Table of Contents

Methylation analysis using PMAAs and dimsyl sodium

(CH3SOCH2--Na+)

Introduction

This is the classical analysis for establishing the linkage pattern of sugar residues. The method has several steps; conversion of the polysaccharide to the polyanion, methylation of all free alkoxy groups with methyl iodide, hydrolysis to monosaccharides, reduction to alditols and acetylation to give partially methylated alditol acetates (PMAAs). The three last steps are similar or identical to those in sugar analysis. The first alkylation step can be obtained in a variety of ways most of them relying on the dimsyl anion, CH3SOCH2-, and some on the hydroxide anion. Thus, the finding of a 2,3,4,6-tetra-O-methyl-hexose in a hydrolysate, detected as the 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-hexitol derivative indicates a terminal hexose residue i.e. a 1-linked residue. Since position 5 is not methylated, the ring must be formed through that hydroxy group and the residue be pyranosidic i.e. have a 6-membered ring.
The presence of a 2,3,6-tri-O-methyl-hexose indicates either a 4-linked residue with a ring fused 1,5 i.e. a pyranosidic residue, or a 5-linked residue with a ring fused 1,4 i.e. a furanosidic residue. There are two ways to establish the presence of the possible furanosidic ring:

Acetamido sugars are normally methylated on the nitrogen to give the N-methyl-acetamido sugar. Occasionally the acetamido sugars is not quantitatively methylated and two peaks are obtained in the GLC chromatogram, one for the N-methylated and one for the unmethylated. Uronic acids give, after hydrolysis and treatment with sodium borohydride, the sodium salts of the acid and these are not volatile. One of the most common problems encountered with methylation analysis is undermethylation i.e. hydroxy groups are not converted to OMe groups but remain as OH-groups.

Flow scheme

Methylation Hydrolysis Reduction Acetylation

Reagents

Full list of chemicals

Procedure

  1. Transfer the dry sample (0.1-1 mg) to a serum flask (5 mL). Add a 1 cm magnetic rod, flush the flask with N2, and seal the flask with a rubber septum.
  2. From now on keep the flask in a fume hood due to odor and toxic fumes. Add 0.5 mL of dry DMSO using a syringe whilst releasing the pressure in the flask by inserting another neede through the septum. Stir the sample for a couple of hours and then sonicate for 30 min. Repeat the stirring and sonication using the same times, if the sample is not dissolved. If still undissolved after 8 h continue anyway, or alternatively add another 0.5 mL DMSO and repeat dissolution procedure.
  3. Add 0.25-0.5 mL 2M dimsyl sodium. Stir at room temperature for at least 5 h or over night if analysis indicates undermethylation.
  4. Freeze the sample and add 0.25 mL MeI. Stir when melted for ca 1 h. Excess pressure may have to be relieved shortly through a needle.
  5. Remove septum and take away MeI by pushing a needle through the septum and applying vacuum, or dry by using a stream of N2. 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.
  6. 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:
      1. 2 mL CH3CN
      2. 2 mL EtOH into a 13 x 100 mm screw cap tube.
    6. Blow down the last 4 mL to dryness.
  7. Hydrolyse in ~0.3 mL 2M TFA 120°C for 2h or 0.5M TFA 100°C over night.
  8. Evaporate solvent and add 1 mL MeOH, evaporate.
  9. Reduce with 0.3 mL fresh solution of NaBH4 for 1 h at 20°C.
  10. 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
  11. Acetylate with 100 µL Ac2O and 100 µL pyridine 100°C 20 min. Add 50 µL of water if problems.
  12. Let the solution cool, evaporate solvent and add 1 mL toluene, evaporate.
  13. 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.

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References