[3] 13C NMR Analysis of Complex Carbohydrates

58

ANALYTICAL METHODS

[3l

O-methylfucosyl-l,2-galactose. The intensity of m/e 282 greater than m/e 250, together with aA~ (m/e 189) and baA~ (m/e 393) indicates that 6-deoxy-hex-hex is linked to the 3 position of the internal hexNTF. Nonreducing terminal hexNTF can eliminate methanol from the 3 position to give m/e 282 greater than m/e 250 (Figs. 8 and 10). In chitotetraose, however, m/e 250 is greater than m/e 282, probably because of contributions from the two internal 4-0 substituted hexNTF residues (Fig. 9).

[3]

13C

NMR Analysis of Complex Carbohydrates

By R. BARKER, H. A. NUNEZ, P. ROSEVEAR, and A. S. SERIANNI General approaches to the analysis of 13C NMR spectra of complex carbohydrates have been well described in an earlier contribution to this series 1 and significant advances have been made in the application of 1H NMR spectroscopy to the elucidation of oligosaccharide structures, la In this chapter we describe applications of ~3C NMR using higher field NMR spectrometers to the study of oligosaccharides containing a number of different monosaccharide units with special attention given to the examination of solution conformations. In principle it might eventually be possible to determine the total structure of a complex oligosaccharide by laC NMR spectroscopy. Such an analysis would identify the component monosaccharides, anomeric configurations, positions of linkages, and conformation of both the monosaccharide residues and of the glycosidic linkages. So far this goal has not been achieved, although laC NMR has proved to be a most valuable addition to traditional chemical and enzymologic approaches to structure elucidation. Four parameters can be obtained from 13C NMR experiments. These are: chemical shifts of 13C nuclei which are characteristic of their chemical nature and environment; spin-spin coupling constants between ~3C and other nuclei with spin which give information on the angles between bonds joining the coupled nuclei; nuclear Overhauser enhancement (NOE) and nuclear relaxation times (TI and TD, both of which can give information on internuclear distances and mobility. 1 H. J. Jennings and I. C. P. Smith, this series, Vol. 50, p. 39. la j. Montreuil, Adv. Carbohydrate Chem. Biochem. 37, 157 (1980) and references cited therein.

METHODS IN ENZYMOLOGY,VOL. 83

Copyright © 1982by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181983-3

[3]

13C N M R OF COMPLEX CARBOHYDRATES

59

TABLE I COMPARISON OF 13C CHEMICAL SHIFTS IN SEVERAL/3-D-GALACTOPYRANOSYL-CONTAINING COMPOUNDS

/3-D-Gal carbon chemical shifts Compound

1

2

3

4

5

6

/3-D-Gal /3-D-Gal OCH3 /3-D-GaI(1 ~ 4)Glc (ct or/3) /3-D-Gal(1 -o 4)GlcNAc (a or/3) a-L-Fuc(1 ~ 2)/3-D-Gal(1 --->4) GIcNAc hexanolamine

