfide bridges and the intervening backbone. The size of the cysteine knot may be quantified by the number of residues comprising the closed ring. In w-conotoxin GVIA, 12 residues form this ring, while this number is 11 and 8 for the squash peptides and kalata B1, respectively. 129 The cysteine knot formed in kalata B1 is the only true knot of the series since, being cyclic, it cannot be unravelled, unlike that of w-conotoxin GVIA and the squash peptides. Aside from the cysteine knot, all of these peptides have as their dominant secondary structural feature a triple-stranded, anti-parallel p sheet. The cysteine knot and /? sheet regions of w-conotoxin GVIA, kalata B1 and CMTI-I superimpose well in three dimensions. A side-by-side view of their global folds, shown in Fig. 25, illustrates the remarkable similarity, with each dispiaying four common structural elements in sequence. These are (1) a peripheral p strand, (2) a connecting region containing turns or a 310helix, but with no overall chain reversal, (3) the other peripheral p strand, and (4) the central p strand. These four segments are separated by three topologically important chain reversals, the positions of which are maintained in the three pep tide^.'^^ The occurrence of the cysteine knotlp sheet motif in peptides from diverse sources, unrelated in primary sequence and with different activities, suggests that it is an energetically favourable and stable structural element. This motif is likely to confer stability to the structure of these peptides, providing a compact and stable scaffold for the presentation of active residues for specific binding interactions. 1293136This exciting outcome from the structural studies suggests the possibility of designing compounds with specific biological activities which can be controlled by engineering strategically placed side-chains on to the structural platform. Already, in the case of EETI-11, this has been achieved in a elementary sense, with the replacement

PHARMACEUTICAL APPLICATIONS OF NMR

A

189

C

Fig. 25. A side-by-side view of (A) w-conotoxin GVIA, (B) CMTI-I and (C) kalata B1 in similar orientations showing the triple-stranded p sheet (ribbon) and disulfide connectivities (ball-and-stick). (Reproduced with permission from Pallaghy et al.

of specific reactive-site residues which alter the specificity of the peptide for trypsin and redirect it towards chymotrypsin or e1a~tase.l~' For novel and natural peptide inhibitors with the cysteine knotlp sheet motif, the potential range in the profile of activities invoked by the introduction of specific side-chains may be limited only by the nature and number of assays available to test biological activity. 4.3. Insulin

The introduction of insulin therapy'38 revolutionized the treatment of type I diabetes mellitus by providing a means of controlling blood sugar levels and ketoacidosis. Insulin therapy, however, is not entirely adequate, and the disease persists as a chronic disorder with associated degenerative complications. Consequently, there is considerable research aimed at improving treatments. Insulin consists of 51 amino acids divided into two peptide chains-the A chain of 21 amino acids and the B chain of 30 amino acids (Fig. 26). These chains are covalently linked by a pair of disulfide bonds between the A7 and B7 and the A20 and B19 cysteine residues. A third disulfide bond occurs within the A chain between the A6 and A l l residues. Insulin shows complex aggregation behaviour in solution. Depending on the pH, concentration and zinc content, insulin may form monomers, dimers, hexamers or aggregates of hexamers (Fig. 27). The aggregation of insulin has been investigated over a range of solution conditions. 13%14' Solvents of low dielectric constant favour dissociation by enhancement of electrostatic repulsions between the molecular units. The structure of the 2Zn insulin hexamer has been well-defined (to 1 . 2 A

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Fig. 26. The amino acid sequence of bovine insulin. In human insulin the A8, A10 and B30 residues are replaced by threonine, isoleucine and threonine, respectively, and in porcine insulin these residues are replaced by threonine, isoleucine and

alanine, respectively. resolution) by X ray crystallographic techniques. 142 Six molecules of insulin are incorporated into the rhombohedra1 2Zn insulin crystal. The hexamer is organized as three equivalent dimers related by a three-fold axis. The dimer constitutes the asymmetric unit. Perpendicular to and intersecting the three-fold axis is an approximate non-crystallographic two-fold axis. Two zinc atoms are situated on the three-fold axis, 1 7 A apart and equidistant from the approximate two-fold axis. In the crystal structure, the A chain has two helical regions from residues A2 to A8 and A13 to A20 which run nearly antiparallel and are joined by a region of extended polypeptide chain. The main secondary structural features of the B chain include a central portion of the a helix from the B9-Bl9 residues as well as a sharp turn from B20 to B23 which brings the C-terminal region Phe B24 and Tyr B26 amino acids into contact with the Leu B15 and B11 residues. This region then lies as an extended chain over the central hydrophobic core of the molecule. The dimer forms when the two monomers pack inverted relative to one another. As a result, the extended C-terminal regions of the B chains of adjacent monomers form an antiparallel P-pleated sheet structure which includes four hydrogen bonds (Fig. 28). NMR spectroscopy has been extensively used in recent years to derive information about the properties and conformations of insulin in solution.

PHARMACEUTICAL APPLICATIONS OF NMR

'

191

monomer

1

1

Fig. 27. The monomeric, dimeric and hexameric forms of the 2Zn porcine insulin crystal. (Reproduced with permission from Blundell et ul.'")

192

D. J. CRAIK, K. J. NIELSEN AND K. A . HIGGINS

Fig. 28. The extended C-terminal regions of the B chain in the 2Zn insulin hexamer forming an antiparallel /3 sheet. (Reproduced with permission from Blundell et t ~ 1 . l ~ ~ )

These studies, which may ultimately assist in the improvement of diabetic therapy, are described below. 4.3.1. ' H N M R studies In common with NMR studies of other proteins, most studies of insulin have utilized the 'H nucleus as a probe. In fact, insulin was among the first Initially the 'H NMR proteins studied using 'H NMR spectro~copy.'~~ spectrum was shown to consist predominantly of several envelopes of peaks.144 Even so, in several instances, spectral changes in these peak envelopes were used to derive information about the conformation in

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193

solution. The broadness of peaks observed in these studies can be attributed to the size of the protein and the large number of overlapped protons. These initial studies were carried out using conditions in which the hexamer and higher aggregates dominate the solution ensemble.

(i) Hexamer. NMR spectroscopy has been used to investigate various features of the insulin hexamer in solution, including conformational changes associated with different hexameric species. Early examples included studies of the 2Zn to 4Zn insulin transformation.144146 A more recent study examined changes associated with the T to R state transition in the 2Zn insulin hexamer. The R state 2Zn insulin hexamer is a new conformational variant first characterized by X ray crystallography. 147 In the R state, residues Bl-B8 of all six subunits take up a helical conformation to form a continuous region of helix from B1 to B19. This change in conformation causes each of the Zn2+ ions to adopt a tetrahedral coordination geometry that comprises three B10 His residues and a single anion or small-molecule ligand. This compares with the T state, in which residues B1-B8 form an extended conformation and the two Zn2+ ions of this hexamer each reside in octahedral ligand fields comprising B10 His residues and three H 2 0 molecules. 'H NMR spectral features have recently been used to distinguish between the T and R states of insulin.'48 The R state hexamers are induced by addition of phenol or cyclohexanol and anions, which have the effect of sharpening the aromatic region of the 'H NMR spectrum. Also evident is the appearance of new resonances between 5.0 and 6.6 ppm. These have previously been attributed to altered anisotropic ring current effects arising from the new R state subunit conformation and the bound phenol molecules. 149 The similarities between the spectra for the cyclohexanol- and phenol-induced conformations indicate that these conformations are similar. Further evidence for changes in the tertiary structure of insulin in the presence of phenol was provided by observation of a decreased rate of amide exchange in 'H NMR studies.'50 In particular, exchange rates of amide protons in the B chain helix (B9-B20) are significantly affected, suggesting either stabilization of the helix or a reduction in the solvent accessibility of the helix in the R state. For Val B18 and Tyr B16, exchange rates of the two amides decrease approximately 400-fold as a result of the ligand-induced conformational change. The conditions necessary to initiate the T to R transition for the copper substituted insulin hexamer were discussed by Brader et al. 15' NMR spectroscopy was used in conjunction with other techniques to characterize adducts of the Cu2+ species. 'H NMR studies established that, in the presence of 100 mM phenol, various Cu2+-insulin hexamers and a Cu'+ complex all possess R state structures. The effect of the addition of various metal ions on the 'H NMR spectrum of insulin has been extensively investigated. Because of the important role

194

D. J . CRAIK, K. J. NIELSEN AND K. A. HIGGINS

of the His B10 residue in metal ion coordination, this residue provides a ~ that in sensitive probe of metal ion interactions. Sudmeier et ~ l . ' ' showed the 3Cd2+ hexamer, (Ins)6(Cd2+)3,two Cd2+ are coordinated to the His B10 residue and the third is coordinated to the Glu B13 residue, while in the (Ins)6(Zn2+),(Ca2') insulin hexamer, the two Zn2+ coordinate to the His 310 residues and the Ca2+ to the Glu B13 site. 'H NMR studies show that the His B10 C2 proton resonance is shifted upfield relative to metal-free insulin. The addition of the paramagnetic shift probe Ni2+ to metal-free insulin caused changes to the spectrum similar to those produced on addition of diamagnetic Zn2+.Is3 At p H 8.610.0, spectra of solutions containing insu1in:zinc in a 6:2 ratio had signals from metal-free insulin as well as 2Zn insulin. Addition of one equivalent of Cd2+ per hexamer was found to bring about almost complete assembly of hexamers. 154 (ii) Aggregation. Insulin aggregation behaviour in solution is complex and has been extensively studied. As indicated in the previous section, metal ions have a significant effect on insulin conformation and aggregation. In particular, addition of zinc to various insulin solutions has been observed to promote spectral line broadening due to increased aggregation.'" Concentration also has a significant effect on the degree of aggregation. Roy et al"' examined the effect of high dilution on the 'H NMR spectrum of human insulin at high p H (8.0 and 9.3). A comparison of the spectrum of human insulin with that of the biologically active mutant insulin (Ser B9 + Asp) allowed the identification of conditions under which insulin is monomeric. It was noted that on dimerization, a change in the insulin conformation was reflected by changes to the spectrum. The methyl signal at 0.105 ppm (observed in monomeric insulin) shifts downfield to 0.45 ppm on dimerization. The highfield shift of the methyl signal in the monomer is due to a conformation in which the Leu methyl group is centred over and in van der Waals contact with the ring of an aromatic side-chain. Dimerization causes a conformational shift which alters this interaction. A general decrease in linewidth was observed on dissociation to the monomer, which correlates with the decrease in molecular size expected on dissociation. Studies at higher pH indicated that at a concentration suitable for 'H NMR (approx. 1 m ~ the ) 2Zn hexamer was largely dissociated to dimer at pH 10.3 and monomer at p H 1O.8.ls3 The 'H resonances of the two His (B10 and B5) residues have been the subject of several studies. 1ss*'61-166 In an early study of spectral changes ~ associated with a change of solution concentration by Bradbury et ~ l . , ' ~two C E proton ~ signals were detected. Later, improved spectral resolution allowed four separate resonances for the two His (B5 and B10) C E protons ~ to be observed.'6s It was suggested that differences which exist between the two monomers in the asymmetric unit in the crystal are retained in solution. These resonances have subsequently been used to determine the degree of aggregation of insulin in solution. 1s9,163

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195

The complex aggregation behaviour of insulin in solution, makes structural analysis using 2D methods very difficult. Consequently, one of the primary aims of many investigations of insulin has been to limit aggregation. This has been attempted using a range of solvent conditions, as well as through the use of various substituted and/or truncated i n s u l i n ~ , ' ~ ~ -as '~" described in the following section.

(iii) Native insufins. Kline and JusticelS7 attempted to minimize protein aggregation without destroying the global structure of monomeric insulin. The complete sequence specific assignments for human insulin in a 35% acetonitrile solution at p H 3.6 were reported. This solvent reduced self-association of the protein, and allowed good quality spectra to be obtained (Fig. 29). Well-established 2D NMR methods for proton resonance assignment and protein structural analysis were used.8 Analysis of the secondary structural features based on observed NOESY cross-peaks indicated that the insulin conformation seen in the crystal structure of the 2Zn hexamer is largely retained in solution. Slowly exchanging protons were observed for seven backbone amide protons and were assigned to positions A15 and A16, and to positions B15-Bl9. There was some suggestion of cis-frans isomerism of the Pro B28 peptide bond. Kline and Justice157noted the presence of a few very broad signals which were attributed to the occurrence of "multiple states that may be exchanging had previously on an intermediate NMR time-scale". Weiss et al. reported large variations in the linewidths of amide resonances in the monomer which was suggested to result from intermediate exchange among conformational substates. These substates may relate to conformational changes observed in different crystal states. Although of interest in relation to insulin dynamics, exchange-mediated line broadening limits the straightforward application of sequential 'H NMR assignment methods. Comparative analysis of the 2D NMR spectra of insulin and des-(B26B30) insulin (DPI) was used to overcome the difficulties associated with variation in linewidths.168 'H NMR spectra were acquired using insulin and DPI solutions of 80%/20% H20/acetic acid."* Although the spectra of the two proteins are very similar, that of DPI exhibits markedly less line broadening, which may be attributed to more rapid motions. Comparative analysis of insulin and DPI spectra was also used to make corresponding assignments of human insulin, which provided information regarding the environments of residues B26B30 in the insulin monomer. The results indicate that insulin consists of a stable folded moiety (DPI) with a C-terminal extension (residues B24B28) that packs as a p strand against the hydrophobic core. Some, but not all, of the interchain NOEs predicted by crystal models are absent, and this suggests flexibility within the structure. These NOEs may be quenched by motion within the protein. An additional insulin-specific sequential connectivity (Thr B28 Ha to Pro

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v c

e

0 N

E

n o n m

0 U

0

m PPm Fig. 29. The aliphatic region of the DQF-COSY spectrum of human insulin at 25°C in 35%/65% CD3CN/D20. (Reproduced with permission from Kline and J ~ s t i c e , ' ~ ' copyright 1990, American Chemical Society.)

B28 Ha2 and Ha1) is observed in the NOESY spectrum and, as expected, is absent in the spectrum of DPI. These NOES establish the geometry of the B27-B28 peptide bond as trans in accordance with crystal models. No minor cross-peaks were observed, the presence of which would indicate cis-trans isomerization of the Thr B27-Pro B28 peptide bond. Slowly exchanging amide resonances are observed for both proteins, suggesting that elements of structure are maintained on the time-scale of hours. A 'H NMR study of insulin (bovine) in 33% TFE at pH 2.4 indicated that the main secondary features identified in the 2Zn crystal structure are maintained in solution. This includes three a-helical regions; however, a

PHARMACEUTICAL APPLICATIONS OF NMR

197

turn in the B chain C terminus was also observed. Corresponding to the a-helical regions A15-Al9 and B13-Bl8, 14 slowly exchanging amide peaks were observed. Under these solution conditions there was no evidence of cis-trans isomerism of the Pro B28 peptide bond.'" Each of these studies suggests that monomeric insulin retains the major secondary conformational features that are present in the crystal structure of 2Zn insulin hexamer. There is some suggestion of multiple conformational states, based on the broadness of the amide peaks. The conformation at the C terminus of the B chain appears to vary under different solvent conditions. (iv) Truncated insulins. In DPI, the last five C-terminal residues of the B chain are removed. Consequently, DPI has a reduced tendency to form aggregates, when compared with native insulin, although it still shows full biological activity when amidated at the B chain C t e r m i n ~ s . ' ~ ~ , ~ ~ ~ Complete assignment of the 'H NMR spectrum of DPI in H 2 0 at pH 1.6-1.7 has been made and its structure subsequently Secondary structural analysis indicated that DPI maintained the same secondary structural elements as the X ray crystal structure of 2Zn insulin i.e. three a-helical regions. Relative to the crystal structure, differences were reported at the N terminus of the B chain (B1-B4).172 Interestingly, unlike other studies of insulin and DPI in various solvent^,^^^^'^^'^^ no slowly exchanging amide protons were identified in this study. This indicates a high degree of flexibility of the protein in solution. Based on observed NOES, the conformation adopted by the DPI in solution is similar to that of molecule 1 (Chinese nomenclature) in the crystal structure of 2Zn insulin. Analysis of simulated NOESY spectra, back calculated from the crystal structure of DPI, showed that the experimental NOESY spectrum is underdetermined. 168~183These calculations were used to suggest that DPI provides a model of a compact, partially folded with the possibility that conformational flexibility is necessary for reorganization of discrete protein surfaces on receptor binding. This has been referred to as the molten globule hypothesis for insulin. 176~177Insulin exhibits conformational variabi1ityl7* but with an essential difference: unlike smaller peptide hormones or their fragments, insulin contains stable elements of secondary structure which pack to define an interior and exterior. (v) Substituted insulins. (a) DPK insulin. DPK insulin contains three amino acid substitutions that alter the two main insulin surfaces involved in self-association. The His B10 -+ Asp substitution destabilizes the hexamer interface, while the Pro B28 .--, Lys and Lys B29+ Pro substitutions destabilize the dimer interface. demonstrated the feasibility of carrying out 2D NMR Weiss et al. studies on DPK insulin in aqueous solution and in the physiological pH

I I

I I

I

I

v)

0 I

V LL

0 0

I

r 0

1 I I C. DKP Insulin

-

I

I

I

1

-.

I

1

7 D. DKP

--I

1

1

1

I

I

I

I

- Insulin

-.I

2-

4 -

8

6

4

2

0

Fig. 30. (A) DQF-COSY spectrum of native human insulin. The absence of spin systems is attributed to antiphase cancellation which arises from broadened linewidths due to intermediate exchange between aggregates. The labelled cross-peaks are assigned to Tyr A19 orthoheta (a), Tyr A14 ortholmeta (b), three Thr CH/3-CH3y (c-e), Ala B14 CHa-CH3P (f), Lys B29 CH26-CH2~(g), and unassigned (h). (B-D) The DQF-COSY, TOCSY and NOESY spectra, respectively, of DKP insulin. The DQF-COSY and TOCSY spectra exhibit the expected number of amino acid spin systems. In the NOESY spectrum, the aromatic-Ha, aromatic-HP and aromatic-methyl regions are indicated (a and b). (Reproduced with permission from Weiss et al. ,179 copyright 1991, American Chemical Society.)

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D. J. CRAIK, K. J. NIELSEN AND K. A. HIGGINS

range. The 'H NMR spectrum of DPK insulin is well resolved with narrow linewidths. Figure 30 shows the improvement in spectra for DPK insulin compared with native insulin. The overall number of non-local NOESY cross-peaks is less than expected, based on the crystal structure, and in this case is attributed to local dynamics. Under the conditions used (D20 at pD 8), no slowly exchanging amide resonances were observed. The rapid kinetics of the amide proton exchange indicates global flexibility in the solution structure. Independent of solvent exchange, a large variation in the linewidths of amide resonances is observed. Similar results were previously reported for native human insulin and DPI in mixed acidorganic solvents and were ascribed to intermediate exchange among conformational substates. These results, obtained at physiological pH, suggest that motions are an intrinsic feature of the insulin fold (in the monomeric state) and are not dependent on the particular conditions used in a study. DPK insulin was 'H and 13C labelled at the C-terminal B23-B30 residues and isotope-edited 2D NOESY and 2D 13C-'H heteronuclear multiplequantum correlation (HMQC) NMR spectroscopy were used to probe the structural and dynamic features of this region of the peptide. These studies showed that the C-terminal region of the B chain remains tethered to the rest of the protein. NOESY interactions indicate that Phe B24 and Tyr B26 each pack against the hydrophobic core. The authors note that although stably maintained in the monomer, these interactions may be broken in the hormone-receptor complex,lso,'sl as suggested by some B25, B26 mutants and by the retained bioactivity of DPI. DPK insulin was also used to probe features of the insulin-receptor complex. Based on comparison of X ray crystallographic structures of insulin and studies of insulin analogues, a putative receptor-binding surface for insulin has been proposed. This includes the Gly A l , Gln A5, Tyr A19, Asn A21, Val B12, Tyr B16, Gly B23, Phe B24 and Phe B25 residues. There is considerable overlap between residues involved in the receptor binding surface and those involved in the dimer-forming surface. Many substitutions were made at the Phe B24 and Phe B25 residues, with receptor binding potency monitored and related broadly to conformational change using 'H NMR spectroscopy as one of several techniques. Figure 31 shows 1D 'H NMR spectra of human insulin, DPK insulin, Ser B25 DPK insulin and Gly B24 DPK insulin. Both human and DPK insulin show full bioactivity. As noted previously, the broad linewidths for human insulin are attributed to intermediate exchange mechanisms and longer correlation times in aggregates. The substituted DPK insulin analogues exhibit improved linewidths and chemical shift dispersion, which indicate that each is stably folded. Relative to DPK insulin, Gly B24 shows substantially less chemical shift dispersion. 2D NMR studies indicate structure unfolding with a disordered B20-B30 region.ls3 The extent of

PHARMACEUTICAL APPLICATIONS OF NMR

201

chemical shift dispersion in the Ser B25 DPK insulin is as great or greater than that of DPK insulin. Weak binding affinity for the Ser B25 insulin was attributed to global stabilization of the structure. (b) Asp B9 insulin. The charge repulsion introduced by the carboxylate anions (at neutral pH) of the aspartate side-chains in the monomermonomer interface reduces or eliminates self-association. At a pH of 1.8 or 1.9, at which these studies were carried out, the repulsive charges are neutralized and a well-defined dimer forms. Sequential assignment and determination of the secondary structural features of the Asp B9 insulin were reported. lS4The NOE data identify three a-helical domains consistent with the secondary structure of the native human 2Zn insulin crystal. Numerous slowly exchanging amide protons support these structural elements and indicate a relatively stable structure. Distance geometry calculations used in a later study showed no detectable difference between the monomers in the Asp B9 insulin dimer. lE5Some of the residues Val B12, Tyr B26 and Phe B25, which occur on the monomer-monomer interface, show considerable line broadening similar to observations made using other insulins. 157,158 The line broadening indicates that these residues exchange between different environments in the molecule, and that the solution structures undergo significant conformational averaging. (c) Gly B24 insulin. The Phe B24+ Gly insulin mutant shows near-native bioactivity.lS6 A recent CD and NMR study of this mutant provides considerable insight into the insulin-receptor i n t e r a ~ t i o n . ’This ~ ~ study highlights differences between the crystal structures of various insulin analogues and the solution structure of the bioactive Phe B24+ Gly mutant. Secondary structural analysis demonstrates the existence of three a-helices in the solution structure of the Gly B24 insulin; however, the well-defined p turn B20-B23 previously observed in crystal and solution structures of insulin is absent. 13C isotope-edited 2D NMR studies indicate disruption of contacts between the B chain P strand and the hydrophobic core. For example in the isotope-edited NOESY spectrum (Fig. 32) the Phe B25 and B26 aromatic resonances exhibit only intraresidue NOEs to the CHa and CHP. No inter-residue NOEs are observed. As a control, isotope-edited NOESY studies were also undertaken on a monomeric insulin analogue with native structure labelled at Tyr B26. Long-range NOEs between the Tyr B26 aromatic protons and the a-helical core were observed. Together with previous NMR and X ray studies on related analogues, the studies described above on the Gly B24 mutant provide a model for identifying sites of conformational changes in insulin on receptor binding. These findings are conveniently summarized in Fig. 33.1E7The two “closed” derivatives, Pro-insulin and mini-Pro-insulin exhibit no activity but have conformations essentially identical to native insulin. By contrast, the Phe B24 mutant, which exhibits high activity, differs in its conformation at the C

202

D. J. CRAIK, K. J. NIELSEN AND K . A. HIGGINS

f7

D. GlyB24 DKP

B. DKP Insulin

1.6

1.2

0.0

0.4

PPm

Fig. 31. The aliphatic region of the 500 MHz 'H NMR spectra of (A) native human insulin, (B) DKP insulin, (C) B25 Ser DPK insulin and (D) B24 Gly DKP insulin. Samples were in 50mM aqueous potassium phosphate buffer at pH 7.4 and at concentrations of approximately 10 mM. The labelled resonances were assigned as Ala B14 HP (a), Thr AS H y (b), Thr B27 and B30 H y (c), Leu B15 Hal (d), He A10 H 6 e and Leu B1.5 and HP (f). (Reproduced with permission from Shoelson et a[. copyright . 1992, American Chemical Society).

0

terminus of the B chain, suggesting that the active receptor-bound conformation of insulin may differ from the crystal structure in this region. (d) His B16 insulin. The B16 Tyr-His insulin remains monomeric in millimolar concentrations in aqueous solution at low pH while retaining 43% biological potency and folding stability. 188 The aromatic and amide resonances of the 1 D 'H NMR spectra of human insulin and selected mutants are shown in Fig. 34. Under the solution conditions used, human insulin is dimeric. The three modifications led to a progressive enhancement of the resolution of the 'H NMR spectrum. The improvement in spectral resolution is best for the His B16 insulin. The results suggest that both the His B25 insulin and DPI exhibit broad lines due to intermediate exchange between monomeric and dimeric states, while the His B16 is monomeric under these conditions.

203

PHARMACEUTICAL APPLICATIONS OF NMR 1

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Fig. 32. "C-edited NOESY spectrum of selectively labelled [I3Ca]Gly B24, [ringI3C6]Phe B25 and [ring-'3C4]Tyr B26. Non-local NOES to Val B12, Leu B15, Ile A2 or Val A3 are not observed, as expected based on the crystal structure. These resonances would be expected to appear in the boxed region. The labelled resonances are assigned as Tyr B26-orth0 to B26 H a (a), Phe B25-ortho to B25 H a (b), Tyr B26-meta to B26 HP (c), Tyr B26-ortho to B26 HP, (d), Phe B25-ortho to B25 HP1 (e and Phe B25-ortho to B25 HP2 (f). (Reproduced with permission from Hua et al. copyright 1992, American Chemical Society).

,'I7

Assignment of the 'H NMR resonances was achieved using wellestablished 2D NMR techniques.* Secondary structural features identified include two a helices in the A chain from A2 to A7 and A12 to A19 connected by an extended strand. The B chain consists of a loop Bl-B8, an a helix B9-Bl9, a /3 turn B20-B23 and an extended strand B24-B30. Observation of broad amide resonances for residues A5, A8, A9, A10, B6, B8, B9, B11 and B12 may be ascribed to the occurrence of multiple conformational states that exchange on an intermediate NMR time-scale. 4.3.2. ''C N M R studies A natural abundance 13C NMR study of insulin was reported by Bradbury and Brown in 1977.lS9In this study, the effect of methylation at the terminal and Lys B29 amine groups on the conformation of insulin in solution was investigated. From an examination of spectra of native and modified insulin, it was concluded that methylation did not produce significant structural change. The effect of solution concentration on the resonances arising from the NE-(CH& groups on Lys B29 was then used to study the degree of aggregation in solution.

204

D. J . CRAIK, K. J. NIELSEN AND K. A. HIGGINS

A. Native Insulin

B. Proinsulin ~

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Fig. 33. Schematic representation of the crystal structure of (A) native insulin, (B) pro-insulin, (C) mini pro-insulin and (D) Gly B24 insulin. Mini pro-insulin is a single-chain analogue with a peptide bond connecting B29 and Al. It is completely inactive and is proposed to represent the “closed state” of the insulin structure. The Gly B24 analogue represents the “open state” in which the hydrophobic surface of the A chain N terminus is exposed. (Reproduced with permission from Hua et al. ,IE7 copyright 1992, American Chemical Society).

