Anal. Chem. 1990. 62. 212R-223R
EMISSION SPECTROMETRY (E2071 Qelante, L. J.; Wilson. D. A.; Hieftb, 0. M. Anal. Chlm. Acta 1988, 215, 99-109. (E208) Matousek, J. P.; Orr, E. J.; Selby. M. Appl. Spectrosc. 1989, 43, 573-576. (E209) Alandarl, J.; Diemi, A. M.; Ouillerme, J. M.; Legrand, Ben-Aim, R. I. Appl. SWChSC. 1989, 43, 681-687. (E210) seltrer,M. D.; Psepmeier, E. H.; Green, R. E. Appl. Spe&osc. 1968, 42, 1039-1045. (E211) Seltzer. M. D.; Green, R. E. Spectrosc. Lett. 1989, 22, 461-470. (E212) Sobering, 0. S.; Bailey, T. D.; Farrar, T. C. Appl. Spectrosc. 1988, 42, 1023-1025. (E213) Mdremed, M. M.; Uchida, T.; Mlnami, S. Appl. Spectrosc. 1989, 43, 129- 134. (E214) Mohemed, M. M.; Uchida, T.; Mlnami, S. Appl. Spectrosc. 1989. 43, 794-800. (E215) Uchida, H.; Masamba, W. R.; Uchida, T.; Smith, E. W.; Wlnefordner, J. D. A@. SpectrosC. 1989, 43, 425-430. (E216) Hwang, J. D.; Masamba, W.; Smith, E. W.; Winefordner, J. D. Can. J . SpeCtroSC. 1988, 33, 156-160. (E2171 mews, W.: Weber, 0.: Tola. G. Fresenius’ Z . Anal. Chem. 1989. 332, 862-865. (E218) Nakahara, T.; Kawakami, K.; Wasa, T. Chem. Express 1988. 3 , 651-654. (E2i9) CUU, K. E.; Camahan, J. W. Appl. Spectrosc. 1988, 42, 1061-1085. (E220) RMere, E.; Mermet, J. M.; Deruaz, D. J . Anal. At. Spectrom. 1988, 3, 551-555. (E221) B e M , A.; Hubert, J. J. Anal. At. SpeCtrom. 1988, 3 , 381-385. (E222) Boudreau, D.; Laverdure, C.; Hubert, J. Appl. Spectrosc. 1989, 43, 458-460. (E223) Sklarew, D. S.; Olsen, K. E.; Evans, J. C. Chromatographia,1989, 27, 44-48. (E224) Jiang, M.; Fielden, P. R.; Snook, R. D. Anal. Proc. (London) 1989, 26, 45-46.
Spark Dlrcharg.8 (E229 Mcrk, E. J.; Scheeline, A. Spectrmhim. Acta, Part B 1989, 44, 1297- 1323. (E226) Sainz. M. A.; Coleman, D. M. Appl. Spectrosc. 1989, 43, 553-558. (E227) Watters, R. L., Jr.; DeVoe, J. R.; Shen, F. H.; Small, J. A.; Ryna, E. Anal. Chem. 1989. 61, 1826-1833. (E228) Kauffman, R. E. Lubr. Eng. 1989, 45, 147-153. (E229) Lunner, S. E. Prm. Chem. Conf. 1988, 41, 106-113. (E230) McDonald, J. T.; Williams, J. C.; Williams, J. C., Jr. Appl. Spectrosc. 1989, 43, 697-702. (E231) Chen, L. Fenxi Ceshi Tongbao 1988 7 (3), 66-68. (E232) Rong, W.; Yang, Y. Yankueng Ceshi 1988, 7 (1). 43-49. (E233 Nhbe, H.; Kurosakl, M.; Kasai, S. Bunseki Kagaku 1988, 37 (1I), T133-T137. (E234) c3ollghtly. D. W.; Montaser, A.; Smith, E. L.; Dwrzapf, A. F Talents 1989, 36, 299-303. (E2351 Rell, L. J.; Kolrtyohann, S. R. Appl. Spectrosc. 1988, 42, 1221-1228. (E236) Raeymaekers, E.; van Espen, P.; Adams, F.; Brcekaert, J. A. C. Appl. Spe~trO~c. 1988, 42, 142-150. (E237) Zheleznova, A. A.; Bel’baeva, N. N.; Tlkhonov, A. A. Zh. Anal. Khim. 1988, 43, 1254-1260. (E238) Grikit, I.A.; Il’enko, V. S.; Kozhevnikova, A. S.; Sokoiova, V. P. Zavod. Lab. 1988, 54 (1l), 48-50. (E239) Karpenko, L. 1.; Fadeeva, L. A.; Shevchenko, L. D. Zavod. Lab. 1988, 54 (4), 48-49. (E240) Kharlamov. I.P.; Pcheikin. A. I.; Karyakin, V. Y.; Semin, M. Nov. v . makt Khlm. Anal. Veshchestv: Mater. Semln. 1989, M , 55-62.