97.7 104.9 104.3 104.1

73.3 71.8 72.5 72.3

74.2 73.9 74.1 73.9

70.1 69.8 70.1 69.9

76.3 76.2 76.9 76.6

62.3 62.1 62.6 62.3

101.1

77.9

75.0

70.6

76.9

61.6

Typically, 13C NMR spectra are obtained using broad-band decoupling of protons. Spectra are relatively simple, with a single sharp resonance for each carbon in the compound. At natural abundance levels, 13C-'3C coupling is not observable. Proton-coupled 13C spectra are much more complex and show the number of hydrogens covalently bonded to each carbon since each I3C resonance is split (150-180 Hz) by covalently bonded hydrogens into n + 1 lines (n = the number of attached hydrogens). Additional splittings or line-broadening (---8 Hz) due to 2- and 3-bond and long-range coupling also add to the complexity of 1H-coupled '3C spectra. Several experimental options are available on most high-field FT spectrometers that facilitate assignment of 13C resonances and of lzC-~H coupling constants. These include: gated 1H decoupling, 2"3 off-resonance 1H decoupling, 4'5 selective ~zC saturation combined with gated 1H decoupling and FT difference spectroscopy, 6 and two-dimensional J spectroscopy/ The latter may be particularly valuable for complex molecules, since it permits lac chemical shifts and 13C-1H coupling of each ~3C nucleus to be displayed separately. Monosaccharide Composition and Position of Linkages. The monosaccharides and simple glycosides have characteristic patterns of 13C chemical shifts (Table I). Generally, a simple sugar or glycoside can be identified 2 O. A. Gansow and W. Schittenhelm, J. Am. Chem. Soc. 93, 4294 (1971). 3 R. Freeman and H. D. W. Hill, J. Magn. Reson. 5, 278 (1971). E. Wenkert, A. O. Clouse, D. W. Gochran, and D. Doddrell, J. Am. Chem. Soc. 91, 6879 (1969). H. J. Reich, M. Jautelat, M. T. Messe, F. J. Weigert, and J. D. Roberts, J. Am. Chem. Soc. 91, 7445 (1969). '~ G. T. Andrews, I. J. Colquhoun, B. R. Doggett, W. McFarlane, B. E. Stacey, and M. R. Taylor, J. Chem. Soc. Chem. Commun., 1979 p. 89. r R. Freeman and G. A. Morris, Bull. Magn. Reson. 1, 5 (1979).

60

ANALYTICAL METHODS

[3]

from its 13C NMR spectrum. The anomeric carbon and the methylene carbon (C-6 in the hexoses) have characteristic resonances well removed from the carbons at other positions. The latter fall in a narrow range between 68 and 78 ppm. Resonances of carbons bearing amino or substituted amino groups occur at higher field (56 - 2 ppm). When substituted by a glycosyl residue, the resonance of the substituted carbon moves downfield generally by 4 to 10 ppm, while resonances of adjacent carbons generally (but not always) move upfield by a small amount (< 1.5 ppm). Because of this, the site of substitution is not always easily established. Resonances of terminal nonreducing sugars are similar to those of the corresponding methyl glycoside or reducing sugar. For example, the 13C resonances of fl-o-galactopyranosyl residues in lactose, N-acetyllactosamine, methyl-/3-o-galactopyranoside, and/3-D-galactopyranose are very similar (Table I). Equally imlSortant, the relative positions of resonances are very similar even when the absolute chemical shifts are different. Although the monosaccharide composition of an oligosaccharide may be difficult to infer from its laC NMR spectrum, it is usually possible, although occasionally tedious, to assign resonances to specific carbons if the composition is known. An excellent example of the assignment of chemical shifts in a series of complex oligosaccharides is provided by Lemieux et al., s who synthesized a group of ABH and Lewis antigenic determinants (up to the pentasaccharide) and assigned ~3C and 1H parameters. Assignment of Anomeric Configuration. In many oligosaccharides, each anomeric carbon gives a discrete ~aC resonance between 95 and 105 ppm. The number of resonances can, with caution, be taken to reflect the number of monosaccharide residues in the structure. The chemical shifts of anomeric resonances can be affected by changes in the structures of both the glycosyl residue and the aglycon (Table I). Even remote changes in structure can have measurable effects (0.1-0.3 ppm), so that the position of anomeric resonances should be interpreted with care in terms of either anomeric configuration or the nature of the glycosyl residue. For nonreducing terminals the resonance of the anomeric carbon will usually be similar to that of the parent methyl glycoside. Generally, resonances of anomeric carbons having the aglycon equatorial are downfield relative to those having an axial substituent. This rule is not upheld in the case of mannopyranosyl or rhamnopyranosyl residues (Table II). These generalities must be applied with caution since the structure of the aglycon and the position of substitution in the aglycon may shift the resonances of anomeric carbons (Table I). s R. U. Lemieux, K. Bock, L. T. J. Delbaere, S. Koto, and V. S. Rao, Can. J. Chem. 58, 631 (1979).