The natural abundance 13C NMR spectrum of (bovine) insulin (pH 10.0) recorded using distortionless enhancement by polarization transfer (DEPT) spectral editing to separate CH, CH2 and CH3 resonances is shown in Fig. 35.”’ 13C NMR spin-lattice relaxation times and NOES were measured in order to determine the relative flexibility of different parts of the 3D structure. The 13C NMR spectrum has been assigned.lgl Observation of 13C resonances for the cis and trans forms of the Pro B28 peptide bond allowed the relative proportions of each isomer to be determined. lg2

PHARMACEUTICAL APPLICATIONS OF NMR

205

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Fig. 34. 1D 'H NMR spectra of (A) human insulin, (B) DPI, (C) His B25 human insulin and (D) His B16 human insulin. Spectra were recorded at pH 2.2-2.3 in 90% H20/10% D20 solution at 302 K. (Reproduced with permission from Ludvigsen d al. ,188 copyright 1994, American Chemical Society.)

To combat the low sensitivity and natural abundance problems associated with I3C NMR spectroscopy, some use has been made of 13C enrichment at specific sites in the protein. Led et al. used I3C-enriched potassium cyanate to modify insulin at the a-amino groups (Al, B1 and Lys B29).lg3 The resulting spectral simplification facilitated the measurement of relaxation times and NOES a t these sites. The data were used to investigate the degree

206

D . J. CRAIK, K. J. NIELSEN AND K. A . HIGGINS

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of aggregation of insulin in solution. Further, Saunders and O f f ~ r d " ~ examined the 13C NMR spectrum of insulin in which the Phe B1 was replaced with a I3C (90%)-enriched Gly residue. 13C isotopic enrichment in residues of the B chain C terminus has been used in studies of substituted His B16 and Gly B24 i n ~ u l i n s . ' ~These ~ ~ ' ~studies ~ were used to investigate the interactions of this region with the remainder of the protein. This was largely accomplished by isotope-edited 2D methods and was discussed in detail above. 4.3.3. Summary Insulin has been extensively studied using NMR spectroscopy. Studies of the insulin hexamer using both 'H and 13C NMR have focused on more general features of conformation or well-resolved resonances such as the aromatic resonances. Conformational change from 2Zn to 4Zn, and from the T to R state of the insulin hexamer, were identified using general features of the 'H

PHARMACEUTICAL APPLICATIONS OF NMR

207

NMR spectrum. Residue-specific assignments of the 13C NMR spectrum of insulin have allowed the identification of cis-trans isomerization in the hexamer. The biologically active monomeric form of insulin ( M , -6000 Da), in theory, should provide a good candidate for sequential assignment and structure determination using well-established 2D NMR techniques.' However, this has been hindered by the complex aggregation behaviour of insulin in solution. Consequently, one of the primary objectives of 'H NMR studies of insulin has been the identification of solution conditions which favour the monomer. This has led to studies of insulin under a variety of solution conditions and using various substituted and truncated analogues. Interestingly, in each case, it was found that the conformation identified from the X ray study of the 2Zn insulin is largely retained in solution. Structurally, the greatest degree of variation between these insulins is in the C-terminal region of the B chain. Broad amide linewidths identified from the 2D studies indicate that regions of insulin are particularly flexible in solution. NMR studies have been important in characterizing the dynamic nature of insulin in solution, and consequently have provided some insight into receptor binding. It appears that future studies of insulin will involve the use of a greater number of substituted insulins to further probe features of receptor binding.

ACKNOWLEDGEMENTS

The authors would like to thank Justine Hill and John Gehrmann for their substantial contributions including helpful comments, assistance with figures and manuscript preparation. We also thank Ann Atkins for her advice and Jackie Wicke for supplying material on protein-ligand interactions. Parts of section 1 and 2 of this article were adapted from reference 14, for which we thank CRC Press. The work originating in the author's laboratory was supported in part by grants from the Australian Research Council and the National Health and Medical Research Council (Australia).

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NOTE ADDED IN PROOF Since this article was written, the structures of several other w-conotoxins (in addition to GVIA reported in Section 4.2.1) have appeared in the literature. These are given in references 195-198.

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NMR Spectroscopy in Forensic Science CHRISTOPHER J. GROOMBRIDGE Metropolitan Police Forensic Science Laboratory, 109 Lambeth Road, London SE1 7LP, UK 1. Introduction 2. Misused Drugs 2.1. Amphetamines and related substances 2.2. Opiate alkaloids 2.3. Cocaine and related substances 2.4. Cannabinoids 2.5. Ergot and other indole alkaloids 2.6. Fentanyls 2.7. Phencyclidine and related substances 2.8. Quinazolinones (methaqualone) 2.9. Anabolic steroids 2.10. Miscellaneous drug substances 3. Toxicology, body fluids 4. Other forensic analysis 4.1. Hydrocarbon fuels, fire accelerants 4.2. Explosives 4.3. Lachrymators 4.4. Fingerprint reagents 5. Magnetic resonance imaging Ac knowledgements References

215 219 221 240 246 256 257 27 1 272 279 280 281 282 284 284 285 286 286 286 288 288

1. INTRODUCTION Forensic: of, pertaining to, or used in a court of law, now spec. in relation to the detection of crime.

Forensic science is a multidisciplinary activity with aspects of many types of basic science, including biology, physics, chemistry and engineering. Active laboratories are listed in the Forensic Science Society guide.' As the definition indicates, the primary purpose of forensic science is to provide evidence for courts of law, and not directly for publication, but articles in refereed journals are an important part of method validation, and individual case histories may also be recorded. Brief descriptions of some types of forensic work were given in a recent issue of Chemistry in ANNUAL REPORTS ON NMR SPECTROSCOPY VOLUME 32 ISBN 0-12-505332-0

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Copyrighl 1996 Academic Press Limited A l l rights of reproducrion in any form reserved

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The purpose of this review is to update the excellent article by Dawson6 on drug analysis, and to review for the first time the limited amount of nuclear magnetic resonance (NMR) work on other applications. The material for this review has partly been located through Chemical Abstracts but also from a cover-to-cover scan of individual journals which are aimed specifically at the forensic science profession, such as the Academy of Forensic Sciences-published Journal of Forensic Sciences. Newsletters and bulletins such as the US Drug Enforcement Administration (DEA) publication Microgram, which may only be accessible by approved forensic laboratories, are not covered here. A familiar part of forensic science involves the examination of microscopic trace evidence (e.g. see refs 3 and 4), the modern development of Locard’s principle* usually quoted as “every contact leaves a trace”. For example, this may be as little as a single clothing fibre (20pm diameter and perhaps less than 10 mm in length) from which 1-100 ng of evidential dyestuff might be extracted. Modern organic gunshot cartridges might leave less than 10 ng of discharge residue on hands and clothing when a gun has been fired. While NMR spectra are of the highest value for chemical identification, these quantities are well below the detection limit of even the highest field instrument. Chromatography techniques (especially gas chromatographymass spectrometry or GC-MS) currently dominate forensic analysis and a recent survey of forensic methods7 omitted NMR altogether. Nevertheless, there have been advances in NMR which have not yet been widely exploited. There is considerable accumulated expertise at sites such as the US Special Testing and Research Laboratory (McLean, VA) and the Drugs Directorate of Health Canada (Ottawa), but other recent publications still describe the use of continuous wave (CW) 60 MHz instrumentation. Superconducting magnets have brought increased spectral separation (Fig. 1) and improvements in sensitivity to the low-microgram range when necessary. Automatic sample changers bring an ease of use which is revelatory for those who have experienced NMR the hard way.’ Perhaps because of infrequent use of NMR, published material does not always adhere to the appropriate standards’ for recording and documenting spectra; for example, tetramethylsilane (TMS) or sodium trimethylsilylpropionate (TSP) are not always added as shift references, yet there may be errors if a solvent peak (especially water HOD) is used as the reference point. Round-robin tests have revealed a lack of consistency, even in experienced NMR laboratories.’’ This applies to a few spectra in the large published data collections, so it is important to scrutinize spectra according to the normal principles of NMR interpretation.

*Edmond Locard (1877-1966),Lyons, France. Pioneer of modern criminalistics.

Fig. 1. 'H NMR spectra of amphetamine base (1) (see Section 2.1 for structure) in CDC13: (a) 60MHz (L. V. Jones, unpublished work); (b) 400 MHz; inset-expansion of aromatic region ( C . J. Groombridge, unpublished work).

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Several ongoing technical developments in NMR have a potential relevance to forensic science, although the abiding problem is likely to be cost. Ever-better sensitivity and component separation are obviously important issues. Microprobe technology (2-3 mm capillary diameters) has been available for some time, but the interest has revived in recent years with new products from a number of manufacturers (e.g. see refs 11-13). Similarly, coupled high-performance liquid chromatography (HPLC)-NMR has developed very rapidly in a few years, such that commercial systems are now in use with several pharmaceutical companies. The potential ly a direct applications-drug metabolite^'^ or i m p u r i t i e ~ ~ ~ - c l e a rhave relevance to forensic drug analysis described below. HPLC-NMR sensitivity is, of course, not as good as alternative techniques but is often adequate, and NMR spectra are especially valuable for identifying crucial aspects of molecular structure, e.g. ring substitution pattern. Preliminary experiments on coupled capillary zone electrophoresis (CZENMR) have suggested 'H detection limits below 100 ng.16 Many formidable technical problems remain to be solved, but forensic scientists are already alert to the high separative potential of CZE. A significant factor in the success of GC-MS for compound identification is the well-developed facility for comparison with software library spectra. Extensive databases also exist for 13C NMR,I7,l8 but proton NMR is more important for forensic science and 'H databases are much less advanced, partly because of field dependence and also because of the awkwardness of coding coupled multiplets which are not first order. The large database systems are known to have some entry errors,17 so these would be used with caution, but the availability of robust software for in-house 'H database construction and searching would be useful. A neglected advantage of NMR is the potential efficiency that spectra can provide identification and quantification at the same time. Quantitative analysis by NMR was often reported up to the 1 9 7 0 ~ , ' ~and ~ * there ~ were several descriptions of forensic applications, but the number of publications has diminished in recent years. There may be a number of reasons for this: the advent of automatic sampling for chromatography has led to high reproducibility, and there is off-the-shelf software tailored for this purpose. NMR is probably still used but not thought worthy of publication; moreover, high-field NMR instrumentation is in greatest demand for the elucidation of new structures. A distinct advantage of NMR is that it is completely linear, and does not require a primary standard of the pure target analyte. This may be important for the analysis of drugs of abuse, since reference materials may not be available from commercial suppliers or in-house without lengthy preparative work. If a drug is encountered intermittently, it is potentially quicker to set up an NMR rather than a chromatography procedure.

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2. MISUSED DRUGS 0, mickle is the powerful grace that lies In plants, herbs, stones, and their true qualities; For naught so vile that on the earth doth live

But to the earth some special good doth give; Nor aught so good, but strain’d from that fair use; Revolts from true birth, stumbling on abuse: Virtue itself turns to vice, being misapplied, And vice sometime’s by action dignified. Within the infant rind of this weak flower Poison hath residence, and medicine power; For this, being smelt, with that part cheers each part; Being tasted, slays all senses with the heart. Two such opposed kings encamp them still In man as well as herbs-grace and rude will; And where the worser is predominant, Full soon the canker death eats up that plant. Friar Lawrence soliloquy; William Shakespeare, Romeo and Juliet ( c . 1595) The largest use of NMR in forensic science has been for the analysis of controlled drugs. Most countries have legislation that deals with dangerous and abused drugs, and this is partly in response to the international obligation associated with Conventions of the United Nations. In Britain, the key Act of Parliament is the Misuse of Drugs Act 1971 ( M o D A ) , ~ ’ - which ~~ has been updated a number of times in response to drug abuse trends and advice from the Advisory Council on the Misuse of Drugs. Drug control laws are, of course, only part of the stratagem for tackling drug abuse. Stockley’s bookz4 gives extensive information on substance abuse, and is superbly illustrated. Some statistics on drugs seized in Britain are summarized in Table 1. Within the MoDA, drug substances are classified (classes A , B and C) according to the perceived potential for social and self-harm, and balanced against the possible medical use. Many of the drugs listed in the Act are specifically named, e.g. cocaine or amphetamine, but others are included by generic definitions, so that the Act cannot be circumvented by changing a substituent group. In other countries (particularly the USA), the equivalent law initially controlled only specific substances, and this was then exploited since it was possible for active drug modifications to be synthesized which were not controlled, at least until the substances were identified and the regulatory schedules amended. The “designer drug” situation has occurred a number of times, particularly in the USA and Canada, and reached a critical point with analogues of

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Table 1. Seizures of controlled drugs in Britain in 1991. Total by HM Customs and

Excise, Police, and other authorities. Drug

Seized 1991

Class A

Cocaine Heroin Lysergic acid diethylamide (LSD)

Methylenedioxy-N-methylamphetamine (MDMA) Class B Amphetamine

Cannabis (herbal) Cannabis resin

1077 kg 493 kg 170 400 doses 365 100 doses

420.7 kg 9525 kg 22 676 kg

Source: Drug Misuse in Britain 1992. ISDD, London, 1993.

the opiate fentanyl (see Section 2.6). The actual designer drug term was coined in reference to fentanyls by Gary Henderson (University of California, Davis).25 This description has been re-used with more vague meaning, but strictly means the deliberate synthesis of a drug to circumvent legislation. Designer modifications continue to be encountered for amphetamines, synthetic opiates, and phencyclidines. Much forensic drug analysis is routine, with the repetitive identification of a few substances. There are a number of ways by which identifications can be made, with GC-MS as currently the most favoured method. However, the analytical scheme must be capable of distinguishing between isomeric and other closely related substances, and this is inherently a strong feature of 'H NMR because of spin-spin coupling. There are instances, for example of amphetamine variations, where MS fragmentation patterns and infrared (IR) spectra show only subtle differences, whereas 'H spectra are unambiguously different (see Section 2.1). Abused drugs are loosely of two types: those of natural origin (or semisynthetic alterations) such as cocaine or heroin, and those which are fully synthetic (e.g. amphetamine or phencyclidine). The synthetic substwhich ances are mostly prepared in some form of clandestine varies in sophistication from pharmaceutical standard to crude kitchen cookery. It is occasionally the task of forensic chemists to investigate laboratory scenes,2G28 and to compile evidence from seized materials (solvents, precursors, reaction mixtures and residues). We have found that NMR spectra can be helpful for identifying precursors and intermediates, since pure standards may not readily be available for comparison. A major theme in forensic analysis is the information that can be gleaned

NMR SPECTROSCOPY IN FORENSIC SCIENCE 1 c f

Si g .

DRTHr I 3 H G - C R L 2

B

221

0

D

cl L

Fig. 2. GC impurity profiling: typical amphetamine Leuckart material (see Section 2.1 for structures). A, amphetamine; B, 4-methyl-5-phenylpyrimidine(21); C , N-benzylamphetamine; D, di-(P-phenylisopropy1)amine (23); E, N-methyl-di-(Pphenylisopropy1)amine;F, N,N-di-(P-phenylisopropy1)formamide(24). (Reproduced with permission from King et a/.%)

from the impurities which remain (often at very high level) in illicit drugs. These impurities may be route-specific, and thus suggest lines of enquiry for police investigation. This has similarities with work which is done in the pharmaceutical industry in order to monitor patent infringement. It has also been shown that impurities can be profiled using GC (e.g. see ref. 29), giving a pattern which enables drug samples to be linked to a common clandestine laboratory or procedure. Profiling (also termed chromatography impurity signature profile analysis, CISPA) was first suggested by StrOmberg3' for amphetamine, and has subsequently been used for many other drug types. An example is shown in Fig. 2. Identification of key impurities is an important part of the validation of profiling, and has usually best been accomplished by the combination of MS and NMR. NMR spectra have been reported for most controlled drugs (e.g. see ref. 31), but this review will concentrate on those substances (mostly class A) where there is clandestine synthesis, or where there has been research into impurity characterization. Some of the earliest forensic NMR work was targeted at barbiturates, but this will not be reviewed again here.

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2.1. Amphetamines and related substances

Phenethylamines are a large class of compounds which have appeared as drugs of abuse progressively since the 1960s. The most common substances are amphetamine (1) and methylamphetamine (2), which are stimulants; chronic use may cause aggression and psychosis. Other compounds have a spectrum of pharmacological properties, with often a degree of hallucination or distorted perception. Most notable are several methylenedioxyamphetamines, also known as “Ecstasy”. In Europe, amphetamine is common but methylamphetamine is unusual; in contrast methylamphetamine (“meth”) has been dominant in North America, Japan and Australia.

Both substances have medical uses and are legitimately manufactured, but the supply of illicit drug comes from clandestine laboratories which produce material of variable purity. Forensic chemical analysis of these drugs requires the distinction between substances which are closely related (often isomers), and between similar compounds which are not harmful and thus not controlled, e.g. ephedrine. Such compounds may have mild stimulant properties, and often occur mixed with amphetamines, or may be sold as “lookalike” fake illicit amphetamine. It is common to make an identification by comparison with a collection of reference standard substances, which have been examined by a full range of techniques, including IR and NMR spectroscopy. This analytical profile approach has given rise to a series of detailed publications from Japanese laboratories (Table 2). Most of the phenethylamines are of synthetic origin, but the earliest known of these drugs was the hallucinogen mescaline (class A) which comes from the small peyotyl cactus, Lophophora williamsii or Anhalonium lewinii. Mescaline was also produced synthetically in the 1960s, but has faded from the current drugs market. More recently there has been a problem with a further natural product, khat (Cutha edulis, see below). Early forensic drug identification usually depended upon chromatography and IR spectrometry, while the use of NMR was barred by sensitivity. However, ’H NMR spectra were reported as drug abuse expanded in the 1960s, and new legislation was introduced throughout the world. This included data for a m ~ h e t a m i n e ~ ’ -and ~ ~ methylamphetamine.3540 The influence of fast internal rotation upon phenethylamine spectra was also appre~iated.~~’~~,~~,~’

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Table 2. NMR spectra of amphetamines. Compound

Spectrum

Ref.

Amphetamine Amphetamine Amphetamine Amphetamine Amphetamine

'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 90 MHz, lanthanide shift reagents 'H 90 MHz 'H270 MHz 'H 200 MHz, chiral reagent 'H 100MHz, I3C 'H 60 MHz, chiral reagent 'H 60 MHz 'H 90 MHz 'H 400 MHz, chiral reagent 'H 400 MHz, chiral derivative

Warren (1970)" Wright (1973)* Smith (1976)' Kram (1977)d Nakahara (1978)'

Amphetamine Amphetamine Amphetamine Amphetamine Amphetamine Amphetamine Amphetamine Amphetamine Amphetamine Amphetamine Amphetamine Methylamphetamine Methylamphetamine Methylamphetamine Methylamphetamine Methylamphetarnine Methylamphetamine Methylamphetarnine Methylamphetamine Methylamphetamine Methylamphetamine synthesis mixture Methylamphetamine Methylamphetamine Methylamphetamine N-Ethy lamphetamine Dimet hylamphetamine Dimethylamphetamine N-Hydroxy-MDA (MDA = 3,4- methylenedioxyamphetamine) N-formyl-MDA MDA MDA 3,4-MethylenedioxyN-methylamphetarnine (MDMA) 3.4-MethylenedioxyN-ethylamphetamine (MDEA) N-Cyano- MDA N-Hydroxy-MDA MDA MDEA MDEA MDA MDMA

lnoue (1978)' Makriyannis ( 1981)g Wainer (1981)* Alm (1982)' Liu (1982)' Ali (1983)k Miyata (1984)' Mori (1990)" Mori (1991)" '3c Gee (1991)O '3C Border (1993)p 'H 6OMHz Warren (1970)" 'H 90 MHz, "C Royer (1975)* 'H 60 MHz Barron (1974)' ' H 60 MHz Kram (1977)d 'H 60 MHz Kram (1977)s 'H 90 MHz, LSR Nakahara (1978)' Inoue (1978)' 'H 90 MHz I3C Alm (1982)' 'H 90 MHz Miyata (1984)' 'H 90 MHz Liu (1982)' 'H 400 MHz, chiral derivative Mori (1991)" 'H 200 MHz, chiral reagent Kram (1992)" 'H 400 MHz, chiral shift reagent LeBelle (1995)" Shimamine (1992y 'H 300 MHz 'H 90 MHz, LSR Nakahara (1978)c 'H 60 MHz Sekine ( 1 9 8 7 p ' H 300 MHz 'H l00MHz 'H 100MHz 'H60 MHz

Shimamine (1993)' Lukaszewski (1978)y Lukaszewski (1978)y Bellman (1970)'

'H 60 MHz

Bailey (1975y

'H 60 MHz

Braun (1980)'"' Braun (1980)bb Braun (1980)'* Avolio (1985)cr Noggle ( 1986)dd Shimamine (1993)" Shimamine (1990)Shimamine (1990)"

' H 60 MHz 'H 'H 'H 'H 'H 'H

60 MHz 60 MHz, chiral reagent 60 MHz 300 MHz 300 MHz 300 MHz

224

C. J. GROOMBRIDGE

Table 2.-cont. Compound

Spectrum

Ref.

S-Methoxy-3,4-MDA 3-Methoxy-4,5methylenedioxyamphetamine MDA

'H 300 MHz

Shimamine (1990)c'

'H 300MHz 'H 90 MHz, I3C 'H 90MHz, I3C 'H 100MHz '~60~1-h 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H~~MHZ 'H 400 MHz 'H 400 MHz

Tanaka (1991)4 Soine (1983)= Soine (1983)" Yamauchi (1986)hh Noggle (1987)" Noggle (1987)" Noggle (1987)" Noggle (1987)" Noggle (1987)" Noggle (1987)" Tanaka (1988yj Tanaka (1988yj

'H 400 MHz

Tanaka (1988y'

2,3-Methylenedioxyamphetamine MDMA (base and HCI) MDEA N-propyl-MDA N-IsopropyI-MDA N-Butyl-MDA N-Isobutyl-MDA N-Neobutyl-MDA MDA MDMA 3-Methoxy-4-hydroxy-MDA (metabolite) 3-Methoxy-4-hydroxy-MDMA (metabolite) MDA MDMA MDEA N-propyl-MDA N-Isopropyl MDA N,N-dimethyl MDA (MDDMA) N-Hydroxy MDA MDMA Ephedrine Pseudoephedrine Phenmetrazine Ephedrine Pseudoephedrine Phenolpropanolamine Phenolpropanolamine Mescaline Phentermine Amfepramone Ephedrine Norephedrine 2,3-Dimethoxyamphetamine 2,4-Dimethoxyamphetamine

2,5-Dirnethoxyamphetamine 2,5-Dimethoxyamphetamine 3,4-Dimethoxyamphetamine 3,5-Dimethoxyamphetamine 2-Methoxyamphetamine 3-Methoxyamphetamine 4-Methoxyamphetamine 2-Methylamphetamine

'H 'H 'H 'H 'H 'H 'H 'H

400 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz

'3c

'H 'H 'H 'H 'H 'H 'H 'H

60 MHz 60 MHz 60 MHz 60 MHz 60 MHz 60 MHz 60 MHz 60 MHz

'3c '3c

'3c

'3c 'H 'H 'H 'H 'H 'H 'H 'H

60 MHz 60 MHz 60 MHz 60 MHz 60 MHz 60 MHz 60 MHz 60 MHz 'H 60 MHz 'H 60 MHz

Tanaka (1988yj Dal Cason (1989p Dal Cason (1989p Dal Cason (1989p Dal Cason (1989)kk Dal Cason (1989)kk Dal Cason (1989)" Dal Cason (1989y Renton (1993)" Portoghese ( 1967yrn Portoghese (1967ym Portoghese (1967)mm Warren (1970)" Warren (1970)" Warren (1970)' Bailey (1970)"" Bellman (1970)' Alm (1982)' Aim (1982)' Alm (1982)' Alm (1982)' Bailey (1971)"" Bailey (1971)" Bailey (1971)"" Shaler (1972p Bailey (1971)"" Bailey (1971y Bailey (1974)qq Bailey (1974)qq Bailey (1974)qq Bailey (1974)qq

NMR SPECTROSCOPY IN FORENSIC SCIENCE

225

Table 2.-cont. Compound

Spectrum ~~~

3-Methylamphetamine 4-Methylamphetamine Mescaline

'H 60MHz 'H 60 MHz

Selegiline (Deprenyl) N-Cy anomethylamphetamine (methylamphetamine pyrolysis)

'H 250 MHz

'H~OMH~ 4-Methyl-2,5-dimethoxyamphetamine (STP) 'H 60 MHz IH, I3C

2,5-Dimethoxy-4-methyl-amphetamine 'H 300 MHz 2,5-Dimethyl-4-bromoamphetamine 'H300 MHz 'H 300 MHz 2.3-Dimethyl-4-ethylamphetamine Cathinone 'H 300 MHz Cathinone 'H 400 MHz, chiral Norephedrine 'H 400 MHz, chiral Cathine 'H 400 MHz, chiral Merucathinone 'H 400 MHz, chiral Merucathine 'H 400 MHz, chiral N-methyl-1-(I ,3-benzodioxol-5-yl)2-butanamine (MBDB) 'H 80 MHz 1-(1,3-benzodioxol-5-yl)-2-butanamine (BIIB) 'H 80MHz Substituted BDBs 'H 80 MHz 'H 80 MHz N-Formyl MDA MBDB 'H 250 MHz, "C Cathinone 'H 400 MHz, "C N-methylcathinone 'H 400 MHz, I3C 4-Bromo-2,5-dimethoxyphenethylamine'H 60 MHz Ephedrine (precursor) 'H 200 MHz Pseudoephedrine (precursor) 'H 200 MHz Chloroephedrine (intermediate) 'H 200 MHz Chloropseudoephedrine (intermediate) 'H 200 MHz cis-l,2-Dimethyl-3-phenylaziridine (impurity) 'H 200 MHz trans- 1,ZDimethyl-3-phenyl aziridine (impurity) 'H 200 MHz N-Formylmethylamphetamine 'H 60 MHz N-Acetylmethylamphetamine 'H 60 MHz N-Propionylmethylamphetarnine 'H 60 MHz N-Cyanomethylamphetamine (pyrolysis product) 'H 270 MHz 2,4,5-Trimethoxyamphetamine 'H400 MHz 2,5-Dimethoxy-4-ethoxyamphetamine 'H 400 MHz 2,5-Dimethoxy-4-propylamphetamine 'H 400 MHz 2,5-Dimethoxy-4methylthioamphetamine 'H 400 MHz 4-Hydroxy-N-ethylamphetamine (metabolite) 'H Methylcathinone (Ephedrone) 'H 300 MHz

Ref. ~

Bailey (1974)¶¶ Bailey (1974)qq Ono (1979)"

Ono (1979)" Podhnyi (1992)Ito (1994)" Shimamine (1989)"" Shimamine (1989)"u Shimamine (1989y Shimamine (1992)'" Dawson (1994)"" Dawson (1994)"y Dawson (1994)"" Dawson (1994)"" Dawson (1994)"" Nichols (1986)": Nichols (1986)"" Nichols (1986)"" Nichols (1986)"" Azafonov (1990)" Togawa ( 1994)yy Togawa (1994)yy Ragan (1985)" Allen ( 1987yaa Allen (1987)""" Allen (1987)'"u Allen (1987)""" Allen (1987)aU' Allen (1987)aU' Sekine ( 1 9 8 7 p b Sekine ( 1987)bbb Sekine (1987)bbb Sekine (1987)bhb Foster (1992)"" Foster (1992)" Foster (1992)"' Foster (1992)"" Makino ( 1989)ddd Zhingel ( 1 9 9 1 y

226

C. J . GROOMBRIDGE

Table 2.-cont. Compound

Spectrum

Ref.