0 t h Excllatlon P a v (E241) Liang, D.; Blades, M. W. Spectrochlm. Acta, Part 8 1989, 44, 1049- 1057. (E242) Liang, D.; Blades, M. W. Spectrochlm. Acta, Part B 1989, 44, 1059- 1063. (E243) Essien. M.; Radziemski, L. J.; Sneddon, J. J . Anal. At. Spectrom. 1988, 3 , 985-988. (E244) Sneddon. J.; Esslen. M.; Radziemski, L. J. Proc. Int. Conf. Lasers 1988, 1987, 808-912. (E245) Archontaki, H. A.; Crouch, S. R. Appl. Spectrosc. 1988, 42, 741-746. (E246) Lin, L. T.; Archibald, D. D.; Honigs, D. E. Appl. Spectrosc. 1988, 42, 477-483. (E247) Iida, Y. Appl. SpeCtroSC. 1989, 43, 229-234. (E248) coche,M.; Berthouce, T.; Mauchien. P.; Camus, P. Appl. Spectrosc. 1989, 43, 646-650. (E249) Kagawa, K.; Nomura, H.; Aokl, K.; Yokoi, S.; Nakajima, S. Bunko Kenkyu 1988, 37, 360-365. (E250) McCaffrey, J. T.; Michel, R. G. W o c h e m . J . 1988, 37, 357-367. (E251) Antunovic, I.H.; Trlpkovic, M.; Radovanov, S.; Andrlc, G. J . Anal. At. Spectrom. 1989, 4 . 593-598. (E252) Sturgeon, R. E.; WIlHe, S. N.; Luong, V.; Berman, S. S.; Dunn. J. G. J. Anal. At. Spectrom. 1989, 4 , 669-872. (E253) Rutherford, M. L.; Clouthler, D. J.; Holier, F. J. Appl. Spechosc. 1989, 43,532-538. (E254) Johnson, E. T.; Mitchell, J. W. Anal. Chim. Acta 1989, 217, 53-60. (E255) Jones, R. W.; McCleiland, J. F. Anal. Chem. 1989, 61, 650-656. (E256) Pourreza, N.; Townshend, A.; Turner, P. S. Anal. Proc. (London) 1988, 25, 244-246. (E257) ACZamil, I . 2.; Townshend, A. Anal. Chim. Acta 1988, 207, 355-359. (E258) Shakr, I.M. A.; Atto, S. Y.; Jawkal, N. A. J. Unhr. Kuwah’Sci. 1988, 15, 269-279. (E259) Celik, A.; Henden, E. AnaMt (London)1989, 174, 563-566. (E2601 Krnak. P.; Hejtmanek, M. Sb. Vys. Sk. Chem.-Technol. Raze, Anal. Chem. 1989, H23, 53-64. SELECTED APPLICATIONS
(Fl) Vaamonde, M.; Alonso, R. M.; Garcia, J.; Izaga, J. J. Anal. At. Spectrom. 1988, 3 , 1101-1103. (F2) Lieser, K. H.; Fey, W. Fresenlus’ Z . Anal. Chem.1988, 331. 350-355. (F3) Chao, K. J.; Chen, S. H.; Yang, M. H. Fresenlus’ 2.Anal. Chem. 1988, 331. 418-422. (F4) Flock, J.; Ohls, K. Fresenius’ 2. Anal. Chem. 1988, 331, 408-412. (F5) Navissi, A. E.; DeWaile, F. E.; Sung, J. F.; Mayer, K.; Dakey, R. J . Environ. Scl. Health, PartA 1988, 23, 823-841. (F6) Jones, J. W. J. Res. Net/. Bur. Stand. 1988, 93, 358-360. (F7) ACSwaidan, H. M. Anal. Lett. 1988, 21, 1469-1475. (Fa) Sakakibura, S.; Uwamino, Y.; Morikawa, H.; Iida, Y.; Tsuge, A.; Ishlzuka, T. Anawst (London) 1989, 114, 1664-1666. (F9) Que Hee. S. S.; Boyle, J. R. Anal. Chem. 1988, 60, 1033-1042. (F10) Balogh, M. P.; Potter, N. M. Anal. Chlm. Acta 1989, 221, 167-171. (F11) Plechaty, M. M.; Olson, E. L.; Scilla, G. J. Talanta 1989, 36, 809-61 1. (F12) Zhang, D. Fenxl Shwanshl 1988, 7 (3), 61-62. (F13) Yang, H.; Zhuang, S.; Chen, 0. Guangpuxue Yu Guangpu Fenxi 1988, 8 (3), 63-65. (F14) Sun, W.; Ren, Y. Fenxl Shiyanshl1988, 7 (2), 31-34. (F15) Qiu, C. Fenxi Ceshl Tongbe0 1988, 7(6), 68-71. (F16) Mochizuki, T.; Sakashita, A,; Iwata, H. Bunsek/ Kagaku 1988, 37, T109-T114. (FIT) Bauer, G.; Rehana, A.; Wegscheider, W.; Ortner, H. M. Spectrmhim. Acta, Part B 1988, 43, 971-982.
Nuclear Magnetic Resonance Spectroscopy Lynn W.Jelinaki
AT&T Bell Laboratories, Murray Hill, New Jersey 07974
INTRODUCTION AND SCOPE NMR spectroscopy continues to be an exceptionally vibrant
field. New developments, novel techniques, ever-more sophisticatad instnunentation, and powerful forces in biophysics and medicine conspire to drive the field forward at a remarkable pace. Recent progress along these lines is highlighted in this review. Areas reviewed are of necessity selective, since the literature concerning NMR spectroscopyis enormous. A search of the Chemical Abstracts files on this subject revealed nearly 6000 publications and more than 240 reviews between January 1989 and March 1990. Rather than simply parroting back the titles 212 R
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and abstracts of a huge number of papers of varying degrees of quality, I have attempted to critical1 analyze trends and directions in fundamental NMR researci and in applications of NMR s ectroscopy. New solutions to problems are discussed anfareas where more research is needed are pointed out. The intent is to provide a concise summary of the NMR highlights of 1989 and to describe im ortant new research directions, rather than to condense of 198% activity in NMR spectroscopy into a review with lots of references but of dubious utility. To thisend, each section contains a brief summary of recent significant highlights, as well as references to important review
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0 1990 American Chemical Society
NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY Lynn W. Jelkld~Iis Head 01 me 0iophysks Research Lkpartment at AT&T Sell Labwalories at Murray Hill. NJ. Ongoing research areas in her department inclvje lundamemai studies on neural netwOrks and neurabiob gy. protein structure and dynamics. bioenergetics. and biomedical engineering. Her own ares 01 research involves the application 01 nuclear magnetic resonance (NMR) and NMR imaging (MRI) 10 ths study 01 bioDhvsicai systems inclu(inq biwd flow and
that there is substantial overlap between the two groups (85-87).
CERAMICS Advanced ceramics can be formed by a sol-gel process in which an organosilicon precursor such as tetraethoxysilane is hydrolyzed to form a gel, which is then fired to produce ceramic objects. Solid-state "Si NMR continues to he useful for characterizing the chemistry of the sol-gel reaction, for investigating the distribution of organic hinder, for quantifying the amount of crystalline and amorphous phases, and for probing local structural order. Useful reviews on this subject are found in ref C1 and C2. NMR imaging has recently been used to study porosity and to detect defects in ceramin (C3). The ceramic object under study is soaked in water for a predetermined amount of time and the water in the voids is imaged by using ima ing techniques developed for medical applications (see IMAtING and SOLID-STATE NMR). Imaging appears to be a useful way to characterize the spatial distribution of defects in ceramics.
CHEMICAL SHIFT AND CHEMICAL SHIFT TENSORS
articles in the field. The interested reader is referred to these cited reviews for more complete and comprehensive information. Topics are organized alphabetically in somewhat fine-grained categories. These categories generally follow the major lines of chemical applications of NMR spectrwopy and reflect the impact NMR has had in these diverse areas of chemistry. Clinical applications of NMR imagin are not covered unless they are largely chemical or molecufar in nature. Several im ortant NMR books have recently been published. In ad&ion to edited books consisting of contributions by multiple authors or compilations of conference proceedings, there are several new NMR hooks of note. These include the third edition of Principles of Magnetic Resonance by Slichter (AI),a reprinting of the 1987 classic, Principles of Nuclear Magnetic Resonance in One and Two Dimensions by Ernst, Bodenhausen, and Wokaun (AZ), and Modern NMR Spectroscopy by Duddeck and Dietrich (A3). These three books are available BS relatively inexpensive paperback editions. Also published recently is the hook One and Two Dimensional NMR Spectroscopy by Rahman (A4). Together with Ahragam (A.51, Bovey's Nuclear Magnetic Resonance Spectroscopy (A6),Wuthrich's NMR of Proteins and Nucleic Acids (An, and books by Derome (As) and Sanders and Hunter (As) aimed at chemists, these new volumes round out the NMR spectroscopist's personal library. There are also two new volumes of Annual Reports on NMR Spectroscopy (AIO, All), two volumes of Advances in Magnetic Resonance edited by Warren (A12, A13), and volume 18 of Nuclear Magnetic Resonance by Webb (A14).