[3]

13C NMR OF COMPLEXCARBOHYDRATES

61

TABLE II 13C CHEMICAL SHIFTS OF ANOMERIC CARBONS IN RELATION TO ORIENTATION OF THE AGLYCON Methyl glycoside

Position of aglycon

C-I chemical shift

a-D-Glc fl-D-Glc c~-D-Gal /3-o-Gal a-o-Fuc /3-D-Fuc a-D-Man /3-D-Man a-L-Rhamno fl-L-Rhamno

Axial Equatorial Axial Equatorial Axial Equatorial Axial Equatorial Axial Equatorial

100.3 t04.3 100.5 104.9 100.5 104.8 102.0 101.9 95.0 94.6

In addition to chemical shift, one-bond coupling between C-1 and H-1 can be used to assign anomeric configuration. In pyranosides, C1-H1 coupling (IJcl_nl) is larger when H-1 is equatorial ( - 1 7 0 Hz) than when H-1 is axial (-160 Hz). This correlation can be applied with confidence, since a large number of compounds have been examined) -ll C1-H1 coupling in furanosides does not vary in the same fashion with anomeric configuration. Ring Conformation. The conformations of pyranosyl rings of the monosaccharides commonly found in complex oligosaccharides have been established through studies of simple glycosides, lz These conformations have characteristic dihedral angles and associated coupling constants between ~H or ~3C separated by three bonds. With the exception of the anomeric proton, observation of ~H-~H coupling in 1H NMR spectra is often hindered by the overlap of resonances. ~C-IH coupling sometimes can be evaluated from 1H-coupled 'aC NMR spectra (see Instrumentation and Methods), although such spectra are complicated by multiple resonances produced by one-bond ~H-'3C coupling. The more useful three-bond couplings are smaller (often <3.0 Hz) and can be obscured or unresolved. One- and two-bond coupling between ~H and laC or between 13C and I'~C (see below) also can give information about conformation. The magnitude of two-bond I3C-IH coupling appears to depend on the relative positions of substituent hydroxyl groups. 9 K. Bock and C. Pedersen, Acta Chem. Scand. Ser. B B31, 354 (1977). ~o K. Bock and C. Pedersen, Acta Chem. Scand. Set'. B B29, 258 (1975). 1~ K. Bock and C. Pedersen, J. Chem. Soc. Perkin Trans. 2, 1974 p. 293. 12 S. J. Angyal, Angew. Chem. Int. Ed. Engl. 8, 157 (1969).

62

ANALYTICAL METHODS

[3]

The observation of three-bond 13C-IH coupling (3Jc-H) in 1H-coupled ~3C spectra is facilitated by the use of higher-field spectrometers (>60 MHz). In monosaccharides and simple glycosides, aJc_a displays a Karplus dependence ranging from - 6 Hz when the dihedral angle is 180°, 0 Hz at 90°, 2 --+ 0.5 Hz at 60° or 120°, and - 5 Hz when the dihedral angle is 0°. 13 Values such as these can be difficult to measure in oligosaccharide spectra, where resonances tend to be broad. Bock and Pedersen 9 have reported three-bond couplings for a model compound, 1,6-anhydro-D-galactopyranose, which has a fixed conformation (4C~). Through selective 1H-decoupling and specific deuteration, eleven 3Jc_H values were assigned and related to dihedral angles. Specific enrichment of monosaccharides with 13C facilitates observation of some 13C-XH couplings by both 1H and ~3C NMR. It also permits the observation of IaC-13C coupling, which can be related to molecular geometry. TM The development of a simple, high-yield synthesis of ~zCenriched monosaccharides ~4-1r and the use of partially purified glycosyltransferases 1s'~'19a to prepare '3C-enriched oligosaccharides has allowed solution conformation of these compounds to be evaluated. Several di- and trisaccharides related to blood group substance H were prepared with ~3C enrichment at various sites (Scheme 1). In singly enriched compounds, ~3C-~3C couplings are observed in the resonances of unenriched carbons. A partial spectrum of fl-D-[1-aaC]Gal(1--~4)/3-D GIcNAc hexanolamine (Scheme 1, 1) is shown in Fig. I. Coupling of 1-~3C to various carbons in the Gal moiety is apparent. The pattern and magnitudes of these couplings (both two- and three-bond) are the same as in fl-o-galactopyranose and its methyl and ethyl glycosides, indicating that the conformation of the galactopyranosyl residue in the disaccharide is similar to that in the monosaccharides. This conclusion is confirmed by the observation at 600 MHz that 1H-1H couplings are virtually identical in the monosaccharide and the disaccharide. Similarly, in the trisaccharide 4, the conformation of the galactopyranosyl residue is maintained (4C0.

