Proc. Nati. Acad. Sci. USAVol. 80, pp. 7192-7196, December 1983Biophysics

Deuterium NMR as a structural probe for micelle-associatedcarbohydrates: D-Mannose

(liquid crystal solvents/potassium laurate/two-dimensional NMR/multiple-quantum NMR)

J. H. PRESTEGARD, V. W. MINER, AND P. M. TYRELLDepartment of Chemistry, Yale University, New Haven, CT 06511

Communicated by Kenneth B. Wiberg, August 15, 1983

ABSTRACT Residual quadrupole splittings for deuterons onperdeuterated a- and (3-D-mannose oriented in a potassium laur-ate liquid crystal have been measured. Multiple quantum two-di-mensional NMR spectroscopy at high magnetic field is used to gainsufficient resolution to assign splittings to specific sites within thesemolecules. The assigned splittings are interpreted in terms of mo-lecular geometries and preferred orientations of the moleculesrelative to the surface of the potassium laurate micelles that makeup the liquid crystalline phase.

The three-dimensional structures of complex carbohydrates andconstituent sugars have become a topic of considerable interestover the last several years. It is now recognized that these mol-ecules play central roles in cellular biology as structural com-ponents, as protective layers, as cell surface receptors, and asantigenic determinants (1, 2). Yet these molecules are amongthe least well characterized of biological macromolecules. Re-cent advances in high-resolution NMR, mass spectrometry, anddiffraction studies (2-6) are providing new primary and sec-ondary structural features, but progress toward an understand-ing of long-range folding in solution or the nature of membranesurface interactions is slow. Improved analytical methods ca-pable of providing such information would be extremely val-uable.

Deuterium NMR has shown substantial promise as a probeof structural properties of both large and small molecules. Thisderives from the fact that this spin one nucleus displays bothmagnetic field-nuclear magnetic moment interactions and elec-tric field gradient-nuclear quadrupole moment interactions. Theformer are defined in the laboratory frame by the applicationof a magnetic field. The latter are defined in the molecular frameby the distribution of electrons in chemical bonds, for example,a deuterium-carbon bond. In ordered systems, the manner inwhich the two interactions combine depends on the orientationof the bond relative to a molecular frame and on an order pa-rameter matrix describing ordering of the molecule relative tothe magnetic field. The interactions lead to two observabletransitions for each site. At high field, for an axially symmetricbond in an axially ordered system, these transitions are split by

3e2qQAVQ = 4h (1 - 3 cos2)Sz. [1]

e2qQ/h is a constant for a given bond type (170 kHz for deu-terium bound to aliphatic carbons), 6 is the angle between thebond and averaging axis, and Sz is an order parameter (7). It isclear that information concerning orientation of molecular bondsor molecular order can be extracted from observation of deu-terium spectra.

Most applications of deuterium spectroscopy to date haveused a known molecular geometry to extract order parameters.In a few liquid crystal studies, molecular geometries have beendeduced (8-11). This normally requires measurements on sev-eral sites in a rigid molecule in order to eliminate, dependingon symmetry, one to five order parameters as unknowns. Res-olution and sensitivity limitations in deuterium NMR usuallyrequire multiple experiments, each with specific site enrich-ment. The associated synthetic tasks are formidable and havelimited structural studies to relatively small molecules.

In what follows, we hope to demonstrate that application ofrecently developed two-dimensional (2D) NMR methods andultra-high-field spectrometers allow extraction of quadrupolecoupling information in even uniformly labeled compounds. Asan example of the extent of applicability, we have chosen a studyof the monosaccharide D-mannose.

Mannose is studied in a liquid crystal formed by orderingpotassium laurate micelles in a magnetic field (12). An abilityto determine a preferred orientation relative to a liquid crystalaxis, or a micelle surface, can be demonstrated. In addition, itcan be demonstrated that the observed set of quadrupole split-tings is consistent with the known crystal structure for this com-pound (13). This set of experiments can be viewed as a steptoward the study of more complex oligosaccharides.

