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Carbohydrates Studied by NMR
Carbohydrates Studied by NMR Charles T Weller, School of Biomedical Sciences, University of St Andrews, UK ã 2017 Elsevier Ltd. All rights reserved.
Symbols J J(v) T1, T2 Dxi
coupling constant spectral density function relaxation constants Huggins electronegativities
Introduction Carbohydrates, the most widely abundant biological molecules, are key components within a wide variety of biological phenomena. Polysaccharides such as glycogen, cellulose and starch have important structural or nutritional roles, but it is the mediation of specific recognition events by carbohydrates that has sparked detailed analyses of structure, conformation and function. The non-invasive, non-destructive nature of the technique is commonly mentioned as the most important reason for analysing carbohydrates by NMR and is of considerable advantage when dealing with small amounts of precious material. This advantage is, however, offset by the relative insensitivity of the technique when compared with other methods such as mass spectrometry or enzymatic approaches that may be more appropriate for specific problems of sequence determination or compositional analysis. The value of NMR analysis is apparent in the additional data obtainable regarding composition, structure, conformation and mobility necessary for understanding recognition processes. The analysis of carbohydrates by NMR is characterized by a modification of existing techniques to address the particular problems of oligosaccharide spectra, namely poor dispersion, heterogeneity, a relatively high degree of interresidue mobility and a lack of conformational restraints. This article will attempt to provide an introduction to a large field of study, and readers are encouraged to look to the Further Reading section for additional information.
Sample Preparation In high-field NMR, the use of high-quality sample tubes can have a significant impact upon the quality of the spectra. For biologically relevant NMR analysis, experiments are generally carried out in aqueous solution. A strong water signal prevents direct analysis of proton signals lying beneath it and, owing to the problems of dynamic range, would reduce the sensitivity with which weak solute signals are detected. If, as is often the case, the exchangeable OH or NH protons are not of interest, then experiments are usually carried out in 2H2O.
This article is reproduced from the previous edition, Copyright 1999, Elsevier Ltd.
158
u torsion angle tc, t i and, t0 rotational, internal and overall correlation times respectively w, c and, v glycosidic torsion angles
Repeated dissolution and evaporation or lyophilization is recommended to reduce the proportion of residual protons remaining. In most cases, two dissolutions into>99.8% 2H2O followed by a final dissolution into>99.95% 2H2O, preferably from a sealed ampoule, should be sufficient. Other precautions such as pre-wetting pipettes, further rounds of dissolution and drying, or the use of a dry box may be used for greater sensitivity or quality. Although extremes of pH or pD (outside the range d 5–8) are to be avoided, buffer solutions are only necessary in two cases: if the molecule is charged and the charge state is relevant to the study; and in the study of acid-labile carbohydrates, such as sialylated oligosaccharides. The use of 1H2O as solvent is necessary in some cases and requires appropriate water suppression techniques. Care must be taken to ensure that the water is free from impurities as well as dissolved gases or paramagnetic species. A deuterated solvent to provide an appropriate lock signal should be added, usually 5–10% 2H2O. Oligosaccharides are not normally soluble in nonaqueous solvents, except for dimethyl sulfoxide (DMSO). Despite the biologically irrelevant environment, this permits the observation of exchangeable protons, and there is also some increase in the proton spectral dispersion. Similar precautions to those noted for the use of 2H2O should be observed, such as the use of high isotopic purity (>99.96%) solvent and repeated dissolution to remove exchangeable protons. To approach optimal line widths, it is often advisable to remove soluble paramagnetic components by passage through a suitable chelator. Degassing to remove dissolved oxygen is also recommended. For acceptable spectra within a reasonable time using a 500 MHz spectrometer, 10 nmol is an approximate lower limit for 1D 1H spectra; for 2D 1H spectra, amounts of 1 m mol are preferable, although spectra with as little as 100 nmol are possible with care. Sample volumes vary between 0.4 and 0.7 ml, depending on the size of the spectrometer RF coils. Temperature should be maintained at a constant level, preferably one or two degrees above room temperature for stability. Sample spinning is not necessary, except in cases where resolution is a particular problem. T1 for protons in carbohydrates is of the order of 0.1–0.5 s, and recycle delays of 1–1.5 s are usually sufficient. Referencing of samples is commonly to acetone at d 2.225 relative to DSS (5,5-dimethylsilapentanesulfonate) at 25 C, or DMSO at 2.5 ppm.
Encyclopedia of Spectroscopy and Spectrometry, Third Edition
http://dx.doi.org/10.1016/B978-0-12-803224-4.00113-8
Carbohydrates Studied by NMR
Analysis of Carbohydrate Structure by NMR The problems of carbohydrate structure addressed by NMR can be divided into four parts: 1. What is the composition of the molecule: what monosaccharides are present, are they in furanose or pyranose configurations, and a or b anomeric forms? 2. What is the nature of the linkages between the monosaccharides: at what positions do the substituents occur, and in what sequence? 3. What is the conformation of the molecule: what conformation or family of conformations are populated about the Oglycosidic bonds and hydroxymethyl rotamers? 4. What are the dynamic properties of the molecule? Whilst the first and second questions can be satisfactorily answered by NMR, they are perhaps better approached in tandem with chemical or enzymatic methods, such as hydrolysis, followed by reduction and analysis of alditol acetates by GC-MS, or digestion with specific glycosidases. Such a concerted approach has been used to successfully determine the composition and configuration of many oligosaccharides and glycans. The unique advantages of NMR in the analysis of carbohydrate structure are only fully apparent in consideration of points 3 and 4. In contrast to the unbranched polymeric nature of polypeptide and nucleic acid chains, carbohydrates may be branched structures, capable of substitution at several points. The monosaccharide constituents are polymerized in nature by
159
a nontemplate directed, enzymatic process; the resulting oligosaccharides are often heterogeneous, differing in detail from a consensus structure. NMR is particularly efficient at investigating the solution conformations, and the dynamic properties of such molecules.
1
H NMR of Carbohydrates
Primary Analysis and Assignment: 1H 1D Spectra The 1H NMR spectrum of the disaccharide galactopyranose b 1–4 linked to glucopyranose (Galpb1–4Glcp, lactose, shown in Figure 1) is shown in Figure 2. Despite the relative simplicity of the molecule, with only 14 proton signals observable, the spectrum is surprisingly complex. This is due to the relatively small chemical shift dispersion of the non-exchangeable ring protons, resonating between d 3 and 4. The small chemical shift dispersal combined with homonuclear spin–spin
Figure 1 Structure of the disaccharide Galpb1-4Glcp, showing the numbering of protons within the sugar rings.
Figure 2 1H 1D spectrum of the disaccharide galactopyranose b1-4glucopyranose at 500 MHz and 303 K in 2H2O. Peak assignments are given for well-resolved peaks, following the numbering of Figure 1. The unresolved proton ‘envelope’ consists of the remaining ring proton resonances. The peak marked ‘HOD’ arises from residual water within the sample.
