A wide range of physical techniques has been used to study the structures of carbohydrates at different levels, i.e., molecular, macromolecular, and supramolecular structures (11). The use of such means as mass spec- troscopy and molecular spectroscopy to elucidate the pri- mary structure of carbohydrates will not be covered in this chapter. The purpose is to include only those physical techniques used for studying the conformation of carbo- hydrates in general, and for probing the higher level struc- tures of polysaccharides. Generally there is a need to combine several physical techniques to provide comple- mentary information about the structure of carbohydrates.
1. X-Ray Diffraction
a. Background
X-ray diffraction and other types of diffraction methods (electron and neutron) have contributed to our understand- ing of the molecular geometry of carbohydrates. Diffraction is essentially a scattering phenomenon. When a monochro- matic x-ray beam travels through a test specimen, a small proportion of the radiation is scattered with mutual rein- forcement of a large number of scattered rays, and the result- ant x-ray intensity in specific directions depends on the arrangement of the scattering atoms within the sample. X-ray scattering techniques are divided into two categories: wide-angle x-ray scattering (WAXS) and small-angle x-ray scattering (SAXS). Typically, SAXS gives information on a scale of ~ a few nanometers and smaller, while WAXS gives information on a scale of 1–1000 nm. WAXS is used to measure crystal structure and related parameters, which is the topic of this section. SAXS will be discussed in the next section together with light and neutron scattering tech- niques.
The diffraction pattern, commonly recorded on photo- graphic film, consists of an array of spots (reflections) of varying intensities, from which structural information for a chemical repeat may be deduced. If a large enough size of crystal can be prepared, it is usually possible to determine the crystal structure and hydrogen bonding to a high degree of accuracy. Information such as repeating unit cell dimen- sions, lattice type, space group symmetry and bond lengths, and valence angles can be derived from the analysis.
b. Monosaccharides and oligosaccharides
For almost all monosaccharides and many oligosaccha- rides with low degrees of polymerization, it is not a major problem to prepare single crystals for x-ray measurement. X-ray characterized structures are available for most of these molecules (3, 12–16). As an example, in the study of mannotriose (O-β-D-mannopyranosyl-(1→4)-O-β-D- mannopyranosyl-(1→4)-O-α-D-mannopyranose) (14), the unit cell was determined as monoclinic with dimensions of
a⫽ 0.1183 nm, b ⫽ 0.1222 nm, and c ⫽ 0.9223 nm, and β⫽ 112.34°; the space group was P21. The crystal struc-
ture includes three water molecules, two of which are involved in hydrogen bonding such that the mannotriose molecules occur effectively as sheets of long parallel chains, with each consecutive sheet having chains lying at approximately right angle to those in a neighboring sheet.
c. Polysaccharides
Large oligosaccharides rarely and polysaccharides never form single crystals that are good enough for classical x-ray crystallography. They tend to form fibers that are amorphous, or at best only partly crystalline, starch being a typical example of the latter. X-ray study of starches mostly measures the degree of crystallinity and identifies different polymorphic forms. To obtain useful x-ray dif- fraction data from other more amorphous non-starch poly- saccharides, oriented fibers or films are used (6). These polycrystalline fibers or films are prepared in such a way that the polysaccharide helices are preferentially oriented with their long axes nearly parallel. The x-ray diffraction intensities then provide information about the helical structures such as repeat spacing of the helix and helix screw symmetry, and if the diffraction pattern is suffi- ciently “crystalline,” the unit cell dimensions and lattice type. However, the x-ray data alone are inadequate to solve a fiber structure, and interpretation requires supplementa- tion with molecular modeling analysis using existing stereochemical information derived from surveys of crys- tal structures of related mono- or oligosaccharides (7, 17).
X-ray fiber diffraction is of great value in the determi- nation of the conformations of polysaccharides. Studies of the (1→3)-β-D-glucan family, curdlan, schizophyllan, and scleroglucan, are good examples. Curdlan is a linear (1→3)-β-D-glucan, whereas schizophyllan and scleroglucan also contain some β-(1→6)-glucosyl branches. These (1→3)-β-D-glucans usually form triple-stranded helices (18). The structure of curdlan (in both hydrated and anhy- drous forms), determined from oriented fibers, assume a right-handed, parallel, six-fold triple-helical conformation. There are interstrand O2…O2 hydrogen bonds in the hexagonal unit cell, with parameters a⫽ b ⫽ 1.441 nm and c⫽ 0.587 nm. The space group is P63 and there is one
helix per unit cell (19). The short-branch substitutions on the main chain primary hydroxyls in schizophyllan and scleroglucan do not seem to affect the fundamental triple- helical structure (20).
