Nuclear Magnetic Resonance Spectrometry - Analytical Chemistry

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Anal. Chem. 1996, 68, 161R-168R

Nuclear Magnetic Resonance Spectrometry Cecil Dybowski* and Martha D. Bruch

Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716-2522 Review Contents Reviews and Books Instrumentation and Theory Solids Surfaces and Catalysis Polymers Biochemistry Proteins Oligonucleotides Polysaccharides Organic Chemistry Literature Cited

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To begin, we emphasize that we have not tried to make an exhaustive survey of the articles and books that deal with NMR spectroscopy published from October, 1993 to October 1995. The field is simply far too large and complex to produce a meaningful survey of that type. We have tried to emphasize certain trends in the field, with references to certain papers and books about them. In that way, we hope to give a schematic picture of the field. One specific omission is a lack of any substantial coverage of the field of imaging; while important, imaging is still primarily used in medical studies, which we do not cover. REVIEWS AND BOOKS More and more books and reviews on the application of NMR spectroscopy to specific areas have continued to appear, and this compilation is not intended to be complete. Rather, we give only a few examples to show the wide variety of publications relating to NMR in various systems. For example, applications in polymer systems have been examined (1), and recent developments in the use of 15N NMR in polymers systems have been reviewed (2), as has the study of polymer-polymer miscibility (3). The characterization of zeolitic acidity with NMR techniques has been reviewed by Pfeifer (4), and this book also includes a review by Maciel of studies of silica and and a review of applications to zeolites by Fyfe. Cross-relaxation has been discussed (5). NMR of body fluids and tissue extracts has been discussed (6), as has its application to cell metabolism (7). An exciting field is the use of NMR in conjunction with other experiments to correlate information. A review on correlation with transport processes has appeared (8), as has a review of the uses of nuclear magnetic resonance to monitor chromatographic separations (9). INSTRUMENTATION AND THEORY More emphasis has been placed on the theory of spectroscopy of quadrupolar nuclei as the ability to detect them has been developed. Thus, exact expressions for spin-7/2 line intensities have been reported (10). A similar study used computer algebraic methods to obtain analytical solutions for spin-7/2 line intensitites (11). Nutation experiments, popularized some years ago for spins 1/ , have been applied to spin 3/ and 5/ (12). Reports of cross2 2 2 polarization of quadrupolar nuclei with protons continue to appear, S0003-2700(96)00007-8 CCC: $25.00

© 1996 American Chemical Society

with the potential of selectively detecting quadrupolar nuclei near isolated spin-1/2 pools (13). Analytical expressions for the central transition of quadrupolar nuclei subject to variable-angle NMR spectroscopy have been reported (14). As an alternative to the dynamic-angle-spinning (DAS) or double rotation (DOR) techniques for obtaining isotropic spectra of quadrupolar nuclei, a recently proposed bidimensional experiment is demonstrated on sodium and aluminum in multicomponent mixtures. The obvious advantage of avoiding the complex mechanical structure of the former techniques makes this a potentially practical means to examine quadrupolar resonances (15). However, the two-dimensional character means it requires a long accumulation time. The correlation of interactions of quadrupolar nuclei with nuclei of spin 1/2 in two-dimensional experiments that combine dynamicangle spinning gives a way to extract correlations based on dipolar coupling between the spins, as demonstrated for the coupling of 23Na with 31P in Na P O (16). 3 3 9 An interesting report recently discussed the use of composite pulses in nuclear quadrupole resonance (NQR) spectroscopy to improve the spectroscopic performance (17). Such sequences seem to offer improvements in NQR similar to the advantages of their use in NMR. A recent report has been made of a laboratory prototype NQR explosives detector that was shown to be able to detect RDX, an explosive (18). A recent report of a novel experiment has appeared that uses flash heating of a sample combined with simultaneous NMR detection to correlate solid and liquid NMR spectra (19). Another recent report from the same group demonstrates the difference between flash heating with a laser and inductive heating with a radio-frequency field (20). The inductive heating apparently gives a more uniform heating of the sample. A similar two-dimensional flash-heating experiment has been applied to address steps in a catalytic reaction (21). These kinds of experiments could provide important, innovative ways to study “fast” processes with NMR spectroscopy. With the desire to obtain resolution from magic-angle spinning and also provide a simulation of the flow characteristics of a chemical reactor, spectroscopists have tried to develop MAS probes which allow simultaneous injection of materials. A report (22) of one such design has been published. An important topic, the effects of instrumental problems such as rf inhomogeneity on quantification in solid-state MAS measurements, has been addressed recently (23). In an attempt to address needs in special circumstances, an unusual flat-coil geometry has been reported, which may be of use in studying peptides and proteins oriented at surfaces (24). Another report emphasizes the properties of loop-gap resonators for improving rf field homogeneity, particularly for MAS experiments (25). Another dramatic improvement in solution NMR has resulted from the design of very small radio-frequency coils. These tiny coils, wrapped directly around a capillary tube, allow 1H NMR Analytical Chemistry, Vol. 68, No. 12, June 15, 1996 161R