la j. A. Schwarcz and A. S. Perlin, Can. J. Chem. 50, 3667 (1972). lza T. E. Walker, R. E. London, T. W. Whaley, R. Barker, and N. A. Matwiy0ff, J. Am. Chem. Soc. 98, 5807 (1976). 14 A. S. Serianni, H. A. Nunez, and R. Barker, Carbohydr. Res. 72, 71 (1979). is A. S. Serianni, E. L. Clark, and R. Barker, Carbohydr. Res. 72, 79 (1979). 10 A. S. Serianni, J. Pierce, and R. Barker, Biochemistry 18, 1192 (1979). lr A. S. Serianni and R. Barker, Can. J. Chem. 57, 3160 (1979). 18 H. A. Nunez and R. Barker, Biochemistry 19, 489 (1980). lap. Rosevear, H. A. Nunez, and R. Barker, Biochemistry 21 (1982), in press. 19a M. L. Hayes, A. S. Serianni, and R. Barker, Carbohydrate Res. 100 (1982), in press.

[3]

~3C N M R OF COMPLEX CARBOHYDRATES

63

z~

o 0

0 I--

GDP

0

FUCOSYL

TRANSFERASE

o..

J J >kLd 0

o°3\

0

I

---'~.~\ ?/

~-~o -r

,-

O.

-I

<

f-

0%

I k=

~,,~\0 "r

',-

6",~_ o

a

~J

=

Ol.A 0

0 J

° ~

t~

0

o m o I,-

o--o

~

~

Z

~

= o

_1 q

~L8

l

oo

%

\~,

0 J

=0

~

gW\

~

8~

=.~

,

v

f-

E 0

...1

u_

FW

.o

E r~

312 + a.

,E

I

.4

o~4o -'r

~4

=

oJo

,

~-

o~".~__g

# 0 0

64

ANALYTICAL METHODS

[3]

1 N

I I

00

78

'

'

'

~

'

I

iNtm74

?2

" '?0

FIG. 1. Partial 90.5 MHz 13C spectrum of fl-D-[1-13C]Gal(l ~ 4)fl-D-GIcNAc hexanolamine. Solution 0.2 M in 2H20 at 20°C, 3000 transients, spectral width 5434 Hz, 0.16 Hz per data point, 45° pulse angle. Chemical shifts are in parts per million downfield from tetramethylsilane. Reprinted with permission from Biochemistry 19, 489 (1980). Copyright 1980 American Chemical Society.

In all cases examined, the conformations of pyranosyl rings of oligosaccharides in solution are similar to those of the respective simple glycosides. Glycosidic Bond Conformation. The conformation about a glycosidic bond is specified by the angles q5 and ~b (Scheme 2). In oligosaccharides, ~b = 0° when H1 and the aglycon carbon are eclipsed; ~ = 0° when the anomeric carbon and the proton on the aglycon carbon are eclipsed. Estimates of ~b and ¢ can be obtained from the three-bond coupling constants between laC and 1H atoms involved in the glycosidic bond; ~b from" 3Jc4,-ri1 and aJc4,_c2; ~bfrom aJcl-n4,, aJcl-c3,, and aJcl_cv. Two-bond coupling between C- 1 and C-4' also provides information about glycosidic bond conformation. 1s.19 Perlin and co-workers 2° measured 3Jc1_i~4, in methyl B-maltoside

OH

GOCH3 \

SCHEME 2. Reprinted with permission from Biochemistry 19, 489 (1980). Copyright 1980 American Chemical Society. 20 A. S. Perlin and G. K. Hamer, in "Carbon-13 NMR in Polymer Science" (W. M. Pasika, ed.), pp. 123-141. Am. Chem. Soc., Washington, D.C., 1979.

[3]

~3C NMR OF COMPLEXCARBOHYDRATES

65

H'~C~o H OH

5.0Hz /

FIG. 2. Linewidth measurements of resonances due to laC-enriched carbons: (A) (X-LFuc(1 --~ 2)/3-D-[1-1zC]GaI(1~ 4)/3-D-GIcNAc hexanolamine; (B) a-L-[1-1aC]Fuc(1 ~ 2)/3-t)[1-~aC]Gal(1 ~ 4)/3-I)-GlcNAc hexanolamine. (C) Natural abundance dioxane. Spectral width 1000 Hz, 0.36 Hz per data point, I000 transients. Reprinted with permission from Biochemistry 21 (1982), in press. Copyright 1982 American Chemical Society.

