Structural Characterisation

In order to elucidate the structure of a polysaccharide, the structural analysis can be approached from three main aspects: 1) constituents, 2) linkage, and 3) sequence. For the mamaku polysaccharide, the constituents and linkage were analysed and the sequence was deduced based on linkages identified and similar polysaccharides.

3.3.1

Monosaccharide Composition

Monosaccharide composition is usually determined by hydrolysing the polysaccharide to its constituent monosaccharides i.e. depolymerisation. Hydrolysis is done using acid or enzymes. High performance liquid chromatography (HPLC) or gas chromatography (GC) is then subsequently used to quantitatively and qualitatively detect the monosaccharide fractions.

3.3.1.1

Acid Hydrolysis

The glycosidic bonds between monosaccharides of a neutral polysaccharide are heat and acid labile. In the presence of a strong acid and heat, these bonds are cleaved and the monosaccharide units are released from the polysaccharide. Concentrated sulphuric acid, hydrochloric acid, and trifluoracetic acid (TFA) can be used for the hydrolysis reaction. TFA is usually used due to its volatility which makes it easy to remove via rotary evaporation after hydrolysis. Generally for an unknown polysaccharide, the material is heated in 1M TFA at 121qC for an hour (Cui, 2005). However, once the monosaccharides are released, it will be subjected to degradation by the hot concentrated acid. Therefore the reaction time, temperature, and acid concentration must be adjusted accordingly such that the reaction conditions are sufficient for complete glycosidic linkage hydrolysis and yet not cause sample degradation. Polysaccharides containing uronic acids i.e. acidic form of the monosaccharide are more difficult to hydrolyse. The linkage between uronic acid residues are more resistant to acid hydrolysis (Cui, 2005), therefore incomplete depolymerisation of the polysaccharide is more likely to occur.

3.3.1.2

Derivatisation (Acetylation)

The monosaccharides obtained after acid hydrolysis are not volatile – therefore in order to analyse the monosaccharide composition using gas chromatography, the monosaccharide fractions have to be made volatile through a process called derivatisation (Cui, 2005). Derivatisation involves converting the neutral monosaccharide into alditol acetates and the acidic monosaccharides into trimethylsilyl (TMS) derivatives as shown in Figure 3.21. The conversion of a neutral monosaccharide into its alditol acetate involves two steps. It is first reacted (reduced) with sodium borohydride in ammonium hydroxide to form an alditol. Excess sodium borohydride is removed by adding acetic acid (which neutralises the sample as well) and methanol, and continuously dried with nitrogen. The alditols are then acetylated by reacting with acetic anhydride at 121qC for a few hours. The resulting dry alditol acetates are dissolved in the solvent methylene chloride prior to GC analysis. Acidic monosaccharides on the other hand are converted to trimethylsilyl (TMS) ethers. The first reaction step is the same as with neutral monosaccharides, i.e. addition of sodium borohydride and subsequent

removal with acetic acid. It is then treated with a mixture of reagents containing pyridine, hexamethyldisilazne and trifluoracetic acid in order to facilitate acetylation (Cui, 2005).

Figure 3.21 – Derivatisation of neutral monosaccharides into alditol acetates and TMS derivatives (adapted from Cui, 2005)

3.3.2

Linkage Analysis

Apart from knowing the types of monosaccharides which make up the polysaccharide, it is also essential to obtain information on how these monosaccharides are linked together throughout the polymer chain. Polysaccharides with the same monosaccharide composition but differently linked will have markedly different properties. The flexibility of the polymer chain depends on the freedom of the glycosidic bonds to rotate and therefore its ability to form ordered/regular or disordered structures. Glycosidic bonds can also be either D- or E-linked, depending on the configuration of the monosaccharide at the anomeric carbon atom (C1) of the residue involved. Again, two monosaccharide units linked at the same positions but having different anomeric configuration (i.e. D- or E-) will result in different properties of the polysaccharide as well.