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Carbohydrates Studied by NMR
coupling gives rise in many cases to non-first-order spectra, complicating the measurement of spin-coupling values. This makes the assignment and analysis of even quite small saccharides quite difficult, and as a consequence the use of spectrometers of field strength 400 MHz or above is recommended. Primarily owing to the electron-withdrawing effect of the ring oxygen, the anomeric (H1) protons give rise to signals lying outside this envelope of ring protons, between d 4.25 and 5.5. Since equatorial protons experience a shift of approximately d 0.5 relative to axial proton signals, a anomeric protons of D-sugars tend to be found between d 4.9 and 5.5, and b protons between d 4.3 and 4.7. The ring protons of each monosaccharide type give rise to characteristic patterns within the envelope of overlapping ring proton resonances. However, incorporation of a monosaccharide residue into an oligosaccharide will cause changes in these chemical shift patterns. For example, substitution at a given carbon causes the signal arising from the attached proton to change by d 0.2–0.5. The glucose residue of the sample shown in Figure 2 is free to mutarotate between a and b forms. The signals arising from these are easily distinguished from those owing to the galactose H1 by the difference in area. The two glucose resonances have a combined area approximately equal to that of the galactose H1 resonance. Anomeric signals typically exhibit characteristic doublets, arising from the 3JH–H H1–H2 coupling, whereas ring protons show more complex multiplet patterns. These couplings are proportional to the dihedral angle between the two protons. Typical values for 3JH–H in carbohydrates are: axial–axial 7–8 Hz, axial–equatorial and equatorial–equatorial 3–4 Hz. During the early 1980s, Vliegenthart and co-workers identified a number of ‘structural reporter groups’, outlined in Table 1. Measurement of the chemical shift values, coupling patterns and line widths of signals arising from these elements can be interpreted to aid in the assignment and interpretation of 1D 1H NMR spectra. Sugar composition can thus be identified, and signals assigned on the basis of chemical shift and characteristic coupling values. The type and number of each can be established by consideration of chemical shifts, coupling patterns and relative integrals. Owing to the poor dispersion of the majority of resonances, this is usually limited to resolved anomeric protons or other structural reporter groups. Despite the development of highly efficient experiments based upon the use of selective excitation to produce edited one-dimensional spectra with more manageable information content, full proton assignment usually relies upon two- or, in some cases, threedimensional techniques.
Table 1
Two-Dimensional Homonuclear Analysis The poor dispersion and strong coupling present in carbohydrate proton spectra pose particular problems for the assignment process. In addition, the characteristics of a given spin system differ for each oligosaccharide and are highly dependant on structure. The use of two-dimensional experiments alleviates these problems.
Coherence transfer methods The correlated spectroscopy (COSY) experiment reduces the degree of resonance overlap by separating resonances into two orthogonal proton dimensions. The 1D spectrum lies along the diagonal, with cross-peaks joining pairs of J-coupled spins. For carbohydrates, assignment can then start with the well-resolved anomeric protons, and continue by stepwise Jcorrelation to the remaining nuclei. This simple process is complicated in many cases by overlap and strong coupling between signals, even in two dimensions. To reduce these difficulties, high-quality spectra are needed, with optimal digital resolution. Sometimes absolute value mode, as opposed to pure phase spectra, can be preferable for easy assignment, despite the theoretical disadvantages of a phase-twist line shape. Carbohydrate line widths are generally narrower than those of proteins and nucleic acids, and the use of absolute value spectra does not significantly diminish the quality of spectra, as seen in Figure 3. The COSY spectrum requires a pseudoecho weighting function to remove dispersive line shapes, reducing the sensitivity of the experiment. If sensitivity is an issue, then an inherently more sensitive experiment, such as the absorption mode DQ-COSY (see following text) should be used. If necessary, linkage positions can be identified using a COSY spectrum of a sample dissolved in DMSO-d6. The hydroxyl protons do not exchange with solvent, and they give rise to observable signals within the spectrum. Substitution removes the hydroxyl proton at that position, which can then be identified by the absence of a hydroxyl proton signal. The COSY experiment may fail to generate the expected cross-peaks, for several reasons: 1. For strongly coupled neighbours, the cross-peak lies close to the diagonal and may not be seen. 2. Pairs of nuclei with small scalar coupling values (3JH–H) will produce low-intensity cross-peaks. Since the active coupling is antiphase in COSY cross-peaks, they will cancel if the value of J is less than the line width. This is particularly noticeable in larger molecules. Common examples are
Structural reporter groups as described by Vliegenthart and co-workers
Sugar type
Proton(s)
Parameter
Information content
All Mannose Sialic acid Fucose Galactose Amino sugars
H1 H2, H3 H3 H5, CH3 H4, H4 N-acetyl CH3
d, J d d d d d
Residue and linkage type Substitution within core region of branched glycan Type and configuration of linkage Type and configuration of linkage: structural environment Type and configuration of linkage Sensitive to small structural variations
Carbohydrates Studied by NMR
161
Figure 3 1H–1H 2D COSY spectrum of galactopyranose b 1-4glucopyranose at 500 MHz and 303 K in 2H2O. A full proton assignment is usually possible from a spectrum like this, and peak identities are shown where space permits. The well-resolved anomeric protons provide a useful starting point for assignment, and subsequent pairs of spin-coupled nuclei are correlated through off-diagonal peaks.
D-mannopyranose b H1–H2 (J<1 Hz) and Dgalactopyranose H4–H5 (J<1 Hz). 3. Unrelated resonances may simply occur at the same position and cannot be distinguished.
Experiments incorporating isotropic mixing sequences The homonuclear Hartmann–Hahn (HOHAHA) experiment now also more commonly known as total correlation spectroscopy (TOCSY) is used to overcome problems of overlap and strong coupling. These experiments give correlations to all other spins within the same coupling network. The second pulse in the COSY sequence is replaced by a spin–lock sequence such as MLEV-17. The spin system becomes strongly coupled and coherence transfer occurs between all nuclei within the coupling network. This illustrates an advantage over experiments such as RELAY-COSY: values of 3JH–H between adjacent nuclei vary, and the use of an isotropic mixing period produces an increased efficiency of transfer. The presence of small couplings between nuclei, reducing the efficiency of transfer, still presents a problem. Resolution in this type of experiment is better than for a COSY spectrum; however, more cross-peaks are generated, producing a more complicated spectrum. This is at first sight hard to interpret; however, proton assignments in each ring can be determined by inspection of a line lying through the frequency of the anomeric proton of the sugar residue. The cross-peaks found along this line can be distinguished by reference to the COSY spectrum, and by consideration of the
multiplet structure. Pyranose rings have an effectively rigid ring geometry, and it is possible to estimate the couplings between adjacent protons based upon the dihedral angle between them. Judicious variation of the length of the isotropic mixing period can also be used to give stepwise correlations along the spinsystem, since shorter periods correlate proportionately smaller fragments. One-dimensional analogues of this experiment, using selective pulses to specifically excite anomeric protons, may be quicker and more efficient, especially when the anomeric protons are well resolved.