spectra to be obtained on sample volumes of less than 50 µL, resulting in a dramatic improvement in sensitivity of the NMR experiment (26). The initial coil design was improved by surrounding the coil with a susceptibility matching fluid, resulting in a coil requiring a sample volume of only 5 µL (27). By reducing the size of the coil, the mass sensitivity, defined as the signal-tonoise ratio per micromole, is increased by a factor of 100 compared to a conventional coil. These microcoils offer hope for analysis of tiny amounts of samples that could not be observed by conventional methods. Improvements in shim coil design have been addressed by using a power-minimization matrix method, resulting in highaccuracy, power-efficient shim coils without the drift associated with conventional shims (28). One of the improvements in instrumentation that has enabled many of the advances in NMR analysis of biopolymers is the increased availability of pulsed-field gradients for high-resolution NMR experiments. Although gradients have been used routinely in magnetic resonance imaging (MRI), they have only received widespread application to solution NMR in recent years. Improved sensitivity in 2D NMR experiments has been achieved by inserting gradient pulses to select specific coherence transfer pathways (29-32). Gradients have also been used to obtain superior water suppression in 2D (33) and 3D (34) NMR experiments on biopolymers in H2O. This increase in sensitivity and improvement in water suppression allows gradients to be used for simultaneous acquisition of 2D 15N-1H and 13C-1H heteronuclear singlequantum correlation (HSQC) data (35). In another study, gradient-enhanced proton-detected heteronuclear NMR experiments were shown to have improved resolution, independent of shimming, compared to nongradient experiments (36). Gradients also have been used to improve the quality of one-dimensional NOE spectra. Conventional methods require subtraction of two spectra, along with concomitant subtraction of artifacts, in order to obtain a NOE difference spectrum. However, gradients eliminate the need for subtraction, resulting in a much cleaner NOE spectrum (37). Pulse field gradients have also been used in diffusionordered 2D NMR experiments. In this technique, a twodimensional spectrum is obtained where one axis represents mobility of the molecule, based on size and hydrophobicity. Hence, individual spectra for components in a mixture can be obtained separately (38). Improvements continue to be made in the use of NMR for online process analysis, and a report has appeared on 1H NMR process analysis of oxygenates in gasoline (39). In another study, 13C dynamic nuclear polarization has been used as a detector for on-line liquid chromatography (40). SOLIDS Generally, the studies of solids have focused on understanding tensor properties of NMR parameters, as seen in the study of 13C in solid trimethylsulfoxonium iodide (41) or in the determination of chemical shift tensor elements of various nuclei (207Pb, 119Sn, 29Si) in a set of closely related compounds. The results were analyzed in terms of the similarities of local environments (42). Recently, a report of new techniques for spectral editing using chemical shift anisotropy dephasing has been demonstrated (43). A recent report highlighted the effects of simultaneous multiple interactions in MAS NMR of a spin 1/2 (44). A new broad-band means of transferring polarization in rotating solids is reported (45). 162R