Methylcathinone 'H 400 MHz, chiral shift reagent LeBelle (1995)" Ephedrine 'H 400 MHz, chiral shift reagent LeBelle (1995)" Pseudoephedrine 'H 400 MHz, chiral shift reagent LeBelle (1995)" Cat hinone 'H 60 MHz Berrang ( 1 9 8 2 p N-Nitrosomethylamphetamine 'H Ando ( 1983ygb: Ephedrine 'H 90 MHz Miyata (1984)' p-Hydroxymethylamphetamine 'H 90 MHz Miyata (1984)' Di-(P-phenylisopropy1)amine (impurity) 'H 300 MHz Huizer ( 1985)hhh 4-Bromo-2,5-dimethoxyamphetamine 'H unspecified Delliou (1983)"' 4-Methylaminorex (U4Euh) '€4 60 MHz Davis (l988j'j' trans-4-Methylaminorex 'H 300 MHz, "C Klein (1989)kkk 'H 300 MHz, I3C Klein ( 1 9 8 9 p cis-4-Methylaminorex 4-Methylaminorex 'H 80 MHz, I3C By (1989)"' 'H 300 MHz, I3C Brewster (1Y91)m"'"' Aminorex 2,5-Dimethoxy-4-ethoxyamphetamine 'H 80 MHz, "C (HCI and base) + precursors By (1990)""" N-Cyanomethyl-Nmethylphenethylamine 'H 270 MHz Sekine (19YO)""" N-Cyanomethyl-N-ethylbenzylamine 'H 270 MHz Sekine (1990)""" 4-Bromo-2,5-dimethoxyamphetamine 'H 60 MHz Shulgin ( 1971).pp 4-Benzylpyrimidine 'H 60 MHz Van der Ark (1Y77)qqq 4-Methyl-5-phenylpyrimidine 'H 60 MHz Van der Ark (1977)yyq 2-Benzyl-2-methyl-5-phenylpyrid-4-one 'H 60 MHz Van der Ark (1977)'" 2,4-Dimethyl-3,5-diphenylpyridine 'H 100 MHz, 13C Van der Ark (1978yIs 2,6-Dimethyl-3,5-diphenylpyridine 'H 100 MHz, I3C Van der Ark (1978)115 4-Methyl-5-phen yl-2(pheny1methyl)pyridine 'H 100 MHz, 13C Van der Ark (1978)"" 2-Methyl-3-phenyl-6Van der Ark (1978)"' (pheny1methyl)pyridine 'H 100MHz, I3C 2.4-Dimethyl-3-phenyl-6'H 100MHz, I3C Van der Ark (1978)"' (phenylrnethy1)pyridine 2,4-Dihydroxy-l,5-diphenyl-4methylpent-1-ene 'H 100, I3C Huizer ( 1981)""' Di-(P-phenylisopropy1)amine 'H 300 MHz Huizer (1985)""" Ephedrine 'H 90 MHz, LSR Nakahara (1978)' Methylephedrine 'H 90 MHz, LSR Nakahara (1978)' Phentermine 'H 90 MHz Nakahara (1978)' Mephentermine 'H 90 MHz Nakahara (1978)' Methoxyphenamine 'H 90 MHz Nakahara (1978)' Dimethoxy-methylthioamphetamines 'H 60 MHz Jacob (1977)""" 2,5-Dimethoxy-4-methyIamphetamine I3C TI study Weintrub (1980)Benzphetamine 'H 90 MHz Niwaguchi ( 1982)yyy 4-Hydroxyamphetamine 'H 90 MHz Niwaguchi ( 1982)yyy 4-H ydroxymethylamphetamine 'H 90 MHz Nigawuchi ( 1982)yYy 3,5-Dimethoxy-4methylthioamphetamine 'H 80 MHz Jacob ( 1981)zzz 4-Met hoxyphenethylamine I3C T I Makriyannis (1978)"" Makriyannis (1978)"" 4-Methoxyamphetamine I3C TI

NMR SPECTROSCOPY IN FORENSIC SCIENCE

227

Table 2.-c:ont. Compound

Spectrum

"C TI 3,4,5-Trimethoxyamphetamine 3,4-Dimethoxy-a-ethylphenethylamine I3C T I 'H 400 MHz 4-Propoxyamphetamine 'H 400 MHz 4-(2-Hydroxypropoxy)amphetamine 4-Ethoxy-2,5-dimethoxyamphetamine 'H 80 MHz 2,4,5-Trimethoxyamphetamine 'H 80 MHz 4-Ethyl-2,5-dirnethoxyamphetamine 'H 80 MHz 4-Chloro-2,5-dimethoxyamphetamine 'H 400 MHz 23-D imethoxy-4methylthioamphetamine 'H 400 MHz 4-Ethoxyamphetamine 'H 80 MHz, I3C 3-Ethoxyamphetamine 'H 80 MHz, I3C 2-Ethoxyamphetamine 'H 80 MHz, I3C 4-Ethoxyamphetamine 'H 400MHz, l3C 3-Ethoxyamphetamine 'H 400 MHz, I3C 2-Ethoxyamphetamine IH 400 MHz, "C 2,5-Dimethoxy-4-ethylamphetamine 'H 60 MHz, chiral reagent Methoxy-N-meth ylamphetamines 'H 90 MHz Dimethoxy-N-methylamphetamines 'H 90 MHz Trimethoxy-N-meth ylamphetamines 'H 90 MHz 4-Me thoxyamphetamine '3C 2,5-Dimethoxyamphetamine '3C 2,5-Dimethoxy-4-methylamphetamine13c isc Trime thoxyamphetamines Methox y-N-methylamphetamines 'H 60 MHz Bromodimethoxyamphetamines 'H 60 MHz Dimethylamphetamines 'H 60MHz I 3 c Methoxyamphetamines Dimethoxyamphetamines '3C Trimethoxyamphetamines '3c Aryl-methyl amphetamines '3C Aryl-dimethyl amphetamines '3c i3C Trimethoxyamphetamines 1 3 c Methoxyamphetamines Dimethoxyamphetamines '3c N-H ydrox yamphetamine 'H 3,4-Dimethoxy-N-hydroxyamphetarnine'H N-H ydroxyamphetamine 'H 90 MHz N-Hydroxy-4-bromoamphetamine 'H 90 MHz N-Hydroxy-4-nitroamphetamine 'H 90 MHz N-Hydroxy-2,3-dimethoxyamphetamine 'H 90 MHz 2.5-Dimethoxy-4methylamphetamine (STP) 'H I 3c Iodo-methoxy-amphetamines Di-( 1-phenylisopropy1)formamide 'H N-Methyl-N-(a-methylphenylethyl) formamide 'H 60 MHz Di-( 1-phenylisopropy1)methylamine IH 60 MHz

Ref. Makriyannis (1978)"" Makriyannis (1978)"" Foster (1993)bbbb Foster (1993)bbbb Dawson (1987)"" Dawson (1987)'" Dawson (1987)'" Dawson ( 1989)dddd Dawson (1989)dddd By (1991)eeee By (1991)eeee By ( 1 9 9 1 y By (1991)ffff By (1991)fm By (1991)ffff Hatzis (1987)gggg Clark ( I 9 8 4 p h h Clark ( 1984)hhhh Clark (1984)hhhh Knittel (1981)"" Knittel (1981)"" Knittel (1981)1'1' Knittel (1981)"" Bailey (1975)"" Bailey (1976)"" Bailey (1977)kkkk Bailey (1983)'" Bailey (1983)"" Bailey (1983)"' Bailey ( 1981)mmmm Bailey ( 1981)mmmm Bailey (1981)"""" Bailey (1981)O""" Bailey ( 1981)0000 Beckett (1975)pppp Beckett (1975)pppP Mourad (1985)qqqq Mourad (1985)qqqq Mourad (1985)qqqq Mourad ( 1985)4qqq Martin (1968)"" Dawson ( 1 9 9 4 p Kram (1979)"" LeBelIe (1973)"""" Bailey (1974)""""

228

C. J. GROOMBRIDGE

Table 2.-cont. Compound

Spectrum

Ref.

N-Formyl methylamphetamine Amphetamine oxime Methyl phenyl aziridine Nitrostyrene precursors Nitrostyrene precursors Nitrostyrene precursors Nitrostyrene precursors MDA impurities MDMA impurities Methylamphetamine impurities 2-Acetoxy-1-phenyl-I-propene I-Benzyl-3-methylnaphthalene

'H 80 MHz, 13C I3C 13C 'H 80 MHz

Dawson (1989)"""" Hawkes (1974)x**x Landow ( 1974)yyyy By (1990)"" Dawson (1991)""""" Dawson ( 1993)bbbbb Bailey (1981)"'cf Bohn ( 1993)ddddd Bohn (1993)ddddd Tanaka (1992)'""' Forbes ( 1992pff Cantrell ( 1988)ggggg

~~~~~

1 3 c

I3C I3C 'H 200 MHz 'H 200 MHz 'H 300 MHz, I3C 'H 300 MHz 'H 300 MHz, I3C ~

~

~

~~~

R. J . Warren, P. P. Begosh and J. E. Zarembo, J. Assoc. Off. Anal. Chem., 1970, 54, 1179. G. E. Wright, Tetrahedron Lett., 1973, 1097. R. V. Smith, P. W. Erhardt, D. B. Rusterholz and C. F. Barfknecht, J. Pharm. Sci., 1976. 65,412. T. C. Kram and A. V. Kruegel, J. Forens. Sci., 1977, 22, 40. Y. Nakahara and T. Niwaguchi, Kagaku Keisatsu Kekyujo Hokoku, Hokagaku Hen, 1978,31, 267. T. Inoue, T. Niwaguchi, T. Niwase and Y. Matsumura, Kagaku Keisatsu Kekyujo Hokoku, Hokagaku Hen, 1978, 31, 261. A . Makriyannis and J. Knittel, Tetrahedron Lerr., 1981, 22, 4631. I. W. Wainer, L. C. Schneider and J. D. Weber, J. Assoc. Off Anal. Chem., 1981, 64, 848. I S . A h , B. Bomgren, H. B. BorCn, H. Karlsson and A. C. Maehly, Forens. Sci. h., 1982, 19, 271. J. H. Liu and J. T. Tsay, Analyst, 1982, 107, 544. A. R. E. N. 0. Ali, Ind. J. Chem., 1983, 22B,762. Y. Miyata and H. Ando, Kugaku Keisutsu Kekyujo Hokoku, Hokagaku Hen, 1984, 37, 215. A. Mori, H. Shiyama, H. Akita, K. Suzuki, T. Mitsuoka and T. Oishi, Xenobiotica, 1990, 20, 629. A. Mori, I. Ishiyama, H. Akita and T. Oishi, Nippon Hoigaku Zusshi, 1991, 45, 1. A. Gee and B. LBngstrom, Acta Chem. Scand., 1991, 45,431. C.L. Border, D. J. Craik and B. P. Shehan, Magn. Reson. Chem., 1993, 31, 222. R.-J. Royer, P. Granger, M-J. Royer-Morrot and F. Humbert, Eur. J . Toxicot., 1975, 8,74. ' R. P. Barron, A. V. Kruegel, J. M. Moore and T. C. Kram, J. Assoc. Off. Anal. Chem., 1974, 57, 1147. ' T. C. Kram, J. Forens. Sci., 1977, 22, 508. ' J. H. Liu, W. W. Ku, J. T. Tsay, M. P. Fitzgerald and S. Kim, J. Forens. Sci., 1982, 27, 39. T. C. Kram and I. S. Lurie, Forens. Sci. Int., 1992, 55, 131. " M. J. LeBelle, C. Savard, B. A. Dawson, D . B. Black, L. K. Katyal, F. Zrcek and A. W. By, Forens. Sci. Int., 1995, 71, 215. " M. Shimamine, K. Takahashi and Y. Nakahara, Eisei Shikensho Hokuko, 1992, 110, 67. M. Shimamine, K. Takahashi and Y. Nakahara, Eisei Shikensho Hokoku, 1993, 111, 66. Y T . Lukaszewski, J. Assoc. Off. Anal. Chem., 1978, 61, 1978. ' S. W. Bellman, J. W. Turczan and T. C. Kram, J. Forens. Sci., 1970, 15, 261. K. Bailey, A. W. By, D. Legault and D. Verner, J. Assoc. OH.Anal. Chem., 1975, 58, 62. " U. Braun, A. T. Shulgin and G. Braun, J. Pharm. Sci., 1980, 69, 192. cc J. Avolio and R . Rothchild, Spectrosc. Lett., 1985, 39, 604. dd F. T. Noggle, J. DeRuiter and M. J. Long, J. Assoc. Off. Anal. Chem., 1986, 69,681. a

'

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229

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ff

OBa

"'

230

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A. M. Van der Ark, A. Sinnema, J. M. Van der Toorn and A. M. A. Verweij, Pharm. Weekblad, 1978, 113, 341. uuu H. Huizer, A. B. E. Theeuwen, A. M. A. Verweij, A. Sinnema and J. M. Van der Toorn, J. Forens. Sci. SOC.,1981, 21, 225. """ H. Huizer, H. Brussee and A. J. Poortman-van der Meer, J . Forens. Sci., 1985, 30, 427. www P. Jacob, G . Anderson, C. K. Meshul, A. T. Shulgin and N. Castagnoli, J. Med. Chem., 1977, 20, 1235. xxx H. J. R. Weintrub, D. E. Nichols, A. Makriyannis and S. W. Fesik, J. Med. Chem., 1980, 23, 339. y y y T. Niwaguchi, T. Inoue and S. Suzuki, Xenobiotica, 1982, 12, 617. "* P. Jacob and A. T. Shulgin, J. Med. Chem., 1981, 24, 1348. Oaaa A. Makriyannis and J. Knittel, QuaSAR Quantitative Structure Acriviry Relationships of Analgesics, Narcotic Antagonists, and Hallucinogens, (ed. G . Barnett, M. Trsic and R. E. Willette). NIDA Res. Monogr., 1978, 22, 464. hhbh B. C. Foster, D. L. Litster, H. S. Buttar, B. A. Dawson and J. Zamecnik, Biopharm. Drugs Dispos., 1993, 14, 709. cccc B. A. Dawvon and H. W. Avdovich, Can. SOC. Forens. Sci. J . , 1987, 20, 29. dddd B. A. Dawson and G. A. Neville, Can. SOC.Forens. Sci. J . , 1989, 22, 195. eeec A. W. By, R. Duhaime and B. A. Lodge, Forens. Sci. Znr., 1991, 49, 159. jfffA. W. By, B. A. Dawson, B. A. Lodge and J. Zamecnik, Can. SOC.Forens. Sci. J . , 1991, 24, 131. ggRg A. Hatzis and R. Rothchild, J. Pharm. Biomed. Anal., 1987, 5 , 119. hhhh C. R. Clark, J. Forens. Sci., 1984, 29, 1056. "" J. Knittel and A. Makriyannis, J. Med. Chem., 1981, 24, 906. 1"' K. Bailey, D. R. Gagnt, and R. K. Pike, J . Assoc. Off. Anal. Chem., 1976, 59, 1162. kkkk K. Bailey, D. R. GagnC, D. Legault and R. K. Pike, J. Assoc. Off. Anal. Chem., 1977, 60, 642. IN' K. Bailey and D. Legault, Org. Magn. Reson., 1983, 21, 391. rnrnrn", K. Bailey and D. Legault, Anal. Chim. Acta, 1981, 123, 75. nnnn K. Bailey and D. Legault, J. Forens. Sci., 1981, 26, 368. Oooo K. Bailey and D. Legault, J . Forens. Sci., 1981, 26, 27. p p p p A. H. Beckett, K. Haya, G. R. Jones and P. H. Morgan, Tetrahedron, 1975, 31, 1531. q444 M. S. Mourad, R. S. Varrna and G. W. Kabalka, J. Org. Chem., 1985, 50, 133. "" R. J. Martin and T. G. Alexander, J. Assoc. Off. Anal. Chem., 1968, 51, 159. "''B. A. Dawson, D. B. Black, W.-W. Sy and K. Graham, Magn. Reson. Chem., 1994, 32, 557. '"' T. C. Kram, J. Forens. Sci., 1979, 24, 596. M. LeBelle, M. Sileika and M. Romach, J. Pharm. Sci., 1973, 62, 862, """" K. Bailey, J . G. Boulanger, D. Legault and S. Taillefer, J . Pharm. Sci., 1974, 63, 1575. wwww B. A. Dawson, H. W. Avdovich, W.-W. Sy, G . A. Neville and L. D. Colebrook, Can. J. Spectrosc., 1989, 34, 107. xxxx G. E. Hawkes, K. Herwig and J. D. Roberts, J . Org. Chem., 1974, 39, 1017. yyyy S. R. Landor, 0. 0. Sonola and A. R. Tatchell, J . Chem. SOC., Perkin Trans. 1, 1974, 1294. ZLZz A. W. By, B. A. Lodge, W.-W. Sy, J. Zamecnik and R. Duhaime, Can. SOC.Forens. Sci. J . , 1990, 23, 91. B. A. Dawson, A. W. By and H. W, Avdovich, M a p . Reson. Chem., 1991, 29, 188. hhhhh B. A. Dawson, A. W. By and H. W. Avdovich, Magn. Reson. Chem., 1993, 31, 104. cccc K. Bailey and D. Legault, Org. Magn. Reson., 1981, 16, 47. ddddd M. Bohn, G . Bohn and G. Blaschke, lnt. J. Leg. Med., 1993, 106, 19. eeeee K. Tanaka, T. Ohmori and T. Inoue, Forens. Sci. Int., 1992, 56, 157. mfiI. J. Forbes and K. P. Kirkbride, J. Forens. Sci., 1992, 37, 1311. gggR.gT. S. Cantrell, B. John, L. Johnson and A. C. Allen, Forens. Sci. Znt., 1988, 39, 39. 'If

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231

The description of methylamphetamine identification by Royer et ~ 2 1 is. arguably typical of the utility of NMR in forensic science: the identification of a substance which routine methods indicate to be unusual. This is also the earliest use of 13C NMR for amphetamines. Pioneering NMR work on the characterization of ring-substituted analogues was done by Bailey, and on synthesis impurities by Kram and Kruegel. There was also some early work in which employed shift reagents in order to increase the spectral separation of mixtures, since illicit material is typically multicomponent, making an individual drug substance difficult to observe at low field. and methylamphetamine38345were added 13C data for later, and 13C NMR became more important with the appearance of substituted phenethylamines. The basic principles of 13C NMR were also described by Alm et al. ,45 with suggested applications to phenethylamine samples, including quantitative analysis. The first synthetic hallucinogenic amphetamine, 2,5-dimethoxy-4methylamphetamine (“DOM” or “STP” (3)) was observed in 1967, and brought the earliest use of NMR to identify a new CH,O

I

CH,b (3) (DOM, STP)

A systematic investigation of methoxy- and methyl-substituted amphetamines was given by Bailey, initially with ‘H ~ p e c t r a then ~ ~ - 13C ~ ~ data,5457 which led to a detailed examination of 13C aromatic additive shift factors-a possible means of anticipating the spectral appearance of unknown drugs, and thus avoiding lengthy synthesis programmes. Methoxy group conformation (in or out of aromatic plane) effects on 13C shifts were also Several other 4-substituted variants of 2,5-dimethoxyamphetamines,(4)(8), have also appeared, mostly in the USA and Canada. NMR has mainly been used to confirm structures suggested by MS and IR, and particularly to identify the ring substitution The highly potent 4-bromo-2,5 dimethoxyamphetamine (“DOB”, 6 ) was prevalent in Britain during 19931994. An additional phenethylamine analogue (“2C-B”) has also been identified. The ring substitution pattern is important for a full identification of such

~ ~

232

C. J. GROOMBRIDGE

(4)

R = Et

(5)

R =

CI

(6) R = Br

(7) R = OEt

CH,d

(8) R = SCH3

drugs, but this can be difficult to accomplish. Information can be obtained from IR or MS, but NMR can give a definitive solution, provided that the identity of any ‘H-silent group (Cl, Br, I, etc.) is determined by other means. The 16 possible 2,4-5-trisubstituted permutations are reduced to three because of the unresolved small ’JHH,then additional NMR experiments distinguish between these. Dawson et u1.58,59 have shown that lanthanide shift b e h a v i o ~ or r ~nuclear ~ ~ ~ ~ Overhauser effect (NOE) difference methods” can be used to prove substituent placement. 3,4-Methylenedioxyamphetamine(MDA) (9) also appeared in America in the 1960s, followed by the N-methyl analogue 3,Cmethylenedioxy-Nmethylamphetamine (MDMA), c. 1972) (10). There was an upsurge in MDMA use in many countries in the 1980s associated with a change in drug culture, which also saw the name “Ecstasy” adopted for MDMA. This term tends also to be used for any of this class, (9)-(12). These are reportedly mood and perception altering but are not strongly hallucinogenic akin to lysergide (LSD). There is evidence that these substances may cause a permanent change to brain transmitters (i.e. neurotoxicity).62 Proton NMR spectra were given for MDA by Bellman et d 3 and other NMR data have been progressively added (see Table 2). The isomer 2,3-methylenedioxyamphetaminehas also been examined64in order to eliminate any misidentification, or an “isomer defence”. ‘H NMR spectra at 90 MHz of 2,3-methylenedioxyamphetamine and MDA were found not to give good discrimination,64 but this is undoubtedly no longer true with higher-field instruments.

(9) R

= H

(MDA)

(10) R = Me

(MDMA)

(11) R = Et

(MDEA)

(12) R = OH

NMR SPECTROSCOPY IN FORENSIC SCIENCE

233

Proton spectra of MDA (9) and N-hydroxy-MDA (12) are but show significant shift differences. The hydroxy proton resonance was unobservably broad, in contrast to earlier measurements for Nhydroxyamphetamine, dimethoxyamphetamines and others (broad resonance 6 5.7-6.3);68,69 the hydrogen bond-breaking dimethylsulfoxide (DMSO) might be a better solvent choice. Presumably, double nitrogen substitution such as N-hydroxy-N-methyl-MDA will give rise to diastereoisomers, but there is not yet a report of any spectroscopic data.

A further structural variation has recently been noted, with the appearance of N-methyl-l-( 1,3-benzodioxo1-5-y1)-2-butanamine(“MBDB”) (13) in Europe. The first alert to this drug came from Germany,70 and tablets have been noted in London since early 1995. Discrimination between this substance and the isomeric 3,4-methylenedioxy-N-ethylamphetamine (MDEA) or N,N-dimethyl-MDA (MDDMA) can be made by careful examination of mass spectra; by contrast, the ‘H NMR spectra are very different7’ (Fig. 3). Synthesis and some ‘H NMR had first been described by Nichols ef a1.,71 but there has also been ‘H and 13C spectroscopic data reported by Azafonov et al.72 In British law, this is a class A controlled substance under the generic definition within the MoDA. Recent developments have seen the appearance of further stimulant phenethylamine substances, synthesized from phenylpropanolamine or ephedrine. Methylaminorex (“U4EuH”) (14) emerged in USA and Canada in 1987, and was identified by a combined spectroscopic approach.73 Subsequently, the existence of cis-trans isomeric forms was demonstrated using ‘H/I3C NMR and IR; mass spectra are virtually i d e n t i ~ a l . ~ ~ . ~ ’ Aminorex (15) also appeared in about 1990, and was similarly identified.76

(14)

RI, R2 = Me, H

(15)

R1, R2

= H, H

233

C. J. GROOMBRIDGE

(33XCH3 'CH,CH,

6.5

6.0

PPM

I 6.5

6.0

PPM

Fig. 3. 'H NMR spectra (400MHz, hydrochlorides in CDC13) of (a) MDEA (11) and (b) MBDB (13). ( C . J . Groombridge, unpublished work.)

Methylcathinone ("ephedrone" or "Jeff") was first encountered as a street drug in the Soviet Union ( c . 1982),77and a collaborative description was given by Zhingel et This substance subsequently started to appear in the USA. Optical isomer analysis has also been reported by LeBelle et

NMR SPECTROSCOPY IN FORENSIC SCIENCE

235

There has been growing concern over the increase in abuse of drug from . ~ ~is a shrub or small tree which is native to the plant khat, Catha e d u l i ~This east Africa and southern Arabia (notably Somalia and Yemen). It is cultivated, and the leaves then used for chewing, yielding an amphetaminelike stimulation which may give comparable problems with chronic consumption. The harvested leaves lose potency in a few days, but rapid air transport has meant that this has been appearing in Europe, USA and Canada.79Use of khat within immigrant communities has not been a serious social p r ~ b l e m , ’but ~ there may be some use as a “club” drug. Reputedly, many tonnes are now shipped through London each year. Legal control of khat has been implemented in the USA and Canada. The MoDA in Britain does not specifically name khat (unlike coca leaves, which are class A), but the two principal components are class C drugs (cathinone (16), cathine (17)).

NMR shift reagent characterization of cathinone stereoisomerism was and Rothchild,*’ and has been expanded with described by Berrang et d.*’ highly detailed studied NMR studies of alkaloid mixtures by Dawson et al. ,** including the newly identified khat substances merucathinone (18) and merucathine (19).

236

C. J. GROOMBRIDGE

Oddly, a-phenethylamine (l-phenylethylamine) has also been appearing in amphetamine seizures in Britain since 1993. This is not a controlled substance, and is widely used as an organic reagent. The individual stereoisomers are marketed as chiral reagents (ChiraSelect, Fluka). 'H data are given by Mills and Rober~on,~' but with a shift reference error; Aldrich library spectra83 are correct. Stereoisomerism of pharmaceuticals is of rapidly increasing importance for commercial production, and although clandestine chemists are not particularly concerned with this aspect, this does impinge on forensic analysis. Measurement of enantiomeric excess may indicate the use of a particular synthetic route or precursor (e.g. methylamphetamine from ephedrine). In a few instances, drug legislation specifically controls a single drug enantiomer, and forensic scientists may then carry out a selective identification. A specific forensic problem exists in the USA where a proprietary nasal decongestant (Vicks Inhaler) and other over-the-counter products contain the non-stimulant (R)-(-)-methylamphetamine. In Britain, this product contains menthol and other ingredients, but no methylamphetamine . Chiral chromatography techniques are now well developed, and are particularly appropriate for repetitive analysis, but numerous NMR procedures are also in Industrial use of NMR is common, but is generally not published. Arguably an NMR method is easier and quicker when such an analysis is performed intermittently, and is also valuable for calibration and validation of HPLC or GC. Rothchild has produced a succession of NMR investigations of drugs using lanthanide shift reagents. Various chiral shift reagent studies have been published for amphetamine and methylamphetamine; these have mostly used lanthanide reagents (see Table 2). As yet, there have been no publications concerning the use of cyclodextrins for amphetamines, although ephedrine and some other phenethylamines have been examined.86 Amphetamines are most often taken orally, but it is known that methylamphetamine is also ingested by smoking, and this may enhance the speed and intensity of the euphoric effect. Some work on the pyrolytic decomposition has consequently been carried out in J a ~ a n . ' ~ - 'NMR ~ was used in conjunction with IR and MS to identify several pyrolysis products, including the novel N-(cyanomethyl)-N-methylamphetamine.89 As previously mentioned, amphetamine and its analogues are mainly produced by clandestine synthesis. A great deal of forensic effort has focused on identifying the reactions used, primarily through the detection of route-indicative impurities which persist in the often crude illicit product. In Britain, amphetamine seizures have been routinely subjected to GC profiling at the Drugs Intelligence Laboratory (Fig. 2) .90 This information may then contribute to police seizure of the clandestine laboratory, and forensic scientists will then examine paraphernalia found at the scene. This

NMR SPECTROSCOPY IN FORENSIC SCIENCE

237

may mean an analysis of precursors and general solvents or reagents, reaction mixtures, product or trace residues. NMR has played a significant part in establishing the structures of synthesis impurities, although there is a recent trend towards the use of GC-MS exclusively. There are many potential synthesis routes described in the general scientific literature, but only a few are believed to be in regular use: (a) Leuckart, (b) reductive amination, (c) oxime, and (d) nitrostyrene. Comprehensive reviews of impurity chemistry have been given by Verweij (amphetamine91.92and methylenedio~yamphetamines~~) and Allen et ul. (amphetamine/methylamphetamine) .94 For amphetamine and methylamphetamine, routes (a)-(c) use a common precursor phenyl-Zpropanone (P2P, or benzyl methyl ketone, BMK). In consequence, P2P was itself made a controlled substance in the USA in 1980. The synthesis of this precursor has subsequently become the target of clandestine activity.” The Leuckart route was extensively studied in the 1970s, for amphetamine in the Netherlands, and “meth” in the USA. The N-formyl intermediate (20) will often carry over into the final product, and is thus Leukart route-indicative. The equivalent N-methyl compound has been studied more extensively by NMR95in order to understand the restricted C- N rotation.