Multiple-quantum homonuclear experiment Incorporation of a multiple-quantum filter into a COSY-type sequence reduces the complexity of the spectrum, and aids in the assignment process. A DQF-COSY, incorporating a double-quantum filter transparent to two or more coupled spins, therefore passes all signals except singlets. Such a sequence has a slightly reduced sensitivity (cross-peak intensity is approximately half that of a comparable COSY), but there is no need for the sensitivityreducing weighting functions that are necessary with COSY. One noticeable feature is the absence of a diagonal, thus crosspeaks lying close to the diagonal can be more easily identified. Incorporation of a triple-quantum filter produces a spectrum with signals due only to three or more coupled spins with two resolvable couplings. There are no singlets or doublets, including anomeric resonances, although suppression is not
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Carbohydrates Studied by NMR
absolute, due to the presence of small couplings along four or more bonds. Under ideal conditions, one would expect to see resonances from only H5 and H6 protons, and this experiment is best used in conjunction with TOCSY for full assignment. Sensitivity is much reduced relative to COSY spectra.
Through-space dipolar interaction methods Although primarily used for conformational analysis, the nuclear Overhauser effect spectroscopy (NOESY) experiment is also helpful in the assignment process. The resulting spectra show correlations between protons coupled by through-space dipolar interactions. Both inter- and intraresidue NOEs help to confirm the consistency of assignments made by the techniques described in the preceding text. Long-range NOEs are not often seen in carbohydrates, and the observation of NOEs between protons on adjacent residues is often evidence of the linkage positions between the two sugars. Through-space dipolar interactions are of most use in the conformational analysis of biomolecules, as described in the section “Conformational analysis”. At 500 MHz, moderate-sized (more than six residues) oligosaccharides lie within the spin-diffusion limit. However, for smaller molecules, as the value of the function o0tc (where o0 is the Larmor frequency of protons, and tc is the correlation time of the molecule) approaches 1 then the value of the NOE tends towards 0. Cross-peak intensities of NOESY spectra of smaller oligosaccharides (2–5 residues) may thus become too small to measure accurately. In such cases, the rotating frame Overhauser effect spectroscopy (ROESY, originally referred to as CAMELSPIN) experiment is commonly used to measure NOE values. To reduce the appearance of TOCSY-like crosspeaks, a low power spin–lock field should be used, and the transmitter carrier offset to the low-field end of spectrum. The offset dependency of cross-peak intensities should also be removed by 90 pulses at either end of spin–lock period. Three-dimensional experiments, often essentially hybrids of simpler sequences such as TOCSY-COSY, have been used successfully to resolve cases of particularly difficult resonance overlap.
Hydroxyl Protons The quest for additional conformational information has led to the investigation of hydroxyl protons in aqueous solution. Samples are dissolved in mixed methanol–water or acetone– water solvents, and analysed in capillary NMR tubes at low (5 to 15 C) temperatures. Chemical exchange of hydroxyl protons is reduced to the point that it is possible to use them as probes of hydration and hydrogen bonding. Distance information can also be extracted from NOESY or ROESY spectra under these conditions.
13
C NMR of Carbohydrates
Primary Analysis and Assignment Initial investigations of the 13C spectra of carbohydrates were of natural abundance samples, although spectra of isotopically enriched samples have become more common in recent years.
13
C spectra are usually acquired with broad band proton decoupling. The resulting spectra are not very complex with, at natural abundance, a single sharp peak for each nucleus, there being no visible carbon–carbon coupling. The value of the carbon–proton coupling is approximately 150–180 Hz, and without proton decoupling this splitting, along with further two- and three-bond couplings, can render the spectrum quite complex. Monosaccharides exhibit characteristic carbon chemical shift patterns. Anomeric carbons typically occur between d 90 and 110 in pyranose rings, C6 (hydroxymethyl) signals between d 60 and 75, and the remaining carbons resonate between d 65 and 110. Substitution at a given carbon causes an approximate shift of up to dþ10. The usefulness of this shift in identifying the position of substitution is complicated by an additional d –1 to 2 shift of the surrounding carbon signals. In addition, the local structure of the saccharide can have a considerable effect upon the chemical shift values. Residue type and substitution patterns can thus be discovered, with care, from measurements of the carbon chemical shifts of a simple sugar. Databases of 13C chemical shift information for large numbers of simple carbohydrates are available for comparison in the literature.
Carbon–Proton and Carbon–Carbon Spin Couplings A consistent estimate of the anomeric configuration is given by the value of the one-bond 1JC–H coupling; b anomers show a value of 160 Hz, and a anomers 170 Hz. Three-bond carbon–proton scalar coupling values, such as 3 JCCCH, or 3JCOCH provide useful indicators of conformation. These couplings can be used to confirm the internal conformation of a saccharide residue, using appropriate Karplus curves. Across the glycosidic link, values of the couplings H1–C1–O–Cx (where x is the aglyconic carbon) and C1–O–Cx–Hx are proportional to the glycosidic dihedral angles f and c, respectively (see section ‘Conformational analysis’). Selective incorporation of 13C has been successfully used to simplify and enhance the measurement of these parameters. Linkage positions can be unambiguously determined by the identification of these couplings using experiments such as the 2D 13C–1H multiple-bond correlation experiment (HMBC). This experiment correlates carbon and proton resonances by means of 3JC–H couplings; 1JC–H correlations are suppressed, leaving only long-range couplings. Those couplings that span the glycosidic bond can thus be identified and measured.
Multidimensional, Heteronuclear Analysis Enrichment with 13C allows the use of a wide range of useful experiments that greatly facilitate the investigation of carbohydrate structure. Proton detected versions of carbon–proton correlation experiments, such as that shown in Figure 4, overcome the sensitivity limitations of observing carbon signals. If the proton assignments are known, assignment of carbon resonances from such spectra is a relatively simple process. The use of carbon nuclei to edit spectra into an orthogonal frequency dimension to overcome overlap has led to the development of a series of useful hybrid 3D or pseudo-3D experiments, such as HCCH-COSY or HMQC-NOESY. Editing spectra in this way has helped to assign the spectra not only of complex glycans in free solution, but also of the attached carbohydrate
Carbohydrates Studied by NMR
163
Figure 4 HSQC 1H–13C correlation spectrum of galactopyranose b1-4glucopyranose at 500 MHz and 303 K in 2H2O. Resonances are labelled with the appropriate assignment for each correlation. In practice, these can be relatively easily identified by comparison with previously assigned proton or carbon chemical shifts. Reproduced by courtesy of Homans SW and Kiddle GR, University of St. Andrews.
chains of glycoproteins, both at natural abundance as well as enriched with 13C and 15N. The use of these heteronuclei allows the carbohydrate resonances to be observed separately from those due to the protein portion of the molecule.