BODY FLUIDS NMR spectroscopy, particularly of protons, is used to examine various human and animal body fluids, including blood, urine, amniotic fluid, and cerehrcrtpinal fluid. Although NMR spectroscopy has not replaced standard clinical chemical analyses in this regard, it is useful in studies of drug metaholism and in toxicological applications. Three useful reviews of this subject are found in references Bl-B3. The analysis of a particular body fluid, plasma, became an important issue after the 1986 publication of the Fossel test (B4), which claimed that the proton linewidths in watersuppressed spectra of the methyl and methylene resonances in plasma reflected whether or not the patient bad malignant cancer. Fossel found that there was a narrowing of lipoprotein-lipid resonances with cancer. This publication generated a huge amount of research and work in this area, and plasma samples from thousands of people were examined by NMR spectroscopy. It is now clear that the Fossel test, as it was originally published, does not have general utility in screening for cancer. It appears that although there are significantly different mean line widths between plasma samples from patients with cancer and plasma samples from controls,
Although the chemical shift is enormously important for structural assignments and conformational analyses (011, our theoretical understanding of the origins of the chemical shift is still incomplete (02). Knowledge of both the orientation and magnitudes of chemical shift tensors is becoming increasingly important as solid-state NMR techniques are used to determine distances and molecular orientations in large molecules such as proteins. Principal values of I3C NMR chemical shift tensors have been reported for 63 substituted benzenes (D3) and Duncan (04) has compiled an invaluable comprehensive and exhaustive list of all published chemical shift tensors. Klein (0.5 correlated ) the principal values of the 31Pshift tensor with bond lengths and bond angles. He found that the chemical shift tensor elements are linearly related to T bond order. These results have impact on further studies of lipids.
CHEMICALLY INDUCED NUCLEAR POLARIZATION AND DYNAMIC NUCLEAR POLAHIZATION Chemically induced dynamic nuclear polarization (CIDNP) continues to be used to characterize excited-state tripleu. mainly in organic molecules ( E l l . Two-dimensional proton N M R methods have recently been applied to photo-CIDNP (E~J. Dynamic nuclear polarization (DNP) techniques are the suhlect of renewed interest, sinre one could theoretically obtain an enhancement of ye, (640:lJhy using the electronir ex. citations in the microwave to drive allowed nuclear transitions. Several groups are wnstructing high.field I)NP spertrumeters and are actively exploring this technique. Although the theoretical maximum enhanrement is G40, the maximum that has been attained so far is less than I O at high fields (EJJ, prohahly because of relaxation effects. Neverthtless. this enhanced sensitivity is likely to make possihle experiments that presently are not feasible. This method will be ezperially useful tur examining trapped intermediates for elertrun transfer or tor studying free radical catalysts.
CLUSTERS Formed of very small numbers of atoms, clusters occupy the regime between single molerules and the bulk. Clusters of metals or semironducton are currently of great excitement berause they provide a test bed for our understanding of elertronir hand structure and other phenomena associated with the bulk. Metallic and semiconductor clusters are just now beginning to he investigated hy NMR spectroscopy. Fur examplr, group Ill-V semiconductnr clustersoiGal' have bpen repared within the pores of a zeolite. hlagic angle spinning !I> N M H ha s been used t o rharartprizr the formation and transformation of size quantized speries ( F I J . Furthermore, ldyXeI\MH uf adsorbed Xe has bern used lo follow the calcination rlrprndence uf platinum rlusters (FZ).Xe was used to monitor hoth the size of the Pt clusters and the rluster precursors as a function of thermal treatment. ANALYTICAL CHEMISTRY. VOL. 62. NO. 12. JUNE 15. 1990
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C e + n insights into cluster formation can be pbtained by studyng biol cal analogues. For example, cadmium clusters occur natural y in the sulfur-containing protein, metallothionein. The three-dimensional structure of cadmium-containing rabbit liver metallothionein has been solved by twodimensional NMR methods, and the NMR structure was compared to the crystal structure (F3).These resulta elucidate possible pathways for Cd cluster formation.
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TWO-DIMENSIONAL (2D) NMR Two-dimensional NMR has attained a ition of premier importance in chemical applications of MR spectroscopy. Its routine use belies the fact that “2D or not 2D” was a topic at the Experimental NMR Conference only a decade a 0. Recent reviews include one by McIntyre and Freeman on fast 2D-correlation spectroscopyand one by Bax (G2) on homonuclear magnetization transfer experiments using isotropic and nonisotropic mixin schemes. A general review by Kriwacki and Pitner (G3)has%een published, updating Benn and Gilnther’s (G4) classic review on the subject. Heterocorrelated NMR techni ues are assuming a role of increasin importance as chemic3 shift assignments become more and more difficult because of the crowded nature of spectra of high-molecular-weightmaterials. Multiple quantum coherences enable one to obtain proton-detected heteronuclear correlations (G5, G6). Chemical shift selective coherence Heteronuclear and transfer has also been demonstrated (0. homonuclear coupling constants are also being recognized as important constraints for structure determinations (GB). A method has been set forth to obtain a m a t e HN-Ha cou ling constants using heteronuclear 2D NMR experiments rG9). 2D NMR ex riments are not without artifacts, and recent attention has E e n afforded this area ((210,Gll). Methods have been set forth to minimize off-resonance effects in ROESY spectra (GI2)and to minimize base-line distortions in hased 2D NMR spectra (G13). 8pecific applications of solution and solid-state 2D NMR to proteins,m natural products, polymers, and nucleic acids are described in other sections of this review. Techniaues specific to the 2D analysis of solid materials are listed uider SOLID-STATE NMR.
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(81)
THREE-DIMENSIONAL (3D) NMR Three-dimensional NMR spectroscopy extends two-dimensional spectroscopy by a pending an additional time period. For example, nuclear 8verhauser enhancements and J-correlations can be measured simultaneously by a 3D experiment. The time to acquire a 3D NMR data set is large and has so far limited the practical utility of the technique to samples that can be examined at fairly high concentrations. Data storage and data manipulation (the processing and visualization of 3D data sets) are less of a problem, given modern computer workstations. Althou h very time-consuming at present, it is likely that 3D N d will soon become a routine NMR technique. Two recent reviews, one by Bax ( H I ) and one by Ernst and co-workers (H2),describe the appearance of spectra, the assignment procedures, and the sensitivity and information content of these experiments. Heteronuclear 3D NMR has been used to obtain sequential backbone assignments in 15N-labeledsta hylococcal nuclease. Resonance overlap was reduced and t e sensitivity is approximately comparable to a regular 2D NOESY experiment (H3). New 3D NMR techni ues are continually being developed. For example, a methodlas been proposed for a 3D heteronuclear multi le- uantum coherence homonuclear Hartmann-Hahn (fiO&HA) experiment (H4). A heteronuclear 3D NMFt experiment has also been described which measures The 3D-HOsmall heteronuclear coupling constants (H5). HAHA experiment has been performed in water and applied to proteins (H6).