Several heteroaromatic compounds were isolated by Van der Ark et ul.96-100using preparative thin-layer chromatography, and identified using combined MS, ‘H and 13C NMR; substances with a proton ortho to a ring nitrogen were distinctive because of the normal downfield shift ( 8 8-10), 4-Methyl-5-phenylpyrimidine(21) was found to be prominent in illicit Leuckart amphetamine.96,100 The Netherlands laboratory later noted a switch to amphetamine

238

C . J. GROOMBRIDGE

production by reductive amination, and reported"' a non-nitrogenous compound (22) which had been produced by an early unsuccessful attempt. Identification presented quite an awkward spectroscopic problem, because of keto-enol interconversion, leading to the disappearance of some proton signals due to solvent deuterium exchange. Verweij has continued to examine impurities from reductive a m i n a t i ~ n , ~ ~ and also to study methylenedioxyamphetamine imp~rities,'~but this has used MS fragmentation patterns exclusively. A further major Leuckart impurity has been reported in a number of studies: the dimeric di-(P-phenylisopropy1)amine (DPIA) (23). Huizer et ul.'02 have pointed out that this is diastereoisomeric, as can be seen from 'H NMR, and that there are 'H shift differences from amphetamine. Kram'03 noted that DPIA will undergo a further N-formylation to give (24), and that this had earlier been misidentified from mass spectrometric evidence. Very recently, Bohn et d 1 0 4 have described extensive work on the isolation of MDA and MDMA impurities, together with MS and 'H NMR identification. Three routes (Leuckart from ketone or carboxylic acid, and H

I

NMR SPECTROSCOPY IN FORENSIC SCIENCE

239

reductive amination) were studied. Leuckart impurities included pyridines and pyrimidines together with the dimer (analogous to DPIA). Reductive amination also produced this dimer. Work in the USA has concentrated more on methylamphetamine, which is prevalent in that region. Direct 'H NMR examination of unseparated methylamphetamine product was considered by Liu,'" but (90 MHz) spectral separation was not capable of identifying the components. This may be considerably improved with higher-field NMR, but capillary G C separation remains unquestionably inherently better. An evaluation of HPLCNMR would be interesting. Noggle and Clark (e.g. see ref. 106) have embarked upon an extensive series of experiments on amphetamine/methylamphetaminemissing MDA synthesis impurities. Use of NMR was mentioned only briefly.'" The nitrostyrene route seems to be more common for ring-substituted amphetamines, and spectral data (lH and 13C) for an extensive number of precursors (25) have been reported. 108-111 H

Clandestine chemists have sought to continue to obtain P2P indirectly, such as by synthesis from phenylacetic acid or allylbenzene. In consequence, forensic laboratories have begun to examine the appropriate chemistry, including some NMR characterization. Two procedures for P2P preparation from phenylacetic acid were examined in great detail by Allen et ~ 1 . NMR ' ~ ~ characterization was mentioned, but without any data. P-Acetoxy-Pmethylstyrene isomers have also been tentatively identified by Forbes and Kirkbride. '13 Several methylamphetamine syntheses use ephedrine as the precursor, so that bulk ephedrine supply has also become regulated in the USA. Two main routes from ephedrine are through the iodo and chloro intermediates and reduction. The abundant literature on reductions has been reviewed by Allen and Cantrell.'14 Kishi et ~ 1 . ~ identified " some HI/red phosphorus impurities, and gave 'H NMR of one: l-phenyl-2-(methylamino)propan-lone. Subsequent w ~ r k ' ' ~ , "has ~ brought modern two-dimensional 'H and 13C-'H techniques to bear for structure elucidation. Aziridine impurities (26) are also known from the chloro intermediate work of Allen and Kiser.'"

240

C . J. GROOMBRIDGE

H

WrCH3 O 'H

Similar amphetamine preparative reactions are possible from an oxime intermediate (27), which also give aziridine by-products. 119~120Oximes are also metabolites of amphetamines, and are formed spontaneously by N-hydroxyamphetamines in alkaline solution. NMR has been used very occasionally for metabolite identification. Nine benzphetamine metabolites were noted by Niwaguchi et al. 12' Two new metabolites of N-ethylamphetamine were synthesized by Makino et al. ,122 2,5-dimethoxy-4-methylthioamphetamines ~ l f o x i d e ' ~and ~ 4-(2-hydroxyprop~xy)amphetamine~*~ were prepared by Foster et al. Finally, Mori et al. 125*126 have demonstrated that some intestinal bacteria are capable of reducing 1-phenyl-2-nitropropane to amphetamine, although with low efficiency. 'H NMR with a chiral shift reagent was used to determine optical purity. 126 2.2. Opiate alkaloids

Opium is the dried viscous exudate from the unripe seed capsules of the opium poppy (Papaver sornniferurn) or other poppy species, which are grown in many countries. Untreated opium contains a mixture of alkaloids, and is still occasionally seen in forensic laboratories, but it is the chemically treated form, heroin, that constitutes the main opiate abuse problem. Morphine (28) is the principal opium alkaloid, constituting perhaps 1-15% by weight, and over 40 other components have been identified, most significantly codeine (29), thebaine (30), papaverine (31) and noscapine (narcotine) (32). These substances and their acetylation derivatives feed

NMR SPECTROSCOPY IN FORENSIC SCIENCE

CH,O

241

OH

OCH,

I

cH30x% CH,O

bCH,

(32)

through into illicit heroin, and the impurity profile can provide evidence for sample comparison, as discussed for other drug types. NMR has been used to characterize many of these substances (Table 3). The heroin chemical derivatization is a simple acetylation reaction, so that the morphine becomes diacetylmorphine (diamorphine) (33). The name “Heroin” was originally the trade name for this substance when it was introduced in 1898 by Bayer Pharmaceutical as a powerful o r “heroic” new analgesic, then thought to be less addictive than morphine. Illicit heroin samples are also often diluted with a range of other substances, particularly caffeine, diazepam, paracetamol, methaqualone and

242

C . J. GROOMBRIDGE

Table 3. NMR data for opiate alkaloids. ~~~~

~

_

_

_

_

_

~

~~

Compound

Spectrum

Ref.

Morphine Codeine Diamorphine 6-0-Monoacetyl morphine Dihydrocodeine Thebaine Dihydromorphine Dihydrocodeinone Diamorphine Diamorphine HCI Thebaine Diamorphine (base and HCI) Diamorphine HCI Diamorphine HCI Morphine 03-Monoacetylmorphine 06-Monoacetylmorphine Diamorphine Codeine Thebaine Morphine Nalorphine Oxymorphone Naloxone Codeine Acetylcodeine Thebaine Codeine Thebaine Morphine

"C I3C 13C

Carroll (1976y Carroll (1976)" Carroll (1976)" Carroll (1976)" Carroll (1976)" Carroll (1976)" Carroll (1976)" Carroll (1976)" Allen (1983)' Chazin (1986)' Chazin (1986)' Cook (1985)d Allen (1985)' Neville (1987)f Neumann (1981)x Neumann (1981)g Neumann (1981)g Neumann (1981)g Terui (1975y Terui (1975)h Glasel (1985)' Glasel (1985)' Glasel (1985)' Glasel (1985)' Okuda (1964)' Okuda (1964)' Okuda (1964)' Riill (1963)k Riill (1963)k

I3C

13C

'T

Isoheroin a-Isoheroin P-Isoheroin y-Isoheroin Thebaine Meconine (noscapine metabolite) Morphine

I3C I3C 'H 200 MHz 'H 400 MHz 'H 400 MHz 'H 250 MHz, I3C 'H 200 MHz 'H 500 MHz 'H 90 MHz 'H 90 MHz 'H 90 MHz 'H 90 MHz I3C I3C 'H 100 MHz and 600 MHz 'H 100 MHz and 600 MHz 'H 100 MHz and 600 MHz 'H 100 MHz and 600 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 100MHz and 600MHz, I3C Glasel (1981)' 'H 200 MHz, I3C Muhtadi (1988)m 'H XOMHz, 13C Hifnawy (1988)" 'H 200 MHz, I3C Al-Yahya (1982)" 'H, I3C Wyatt (1981)p 'H 200 MHz, I3C Muhtadi (1981)q 'H 200 MHz, micelle binding Yushmanov (1994)' 'H 90 MHz Beazley (1985)' 'H 90 MHz Medina (1989)' 'H 90 MHz Medina (1989)' 'H 90 MHz Medina (1989)' 'H 100 MHz Eppenberger (1968)' 'H 90 MHz Tsunoda (1979)" 'H 150 MHz, l3C Brown (1983)w

Impurities Cyanoheroin A'5.'6-Didehydroheroin A'6.'7-Dehydroheroin

'H 200 MHz 'H 200 MHz 'H 200 MHz

Morphine Papaverine Noscapine Diamorphine Codeine Papaverine

Allen (1983)b Allen (1983)' Allen (1983)'

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243

Table 3. cont. Compound N-acetylnornarcotine (2)-N- Acetylanhydronornarceine (E)-N- Acetylanhydronornarceine (IR,9S)-l-Acetoxy-N-acetyl-l,9dihydroanhydronornarceine

Spectrum

Ref.

'H 200 MHz 'H 200 MHz 'H 200 MHz

Allen (1984)x Allen (1984)x Allen (1984)x

'H 200 MHz

Allen (1984)"

'H 200 MHz

Allen (1984)"

'H 200 MHz 'H 200 MHz 'H 200 MHz

Allen (1984)x Allen (1984)" Moore ( 1984)y

'H 200 MHz 'H 200 MHz 'H 200 MHz

Moore (1 984)y Moore ( 1984)y Moore (1984)y

'H 250 MHz, 13C 'H 250 MHz, I3C 'H 250 MHz, I3C

Cook (1985)d Cook (1985)d Cook (1985)d

(1R.9R)-l-Acetoxy-N-acetyl-l,9di hydroanhydronornarceine

(E)-3-[2-(2-N-methylacetamido)ethyl)4.5-methylenedioxy-6methoxyphenyl]acrylic acid N-Acetylnorlaudanosine A''.l6-Dide hydroheroin

15-HFB-A1s~'6-dihydroheroin (HFB = heptafluorobutyrate) Noscapine 4-HFB-A3.4-didehydronoscapine Pyrolysis products 6-O-Monoacet ylmorphine N.6- Diacetylnormorphine N- Acetylnorheroin

F. I. Carroll, C. G. Moreland, G . A. Brine and J. A. Kepler, J . Org. Chem., 1976, 41, 996. A. C . Allen, J. M. Moore and D. A. Cooper, J . Org. Chem., 1983, 48, 3951. W. J . Chazin and L. D. Colebrook, J . Org. Chem., 1986, 51, 1243. C. E . Cook and D. R. Brine, J . Forens. Sci., 1985, 30, 251. ' A. C . Allen, D. A. Cooper and J. M. Moore, J . Forens Sci., 1985, 30, 908. G . A . Neville, I. Ekiel and I. C. P. Smith, Magn. Reson. Chem., 1987, 25, 31. g H. Neumann and G. Vordemaier, Archiv. Kriminol., 1981, 167, 33. Y. Terui, K. Tori, S. Maeda and Y. K. Sawa, Tetrahedron Left., 1975, 2853. ' J. A. Glasel and H. W. Reiher, Magn. Reson. Chem., 1985, 23, 236. S. Okuda, S. Yamaguchi, Y. Kawazoe and K. Tsuda, Chern. Pharm. Bull., 1964, 12, 104. T. RUN, Bull. SOC. Chim. Fr., 1963, 586. J . A. Glasel, Biochem. Biophys. Res. Commun., 1981, 102, 703. F. J. Muhtadi Anal. Profiles Drug Subst., 1988, 17, 259. M. S. Hifnawy and F. J. Muhtadi, Anal. Profiles Drug Subst., 1988, 17, 367. M. A. Al-Yahya and M. M. A. Hassan, Anal. Profiles Drug Subst., 1982, 11, 407. P D. K. Wyatt and L. T. Grady, Anal. Profiles Drug Subst., 1981, 10, 357. 4 F. J. Muhtadi and M. M. A. Hassan, Anal. Profiles Drug Subst., 1981, 10, 93. V. E. Yushmanov, J. R . Perussi, H. Imasato and M. Tabak, Biochim. Biophys. Acta, 1994, 1189, 74. W. D. Beazley, J . Forens. Sci., 1985, 30, 915. ' F. Medina, J . Forens. Sci., 1989, 34, 565. '' U. Eppenberger, M. E. Warren and H. Rapoport, Helv. Chim. Acta, 1968, 51, 381. " N. Tsunoda and H. Yoshimura, Xenobiotica, 1979, 9, 181. C. E. Brown, S. C. Roerig, J. M. Fujimoto and V. T. Burger, J . Chem. SOC., Chern. Commrm., 1983, 1506. A. C. Allen, D. A. Cooper, J. M. Moore, M. GIoger and H. Neumann, Anal. Chem., 1984, 56, 2940. Y J. M. Moore, A. C. Allen and D. A. Cooper, Anal. Chem., 1984, 56, 642. a

'

244

C. J. GROOMBRIDGE

(33) R3,R6 = C0.CH3 (34) R6 = CO.CH,, R3 = H

(35) R3 = CO.CH,, R6 = H OR6

phenobarbitone. An average purity is below 40%. Abuse is by injection or smoking-inhaling the fumes when heroin base is heated on tinfoil.24 Early NMR studies of morphine-related substances omitted morphine or diamorphine but established some of the basic 'H features of the four-ring structure.127-131 Full 13C assignments for diamorphine were given by Carroll et uL.,'~ and 'H interpretation for morphine was made when high-field instrumentation became available. 133,134 A highly detailed analysis for morphine and diamorphine was subsequently assembled by Neville et al. 135 using two-dimensional COSY (correlation spectroscopy) and partial simulations (Fig. 4). Neumann and V ~ r d e r m a i e r have ' ~ ~ explicitly considered the possible value of using 'H NMR for forensic analysis of impure illicit heroin samples. Other work is summarized in Table 3. The question of isomer discrimination seems to have been less urgent for heroin than for other substances; however, B e a ~ l e y 'and ~ ~ M e d i ~ ~have a'~~ prepared three diastereoisomers, and have shown that these give subtly different 'H NMR, IR and mass spectra. Two substances are often detected in illicit heroin: 6-Umonoacetylmorphine (6-U-MAM) (34), from incomplete acetylation, and 3-U-monoacetylmorphine (3-U-MAM) (35), from decomposition through hydrolysis; it is notable that diamorphine has a metabolic half-life of less than 10min because of rapid hydrolysis. 'H NMR data has been given by Neumann and V ~ r d e r m a i e r 'and ~ ~ Neville et ~ 1 . (Fig. ' ~ ~ 4). Sy et al. have described the synthesis and characterization of 3-0-MAM'39 and 6-U-MAM.'40 Several other minor heroin components have been identified using MS, 'H NMR and IR spectroscopy (Table 3), and this information is used for sample comparison or profiling. Part of this research has been through international collaboration between the US D E A Special Testing and Research Laboratory and the Bundeskriminalamt, Wiesbaden, in Germany,I4' although there has been less research activity recently as the profiling protocol has become established, and attention has shifted to the problems of cocaine or synthetic phenethylamines. Allen and co-workers have identified and given 'H NMR data for A15,16-didehydroheroin,142 A'6"7-dehydroheroin,142 two thebaine rearrangement products,'43

NMR SPECTROSCOPY IN FORENSIC SCIENCE

6.5

1

I

6.0

5.5

5.0

I

4.5

4.0

245

I

3.5

3.0

2.5

2.0

1.5

PPm

Fig. 4. 'H NMR spectra (500 MHz) of morphine (28), 3-0-acetylmorphine ( 3 - 0 MAM) (35), 6-0-acetylmorphine (6-0-MAM) (34) and diacet lmorphine (heroin) (DAM) (33). (Reproduced with permission from Neville et al.13')

n ~ s c a p i n e , 'N-acetylnorn~scapine,'~~ ~~ N-acetylnorla~danosine~~~ and four anhydronarceine compounds.'41 The chemistry associated with heroin smoking has been studied by Cook and Brine,'46 who found acetyl migration products (6-O-MAM, Nacetylnordiamorphine and N,O6-diacety1normorphine), and some evidence for D ring cleavage. There are many semisynthetic opiate substances which are in medical use, and this continues to be an active area of research, which includes NMR spectroscopic characterization. We will not attempt to review this extensive literature, although it is useful as a background to spectroscopic interpretation. Assignments have recently been detailed for buprenorphine and diprenorphine.147,148

246

C. J. GROOMBRIDGE

2.3. Cocaine and related substances

Cocaine was the first local anaesthetic to be discovered, but it is also a strong central nervous system stimulant, and its abuse has risen considerably in recent years in many parts of the world. Cocaine users may develop toxic psychoses, irrationality, paranoia and proneness to vi01ence.l~~ Withdrawal may lead to severe depression and dysphoria, and this is addictive in some instances, according to the “overpowering desire” medicolegal definition. Cocaine is a tropane alkaloid (36), a group which also includes atropine (hyoscyamine) and scopolamine (hyoscine) , which have valuable medical uses but have been noted historically as poisons (e.g. the notorious Dr Crippen case, London, 1910). There have been local instances of the abuse of plant material containing hyoscyamine (henbane Hyoscyarnus niger15’ or thorn apple Datura ~trarnoniurnl~~).

Although there are routes for total synthesis, and a few illicit laboratories have been reported in the USA,’52 cocaine is primarily obtained from the leaves of the shrubs Erythoxylum coca or Erythoxylurn novogranatense cultivated in South America. The leaves yield 0.5-1.5% of alkaloids, of which cocaine is the principal substance (more than 75%, depending on the variety). 15* The extraction is carried out in clandestine laboratories close to the growing regions. The minor alkaloids have a considerable forensic significance, as discussed below. Cocaine is encountered by forensic analysts as the salt form, cocaine hydrochloride, or as the free base, also known as “crack”. The hydrochloride form is commonly diluted with a carbohydrate (e.g. glucose) and sometimes also with another local anaesthetic such as lignocaine, so that the resulting white powder may contain &loo% of cocaine. Crack cocaine tends to be relatively pure; it is usually a waxy solid which melts and forms an aerosol for smoking more easilyZ4-the solid is heated in an improvised pipe so that the fumes can be inhaled. By some definitions, “free base” is prepared by solvent extraction, whereas “crack” is formed by direct precipitation. Early ‘H NMR measurement^'^^'^^^ were only partially assignable by inspection, but it was realized that the C3 proton resonance provided a

NMR SPECTROSCOPY IN FORENSIC SCIENCE

1 5.5

247

3

5.0 PPM

6

6 7

Fig. 5. 'H NMR spectrum (400MHz) of cocaine base (36). See ref. 156 for assignments.

feature which unambiguously distinguished cocaine from its C2/C3 diastereoisomers (allococaine, pseudococaine and allopseudococaine) in a very simple manner. This is a practical application of the Karplus equation. More detailed 'H assignment has been gradually achieved as higher-field spectra have been obtained (see refs 155-157 and Table 4), as shown in Fig, 5 , although there remain some ambiguities of interpretation of the H6 and H7 proton resonances because of very tight coupling. 156,157 A full assignment is not needed for the use of an NMR spectrum in a forensic context, but is

248

C. J. GROOMBRIDGE

Table 4.

NMR data for cocaine and related substances.

Compound

Spectrum

Cocaine

'H 100 MHz, chiral shift reagent 'H 60 MHz ' H 90 MHz, I3C

Cocaine Cocaine Cocaine Cocaine Cocaine Cocaine Cocaine Cocaine Cocaine Cocaine Cocaine Cocaine (base and Cocaine (base and Cocaine Cocaine (base and Cocaine Cocaine HCI Cocaine Cocaine HCI Cocaine HCI Cocaine-ND, Cocaine Cocaine HCI Cocaine (base and

"c

zc

I

''c

HCI) HCI) HCI)

HCI)

' H 90 MHz 'H 60 MHz, lanthanide chiral shift reagent 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 90 MHz 'H 250 MHz, "C

Ref

Jochims (1967)" Sinnema (1968)' Stenberg (1977)' Taha (1977)d Taha (1978)' Baker (1978)' Lopez (1978)g

Kroll (1979)h Singh (1979)' Siegel (1980)' Lukaszewski (1980)' Allen (1981)' Carroll (1982)" '3c Avdovich (1983)" 'H 200 MHz, "C Valensin (1985)O 'H 60 MHz, 'C Muhtadi (1986)' 'H 400 MHz Chazin (1986)q 'H 250 MHz, quantification Sanchez Gonzalez (1987)' I5N Ciimirik (1987)" 'H 400 MHz, I3C Glaser (1988)' 'H 250 MHz, NOE Mosquera (1989)" 'H 90 MHz Novik (1989)" 'H Garro Galvez (1990)"' I 'C Leete (1991)x 'H 300 MHz Mills (1993y

Diastereoisomers

Pseudococaine Pseudococaine Pseudococaine Pseudococaine (base and HCI) Pseudococaine Allococaine Allococaine Allococaine Allopseudococaine Allopseudococaine Allopseudococaine (base and HCI) Allopseudococaine

' H 60 MHz ' H 60 MHz 'H 90 MHz ' H 250 MHz, "C ' H 300 MHz 'H 60 MHz 'H 90 MHz ' H 250 MHz, 'C 'H 60 MHz 'H 90 MHz ' H 250 MHz. ''C 'H 300 MHz

Sinnema (1968)' Siegel (1980)' Allen (1981)' Carroll (1982)" Mills (1993)Y Sinnema (1968)' Allen (1981)' Carroll (1982)'n Sinnema ( 1968)h Allen (1981)' Carroll (1982)"' Mills (1993)v

'H 'H 'H 'H 'H

Moore (1973)' Moore (1973)' Green (1974)"" Lukaszewski (1980)k Lukaszewski ( 1980)~

Impurities

trans-Cinnamoylcocaine cis-Cinnamoylcocaine p-Truxinic acid Ecgonine methyl ester Ecgonidine methyl ester Diphenylcyclobutanedicarboxylic acids

60 MHz 60 MHz 60 MHz 60 MHz

'H 200 MHz

Moore (1987)'"

N M R SPECTROSCOPY IN FORENSIC SCIENCE

Table 4.-cont. Compound

Spectrum

cis-Cinnamoylcocaine Pseudoecgonine HC1 Ecgonine HCI N-Formylnorcocaine Ecgonine methyl ester cis-Cinnamo ylwcaine rrans-Cinnamoylcocaine Norcocaine N-Benzoylnorecgonine methyl ester N-Form ylnorcocainc N-Formylnorcocaine N-Benzoylnorecgonine methyl ester 1-Hydroxytropacocaine

'H 'H 'H 'H I

'c

80 MHz, "C 60 MHz 60 MHz 200 MHz

I 'C

I

zc

'H 300 MHz, "C 'H 300 MHz, "C 'H 300 MHz. I3C 'H 400 MHz 'H 400 MHz 'H. 13C

Ref. By (1988)'" Casale ( 1990)"d Casale (1990)"" Brewer (1991)" Leete (1991)" Leete (1991)" Leete (1991)" Ensing (1991)rf Ensing (1991)fl Ensing (1991p LeBelle (1991)xb' LeBelle (1991)gg Moore (1994)hh

Metabolites

Norcocaine Benzoylecgonine Norcocaine Benzoylecgonine Ecgonine Norcocaine Benzylnorecgonine Norecgonine Hydroxymethoxycocaine Hydroxycocaine Benzoylecgonine ethyl ester (cocaethylene)

'H 60 MHz 'H 60 MHz 'H 60 MHz "C

'H 200 MHz 'H 200 MHz

Stenberg (1976)" Stenberg (1976)" Borne (1977)" Baker (1978)' Baker (1978)r Baker (1978)' Baker (1978)' Baker (1978)' Smith (1984)kk Smith (1984)"

'H 300 MHz

Brzezinski (1992)'""'

'H 90 MHz 'H unspecified 'H 'H IH 'H 300 MHz, "C 'H 300 MHz, "C 'H 300 MHz. "C

Novak (1984)"" Sisti ( 1 9 8 9 ) Sisti (1989)"" Sisti (1989)"" Nakahara (1991)pp Newman (1994)qq Newman (1994)qq Newman (1994)qq

1 3 c

Wenkert (1974)" Wenkert (1974)" Wenkert (1974)" Simeral (1974)" Simeral (1974)"" Simeral (1974)"' Simeral (1974)" Simeral (1974)"" Feeney (1977)" Feeney (1977)" Stenberg (1977)'

I3c 1

'c

1 3 c

"c

Pyrolysis products

Methyl 4-(3-pyridyl) butyrate Benzoic acid N-Methylpyrrole Methyl 3-butenoate Ecgonidine methyl ester Ecgonidine methyl ester Ecgonidine ethyl ester Norecgonidine methyl ester Other tropane alkaloids

Atropine Tropacocaine Scopolamine Tropine Tropic acid Atropine Scopolamine Methyl atropine Atropine sulfate Scopolamine Atropine

l3c

'C 13C

1 . 7 ~ l3c

"C I

'c

'H 270 MHz, "C 'H 270 MHz, 13C 'H 90 MHz, I3C

249

250

C. J. GROOMBRIDGE

Table 4.-cont. Compound

Spectrum

Tropine Atropine Scopolamine Atropine methonitrate Atropine Scopolamine Tropacocaine Ecgonine methyl ester Tropine Tropinone Tropine N-oxide Tropacocaine Hyoscamine Atropine Scopolamine Tropic acid Benzoyltropeine (heroin diluent) N-Allylnorcocaine 3-Aminomethyl-2-methoxycarbonyl-8methyl-8-azabicyclo[3.2.l]oct-2-ene (synthetic cocaine impurity)

1 3 c

3-Benzoyloxy-2-methoxycarbonyl-8methyl-8-azabicyclo[3.2. IIoct-2-ene 3-Benzoyloxy-8-methy1-8azabicyclo[3.2.1]oct-2-ene Pseudoecgonine methyl ester Allopseudoecognine methyl ester Alloecgonine Alloecgonine methyl ester Allococaine Allopseudococaine Ecgonine HCI Ecgonine ethyl ester Scopolamine Scopolamine H y oscy am in e Scopolamine Homatropine Atropine Scopolamine Scopolamine metabolites Ecgonine methyl ester Pseudoecgonine methyl ester Alloecgonine methyl ester Allopseudoecgonine methyl ester Hyoscyamine Scopolamine Benzoylecgonine Ecgonine HCI

Ref.