2. Light, X-Ray, and Neutron Scattering
a. Background
The principles on which light, x-ray, and neutron scattering depend are basically similar and can be treated by the same fundamental sets of equations. For all three modes of scat- tering, angular dependence of the normalized scattering
intensity provides information on the size and shape of the macromolecules. The resolving power of scattering tech- niques is related to the wavelength of the scattered radiation (21). The wavelengths are 0.1–0.3 nm for SAXS, 0.2–1.0 nm for small angle neutron scattering (SANS), and ~500 nm for light scattering. Conventional light scattering typi- cally reveals only the global dimensions of a macromole- cule, which may be tens to hundreds of nanometers for a typical polysaccharide. SAXS and SANS can probe molec- ular structures at closer ranges of about 2–25 nm (22). SANS may additionally observe the Gaussian behavior of polymer chains in their own bulk (solid), which conven- tional light scattering cannot. Light scattering is effective in measuring the angular dependence of intensity typically in the range 30° to 135°. SAXS can be carried out at very small angles, typically less than 1°, and is thus superior for the determination of the size and shape of macromolecules, but it is less convenient for the determination of molecular weight and second virial coefficient.
b. Application to polysaccharides
Scattering measurements can be carried out in two modes, static and dynamic. The former measures the average scattering intensity within a selected time period, whereas the latter measures the fluctuation of the intensity over time. From static measurements, the weight average molecular weight (Mw), z-average radius of gyration (Rg),
and the second virial coefficient can be extracted. From dynamic measurement, the translational diffusion coeffi- cient is obtained from which the hydrodynamic radius (Rh) can be determined. The parameter,ρ = Rg/Rh, may
provide information on the architecture of the macromol- ecules and their aggregates (23). From the combination of static and dynamic scattering data, other information may be derived including the linear mass density, Kuhn seg- ment length, and polydispersity index. To obtain as much structural information as possible, experimental data from scattering are usually processed and presented through various plots, and need to be interpreted using molecular model such as the worm-like chain model (23).
Light scattering was applied to study the solution properties of amyloses and the retrogradation of amyloses as early as the 1960s (24–26). A typical flexible chain behavior was observed for high-molecular-weight amy- loses in freshly prepared aqueous solutions. With decreas- ing molecular weight, the tendency to aggregate increased considerably so that a stable aqueous solution could not be prepared. The many studies on amylopectin and glyco- gen demonstrated how scattering techniques may be used for investigating the branching behavior of polysaccha- rides (27). The branching nature of amylopectin and glycogen can be detected clearly by light scattering from the Zimm plot, which shows an upturn (28, 29).
Scattering techniques can be used to probe the confor- mational transition of polysaccharides in solution. For
example, the thermal transition evident in low ionic strength xanthan solutions was followed by light scattering (30). It was observed that the apparent hydrodynamic radius signif- icantly decreases with increasing temperature in the vicinity of the helix-coil transition temperature. As discussed above (Section II.C.2), light scattering is also useful in investigat- ing aggregation properties of polysaccharides.
3. Chiroptical Methods
a. Background
Optical activity is one of the most readily and often meas- ured physical properties of carbohydrates. Carbohydrates contain several similarly substituted asymmetric carbon atoms and are therefore all optically active. The optical activity can be determined by optical rotation (OR), opti- cal rotatory dispersion (ORD), and circular dichroism (CD). OR is measured by a polarimeter at a single wave- length, usually the sodium D line (589 nm), and expressed as specific (or molecular) rotations [α]D. A number of approaches, all of them empirical in nature, have been devised to interpret the relationship between the measured optical rotations and structural features of carbohydrates (31). Specific rotations are used extensively to character- ize new derivatives and to recognize known ones. Instead of using a single wavelength, optical rotatory dispersion measures the optical rotation angle (ϕ) over a wide range of wavelengths, and circular dichroism measures the dif- ferential absorption of right- and left-circularly polarized light as a function of wavelength. Both ORD and CD spectra can exhibit marked changes in slope in the vicin- ity of the absorption maximum of a chromophore attached to the chiral center, known as the Cotton effect.
b. Optical rotation
In a monosaccharide molecule, several chiral carbons contribute to the overall optical rotation, but the configu- ration of the carbon atoms attached to the ring oxygen atom have the greatest influence on the overall rotation value. For many monosaccharides and reducing oligosac- charides, the initial optical rotation in aqueous solutions changes with time until reaching a constant value. This phenomenon is known as mutarotation, most often the outcome of interconversion between αand βring isomers, until reaching an equilibrium.