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More and more, one sees reports of NMR of “esoteric” nuclei, such as the measurement of relaxation of 125Te in R-TeO2 (46), or 85,87Rb in Rb3C60 (47), or 183W in tungstate complexes (48) or 203,205Tl (49), or 63,65Cu (50), or 9Be and 11B (51) in high-T c superconductors. Yet another example is given by the study of the 51V resonance in Ca VO3-y compounds (52). 115In NMR is reported for AuIn2 at extremely low temperatures to look for magnetic ordering (53). In certain cases, systems with superconductive materials such as LaPb2 can be addressed by study of, in this case, the 207Pb resonance (54). Organic matter gathered from various places has been examined. For example, 13C CPMAS NMR has been used to study decomposition of lignocellulosic wastes from maple over periods of months (55), and the NMR properties of cellulose films have been described (56), as have spruce and beech (57), cork (58), and tropical hardwoods (59). Bound anilazine in soil can be detected, but only if specifically enriched (60). Analysis of soils of different ages from Kiel, Germany, for example, shows differences in the composition with 13C CPMAS NMR (61), and this difference can be understood in terms of the different chemistry and biology occurring in these different systems. An interesting application of solid-state NMR spectroscopy in archaeology is the study of carbonized residues on pottery to determine the dietary habits in prehistoric times (62). Similarly, a recent examination of the 13C solid-state NMR of extant and extinct lycopods and gymnosperms demonstrates that this technique can allow one to observe differences in fossil compositions, thereby inferring certain qualities of the materials (63). Related to this is the apprearance of NMR studies of high-quality antique paper (64). NMR studies of curing of urea-formaldehyde have shown a correlation between the 13C NMR spectra and the particleboard strength (65). Such correlations may provide useful connections of spectral parameters to practical parameters. Another interesting application of NMR in the solid state is the investigation of conducting glasses, where structure is probed by the silicon chemical shift (66) and the study of ion motion in such materials as R-PbF2 (67). The magic-angle-turning (MAT) 13C experiment (68) for extracting complex spectral features that are highly overlapped has been applied recently to coal and a naphthalene-derived pitch (69). Such experiments greatly simplify the determination of the various components of a complex spectrum. SURFACES AND CATALYSIS 13C NMR of adsorbed acetone has been reported (70), in which the chemisorbed acetone is detected in various environments including ZSM-5 and dealuminated faujasites. NMR parameters sense the structure and properties of the adsorbed complex. 13C NMR has been used to follow reactions of 2-propen-1-ol over H-ZSM-5 to confirm the production of propanal and its condensation products (71). Similarly, 1H MAS NMR studies have addressed the chemical state of zeolites through sensing the state of the surface hydroxyls (72). The carbon spectra were of adsorbed acetone, trichloroethylene, and carbon tetrachloride on various materials, including a a whole soil (73). Substantial chemical shift differences are seen, and it is suggested these are related to the strength of hydrogen bonding on the material. Benzene adsorbed in a variety of materials continues to be studied. For example, carbon NMR spectra were analyzed to

determine the exchange processes in benzene adsorbed in a CaLSX zeolite (74). Incorporation of deuterium into the surface sites has allowed the investigation of Brønsted acid sites in Y zeolite, mordenite, and ZSM-5 (75). The authors give characteristic parameters for deuterons in various situations. Deuterium and aluminum NMR studies of acidity effects in various zeolites are also reported by the Leipzig group (76). In an effort to understand the quantitative ability of 13C NMR, the Namur group has investigated model compounds adsorbed on a catalyst (77). Their conclusions are that not all coke is observable under the conditions of the particular NMR experiment they used. Such results indicate that care must be exercised in interpreting such spectra as representing the concentrations of carbons in such samples. An area of some recent activitiy is the application of NMR to investigate the surface phase of a chromatographic system (78). A review of various NMR techniques applied to silica surfaces has described the current state of affairs in studying the silanols and silicon portions of silylated surfaces (79). 129Xe NMR continues to be used to probe microporous materials such as catalysts, clathrates, and polymers. A novel experiment uses spin exchange with optically pumped rubidium in the gas phase to create a large xenon polarization; the hyperpolarized xenon is then swept into a sample where it is used to detect the local environment. A recent example is the application of this technique to the study of TiO2-supported V2O5 catalysts (80). Xenon continues to be studied when it is in contact with various microporous materials, such as ALPO-11 (81). An especially nice piece of work is a study of clustering of xenon in zeolite A (82). The effects of coking have been observed through xenon NMR in a dealuminated Y zeolite (83). Xenon adsorbed in alumina fibers has also been examined with temperaturedependent NMR (84). The explanation of the temperature and pressure dependence of the xenon chemical shifts requires two processes to account for the observed dependence. The effects of coadsorbates on the NMR spectroscopy of xenon in zeolites has been addressed, which gives clues to the interaction between xenon and the coadsorbate molecule (85). A recent report of xenon NMR as a function of temperature for the gas in contact with platinum particles in a Na Y zeolite shows heterogeneity of the electronic environment (86). The study of galliated zeolites with NMR of adsorbed xenon gas has been reported (87). Diffusion of xenon using pulsed-field-gradient techniques allows one to follow exchange processes of xenon in a zeolite with xenon in the gaseous phase (88). The question of the effects of interparticle diffusion on the xenon NMR of the adsorbed gas has been addressed using a blocking agent to trap xenon in a single particle (89). The investigation of cations in zeolites is important, and NMR was applied to study 23Na ions in zeolite ω recently and compared to the sodium NMR in gmelinite (90) and the examination of sodium ions in cation-exchanged sodium Y zeolite (91). Overlapping signals in the sodium spectrum of zeolite ω have been shown to be separated by the 2D nutation experiment or by variableangle-spinning techniques (92). POLYMERS Both liquid-state and solid NMR have been used to address problems in polymer science. For example, ethylene-propylene