I3c

Taha (1977)d Taha (1977)d Taha (1977)d Taha (1977)d Taha (1978)d Taha (1978)d Singh (1979)' Singh (1979)' Avdovich (1983)" Avdovich (1983)" Avdovich (1983)" Avdovich (1983)" Avdovich (1983)" Avdovich (1983)" Avdovich (1983)" Avdovich (1983)" Mari (1984)"" Baker (1978y

' H 200 MHz

Cooper (1984)""

'H 200 MHz

Cooper (1984)""

1 3 c

l3c I3C

I3c I3c ' H 60 MHz 'H 60 MHz

I3c I3c 13C

I3c

I3c I3c I3c '3c

'H 80 MHz

'H 'H 'H 'H 'H

200 MHz 250 MHz 250 MHz 250 MHz 250 MHz 'H 250 MHz ' H 250 MHz

'H 'H ' H 300 MHz, "C

'H 200 MHz, l3C 'H 200 MHz, I3C 'H, I3C ' H 60 MHz, 13C ' H 100MHz ' H 100 MHz ' H 60 MHz ' H 60 MHz 'H 60 MHz 'H 60 MHz 'H 400 MHz, I3C 'H 400 MHz, I3C 'H 300 MHz 'H 300 MHz

Cooper (1984)"" Carroll (1991)"" Carroll (1991)"" Carroll (1991)"" Carroll (1991)ww Carroll (1991)"" Carroll (1991)"" Garro Galvez (1990)" Garro Galvez (1990)" Sarazin (1991)" Glaser ( 1993)yY Muhtadi (1994)" Muhtadi (1990)""" Muhtadi (1987)hhh Al-Badr (1985)"' Wada ( 1991)ddd Wada (1991)"dd Sinnema (1968)' Sinnema (1968)b Sinnema (1968)h Sinnema (1968)b Watson ( 1 9 9 3 y Watson (1993)"' Mills (1993)Y Mills (1993)y

NMR SPECTROSCOPY IN FORENSIC SCIENCE

251

Table 4.-cont. Compound Ecgonine methyl ester Propylbenzoylecgonine

Spectrum

Ref.

'H 300 MHz I H 300 M H ~

Mills ( 1993)y Mills (1993)"

'H 90 MHz ' H 90 MHz 'H 90 MHz 'H 90 MHz I H 90 MHZ 'H 270 MHz, 13C 'H 360 MHz, I3C 'H 200 MHz, "C

Lopez (1978)R Lopez ( 1Y7X)8 Lopez (l978)R Lopez (1978)R Lopez (1978)R Wilson (1990)"'

Other local anaesthetics

Tetracaine (= amethococaine) Carbocaine Procaine Lignocaine Benzocaine Bupivacaine Tetracaine Lignocaine Dibucaine Benzocaine Lignocaine Benzocaine Procaine HCI Procainamide Cinchocaine Tetracaine

'H 60MHz. "C 'H 60 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz

Riaz (1989)ggRp

Groningsson (1985)hhh Padmanahhan (1983)"' Ali (1983)"' Mills (1993)y Mills (1993)y Mills (1993)y Mills (1993)Y Mills (1993)Y Mills (1993)y

J. C . Jochims, G . Taigel and A . Seeliger, Tetrahedron Left., 1967, 20. 1901. Sinnema, L. Maat, A. J. van der Gugten and H. C. Beyerman, Rec. Trav. Chim. Pays-Bas, 1968, 87. 1027. ' V. I . Stenberg, N. K. Narain and S. P. Singh, J . Heterocyclic Chem., 1977, 14, 225. A. M. Taha and G. Riicker, Egypt. J . Pharm. Sci, 1977, 18, 59. A. M. Taha and G . Riicker, J . Pharm. Sci., 1978, 67, 775. J. K. Baker and R . F. Borne, J . Heterocyclic Chem., 1978, 15, 165. A . Lopez and G . Quaglia Strano, Zacchia, 1978, 14, 158. ' I J . A . Kroll, J . Forens. Sci.. 1979, 24, 303. ' S. P. Singh, D. Kaufrnan and V. I. Stenberg, J . Heterocyclic Chem., 1979, 16, 625. J . A . Siege1 and R . A . Cormier, J . Forens. S c i . , 1980, 25, 357. T. Lukaszewski and W. K . Jeffery, J . Forens. Sci.. 1980, 25, 499. ' A. C. Allen, D. A . Cooper, W. 0. Kiser and R. C. Cottrell, J . Forens. Sci., 1981, 26, 12. "' F. I . Carroll, M. L. Coleman and A . H. Lewin, J . Org. Chem., 1982, 47, 13. '' H. W. Avdovich and G. A. Neville, Can. J . Spectrosc., 1983, 28, 1. G . Valensin, E . Gaggelli. N. Marchettini and I. Barni Comparini, Biophys. Chem., 1985, 22, a

' A.

'

77.

F. J . Muhtadi and A. A . Al-Badr, Anal. Profiles Drug Subsf., 1986, 15, 151. W. J . Chazin and L. D. Colebrook, J . Org. Chem., 1986, 51, 1243. A . Sanchez Gonzalez and E. Uriarte, Farmaco (Ed. Prat.), 1987, 42, 281. J . Ciimarik and A LyEka, Pharmazie. 1987, 42, 697. ' R . Glaser, Q.-J. Peng and A . S . Perlin, J . Org. Chem., 1988, 53, 2172. R. A . Mosquera and E . Uriarte, J . Mol. Sfrucf.,1989, 195, 325. " M. Novak and C . A . Salemink, Tetrahedron, 1989, 45, 4287. J . M. Garro Galvez and A. P. d e Abram, Bol. Soc. Quim. Peru, 1990, 56, 12. E. Leete, J. A . Bjorklund, M. M. Couladis and S. H. Kim, J . A m . Chem. Soc., 1991, 113, 'I

-I

9286.

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C. J . GROOMBRIDGE

T. Mills 111 and J . C. Roberson, Instrumental Data for Drug Analysis, 2nd edn. CRC Press, Boca Raton, 1993. ' J. M. Moore, J . Assoc. Off.Anal. Chem., 1973, 56, 1199. u" B. S. Green and M. Retjo, J . Org. Chem., 1974, 39, 3284. hh J . M. Moore, D . A . Cooper, I. S. Lurie, T. C. Kram, S. Carr, C. Harper and J . Yeh, J . Chromatogr., 1987, 410, 297. '' A. By, B. A. Lodge and W.-W. Sy, Can. SOC. Forens. Sci. J . , 1988, 21, 41. dd J. F. Casale, Forens. Sci. Int., 1990, 47, 277. ee L. M. Brewer and A . Allen, J . Forens. Sci., 1991, 36, 697. ff J . G . Ensing and J. C. Hummelen, J . Forens. Sci., 1991, 36, 1666. Rg M. J . LeBelle, B. Dawson, G . Lauriault and C. Savard, Analyst, 1991, 116, 1063. ''I J. M. Moore, P. A. Hays, D . A. Cooper, J. F. Casale and J. Lydon, Phytochem., 1994,36, 357. " V. I. Stenberg, N. K. Narain, S. P. Singh and S . S. Parmar, J . Heterocyclic Chem., 1976, 13, 363. IJ R. F. Borne, J . A . Bedford, J. L. Buelke, C. B. Craig, T. C. Hardin, A. H. Kibbe and M. C. Wilson, J . Pharm. Sci., 1977, 66, 119. kk R. M. Smith, M. A. Poquette and P. J. Smith, 1. Anal. Toxicol., 1984, 8, 29. 'I R. M. Smith, J . Anal. Toxicof.,1984, 8, 35. mm M. R. Brzezinski, C. D. Christian, M.-F. Lin, R. A. Dean, W. F. Bosron and E. T. Harper, Synrh. Commun., 1992, 22, 1027. "" M. Novak and C. A. Salemink, Bull. Narc., 1984, 36(2), 79. N. J. Sisti, F. W. Fowler and J. S. Fowler, Tetrahedron Left., 1989, 30, 5977. p P Y. Nakahara and A . Ishigami, J . Anal. Toxicol., 1991, 15, 105. 44 A. H. Newman, A. C. Allen, J . M. Witkin, S. Izenwasser, D . Mash and J . L. Katz, Med. Chem. Res., 1994, 4, 93. '' E. Wenkert, J. S. Bindra, C.-J. Chang, D. W. Cochran and F. M. Sohell, Acc. Chem. Res., 1974, 7 , 46. .',' L. Simeral and G . E. Maciel, Org. Magn. Reson., 1974, 6, 226. J . Feeney, R . Foster and E . A. Piper, J . Chem. SOC., Perkin Trans., 1977, 2016. u' F. Mari, E . Bertol and M. Tosti, Bull. Narc., 1984, 36(1), 59. "" D. A. Cooper and A. C. Allen, J . Forens. Sci., 1984, 29, 1045. w w F. I. Carroll, A. H. Lewin, P. Abraham, K. Parham, J. W. Boja and M. J. Kuhar, J . Med. Chem., 1991, 34, 883. xx C. Sarazin, C. Goethals, J.-P. Stguin and J.-N. Barbotin, Magn. Reson. Chem., 1991, 29, 291. .w R. Glaser, Magn. Reson. Chem., 1993, 31, 335. z L F. J. Muhtadi, Anal. Profiles Drug Subst. Excipients, 1994, 23, 153. U*" F. J. Muhtadi and M. M. A . Hassan, Anal. Profiles Drug Subst., 1990, 19, 477. hhh F. J. Muhtadi, M. M. A . Hassan and A . F. A. Afify, Anal. Profiles Drug Subst., 1987, 16, 245. cL.c A. A. Al-Badr and F. J . Muhtadi, Anal. Profiles Drug Subst., 1985, 14, 325. ddd S. Wada, T. Yoshimitsu, N. Koga, H . Yamada, K. Oguri and H . Yoshimura, Xenobiotica, 1991, 21, 1289. ccc A. B. Watson, I. K. A . Freer. D . J . Robins and N. J . Walton, J . Nar. Prod., 1993, 56, 1234. T. D . Wilson, Anal. Profiles Drug Subst., 1990, 19, 59. zg8 M. Riaz, Anal. Profiles Drug Subst., 1989, 18, 379. 'zh'' K. Groningsson, J.-E. Lindgren, E. Lundberg, R . Sandberg and A. Wahlen, Anal. Profiles Drug Subst., 1985, 14, 207. iii G . R. Padmanabhan, Anal. Profiles Drug Subst., 1983, 12, 105. jiJ S. L. Ah, Anal. Profiles Drug Subst., 1983, 12, 73. Y

fff

NMR SPECTROSCOPY IN FORENSIC SCIENCE

253

desirable for the interpretation of any structural variation that might be encountered. Valensin et af.’ss have studied cocaine in DMSO solution using ‘H and 13C techniques, but there are some puzzling aspects of their interpretation: cocaine H6/H7 appear to have been assigned to the solvent DMSO-dS peak, and some two-dimensional NOE spectroscopy (NOESY) ‘H data were described only briefly, but apparent correlations may be artifacts due to tl noise. Chazin and Colebrook’s6 (Fig. 5 ) gave tentative H6/H7 assignments, and a full interpretation was proposed by Mosquera and UriartelS7 from NOE difference measurements, although these may be difficult to analyse in Other measurements are summarized in strong-coupling situations. lSx Table 4. As mentioned above, ‘H NMR spectra have been used t o characterize the cocaine d i a s t e r e o i s ~ m e r s , ’and ~ ~ there has also been some work on optical isomer analysis in response to forensic requirements. Cocaine from plant is I-cocaine; in the USA in the mid-1970s the law was framed as follows: “Coca leaves, . . ., and any salt, compound, derivative, or preparation thereof which is chemically equivalent to or identical with any of these substance^".'^^ A spurious loophole defence was possible (“isomer defense”), arguing that the analytical procedures were not able to determine the presence of the 1 form of cocaine. This forced forensic scientists to develop synthetic procedures and characterizations of the full set of stereoisomers. The law was changed in 1984 to state that cocaine and all its isomers are controlled (see ref. 159). In Britain the interpretation of the court was that “cocaine” was a generic term which included stereoisomers ( R v. Greensmith, 1983). Jochims et aI.lS3 made one of the earliest uses of a chiral shift reagent, 1-phenylethanol, shortly after the first description of this effect by Pirkle,16’ and this was subsequently adopted by some forensic laboratories. Krol1161 also described a lanthanide reagent method which has been in active casework use. As discussed for amphetamine, forensic chemists are often asked to compare drug samples in order to provide information on origin, manufacturing procedures and subsequent adulteration. This may also be used as evidence for prosecution for trafficking or supply offences. This is extremely important for cocaine, particularly in North A m e r i ~ a . ’As ~ with other drugs, capillary gas chromatography has been found to be the most suitable method for routine impurity profiling, but component identification is an important part of the analytical validation. Over 30 cocaine impurities have now been identified as minor components co-extracted from the coca plant, or produced by chemical treatment such as permanganate oxidation, which has been used in some clandestine laboratories. In many instances, impurity identification has been by MS, but ‘H NMR has frequently been used. Cinnamoylcocaines (37)and (38) are generally the most prominent minor

254

C. J. GROOMBRIDGE

FOOCH,

alkaloids, and it was shown by Moore’62 that both cis (37) and trans (38) isomers are present. The relative contents of these compounds have been used to give sample comparisons, 163 but latterly extensive profiles have been used. All 11 isomers of the diphenylcyclobutanedicarboxylic acid esters (the truxillines, (39)) have been detected or tentatively observed in illicit cocaine samples.’64 Hydrolysis of the truxillines produces the parent acids (the head-to-tail truxillic (40) and head-to-head truxinic (41)), and some of these have also been detected. A systematic investigation of these acids was given including ‘H NMR of synthesized reference substances by Moore er al. (Table 4). Hygrine (42) and cuscohygrine (43) are also known impurities. Some NMR data have been reported by Endo et ~ 1 . and l ~ ~Leete et ~ 1 . ‘ ~ ~ Some cocaine production includes a permanganate purification or bleaching stage, and it has been shown that this can result in oxidation and N-benzoylnorecgonine impurities, including N-formylnor~ocaine,~~’-~~~ methyl e ~ t e r l ~ from * ” ~ N+O ~ migration.

NMR SPECTROSCOPY IN FORENSIC SCIENCE

255

HOOC

OOH

COOH

Very recently, the knowledge of individual Erythoxylum varieties has been extended by greenhouse c u l t i ~ a t i o n . ' ~1-Hydroxytropacocaine ~) has been newly isolated from such experiments. L71 Other impurities identified with the aid of NMR are listed in Table 4. Solvents used in cocaine extraction may also be found in forensic samples, and headspace GC is often a favoured method for the analysis of volatile ' ~ ~shown that 'H NMR can also be used; organics, but Avdovich et ~ 1 . have these are often small molecules which give very sharp peaks because of short motional correlation times. NMR is similarly used by some pharmaceutical companies for product solvent residue quality control. The trend towards cocaine abuse by smoking has brought the possibility that drug users are ingesting pyrolysis decomposition products; consequently there have been a number of experiments either with medically supervised volunteers or with apparatus designed to mimic smoking habits. The decomposition is temperature, flow and gas (air/N2) dependent. 'H NMR was used to identify methyl 4-(3-pyridyl) b ~ t y r a t e , ' benzoic ~~ acid,'74 N - r n e t h y l p y r r ~ l e , methyl '~~ 3 - b ~ t e n o a t e 'and ~ ~ methyl e~gonidine.'~'Salemink also prepared cocaine-[ND3] in order to clarify the GC-MS results.'76

256

C. J. GROOMBRIDGE

Methyl ecgonidine (anhydroecgonine methyl ester, AEME) (44) has been reported both in smoke tests and in urine; this substance can be produced as a G C injection port artifact, and had previously been identified using MS and 'H NMR.'77 A detailed study has recently been given by Newman el a ~178 .

The metabolism of cocaine is rapid and extensive, so that forensic toxicologists may only observe metabolites in body fluids. Spontaneous F00CH3

hydrolysis produces the primary metabolite, benzoyl ecgonine, whereas ecgonine methyl ester is produced by enzyme catalysis. A range of other minor metabolites is known, and NMR data are summarized in Table 4. Smith et a1.17y,180were able to identify a number of hydroxybenzoyl and hydroxyrnethoxybenzoyl substances by comparison with synthesized compounds for which the ring substitution has been determined by NMR. It was first noted by Rafla and Epstein"' that an additional metabolite is produced when alcohol drinking is combined with cocaine abuse. In vivo transesterification leads to the ethyl analogue, benzoylecgonine ethyl ester, which is also referred to (illogically) as cocaethylene. Synthesis and NMR characterization has been reported by Harper and co-workers.'82 2.4. Cannabinoids

Cannabis is obtained from the Cannabis sativa plant, perhaps best known from the ubiquitous leaf-shape symbol. Drug material may occur in a number of different forms, particularly herbal cannabis (marijuana, the flowering or fruiting parts of the plant) or cannabis resin (exuded by the flowering tops but with less plant debris). Cannabis is a mild hallucinogen and induces a light-headed euphoric intoxication. It is considered to have an incapacitating effect on activities such as driving. Cannabis continues to be the most controversial of the illegal drugs, but it is a class B controlled drug under the MoDA in Britain, or comparable legislation in other countries, and it is consequently frequently the task of forensic scientists to examine cannabis material. The yield of cannabinoids from C. sativa is highest when this is grown in hot conditions, so cannabis is primarily imported from growing regions in Asia, Africa or the West Indies. However, there have often been attempts

NMR SPECTROSCOPY IN FORENSIC SCIENCE

257

(45)

at clandestine cultivation in cooler climates such as Britain, latterly using supposed high-yield varieties ("skunkweed") or artificial conditions (e .g. indoor hydroponics). The primary psychoactive substance in cannabis was identified in 1964 using 'H NMR by M e c h ~ u l a m 'as ~ ~A9-tetrahydrocannabinol (45). Further detailed spectroscopic interpretations have been given by Archer et al. 184 and Makriyanni~."~ 13CNMR data have also been reported by Wenkert and Archer. '863'87 Several research groups have produced series of publications on cannabis chemistry over some years, for example: Mechoulam (Jerusalem), Shoyama (Kyushu), Crombie (Nottingham) , Turner (Mississippi) and Harvey (Oxford). Extensive research has identified more than 100 other cannabis substanceslB8 (which may also have bioactivity). Again, 'H NMR was the main method by which the structures of these substances were elucidated, as summarized in Table 5. The comparison of cannabis samples by the profiling of major and minor components has been used for drugs intelligence,'89 but this is possibly less important than for synthetic and semisynthetic drugs, at least partly because the external appearance is more characteristic. NMR is certainly not appropriate for routine cannabis analysis, except indirectly through the authentication of pure substances used in chromatography or immunoassay. The rare exception has been for an instance of clandestine tetrahydrocannabinol synthesis. 190 Proton and 13C NMR and MS were used to identify the reagents and products. 2.5. Ergot and other indole alkaloids Domestic Medicine 3599. Ergot.-In the form of the liquid extract, this drug is useful in an eminent degree in cases of blood-spitting or flooding after confinement. For the former it may be given in doses of fifteen drops every three hours; for the latter a teaspoonful, to be repeated in a quarter of an hour if necessary.

Mrs. Beeton's Book of Household Management (1888)

258

C. J. GROOMBRIDGE

Table 5.

NMR spectra of cannabinoids.

Compound

Spectrum

Ref.

Cannabinol-0-p-o-glucopyranoside 9'-Hydroxycannabinol-O-~-~-glucopyranoside (- )-A'-Tetrahydrocannabinol (-)-AX-Tetrahydrocannabinol ( +)-AR-abn-Tetrahydrocannabinol ( +)-cis-A'-Tetrahydrocannabinol Cannabinol ketones Cannabinol Cannabinolic acid, methyl ester Cannabidiol Cannabidiolic acid, methyl ester Cannabispiran Deh ydrocannabispiran P-Cannabispiranol (3-[2-(3-Hydroxy-4-methoxyphenyl)ethyl]5-methoxyphenoI Canniprene Cannabitetrol Synthetic cannabinoid CP-47, 497 (+)-Cannabidiol (+)-A'-Tetrahydrocannabinol (+)-Cannabidiol dimethyl ether A*-Tetrahydrocannabinol Cannabichromene Tetrahydrocannabinol carboxylic acid A9,' '-Tetrahydrocannabinol A"-Tetrahydrocannabinol A9-Tetrahydrocannabinol Olivetol (precursor) Citral (precursor) Cannabidol A'-&Tetrahydrocannabinol A'--trans-Tetrahydrocannabinol A8-trans-Tetrahydrocannabinol Cannabispirone Cannabispirenone Cannabichromene Cannabidiol Cannabinol Cannabispiran Cannabicyclol Cannabigerol Ax-Tetrahydrocannabinol A'-Tetrah ydrocannabinol Citral (precursor) Olivetol (precursor)

'H 270 MHz 'H 270 MHz

Tanaka (1993)" Tanaka (1993)"

I3c

Archer (1977)h Archer (1977)b Archer (1977)b Archer ( 1977)b Archer (1977)' Mechoulam (196S)c Mechoulam (1965)' Mechoulam (1965)' Mechoulam (1965)' ElSohly ( 1982)d ElSohly ( 1982)d ElSohly ( 1982)d ElSohly ( 1982)d

1 3 c

I3C '3C I3C 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz, 'H 60 MHz, 'H 60 MHz, 'H 60 MHz,

I3C l3C I3C "C

'H 60 MHz, "C 'H 300 MHz 'H 300 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 500 MHz 'H 250, 500 MHz 'H 'H 90 MHz 'H 90 MHz 'H 200 MHz 'H 200 MHz, I3C 'H 200 MHz 'H 200 MHz 'H 300 MHz, I3C 'H 300 MHz, 13C 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz

ElSohly ( 1982)d ElSohly (1984)' Xiang-Qun Xie (1994)' Mechoulam (1972)" Mechoulam (1972)" Mechoulam (1972)g Petrzilka (1970)h Korte (1970)' Korte (1970)' Makriyannis (198s)' Makriyannis (1985)' Kriwacki (1989)k Churchill (1983)' Churchill (1983)' Churchill (1983)' Churchill (1983)' Churchill (1983)' Churchill (1983)' Bercht (1976)"' Bercht (1976)"' Mills (1993)" Mills (1993)'' Mills (1993)" Mills (1993)" Mills (1993)'' Mills (1993)" Mills (1993)" Mills (1993)" Mills (1993)" Mills (1993)"

NMR SPECTROSCOPY IN FORENSIC SCIENCE

259

" H. Tanaka, S. Morimoto and Y. Shoyama, J . Nut. Prod., 1993, 56, 2068. R. A . Archer, D. W. Johnson, E. W. Hagaman, L. N. Moreno and E. Wenkert, J . Org. Chem., 1977, 42, 490. R. Mechoulam and Y. Gaoni, Tetrahedron, 1965, 21, 1223. H. N. ElSohly and C. E. Turner, Bull. Narc., 1982, 34(2), 51. ' H. A . EISohly, E. G. Boeren, C. E. Turner and M. A. EISohly, The Cannabinoids: Chemical Pharmacologic, and Therapeutic Aspects (ed. S . Agurell, W. L. Dewey and R. E. Willete), p. 89. Academic Press, London. 1984. Xiang-Qun Xie, De-Ping Yang, L. S. Melvin and A. Makriyannis, 1. Med. Chem., 1994, 37, 1418. R. Mechoulam, P. Braun and Y. Caoni, J . Am. Chem. S O C . , 1972, 94, 6159. T. Petrzilka, The Botany and Chemistry of Cannabis (ed. C. R. B. Joyce and S. H. Curry), p. 79. Churchill, London, 1970. F. Korte, The Boiany and Chemisrry of Cunnabis (ed. C . R. B. Joyce and S. H. Curry), p. 119. Churchill, London, 1970. A. Makriyannis, S. Fesik and R. Kriwacki, New Methods Drug Res., 1985, 1, 19. R. W . Kriwacki and A. Makriyannis, Mol. Pharmacol., 1989, 35, 495. K. T. Churchill, J . Forens. Sci., 1983, 28, 762. C. A. L. Bercht, J. P. C. van Dongen, W. Heerma, R . J. J. Ch. Lousberg and F. J. E. M. Kiippers, Tetrahedron, 1976, 32, 2939. " T. Mills I11 and J. C. Roberson, Insirumentnl Data for Drug Analysis, 2nd edn. CRC Press, Boca Raton, 1993.

'

'

' '

Ergot is the dried material from the parasitic fungus Claviceps purpurea which grows on rye and other grain. While raw ergot may not now be part of the medicine cabinet of every well-managed household, pure derivatives have a number of valuable medical uses, such as the treatment of severe migraine and of Parkinson's disease. Ergot yields four main alkaloid classes: clavines, lysergic acids, lysergic amides and ergot peptides. Ergot alkaloids have also been found in many plant species, with the Convofvuluceae (morning glories) also having mixed lysergic acid substances. The drug of abuse derived from ergot is lysergic acid diethylamide (LSD, or lysergide) (46), which is obtained by amidation

dLH3 CON(CH,CH

13

14

4

HN

2

J2

260

C. J . GROOMBRIDGE

Table 6 . NMR spectra of ergot alkaloids. Compounds

Spectrum

13C, 'H 400 MHz Lysergide (LSD) I3C, 'H 400 MHz Lysergic acid methylpropylamide N6-Demethyllysergic acid diethylamide 'H 60 MHz ' H 270 MHz Ergotamine ' H 400 MHz Ergotamine tartrate 'H 400 MHz Ergocristine ' H 400 MHz Ergocryptine ' H 400 MHz Ergometrine maleate ' H 400 MHz Methysergide maleate 'H 400 MHz Bromocriptine mesylate 'H 400 MHz Pergolide mesylate 'H 400 MHz Dihydroergotamine mesylate 'H 400 MHz Dihydroergocristine mesylate ' H 400 MHz Dihydroergocornine mesylate 'H 200 MHz, 13C 9,lO-Dihydrolysergic acid methyl ester 'H 200 MHz, I3C 6-nor-9.10-Dihydrolysergicacid methyl ester 6-(5-p-Chlorobenzoyl-4-n-propyl-thiazol-2-yl) 'H 200 MHz, 13C dihydrolysergic acid 6-(5-p-Bromobenzoyl-4-ethyl-thiazol-2-yl) 'H 200MHz, I3C dihydrolysergic acid 6-(6-MethyI-4H-1,3-thiazin-4-on-2-yl) 'H 200 MHz, 13C dihydrolysergic acid 10~u-Methoxy-9,10-dihydrolysergic acid methyl ' H 200 MHz, 13C ester Agroclavine 'H 220 MHz, I3C Elymoclavine 'H 220 MHz, I3C Festuclavine 'H 220MHz. 13C Fumigaclavine 'H 220 MHz, 13C Lysergine I3C 1 3 c Isolysergine Methyl lysergate 13C Ergonovine l3C Ergonovinine '3C "c Ergotamine Ergocryptinine I3C Lysergic acid dimethylamide 'H 220MHz Terguride 'H 400 MHz, I3C Methyl lysergate 'H 200 MHz Methyl isolysergate 'H 200 MHz Isofumigaclavine 'H 200 MHz Lysergide 'H 470 MHz N(6)-Ethyl lysergide ' H 470 MHz N(6)-n-Propyl lysergide 'H 470 MHz N(6)-Isopropyl lysergide 'H 470 MHz N(6)-n-Butyl lysergide 'H 470 MHz N(B)-AIIyI lysergide 'H 470 MHz N(6)-(2-Phenylethyl) lysergide 'H 470 MHz N(6)-Cyano lysergide 'H 470 MHz N(6)-nor-Lysergide 'H 470 MHz

Ref. Neville (1992)" Neville (1992)" Nakahara (1971)h Pierri (1982)' Casy (1994)" Casy (1994)" Casy (1994)" Casy (1994)" Casy (1994)d Casy (1994)" Casy (1994)" Casy (1994)d Casy (1994)" Casy (1994)" Seifert (1989)' Seifert (1989)' Seifert (1989)' Seifert (1989)' Seifert (1989)' Seifert (1989)' Bach (1974)' Bach (1974)' Bach (1974)' Bach (1974y Bach (1974)' Bach (1974)' Bach (1974)' Bach (1974)' Bach (1974)' Bach (1974)' Bach (1974)' Bailey ( 1972)R HuSak (1993)h Ninomyia (1985)' Ninomyia (1985)' Ninomyia (1985)' Hoffman (1985)' Hoffman (1985)' Hoffman (1985)' Hoffman (1985)' Hoffman (1985)' Hoffman (1985)' Hoffman (1985y Hoffman (1985)' Hoffman (1985)'

NMR SPECTROSCOPY IN FORENSIC SCIENCE

261

Table 6.-cont. Compounds

Spectrum

Ref.