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DATA BASES Man companies maintain internal data bases for NMR spectrddata, and several microcomputer software pack es exist for storing, simulating, and calculating NMR spectra These abilities, especially when used in conjunction with other physiochemical data, greatly facilitate determining the structure of unknown compounds. Chemical Abstracts Service (Columbus, OH) has an electronically searchable 13C NMR
71).
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data base currently accessible through STN International. Partial substructure searches can be performed on this data base. Numeric data bases for chemical analysis have been recently reviewed (121, although in this review the primary emphasis is on mass spectroscopy, rather than on NMR. A llB data base for personal computers has been described recent1 (13). In adhition to data bases containing chemical shift data, there is a growing concern that the coordinates for NMRderived structures be promptly deposited in the Brookhaven structural data bank, as is currently the practice for protein and nucleic acid crystal structures.
DATA PROCESSING, AUTOMATIC ASSIGNMENT TECHNIQUES, AND OBTAINING THREE-DIMENSIONAL STRUCTURAL DATA FROM NMR DATA The Fourier transform is the mainstay of modern NMR spectroscopy and a recent handbook has been published on the subject ( J l ) . Many methods to enhance the appearance of spectra have been borrowed from existing technology in processing. These include the maximumentropy the maximum liklihood method (531,and linear predictive coding (J4). Although these methods generally enhance the appearance of spectra, they must be used with care (J5). Some work has been performed on automating the extraction, data management, and assignment process in 2D NMR data. This is a difficult problem and progress has been slow, especially because humans are generally surperior at problems in pattern and character recognition. However, as 3D NMR becomes more commonly utilized, the increased resolution and the attendant increase in the amount of information will provide a strong driving force for the development of even more sophisticated automated routines. Recent work in automated data extraction has been described using relayed proton-proton-carbon magnetization transfer (8) and uniform 15Nlabeling (57).The uniform 15Nlabeling means that crosspeaks in the 2D spectra can be automatically sorted and spin systems can be readily identified. An automated routine has also been described which elucidates Jconnectivities in proton spectra (J8)and another one for the assi ment of proton resonances using 2D HOHAHA and NO&Y spectra (J9).Very promising preliminary work has been described in which stereospecific proton NMR assignments are made automatically (JlO). A fair amount of progress has been made on automating the main chain directed (MCD) assignment algorithm (Jll)(see PROTEIN STRUCTURE). Once NMR assignments have been made, coupling constants and nuclear Overhauser enhancements are used as constraints for structural determination. This rocess has been reviewed by Gronenborn and Clore (5127. Several methods are currently employed. These include distancegeometry calculations (J13),a complete relaxation matrix approach, and a back-calculation refinement procedure similar to that used in X-ray crystallography. These methods are usually used with Monte Carlo techniques to avoid local minima and in conjunction with some type of energy minimization routine using constrained molecular dynamics. An alternative approach toward the problem of determining three-dimensional structures from constraints has recently been set forth by Jardetzky (J14). It uses an artificial-intelligence-based expert system called PROTEAN for problem extraction to various levels of structural abstraction, for local constraint satisfaction, and for heuristic control techniques to reduce computational demands.
%Fig,
EARTH SCIENCES AND THE ENVIRONMENT Until recently, the primary application of NMR spectrosco y to the earth sciences was in characterization of coal. SoEd-state NMR found an important niche in this area, since coal is an extremely complicated and heterogeneous material that does not readily yield to more traditional analytical techniques. The main problems and optimum NMR techniques for obtaining information about coal have been largely worked out (Kl), and emphasis is now being placed on the analysis of rocks and minerals (K2).For example,,proton NMR has been used to study hydrogen bonding in dissolved
NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
water in magmas (K3) and solid-state%i N M R has been used to follow speciation changes in quenched silicate liquids (K4). We are resently in an era of increased environmental awareness. %MR spectroscop has already found much utility in agriculture (see PLANTST and is beginning to be investigated in the stud of environmentally contaminated sediments. l W d NMI$ has been used to study cadmium speci.The utility ation in soils contaminated by heavy metals (K5). of multinuclear NMR and NMR imaging for enwronmental research is relatively unexplored.
FOOD CHEMISTRY NMR is used for many facets of food chemistry, including morphology, dynamics, and chemical analysis of food materials (LI).It is also useful for wine analysis, where 13CNMR can detect the sugars, alcohols, or anic acids, amino acids, and possible adulteration by di(etgy1ene gl col) (L2). The SNIF-NMR (site-specificnatur&isotope abundance) method is basically un aralleled by other methods for establishin product aut enticjty and uality (L3). In this method, fI/D ratios are acquired for t e components of interest (ethanol, for exam le). The H/D ratios of bio-made materials will vary accoring to the method of synthesis because of the isotope effect of deuterium, and site-specificH/D ratios are almost a fingerprint for the origin of the material.
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HIGH Tc SUPERCONDUCTORS Since their discovery only three years ago, high T,superconductors have been the subject of intense investi ation by large numbers of scientists, worldwide. Althou a large number of new high-temperature superconductorskve been synthesized, most NMR studies have centered around the La-Sr-Cu-0 (T = 38 K for Lal Sr 16Cu04),Y-Ba-Cu-0 (T,= 92 K for qBa2Cu 07),Tl-#a-ka-Cu-O, and Bi-PbSr-Ca-Cu-O compounds. Possessing a number of NMRactive nuclei, these materials have been a rich arena for N M R (and NQR) investigations. A number of excellent recent The renews have summarized the work in this area (MI-M5). results from copper and 1 7 0 NMR are particularly interesting. The hi h T su erconductors have two nonequivalent co per sites, t t e Ch2yplane sites and the Cu(1) chain sites. i o t h sites have an anomalous T temperature dependence above T ,which has been a t t r i b u d to antiferromagneticfluctuations The relaxation $the copper d-spins in the normal state (M5). of the Cu plane sites is anomalous both above and below T,. This su gests that the behavior is not that of a nonmagnetic metal Aove T,,nor of a BCS superconductor below T,. However, BCS behavior is observed in the 1 7 0 NMR relaxation times measured just below T, (M3). Oldfield has written a comprehensive review summarizing 170NMR studies on high-temperature superconductors (M4).