Methylation is the most widely used method to determine glycosidic linkage positions present in polysaccharides. A typical methylation test would give information on the molar percentage of the various linkage types present within the polysaccharide. However, results would not show the sequence of monosaccharides or the anomeric configuration of the glycosidic bonds. Methylation involves four main steps: 1) conversion of all free hydroxyl groups into methoxyls, 2) acid hydrolysis of inter-glycosidic linkages, 3) reduction and acetylation to give volatile partially methylated alditol acetates (PMAA) and lastly, 4) analysis with gas chromatography coupled with a mass spectroscopic detector (GC-MS) (Cui, 2005). The conversion of hydroxyl groups into methoxyls can be done using dry powdered sodium hydroxide (NaOH) and methyl iodide (CH3I) (Cui, 2005). Trifluoracetic acid (TFA) is used to hydrolyse the methylated polysaccharide into

monosaccharides, which are then deuterised with sodium borodeuteride into partially methylated alditols. A deuterium atom is introduced at C1 of the monosaccharide, which distinguishes C1 from C6 carbon. The final step is acetylation of the alditols into partially methylated alditol acetates (PMAA), for which the oxygen atoms involved in the glycosidic linkage are acetylated. The PMAAs are then qualitatively and quantitatively analysed using GC-MS.

3.3.3

Nuclear Magnetic Resonance (NMR)

Nuclear magnetic resonance is one of the most common and useful techniques for determining structures of polysaccharides. It is often used as a complementary technique in addition to the previously described chemical methods to identify and quantify monosaccharide constituents, sequence distribution, detect functional groups and intermolecular interactions etc. Nuclear magnetic resonance is based on the principle of nuclear spin. When placed in a strong magnetic field, the positively charged atom protons align either in the same direction or opposite direction to the field. The action of alignment is known as the intrinsic angular momentum of the atom i.e. nuclear spin. The difference in alignment directions results in two states separated by an energy difference 'E. The 'E and therefore resonance frequency also depends on the chemical environment of the nucleus in a molecule, an effect known as the chemical shift. The intensity of the frequency/chemical shift (in ppm) can be calculated based on the strength of the magnetic field. Different protons within the molecule will emit different energies at specific frequencies, depending on its environment e.g. shielding by electrons which gives an NMR spectrum unique to the molecule (Balci, 2005).

For polysaccharides, hydrogen and carbon are the most abundant elements in the molecule with NMR active isotopes 1_{H and} 13_{C respectively, with} 1_{H abundance close to 100% but relatively lesser for} 13_{C of 1.1%.}

Internal chemical shift references are used to ‘calibrate’ frequencies e.g. 1,4-dioxane in D2O for 13C NMR

where the chemical shift is 67.40 ppm. The number of monosaccharide units in the repeating unit can be known by counting the resonances in the anomeric region (4.4-5.5 ppm). Common hexoses e.g. glucose show up here as well as in chemical region of 95-110 ppm of 13_{C NMR spectra (Jonsson, 2010). Other resonances}

may reveal -CH3 groups (1 ppm) or presence of N-acetyl (2.0 ppm) and/or O-acetyl groups (2.1 ppm)

(Zaccheus, 2012). Monosaccharides have complicated NMR spectra, and therefore even more so for polysaccharides. The macromolecular structure of high molecular weight polysaccharides tumble slowly in solutions (on top of high viscosity effects), which causes the excited spins to relax more rapidly, translating to broad resonances which are difficult to singularise (Cheng & Neiss, 2012). The interpretation of NMR spectra is often based on reference spectra (assignments based on literature review), since the NMR shifts show general trends for carbon atoms or protons at a particular ring position (Table 3.3).

Table 3.3 – Key 1_{H and} 13_{C chemical shifts for nuclei of polysaccharides (adapted from Perlin & Casu, 1982)} 1_{H Shift (ppm)} 13_{C Shift (ppm)} CH3C ~1.5 CH3C ~15 CH3CON 1.8-2.1 CH3COH) 20-23 CH3CO2 2.0-2.2 CH3CO2) CH(NH) 3.0-3.2 CH2C 38 CH3C 3.3-3.5 CH3O 55-61 H-2 to H-6’ 3.5-4.5 CH(NH) 58-61 H-5 4.5-4.6 CH2OH 60-65 H-1 (ax) 4.5-4.8 C-2 to C-5 65-78

H-C(OH)2 5.2 C-1 (ax-O, red) 90-95

HO 5.0-5.4 C-1 (eq-O, red) 95-98

H-1 (eq) 5.3-5.7 C-1 (ax-O, glyc) 98-103

H-CO2 5.9 C-1 (eq-O, glyc) 103-106

C-1 (fur) 106-109

COOH 174-174

COOR 175-180

Ax: axial; eq: equatorial; red: reducing; glyc: glycosidic; fur: furanosyl