( - 3 . 0 Hz), methyl/3-cellobioside (4.3 Hz), and cyclohexaamylose (4.8 Hz). Selective deuteration was used to eliminate interfering proton resonances and couplings. These values correspond to dihedral angles (tk) of 45-50 °, 30°, and --- 10°, respectively. In cyclohexaamylose, 3Jc4,_r, = 5.2 Hz corresponding to (h = - 1 0 °. From these data, it appears that the solution conformations of the glycosidic bonds in methyl/3-cellobioside and cyclohexaamylose are similar to those found in the crystalline state. Methyl/3-maltoside, however, has a substantially different conformation in solution (~b = ~k - 45 °) than in the crystal (4) -- ~b ~ 10°).20 In di- and trisaccharides containing D-[ l-~3C]galactopyranosyl residues linked /3-(1 ~ 4) to glucosyl or 2-acetamido-2-deoxyglucosyl residues, 3J~.1_c3, = 0 Hz, 3Jc1_c5, < 1.5 Hz, and 3Jc1_~4, = 4.9 Hz, indicating that the dihedral angle qJ = 10°.ls'19,laa The C1-CY dihedral angle is ~ l l 0 °, while the C1-C5' angle is~1300. TM The dihedral angle ~b was estimated to be 45 ° from 3Jc2_c4, = 3.1 Hz observed in the ~aC spectrum of [2-13C] lactoside and from 3Jc4,_m = 3.8 Hz observed in the ~H spectrum of [4,_13C]lactoside. ~9~ Studies of blood group substance H (Scheme 1, 4) enriched with ~aC at specific sites provided estimates of ~b and 0 for the a-L-fucopyranosyl glycosidic bond to 2-OH of the galactopyranosyl residue. Two samples of J3C-enriched trisaccharide ~9 were prepared; one contained [I-~3C]Gal (spectrum A, Fig. 2), and the other contained [1-~3C]Gal and [1-1aC]Fuc (spectrum B, Fig. 2). In the latter, resonances of the enriched carbons are

66

ANALYTICALMETHODS

[3]

broadened b y - 2 . 5 Hz, indicating coupling between these nuclei. The singly enriched sample serves as a control for the linewidth in the absence of coupling, and dioxane (spectrum C, Fig. 2) in both samples provides an internal check of spectrometer performance. From the value of 3JclGal-CiFu c the dihedral angle between C-I Gal and C-1 Fuc must be - 6 0 ° (or 120°), giving 0 --" 0° -+ 15°. 3Jc2Ga~-atFu~ = 3.5 Hz was obtained from the 1H NMR spectrum at 180 MHz, indicating that ~b ~ 55°. These values are in reasonable agreement with the angles proposed by Lemieux et al. 8 on the basis of hard-sphere energy calculations and proton chemical shifts (6 -~ 40°, ~ = 20°) for the isomeric trisaccharide having a/3-DGal(1 ~ 3)/3-D-GlcNAc rather than/3-D-Gal(1 ---, 4)/3-o-GlcNAc core. Perlin and co-workers, 2° Bock and Pedersen, a and Walker et al. laa have shown that two-bond coupling between C-1 and H-2 (or C-2 and H-l) in pyranosyl rings depends on the positions of hydroxyl and other substituents at C-1 and C-2. Nunez and Barker 18 have proposed a similar dependence for 13C-x~C coupling through the glycosidic bond and through the ring oxygen. The presence of O or C anti to the coupled atom makes a positive contribution, whereas gauche substituents make negative contributions. Book and Pedersen 9 demonstrated that a vectorial analysis gave reliable predictions of couplings. In the case of 2Jc_c through a glycosidic bond, there is presently insufficient evidence to establish whether a similarly useful correlation exists. Nuclear

Overhauser Enhancement

and Relaxation

Measurements.