IMAGING TECHNIQUES One of the frontiers of NMR imaging involves the fourth dimension-that of chemical information. The study of diffusion and perfusion is receiving increasin attention (NI, N2). Furthermore, techni ues such as D R J d ( d e p t h rpolyed surface coil spectroscopj and ISIS (image-selected in vivo spectrosco y) have been widely to produce chemical information. I&w techniques have been developed that further enhance the chemical information obtained from imaging, both for the abundant nucleus (e .,protons) and for less abundant nuclei. For example, tailor$ excitation techniques have been described for chemical-shift-sensitiveimaging, which produce spectral simplifiction (N3). Images that correspond to signals from coupled spins only can be obtained b multiple echo acquisitions (N4),and spatially localized COgY spectra have been obtained by using a surface coil and phase-encoding magnetic field gradients (N5). New techniques in biomedical magnetic resonance are described in a book by Chen and Hoult (N6). Less abundant nuclei can be observed either directly or indirectly. Localized lSC NMR spectra have been obtained on humans (N7). Proton decoupling and nuclear Overhauser enhancement were used to increase sensitivity, just as they are used in high-resolution solution-state NMR spectroscopy. A new technique has been described for volume-selective single-scan spectral editing (N8)and it has been applied to the editing of proton signals for the indirect detection of coupled x-nuclei (N9).
INORGANIC MATERIALS Solution-state NMR is very useful for determining the dynamic properties of ligand rearrangements in inorganic and organometallic compounds, and solid-state NMR has been very valuable in investigating defects in inorganic materials. The area of NMR spectroscop of the early transition metals has been reviewed recently (81). Proton chemical shifts in a series of pol crystalline trihydrogen selenites have been obtained an interpreted in terms of hydrogen-bonding structures (02). Interstitial mobility or diffusion (03)and defects in inorganic materials can be studied by solid-state N M R (04). Fast i o n i d y conductive chalcogenideglasses have also been c h a r a c t e d by solid-state NMR, particularly those that contain ?3i (05, 06). Inor anic materials are often where one finds applications of the k M R of the “less common” nuclei. For example, %C1 NMR was used to characterize the relaxation of the chlorine and 77SeNMR has been used to nucleus in K20sCI, (07) characterize selenium iodine compounds (08).
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INSTRUMENTATION At present, 14.1-T (600MHz for protons) NMR spectrometers are commercially available for hi h-resolution solution-state NMR experiments, and soli8state NMR spectrometers are commercially available with reliable and routine high power proton decoupling and magic angle spinning. Cfical imagers are present in essentially every major medical center, and animal-sized imagers and microimagingaccessories are also available. The technical data for various NMR spectrometers are summarized in a review by Roth (PI). NMR in the analytical laboratory is now a high through-put technique, complete with automated sample changers (P2). The current forefronts of research in NMR instrumentation center around three main areas that should have major impact on the inevitable quest to improve the sensitivity of NMR measurements. dc SQUIDS have been used as preamplifiers for NMR measurements (P3), which greatly improve the sensitivity. Furthermore, dynamic nuclear polarization (DNP) at hi h magnetic fields (see section on CIDNP AND DNP) shoufd provide important structural information for those samples that possess stable free radicals. Finally, dynamic an le spinning (DAS) and double rotation (DOR) are new teckques which not only eliminate the first-order interactions (chemicalshift anisotropy and dipoledipole interactions) but also average the second-order quadrupolar broadenin . They thus provide hi6h-resolution spectra of “difficult”qua&polar nuclei such as 0 (P4). In DAS, the sample is spun successively about two “magic” angles, whereas in DOR the sam le is spun simultaneously about two axis by a “spinner withn a spinner”. These techniques have recently been demonstrated for 170in labeled mineral samples (P5). IN VIVO SPECTROSCOPY NMR spectroscopy has lon been applied to problems in biomedical research. 31PN h , in particular, has been especially valuable in uncovering the details of metabolic pathways. Many excellent reviews have been written on the subject (81-84). They summarize many of the im ortant contributions of NMR spectroscopy to our understanlig the chemistry and biochemistry of livin systems. We are now entering an era in which in vivo MR spectroscopy may become clinically valuable (85, 9 6 ) and even routine. Particularly in the area of cardiology, ‘P NMR spectroscopy may be useful for obtaining biochemial and clinically diagnostic 88). For example, information about cardiac disease (87, myocardial creatine kinase exchange rates and 31PNMR relaxation rates have been measured in intact animals (89). NMR spectroscopy has also been used extensively for detailing which biochemical metabolites can be observed in tumors in vivo. Both ‘H and 13Cspectrosco ic studies have been performed, as well as the more common 8P investigations (810). Of particular interest is the response of tumors to chemotherapeutic agents such as 5-fluorouracil, as well as determining the actual concentration of the at the tumor site (811).Altho h changes are observed in t e aP spectra of tumors during t e course of treatment, these changes are still poorly understood (Q12)and will undoubtedly require further studies on carefully controlled animal models. Brain chemistry is also an area of interest. N-Acetyl+ aspartic acid, a compound prominent in the proton NMR
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s ectra of the brain, is the subject of a recent review (813). 8nce it is r e w t e d by reserpine and other exogenous drugs, it may rovlde a valuable marker for understanding dru metabokm. In addition to Droton NMR. 13C,31P,and NMR have also been used i i drug metabblism studies and to identify dru metabolites in biological tissues and fluids (see BODY FL~JIDS)( 8 1 4 ) . ' With the eneral avdbility of high field ets, studies of biolcgidy im ' rtant ions such as sodium z t h i u m have example, lithium transport across human become routine. erythrocyte membranes can be measured by lithium-7 NMR spectroscopy (815). Sodium NMR and even sodim imaging can be readily performed. Important questions involve the differentiation of the intracellular sodium from the sodium preeent in the extracellular milieu. Several methods have been advanced to accomplish this. Intracellular sodium and extracellular sodium can be differentiated from each other by the addition of shift reagents to the extracellular compartment ( Q I S ) , and multiple quantum coherences can be used to selectively observe the si als from intracellular sodium, only. One can also obtain in ormation about the amount of intracellular ions such as sodium and calcium by the application of fluorine-labeled compounds, whose chemical shifts change when they com lex calcium or sodium. These compounds are generally i n d u c e d to cells in the ester form, where they are membrane soluble and cross the membrane. Once inside the cell they are converted to the acid form by intracellular esterases, and the excess re ent can be washed away. Integration of the lSF si alsyrom the com lexed and uncomDlexed mecies Drovigs an estimate of t e amount of intracellular Ions (QI~). Although the possible,clinical applications of NMR spectroscopy tb d i v o s i s and treatment monitoring are currently under intense investigation, in vivo spectroscopy continues to be applied to deepen our understanding of metabolic pathways. For exam le, NMR has also been used to study muscle contraction, indin that glycogenolysis in contracting muscle can be regulated goth by calcium regulation at the phosphorylase step and phosphate roduct regulation at the hosphofructokinasestep ( Q I S ) . N h spectroscopy has also k e n applied to areas as diverse as parasite metabolism (819) and bioreactor performance (820). It has been established that NMR monitoring is very useful for the optimization of mammalian cell bioreactors. Cellular physiology and metabolism is monitored in situ, in reasonable times with minimum perturbation of the sample. This type of application is of increased importance as more and more commercial products come to rely on mammalian cell culture systems.