polymer structure was determined as a function of the catalyst type using NMR (93). From NMR spectra, one group was capable of determining the methylene-ether bridge concentration of a melamine-formaldehyde resin quantitatively (94). In another, different way of using NMR, the magnetic alignment of side-chain liquid crystal polymers was investigated with NMR. The shift of the resonance at the phase transition was attributed to preferential alignment of the chains, giving weight to a particular orientation of the chemical shift tensor. Similarly, radiation cross-linking has been studied with NMR (95). Changes in line width were interpreted as changes in the mobility of the polymer segments. Mobility at the interface of a block copolymer of nylon-6 and poly(dimethylsiloxane) was studied with deuterium NMR and shown to be consistent with dynamic mechanical studies of mobility (96). Highly conductive polymers also can be investigated with NMR, as was done with polyaniline (97). Investigation of interactions between components in polymer blends continues to be an active area of research. Measurement of relaxation times by solid-state NMR techniques has been used to probe blend miscibility and domain sizes in a variety of polymer blends, including propylene-carbon monoxide alternating copolymer and poly(methyl methacrylate) (98), polycarbonate and poly(ethylene terephthalate) (99), oligo(p-phenylenevinylene) and polystyrene (100), and polystyrene with styrene-butadiene block copolymers (101). In an interesting study of blends of polyisoprene and 1,2 polybutadiene, two-dimensional solid-state experiments were used to observe specific interactions between the polyisoprene methyl group and the polybutadiene side chain (102). These interactions, manifested in the form of nuclear Overhauser effects (NOE) observed at temperatures above the glass transition temperature, Tg, are responsible for the high degree of miscibility in these blends. 129Xe NMR also has been used to probe polymer morphology (103), including the use of 2D 1H-129Xe correlation spectroscopy (104). 129Xe NMR spectroscopy is important in the study of micropores in materials, and a recent report (105) demonstrates how this technique may be used to interrogate the state of micropores in poly(arylcarbinols). The results are consistent with other methods of investigation of these materials. Various spectral editing routines have been used to determine the structure of polymers. For example, the proton-detected heteronuclear multiple-quantum correlation (HMQC) was employed to deduce resonance assignments in block polyisobutylene-polybutadiene copolymer (106). Two-dimensional 13C-1H NMR has been used to characterize the stereochemical structure of poly(acrylic acid) (107), and 2D 13C INADEQUATE NMR has been used to probe the termination mechanism of styrene freeradical polymerization (108). In one particularly nice application of solid-state NMR, the contributions to the 13C CP MAS spectrum from crystalline and noncrystalline components of ethylene-vinyl alcohol copolymer were observed separately (109). The use of the rotational-echo double-resonance (REDOR) technique for measuring the position of water in polycarbonate demonstrates the use of this technique for a technologically significant problem (110). Chemically resolved images of polymers have been reported (111). New techniques for spatial encoding of solids are being reported (112). Analytical Chemistry, Vol. 68, No. 12, June 15, 1996