MATERIALS AND METHODSD-Mannose was separated from a perdeuterated algal sugarmixture (98% 2H; MSD Isotopes, Montreal, Canada) on a di-ethylaminoethylcellulose column with a sodium borate buffer(0.2 M, pH 7) containing 2 M propylene glycol (14). Borate saltsin the fractions collected were converted to boric acid by usinga cation-exchange resin, Dowex 50, and boric acid was removedas a methyl ester by repeated evaporation of added methanol.Propylene glycol was removed under reduced pressure at 50'C.The residual D-mannose was identified by its optical rotation(+ 14.50) and migration in thin-layer chromatography (15).

Potassium laurate was prepared by addition of an equimolaramount of lauric acid (Eastman) to a solution of 2.5 M KOH inethanol. The potassium laurate formed was recrystallized fromethanol.A liquid crystal phase composed of -35% potassium laurate,

2% KCl, and 63% H20 was prepared as described by Forrestand Reeves (16). Approximately 15 mg of mannose in 0.04 mlof H20 was added to 1.5 ml of this phase. The sample in a 10mm NMR tube was placed in an 11.7-T superconducting mag-net and allowed to equilibrate at 318 K for -1 hr.

Deuterium spectra were acquired by using a Bruker WM-500 spectrometer at a frequency appropriate for an 11.7-T field

Abbreviations: 2D, two-dimensional; SECSY, scalar coupling correlated2D spectroscopy.

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(76.73 MHz). One-dimensional 2H spectra of liquid crystalsamples are the result of 600 transients at 0.5-sec intervals us-ing 900 pulses of 40 lusec, and one-dimensional 2H spectra ofsolution samples are the result of 500 transients at 0.2-sec in-tervals. 2D multiple quantum spectra were acquired by usingthe pulse sequence 90-T/2-180-T/2-(45) 90-t,-90-t2 and ap-propriate phase cycling to provide quadrature detection, elim-inate one-quantum signals, and eliminate artifacts associatedwith pulse imperfections (17, 18). The delay, x, was chosen ap-proximately equal to one-half the reciprocal of the largest split-ting, 0.3 msec, and an extra delay of 0.2 sec was allowed be-tween acquisitions, which occur during t2. A data set containing64 incremented time delay points for a t1 acquisition time of0.05 sec, by 1,024 incremented time delay points for a t2 ac-quisition time of 0.12 sec, was acquired by using 128 scans perspectrum. The resulting data were transformed by using aGaussian weighting function in both dimensions.

Proton NMR spectra of mannose at 500 MHz in aqueous so-lution were acquired for comparison and assignment purposes.A 50 mM mannose sample in 2H20 was examined by using sca-lar coupling correlated 2D spectroscopy (SECSY) and 2D J-re-solved spectroscopy (19, 20).The scalar coupling correlated spectrum represented a 1,024

by 512 data set acquired in 3.4 hr, and the -resolved spectruma 2,048 by 32 data set acquired in 3.7 hr. Sequential connec-tivities in the SECSY spectrum, beginning at each of the well-resolved anomeric protons, were used to assign the spectrumof each mannose anomer. The J-resolved 2D spectrum allowedprojection onto thef2 axis to provide a completely homonucleardecoupled proton spectrum for comparison to deuterium spec-tra.

RESULTSFig. 1 shows the deuterium NMR spectrum of D-mannose ina potassium laurate liquid crystal. The observable resonancescover a range of 2,500 Hz. This is approximately 20 times thechemical shift range expected for mannose deuterons, sug-gesting that the spectral complexity results predominantly fromsuperposition of quadrupole doublets with different couplingconstants. Mannose has seven nonexchangeable deuterons, andit exists in an anomeric equilibrium of 65% a anomer and 35%(3 anomer. With two lines per site, we would therefore expecta maximum of 28 lines in the spectrum. Discounting the linein the center from an unoriented fraction in the sample, and a

1 kHz

FIG. 1. The 76.7-MHz deuterium NMR spectrum of perdeuteratedD-mannose in a potassium laurate liquid crystal (52 mM; 318 K).

pair of lines expected from natural-abundance deuterium inwater, 23 lines are resolved. To extract structural information,these lines must be paired into quadrupole doublets and doub-lets must be assigned to specific sites.