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ISOTOPE EFFECTS Isotopic substitution of atoms causes very small chemical shifts that can be resolved at high field strengths and under conditions of excellent homogeneity. For example, the presence of a deuterium, rather than a proton, causes the '3c resonance to shift in a perceptible manner (RI). These small isotope shifts have been very useful in determining reaction pathways of oxygen, since the isotope effect of 170and of '80 can be distinguished. Isotope effects form the sub'ect of a recent review in the NMR-Basic Principles and hrogress Series (R2).
LIPIDS Deuterium NMR spectroscopy has been particularly effective in the study of lipids, protein-lipid interactions, and the effect on mobility that results when cholesterol and pther s cies are added to the membrane (SI). The application of E t e r i u m NMR as a monitor of the organization and dynamics at the surface of glycolipids has recently been reviewed (S2).NMR has also been applied to the study of lipid and fatty acid metabolism (S3). Multinuclear N M R has been used to study micellar solutions (S4) and model microemulsions formed by ternary mixtures of, for example, sodium oleatebutanol-water (ss). (See also the following section on LIQUID CRYSTALS.I
LIQUID CRYSTALS Liquid crystals are a rich material for NMR investigations. Under certain conditions they form macroscopicallyoriented structures and have found wide commercial utility in appli216R
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cations as diverse as liquid tal displa and the precursor for forming ultrahigh-strespolymer &rs such as Kevlar. Although early NMR studies involved interpretation of the spectra of small molecules oriented in liquid crystalline hosts (TI), current investigations center around the NMR characterization of the li uid crystals, themselves. Deuterium NMR has been very useyul in this re ard, since orientational order parameters can be determined kom the quadrupolar splittings and dipolar couplings (2'2). NMR is also used to quantify the field-induced ordering in the isotro ic hase of mesogenic molecules (7'2). Two-dimensional N h R exchange s ectroscopy has been applied to the study of liquid c r y s d i n e solutions (T3),and a new method has been set forth which uses Droton diffusion measuremenh to characterize chiral liquid crystals (2'4). Polymeric liquid crystals, both main-chain and side-chain liquid crystals, are currently of great interest, and a recent review describes the advantages of using solid-state 13CNMR to characterize these materials (T5).
LOW-TEMPERATURE NMR NMR at very low temperature extremes has been traditionally used to uncover new physics, particularly about quantum tunneling ( V I ) . Dipolar-driven N M R has been used at low temperatures to study molecular motion ( V 2 ) ,and low-temperature nuclear orientation has been used to study magnetism in solids (U3). In an effort to determine the mechanisms for NMR relaxation in solids below 1 K ( V 4 ) , Waugh and co-workers discovered that 3He causes extremely efficient surface relaxation (US).One of the very exciting future prospects in ultralow tem rature N M R concerns the abh to use relaxation generated 3He at the surfaces of materi to study surface atoms at low concentrations and to follow spin diffusion into the materials (US). S i c e the sensitivity increases dramatically at low temperatures due to the Boltnnann factor, this method may have a very important impact on future studies of clusters, catalysts, and other surface phenomena.
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METALS Nuclear techniques, including NQR, NMR, among others, have been used to study metals and all0 s and have found wide application in metallurgy. These dkelopments have been reviewed ( V I ) . Nuclear spin relaxation rates in quadrupolar nuclei have been used to construct a model which describes the thermodynamic pro rties of liquid metal alloys (V2),and hydrogen in metals anSehydrides has been studied at very high tem eratures (V3). (See also the section on INORGANIC M~TERIALS.)
NATURAL PRODUCTS NMR is indispensable for natural products structure elucidation. Derome has written an important review on the application of 2D NMR spectroscopy to structure determiCom rehensive books and nation of natural products (WI). reviews have been published on the '$NMR spectra of flavonoids (W2),on the structural analysis of ecdysteroids (W3), and on the analysis of steroids (W4).
NUCLEIC ACIDS AND DRUGNUCLEIC ACID INTERACTIONS 'H, 2H,and 31PNMR have been used to study nucleic acids
and drug-nucleic acid interactions. Two-dimensional NMR is used routinely for the sequential resonance assignment in short and medium-length oligonucleotides, and nuclear Overhauser enhancements and couplin constants are used to introduce constraints for the compfete three-dimensional structure determination of these materials ( X I ) . DNA oligomers have been intensively studied, and some of the current focus is now turning to unusual forms such as triple strands ( X 2 ) ,to lesioned DNA ( X 3 ) ,and to RNA structures. The RNA studies are more difficult than the DNA structures, and 13C NMR may become an important techni ue in this area. The interaction of antitumor drugs wi& nucleic acids continues to be an active area of research, and an important review has been written by Pate1 on NMR studies of echinomycin and chromomycin complexes with DNA ( X 4 ) . An independent detailed analysis of the conformation of chromomycin and dechromose-A chromomycin as it binds to (d-
NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
[ATGCAT],) has also been-published (X5). These two antitumor agents do not bind in the same manner to the DNA, suggesting that the function of the sugar moieties in chromomycin is to enable the molecule to assume the compact conformation it needs so it will be recognized at the DNA bindin site. AMR has been used to study the binding of platinum anticancer drum to oligonucleotide models of DNA (X61, suggeating that h e plathi-um binds to guanine5 and adenine-8.
activation determined from these measurements reflects the ionic character of the transition state.
PARAMAGNETIC CONTRAST AND SHIFT REAGENTS
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OPTICALLY DETECTED NMR (ODNMR) Optically detected NMR (ODNMR) has found an important niche in the study of defect structures in GaAs, PGa, and Gap. These studies have been recently reviewed (YI-Y3).
ORGANIC MOLECULES AND REACTION MECHANISMS NMR continues to enjoy widespread utility in the charac-
terization of small organic molecules. It is used for conformational analysis, the atudy of steric crowding effects, and other physical organic studies. Furthermore, paramagnetic shift reagents are often the method of choice to routinely establish0 tical uri (seePARAMAGNETICCONTRAST AND sH&T REA&NTs). Several new compilations of NMR data of organic molecules have a eared, includin a reprint of a 1982 book by Memory and #ilson on the N h of aromatic compounds (21).The conformational analysis of enones has also recently been reviewed (22,231. NMR can be used to determine the structure of reaction products and thereby to establish particular reaction mechanisms. If the reaction is slow enough or if the intermediates can be trapped, the reaction mechanism can be followed directly. For example, INEPT has been used to monitor polarization transfer from para hydro en to by oxidative addition (24),and deuterium NMR 2 b exchange spectroscopy in liquid crystalline solutions has been applied to study the Cope rearran ement in bullvalene (25). Nutation NMR spectroscopy as been used to determine the carbon-carbon bond len hs and bond angle in a reactive intermediate, the tert-buty cation (26). Although ‘H, ,H, and 13Cstudies are the most common for organic molecules, the “less common” nuclei have also been applied. For example, 170NMR spectroscopy is very useful for assessin steric rturbation of structure in organic compounds (28and l& spectroscopy has been used to study structure, stereochemistry, and binding phenomena (28).3H NMR has been em loyed to determine the confi uration of stereogenic (chiraly methyl oups comprised of CHDTX where X is a leaving group E9),D is deuterium, and T ii tritium.