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BIOCHEMISTRY One very active area of research in recent years has been the application of NMR spectroscopy to the characterization of biopolymers in solution. A thorough discussion of all the work in this area during the last two years is beyond the scope of this review. Instead, emphasis will be placed on the most noteworthy papers in this area, with more routine work summarized briefly. Proteins. Determination of the solution structure of proteins has become almost routine, primarily due to two factors: (1) increases in the performance and availability of 3D and 4D heteronuclear NMR techniques and (2) increased availability of expression systems capable of generating milligram quantities of proteins uniformly (or selectively) labeled with 13C, 15N, and/or 2H. Heteronuclear multidimensional NMR methods, reviewed by Clore and Gronenborn (113), have been used to determine the solution structure of many proteins during the last two years (114). The increased use of labeled proteins has led to investigations of the correlation between protein secondary structure and 13C (115) or 15N (116) chemical shifts. For proteins too big to be amenable to direct analysis by NMR, the solution structures of individual domains of many proteins have been determined by NMR (117). 1H and 19F NMR methods for obtaining structural information on proteins larger than 25 kDa have been reviewed (118), and the utility of chemical shifts for protein structure determination has been addressed (110). The accuracy of protein structures determined by NMR has been an active area of research. Garrett et al. demonstrated that the quality of protein structures can be improved significantly with minimal increase in computation time by including accurate vicinal coupling constants in the computational algorithms (120), while James has explored ways to refine protein structures by using more accurate interproton distances (121). The precision and accuracy of protein structures determined by NMR has been assessed by Zhao and Jardetzky (122). For situations where accurate NMR data are not available, Connolly et al. have explored methods for estimation of protein structure from incomplete and approximate distance restraints, as low as 0.5 long-range restraint/ residue (123). The proliferation of protein structures by NMR has provided numerous opportunities for comparison of solution structures determined by NMR and crystal structures determined by X-ray diffraction (124). In particular, interleukin-4 provides a nice example for comparison of solution and crystal structures since the structure of this protein has been determined independently by four different groups: two solution structures generated from NMR data and two X-ray crystal structures. These four structures have been compared by Smith et al. (125). The increased availability of 15N- or 13C-labeled proteins has led to more prevalent use of heteronuclear relaxation studies to study protein dynamics in solution, and these methods have been reviewed (126). More recent applications include the use of 15N relaxation data to probe molecular motion in Escherichia coli ribonuclease HI (127), hen egg white lysozyme (128), and calcium-free calmodulin (129). In other studies, the model-free approach for interpretation of 15N (130) and 13C (131) relaxation data for proteins has been evaluated. The dynamic effects of ligand binding on protein mobility have been examined for FK506 binding protein (132) and HIV protease (133). In a careful study of polypeptide dynamics, Pascal and Cross have observed a correlation between molecular dynamics in solution and disorder 164R

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in the X-ray structure of gramicidin (134). The use of solid-state deuterium NMR to study dynamics (as has been done in synthetic polymers and lipid bilayers) has been applied to the motion of L-phenylalanine bound to carboxypeptidase (135). The simulation of the NMR line shape is an essential feature of such studies. Determination of the structure and dynamics of a protein is a necessary first step for investigation of more interesting biochemical questions involving interactions between proteins and other molecules. Multidimensional NMR has been used to probe protein-protein interactions (136), protein-peptide interactions (137), protein-DNA interactions (138), and protein-ligand interactions (139). Interactions between proteins and ligands also have been probed by using transferred nuclear Overhauser effect (trNOE) experiments to determine the structure of bound ligands (140). Ligand binding has also been studied in the solid state by elegant experiments REDOR experiments to measure internuclear distances between selectively labeled nuclei in protein-ligand complexes (141). Several reviews of the use of NMR to probe protein-ligand interactions have appeared recently (142). Solid-state rotational resonance was used to elucidate the structure of specifically labeled amyloid fibrils (143). From the constraints imposed by the dipolar couplings among nuclei, a structure can be proposed in which the peptide adopts a highly pleated β sheet. In a recent application of rotational resonance NMR spectroscopy to a crystalline 11-residue peptide, rotational resonance MAS spectroscopy was shown to give distance measurements among spins with an accuracy on the order of a few tenths of an Angstrom (144). Such measurements hold promise to give structural and geometric information in these systems. Similarly structural information on an inhibitor bound to thermolysin was obtained from REDOR studies of specifically lablled systems (145). Inverse-detected heteronuclear 2D NMR of unusual nuclei has been used in some clever studies of metal binding in proteins. In one study, 1H-111Cd HMQC spectroscopy was used to probe the metal cluster topology of lobster metallothionein-1 through observation of cadmium-cysteine connectivities (146). In another study, 1H-77Se heteronuclear multiple-bond correlation (HMBC) spectroscopy was used to determine Se-methionine residues in selenomethionyl calmodulin (147). These experiments demonstrate the power of inverse detected 2D NMR to observe nuclei that are difficult to observe directly. Proteins retain many associated water molecules in the crystalline state, and bound water molecules in the interior of the protein are routinely observed by X-ray diffraction. Some of these bound water molecules can also be observed in solution by NMR, but the significance and exact nature of these water molecules has been somewhat controversial. Several recent studies have used 17O NMR to characterize these bound water molecules. Flesche et al. have used triple-quantum-filtered 17O NMR to selectively observe reorientationally hindered water in biological tissue (148), while Denisov et al. have examined 17O and 2H relaxation times to determine the residence times of buried water molecules in BPTI (149). In a similar study, 17O and 2H relaxation data were used to observe calcium-coordinated water in calbindin D9K (150). As discussed above, NMR has been used extensively for analysis of protein structure and dynamics. These studies pave the way for experiments designed to probe the folding pathway of a protein, and much progress has been made in this area. NMR