Lysergic acid dipropylamide 10-Methoxy dihydrolysergic acid methyl ester 10-Methoxy dihydrolysergamides 1-N-Methyl dihydrolysergamides Lysergide Lysergide a-Ergocryptine a-Ergocryptine CH2 Bromocryptine Bromocryptine CH2 Ergometrine (ergonovine) maleate Ergotamine tartrate Lysergic acid Lysergide Lysergic acid methylpropylamide Bromocriptine mesylate Bromo-LSD Methylergometrine Dihydroergotamine Terguride and metabolites Pergolide mesylate Ergohine Ergobinine Lysergic acid ethyl vinyl amide (LSD metabolite) 13-Hydroxy-LSD (LSD metabolite) Ergobalansine Ergo balansinine Ergocristine a-Ergocryptine Lysergic acid ethylvinylamide (metabolite) Lysergic acid ethyl-2-hydroxyethylamide (metabolite) Dihydrolysergic acid diethylamide Dihydroisolysergic acid diethylamide Lysergene Lysergine Isolysergine Festuclavine Pyroclavine 10-Methoxydihydrolysergic acid amides Lysergide 2-Bromo-LSD 2-10do-LSD

'H 60 MHz 'H 270 MHz, I3C 'H 90 MHz, I3C 'H 90 MHz, I3C 'H 100MHz 'H 60 MHz 'H 300 MHz, I3C 'H 300 MHz, I3C 'H 300 MHz, I3C 'H 300 MHz, I3C 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz ' H 300 MHz 'H 500 MHz 'H 300 MHz 'H 400 MHz 'H 200 MHz

Bailey (1973)k Zetta (1975)' Zetta (1977)" Zetta (1977)"' Sapper (1976)" Bellman (1970)" Szantay (1994)" Szantay (1994)" SzBntay (1994r Szantay (1994)p Mills (1993y Mills (1993)q Mills (1993y Mills (1993)q Mills (1993)q Mills (1993)q Mills (1993)q Mills (1993)q Mills (1993)q Miyamoto (1993)' Sprankle (1992)" Crespi Perellino (1993)' Crespi Perellino (1993)'

'H 'H 'H 'H 'H 'H 'H 'H

Inoue (1980)" Inoue (1980)" Powell (1990)" Powell (1990)" Stuchlik (1982)w Flieger (1984)x Ishii (1980)Y Ishii ( 1980)y

+ +

N~-AJI~I-LSD N6-Ethyl-LSD N6-Propyl-LSD 12-Hydroxy-LSD

200 MHz 200 MHz 300 MHz 300 MHz

90 MHz 90 MHz

'H 90 MHz 'H 90 MHz 'H 90 MHz 'H .90 MHz 'H 90 MHz 'H 90 MHz 'H 90 MHz 'H 60 MHz 'H 90 MHz 'H 90 MHz 'H 90 MHz 'H 90 MHz 'H 90 MHz 'H 90 MHz 'H 90 MHz

Nakahara (1977)' Nakahara (1977)' Nakahara (1977)' Nakahara (1977)' Nakahara (1977)' Nakahara (1977)" Nakahara (1977)' Vigevani (1971)ua Moretti-Rojas (1983)bb Moretti-Rojas (1983)" Moretti-Rojas (1983)*' Niwagauchi (1976)" Niwagauchi (1976)" Niwagauchi (1976)c' Siddik (1979)dd

262

C. J. GROOMBRIDGE

Table 6.-cont. Compounds

Spectrum

Ref.

6-Methyl-aceto~ymethyI-A~~'~-ergolene + N-oxide Ergosine Ergosinine Ergosine Ergosinine 1-Methylpropylamide LSD isomer Ergonovine maleate Bromocriptine mesylate Pergolide (2-(methylthio)agroclavine Agroclavine cis-Paspalic acid 10-Hydroxy-cis-paspalic acid amide 10-Hydroxy-trans-paspalic acid amide a-Ergocriptine Dihydroergotamine 2-Aza-dihydroergotamine

'H 200 MHz 'H, "C 'H, I3C 'H 'H 'H200 MHz 'H 100MHz, I3C ' H 90 MHz, l3C 'H 300MHz 'H 400 MHz, I3C 'H 400 MHz, I3C 'H 400 MHz, I3C 'H 400 MHz, I3C ' H 400 MHz, "C 'H360MHz 'H 360 MHz

Ballabio (1992)" Kidric (1985)'/ Kidric (1985p Hadzi (1987)gK Hadzi ( 1987yg Oberlender (1992)"h Reif (1982)" Giron-Forest (1979)" Tupper (1993)kk Flieger (1993)" Flieger (1993)" Flieger (1993)" Flieger (1993)" Spassov (1990)Stadler (1981)"" Stadler (1981)""

G . A. Neville, H. D. Beckstead, D. B. Black, B. A . Dawson and J.-C. Ethier, Can. J . Appl. Spectrosc., 1992, 37, 149. Y . Nakahara and T. Niwagushi, Chem. Pharm. Bull., 1971, 19, 2337. ' L. Pierri, I. H. Pitman, I. D. Rae, D. A. Winkler and P. R. Andrews, J . Med. Chem., 1982, 25, 937. A. F. Casy, J . Pharm. Biomed. Anal., 1994, 12, 27. " K. Seifert, H. Meyer, S. Hartling and S. Johne, Tetrahedron, 1989, 45, 7291. N. J. Bach, H. E . Boaz, E. C. Kornfeld, C.-J. Chang, H. G. Floss, E. W. Hagaman and E. Wenkert, J . Org. Chem., 1974, 39, 1272. K. Bailey and A. A. Grey, Can. J . Chem., 1972, 50, 3876. M. HuSak, B. Kratochvil, P. Sedmera, J. Stuchlik and A . Jegorov, Collect. Czech. Chem. Commun., 1993, 58, 2944. ' I. Ninomyia, C. Hashimoto, T. Kiguchi and T. Naito, J . Chem. Soc., Perkin Trans. 1, 1985, 941. j A . J. Hoffman and D. E. Nichols, J . Med. Chem., 1985, 28, 1252. K. Bailey, D Verner and D. Legault, J . Assoc. Off. Anal. Chem., 1973, 56, 88. L. Zetta and G. Gatti, Tetrahedron, 1975, 31, 1403. L. Zetta and G. Gatti, Org. Magn. Reson., 1977, 9, 218. " H. Sapper and W. Lohmann, Mol. Pharmacol., 1976, 12, 605. S. W. Bellman, J . W. Turczan and T. C . Kram, J . Forens. Sci., 1970, 15, 261. C. Szantay Jr., M. Bihari, J. Brlik, A . Csehi, A. Kassai and A. Aranyi, Acta Pharm. Hung., 1994, 64, 105. 4 T. Mills 111 and J. C. Roberson, Insfrumental Data for Drug Analysis, 2nd edn. CRC Press, Boca Raton, 1993. Y . Miyamoto, K. Washio, H. Nakashima, H . Tsutsui, R. Yanase and H. Azurna, Takubursu Dotai, 1993, 8, 1017. D. J. Sprankle and E. C . Jensen, Anal. Profiles Drug Subst. Excipients, 1992, 21, 375. ' N. Crespi Perellino, J. Malyszko, M. Ballabio, B. Gioia and A. Minghetti, J . Nar. Prod., 1993, 56. 489. I* T. Inoue, T. Niwaguchi and T. Murata, Xenobioiica, 1980, 10, 343. a

'

'

NMR SPECTROSCOPY IN FORENSIC SClENCE

263

" R . G. Powell, R. D. Plattner, S. G. Yates, K. Clay and A. Leuchtmann, J . Naf. Prod., 1990, 53, 1272. J. Stuchlik, A. Krajicek, L. Cvak, J . Spacil, P. Sedmera, M. Flieger, J . Vokoun and Z. Rehacek, Collect. Czech. Chem. Commun., 1982, 47, 3312. M. Flieger, P. Sedmera, J. Volkoun, Z. Rehacek, J. Stuchlik, Z. Malinka, L. Cvak and P. Harazim, J . Nar. Prod., 1984, 47, 970. H. Ishii, T. Niwaguchi, Y . Nakahara and M. Hayashi, J . Chem. Soc., Perkin Truns. 1, 1980, 902. ' Y. Nakahara, T. Niwaguchi and H. Ishii, Chem. Pharrn. Bull., 1977, 25, 1756. OU A. Vigevani and E . Gandini, Chim. Ind. (Milan), 1971, 53, 841. bb I. Moretti-Rojas, E. G . Ezrailson, L. Birnbaumer, M. L. Entman and A . J. Garber, J . Biol. Chem., 1983, 258, 12499. '' T. Niwagauchi, Y . Nakahara and H. Ishii, Yakugaku Zasshi, 1976, 96, 673. dd Z. H. Siddik, R. D. Barnes, L. G . Dring, R. L. Smith and R. T. Williams, Biochem. Pharmacol., 1979, 28, 3081. '' M. Ballabio, P. Sbraletta, S. Mantegani and E. Brambilla, Tetrahedron, 1992, 48, 4555. f f J. Kidric, D. Kocjan and D. Hadzi, Croat. Chem. Acta, 1985, 58, 389. gg D. Hadzi, J. Kidric, D . Kocjan and M. Hodoscek, J . Serb. Chem. SOC., 1987, 52, 617. R . Oberlender, R. C. Pfaff, M. P. Johnson, Xuemei Huang and D. E. Nichols, J . Med. Chem., 1992, 35, 203. j i V. D. Reif, Anal. Profiles Drug Subst., 1982, 11, 273. ji D. A. Giron-Forest and W. D. Schonleber, Anal. Profiles Drug Subst., 1979, 8, 47. kk D. E. Tupper, I. A. Pullar, J. A. Clemens, J. Fairhurst, F. C. Risius, G . H. Timms and S. Wedley, J . Med. Chem., 1993, 36, 912. " M. Flieger, P. Sedmera, V. Havlifek, L. Cvak and J. Stuchlik, J . Nut. Prod., 1993, 56, 810. m'x S. L. Spassov, M. F. Simeonov, B. P. Mikhova and P. S. Denkova, J . Mol. Struct., 1990, 217, 169. n n P. A . Stadler, E . Stiirmer, H. P. Weber and H.-R. Loosli, Eur. J . Med. Chem.-Chim. Therap., 1981, 16, 349.

of lysergic acid. Few drug substances can have had such a startling discovery as that for LSD in 1943, which has been vividly described by Hofmann.lgl Ergotamine is thought to be the most likely initial precursor for illicit synthesis. The abuse of LSD for its hallucinogenic quality became extensive in the 1960s, but declined after controls were introduced worldwide; it is a class A drug in Britain. However, in the latter part of the 1980s there was again a sudden surge in the use of the drug. Britain has been reported to have the LSD is currently encountered in two greatest demand for LSD in Europe. lY2 main forms: small tablets (microdots) and, more commonly, impregnated paper or card squares which usually bear a printed design (Fig. 6); over 200 designs are known. The potency of LSD is remarkably high: there may be a perceptible effect from 10 pg, and street samples are currently found to contain 25-100 p g per dose. The forensic analysis of LSD has a number of difficulties, because of its instability to light or high temperatures and moisture. It is well known

264

C . J. GROOMBRIDGE

Fig. 6. Example LSD paper square doses, from a police seizure in London in 1993. The printed motif resembled a postage stamp design, and alluded to the discovery of the hallucinogenic properties of LSD (see ref. 191).

that direct GC can suffer from unreproducibility. In England, analyses are carried out using HPLC. The low unit content of LSD is a potential problem for NMR, but detection of below 1Opg is feasible for 400MHz 'H using 1-2 h accumulation. 193 The NMR literature on ergot substances is quite extensive, but is rather scattered (Table 6). The industrial production has been concentrated in eastern Europe, and there has been a noticeable recent revival in ergot chemistry activity. This has included several full NMR spectroscopic interpretations, which add important background data to any forensic application. 'H spectra of LSD have been given in a few publications, but the only assignment is that suggested by Hoffman and which is directly based on the earlier highly detailed work of Bailey and Grey.195 The latter study examined the less potent (and thus safer) dimethylamide analogue using 220MHz homodecoupling measurements. It was found that the free base of this substance adopts a solution conformation which resembles that

NMR SPECTROSCOPY IN FORENSIC SCIENCE

265

found by crystallography for LSD iodobenzoate. Rings C and D both adopt a half-chair configuration, with the D ring in a “flap-up” mode (47); key evidence for this came from the coupling constants of H8 to the two H7 protons (although the amide methyl singlets partially obscured some of the central part of the spectrum). Published spectra of LSD free base31v196 indicate that the same interpretation is valid for LSD. The spectra are made more complex because the ergoline framework has significant long-range coupling pathways. 195 The flap-up conformation is unusual when compared to other ergot alkaloids, which invariably have the inverted D ring form, again as revealed by H8/H7 couplings. The reason is believed to be intramolecular hydrogen bonding. The C8 epimer isolysergic acid dimethylamide was found to exist in the more common flap-down conformation.195 Proton shift differences between lysergamides and isolysergamides were found to be small (0.2-0.3 ppm), with 0.2 ppm at the epimer centre (H8), but also similar shifts at the more distant H5 and H4P. This can be explained if the ring D flip is accompanied by inversion of the N6 lone pair. Proton spectra of LSD in aqueous solution are more poorly resolved than for the free base,’96 and it is apparent that there is some variation from sample to sample (Fig. 7).’96 LSD N6 protonation occurs at near-neutral pH (pK, 7.5),19’so presumably the variation reflects small changes in pH. These proton NMR data were also important in revealing a problem with a supposedly pure reference standard of LSD tartrate.196 Proton peak intensities showed that this sample contained a stoichiometric excess of tartrate (65%). It is customary for drug analysis laboratories to use certified commercial samples for the calibration of quantitative analyses. Clearly, such a sample would lead to the overestimation of LSD quantity, but NMR does provide a calibration-free method by which most substances can be compared to a common standard. 13Cspectra of LSD (free base and tartrate) have been reported by Neville

266

C . J. GROOMBRIDGE LSD BASE / CDCL3

7.0

6.0

5.0

4.0 PPY

FROM

3.0

LSO TARTRATE /I 020

USP LOT I

2.0

USP

1.0

0.0

LOT I

HOD

A

/ PPH Fig. 7. 'H NMR spectra of LSD (46): (a) LSD base in CDCl,; (b) LSD tartrate USP Lot 1 in D,O; (c) LSD tartrate Sandoz Lot 79001 in D20. (Reproduced with permission from Neville et al. 196)

NMR SPECTROSCOPY IN FORENSIC SCIENCE

267

(c) N. H3

1

7.0

6.0

5.0

4.0

PPM

3.0

2.0

i.0

0.0

Fig. 7.-contd.

et al. ,Ig6 and there were earlier partial data and assignments given by KidriE and Kocjan.”’ Shift differences between LSD and iso-LSD were up to 5.5 ppm. As discussed for other drug substances, forensic evidence is sometimes challenged in court by an isomer defence. In the USA in the early 1980s it was realized that existing chromatographic analytical techniques would indeed have problems in distinguishing between LSD and non-controlled isomer, lysergic acid methylpropylamide (LAMPA). It is not clear from the scientific literature if this was actually used in court; both LSD and LAMPA are controlled substances in Britain. In consequence, there was vigorous effort to develop improved methods, and several chromatographic schemes were found. MS fragmentation patterns were shown to have significant small and some NMR data have also been reported. 1y5~196,200 As differences, lYy expected, the ‘H spectra give a simple distinction between LSD and the methyl-n-propylamide. There have been no data for the alternative methyliso-propyl isomer. Some of the procedures for the synthesis of LSD cause the epimerization, so that unless the illicit synthesis has been followed by the separation of LSD and iso-LSD, both substances will appear in the final product. Lysergic

268

C . J. GROOMBRIDGE

acid may be isolated in clandestine laboratories, but the only published NMR spectrum of this compound appears in the extensive Mills and Roberson ~ol l e c tio n .~ ' NMR spectra have been reported for a range of other ergot alkaloids (Table 6). Pierri et ~ 1 . ~ "gave a detailed 'H spectral analysis of ergotamine and its C8 epimeric counterpart, ergotaminine, and discussed the factors controlling the conformation. Recently, Casy202 has given a highly detailed study of a range of ergot derivatives, with the emphasis on compounds which are in therapeutic use (ergotamine, ergocristine, ergocriptine, ergometrine, methysergide, bromocriptine, pergolide, dihydroergotamine, dihydroergocristine and dihydroergocornine). This report highlighted the features in the 'H spectra which are of value for rapid identification of these substances; this was explicitly phrased as a diagnostic scheme for the identification of substances as in the forensic context. Sample quantities of approximately 10mg were used, which is rather larger than would be forensically useful, but this can be reduced to microgram levels without excessive accumulation time. 193 It was also noted2'* that, in general, proton spectra of the N6 ammonium salt forms often gave relatively poorly resolved spectra, compared to those of the free bases; assignments were still, however, achievable using COSY-45. The most probable cause was considered to be conformation exchange of an appropriate time-scale. A companion paper203 has also discussed MS techniques, which are more familiar to forensic drug analysts. Relatively little chemistry related to LSD has been published in recent years, because of controls on these substances and precursors, and medical applications have ceased. However, some exploratory synthetic work was continued in Japan, particularly through N6 demethylation and s ~ b s t i t u t i o n Proton . ~ ~ ~ NMR ~ ~ ~ was routinely used for product characterization. Some derivatives have also been synthesized by Nichols and N6 alkyl substitutions and branched and ringc o - w ~ r k e r s , ' " ~including ~~~ closed dialkylamides. 'H NMR data were reported. Given the relatively low sensitivity of NMR, it is perhaps surprising to find that there have nevertheless been several experiments which have determined the structures of LSD metabolites, albeit by in vitro cell culture. This is still a relatively poorly understood area because of the low active dose. The 'H spectrum is directly indicative of typical metabolic processes (N-demethylation and N-deethylation, aromatic hydroxylation). Siddik et al. 209 synthesized 12-hydroxy-LSDt and identified the aromatic substitution position using 'H NMR. This substance was then identified by chromatographic comparison as a metabolic product in perfused rat liver. Ishii et ~ 1 . ~ "identified lysergic acid ethylvinylamide and lysergic acid ethyl(2-hydroxyethy1)amide as microbial cell culture metabolites. A further metabolite, 13-hydroxy-LSD, was identified by MS and 'H NMR on

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material isolated by preparative chromatography of liver culture extracts,211 although this was described as a large-scale experiment. Many other indole alkaloids are also known to have hallucinogenic effects and have been abused. and Fzve thus been deemed necessary to regulate. These include a number of tryptamine derivatives, either of natural origin or produced synthetically (Table 7). The best known natural substances are psilocin (48) and psilocybin (49) (from Psilocybe mushrooms), and bufotenine (50) (a major component in the skin venom of several toad species) .212 Illicit drug material has shown a recent increase, particularly in New York as described by Chamakura.212 Psilocin and bufotenine are isomers, and give rather similar MS fragmentation patterns, but can be distinguished from small differences in GC retention time. These isomers should be distinguishable by the 'H NMR coupling patterns, however the only bufotenine spectrum3' is unclear (it is basically only solvent peaks). Many other indole spectra ('H and I3C) have been reported as part of research into natural product isolation. This includes 13C shift data of and 'H spectra of N , N N-methyl- and N,N-dimethyltryptamine,213 d i m e t h y l t r ~ p t a m i n e .A ~ ~very ~ extensive review of indole 13C NMR has been given by M ~ r a l e s - R i o s , ~and ' ~ Ranc and Jurs216 have developed models for the prediction of 13C shifts for this class of compounds.

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Table 7. NMR spectra of selected indole alkaloids. Compound

Spectrum

Ref.

Dimethyltryptamine Bufotenine 5-Hydroxytryptamine Gramine Dimethyltryptamine Diethyltryptamine Ethyltryptamine 3-Indolyglyoxyldimethylamide (tryptamine precursor) 3-Indolylglyoxyldiethylamide (tryptamine precursor) Dimethyltryptamine Diethyltryptamine Psilocin Psilocybin 3-Indol ylglyoxylpyrrolidide N - [l-Hydroxy-2-(3-indolyl)ethyl]pyrrolidine N[2-(3-Indolyl)e thyllpyrrolidine 1-(N,N-Diethylamino)-2-(3-indolyl)ethan1-01 Grarnine N,N-Dimethyltryptamine N,N-Dimethyltryptamine N,N-Dimethyltryptamine N-Methyltryptamine

'H 'H 'H 'H 'H 'H 'H

Sapper (1976)" Sapper (1976)" Sapper (1976)u Sapper (1976)" Bellman (1970)' Bellman (1970)b Bellman (1970)'

N-Formyl-N-methyltryptamine Strychnine Strychnine 22-Hydroxystrychnine (metabolite)

100MHz 100MHz 100MHz 100MHz 60MHz 60 MHz 60 MHz

'H 60 MHz

Bellman (1970)'

'H 'H 'H 'H 'H 'H 'H 'H 'H I 'C

Bellman (1970)' Ono (1979)' Ono (1979)' Ono (1979)' Ono (1979)' Cowie (1982)" Cowie (1982)" Cowie (1982)" Cowie (1982)" Wenkert (1974)' Wenkert (1974)' Reyna Pinedo (1994)' Poupat (1976)g Poupat (1976)r Poupat (1976)g Blechta ( 1976)h Craig (1986)' Tanimoto (1991)'

60 MHz 60 MHz 60 MHz 60 MHz 60 MHz 60 MHz 60 MHz 60 MHz 60 MHz

I3C 'H 200 MHz 13C "C 'H 60 MHz, I3C ' H 400 MHz, 13C 'H 400 MHz ' H 270 MHz

* H. Sapper and W. Lohmann, Mol. Pharmacol., 1976, 12, 605. ' S. W. Bellman, J. W. Turczan and T. C. Kram, J . Forens. Sci.,1970, 15, 261. M. Ono, Nippon Hoigaku Zasshi, 1979, 33, 339. " J. S. Cowie, A. L. Holtham and L. V. Jones, J . Forens. Sci., 1982, 27, 527. ' E. Wenkert, J. S. Bindra, C.-J. Chang, D. W. Cochran and F. M. Schell, Acc. Chem. Res., 1974, I, 46. V. Reyna Pinedo and V. Torpoco Carmen, Bol. SOC. Quim. Peru, 1994, 60, 21. C. Poupat, A. Ahond and T. SCvenet, Phycochem., 1976, 15, 2019. '*V. Blechta, F. del Rio-Portilla and R. Freeman, Magn. Reson. Chem., 1994, 32, 134. ' D . A. Craig and G. E. Martin, J . Nar. Prod., 1986, 49, 456. ' Y. Tanimoto, T. Ohkuma, K. Oguri and H. Yoshimura, Xenobiotica, 1991, 21, 395.

A highly unusual tryptamine substance (tetramethylene tryptamine (51)) was identified by Cowie et al?' using MS and 'H NMR. A second component was identified as N-[ 1-hydroxy-2-(3-indoly1)ethyl]pyrrolidine, which was demonstrated to be an intermediate from incomplete reduction. The analogous impurity for diethyltryptamine was also prepared.

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Strychnine, the major alkaloid of Strychnos nux-vornica, is also a complex indole which impinges on forensic chemistry: intermittently as a poison, although it has been found as a diluent in illicit heroin. Extensive NMR data have been published, although not for analytical reasons but because the complex polycyclic structure provides a challenging test for modern 'H and 13C technique^.^^^^^'^ An early NMR method has been given for the assay of strychnine in S . nux-vomica seeds.220NMR has also been used to identify a new metabolite, 22-hydroxystrychnine, isolated from guinea-pig liver microsomes. 221

2.6. Fentanyls The abuse of fentanyl compounds as heroin-like substitutes began in 1979 in the USA, and some of the events surrounding this problem have been reviewed by Kram et ~ 1 and. Henderson.223 ~ ~ ~ Fentanyl, N(l-phenethyl-4piperidy1)propionanilide (52), is a narcotic analgesic which is widely used as a surgical and veterinary anaesthetic. This substance, and a range of derivatives, are extremely potent, with activities over 100 times greater than morphine, and a key factor is that respiratory depression occurs at low dosage. The first indications of this form of drug abuse were from a large number of fatalities which had every indication of opiate overdose, but the cause could not be found at that time from post-mortem toxicology or drug

Q

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(53) China White

paraphernalia examination. These drug overdoses were soon related to drug material being sold as “China White” or occasionally “synthetic heroin” or “super heroin”, which presented a particularly urgent and difficult analytical chemistry problem. The active substance in China White was identified as a-methylfentany1222 (53) using the combination of GC-MS and ‘H NMR. The NMR data (Fig. 8) were particularly important for establishing the structure as the a-methyl derivative, and this is arguably the most important instance of the use of NMR in forensic science. Additional NMR data were later given by Suzuki et al.224 Similar substances had been prepared and patented by J a n ~ s e n , ~ ~ ~ but not produced for pharmaceutical use, consequently it was not encompassed by US drug laws at that time. a-Methylfentanyl was placed on the schedules of controlled substances in 1981, but a series of further fentanyl designer drugs continued to appear in 1981-1985 ,223 and an extensive analytical study has been prepared by Cooper et a1.226Clandestine laboratories continue to be found in the USA. Over 100 deaths have been shown to be due to fentanyl analogue abuse.227 Other NMR data are collected in Table 8. As a digression, NMR piayed no part in the identification of a different type of synthetic opiate, N-methyl-4-phenyl-4-propoxypiperidine(MPPP) (54), which also caused serious problems in the 1 9 8 0 because ~ ~ ~ ~ of the contamination with the impurity N-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) (55), which was found to. induce Parkinson’s disease. ‘H and I3C spectra have been subsequently given by Casy et a1.22y

2.7. Phencyclidine and related substances

Phencyclidine (1-[1-phenylcyclohexyl]piperidine, PCP) (56) is an analgesic which was marketed by Parke Davis from 1958 to 1967 as an anaesthetic,

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273

4 7.0

5.0

4.5

3.5

3.0

2.5

2.0

1.5

1.0

(PPm) 6

Fig. 8. ‘H NMR spectrum (200MHz) of hydrochloride salt prepared from alkaline extract of a “China White” exhibit (a-methylfentanyl (53). (Reproduced with permission from Kram et ~ 1 . ~ ~ ’ )

I

Me

but was withdrawn from medical use as it became apparent that there were unpredictable adverse effects. It is as a hallucinogen and stimulant that it became a drug of abuse from about 1967, primarily in North America, although it seems mercifully rare so far in Europe. It has also become apparent that it has neurotoxic effects, which can lead to “a psychosis clinically indistinguishable from s c h i ~ o p h r e n i a ” . ~ ~ ~

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Supplies of phencyclidine for the illicit drug market come from clandestine laboratories, and it may be the task of forensic chemists to assess such laboratories when they are found by law enforcement agencies. Allen et af.231have recently reviewed the chemistry of the synthesis of phencyclidine and its analogues. The supply of the potential precursor chemicals is monitored, and in consequence an extensive range of analogues have also appeared as street drugs-usually, of course, without the knowledge of the drug user. Table 8.

NMR spectra of fentanyls.