In principle, chemical shift information, although a smallcomponent in the dispersion of lines in Fig. 1, could be usedto make assignments. 2D NMR spectroscopy has proven veryuseful in separating spectral parameters such as spin-spin cou-pling and chemical shift in high-resolution 1H spectra (20). Itis possible to devise a method that similarly allows separationof quadrupole coupling and chemical shift along different fre-quency axes. The spectrum shown in Fig. 2 was acquired byusing multiple quantum spectroscopy, which offers improvedchemical shift resolution at the expense of some additionalspectral acquisition time (21-23). Use of multiple quantumspectroscopy for chemical shift resolution in deuterium spectrais based on the fact that quadrupole interactions, regardless ofbond angle, contribute equally to perturbation of m = + 1 spinstates of the deuterium nucleus (21). A double quantum tran-sition from m = 1 to m = -1, therefore, is independent of anyfirst-order quadrupole splitting. Moreover, chemical shifts arescaled up by a factor of 2 from their single quantum values.Since double quantum coherence is detected indirectly, it be-comes thef, axis (vertical in our plot) and the directly observedone-quantum spectrum becomes the f2 axis (horizontal in ourplot).

In the lower portion of Fig. 2, peaks are displayed by usingcontours and, viewed in three dimensions, would come ver-tically out of the page. Their position along the f" axis is 2xchemical shift, and their position along thef2 axis reflects bothchemical shift and quadrupole coupling. A projection onto thef2 axis is included for comparison to the deuterium spectrum inFig. 1. General features are retained except for loss of signalsfrom unoriented species. The pulse sequence used does notexcite double-quantum coherence in the degenerate lines fromunoriented species, so these are systematically eliminated fromthe spectrum. It is clear in the 2D plot that resonances occurin pairs, many falling on a single resolved horizontal line. Quad-rupole splittings can be measured directly from the separationof each pair of lines.

Projection of the 2D data of Fig. 2 onto the fi axis gives apure chemical shift spectrum. This is shown in Fig. 3, spec-trum a. For comparison, a deuterium spectrum of mannose inH20 is displayed in spectrum b. Aside from intensity anomaliesand some displacement of the water resonance near 4.7 ppm,the spectra are quite similar, with the multiple quantum pro-jection showing somewhat improved resolution. This occursdespite the fact that for the multiple-quantum spectrum, themolecule is in a more viscous, liquid crystal environment.

Assignment of deuterium resonances is best accomplishedby comparison to proton spectra of the same molecule in thesame environment. Chemical shifts, because they arise fromthe electronic structure of the molecules, are within experi-mental error identical for most deuterium and proton spectra.The proton spectra of solution samples are easily assigned byusing scalar coupling correlations as outlined in our previouswork (4). Assuming environmental effects are small, they shouldbe adequate for assignment of liquid crystal spectra. Assign-ments for a- and ,3-mannose are given in Table 1. In most cases,they agree qualitatively with assignments made previously formannose-containing oligosaccharides (24). A proton spectrumobtained by suitable projection of a J-resolved data set is in-cluded in Fig. 3, spectrum c, along with assignments. Thisspectrum does not display any 'H-'H coupling and hence hasone resonance per chemically distinct site, much as the solutiondeuterium spectrum does. It has far greater resolution than the

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FIG. 2. Contour plot and pro-jection onto the f2 axis of a two-quantum deuterium spectrum ofthe D-mannose sample described inthe legend of Fig. 1.

deuterium spectra, but it appears that the envelope of protonlines coincides with the partially resolved lines in the deuteri-um spectrum except for the water resonance near 4.7 ppm, whichexperiences larger isotope- and environment-dependent shifts.