has provided evidence for the existence of partially folded intermediates known as molten globules, and several of these have been characterized recently by NMR (151). Early intermediates in the folding pathway of hen egg white lysozyme were analyzed by a clever combination of NMR and mass spectrometry (152). In these experiments, the heterogeneous folding intermediates observed for the partially folded protein were separated by mass spectrometry, allowing different types of folded structures, which are indistinguishable by NMR, to be observed separately. These studies clearly showed that the R domain of lysozyme can fold independently from the β domain. In another interesting study of protein folding, the folding kinetics of circularly permutated forms of the R-spectrin SH3 domain were determined (153). This study shows that the permutated forms of the protein fold into approximately the same structure and have similar stabilities. However, the rate constants for folding and unfolding various mutants are very different. Another study of protein folding examined the proton-exchange kinetics of partially unfolded RNase A in order to understand how protein unfolding enables amide exchange by exposing otherwise buried protons (154). This study demonstrated that exposure of amide protons proceeds in two stages: a “global unlocking” step to partially expose all amide protons, followed by local unfolding processes that expose distinct, individual amide protons. Progress also has been made in structure determination of polypeptides bound to lipids, primarily through judicious use of isotopic labels. Established techniques such as trNOE can be used in favorable cases to study polypeptides bound to deuterated lipids, as was done for mellitin bound to perdeuterated phophatidylcholine vesicles (155). Other studies have used 13C-enriched peptides (156) or lipids (157) to determine the structure of bound polypeptides from 13C NMR experiments. Similarly, 19F NMR was used to study interactions between a fluorinated inhibitor and small, sonicated unilamellar DMPC vesicles (158). One very promising method proposed recently for structure determination of lipid-bound polypeptides involves high-speed magic-angle spinning. Studies on gramicidin A in DMPC lipids demonstrate that, at sufficiently fast spinning speeds (>10 kHz), relatively sharp peptide resonances can be observed (159). This is a very important observation since direct observation of bound peptide resonances is not possible at lower spinning speeds, so these studies are the first reports of direct observation of bound peptide resonances. Oligonucleotides. Structure determination of various DNA fragments continues to be an active area of research (160), and a comprehensive discussion of all the recent papers in this area is beyond the scope of this review. However, there have been some recent studies on unusual forms of DNA that deserve mention. The structure of a pyrimidine-purine-pyrimidine triplex has been determined (161), as has that of a dimeric hairpin quadruplex (162). The structure of a DNA four-way junction has been determined (163), and the binding of both DNA four-way junctions and duplex structures to the protein, called determining factor SRY, which is a genetic “master switch” for male development in mammals, has been studied by NMR (164). In another study, the relative stability of the two crossover isomers in immobile Holliday junctions was found to depend upon the sequence at the junction (165). Another important area of research is the nature of drug-DNA binding, and several recent studies explore the effects of drug binding on the DNA structure (166).