Similar to monosaccharides, oligo- and polysaccha- rides have optical activity. With advances in the under- standing of carbohydrate stereochemistry, it has become generally recognized that the overall optical rotation is determined more by the relative orientation of adjacent monosaccharide residues (defined by dihedral angles) than by the additive contributions from each asymmetric center. The optical activity of these is therefore beyond those aris- ing from the simple monosaccharides, but is rather associ- ated with the conformation of larger molecules or
macromolecules. Stevens and co-workers developed a chi- roptical technique to investigate disaccharide conforma- tion (32), based upon the estimates of variation in the optical activity of a particular disaccharide as a function of its glycosidic conformation. A number of disaccharides, including sucrose, maltose and cellobiose, have been char- acterized using this method (33).
The optical rotation of polysaccharides at long wave- lengths is usually dominated by the optical activity of the polymer backbone. Measurement of optical rotation at long wavelengths remains a standard and practical tech- nique for polysaccharide systems despite the advent of ORD and CD instruments. For example, OR is used fre- quently for monitoring the progress of cooperative con- formational transitions of polysaccharides (34).
c. Circular dichroism and optical rotatary dispersion
Monosaccharides of most food carbohydrates exist in cyclic forms, thus do not possess the unsaturated chro- mophores necessary to display a Cotton effect at long wavelengths. In the absence of unsaturated chro- mophores, two very short wavelength transitions associ- ated with conformational transitions of carbohydrate backbone may be used (35–37). These can be observed by modern vacuum UV polarimetry. One such transition is centered near 175 nm, attributed to the n→σ* transitions of the acetal oxygen atoms. The second is usually found around 150 nm and is closely related to the optical rota- tion at long wavelengths. CD and ORD experiments show that the variation in intensity of these two bands in poly- saccharides is correlated to their composition and confor- mation (38). Thus, CD and ORD offer powerful tools to study structural and conformational transitions.
Some polysaccharides contain chromophores that absorb at substantially longer wavelengths than the poly- meric backbone and thus give significant CD and ORD bands at wavelengths above ~185 nm. Examples are acyl and pyruvate ketal constituents and the carboxyl groups. In these cases, the CD spectra are close to those of the iso- lated monosaccharides, with little direct influence from the chain geometry. Since CD is very sensitive to the local environment of chromophores, conformational changes caused by, for example, specific site binding of uronate segments are usually accompanied by dramatic changes in CD spectra (39). This provides an alternative approach to study the gelation mechanisms of polysaccharides con- taining carboxyl groups such as alginate, pectin, xanthan, and gellan (40, 41).
4. Microscopy Techniques
a. Background
Direct imaging of polysaccharides using microscopy provides an important additional method for physical
characterization of polysaccharides. Two types of micro- scopy are especially of interest. First, electron microscopy (EM) is the traditional type, like light microscopy, but instead uses an electron beam to probe smaller structures than possible with light. Atomic force microscopy (AFM) senses forces such as electrostatic, magnetic, capillary, or van der Waals forces, as the molecular surface is approached by a probe. EM has considerable power to study supramolecular assemblies such as starch granules and mixed structures such as composite gels, whereas AFM has wide potential applications in investigating the structures of single molecules, as well as supramolecular assemblies and gel networks.
b. Electron microscopy
In EM, an electron beam produced from an electron gun is employed as an illuminating source instead of visible light. In transmission electron microscopy (TEM), when a fine electron beam hits the specimen, the electrons are trans- mitted after a series of interactions with the specimen, and then magnified to produce the image on a fluorescent screen or a photographic film. In scanning electron microscopy (SEM), the secondary electrons originating from ionization of the specimen atoms by the incident pri- mary electrons are collected by an electron detector. The incident beam is scanned over a small area corresponding to the area of the micrograph. EM gives a better resolution than light microscopy because the wavelength of an elec- tron beam is shorter than that of visible light.
A critical part of electron microscopy is adequate preparation of the specimen to minimize structural changes and to avoid artifacts. In most cases, the samples are exposed to a series of treatments prior to observation such as dehydration (or solidification), sectioning, and coating with electrical conducting materials. Thus, the image shapes obtained from the specimens may differ from their true shapes in the hydrated state.