Comparison of the proton spectrum with the projection ofthe multiple quantum deuterium spectrum, and the 2D plotitself, allows the assignment of quadrupole splittings to severalspecific sites. For the anomer, recognizable in part from lowerresonance intensities, these assignments include the C1 (ano-meric), C5, C4, and C2 sites. For the a anomer, assignmentsinclude the C1 (anomeric) and C2 sites. Quadrupole splittings

a

for these sites have been marked in Table I to indicate that theyare primary assignments.From the above assignments of quadrupole splittings to spe-

cific sites, it is possible to begin a search for structural and or-

dering information. In the present case, we will assume thatring protons are rigidly oriented with respect to one anotherand that molecular motions are axially symmetric. All quad-rupole splittings are therefore reduced by the same order pa-rameter (see Eq. 1). We scale all assigned splittings with re-

spect to the largest and then proceed to search for an orientationof the ordering axis in the molecular frame that reproduces thescaled splittings. Eq. 1 was used to calculate splittings for eachof the assigned deuterons at each axis orientation, and thesewere compared to observation. The calculation requires aknowledge of the relative orientation of each carbon-deuteri-um bond. In the case of the a anomer, a crystal structure existsfrom which the relative orientations can be derived (13). Forthe anomer, the same relative orientations were assumed to

l Il I I I I I I I I I I II I

5 4 3ppm

FIG. 3. One-dimensional proton and deuterium chemical shiftspectra of D-mannose. Spectrum a, projection of the 2D data set of Fig.2 onto thef{ axis. The chemical shift scale is actually twice that shownon the figure for this spectrum. Spectrum b, deuterium spectrum of 13mM D-mannose in H20. Spectrum c, projection of a J-resolved protonspectrum of a 50 mM D-mannose sample in 2H20.

Table 1. Spectral assignment and quadrupole splittings of a and,8 anomers of D-mannose

Chemical Observed Calculatedshift,* splitting, splitting, Difference,

Deuteron ppm Hz Hz Hzp1 4.89 2,069t 1,961 -108P2 3.92 2,178t 2,297 119P3 3.63 1,851 1,950 99(34 3.56 2,349t 2,349 0(35 3.37 2,178t 1,998 -180(36 3.72 824 1,769(36' 3.88 824 12,668

al 5.17 856t 651 -205a2 3.91 2,582t 2,582 0a3 3.83 1,618 1,495 -123a4 3.66 1,213t 1,201 -12aS 3.80 1,758 1,776 18a6 3.85 716 J 1,984a6' 3.75 264 1,670

* Chemical shifts are from proton spectra and are reported relative to2,2-dimethyl-2-silapentane-5-sulfonate.

tQuadrupole splittings assigned directly from chemical shift corre-lations.

I II6 t0 ' 0 tlaI1

3 ppm

500 Hz t2

I II I

a 0

!I

II

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exist for all except the anomeric proton. The 8 anomeric protonwas assumed to lie along the carbon-oxygen bond of the ano-meric hydroxyl of the a anomer. The search was a computer-assisted search of a grid covering approximately 00 ' 4), '1800 in approximately 40 steps. Agreement within 10% for eachmeasured splitting was sought.

For the 8 anomer, a best solution is found with the directionof the ordering axis as indicated in Fig. 4. The axis is nearly inthe plane of the ring and nearly bisects the bond angle of thering oxygen. Because the (1 - 3 cos2O) function is unchangedon inversion for all bonds,. the solution is twofold degenerate-i.e., the direction of the ordering axis can be reversed.