Compound

Spectrum

Ref.

a-Meth ylfentanyl Fentanyl a-Methylfentanyl p-Fluorofentanyl cis-3-Methylfentanyl 2-Methylfentanyl N - [1-3-Phenylpropyl)-4-piperidyl]-Npheny lpropanamide N - [1-(2-Phenylethyl)-4-piperidylI-Nphenylacetamide 3-Methylfentanyl a,3-DimethylfentanyI cis-3-Methylfentanyl trans-3-Methylfentanyl a-Methylfentanyl Fentanyl Fentanyl acetyl analogue Fentanyl methyl analogue Fentanyl methyl acetyl analogue Fentanyl 1-m-methylphenyl analogue Fentanyl 1-m-methylphenyl acetyl analogue Fentanyl 1-o-methylphenyl acetyl analogue Fentanyl I-p-methylphenyl analogue Fentanyl I-p-methylphenyl acetyl analogue Fentanyl propyl analogue Fentanyl propyl acetyl analogue Fentanyl o-tolyl analogue Fentanyl m-tolyl analogue Fentanyl p-tolyl analogue Fentanyl o-tolyl analogue Fentanyl m-tolyl acetyl analogue Fentanyl p-tolyl acetyl analogue Sufentanil Sufentanil citrate

'H 'H 'H 'H 'H 'H

200MHz 200 MHz 200 MHz 200 MHz 200MHz 200 MHz

Kram (1981)" Cooper (1986)' Cooper (1986)' Cooper (1986)' Cooper (1986)' Cooper (1986)'

'H 200 MHz

Cooper (1986)'

'H 'H 'H 'H 'H 'H 'H 'H 'H 'H 'H

Cooper (1986)' Van Bever (1974)' Van Bever (1974)' Suzuki ( 1986)" Suzuki (1986)" Suzuki (1986)" Mills (1993)' Mills (1993)' Mills (1993)' Mills (1993)' Mills (1993)'

200MHz 100MHz 60 MHz 100MHz 100MHz 100MHz, I3C 300 MHz 300 MHz 300 MHz 300 MHz 300MHz

' H 300 MHz

Mills (1993)'

'H 300 MHz 'H 300MHz

Mills (1993)' Mills (1993)'

'H 'H 'H 'H 'H 'H 'H 'H 'H 'H 'H

Mills (1993)p Mills (1993)' Mills (1993)' Mills (1993)p Mills (1993)' Mills (1993)' Mills (1993)' Mills (1993)' Mills (1993)' Mills (1993)p Mills (1993)'

300MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300MHz

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Table 8.-cont. Compound

Spectrum

Alfentanil Carfentanil a-Methylfentanyl (China White) P-Methylfentanyl a-Methylfentanyl acetyl analogue p-Fluorofentanyl 3-Fluorofentanyl analogue 2-Fluorofentanyl acetyl analogue 3-Fluorofentanyl acetyl analogue

'H 300 MHz 'H 300 MHz 'H 300 MHz ' H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz 'H 300 MHz

Ref Mills (1993)" Mills (1993)' Mills (1993)' Mills (1993)' Mills (1993)" Mills (1993)' Mills (1993)" Mills (1993)' Mills (1993)'

T. C. Krarn, D. A. Cooper and A. C. Allen, Anal. Chem., 1981, 53, 1379A. D. Cooper, M. Jacob and A. Alle, J. Forens. Sci., 1986, 31, 511. W. F. M. Van Bever, C. J. E. Niemegeers and P. A. J. Janssen, J. Med. Chem., 1974, 17, 1047. S. Suzuki, T. Inoue and C. Kashirna, Chem. Pharm. Bull., 1986, 34, 1340. ' T. Mills 111 and J. C. Roberson, Instrumental Data for Drug Analysis, 2nd edn. CRC Press, Boca Raton, 1993.

Several NMR studies have focused on basic chemistry rather than forensic identification, particularly on the conformational equilibrium of modified phencyclidines, in the hope that its receptor binding could be controlled. A range of NMR data is summarized in Table 9. The two saturated rings give rise to fairly complex overlapping resonances have attempted to unravel. in the 'H spectrum, which only Eaton et dZ3'

(57)

They have also shown that there are molecular motions which broaden the spectrum of the free base at room temperature. The I3C spectrum was relatively easily u n d e r ~ t o o d , ' and ~ ~ it has been found that the chemical shifts are indicative of the phenyl ring configuration233 which is solvent- and temperature-dependent . The products of clandestine phencyclidine synthesis are known often to be impure, with a considerable residue of the toxic intermediate, 1piperidinocyclohexanecarbonitrile(PCC) (57). This has also been characterized by p r ~ t o n ' ~ ~ , and ' ~ ' carbon NMR.23X 2349235

276

C. J . GROOMBRIDGE

Table 9. NMR of phencyclidine and analogues. Compound

Spectrum

Ref.

Phencyclidine (PCP) t-Butylphencyclidine Phencyclidine Methylphencyclidine t-Butylphencyclidine Phencyclidine Tenocyclidine (TCP) 1-Piperidinocyclohexanecarbonitrile (PCC, precursor) 1-(1-Cyanocyclohexy1)piperidine 1-(1-Cyanocyclopenty1)piperidine 1-(1-Cyanocyclohexyl)pyrrolidine 1-( 1-Cyanocyclopenty1)pyrrolidine 1-(1-Cyanocyclohexy1)ethylamine 1-(1-Cyanocyc1opentyl)ethylamine 1-(1-Cyanocyclohexy1)morpholine 1-(1-Cyanocyclopenty1)morpholine

'H 60 and 100 MHz 'H 60 and 100 MHz 13C 13C 'H (brief) 'H (brief)

Mousseron (1968)p Mousseron (1970)' Geneste (1975)' Geneste (1975)' Geneste (1975)' Bailey (1976)d Bailey ( 1976)d

'H (brief) 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz 'H 60 MHz

Bailey (1976)' Gagne (1977)' Gagne (1977)' GagnC (1977)' Gagne (1977)' GagnC (1977)' GagnC (1977)' Gagne (1977)' Gagne (1977r

'3C

N-Ethyl-1-phenylcyclohexylamine (PCE, cyclohexamine) 'H 60 MHz N-Alkyl-1-phenylcyclohexylamine 'H 80 MHz (brief) I3C Phencyclidine 13C Range of analogues Phencyclidine 'H 60 MHz, 13C Tenocyclidine 'H 60 MHz, I3C Range of analogues 'H 60 MHz, I3C I3C Phencyclidine 1 3 c Tenocyclidine I3c Cyclohexamine Range of analogues I3C 1-( 1-Piperidiny1)cyclohexanecarbonitrile 13C (PCC) I3C Range of precursors 1-[1-(1,l '-Biphenyl-4-yl)cyclohexyl]piperidine 'H 100MHz 1-[1-(Phenylethyl)cyclohexyl]piperidine 'H 100MHz 1,1'-( 1,4-PhenylenedicyclohexyIidene)'H 1OOMHz bis[piperidine] 1-( l-Phenylcyclohexyl)-4-methylpiperidine 'H 60 MHz, I3C Phencyclidine 'H 300 MHz Nitrophencyclidine 'H 300 MHz Adamantyl analogue 'H 300 MHz Phencyclidine I3C 13C 4-Methylphencyclidine 4-Methylphencyclidine 'H 60 MHz 4-Fluorophencyclidine 'H 60 MHz 4-Methoxyphencyclidine 'H 60 MHz 4-Chlorophencyclidine 'H 60 MHz 4-Meth ylthiophencyclidine 'H 60 MHz 2,3-Benzo analogue 'H 60 MHz Range of analogues 13C

Bailey (1978)bh Bailey (1979)g Brine (1979)h Brine (1979)h Geneste (1979)' Geneste (1979)' Geneste (1979)' Bailey (1980)' Bailey (1980)' Bailey (1980)' Bailey (1980)' Bailey (1981)& Bailey (1981)& Jones (1981)' Jones (1981)' Jones (1981)' Soine (1982)"' Eaton (1983)" Eaton (1983)" Eaton (1983)" Manoharan (1983)" Manoharan (1983)O Costa (1983)p Costa (1983)p Costa (1983)" Costa (1983)p Costa (1983)p Costa (1983)p Brine (1983)q

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Table 9.-cont. Compound Range of analogues N-Ally1 analogues Hydroxyl analogues Ketamine Phencyclidine Tenocyclidine Range of analogues Phencyclidine Phencyclidine Tenocyclidine

N-Ethyl-1-phenylcyclohexylamine N-Cyclohexylidene ethylamine N-Ethylcyclohexanecarbonitrile 2-Methyl analogue 3-Methyl analogue 4-Methyl analogue Benzyl analogue Phencyclidine 1-Phenylcyclidine-N-ethylanalogue Phenylcyclidine-4-hydroxyanalogue Phencyclidine morpholine analogue Phencyclidine pyrrolidine analogue 1-Phencyclohexylamine N-Phencyclopentylamine Phency clopentylpyrrolidine Piperidinocyclohexane carbonitrile 1-[1-(2-Thienyl)cyclohexyl]morpholine 1-[ 1-(2-Thienyl)cyclohexyl]piperidine 1-[1-(2-Thienyl)cyclohexyl]pyrrolidine

Spectrum

Ref.

1 3 c

'H 80 MHz I3C 1 3 c

'3C '3C 'H 300 MHz I3C solid I3C 1 3 c

'H 'H 'H 'H 'H 'H 'H 'H 'H 'H 'H 'H 'H 'H 'H 'H 'H 'H 'H

90 MHz 90 MHz 90 MHz 80 and 400 MHz, 80 and 400 MHz, 80 and 400 MHz, 80 and 400 MHz, 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz 300 MHz

300MHz

13C I3C 13C I3C

Brine (1984)' Kalir (1984)" Kamenka (1985)' Kamenka (1985)' Kamenka (1987)" Kamenka (1987)" de Costa (1988)" Carroll (1988)w Kamenka (1988)" Kamenka (1988)" Krefft (198Y)y Krefft ( 1989)y Krefft ( 198Y)y Lodge (1992)" Lodge (1992)' Lodge (1992)' Lodge (1992)" Mills (1993)"" Mills (1993)a" Mills (1993)a" Mills (1993)"" Mills (1993)"" Mills (1993)aU Mills (1993)" Mills (1993)"" Mills (1993)"" Mills (1993)"" Mills (1993)"" Mills (1993y

M. Mousseron, J.-M. Bessiere, P. Geneste, J.-M. Kamenka and C. Marty, Bull. Soc. Chim. Fr., 1968, 3803. M. Mousseron, J.-M. Kamenka and M. R. Darvich, Bull. Soc. Chim. Fr., 1970, 1435. ' P. Gcneste and J.-M. Kamenka, Org. Magn. Reson., 1975, 2, 579. K. Bailey, D. R. Gagnt and R. K. Pike, J . Assoc. Off. Anal. Chem., 1976, 59, 81. K. Bailey, A. Y.K. Chow, R. H. Downe and R. K . Pike, J . Pharrn. Pharmacol., 1976, 28, 713.

D. R. Gagne and R. K. Pike, J . Assoc. Off. Anal. Chern., 1977, 60, 32. K. Bailey and D. Legault, J . Assoc. Off. Anal. Chem., 1979, 62, 1124. G. A. Brine, E. E. Williams, K. G. Boldt and F. I . Carroll, J . Heterocyclic Chem., 1979, 16, 1425. ' P. Geneste, J.-M. Kamenka, S. N. Ung, P. Herrmann, R . Gouda1 and G . Trouiller, Eur. J . Med. Chem.-Chim. Therapeut., 1979, 14, 301. K. Bailey and D. Legault, Anal. Chim. A c f a , 1980, 113, 375. K. Bailey and D. Legault, Org. Magn. Reson., 1981, 15, 68. L. A. Jones, R. W. Berver and T. L. Schmoeger, J . Org. Chem., 1981, 46, 3330. "' W. H. Soine, R. L. Balster, K. E. Berglund, C. D . Martin and D. T. Agee, J . Anal. Toxicof.,1982, 6 , 41. T. A. Eaton, K. N. Houk, S. F. Watkins and F. R. Fronczek, J . Med. Chern., 1983, 26, 479.

f

6

'

'

278

C. J. GROOMBRIDGE

Table 9.-cont.

" M. Manoharan, E. L. Eliel and F. I . Carroll, Tetrahedron Lett., 1983, 24, 1855. P

J . F. Costa and T. J . Speaker, J . Anal. Toxicol., 1983, 7 , 252.

G. A. Brine, K. G . Boldt, M. L. Coleman, E. E. Williams, T. M. Krcelic and F. I . Carroll, Phencyclidine arid Related Arylcyclohexylamines: Present and Future Applications (ed. J. M. Karnenka, E. F. Domino and P. Geneste). NPP Books, Ann Arbor, 1983. G . A. Brine, K. G. Boldt, M. L. Coleman and F. I . Carroll, J . Heterocyclic Chem., 1984, 21, 71. ' A. Kalir, S. Teomy, A. Amir, P. Fuchs, S. A. Lee, E. J. Holsztynska, W. Rocki and E. F. Domino, J . Med. Chem., 1984, 27, 1267. ' J.-M. Kamenka, M. Michaud, P. Geneste, J . Vignon and R. Chicheportiche, Eur. J . Med. Chem.-Chim. Ther., 1985, 20,419. '' J.-M. Kamenka and R. Chicheportiche, Eur. J . Med. Chem., 1987, 22, 193. " B. R. de Costa, C. George, T. R. Burke, M. F. Rafferty, P. C. Contreras, S. J. Mick, A. E. Jacobson and K . C . Rice, J . Med. Chem., 1988, 31, 1571. F. I. Carroll, G. A. Brine, K . G. Boldt, S. W. Mascarella, C. G. Moreland, S. J. Sumner, J. P. Burgess and E. 0. Stejskal, Sigma and Phencyclidine-Like Compounds as Molecular Probes in Biology (ed. E. F. Domino and J.-M. Karnenka). NPP Books, Ann Arbor, 1988. J.-M. Karnenka and R. Chicheportiche, Sigma and Phencyclidine-Like Compounds as Molecular Probes in Biology (ed. E. F. Domino and J.-M. Kamenka). NPP Books, Ann Arbor, 1988. R. H. Krefft, S. S. Masumoto and T. V. Caldwell, J . Forens. Sci., 1989, 34, 1266. ' B. A . Lodge, R. Duhaime, J . Zarnecnik, P. MacMurray and R Brousseau, Forens. Sci. Int., 1992, 55, 13. T. Mills I11 and J. C. Roberson, Instrumental Data for Drug Analysis, 2nd edn. CRC Press, Boca Raton, 1993. hh K. Bailey, J . Pharm. Sci., 1978, 67, 885 'I

Jones et al.239have carried out a detailed study of the Kalir phencyclidine synthesis process using HPLC, which indicated 13 significant impurities in typical PCP products. Using preparative chromatography three compounds were isolated for MS and 'H NMR identification. Analytical measurements have been compiled for phencyclidine analogues as these have been encountered (e.g. cyclohexamine (58) and tenocyclidine (59)), and in some instances additional substances have been prepared and examined in an attempt to anticipate future trends. Much of the forensic

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characterization of new analogues has been carried out in Canada (Drugs Directorate of Health Canada, Ottawa), where there has been a regional problem with these substances. The structural variants have involved alterations to each of the three phencyclidine rings,24” as in the original commercial research. The nitrile intermediates have also been e ~ a m i n e d . ~More ~ ” ~ ~recent ~ street drugs have been shown to have alkyl-substituted phenyl or piperidine rings.2413242 Even low-field proton NMR spectra give good distinction between phenyl and thienyl analogues,240 and aromatic/aliphatic peak integrals were of use for reasonably pure substance^.^^^.^^ There do not seem to be any subsequent high-field analyses, although an extensive set of spectra are given by Mills and R ~ b e r s o n . ~ An ’ extensive collection of 13C spectra have been omp piled,'^^-^^' and give full discrimination between any of these substances, but the relatively low sensitivity has meant that this is of most use for the authentication of reference substances, or for an unexpected new appearance. Two further related substances, ketamine and tiletamine, also appear in drug seizures, even in Britain where PCP is rare, and are thought to originate from the diversion of medical or veterinary anaesthetic supplies. Some NMR data are listed in Table 9.

2.8. Quinazolinones (methaqualone)

A number of quinazolinone substances are known as drugs of abuse, of which methaqualone (60) and mecloqualone (61) have been the most frequently encountered in forensic laboratories. Both substances are manufactured as proprietary pharmaceuticals, classed as hypnotics. A third substance, nitromethaqualone, has also been marketed in Europe. Some illicit supplies originate from the diversion of prescription tablets, but in the USA there have been several instances of clandestine synthesis.2628 Methaqualone has, in the past, often been found as a diluent in illicit heroin samples. Daenens and Van B ~ v e n ~gave ~ ’ IR, MS and ‘H NMR spectra for these ’ ~ ~ prepared four series of three main quinazolinones. Dal Cason et ~ 1 . have

280

C . J. GROOMBRIDGE

methaqualone analogues: each isomer for methyl, fluoro, chloro, bromo and iodo monosubstituted compounds. MS, IR, and 'H NMR were reported. Each of the compounds gave visually different 90 MHz spectra, although these were too complex to be interpreted. Angelos and MeyersZ5' have progressed to an examination of the possible substances encountered in clandestine methaqualone synthesis. ClarkZ5l has described the analytical characterization of four nitromethaqualone isomers, including 'H NMR. The usual ortho shift effect of a nitro aromatic substituent leads to moderately well-resolved 90 MHz 'H spectra. ~ ' described the identification of a new Recently, Angelos et ~ 1 . ~have methaqualone analogue from a drug seizure: 2-methyl-3-(2,4-dimethylphenyl)-4(3H)-quinazolinone (methyl methaqualone) (62).

(62)

The benefit of higher-field (300 MHz) Fourier transform NMR was evident, in that well-resolved first-order coupled resonances were obtained. Two-dimensional COSY was used in the normal manner to correlate coupled resonances. Since the 'H spectrum indicated the substitution was not symmetrical, only two permutations remained, and NOESY correlations then unambiguously confirmed structure (62), rather than the 2,5dimethylphenyl isomer. Final confirmation was made by synthesis and the usual MS, but the authors pointed out the improved efficiency in eliminating the need for the synthesis of all of the possible isomers. Current state-of-theart for the elucidation of new structures by NMR would probably suggest the use of I3C spectra, plus one-bond and long-range 13C-lH correlation spectra, although forensic casework samples may not always permit an adequate sample quantity. 2.9. Anabolic steroids

Anabolic steroids have been a problem in sports regulation for many years, and there has been growing concern for the harmful effects of their more widespread misuse. In consequence, various countries have now included these substances within the scope of drug abuse law. In Britain, the process of bringing anabolic steroids under the control of the MoDA was initiated in October 1994, but depends on Parliamentary approval in the near future. It

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is additionally proposed that polypeptide hormones (e.g. human chorionic gonadotrophin) will also be controlled, plus the anabolic and stimulant clenbuterol. GC-MS is the most important technique for steroid metabolite detection in sports medicine, but the new controls will mean that bulk quantities of steroids must be analysed, and there is no reason why NMR should not be used when GC-MS has difficulties, and it should not be overlooked as a potential quick identification technique with inherently good capacity to recognize “designer” modifications. Extensive 13C spectroscopic data of steroids have been reviewed by Blunt and st other^^'^ and by Smith.254 Full assignments are generally easily achieved. Proton spectra are considerably more complex, but the introduction of two-dimensional IH-’H and 13C-lH techniques has led to the full interpretation of the spectra of many steroids (e.g. see refs 255-258). The highly coupled “methylene envelope” is surprisingly informative as a visual pattern. Kirk et al.257 have extensively discussed the analytical value of ‘H NMR, and have stated that “AS a ‘finger-printing’ method, we now regard high-field NMR as more detailed and reliable than the long-established IR spectroscopy.” This refers to the different problem of steroid natural product isolation, but they did also note that contaminated samples could still be identified. So far, there are very few instances of the consideration of NMR for forensic steroid analysis. Bailey et ~ 1 . and ~ ’ Urich ~ et ~ 1 . ~ included ~’ ‘H data with their MS analysis of 1Bmethylprednisone acetate. Chiong et a1.261gave ‘H spectra for three anabolic steroids (nandrolone stanozolol and testosterone) but without comment. Fourier transform IR was nominated as the favoured technique for the few steroids which did not behave well in GS (hydrocortisone, prednisolone oxymetholone and stanozolol). Proton spectra of a number of steroids have now been added to the Mills and Roberson series.31 Spectra have recently been reported for danazol and a minor isomeric contaminant;262 but this does not correlate with the library spectrum.31 NMR has occasionally been used as an adjunct to GC-MS for the elucidation of the structure of steroid metabolites (e.g. see ref. 263), through the synthesis of proposed species. More frequently, only MS data are obtained, and some proposed structures are considered only tentative.264 2.10. Miscellaneous drug substances The pz agonist clenbuterol (63) has also been included in the list of suggested controlled substances, since it is also used to enhance muscle bulk. It is known to have been used in livestock production, and has already been prohibited for this use in the European Community. This has led to the

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use of related compounds (cimaterol, mabuterol and salbutamol) as replacements, and Leyssens et ul.265 have reported the identification of five completely new substances which have appeared as “designer” analogues. The structure elucidation was achieved using MS and ‘H NMR.

4-Hydroxybutyric acid (y-hydroxybutyric acid (GHB, or “GBH”)) is a natural low-level human metabolite, but has also been noted as an abused substance. It is not, however, controlled by drug legislation in Britain. The reason for the abuse is somewhat confused: partly it has been sold to bodybuilders as an anabolic agent (although there is no medical evidence for this), and it has also been suggested that it induces an “Ecstasy”-like euophoric effect, and has been sold as a “club” drug. “GBH” samples have been submitted to this laboratory for identification. Proton and 13C data for GHB sodium salt are given in the Aldrich text.83 Underground literature describes the preparation of GHB from y-butyrolactone, for which spectroscopic data are also a~ailable,’~ and as is well known, GHB will spontaneously revert to the lactone in aqueous solution. Both substances are available commercially.

3. TOXICOLOGY, BODY FLUIDS Toxicology is one of the best known aspects of forensic science, with a particular general fascination with historical accounts of poisonings. The modern forensic toxicology laboratory is concerned less often with poisons than with the involvement of drugs in incidents such as murder, sexual assault or other crime.’ Thus, forensic toxicologists will examine body fluids and tissues to detect and measure drugs or poisons in a range of cases. This also includes the assessment for alcohol level of blood or urine from motorists who are suspected of Road Traffic Act drink-driving offences. The concentrations of target substances vary quite widely; from tens of milligrams per millilitre for ethanol in blood, to nanograms per millilitre for drugs or their metabolites, reaching perhaps 10-100 pg/ml in extreme cases of overdose or of body-packing (drug trafficking by concealment in the stomach or other body cavity).

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Typical analytical methods are immunoassay for preliminary screening, and chromatography (HPLC, GC or GC-MS) for confirmation,’ since these have the high sensitivity to be able to detect small quantities of substances in complex biological matrices. These are generally beyond the sensitivity limitation of even state of the art NMR spectroscopic equipment. Nevertheless, there is a great deal of current research activity into the medical use of NMR for biological fluid a n a l y ~ i s , ’ ~ ~and , ~ ~there ’ have been a few trial experiments for substances of forensic relevance. The proponents of the technique emphasize the capacity for simultaneous fully quantitative determination of a spread of the higher concentration metabolites with minimal need for sample pretreatment (no derivatization). Proton detection limits are considered to be of the order of 1Opg in whole blood or urine, but are limited by the signals from endogenous metabolites (the so-called chemical noise level), so research has led on to sample work-up by solid phase extraction, and to coupled HPLC-NMR.14 Glucose is the endogenous metabolite of highest concentration (-1 mg/ml in blood), and may very occasionally be forensically significant. If a diabetes sufferer fails in the dietary and insulin self-management of the metabolic carbohydrate level, then hypoglycaemia brings drowsiness and a loss of self-control. If there is an attempt to drive a vehicle, this may lead to prosecution under the Road Traffic Act.268There are several clinical glucose assay methods, but it has also been shown that ‘H NMR can be used for q~antification.’~~ A detailed diabetes metabolic study has also been described by Nicholson et ~ 1 . ~ ~ ’ Ethanol is the most commonly analysed intoxicant substance, with the widespread use of robust field-testing devices such as the Breathalyzer and Intoximeter. Toxicology laboratories may be called upon to analyse blood or urine samples for further confirmation, or to aid the interpretation in terms of dose and time of ingestion. Headspace G C then provides an efficient method. Several recent studies have examined the use of ‘H NMR to demonsidentify and quantify ethanol in urine and serum, Pappas et trated good agreement between 300MHz ‘H and G C data in the range 0.4-4 mg/ml which is comparable to the normal working range. Ethanol and associated metabolites (acetate, acetone and P-hydroxybutyrate) were simultaneously assayed by Davin et ul.272using 400 MHz ‘H NMR. Harada et monitored ethanol levels in human saliva following beer or whisky ingestion. This may be (as described by a colleague of the author) “a sledgehammer to crack a nut”, but it is possible to see that an NMR sample could remain sealed after collection which would be beneficial in safety and integrity aspects. Additionally, ethanol measurement can be performed using lower-cost NMR instrumentation such as rapid scan correlation CW NMR.274 Ethanol has also been monitored in vivo as part of medical research studies with humans and monkeys.2757276Ethyl glucuronide has

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recently been identified as a minor ethanol metabolite, and its synthesis described.'" Pappas et uf.278have also shown that 'H NMR can be used to detect other substances (isopropanol, acetone and methanol) quantitatively in serum. Exploratory measurements have also been reported for other types of toxicology. Riicker et ~ 1 . ~ ~ 'gave a remarkably early description of drug detection in autopsy material (blood, bile and brain)-the recent activity in biofluid NMR did not begin until about 1982.266Miyata and AndoZ8' were able to measure methylamphetamine in human urine following intravenous administration (150 mg). However, the detection limit using 90 MHz Fourier transform instrumentation was found to be approximately 70 pg/ml. Harada et af.281measured the concentrations of a number of endogenous metabolites in extracts from rat tissues, with a view to estimating postmortem time. Fineschi et af.282have also considered the use of 'H NMR for measuring post-mortem metabolite changes in rat muscle, finding that several of the identified metabolites increased then decreased in concentration, but that ratios could be used to deduce the time of death. GyorffyWagner et af.283have found that animal brain tissue water T2 relaxation time shows a slight dependence on post-mortem duration. There have been a few instances of the use of NMR to detect poisons, specifically organophosphorus pesticides where 31P is available. Dickson et uf.284 demonstrated the 31P identification and quantification of the herbicide glyphosate in blood, urine and liver extracts from two suicide victims. Miyata et ~ 1 . ~reported ' ~ 31P chemical shift data for 23 organophosphorus pesticides. Hill et uf.286gave a case history of a poisoning epidemic in Sierra Leone, West Africa. MS and 'H NMR were used to obtain urgent identification of the toxic contaminant (parathion) in bread samples. Each of the NMR reports above have involved direct detection of appropriate substances, but an alternative approach in biofluid NMR has been to infer the status from general changes to the 'H spectrum. A description of this approach for veterinary regulation has been given by Lommen and G r ~ o t This . ~ ~demonstrated ~ that the lactate-creatine ratio could be used to screen urine samples for the use of illegal growth promoters (clenbuterol and oestradiol). Lastly, Vine et ~ 1 . have ~ ~ ' reported the identification of two contaminant substances in post-mortem blood samples using MS and 1H-13CNMR; these were shown to originate from sampling bottle rubber seals. 4. OTHER FORENSIC ANALYSIS 4.1. Hydrocarbon fuels, fire accelerants

Hydrocarbon liquids are encountered as forensic evidence primarily as flammable accelerants in arson cases, but also occasionally as contact trace

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residues of oil or grease in other types of case. The analytical method of choice in most instances would be some form of GC, since these hydrocarbons are generally mixtures of homologous series of molecules. The NMR literature of fossil fuel analysis is quite extensive (e.g. see refs 289 and 290) but tends to relate to heavy involatile fractions (e.g. bitumen, or even solid fuels such as coal) since peak integrals can give an average picture of the total functional group composition. There appear to be just two publications of direct forensic application of NMR: Bryce et af.2’1 described some preliminary tests of low-field ‘H NMR for typical accelerant liquids (gasoline, kerosene, diesel, paint thinner and charcoal lighter fuel), together with some casework comparisons. Renzoni et af.292 gave a brief account of the detection of gasoline adulteration with methanol, and mentioned that this had resulted in prosecution. It is also relevant that there have been some recent report^^'^,^'^ of the testing of NMR as a simple method for measuring the content of octane improver oxygenates in unleaded petrol; several compounds are permitted additives, and these might be useful for distinguishing petrol batches. 4.2. Explosives

The analysis of explosives is frequently one of the most imperative types of forensic science investigation. The analysis of post-explosion debris can indicate the type of explosive used, and contact traces may link individuals to bombing activity. This kind of analysis requires exceptionally sensitive and specific techniques, such as HPLC-MS, GC-thermal energy analysis (TEA) or GC-electron capture detection (ECD).2’5 NMR is hopelessly insensitive by several orders of magnitude for trace detection, but has occasionally found a usage in the characterization of bulk explosive samples, and this has proved valuable in the calibration of other procedures. The number of references is relatively few, but it would be surprising if there were not more in the form of restricted industrial or government reports. Gehring2’6 described impurity identification for trinitrotoluene (TNT) samples. Brief reviews of NMR work have been given by B e ~ e r i d g e ~and ’~ Yinon and Z i t r i ~ ~ .The ~ ” latter authors have actively researched this area, with studies on actual casework samples containing pentaerythritol tetranitrate (PETN) and cyclotrimethylene trinitramine (RDT).2’8*2’’ Very recently, Burns and Lewis3”” have examined a range of industrial gelignite samples. ‘H NMR (500 MHz) permitted the identification and quantification of multiple nitroglycerine and nitroaromatic components, and were capable of providing batch fingerprinting. Comparisons were made with samples seized from terrorist-related incidents. Of related significance is the current research activity into explosive detection by 14N nuclear quadrupole resonance (NQR) .301Prototype devices have been constructed for airport-style baggage scanning. Drug trafficking might also be detectable, since the main bulk drugs are nitrogenous.

