Table of Contents

Uronic acid degradation (with direct remethylation)

Introduction

Uronic acid degradation is a method to establish connectivities to a uronic acid. If the experiment succeeds you should at least obtain information of the residue that the uronic acid is linked to (i.e. the residue to the right). In fortunate cases one or sometimes two residues that are linked to the uronic acid can be deduced. The experiment always starts with permethylation of the polysaccharide and it is of importance that the carboxy group is methylated and that it is kept as the methyl ester. The permethylated polysaccharide is then treated with base, either dimsyl sodium in DMSO or sodium alkoxide in alcohol. H-5 is then abstracted and the 4-O-substituent eliminated (1). Elimination of the O-3 substituent from the liberated sugar is an analogous b-elimination which is called pealing (2). Sometimes isolation, purification and mild acid hydrolysis of the product is performed but mostly not. Then, methyl iodide or trideuteriomethyl iodide is added and the liberated sugars labelled.
Normally the experiment is run with three resulting samples, one part is withdrawn after the first methylation, another after the base treatment, and a third after the remethylation. All samples should be run according to step 8-14, i.e. hydrolysed, reduced and acetylated. Only shortage of material is a good excuse for not doing all three.

The fictive case above gives a methylation analysis with 2,4,6-Me3-Glc and 2,3,6-Me3-Glc. A hydrolysate after the base treatment gives only some 2,4,6-Me3-Glc and the remethylation product gives 2,4,6-Me3-3-CD3-Glc only. Thus, it can be concluded, after the base treatment, that a 4-substituted Glc is linked to the uronic acid, because the disappearance of 2,3,6-Me3-Glc. After the last analysis, i.e. the remethylation it can be concluded that the uronic acid was linked to the 3-position of a Glc residue because of the appearance of 3-O-CD3 derivative.

Reagents

Full list of chemicals

Procedure

  1. Methylate the polysaccharide (0.5-2 mg, see appropriate description).
  2. - If the polysaccharide is to be base degraded directly, thoroughly evacuate all excess methyl iodide from the DMSO coming from the methylation.
    1. If the methylated polysaccharide is purified and dried, dissolve in DMSO (1 mL).
  3. Add a crystal of p-toluenesulfonic acid and 100 µL of 2,2-dimethoxypropane, stir for 1h at room temperature.
  4. Add dimsyl sodium (2M, 0.5 ml) sonicate for 15 min (optional) and leave at room temperature for 12h (overnight).
  5. Freeze the sample and add 250 µL MeI or CD3I. Stir, when melted, for ca 1 h. Excess pressure may have to be relieved shortly with needle.
  6. Remove septum and blow off excess MeI and take away last traces of MeI with vacuum, or push two needles through the septum and blow air through. A quick way of removing the MeI is to transfer the sample to a conical flask and to put it on an rotary evaporator. Dilute then with an equal volume of H2O
  7. Sep-Pak
    1. Precondition: 10 mL EtOH, 2 x 2 mL H2O
    2. Apply sample in DMSO/H2O 1:1
    3. Rinse vial with 1 mL DMSO/H2O 1:1
    4. Rinse SepPak: 8 mL H2O, 8 mL 15% CH3CN, keep the washings until you know where your sample is.
    5. Elute methylated carbohydrate: 2 mL CH3CN, 2 mL EtOH into a 13 x 100 mm screw cap tube.
    6. Blow down the last 3 mL to dryness.
  8. Hydrolyse in 0.3 mL 2M TFA 120°C for 2h or 0.5M TFA 100 °C over night
  9. Evaporate solvent and add 1 mL MeOH, evaporate.
  10. Reduce with 0.3 mL fresh solution of NaBH4 for 1 h at 20 °C.
  11. Quench with glacial HOAc, evaporate with 2-3 x 0.5 mL 10% HOAc in MeOH (or less) 2-3 x 0.5 mL MeOH
  12. Acetylate with 100 µL Ac2O and 100 µL pyridine 100 °C 20 min. Add 50 µL of water if problems)
  13. Let the solution cool, evaporate solvent and add 1 mL toluene, evaporate.
  14. Partition between 0.5 mL H2O and 0.5 mL EtOAc, transfer organic phase to sample tube. Repeat. Concentrate to ca 0.2 mL, filter through glass down to sample tube.

Comments

Test substances, Standards