Information can be obtained from EM on how macro- molecules associate into supramolecular assemblies, and under favorable conditions, form gel networks (42). EM was used to monitor the conformational changes of poly- saccharides that often initiate gelation such as coil-helix transitions (42). Direct visualizing of the structure of gel networks using EM has helped the understanding of struc- ture-function relationships of polysaccharide gelation. In addition to these qualitative assessments of structural fea- tures, it is also possible to quantify properties like contour length, persistence length, linear mass density, and thickness of strands, using advanced image analysis sys- tems (42, 43). Polysaccharides like xanthan and various β-D-glucans, all with a persistence length in the order of 100 nm, are ideally suited for such EM investigations. Since EM only provides a two-dimensional projection of the specimen, it is important to compare the parameters derived from EM with those obtained from other physical
techniques, or from specimens prepared by different tech- niques.
c. Atomic force microscopy
AFM is still a relatively new form of microscopy and has only been applied to the study of biopolymers since the late 1990s. It generates images by sensing the changes in force between a probe and the sample surface as the sam- ple is scanned. Using a variety of probing methods (44), a three-dimensional image with sub-nanometer resolution of the surface topography of tested samples can be produced (45). Thus, AFM affords an opportunity to directly image individual molecules and the helical structures of polysac- charides with minimal sample preparation (44, 46, 47). The polysaccharides are simply deposited from aqueous solution onto the surface of freshly cleaved mica, air dried, and then imaged directly under appropriate liquid (45).
For highly flexible polysaccharides such as dextrans, the AFM images show globular structures representing time-averaged pictures of the random coil structure. For more extended polysaccharides, such as xanthan and β-D-glucans, the AFM images may be quantified to yield persistence length, contour length, and its distributions (48). The dimensions observed by AFM are often larger than those derived from conventional techniques (44). This is believed to be due to the polymer-surface interac- tions which occur when the molecules are absorbed onto the mica surface prior to observation. AFM can be used to investigate the nature of association in junction zones, and also the overall structure of gel networks (49–51).
The use of EM and AFM has led to an improved understanding of the functional properties of polysaccha- rides at a molecular level. Furthermore, the ability to pro- vide direct information about heterogeneity makes microscopy not simply complementary to other physical techniques, but also indispensable for obtaining additional detailed structural information.
5. Nuclear Magnetic Resonance
a. Background
Nuclear magnetic resonance (NMR) spectroscopy provides detailed structural information of carbohydrates, such as identification of monosaccharide composition, elucidation of αor β configurations, and establishment of linkage pat- terns and sequence of the sugar units in oligosaccharides and polysaccharides. Recent advances in two-dimensional NMR techniques allow the elucidation of some polysac- charides without chemical analysis (52). NMR can also be used to determine the conformation and chain stiffness/mobility of oligosaccharides and some polysac- charides in solution and to monitor coil-helix transitions and gel formation (53).
The principle of NMR spectroscopy is based on the magnetic property of the nucleus in atoms associated with
spins. The most useful nuclei in carbohydrate research are
1H and 13C, which by absorbing radio frequency energy in
a strong magnetic field, jump to higher energy levels. Spins at the higher energy levels tend to relax to lower energy levels, and the transitions are dependent on the magnetic field strength in the local environment of the nucleus. Therefore, every nuclear spin in a molecule is influenced by the small magnetic fields of the nuclei of its nearest neighbors. Hence, the signal released by the nucleus reveals structural information of the nucleus in specific environment. The analysis of these individual sig- nals relative to a standard, expressed by chemical shift and spin-coupling between nuclei, can yield detailed informa- tion on the structure and shape of molecules.
One-dimensional NMR experiments are limited to the portrayal of response intensity as a function of the obser- vation frequency under the applied field. Two-dimen- sional NMR techniques utilize a second frequency domain, which greatly expands the information contained in the spectrum. The introduction of this second domain allows correlations to be established and hence connectiv- ity information can be obtained. These are very useful in determining molecular structures, particularly of complex oligosaccharides and polysaccharides. For example, COSY (COrrelation SpectroscopY) and TOCSY (TOtal Correlation SpectroscopY) are used to establish connec- tivities around monosaccharide rings. Long-range correla- tion experiments, such as Nuclear Overhauser Effect (NOE), a through-space phenomenon, can be used in the study of shape and conformation. Long-range het-