Calculated quadrupole splittings based on Eq. 1 for the di-rector axis in Fig. 4 are presented.in Table 1, along with ob-served values. The orientation.of .the 8 anomer-derived fromprimary assignments allows assignment of a doublet -centered,at 3.6 ppm to the C3 ring deuteron based on its quadrupolesplitting. Splittings of 6 and 6' deuterons predicted from theabove-orientation and crystal structure conformation do not cor-respond to observable splittings in appropriate chemical shiftregions for these deuterons. This suggests rapid rotational av-

eraging about the C5-C6 bond. Excluding data for the 6 and6' deuterons, a root-mean-square (rms) deviation of 130 Hz iscalculated by using the observed and predicted values of quad-rupole splittings. A second orientational solution for the 8 an-

omer, with the- same resonance assignments and a rms devia-tion only 10% larger, can be found. The director .axis for thissolution is rotated approximately 140 from that of the first so-lution but is still nearly in the plane- of the ring. Other solutionshave rms deviations substantially larger.The additional assignment of resonances for the anomer

frees the way for assignments of the C4 resonance of .the a an-

omer. Together with C1 and C2 assignments, this allows a searchfor an orientation of the a.anomer. A single solution was foundwhich also predicted two additional quadrupole splittings foundin the poorly resolved C3, C5, C6 region of the spectrum. Thissolution, which has an rms deviation value of 120 Hz, is de-picted in Fig. 4. Again, the director is nearly in the plane ofthe ring, but its rotation relative to the bisector of the ring ox-

ygen bond is somewhat different. No other solutions with rmsdeviation values-less than 250 Hz were found for the a anomer.

Assignment of the C3 and C5 a anomer resonances on thebasis of quadrupole couplings leaves only the 6 and 6' reso-.nances for each anomer unassigned. The four remaining quad-rupole doublets have been assigned to each-anomer on the basisof relative intensities and are included in.Table 1.

Lk,] HoLa_

-Mannose

The orientation.of the director axis for each anomer is mostrelevant when compared to the direction of the ordering axisfor micelles in the liquid crystals themselves. The direction ofthe ordering axis for an axially symmetric system relative to themagnetic field can be readily deduced by watching the timecourse of change in the liquid crystal spectrum as it goes fromrandom orientation to fully ordered (12). For potassium laurate,the ordering axis is parallel to the magnetic field. At early times,the spectrum from the D-mannose anomers is a superpositionof apparently axially symmetric powder patterns with singu-larities corresponding-to director orientations of 00 and 900 nearthe extremes and central portions of the spectrum, respec-tively. The resolved doublets grow from the extremes, indi-cating a director orientation at 00 relative to the field and par-allel to-the director axes for the micelles themselves. Orientationalpreference of both anomers relative to this axis is modest, withcalculated order parameters of 0.030 and 0.033 out of a max-imum of 1.00 for 83 and a anomers, respectively. It is note-worthy that both exhibit similar degrees of order while the di-rection of the ordering axis in the molecular frame changessignificantly.

DISCUSSIONThe above results suggest that it is possible to characterize bothstructure and orientation of multiply labeled molecules fromdeuterium spectra in ordered media. In the cases presented,basic structures are known from x-ray diffraction work (13), andthe fact that we were able to find an orientation that reproducesmost splittings by using carbon-deuterium bond vectors de-rived from the crystal structure shows the solution structure tobe in approximate agreement with the crystal structure. Thedeparture of calculated from observed quadrupole splittings is,however, well beyond experimental precision (±20 Hz). Thereare several possible.reasons for this: the solution structure maybe slightly different from the crystal structure, the protons maynot be located with sufficient precision in the x-ray structure,or the axially symmetric model assumed for the quadrupolesplitting analysis may have been inadequate.

Largest departures from calculated splittings occur for the 6and 6' deuterons. A departure from the crystal structure con-formation is very likely at this point through -rotation about theC5-C6 bond. The pair of splittings observed also need notrepresent a single conformer because averaging of spectral pa-rameters will occur for any rotational isomerization on a timescale shorter than =0.1 msec. The simplest model would beone of free rotation about the C5 C6 bond. Assuming the

FIG. 4. The a and 3anomersofD-mannose, showing preferredorientation in the liquid crystalmedium. Carbon atoms, but notoxygen or hydrogen atoms, are-numbered. The molecules rotatefreely about the axis shown, andthe rotation angle depicted is for

a-Mannose viewing convenience only.