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ORIENTED MOLECULES With the revalence of very high field NMR spectrometers, the field-inluced orientation of a molecule in the magnetic field must be considered. Although it has long been known that molecules such as hemoglobin and rigid rod molecules such as DNA will orient in the field, it is less appreciated that small organic molecules will also undergo a perceptible field-induced orientation. The study of small molecules oriented in high magnetic fields has been pioneered by Bothner-By (AAI, AA2).
ORGANOMETALLIC AND RELATED COMPOUNDS Both solution and solid-state NMR have been used to characterize organometallic compounds. Recent reviews discuss solid-state NMR experiments on lisSn and rmPb organochalco enides in which J-coupling is observed in the solid state (BBIf the study of phos horane iminato complexes of transition metals (BBZ),and NbR of oriented organomercury compounds (BB3). Organosilicon and silicon framework compounds are readily characterized by NMR. Williams has recently reviewed this area (BB4). %i double-quantum coherence spectroscopy (INADEQUATE) has been shown to be an efficient method for the structure elucidation of silicon frameworks (BB5). Dynamic NMR spectroscopy has been used to study the rates of bond dissociation in organometallic and coordination compounds (BB6). It has been shown that the entropy of
Once invaluable as agents for shifting resonances apart for structural determinations, the use of parama netic shift reagents has largely been supplanted by the availabity of high magnetic field stren hs, coupled with powerful 2D and 3D NMR methods ( C C I r However, chiral shift reagents remain the method of choice for routine1 assessing optical purity. The N M R of paramagnetic speciesKas been recently reviewed (CC2). The research frontier for Paramagnetic reagents is now in the realm of contrast enhancement agents for ma netic resonance imaging (MRI) (CC3). The possibilities and roblems associated with contrast enhancement in MRI are &cussed in terms of recent results on the relaxometry of paramagnetic agents in a review by Koenig (CC4). One of the major problems with paramagnetic contrast agents is their attendant toxicity. Gadolinium complexes, particularly Gd3+chelates with DTPA and with DOTA, have emerged as diagnostically important reagents (CC5, CC6). Also important in this regard are the superparamagnetic iron oxide contrast agents. Recent work has involved the influence of pulse sequence on the relaxation effects of this contrast agent (CC7) and methods for pulse sequence o timization when using this agent to detect liver cancer (8C8). Methods for obtaining images re resenting the distribution of paramagnetic molecules in sorution by EPR enhancement of the NMR image are the subject of a recent patent application (CC9). Rather than introducing exogenous paramagnetic reagents, one would ultimately like to use naturally occurring paramagnetic species to introduce image contrast. Work along these linea is just beginning. For example, manganese has been visualized in the basal anglia of primates by MRI (CCIO). Furthermore, 0 awa ( 8 C I I ) has used gradient echo techniques to ampli y the image contrast that accompanies the production of paramagnetic deoxyhemoglobin. Since localized high concentrations of deoxyhemoglobin are produced when regions in the brain are involved in neural activity, this method, taken together with local changes in relaxation and in blood flow, may be useful for providing PET-like neural activity maps.
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PLANTS AND PLANT PRODUCTS NMR has been widely used in agriculture, and the subject forms the topic of a book edited by Pfeffer and Gerasimowicz (DDI). Solid-state NMR has been particularly important, especially in the characterization of lignin and its condensation reactions ( 0 0 2 , 0 0 3 )and in the analysis of other plant cell wall constituents, such as cutin. Imaging studies have also been applied to plants. For example, the study of water transport and transpiration in lants is of paramount importance and is just beginning to e studied by microscopic NMR imaging techniques. Furthermore, NMR imaging has been used to detect knots or flaws in lumber. Information about the location and extent of knots can be used to optimize the way that valuable woods are cut.
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POLYMERS Much of what we know about synthetic polymer structure and dynamics derives from solution and solid-state NMR studies of these materials. Solution-state NMR usually provides the most detailed microstructural information, whereas solid-state NMR provides relevant information about internal molecular motions and dynamics. The a plication of both solution-state and solid-state NMR to syntietic polymers has been critically reviewed by Heatley ( E E I ) . Imaging is now beginning to be explored for ita abilit to rovide new information about synthetic polymers. &e fo?lowing sections highlight recent work in solution NMR, solid-state NMR, and imaging of synthetic polymers. Solution-state NMR is especially useful for establishing comonomer sequence distributions and reaction probability information, which are important direct inputs into synthetic optimization of polymerizations (EEZ). NMR can be used to quantify the amounts of various chain branches in ethylene ANALYTICAL CHEMISTRY, VOL. 62. NO. 12, JUNE 15, 1990
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copolymers (EE3). Since the melt properties of ethylene copolymers depend strongly on the number and composition of the branches, such information is essential in optimizing composition. Both 2D and 3D NMR have been used to establish stereochemical configuration and conformation of synthetic polymers in solution (EE4). The relationship belymer microstructure and polymer conformation is of a recent book by Tonelli (EE5). The types, number, and distribution of cross-link sites in a polymer also profoundly affect its properties. The a plication of NMR to cross-linked polymer systems is the su ject of a recent comprehensive review by Andreis and Koenig (EE6). A new book has ap eared on the subject of the structure and dynamics of b u l l polymers b NMR methods (EE7). Since moat synthetic polymers are &imately used in the solid state, NMR s ectroscopy can rovide the link between internal moelcu ar motions and ulk macroscopic pro erties. Furthermore, some polymers, such as many of the po ymeric conductors (EE8),are not soluble and therefore are unable to be observed by solution-state NMR spectroscopy. Spiess has recentl pioneered 2D NMR methods for studying ultraslow mo ecular motions in polymers (EE9, EE10). Polymers present special problems for the application of imaging (EE11).Standard imaging techniques, with reduced echo times, can be applied to polymer problems having to do with mobile components, such as water or plasticizer i pol mer curing, and elastomer uniformit For examz,e?i an8T2 relaxation time information has een used to investigate adhesive bonded structures us' N M R imaging. If one of s y m e r s , one must resort wants to image more 'solid" t to some scheme to narrow t E n e w i d t h s . Both multipulse line narrowing and magic angle s inning ap roaches have been usedsuccessfdl forimagin poymers. A vancesalo lines are descriged more fu!ly under the section on STATE NMR.
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PROTEIN DYNAMICS One of the ultimate objectives of studying protein dynamics is to relate the structure of the rotein to its function. NMR spectroscopy is used extensive y for this purpose, and two recent reviews have been published on this subject (FF1,FF2). Nitrogen-15 inverse detection heteronuclear NMR spectroscopy has been ap lied to sta hylococcal nuclease to study protein dynamics a ong the ackbone (FF3), and rapid motions in spectrin have been quantified by NMR s ectrosco y (FF4). Deuterium NMR in solution and uagupolar r e k a t i o n have been used to study heme mobi ity in myoglobin (FF5) and roton NMR has been used to investigate the nature of i n i d formed (metastable) complexes between apomyoglobin and emin (FF6).