Conformational Analysis Measurement of 3JH–H couplings shows that pyranose rings do not show any large degree of internal flexibility, except for pendant groups such as the hydroxymethyl in hexopyranoses. Interpretation of these uses the Haasnoot parametrization of the Karplus equation for 3JH–H couplings in HCCH portions of the saccharide ring: 3
JHH0 ¼ 13:22 cos 2 y 0:99 cos y X þ i Dwi ½0:87 2:46 cos 2 ðxi yÞ
[1]
where y is the HCCH0 torsion angle, Dwi are the Huggins’ electronegativities of the substituents relative to protons and x is either þ1 or 1 depending upon the orientation of the substituent. If stereo-specific assignments have been made, orientation about hydroxymethyl rotamers can be determined by similar parametrizations. Conformational variability in pyranoside oligosaccharides is mostly owing to variations about the glycosidic torsion angles f and c and, for 1!6 linkages the o angle (Figure 5). NMR analysis of carbohydrate conformation thus concentrates upon determining the orientations about these dihedral angles. This is made difficult by the lack of conformational information available: even in the most favourable conditions, a maximum of around three NOEs are seen between each pair of residues in oligosaccharides, and observation of long-range
Figure 5 A 1!6 linked disaccharide showing the dihedral angles f, c and o, about which conformational variation is greatest.
correlations between different parts of the molecule is extremely unlikely. Interresidue correlations in NOESY or ROESY spectra can be used to determine distance information by making use of the fact that the intensity of the cross-peak is proportional to the inverse sixth power of the distance between the two nuclei. The relatively inflexible ring geometry of pyranose rings allows intraresidue NOEs to be used to calibrate adjacent intraresidue correlations. When implementing such experiments it is important to ensure that the initial rate approximation holds. The motion of the molecule, both internal and overall, also affects the intensity of the NOE; thus interpretation of NOE values must take into account the motional model used. Since
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Carbohydrates Studied by NMR
it is now clear that the majority of carbohydrate structures are flexible to some degree, oscillating through an ensemble of related conformations, this can be a problem. Flexibility is understood to be particularly marked about 1!6 linkages owing to rotation about the additional dihedral angle, o. The use of three-bond 3JC–H couplings as conformational probes has been described, using the Tvaroska parametrization of the Karplus relationship: 3
JðC, HÞ ¼ 5:7 cos 2 y 0:6 cos y þ 0:5
[2]
where y is the HCOC torsion angle. However, in a similar situation to that described for NOE restraints, these analyses have been hampered by the extent to which these values are subject to conformational averaging. Because of these problems, angular information derived from 3JC–H values is perhaps better used for evaluating the quality of conformational ensembles. More recent work has addressed the use of 13C within isotopically enriched compounds to derive additional conformational parameters, such as 13C–13C scalar couplings, and 13 C–1H or 13C–13C NOEs. These approaches have yielded useful results, but have been hampered by the lack of a wellestablished Karplus-type relationship for 13C–13C scalar coupling. Investigators have thus made considerable use of theoretical methods such as molecular mechanics to complement the limited experimental data available.
Dynamics The time-averaged nature of many NMR-derived parameters, and the degree to which it affects oligosaccharide conformations has made it necessary to attempt to measure the degree of mobility within oligosaccharides. Since the relaxation of protonated carbons is almost entirely due to the directly attached proton it is possible to measure relaxation parameters from them that are not affected by variations in internuclear distance. Investigation of carbohydrate dynamics has been approached by molecular modelling in combination with the measurement of relaxation rates, including the T1, T2 of 13C and 1H, as well as 1H–1H and 13C–1H NOE values, to define the spectral density, given by JðoÞ ¼
ð2=5Þtc ð1 þ otc Þ2
[3]
where tc denotes the rotational correlation time. These measurements have been interpreted by the Lipari–Szabo modelfree approach, in which molecular motion is separated into overall and internal motions, related to the overall and internal correlation times t0 and ti: J ðoÞ ¼
S2 ð2=5Þt0 ð1 S2 Þð2=5Þt þ ð1 þ ot0 Þ2 ð1 þ otÞ2
[4]
where 1/t¼1/t0þ1/ti. S2 is a measure of the degree of internal reorientation, varying in value from 0 for isotropic reorientation to 1 for no internal motion. These measurements can then be used to derive dynamic models of carbohydrates and oligosaccharides. It is now generally accepted that carbohydrates are dynamic molecules with
internal motions that occur on a time-scale faster than the overall motion of the molecules.
Application to Complex Carbohydrates NMR spectroscopy has been applied extensively to the identification of complex carbohydrates of biochemical interest. These include bacterial cell wall capsular polysaccharides, complex polysaccharides from a variety of plant origins, the branched oligosaccharides from glycoproteins, and lipid–sugar conjugates such as gangliosides and lipopolysaccharides. One active area of research is the study of carbohydrate–protein interactions, and here with a specific focus on cancer mechanisms, the conformational studies of glycoconjugates related to anti-tumour vaccines have been reported as well as the conformational analysis of glycosaminoglycans and their interactions, such as between heparin and fibroblast growth factor. For polysaccharide vaccines, NMR spectroscopy has been used as part of the quality control process. As well as proving the identity of the individual sugar units and the various linkage positions, NMR spectroscopy can be used for the detailed study of molecular conformations through the interpretation of spin–spin couplings and nuclear Overhauser effects.
See also: NMR Methods, 13C; NMR Parameter Survey, 13C; NMR Spectroscopy of Nucleic Acids, Historical Overview; Nucleic Acids Studied by NMR Spectroscopy; Plant Science Applications of NMR; Structural Chemistry Using NMR Spectroscopy, Organic Molecules; Two-Dimensional NMR.
Further Reading Bush CA (1996) Polysaccharides and complex oligosaccharides. In: Grant DM and Harris RK (eds.) Encyclopaedia of Nuclear Magnetic Resonance, vol. 6, pp. 3746–3750. Chichester: Wiley. De Marco ML (2008) Structure glycobiology: A game of snakes and ladders. Glycobiology 18: 426–440. Homans SW (1993) 1H NMR studies of oligosaccharides. In: Roberts GCK (ed.) NMR of Macromolecules: A Practical Approach, pp. 289–314. vol. 134 of Rickwood D and Hames BD (series eds.) Practical Approach Series. Oxford: Oxford University Press. Homans SW (1993) Conformation and dynamics of oligosaccharides in solution. Glycobiology 3: 551–555. Hounsell EF (1995) 1H NMR in the structural and conformational analysis of oligosaccharides and glyco-conjugates. Progress in Nuclear Magnetic Resonance Spectroscopy 27: 445–474. Jones C (2005) NMR assays for carbohydrate- based vaccines. Journal of Pharmaceutical and Biomedical Analysis 38: 840–850. Serianni AS (1992) Nuclear magnetic resonance approaches to oligosaccharide structure elucidation. In: Allen HJ and Kisalius EC (eds.) Glycoconjugates: Composition, Structure and Function, pp. 71–102. New York: Marcel Dekker. Tvaroska I (1990) Dependence on saccharide conformation of the one-bond and threebond coupling constants. Carbohydrate Research 206: 55–64. van Halbeek H (1994) NMR developments in structural studies of carbohydrates and their complexes. Current Opinion in Structural Biology 4: 697–709. van Halbeek H (1996) Carbohydrates and glycoconjugates. In: Grant DM and Harris RK (eds.) Encyclopaedia of Nuclear Magnetic Resonance, vol. 2, pp. 1107–1137. Chichester: Wiley. Vleigenthart JFG, Dorland L, and van Halbeek H (1983) High resolution spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins. Advances in Carbohydrate Chemistry and Biochemistry 41: 209–374.