Lemieux et al. s have used nuclear Overhauser enhancement (NOE) and T1 determinations to evaluate conformations in blood group oligosaccharides. In the NOE technique, a specific proton is saturated with a perturbing radiofrequency, and the enhancement in the resonances of adjacent protons is measured. The degree of enhancement is inversely proportional to the sixth power of the distance separating the irradiated and observed nuclei. Enhancements of 4-19% were observed, corresponding to distances of approximately 3.2-2.2 .~. Spin-lattice relaxation times (Tx) of 13C nuclei are readily measured (see Instrumentation and Methods). Generally, they increase with increased flexibility in the structure 2~'2z and can be used to evaluate the motional properties of carbohydrates in solution, xaa~ 1H relaxation times have been used s to estimate interproton distances on the assumption that the principal determinant of the relaxation rate (1/TO is the distance between a specific proton and all other protons in the molecule (i.e., 1/T~ = C

zl j. M. Berry, L. D. Hall, and K. F. Wong, Carbohydrate Res. 56, C16 (1977). ~2 A. Allerhand and D. Doddrell, J. Am. Chem. Soc. 93, 2777 (1971).

[3] Instrumentation

13C NMR OF COMPLEXCARBOHYDRATES

67

and Methods

Although NMR instrumentation and computer-assisted techniques for data acquisition and processing have developed rapidly, the general procedures described by Jennings and Smith I still apply. Developments are reviewed annually, 2a and many reference texts have been published, z4 Commercial spectrometers for routine use operate at frequencies from 60 to 400 MHz for 1H (15 to 100 MHz for 13C). In the United States, regional facilities partially supported by the National Science Foundation and the National Institutes of Health z5 provide access to such instruments. Higher-field spectrometers are being developed and, at this time, 500 and 600 MHz 1H NMR spectrometers are accessible. 2~ Higher field strengths increase sensitivity and resolution, and decrease the time required to obtain a useful spectrum. Obtaining a Spectrum. laC N M R spectra are usually obtained with broad-band ~H decoupling. For aqueous solutions of oligosaccharides, it can be difficult to achieve complete ~H decoupling without excessive heating, and broad lines may result. An improved decoupling method has been described/7 A useful spectrum usually can be obtained in 12 hr (-40,000 transients) with a 20-MHz spectrometer using 1.5 ml of a 10 mM solution in an 8-mm tube. If the same sample is observed at 100 MHz, an equivalent spectrum would be obtained with 200 transients ( - 4 min). The quality of the spectrum will be improved by use of a micro cell and by increasing the sample concentration, provided that this does not produce a highly viscous solution. Several key instrumental parameters for FT operation must be selected. These are pulse angle, acquisition time, delay time, and spectral width. Pulse angle refers to the time during which radiofrequency (rf) power is applied to the nuclei to be observed. It is expressed as an angle in degrees or as a time in microseconds. The greater the pulse angle, the longer it takes nuclei to relax before a second pulse can be applied. The time required for the effect of an rf pulse to be dissipated is also determined by the spin-lattice relaxation time (TI) of the nuclei. Since it is ~3 Nuclear Magn. Reson., Vols. 1-8, The Chemical Society, Burlington House, London,

1972-1979. 24 M. L. Martin, J.-J. Delpuech, and G. J. Martin, "Practical NMR Spectroscopy." Heyden, Philadelphia, 1980. 2s Information concerning these facilities can be obtained from Division of Research Resources, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Maryland 20205. 26 500 MHz: National NMR Facility for Biomolecular Research, Francis Bitter National Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; 600 MHz: NMR Facility for Biomedical Studies, Carnegie-Mellon Institute, Pittsburgh, Pennsylvania 15213. 2r V. J. Basus, P. D. Ellis, H. D. W. Hill, and J. S. Waugh, J. Magn. Resort. 35, 19 (1979).

68

ANALYTICALMETHODS

[3]