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PROTEIN FOLDING Determinin the pathway throu h which a linear string of amino acids aiopts its properly fohed protein conformation is a topic of intense interest. NMR is an essential tool in the study of protein dynamics and foldin , and its use in this regard has recently been reviewed ( G 8 I ) . A new method for determining the structure of partially folding intermediates has been pioneered by Englander and co-workers. It promises to become an extremely useful tool for uncoveriy folding intermediates as well as for allowing one to determine regions of binding. The technique involves a scheme in which the protein is introduced, perha s by sto ped flow techni ues, into a reaction vessel in whic it is in&ced to begin foaing and where the artiall folded material can unde o proton-deuterium excEange. h e reaction is then quench2 and the protein can be analyzed by 2D NMR methods.
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PROTEIN AND PEPTIDE STRUCTURE AND BINDING One-dimensional NMR is valuable for establishin interaction and bind' data for peptides and proteins. %or exam le, 'Wd N a was used to characterize a 1:l cadmium adfuct with a peptide from an HIV-1 nucleic acid binding protein ("1). Two- and three-dimensional NMR spectroscopies are the methods of choice to make resonance assignments, to determine protein structures, and to uncover mechanisms of the 218R
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interaction of substrates with e es. A number of excellent reviews have recently a p p e a r x y Wiithrich (HH2), GroHH4), Bax ("9, Davies and nenborn and Clore ("3, and in Methods in EnzyParkes (HH6), Mildvan (HH7), mology (HH8). Several new methods for making resonance assignments are being pioneered and are becomi increasin ly important as ever-larger molecules are s t u d i s by NM#. Markley has developed a Wspin system directed a roach using uniformly isoto labeled proteins ("9). The fingerprint region assigned through the combined use of four 2D excan periments. The 13C-spinsystem directed method overcomes assignment ambiguities. Uniform labeling with 16N and subsequent zero and double quantum spectra reduce the broadening caused by dipolar coupling to the nitrogen nucleus, thereby producing spectra with narrowed proton resonances
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Englander and co-workers have recentl developed an algorithm called the main chain directed (hCD) method for assigning resonances from 2D N M R spectra (HHII). The first step involves detecting connected NH, C and C, resonances. The NOESY spectrum is then sear%ed to fin1 these defined connectivities. The NH-C C, are thus rouped into sets which correspond to secon& structural e ements. These ou s can be related to the protein amino acid seuencefy t i e identificationof simple side chain spin systems. %his method was demonstrated on cytochrome c.
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PULSE SEQUENCES The development of new pulse sequences continues at the forefront of NMR research. Radloff and Ernst have written a review of pulse se uences desi ned for the discrimination of spin systems base! on the topofogy of their scalar couplin networks (111). Both total spin coherence and average! evolution filters are described. A number of new pulse sequences have appeared over the past year. For example, J-spectral pulse sequences have been described for the magnetization preparation for coupled relaxation studies (112)and s in-echo techni ues have been used to measure the scalar re axation of mu tiple-quantum coherences (113). DANTEZ has been proposed as a new approach for accurate frequency selectivity using hard pulses (114), and SHIPPED, for selective heteronuclear inverse POlarization transfer proton editing, has been used (115). A selective pulse se uence for coherence transfer has been developed (116) an] INEPT has been used for nonselective polarization transfer in l70NMR (Z17). Shaped rf pulses with a tailored phase profile have been used for homonuclear coherence transfer experiments (ID),and the Goldman-Shen NMR pulse sequence has been modified (119). Pulse sequences s ecific-to solid sam les are described in the section on SOLIJ~STATE NMR T~CHNIQUES.
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RELATIONSHIP BETWEEN NMR DATA AND DATA FROM X-RAY CRYSTALLOGRAPHY The relationshi between solution-state protein structures derived from N& data and the solid-state structure determined from X-ray crystallography has been pioneered by Wuthrich, who has written recent reviews on the subject (JJ1, 552). In most cases the solution and crystal structures of proteins are similar; however the NMR structural data pinoint re ions of great flexibility or disorder. Furthermore, EN N& studies of a-lytic protease have resolved regions of discre ancy between X-ra and NMR data (JJ3). It has also been $emonstrated that tKe NMR data uncovered mistakes in the original chemical sequence determination of certain proteins. A compilation of complementary NMR and X-ray studies have been published in a recent volume of the Trans. Am. Crystallogr. Accoc. (JJ4). Results from solid-state NMR and X-ray have also been compared in a book by Etter and co-workers (JJ5) and the use of both solid-state NMR and X-ray crystallo aphy for (See studying molecular r ition has been described &). also the section on F N - S P I N COUPLING.)
RELAXATION Relaxation studies form an important part of the characterization of materials, reactions, and processes, and their use is described in application-specific sections of this review (e.g.,
NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
POLYMERS). However, a recent review of relaxation in liquids and gases has compiled data from many sources (KK1).
SOLID-STATE NMR TECHNIQUES Solid-state NMR is used routinely to characterizematerials, and s~ecificaDDlications are described in other sections of this review. For oiher 8 ecific solid-state NMR techniques, see the sections of INSgRlJMENTATION, LOW-TEMPERATURE NMR. POLYMERS, and ZERO FIELD NMR. Chmelka and Pines have written an excellent review on recent developments in NMR of solids (LL1). Their article describes the most recent progress made in m ic angle spinning, multiple-quantum spectroscopy, 2D N& of solids, ima ‘ng of solids, and NMR a t extremes of temperature. Zero k l d techniques are also discussed. A comprehensive review has been written b Dupree (LL2),and Lian has reviewed multiple pulse NMR The distribution of intensity into rotational sidebands continues to be a problem for quantification in solid-state NMR s ectroscopy. This problem has been largely solved by recent evelopments in m c angle spinners, which can now spin at speeds of about 20 Hz (LL4). However, techniques have been described in which the true intensities can be recovered while using TOSS (total suppression of spinning sidebands) (LL5) and TOSS has been applied to nAl spectra (LL6). Asynchronous MASSLF (magic angle spinning local field) spectroscopy has been roposed as a method for assigning solid-state 13CCPMAJspectra (LL7),and an experiment has been demonstrated in which chemical shift anisotropy linesha es can be determined in a 2D MAS NMR experiment (LU!.Fre uency-switched pulse sequences have been used for homonujear decoupling and dilute spin NMR (LL9) and variable frequency ulses have been used in wide-line NMR s ectroscopy (fL10).DIPSI-2 has been pro osed for broag-band homonuclear cross polarization in (LLII),Bliimich has described nonlinear incoherent 2D spectroscopy using 2D time-domain CW techni ues (LL12). Two related solid-state NMR techniques arexeing developed that rovide accurate distances over a