Symbols J J(v) T1, T2 Dxi
coupling constant spectral density function relaxation constants Huggins electronegativities
Introduction Carbohydrates, the most widely abundant biological molecules, are key components within a wide variety of biological phenomena. Polysaccharides such as glycogen, cellulose and starch have important structural or nutritional roles, but it is the mediation of specific recognition events by carbohydrates that has sparked detailed analyses of structure, conformation and function. The non-invasive, non-destructive nature of the technique is commonly mentioned as the most important reason for analysing carbohydrates by NMR and is of considerable advantage when dealing with small amounts of precious material. This advantage is, however, offset by the relative insensitivity of the technique when compared with other methods such as mass spectrometry or enzymatic approaches that may be more appropriate for specific problems of sequence determination or compositional analysis. The value of NMR analysis is apparent in the additional data obtainable regarding composition, structure, conformation and mobility necessary for understanding recognition processes. The analysis of carbohydrates by NMR is characterized by a modification of existing techniques to address the particular problems of oligosaccharide spectra, namely poor dispersion, heterogeneity, a relatively high degree of interresidue mobility and a lack of conformational restraints. This article will attempt to provide an introduction to a large field of study, and readers are encouraged to look to the Further Reading section for additional information.
Sample Preparation In high-field NMR, the use of high-quality sample tubes can have a significant impact upon the quality of the spectra. For biologically relevant NMR analysis, experiments are generally carried out in aqueous solution. A strong water signal prevents direct analysis of proton signals lying beneath it and, owing to the problems of dynamic range, would reduce the sensitivity with which weak solute signals are detected. If, as is often the case, the exchangeable OH or NH protons are not of interest, then experiments are usually carried out in 2H2O.
This article is reproduced from the previous edition, Copyright 1999, Elsevier Ltd.
158
u torsion angle tc, t i and, t0 rotational, internal and overall correlation times respectively w, c and, v glycosidic torsion angles
Repeated dissolution and evaporation or lyophilization is recommended to reduce the proportion of residual protons remaining. In most cases, two dissolutions into>99.8% 2H2O followed by a final dissolution into>99.95% 2H2O, preferably from a sealed ampoule, should be sufficient. Other precautions such as pre-wetting pipettes, further rounds of dissolution and drying, or the use of a dry box may be used for greater sensitivity or quality. Although extremes of pH or pD (outside the range d 5–8) are to be avoided, buffer solutions are only necessary in two cases: if the molecule is charged and the charge state is relevant to the study; and in the study of acid-labile carbohydrates, such as sialylated oligosaccharides. The use of 1H2O as solvent is necessary in some cases and requires appropriate water suppression techniques. Care must be taken to ensure that the water is free from impurities as well as dissolved gases or paramagnetic species. A deuterated solvent to provide an appropriate lock signal should be added, usually 5–10% 2H2O. Oligosaccharides are not normally soluble in nonaqueous solvents, except for dimethyl sulfoxide (DMSO). Despite the biologically irrelevant environment, this permits the observation of exchangeable protons, and there is also some increase in the proton spectral dispersion. Similar precautions to those noted for the use of 2H2O should be observed, such as the use of high isotopic purity (>99.96%) solvent and repeated dissolution to remove exchangeable protons. To approach optimal line widths, it is often advisable to remove soluble paramagnetic components by passage through a suitable chelator. Degassing to remove dissolved oxygen is also recommended. For acceptable spectra within a reasonable time using a 500 MHz spectrometer, 10 nmol is an approximate lower limit for 1D 1H spectra; for 2D 1H spectra, amounts of 1 m mol are preferable, although spectra with as little as 100 nmol are possible with care. Sample volumes vary between 0.4 and 0.7 ml, depending on the size of the spectrometer RF coils. Temperature should be maintained at a constant level, preferably one or two degrees above room temperature for stability. Sample spinning is not necessary, except in cases where resolution is a particular problem. T1 for protons in carbohydrates is of the order of 0.1–0.5 s, and recycle delays of 1–1.5 s are usually sufficient. Referencing of samples is commonly to acetone at d 2.225 relative to DSS (5,5-dimethylsilapentanesulfonate) at 25 C, or DMSO at 2.5 ppm.
Encyclopedia of Spectroscopy and Spectrometry, Third Edition
http://dx.doi.org/10.1016/B978-0-12-803224-4.00113-8
Carbohydrates Studied by NMR
Analysis of Carbohydrate Structure by NMR The problems of carbohydrate structure addressed by NMR can be divided into four parts: 1. What is the composition of the molecule: what monosaccharides are present, are they in furanose or pyranose configurations, and a or b anomeric forms? 2. What is the nature of the linkages between the monosaccharides: at what positions do the substituents occur, and in what sequence? 3. What is the conformation of the molecule: what conformation or family of conformations are populated about the Oglycosidic bonds and hydroxymethyl rotamers? 4. What are the dynamic properties of the molecule? Whilst the first and second questions can be satisfactorily answered by NMR, they are perhaps better approached in tandem with chemical or enzymatic methods, such as hydrolysis, followed by reduction and analysis of alditol acetates by GC-MS, or digestion with specific glycosidases. Such a concerted approach has been used to successfully determine the composition and configuration of many oligosaccharides and glycans. The unique advantages of NMR in the analysis of carbohydrate structure are only fully apparent in consideration of points 3 and 4. In contrast to the unbranched polymeric nature of polypeptide and nucleic acid chains, carbohydrates may be branched structures, capable of substitution at several points. The monosaccharide constituents are polymerized in nature by
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a nontemplate directed, enzymatic process; the resulting oligosaccharides are often heterogeneous, differing in detail from a consensus structure. NMR is particularly efficient at investigating the solution conformations, and the dynamic properties of such molecules.
1
H NMR of Carbohydrates
Primary Analysis and Assignment: 1H 1D Spectra The 1H NMR spectrum of the disaccharide galactopyranose b 1–4 linked to glucopyranose (Galpb1–4Glcp, lactose, shown in Figure 1) is shown in Figure 2. Despite the relative simplicity of the molecule, with only 14 proton signals observable, the spectrum is surprisingly complex. This is due to the relatively small chemical shift dispersion of the non-exchangeable ring protons, resonating between d 3 and 4. The small chemical shift dispersal combined with homonuclear spin–spin
Figure 1 Structure of the disaccharide Galpb1-4Glcp, showing the numbering of protons within the sugar rings.
Figure 2 1H 1D spectrum of the disaccharide galactopyranose b1-4glucopyranose at 500 MHz and 303 K in 2H2O. Peak assignments are given for well-resolved peaks, following the numbering of Figure 1. The unresolved proton ‘envelope’ consists of the remaining ring proton resonances. The peak marked ‘HOD’ arises from residual water within the sample.