common to collect data from several thousand pulses (time-averaged), the pulse angle and the frequency of pulsing are important variables. The most efficient pulse angle can be computed from cos 0 = e -~/rl, where 0 = optimal pulse angle, r = acquisition time + delay time, and T1 = spinlattice relaxation time of the nucleus. 28 The acquisition time is the time required to complete one sweep of the spectral range. It is determined by the width of the spectrum being observed (in hertz) and the number of computer points used. If the acquisition time is too short to permit complete relaxation, a delay time can be entered before the next pulse is applied. T1 values for carbons in oligosaccharides in aqueous solution generally are <1.0 sec, and are often 0.3-0.4 sec. s The number of data points per hertz (digital resolution) is an important determinant of spectral resolution. To observe small coupling constants (<2 Hz), this number should be selected to ensure that the intrinsic resolution of the spectrometer determines the observed linewidths. When the collected data are transformed, a line-broadening factor can be selected to improve S/N in the spectrum. If high resolution is required, line-broadening should be kept to a minimum. Sample Preparation. For high-resolution spectroscopy, especially for T1 measurements, solutions must be free of paramagnetic impurities and particulate matter. Neutral sugar solutions can be deionized with ionexchange resins [Dowex 50 (H ÷) and Dowex 1 (OAc-)] and passed through a small column (1 ml) of chelating resin (Chelex-100) into an NMR tube. The form of chelating resin is important, since the pH of solutions can vary from 3 to 10 after treatment, depending on whether the H ÷ or Na ÷ form is used. Alternatively, EDTA (<3 mM) can be added. Samples should be flushed with N~ or Ar to remove 02 prior to analysis. If T1 values are >5 sec, additional precautions must be taken. ~9,a° If 2H20 is used, it should be carefully purified for T1 studies. 3° NOE and TI Determinations. Nuclear Overhauser enhancement determinations should be made from spectra having high S/N values. 1Hdecoupled 13C spectra are normally obtained with NOE to enhance S/N. Spectra without NOE can be obtainedJ a however, and these spectra can be used for NOE measurements, provided that adequate time is allowed for relaxation. T~ determinations are affected by temperature, solution concentrations, and solvent composition. Pulse sequences for the determination of zs R. E. Ernst, Adv. Magn. Reson., 2, 1 (1966). 2g G. C. Levy and I. R. Peat, J. Magn. Reson. 18, 500 (1975). 30 H. Pearson, D. Gust, I. M. Armatage, H. Huber, J. D. Roberts, R. E. Stark, R. R. Void, and R. L. Void, Proc. Natl. Acad. Sci. U.S.A. 72, 1599 (1975).

[4]

PROTONNMR OF GLYCOSPHINGOLIPIDS

69

T, have been compared, ~9'~1 and suitable sequences are described in spectrometer manuals. Aids to Spectral Analysis. Software and instrumental options that can assist in the assignment of '3C resonances and in observing ' a C - ' H couplings include selective ' H irradiation, 9 gated ' H decoupling, 2'3 offresonance 1H irradiation, 4'~ two-dimensional J spectroscopy, r and selective '3C saturation allied with gated 'H decoupling and FT difference spectroscopy. 6 Several of these programmable options are provided in the software libraries of most spectrometers. Quantitation. The main factors influencing quantitation by 13C are differences in NOE and /'1 values between various carbon nuclei. Approaches to quantitation have been reviewed by Shoolery. 32 Spectral Simulation. Computer programs for spectral simulation of systems containing up to seven nuclei are provided with most spectrometers. Complex spectra can often be resolved by iteration of an approximated spectrum to fit the observed spectrum. Spectra of compounds with more than seven nuclei can sometimes be dealt with by simulation of segments. '~ E. D. Becker, J. A. Ferretti, R. K. Gupta, and G. H. Weiss, J. Magn. Resort. 37, 381 (1980). 32 j. N. Shoolery, Prog. Nucl. Magn, Reson. Spectrosc. 11, 79 (1977).

[4] A n a l y s i s o f G l y c o s p h i n g o l i p i d s b y H i g h - R e s o l u t i o n P r o t o n Nuclear Magnetic Resonance Spectroscopy

By JANUSZ DABROWSKI,

PETER H A N F L A N D , and H E I N Z EGGE

When analyzing an unknown glycosphingolipid, the following aspects of the primary structure have to be clarified: (a) the sugar composition; (b) the anomeric configuration; (c) the conformation of the sugar rings; (d) the sequence; and (e) the sites of glycosidic linkage. The secondary structure of the oligosaccharide chain, i.e., the relative spatial orientation of the saccharide residues, is also of great interest, as it most probably contributes to the immunological specificity of glycosphingolipids. Some of these features can be elucidated by analytical and biochemical methods treated in other contributions to this volume. Because of the small amount of material obtainable from some sources, its loss due to destructive methods is a serious disadvantage. Nuclear magnetic resonance (NMR) is a nondestructive method capable of furnishing many-sided structural information; it is therefore highly desirable fully to exploit its potential,

METHODS IN ENZYMOLOGY, VOL. 83

Copyright © 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181983-3