CHAPTER 6 PURIFICATION AND STRUCTURAL CHARACTERISATION OF MAMAKU
6.2.3 Structural Analysis
6.2.3.1
Constituent Sugar Analysis
The principle of constituent sugar analysis is to determine the monosaccharide composition of the polysaccharide by i) hydrolysing (breaking down) the polysaccharide, ii) converting the monosaccharides into forms appropriate for analysis (reduction and acetylation), and then iii) identifying the constituent monosaccharides qualitatively and quantitatively. More details on the theory of these individual reactions can be found in the experimental techniques section (Chapter 3).
The constituent monosaccharide composition of the purified mamaku polysaccharide was determined by three complementary methods either with different hydrolysis methods or analytical techniques:
Method 1: Samples (1 mg, in duplicate) were hydrolysed with aqueous trifluroacetic acid (TFA) (2 M, 500 μL, containing 0.406 mg/ml myo-inositol, 120°C, 1 h), filtered (0.2 Pm, hydrophilic PTFE) hydrolysates, dried and neutralised by addition of 2 M NH4OH (200 PL). The neutralised hydrolysates were then reduced, acetylated
and analysed by GC-MS as described below. Weight calibration constants were determined from a seven sugar standard mix (rhamnose, fucose, arabinose, xylose, mannose, galactose & glucose; derivatised at the same time as the samples) following the TAPPI standard method T 249 cm-85 (1985) for quantification of monosaccharides in the sample by comparing peak areas and retention times of standards to sample. The TAPPI standard method T 249 is the standard for determining carbohydrate composition of extractive-free wood and wood pulp by gas-liquid chromatography. Myo-inositol of a known quantity was used as an internal standard in mass spectrometry to minimise inaccuracies in sample quantification. Monosaccharide yields were based on the mean values of duplicate samples and were expressed as weight percent anhydro-sugar because this is the form of sugar present in a polysaccharide.
Method 2: Samples (1 mg) were hydrolysed with methanolic acid (3 N, 500 PL, 80qC overnight) and aqueous TFA (2 M, 500 PL, containing 0.86 mg/ml D-allose as an internal standard, 120qC, 1 h). The hydrolysates were reduced and acetylated to form alditol acetates and analysed by GC-MS as described below
Method 3: The monosaccharides resulting from hydrolysis with methanolic acid and aqueous TFA from method 2 were analysed by high-performance anion-exchange chromatography (HPAEC). Samples (20 μL) dissolved in distilled water (0.5 mg/ml) were separated at 30 °C on a CarboPac PA-1 (4 x 250 mm) column equilibrated in 25 mM NaOH. Samples were eluted with simultaneous gradients of NaOH (25–10 mM from 0–
10 min, then 10–100 mM from 10–30 min and held to 55 min) and sodium acetate (0–500 mM NaOH from 30–55 min) at a flow rate of 1 ml/min and monitored by pulsed amperometric detection, using the Dionex standard carbohydrate waveform.
Reduction Sodium borodeuteride (NaBH4, 2M) in 2M NH4OH (200Pl) was added to reduce (i.e. convert the
acetal to corresponding alcohol groups) the hydrolysed samples overnight at 25qC. Glacial acetic acid (3 x 50Pl) was added to stop the reaction. Acetic acid (5% v/v) in methanol (2x500Pl) was added to remove borate as volatile trimethylborate and the samples were evaporated to dryness under 40qC air. Methanol was added and the samples were again dried under 40qC air (repeated twice). A dry white crystalline residue was obtained.
Acetylation Glacial acetic acid (40Pl), ethyl acetate (200Pl), acetic anhydride (600Pl) and 60% v/v perchloric acid (23Pl) were added to the sample and mixed gently. The tubes were left to stand at room temperature for 15 minutes. Distilled water (2ml) and 1-methlyimidazole (40Pl) were added to decompose the acetic anhydride. Dichloromethane (DCM, 2ml) was added to the tubes, shaken and centrifuged at 1120g for 5 minutes to extract the alditol acetates from the aqueous phase. The top layer was removed using a Pasteur pipette and discarded. The DCM phase was washed, successively, with 0.5M sodium bicarbonate (Na2CO3, 2ml) and 2 x
water (2ml) and centrifuged at 1120g for 5 minutes to wash the DCM phase. The washed DCM phase containing the alditol acetates was evaporated to dryness under 40qC air. Acetonitrile (500Pl) was added to remove any residual water and then evaporated to dryness. Residues were re-suspended in an appropriate volume of acetone and run on GC-MS.