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C5-C6 bond orientation to be correct, splittings of 640 Hz forboth 6 and 6' deuterons would be predicted, in reasonableagreement with observation for the (3 anomer. For the a an-omer, splittings of 1,000 Hz would be predicted. The smallerand unequal couplings observed suggest an equilibrium amongdiscrete rotational isomers to be a better model.

While we choose not to push our data to more definitivestructural models, the data illustrate the potential for obtainingsuch information in environments more closely approximatingsolution or surface associated states. In fact, a few recent pub-lications-have exploited residual dipolar splittings or quadru-pole splittings to address conformational averaging in nonrigidmolecules (25, 26).

Even within the more rigid portion of the ring, deviationsof calculated splittings from observation are well beyond ex-perimental error. It is possible that deviations beyond this rangerepresent a less than perfect agreement with the crystal struc-ture. The 200-Hz departure in the case of the 1 deuteron of thea anomer, for example, corresponds to deviation of only 120 incarbon-deuterium bond orientation. In principle, it is possibleto use deuterium spectra to determine bond angles and bondlengths for rigid molecules to very high precision (26).

It is, however, more likely that there is some residual con-formational averaging within the ring or that the axially sym-metric orientation model is inadequate. A model with a slightlylower degree 'of motional symmetry, one having a C2v sym-metry, would require two parameters to characterize order, oneto characterize axial order and another to characterize asym-metry in rotation about that axis (2). Introducing the possibilityof asymmetry in motion, retaining the same direction for theprincipal orientation axis, and orienting additional axes paralleland perpendicular to, the ring surface results in no reduction inthe rms deviation. Fit might be improved further by allowingthe orientation axes, the order parameter, and the asymmetryparameter to vary simultaneously, but given that the first at-tempt gave no improvements, we feel that some slightly mod-ified conformation or some conformational averaging is morelikely to have produced the observed deviations.The orientation of the sugar molecules in the liquid crystal

phase is a unique piece of information. The order parametersobserved are not particularly large, 0.03, but do define a pre-ferred molecular orientation. The molecules themselves do notorient significantly in a magnetic field, so this order must beviewed as the result of liquid crystal order and order of themolecule relative to the surfaces of potassium laurate micellesforming the liquid crystal phase. With deuterium splittings frommethylenes in the fatty acyl chains used to estimate the orderin the liquid crystal itself (16), order parameters with respectto the micelle surfaces would be in the range of 0.10.The micelles in potassium laurate phases have been char-

acterized as rod-shaped micelles with cylinder axes parallel tothe magnetic field (16). The director axes for the a and 8 an-omers are, therefore, parallel to the micelle long axis in bothcases. Examination of molecular models shows that both mol-ecules are slightly flattened perpendicular to the director axis.This is more pronounced in the P anomer, in which one surfaceis decidedly-hydrophobic. On conversion to the a anomer, themolecule both loses this hydrophobic side and becomes morespherical. One might expect both steric factors and more spe-cific intermolecular bonding to contribute to molecular order.The preferred orientation is consistent with the flattened sidesorienting toward micelle surfaces as might be expected if stericfactors dominated. Since this asymmetry changes on conver-sion to the a anomer, one might expect less order for the a iso-

mer. In comparingta to (3, however, it is the orientation of thedirector in the ring plane and not the magnitude of the orderparameter that changes. Also, we note that asymmetry of mo-tion for both molecules is small. Interplay of-a number of fac-tors, including specific hydrogen bonds and coulombic forces,must therefore also contribute. While it is not possible to de-lineate contributions on the basis of the two examples pre-sented here, an expanded study could provide some interestinginformation on surface associations of carbohydrates.The work presented here leaves unanswered several ques-

tions relating to applicability to larger carbohydrates. Resolu-tion may degrade with increased molecular weight, making as-signments more difficult, and an assumption of axial symmetrymay be inappropriate for many molecules, necessitating a largernumber of resonance assignments. Nevertheless, a demon-strated ability to work with multiply labeled compounds greatlyreduces the effort necessary to obtain structural information fromdeuterium spectra. We anticipate a substantial expansion ofdeuterium NMR structural studies in the future..This work was supported by a grant from the National Institute ofGeneral Medical Sciences, GM-19035, and benefited from NMR fa-cilities made available through the National Science Foundation Re-gional Instrument Facility at Yale, CHE-791620.