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coupling gives rise in many cases to non-first-order spectra, complicating the measurement of spin-coupling values. This makes the assignment and analysis of even quite small saccharides quite difficult, and as a consequence the use of spectrometers of field strength 400 MHz or above is recommended. Primarily owing to the electron-withdrawing effect of the ring oxygen, the anomeric (H1) protons give rise to signals lying outside this envelope of ring protons, between d 4.25 and 5.5. Since equatorial protons experience a shift of approximately d 0.5 relative to axial proton signals, a anomeric protons of D-sugars tend to be found between d 4.9 and 5.5, and b protons between d 4.3 and 4.7. The ring protons of each monosaccharide type give rise to characteristic patterns within the envelope of overlapping ring proton resonances. However, incorporation of a monosaccharide residue into an oligosaccharide will cause changes in these chemical shift patterns. For example, substitution at a given carbon causes the signal arising from the attached proton to change by d 0.2–0.5. The glucose residue of the sample shown in Figure 2 is free to mutarotate between a and b forms. The signals arising from these are easily distinguished from those owing to the galactose H1 by the difference in area. The two glucose resonances have a combined area approximately equal to that of the galactose H1 resonance. Anomeric signals typically exhibit characteristic doublets, arising from the 3JH–H H1–H2 coupling, whereas ring protons show more complex multiplet patterns. These couplings are proportional to the dihedral angle between the two protons. Typical values for 3JH–H in carbohydrates are: axial–axial 7–8 Hz, axial–equatorial and equatorial–equatorial 3–4 Hz. During the early 1980s, Vliegenthart and co-workers identified a number of ‘structural reporter groups’, outlined in Table 1. Measurement of the chemical shift values, coupling patterns and line widths of signals arising from these elements can be interpreted to aid in the assignment and interpretation of 1D 1H NMR spectra. Sugar composition can thus be identified, and signals assigned on the basis of chemical shift and characteristic coupling values. The type and number of each can be established by consideration of chemical shifts, coupling patterns and relative integrals. Owing to the poor dispersion of the majority of resonances, this is usually limited to resolved anomeric protons or other structural reporter groups. Despite the development of highly efficient experiments based upon the use of selective excitation to produce edited one-dimensional spectra with more manageable information content, full proton assignment usually relies upon two- or, in some cases, threedimensional techniques.
Table 1
Two-Dimensional Homonuclear Analysis The poor dispersion and strong coupling present in carbohydrate proton spectra pose particular problems for the assignment process. In addition, the characteristics of a given spin system differ for each oligosaccharide and are highly dependant on structure. The use of two-dimensional experiments alleviates these problems.
Coherence transfer methods The correlated spectroscopy (COSY) experiment reduces the degree of resonance overlap by separating resonances into two orthogonal proton dimensions. The 1D spectrum lies along the diagonal, with cross-peaks joining pairs of J-coupled spins. For carbohydrates, assignment can then start with the well-resolved anomeric protons, and continue by stepwise Jcorrelation to the remaining nuclei. This simple process is complicated in many cases by overlap and strong coupling between signals, even in two dimensions. To reduce these difficulties, high-quality spectra are needed, with optimal digital resolution. Sometimes absolute value mode, as opposed to pure phase spectra, can be preferable for easy assignment, despite the theoretical disadvantages of a phase-twist line shape. Carbohydrate line widths are generally narrower than those of proteins and nucleic acids, and the use of absolute value spectra does not significantly diminish the quality of spectra, as seen in Figure 3. The COSY spectrum requires a pseudoecho weighting function to remove dispersive line shapes, reducing the sensitivity of the experiment. If sensitivity is an issue, then an inherently more sensitive experiment, such as the absorption mode DQ-COSY (see following text) should be used. If necessary, linkage positions can be identified using a COSY spectrum of a sample dissolved in DMSO-d6. The hydroxyl protons do not exchange with solvent, and they give rise to observable signals within the spectrum. Substitution removes the hydroxyl proton at that position, which can then be identified by the absence of a hydroxyl proton signal. The COSY experiment may fail to generate the expected cross-peaks, for several reasons: 1. For strongly coupled neighbours, the cross-peak lies close to the diagonal and may not be seen. 2. Pairs of nuclei with small scalar coupling values (3JH–H) will produce low-intensity cross-peaks. Since the active coupling is antiphase in COSY cross-peaks, they will cancel if the value of J is less than the line width. This is particularly noticeable in larger molecules. Common examples are
Structural reporter groups as described by Vliegenthart and co-workers
Sugar type
Proton(s)
Parameter
Information content
All Mannose Sialic acid Fucose Galactose Amino sugars
H1 H2, H3 H3 H5, CH3 H4, H4 N-acetyl CH3
d, J d d d d d
Residue and linkage type Substitution within core region of branched glycan Type and configuration of linkage Type and configuration of linkage: structural environment Type and configuration of linkage Sensitive to small structural variations
Carbohydrates Studied by NMR
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Figure 3 1H–1H 2D COSY spectrum of galactopyranose b 1-4glucopyranose at 500 MHz and 303 K in 2H2O. A full proton assignment is usually possible from a spectrum like this, and peak identities are shown where space permits. The well-resolved anomeric protons provide a useful starting point for assignment, and subsequent pairs of spin-coupled nuclei are correlated through off-diagonal peaks.
D-mannopyranose b H1–H2 (J<1 Hz) and Dgalactopyranose H4–H5 (J<1 Hz). 3. Unrelated resonances may simply occur at the same position and cannot be distinguished.
Experiments incorporating isotropic mixing sequences The homonuclear Hartmann–Hahn (HOHAHA) experiment now also more commonly known as total correlation spectroscopy (TOCSY) is used to overcome problems of overlap and strong coupling. These experiments give correlations to all other spins within the same coupling network. The second pulse in the COSY sequence is replaced by a spin–lock sequence such as MLEV-17. The spin system becomes strongly coupled and coherence transfer occurs between all nuclei within the coupling network. This illustrates an advantage over experiments such as RELAY-COSY: values of 3JH–H between adjacent nuclei vary, and the use of an isotropic mixing period produces an increased efficiency of transfer. The presence of small couplings between nuclei, reducing the efficiency of transfer, still presents a problem. Resolution in this type of experiment is better than for a COSY spectrum; however, more cross-peaks are generated, producing a more complicated spectrum. This is at first sight hard to interpret; however, proton assignments in each ring can be determined by inspection of a line lying through the frequency of the anomeric proton of the sugar residue. The cross-peaks found along this line can be distinguished by reference to the COSY spectrum, and by consideration of the
multiplet structure. Pyranose rings have an effectively rigid ring geometry, and it is possible to estimate the couplings between adjacent protons based upon the dihedral angle between them. Judicious variation of the length of the isotropic mixing period can also be used to give stepwise correlations along the spinsystem, since shorter periods correlate proportionately smaller fragments. One-dimensional analogues of this experiment, using selective pulses to specifically excite anomeric protons, may be quicker and more efficient, especially when the anomeric protons are well resolved.