Until recently, most of the NMR studies of oligonucleotides were directed toward DNA sequences. However, interest has increased in RNA sequences, and several studies have employed multidimensional NMR to determine the structure of RNA fragments (167). These studies have been made easier by improvements in methods for preparing 13C-labeled ribonucleotides (168). The improved sensitivity of heteronuclear multidimensional NMR experiments has opened up new avenues for analysis of labeled nucleotides, including the use of heteronuclear coupling constants to determine sugar conformation and glycosidic torsion angles (169). In addition, novel heteronuclear correlation experiments have been used to make assignments in labeled RNA sequences (170). 31P NMR also is useful for analysis of oligonucleotides, and the utility of 31P NMR as a probe of structure in ribonucleotide sequences has been examined (171). In addition to analysis of pure DNA or pure RNA sequences, several recent studies have been directed toward structure determination of DNA-RNA hybrid duplexes (172). Polysaccharides. 1H and 13C NMR spectroscopy continues to be invaluable for primary structure determination and conformational analysis of naturally occurring polysaccharides (173), and several reviews of NMR analysis of polysaccharides have appeared (174). In addition, heteronuclear 2D NMR methods for analysis of proteins and carbohydrates have been combined to enable complete assignment of the carbohydrate resonances in an intact glycoprotein (175). More interestingly, the interactions between an oligosaccharide and duplex DNA have been explored (176), and two distinct bonding modes were identified. In another study of oligosaccharide binding, the solution structure of a trisaccharide-antibody complex was determined from trNOE experiments (177). One of the most exciting developments in NMR analysis of carbohydrates is the use of water suppression techniques in supercooled aqueous solutions in order to observe the hydroxyl resonances (178). The chemical shifts and coupling constants of these hydroxyl resonances provide structural information not available from more traditional studies in D2O. ORGANIC CHEMISTRY Analysis of solutions by NMR is ubiquitous throughout all areas of NMR, and no one review could cover all of the recent papers in this area. Some of the more noteworthy studies reflect the increased attention given to multinuclear NMR, including observation of less traditional nuclei. One such nucleus that has received much attention is the quadrupolar nucleus 17O (I ) 5/2). Despite the low natural abundance (0.037%) and broad resonances associated with 17O, a number of papers involving 17O NMR have appeared recently, including studies of vinyl ethers (179), steroids (180), butadienes (181), and ozonides (182). In another study, 3He NMR was used to study derivatives of C and C fullerene 60 70 derivatives containing endohedral helium (183). Observation of various metal nuclei by NMR have been used to characterize organometalic compounds, including 77Se (184), 125Te (185), and 119Sn (186). One particularly interesting application involves the use of chiral derivatizing agents to convert enantiomeric mixtures into diastereomers, which can then be observed separately by NMR. This approach has been used to determine the enantiomeric excess in a mixture through comparison of the relative intensities of the two diastereomer signals in quantitative 119Sn (187) or 77Se (188) NMR spectra. Analytical Chemistry, Vol. 68, No. 12, June 15, 1996