1. Sharon, N. & LUs, H. (1982) Mol. Cell. Biochem. 42, 167-187..2. Hakomori, S. (1981) Annu. Rev. Biochem. 50, 733-764.3. Brisson, J. & Carver, J. (1983) Biochemistry 22, 1362-1368.4. Prestegard, J. H., Koerner, T. A. W., Jr., Demou, P. C. & Yu, R.

K. (1982)J. Am. Chem. Soc. 104, 4993-4995.5. Jeffrey, G. A., Wood, R. A., Pfeffer, P. E. & Hicks, K. B. (1983)

J. Am. Chem. Soc. 105, 2128-2133.6. Egge, H. & Hanfland, P. (1981) Arch. Biochem. Biophys. 210, 396-

404.7. Abragam, A. (1962) The Principles of Nuclear Magnetism (Clar-

endon, Oxford), pp. 232-234.8. Emsley, J. H. '& Lindon, J. C. (1975) NMR Spectroscopy Using

Liquid Crystal Solvents (Pergamon, New York).9. Opella, S. J. (1982) Annu. Rev. Phys. Chem. 33,533-562.

10. Brown, G. H.,- Doane, J. W, & -Neff, V. D. (1971) A Review of theStructure and Physical Properties of Liquid Crystals (CRC,Cleveland, OH).

11. Khetrapal, C. L. & Kunwar, A. C. (1982) in Nuclear MagneticResonance, ed. Webb, G. A. (Alden, Oxford), Vol. 11, pp. 248-263.

12. Forrest, B. J. & Reeves, L. H. (1981) Chem. Rev. 81, 1-14.13. Longchambon, F., Avenel, D. & Neuman, A. (1976) Acta Crys-

tallogr. Sect. B 32, 1822-1826.14. Walborg, E. F., Jr., Kondo, L. E. & Robinson, J. M. (1975)

Methods EnzymoL. 41, 10-21.15. Iwakawa, J., Kobatake, H., Suzuki, I. & Kushida, H. (1980) J.

Chromatogr. 193, 333-337.16. Forrest, B. J. & Reeves, L. H. (1981)J. Am. Chem. Soc. 103, 1641-

1647.17. Bodenhausen, G. (1981) Prog. NMR Spectrosc. 14, 137-173.18. Miner, V. W., Tyrell, P. M. & Prestegard, J. H. (1983) J. Magn.

Reson. 55, in press.19. Nagayama, K., Kumar, A., Wuthrich, K. & Ernst, R. R. (1980) J.

Magn. Reson. 40, 321-334.20. Bodenhausen, G., Freeman, R., Morris, G. & Turner, D. L. (1978)

J. Magn. Reson. 31, 75-95.21. Miner, V. W. & Prestegard, J. H. (1981) J. Am. Chem. Soc. 103,

5979-5981.22. Jaffe, D., Vold, R. R. & Vold, R. L. (1982) J. Magn. Reson. 46,

475-495.23. Warren, W. S. & Pines, A. (1981)J. Chem. Phys. 74, 2808-2818.24. Winnik, F. M., Brisson, J. R., Carver, J. R. & Krepinsky, J. J.

:(1982).Carbohydr. 'Res. 103, 15-28.25. Moseley, M. E., Poupko, R. & Luz, Z. (1982)J. Magn. Reson. 48,

354-360.26. Bechtold, W. E. & Goldstein, J. H. (1981) Chem. Phys. Lett. 83,

401-404.

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