Multiple-quantum homonuclear experiment Incorporation of a multiple-quantum filter into a COSY-type sequence reduces the complexity of the spectrum, and aids in the assignment process. A DQF-COSY, incorporating a double-quantum filter transparent to two or more coupled spins, therefore passes all signals except singlets. Such a sequence has a slightly reduced sensitivity (cross-peak intensity is approximately half that of a comparable COSY), but there is no need for the sensitivityreducing weighting functions that are necessary with COSY. One noticeable feature is the absence of a diagonal, thus crosspeaks lying close to the diagonal can be more easily identified. Incorporation of a triple-quantum filter produces a spectrum with signals due only to three or more coupled spins with two resolvable couplings. There are no singlets or doublets, including anomeric resonances, although suppression is not
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Carbohydrates Studied by NMR
absolute, due to the presence of small couplings along four or more bonds. Under ideal conditions, one would expect to see resonances from only H5 and H6 protons, and this experiment is best used in conjunction with TOCSY for full assignment. Sensitivity is much reduced relative to COSY spectra.
Through-space dipolar interaction methods Although primarily used for conformational analysis, the nuclear Overhauser effect spectroscopy (NOESY) experiment is also helpful in the assignment process. The resulting spectra show correlations between protons coupled by through-space dipolar interactions. Both inter- and intraresidue NOEs help to confirm the consistency of assignments made by the techniques described in the preceding text. Long-range NOEs are not often seen in carbohydrates, and the observation of NOEs between protons on adjacent residues is often evidence of the linkage positions between the two sugars. Through-space dipolar interactions are of most use in the conformational analysis of biomolecules, as described in the section “Conformational analysis”. At 500 MHz, moderate-sized (more than six residues) oligosaccharides lie within the spin-diffusion limit. However, for smaller molecules, as the value of the function o0tc (where o0 is the Larmor frequency of protons, and tc is the correlation time of the molecule) approaches 1 then the value of the NOE tends towards 0. Cross-peak intensities of NOESY spectra of smaller oligosaccharides (2–5 residues) may thus become too small to measure accurately. In such cases, the rotating frame Overhauser effect spectroscopy (ROESY, originally referred to as CAMELSPIN) experiment is commonly used to measure NOE values. To reduce the appearance of TOCSY-like crosspeaks, a low power spin–lock field should be used, and the transmitter carrier offset to the low-field end of spectrum. The offset dependency of cross-peak intensities should also be removed by 90 pulses at either end of spin–lock period. Three-dimensional experiments, often essentially hybrids of simpler sequences such as TOCSY-COSY, have been used successfully to resolve cases of particularly difficult resonance overlap.
Hydroxyl Protons The quest for additional conformational information has led to the investigation of hydroxyl protons in aqueous solution. Samples are dissolved in mixed methanol–water or acetone– water solvents, and analysed in capillary NMR tubes at low (5 to 15 C) temperatures. Chemical exchange of hydroxyl protons is reduced to the point that it is possible to use them as probes of hydration and hydrogen bonding. Distance information can also be extracted from NOESY or ROESY spectra under these conditions.
13
C NMR of Carbohydrates
Primary Analysis and Assignment Initial investigations of the 13C spectra of carbohydrates were of natural abundance samples, although spectra of isotopically enriched samples have become more common in recent years.
13
C spectra are usually acquired with broad band proton decoupling. The resulting spectra are not very complex with, at natural abundance, a single sharp peak for each nucleus, there being no visible carbon–carbon coupling. The value of the carbon–proton coupling is approximately 150–180 Hz, and without proton decoupling this splitting, along with further two- and three-bond couplings, can render the spectrum quite complex. Monosaccharides exhibit characteristic carbon chemical shift patterns. Anomeric carbons typically occur between d 90 and 110 in pyranose rings, C6 (hydroxymethyl) signals between d 60 and 75, and the remaining carbons resonate between d 65 and 110. Substitution at a given carbon causes an approximate shift of up to dþ10. The usefulness of this shift in identifying the position of substitution is complicated by an additional d –1 to 2 shift of the surrounding carbon signals. In addition, the local structure of the saccharide can have a considerable effect upon the chemical shift values. Residue type and substitution patterns can thus be discovered, with care, from measurements of the carbon chemical shifts of a simple sugar. Databases of 13C chemical shift information for large numbers of simple carbohydrates are available for comparison in the literature.
Carbon–Proton and Carbon–Carbon Spin Couplings A consistent estimate of the anomeric configuration is given by the value of the one-bond 1JC–H coupling; b anomers show a value of 160 Hz, and a anomers 170 Hz. Three-bond carbon–proton scalar coupling values, such as 3 JCCCH, or 3JCOCH provide useful indicators of conformation. These couplings can be used to confirm the internal conformation of a saccharide residue, using appropriate Karplus curves. Across the glycosidic link, values of the couplings H1–C1–O–Cx (where x is the aglyconic carbon) and C1–O–Cx–Hx are proportional to the glycosidic dihedral angles f and c, respectively (see section ‘Conformational analysis’). Selective incorporation of 13C has been successfully used to simplify and enhance the measurement of these parameters. Linkage positions can be unambiguously determined by the identification of these couplings using experiments such as the 2D 13C–1H multiple-bond correlation experiment (HMBC). This experiment correlates carbon and proton resonances by means of 3JC–H couplings; 1JC–H correlations are suppressed, leaving only long-range couplings. Those couplings that span the glycosidic bond can thus be identified and measured.
Multidimensional, Heteronuclear Analysis Enrichment with 13C allows the use of a wide range of useful experiments that greatly facilitate the investigation of carbohydrate structure. Proton detected versions of carbon–proton correlation experiments, such as that shown in Figure 4, overcome the sensitivity limitations of observing carbon signals. If the proton assignments are known, assignment of carbon resonances from such spectra is a relatively simple process. The use of carbon nuclei to edit spectra into an orthogonal frequency dimension to overcome overlap has led to the development of a series of useful hybrid 3D or pseudo-3D experiments, such as HCCH-COSY or HMQC-NOESY. Editing spectra in this way has helped to assign the spectra not only of complex glycans in free solution, but also of the attached carbohydrate
Carbohydrates Studied by NMR
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Figure 4 HSQC 1H–13C correlation spectrum of galactopyranose b1-4glucopyranose at 500 MHz and 303 K in 2H2O. Resonances are labelled with the appropriate assignment for each correlation. In practice, these can be relatively easily identified by comparison with previously assigned proton or carbon chemical shifts. Reproduced by courtesy of Homans SW and Kiddle GR, University of St. Andrews.
chains of glycoproteins, both at natural abundance as well as enriched with 13C and 15N. The use of these heteronuclei allows the carbohydrate resonances to be observed separately from those due to the protein portion of the molecule.