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Other multinuclear studies illustrate the versatility and wide use of two-dimensional NMR techniques. For example, indirect detection methods have been used to assist in correlations between protons and 77Se (189) or 109Ag (190), and a recent report demonstrates that 2D 13C-207Pb correlation spectroscopy can facilitate assignment of organolead compounds (191). Cecil Dybowski is professor of physical chemistry at the University of Delaware. He received B.S. and Ph.D. degrees from the University of Texas at Austin. His research interests include all aspects of multinuclear solid-state NMR spectroscopy, with special emphasis on surface chemistry and heterogeneous catalysis. He has over 120 publications covering a variety of topics from NMR and mass spectrometry to inelastic electron tunneling spectroscopy, including two books on NMR spectroscopy. He is a lecturer in the American Chemical Society Shortcourse on NMR spectroscopy and a frequent tour speaker for the American Chemical Society and the Society for Applied Spectroscopy. He is a member of the editorial boards of Solid State Nuclear Magnetic Resonance, Magnetic Resonance Reviews, and Applied Spectroscopy. He currently serves as Associate Editor of Applied Spectroscopy. Martha Bruch is the manager of the NMR facility in the Department of Chemistry and Biochemistry at the University of Delaware where she engages in collaborative research involving the application of NMR spectroscopy to solve a wide range of chemical and biochemical problems. She received her Ph.D. in physical chemistry from the University of Delaware in 1984. Past and current research projects include microstructure analysis of synthetic polymers, determination of degree of mixing in polymer blends, structure determination of peptides and oligosaccharides, conformational analysis of small organic molecules, and analysis of chemically modified silica surfaces by solid-state NMR. She has published over 25 papers on various applications of NMR. Dr. Bruch has been co-organizer of the annual Blue Hen NMR Symposium at the University of Delaware. LITERATURE CITED (1) (a) Fawcett, A. H. Nucl. Magn. Reson. 1994, 23, 306-327. (b) NMR Spectroscopy of Polymers; Ibbett, R. N., Ed.; Blackie: Glasgow, UK, 1993. (2) Andreis, M.; Koenig, J. L. Adv. Polym. Sci. 1995, 124, 191237. (3) Veeman, W. S.; Maas, W. E. J. R. NMR 1994, 32, 127-162. (4) Pfeifer, H. NATO ASI Ser., Ser. C 1994, 444, 255-277. (5) Peng, J. W.; Wagner, G. Methods Enzymol. 1994, 239, 563596. (6) Bell, J. D.; Preece, N. E.; Parkes, H. G. NMR Physiol. Biomed. 1994, 221-236. (7) Zupke, C.; Foy, B. Curr. Opin. Biotechnol. 1995, 6, 192-197. (8) Johnson, C. S., Jr. Understanding Chem. React. 1994, 8, 544588. (9) Lightfoot, E. N.; Athalye, A. M.; Coffman, J. L.; Roper, D. K.; Root, T. W. J. Chromatogr. 1995, 707A, 45-55. (10) Man, P. P.; Tougne, P. Mol. Phys. 1994, 83, 997-1009. (11) Ageev, S. Z.; Sanctuary, B. C. Mol. Phys. 1995, 84, 835-844. (12) Ding, S.; McDowell, C. A. J. Magn. Reson., Ser. A 1995, 112, 36-42. (13) Kolodziejski, W.; Corma, A. Solid State Nucl. Magn. Reson. 1994, 3, 177-180. (14) Ajoy, G.; Ramakrishna, J. Solid State Nucl. Magn. Reson. 1995, 4, 77-100. (15) Frydman, L.; Harwood, J. S. J. Am. Chem. Soc. 1995, 117, 53675368. (16) Jarvie, T. P.; Wenslow, R. M.; Mueller, K. T. J. Am. Chem. Soc. 1995, 117, 570-571. (17) Ageev, S. Z.; Isbister, D. J.; Sanctuary, B. C. Mol. Phys. 1994, 83, 193-210. (18) Garroway, A. N.; Buess, M. L.; Yesinowski, J.; Miller, J. B.; Krauss, R. A. Proc. SPIE-Int. Soc. Opt. Eng. 1994, 2276, 139149. (19) Ferguson, D. B.; Krawietz, T. R.; Haw, J. F. Chem. Phys. Lett. 1994, 229, 71-74. (20) Ferguson, D. B.; Haw, J. F. Anal. Chem. 1995, 67, 3342-3348. (21) Ernst, H.; Freude, D.; Mildner, T.; Wolf, I. Z. Phys. Chem. (Munich) 1995, 189, 221-228. (22) Hunger, M.; Horvath, T. J. Chem. Soc., Chem. Commun. 1995, 1423-1424. (23) Campbell, G. C.; Galya, L. G.; Beeler, A. J.; English, A. D. J. Magn. Reson., Ser. A 1995, 112, 225-228. (24) Nielsen, N. C.; Daugaaard, P.; Lagner, V.; Thomsen, J. K.; Nielsen, S.; Sorensen, O. W.; Jakobsen, J. H. J. Biolmol. NMR 1995, 5, 311-314. (25) Larsen, F. H.; Daugaard, P.; Jakobsen, H. J.; Nielsen, N. C. J. Magn. Reson., Ser. A 1995, 115, 283-286. (26) Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Anal. Chem. 1994, 66, 3849-3857. (27) Olson, D. L.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Science 1995, 270, 1967-1970. (28) Hoult , D. I.; Deslauriers, P. J. Magn. Reson., Ser. A 1994, 108, 9-20. (29) Rinaldi, P. L.; Keifer, P. A. J. Magn. Reson., Ser. A 1994, 108, 259-262. 166R

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