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4.3. Lachrymators

Chlorobenzmalonitrile (“CS”) and chloroacetophenone (“CN”) are lachrymator (tear gas) substances which have been used for riot control, and are also components in some personal protection sprays. Other components include ally1 isothiocyanate (mustard oil) and capsaicins (chilli pepper extracts). In Britain these spray devices are considered to be offensive weapons, and are prohibited under the Firearms Act 1968 and Firearms (Amendment) Act 1988. GC-MS appears to be a favoured method of confirming the contents of lachrymator sprays, and is sufficiently sensitive for possible application to traces from clothing. However, some NMR data have also been reported. Identification and quantification of benzmalonitrile was reported by Hassan et ~ 7 1 . ~and ” ~ ‘H spectra of CS and CN were given by Ferslew et ~ 7 1 . ~Spectra ’~ of capsaicins have been given by Gannett et and Lin et a1.;305the latter giving full ‘H and I3C assignments derived from COSY, heteronuclear multiple-quantum spectroscopy (HMQC), and heteronuclear multiple bond coherence (HMBC). 4.4. Fingerprint reagents

A major part of criminal investigation involves the detection and identification of fingerprints, and it has been known for some time that it is possible to use a range of chemical reagents to enhance prints which are not visible without treatment (latent prints). The use of ninhydrin was described in 1954, and has since become widely adopted for forensic examination. A number of scientists have been working on the synthesis of new or modified compounds for potential improved performance, and have intermittently reported some NMR characterization. A range of structural modifications to ninhydrin have been described by Almog et al.306308 Some new compounds and ‘H NMR were also reported by Takahatsu et Considerable advances in fingerprint development have been made by using laser-induced f l u o r e s ~ e n c e ; ~consequently, ’~ much of the ninhydrin-analogue research has been directed at fluorescence enhancement. Almog et have also prepared a range of nitrobenzofurazan (NBD) compounds.

5. MAGNETIC RESONANCE IMAGING

Many hospitals and clinics are now being equipped with magnetic resonance imaging (MRI) body scanners, because the capacity to form highly detailed

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Fig. 9. Post-mortem magnetic resonance imaging. (A) Sagittal and (B) horizontal planes of sections of brain with perforating gunshot wound of diencephalon of a 20-year-old man. Death was essentially immediate; homicide. (Reproduced with permission from Harris.313)

three-dimensional images of soft tissues, especially the brain, has been of great benefit in the diagnosis of disease, especially cancer. Clearly, the possibility exists that the same imaging technology could also be used in forensic medicine or pathology. However, so far there have been relatively few instances of the use of MRI for forensic purposes. This is presumably partly due to the high cost and limited access to MRI, but the benefit of non-invasive imaging is somewhat diminished post-mortem. 1 mentioned . ~ ~the ~use of MRI and computerized Hashimoto et ~ tomography scans to confirm the injuries induced by exposure to carbon monoxide from a gas cooker leakage incident. Harris313 has pointed out that autopsy photographic evidence may be inadmissible in some US courts on the grounds that evidence should not be “gruesome, inflammatory or prejudicial”. In this circumstance, proton MRI can provide clear abstract diagrams, but which leave little doubt of the nature of fatal injuries, such as the bullet wound in Fig. 9. MRI has also been used by Wallace et ~ 1to confirm . ~ the~ precise ~ cause of death in two instances of execution by hanging. The sagittal inversionrecovery image revealed clear spinal ligament and disk disruption (Fig. 10). Further post-mortem imaging examples have been given by Boyko et ufS3I5 Other recent medical imaging experiments have been aimed at understanding the effects of drug stimulation on the brain, either using human volunteers or with laboratory animals.3163317

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(b)

Fig. 10. Post-mortem magnetic resonance imaging of the spinal cord and brain stem from a case of judicial hanging. (Case 2: submental knot placement). (a) Midline sagittal T1-weighted image shows distraction and posterior displacement of C-2 in relation to C-3. (b) Midline sagittal gradient-echo image again shows posterior displacement and distraction of C-2 in relation to C-3. The C2-3 disk remains with the C-2 body (arrowhead). The inferior portion of the spinal cord abuts the C2-3 disk (straight arrow). The superior portion of the spinal cord now lies posterior to the odontoid process (curved arrow). (Reproduced with permission from Wallace et af .3'4)

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NOTE ADDED IN PROOF

We are grateful to Dr G. Vordermaier (Institut fur Polizeitechnische Untersuchungen, Berlin) for drawing our attention to the research which has been carried out at the Forensic Science Institute (Wiesbaden), and to an earlier brief review of NMR in forensic science.318 Impurities from illicit mescaline synthesis have been d e ~ c r i b e d , ~and ” the use of ‘H NMR for the analysis of gasoline samples has also been extensively developed to complement GC technique^.^^^,^^^ Other substances examined have included gun lubricants and other e ~ p l o s i v e s , and ~ ~ ~adhesive - ~ ~ ~ tape plasticizers.

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Index Absorbing boundaries 71-5 Absorbing cylinder 73 Absorbing planes 71-3 Absorbing spheres 73-5 P-Acetoxy-P-methylstyrene 239 Adenosine triphosphate (ATP) 148-9 ”AINMR 36 Albacore tuna 38 Algal cellulose 16 Alginic acids 16 Ally1 isothiocyanate 286 Alurninium-catechin 36 Aluminium-fluorine 36 Aluminium-organic acid 36 Aluminium-phenolic acid 36 Amino acids 36 Amitriptyline 153 Amphetamines 22240 13CNMR 231 ‘HNMR 217 Amylodextrin 15 Amylopectin 20 branching 15 CP-MAS 20 Amylose 23 CP-MAS 19 Anabolic steroids 280-1 Analytical methods 29-43 Anhalonium lewinii 222 Anisotropic diffusion 77-9 Anomalous diffusion 76-7 Anthocyanins 37 Apokedarcidin 154 Apple, 13C NMR 34 Apple cell walls, cellulose in 25 Ascomycin 154 Ascorbic acid 41 Aspergillus awamori 17 Aspergillus japonicus 17 Atlantic salmon 38 Authentication of foods 30 Average propagator 67

Bo gradients 55-8,100-8,112,122-4, 1254, 153 B1 gradients 55-8, 108-9, 112, 117, 124,126, 134 Bacillus subfilis chorismate rnutase (CM) 155-8 Background gradients 99-100 reduction of effects of 100-1 Baker’s yeast fermentation 42 Banana 28-29, 34 BE18257B 172, 178 Bean cell walls 24 Beer, ethanol content 33 Benzaldehyde 30 Bidimensional ‘H NMR 15 Binding 130 Biopolymers 12-29 1 7 0 relaxation studies 3 Z-spectroscopy 28-29 Biot-Savart law 122 2,3-Bisphosphoglycerate (DPG) 130 Bloch equations 64 Bloch-Torrey equations 6-9 Body fluids 2 8 2 4 Boundary conditions 80-95 BQ123 1 7 S 9 Breathalyzer 283 4-Bromo-2,5methylenedioxyamphetamine (“DOB’’) 231 BSA (bovine serum albumin) 120 Butaclamol 151 Butylated hydroxyanisole (BHA) 40 Butylated hydroxytoluene (BHT) 40 C6-bound phosphate 19 13CNMR amphetamine 231 cannabis 257 exchange conditions 156 food authentication 30

300

INDEX

13C NMR-cont. forensic science 218 fruits 34 insulin 203-6 lipids 38 MAS methods 33,36 methylamphetamine 231 plant cell walls 25 polysaccharides 15-17, 19 solid state 36, 37 sulfation 16 Calmodulin 154 Cannabinoids 256-7 Cannabis sativa 256 Capillary zone electrophoresis (CZENMR) 218 Capsaicins 41, 286 Carbohydrates 17, 36 y-Carboxyglutamate (Gla) 180 Carr-Purcell sequence 101 Carr-Purcell-Meiboom-Gill sequence 101 Carrageenans 15 P-carrageenan 16 gels 21 K-carrageenan 21 polysaccharides 20 Carrots 37 p-Casein 15 Catechins 36 Catenella nipae 16 Catha edulis 235 ‘“CdNMR 41 Cell morphology 9 Cellulose 24 in apple cell walls 25 methyl and hydroxylpropylmethyl derivatives of 23 microfibrils 25 Chapman-Kolmogorov equations 84 Chemical shifts 27, 156 anisotropy of 25 Cherries 37 Chilling injury (CI) 37 Chiral chromatography techniques 236 Chloroacetophenone (“CN”) 286 Chlorobenzmalonitrile (“CS”) 286

Cholesterol 39 C-hordein 13, 14 Chorismate 155 Chromatography impurity signature profile analysis (CISPA) 221 Chromoprotein 154 Cimaterol 282 Cinnamoylcocaines 2 5 3 4 Clenbuterol 281 CMPG sequence 4 Cocaine, and related substances 246-56 Coffee 36-7 Coherence selection 110, 114, 134 Collagen 41 Community Bureau of Reference (BCR) 31 Computerized tomography 287 Conotoxins 180-9 3D structure of w-conotoxin GVIA 182-5 peptides structurally related to wconotoxin GVIA 187-9 structure-activity studies on wconotoxin GVIA 185-7 Constant time, pulse and gradient amplitude diffusion experiment (GTPG) 104 CONTIN 119 Conus geographus 182 Conus magus 182 Conus striatus 182 COSY (correlation spectroscopy) 1I& 11, 117, 154 CP-MAS 17-18,27 amylopectin 20 amylose 19 apple cell walls 25 CPMG spin echo sequence 33 Cross-polarization-magic angle spinning (CP-MAS). See CP-MAS; Magic angle spinning (MAS) Cross-relaxation NMR 28-29 ‘33CsNMR 21 CsA-CyP complex 160-1 Cuscohygrine 254 Cutin 27 Cyclophilin (CyP) 159

INDEX

Cyclosporin A 154, 159 Cyclothrimethylene trinitramine (RDT) 285 Cysteine-rich proteins 12 DANTE 117 DANTE-like sequences 113 Datura stramonium 246 DEBOG 117 Debye-Porod law 95 Deconvolution 32 Des-(B26-B30) insulin (DPI) 195-7 “Designer drugs” 219-20 Deuterium NMR 30 see also ’H NMR Dextran 23 DHA 38-9 Diabetes mellitus type I 189 Dietary fibres 23-4 Differential diffusion method 115 Differential scanning calorimetry (DSC) 4 Diffusion 118, 128-33 anisotropic 77-9 anomalous 76-7 differential method 115 free 59-63, 69, 79 isotropic 59, 64, 77 restricted 59-63, 128-30 signal attenuation correlation with 63-6 see also Self-diffusion Diffusion coefficient 59, 61-3,83,89, 98, 115, 121 Diffusion equation 59 Diffusion measurement 58-95, 128 pulse sequences for 100-9 Diffusion-ordered two-dimensional experiments 119 Diffusion tensor 59 Diffusive diffraction 67-9 3’,4’-Dihydroxyacetophenone 37 Dilute solutions 145 N,N-dimethyl-MDA (MDDMA) 233 Dimethyl sulfoxide. See DMSO Di-(P-phen ylisopropy1)amine (DPIA) 238

301

DISCRETE 119 Distortionless Enhancement by Polarization-Transfer (DEPT) 106 DMSO 3, 19, 152, 172, 173, 176, 177, 178,233,253 DNA-binding Hu protein 154 DNA complexes 158 DNDS 92 DNDS-treated cells 91 DOSY (diffusion-ordered spectroscopy) 81, 119, 133 Drink-driving offences 282 Drug abuse, types of drug in 220 Drug analysis 219-82 Drug conformations 151-3 Drug design and development 144, 145 analogue-based 147, 148 design or discovery phase 147 NMR techniques 149-51 receptor-based 147 schematic illustration 147 Drug development 147-9 Drug-DNA complexes 158 Drug substances, classification 219 DRYCLEAN 115 Dynamical mechanical thermal analysis (DMTA) 4

Eddy currents 98 reduction of effects of 1 0 1 4 Electron microscopy 36 Electrophoretic mobility 118, 121 Electrophoretic NMR (ENMR) 109, 119-21, 134 Endothelins (ET) 162-89 comparison of NMR and X ray structures of ET-1 169-72 3D structure of cyclic pentapeptide ET antagonists 172-9 3D structure of ET-1 and related peptides 166-9 EPA 38-9 Equilibrium spin density 66 Ergot 257-71 Erythoxylum coca 246 Erythoxylum novogranatense 246

302

INDEX

Ethanol as intoxicant 283 content of wines and beer 33 Eucheuma gelatinae 15, 16 Eucheuma muricatum 16 Eucheuma speciosa 16 Exchange 84-8, 156 Explosives analysis 285

"FNMR 36,37 Factorization ansatz 61 Fats 39 Fentanyls 220, 271-2 Fick's first and second laws 59 Field frequency locking 126-7 Field gradient pulses 99 Field gradients 60 Fingerprint reagents 286 Fire accelerants 284-5 Firearms Act 1968 286 Firearms (Amendment) Act 1988 286 Fish oils 39 FK-506 154 Food adulteration 30 Food quality and MRI 12 Food science 1-49 reviews 2 see also Biopolymers Foods authentication of 30 magnetic resonance imaging (MRI) of 9-12 water in 3-12 Forensic science 215-97 Fossil fuel analysis 285 Fourier transform infrared (FTIR) spectroscopy 36 Fractals 76-7 Free diffusion 59-63, 69, 79 Free induction decay (FID) 19, 24, 61, 121 Frequency domain 113 Friedelin 37 Fringe (or stray) field methods 105 Fruits 8, 9, 31, 34, 37 Furcellaria lumbricalis 15

Gaussian phase distribution (GPD) approximation 63,65-6,70-1,77, 79-80 GC-MS drug analysis 237 lachrymators 286 steroid metabolite detection 281 Gelatinization of starch 17 Gels 17-24 Glucose 33, 283 Gluten 13 Glyphosate 284 Gradient coils 98-9 Gradient measurements 76 Gradient NMR 51-142 reviews 53 specific examples 128-34 Gradient pulses imperfect 96-8 mismatched 98 rectangular 96 Gradients Bo 55-8,100-8, 112,1224, 125-6, 153 B1 55-8, 108-9, 112, 117, 124, 126, 134 background gradients 99-101 calibration 125-6 coil design 122-4 in high-resolution NMR 55, 109-21 inhomogeneous background 99 magnetic 99 non-homogeneous 95-100 planar 58 radial 58 technical aspects of production 121-8 Grapes 34,37 Green's function 66, 85 Guargum 21

'H NMR 15-19, 29,33,35,37,3843, 159, 166, 173, 196-7, 222 amphetamine 217 bidimensional 15 BQ123 173 cannabis 257

INDEX

carrageenans 15 cocaine 246,253 ET-1 166 insulin 192-203 sulfation 16 see also Proton NMR 2H NMR 40,42 see also Deuterium NMR Hahn spin echo 60,100 Halothane 42 Heart muscle 41-2 Heroin 241,244 Heterogeneous systems 17-24 water relaxation measurements in 58 Heteronuclear multiple bond coherence (HMBC) 286 Heteronuclear multiple-quantum correlation (HMQC) 134 Heteronuclear multiple-quantum experiments 105 Heteronuclear multiple-quantum spectroscopy (HMQC) 286 Heteronuclear 3D and 4D NMR experiments 154 Hexadecyltrimethylammonium bromide (CTAB)-sodium salicylate-water viscoelastic micellar system 133 High degree of esterification (HDE) 22 High molecular weight subunits 13 High-performance liquid chromatography (HPLC)NMR 218 High-resolution NMR 15, 17, 25, 33, 36, 37, 144 applications 133-4 gradients in 55, 109-21 HIV-1 matrix protein 154 Homonuclear multiple-quantum experiments 105 Hydration numbers 130 Hydrocarbon fuels 284-5 Hydrogen bonds 14 Hydroxybenzoyl 256 4-Hydroxybutyric acid (GHB) 282 Hydroxymethoxybenzoyl 256 Hygrine 254

303

Hyoscyamine 246 Hyoscyamus niger 246 Hypophosphite 87

1271 NMR 20-1 Ice, relaxation times 7 Ileostomy effluent 37 Indole alkaloids 257-71 Inorganic phosphate 148-9 Instrumentation requirements 145-6 Insulin 189-207 aggregation 194-5 B9 Asp 201 13C NMR 203-6 DPK 197-201 DQF-COSY spectrum 196,199 GlyB24 201-2 hexamer 1 9 3 4 HisB16 202-3 'H NMR 192-203 native 195-7 properties and conformations solution 190-2 residues containing aromatic rings 194-5 structure 189-90 substituted 197-203 therapy 189 truncated 197 Internal gradients 99-100 Intoximeter 283 Inverse Heteronuclear Correlation Spectroscopy (IHETCOR) 106 Ischaemia 148-9 Isotope editing 159-61 Isotropic diffusion 59, 64,77

Jcoupling

95

Karplus equation 247 Khat 222, 235 Konjac glucomannan (KGM) 23 Konnyak 23

304

INDEX

Lachrymators 286 Lactate-creatine ratio 284 Larmor equation 55 Larmor frequency 56 Lattice correlation function 89 LED (longitudinal eddy current delay) pulse sequence 101,120,121 Lenz’s law 98 Ligand-protein interactions 148 Lipase-catalysed esterification reactions 39 Lipids, NMR studies 37-40 Liquid crystals 130-1 Lithium ch1oride-N-Ndimethylacetamide 16 Locust bean gum 21 Lophophora williamsii 222 LSD 232,259,263,264,265,267,268 Lysergic acid diethylamide. See LSD Lysergic acid methylpropylamide (LAMPA) 267 Lysergide. See LSD

MASSEY method 96, 99, 103 Meat 41-2 P-Mercaptoethanol 120 Mescaline 222 Methaqualone 279-80 Methodology advances 146-7 Methylaminorex 233 Methylamphetamine 222,236 I3CNMR 231 Methylated pectic disaccharide 16 N-Methyl-1-( 1,3-benzodioxol-5-y1)-2butanamine (“MBDB”) 233 Methylcathinone 234 Methyl ecgonidine 256

3,4-Methylenedioxyamphetamine (MDA) 232 3,4-Methylenedioxy-Nmethylamphetamine (MDMA) 232 a-Methylfentanyl 272 N-Me thyl-4-phenyl-4-propoxypiperidine (MPPP) 272

N-Methyl-4-phenyl-l,2,3,6Mabuterol 282 Macromolecular NMR 148 Macromolecular systems 80-2 Macromolecules 131-3, 134, 155 Magic angle spinning (MAS) 29, 30, 33-6 see also CP-MAS Magnetic field gradient pulses 102 Magnetic gradients 99 Magnetic resonance imaging (MRI) food quality 12 food surfaces 11 foods 9-12 forensic applications 286-7 one-dimensional imaging protocols 11 temperature mapping 10 Magnetization fluid 63-4 Maillard reaction 42 Many body effects 84-8 Margarine 32 Mass spectrometry 36 Mass spectrometry-NMR combination 221

tetrahydropyridine (MPTP) 272 Microprobe technology 218 Microvolume probes 145-6 Milk 41 Milk proteins 15 Misuse of Drugs Act 1971 (MoDA) 219 Mobility ordered two-dimensional NMR (MOSY) 119, 121 Moisture content determination 30 Molecular motion imaging 67-9 Monte Carlo method 66 Morphine 240,244 MOSY (mobility ordered twodimensional NMR) 119, 121 Multidimensional NMR 154 Multiple spin echoes (MSE) 107

N-Nitrosodimethylamine 29 23Na NMR 21,22 NOESY (NOE spectroscopy) 158-9,166,253 Non-ionic micelles 95 Non-ionic polymer 95

13, 154,

INDEX

Nuclear Overhauser enhancement (NOE) 144, 145, 151, 154, 167 Nuclear quadrupole resonance (NQR) 285 Nuclear spins 55-8 Numerical simulations 66 relaxation studies, sugar and biopolymer systems 3 Obstruction 83, 128-30 Oil content determination 30 Oils 39 Oldenlandia ajjinis 188 Oiigosaccharides 16 Opiate alkaloids 240-5 Opium 240 Ordering/disordering processes 19 Organophosphorus pesticides 284 170

31PNMR 18, 19,31,42, 148 Peas, phospholipid contents in 31 Pectins, gelation of 22 Pentaerythritol tetranitrate (PETN) 285 Peptides drugs 162 squash 188-9 structurally related to o-conotoxin GVIA 187-9 Persimmon 34 Pesticide 37 Pharmaceutical applications 143-213 Phase cycling 101, 110, 134 Phase distortion, reduction of effects of 101-4 Phencyclidine and related substances 272-9 a-Phenethylamine 236 Phenethylarnines 222 Phenols 37 Phenylalanine 155 1-Phenylethanol 253 Phosphates 19 Phosphocreatine (PCr) 148-9 Phospholipid contents in peas 31 Phosphorus NMR 42 see also 31PNMR

305

Phytochemicals 40-1 Pigments 39 1-Piperidinocyclohexanecarbonitrile (PCC) 272,275 Plant cell walls 24-7 Plant materials, water relaxation measurements in 8-9 Plugflow 69 POC13 19 Poisons 284 Poly(buty1acrylate) 129 Polydispersity 80-2 Polymeric microgels 82 Polymers 80-2, 131-3 Polysaccharides 20, 27 cell wall 25 in solution 15-17 reversibly gelling 20 Polyunsaturated fatty acids 39 Pore-hopping formalism 94 Porous media 88-95,131 Potato cell wall 27 Potato starch maltodextrins 20 Powder diffractometry 36 Power supplies 124 Pre-emphasis method 98-9 Prephenate 155 Principal components analysis (PCA) 31 Process applications 30-1 Procyanidins 37 Protein 37 NMR studies 146 research 12-15 structure determination 153-4 Protein-ligand complexes 15442 Proteins, cysteine-rich 12 Proton NMR 24, 32,36 see also 'H NMR Proton relaxation 13 Proximate analysis method 30 Pu-erh tea 36 Pullulan 23 Pulse sequences for diffusion measurement 100-9 Pulsed field gradient (PFG) NMR 545,60-1,65, 67-9, 75, 80,82, 95, 96, 121, 122, 127, 130

306

INDEX

Pulsed field gradient (PFG) spin echo amplitude 66

Quadrature detection 110 Quinazolinones 279-80 Rapid scan correlation (RSC) NMR 303 87RbNMR 21 Red blood cells 84, 87, 90, 92, 130 Reflecting boundaries 69-71 Reflecting cylinder 70 Reflecting planes 69-70 Reflecting sphere 70 Relaxation parameters 27 Relaxation studies 5-8 Relaxation theory 5 Relaxation times 5-7, 9, 21, 22, 31, 35, 37,99-100, 152 Restricted diffusion 59-63, 128-30 Reverse Fourier transformation 32 Rigid lattice limit (RLL) 20 Road Traffic Act 282,283 ROESY spectrum 173

Sage 40 Salbutamol 282 Sample shimming 126-7 Sample tubes 145 Sarafatoxins 168-9 Scattering centres 94 Scleroglucan 22 SDS (sodium dodecyl sulfate) 120 Selective excitation 115 Selectivity 112-14 Self-diffusion 83, 129 Self-diffusion coefficient 77 Sephadex microspheres 7 Short gradient pulse (SGP) approximation 63,65,66, 69-71, 73,77,79-80 Signal attenuation correlation with diffusion 63-6 Signal-to-noise ratio 105, 110 Single-shot method 107-8

Size distributions of restricting geometry 82-3 Small-molecule NMR 148, 153 Sodium content in processed foods 21 Sodium dodecyl sulfate (SDS) 120 Sodium trimethylsilylpropionate (TSP) 216 Solid state 13CNMR 36, 37 Solids 17-24 Solute-biopolymer systems, water in 3-5 Solvent suppression 11417, 134 Spectral editing 112-14 Spectral simplification according to mobility 118-21 Spices 40-1 Spin-lattice relaxation 25, 42 SPLMOD 119 Squash peptides 188-9 Starch 28 gelatinization and retrogradation processes 17 Starch gels 18 Starch granule 19 Steady gradient methods 104 Stejskal and Tanner PFG sequence 601 Stimulated echo pulse (STE) 60, 100 Stokes-Einstein equation 81 Strychnine 271 Strychnos nux-vomica 271 Suberin 27 Sugar, I7O relaxation studies 3 Sugar-biopolymer systems 3 Sugars in ripeness 31 Superconducting magnets 105,216 Surfactants 130-1 Tagetes rninuta oil 38 Tea 36-7 Teargas 286 Temperature control 127-8 Temperature mapping 10 Tetramethylsilane (TMS) 216 Titrations of complexes 155-8 TOCSY (total correlation spectroscopy) 154, 160, 183 Toxicology 2 8 2 4

INDEX

Transferred NOE (TRNOE) technique 161-2 Transport 130 Trimethoprim-dihydrofolate reductase complex 154 Trinitrotoluene (TNT) 285 Truxillines 254 Two-dimensional (2D) NMR 144 Tyrosine 155 Vegetable oils 39 Vegetables 8, 9, 37 Vernonia galamensis 38 Vigna radiata 25 Water binding 3 content determination 30 in foods 3-12 in oil in water (W/O/W) 33 in solute-biopolymer systems 3-5 mobility 3 Water-collagen interaction 41

307

Water molecule binding 134 Water proton relaxation behaviour 6 Water relaxation measurements in heterogeneous systems 5-8 in plant materials 8-9 Water resonance presaturation 146 Watergate sequence 115-17 Wheat bran 17 Wheat starch 17 Wheat starch gels 17 Wiener-Kintchine theorem 67 Wines 36-7 ethanol content 33 WISE spectra 18

Xanthan 21

Zero-quantum coherences 106-7 Zero-quantum filtering 113 Zero-quantum spectra 106 2-spectroscopy, biopolymer applications 12,18, 28-29

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