GC-MS The alditol acetate derivatives were separated by GC on an Agilent HP-5MS fused silica capillary column (30m x 0.25mm i.d., 0.25Pm film thickness; Agilent, Santa Clara, CA) with the GC oven programmed from 50qC (held for 1 minute) to 130qC at a rate of 25qC/min, then to 230qC at a rate of 3qC/min and detected by MS using a Hewlett Packard 5973 MSD. Identifications were based on peak retention times and by comparison of electron impact mass spectra with standard spectra.
6.2.3.2
Glycosyl Linkage Analysis
Prior to glycosyl linkage analysis, uronic acid and methylesterified uronic acid residues were reduced using a two-step carboxyl reduction method as described by Sims and Bacic (1995) (procedures below; illustrated in Figure 6.2). Carboxyl-reduced samples (1 mg, in duplicate) were methylated (i.e. conversion of –OH to –OCH3
groups with methyl iodide) with the method of Ciucanu and Kerek (1984) except that samples were dispersed in DMSO (200 μl). After extraction into chloroform, the methylated samples were hydrolysed with 2.5 M TFA, reduced and acetylated before analysis by GC-MS as described above (Figure 6.3).
Figure 6.2 – Carboxyl reduction of methylesterified and non-methylesterified uronic acids (adapted from Pettolino, Walsh, Fincher, & Bacic, 2012)
Carboxyl Reduction of Uronic Acids
1st reduction Freeze-dried purified mamaku (10mg) was suspended in imidazole-HCl buffer (500mM, 10ml, pH 8.0) and cooled with ice to 4qC. Aliquots (3 x 1ml) of freshly prepared sodium borodeuteride (NaBD4) in
imidazole-HCl buffer was added to the solution at 10 minute intervals and then left to stand on ice for an hour. The samples were then brought to room temperature and left to stand for another hour. Excess NaBD4 was
destroyed by dropwise addition of glacial acetic acid (300Pl) until the fizzing stopped. The samples were dialysed against distilled water in a 2000 MWCO membrane for 24 hours with 2 dialysate changes. The samples were freeze-dried.
2nd reduction Samples subjected to the first reduction were then prepared for a second reduction step. The freeze-dried samples were suspended in distilled water (1ml) and MES-KOH (0.2M, 200Pl, pH 4.75) was added. Carbodiimide reagent (1-cyclo-hexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulphonate, 400Pl, 500mg/ml) was added and then the samples were sonicated in ice water for 30 minutes to aid dispersal. The samples were heated for 3 hours at 30qC. Tris-HCl (2M, 1ml, pH 8.0) was added and the samples were cooled to 4qC. Sodium borodeuteride (NaBD4; 70mg/ml) was added to two sample tubes while sodium borohydride
(NaBH4; 70mg/ml) was added to another two sample tubes to further reduce the sample for 18 hours at 4qC.
Excess reductant was destroyed by dropwise addition of glacial acetic acid (150Pl) until the fizzing stopped. The samples were dialysed against distilled water in a 2000 MWCO membrane for 24 hours at 4qC with two dialysate changes. The samples were freeze-dried and weighed.
Figure 6.3 – Summary of glycosyl linkage analysis based on methylation, hydrolysis, reduction and acetylation reactions (adapted from Pettolino, et al., 2012)
6.2.3.3
NMR spectroscopy
Purified mamaku polysaccharide was exchanged with deuterium by freeze-drying with D2O (99.9 atom%) three
times. Samples were dissolved in D2O and 1H and 13C (both 1H coupled and decoupled) spectra were recorded
on a Bruker Avance DPX-500 spectrometer at 90qC. The 1Hand 13C chemical shifts were measured relative
to an internal standard of Me2SO (1H, 2.70 ppm; 13C, 39.5 ppm)(Sims & Furneaux, 2003). Assignments were
made from double quantum filtered (DQF) correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC) COSY, HMQC total correlated spectroscopy (TOCSY) and DEPT-135 (distortionless enhancement by polarization transfer with pulse (M3) flip angle of 135q) experiments and by comparing the
spectra with published data (Appendix C Table C1 and C2).