Conformational Analysis Measurement of 3JH–H couplings shows that pyranose rings do not show any large degree of internal flexibility, except for pendant groups such as the hydroxymethyl in hexopyranoses. Interpretation of these uses the Haasnoot parametrization of the Karplus equation for 3JH–H couplings in HCCH portions of the saccharide ring: 3
JHH0 ¼ 13:22 cos 2 y 0:99 cos y X þ i Dwi ½0:87 2:46 cos 2 ðxi yÞ
[1]
where y is the HCCH0 torsion angle, Dwi are the Huggins’ electronegativities of the substituents relative to protons and x is either þ1 or 1 depending upon the orientation of the substituent. If stereo-specific assignments have been made, orientation about hydroxymethyl rotamers can be determined by similar parametrizations. Conformational variability in pyranoside oligosaccharides is mostly owing to variations about the glycosidic torsion angles f and c and, for 1!6 linkages the o angle (Figure 5). NMR analysis of carbohydrate conformation thus concentrates upon determining the orientations about these dihedral angles. This is made difficult by the lack of conformational information available: even in the most favourable conditions, a maximum of around three NOEs are seen between each pair of residues in oligosaccharides, and observation of long-range
Figure 5 A 1!6 linked disaccharide showing the dihedral angles f, c and o, about which conformational variation is greatest.
correlations between different parts of the molecule is extremely unlikely. Interresidue correlations in NOESY or ROESY spectra can be used to determine distance information by making use of the fact that the intensity of the cross-peak is proportional to the inverse sixth power of the distance between the two nuclei. The relatively inflexible ring geometry of pyranose rings allows intraresidue NOEs to be used to calibrate adjacent intraresidue correlations. When implementing such experiments it is important to ensure that the initial rate approximation holds. The motion of the molecule, both internal and overall, also affects the intensity of the NOE; thus interpretation of NOE values must take into account the motional model used. Since
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Carbohydrates Studied by NMR
it is now clear that the majority of carbohydrate structures are flexible to some degree, oscillating through an ensemble of related conformations, this can be a problem. Flexibility is understood to be particularly marked about 1!6 linkages owing to rotation about the additional dihedral angle, o. The use of three-bond 3JC–H couplings as conformational probes has been described, using the Tvaroska parametrization of the Karplus relationship: 3
JðC, HÞ ¼ 5:7 cos 2 y 0:6 cos y þ 0:5
[2]
where y is the HCOC torsion angle. However, in a similar situation to that described for NOE restraints, these analyses have been hampered by the extent to which these values are subject to conformational averaging. Because of these problems, angular information derived from 3JC–H values is perhaps better used for evaluating the quality of conformational ensembles. More recent work has addressed the use of 13C within isotopically enriched compounds to derive additional conformational parameters, such as 13C–13C scalar couplings, and 13 C–1H or 13C–13C NOEs. These approaches have yielded useful results, but have been hampered by the lack of a wellestablished Karplus-type relationship for 13C–13C scalar coupling. Investigators have thus made considerable use of theoretical methods such as molecular mechanics to complement the limited experimental data available.
Dynamics The time-averaged nature of many NMR-derived parameters, and the degree to which it affects oligosaccharide conformations has made it necessary to attempt to measure the degree of mobility within oligosaccharides. Since the relaxation of protonated carbons is almost entirely due to the directly attached proton it is possible to measure relaxation parameters from them that are not affected by variations in internuclear distance. Investigation of carbohydrate dynamics has been approached by molecular modelling in combination with the measurement of relaxation rates, including the T1, T2 of 13C and 1H, as well as 1H–1H and 13C–1H NOE values, to define the spectral density, given by JðoÞ ¼
ð2=5Þtc ð1 þ otc Þ2
[3]
where tc denotes the rotational correlation time. These measurements have been interpreted by the Lipari–Szabo modelfree approach, in which molecular motion is separated into overall and internal motions, related to the overall and internal correlation times t0 and ti: J ðoÞ ¼
S2 ð2=5Þt0 ð1 S2 Þð2=5Þt þ ð1 þ ot0 Þ2 ð1 þ otÞ2
[4]
where 1/t¼1/t0þ1/ti. S2 is a measure of the degree of internal reorientation, varying in value from 0 for isotropic reorientation to 1 for no internal motion. These measurements can then be used to derive dynamic models of carbohydrates and oligosaccharides. It is now generally accepted that carbohydrates are dynamic molecules with
internal motions that occur on a time-scale faster than the overall motion of the molecules.
Application to Complex Carbohydrates NMR spectroscopy has been applied extensively to the identification of complex carbohydrates of biochemical interest. These include bacterial cell wall capsular polysaccharides, complex polysaccharides from a variety of plant origins, the branched oligosaccharides from glycoproteins, and lipid–sugar conjugates such as gangliosides and lipopolysaccharides. One active area of research is the study of carbohydrate–protein interactions, and here with a specific focus on cancer mechanisms, the conformational studies of glycoconjugates related to anti-tumour vaccines have been reported as well as the conformational analysis of glycosaminoglycans and their interactions, such as between heparin and fibroblast growth factor. For polysaccharide vaccines, NMR spectroscopy has been used as part of the quality control process. As well as proving the identity of the individual sugar units and the various linkage positions, NMR spectroscopy can be used for the detailed study of molecular conformations through the interpretation of spin–spin couplings and nuclear Overhauser effects.
See also: NMR Methods, 13C; NMR Parameter Survey, 13C; NMR Spectroscopy of Nucleic Acids, Historical Overview; Nucleic Acids Studied by NMR Spectroscopy; Plant Science Applications of NMR; Structural Chemistry Using NMR Spectroscopy, Organic Molecules; Two-Dimensional NMR.
Further Reading Bush CA (1996) Polysaccharides and complex oligosaccharides. In: Grant DM and Harris RK (eds.) Encyclopaedia of Nuclear Magnetic Resonance, vol. 6, pp. 3746–3750. Chichester: Wiley. De Marco ML (2008) Structure glycobiology: A game of snakes and ladders. Glycobiology 18: 426–440. Homans SW (1993) 1H NMR studies of oligosaccharides. In: Roberts GCK (ed.) NMR of Macromolecules: A Practical Approach, pp. 289–314. vol. 134 of Rickwood D and Hames BD (series eds.) Practical Approach Series. Oxford: Oxford University Press. Homans SW (1993) Conformation and dynamics of oligosaccharides in solution. Glycobiology 3: 551–555. Hounsell EF (1995) 1H NMR in the structural and conformational analysis of oligosaccharides and glyco-conjugates. Progress in Nuclear Magnetic Resonance Spectroscopy 27: 445–474. Jones C (2005) NMR assays for carbohydrate- based vaccines. Journal of Pharmaceutical and Biomedical Analysis 38: 840–850. Serianni AS (1992) Nuclear magnetic resonance approaches to oligosaccharide structure elucidation. In: Allen HJ and Kisalius EC (eds.) Glycoconjugates: Composition, Structure and Function, pp. 71–102. New York: Marcel Dekker. Tvaroska I (1990) Dependence on saccharide conformation of the one-bond and threebond coupling constants. Carbohydrate Research 206: 55–64. van Halbeek H (1994) NMR developments in structural studies of carbohydrates and their complexes. Current Opinion in Structural Biology 4: 697–709. van Halbeek H (1996) Carbohydrates and glycoconjugates. In: Grant DM and Harris RK (eds.) Encyclopaedia of Nuclear Magnetic Resonance, vol. 2, pp. 1107–1137. Chichester: Wiley. Vleigenthart JFG, Dorland L, and van Halbeek H (1983) High resolution spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins. Advances in Carbohydrate Chemistry and Biochemistry 41: 209–374.