Table of Contents

Methylation with sodium hydroxide


This is an alternative way of performing linkage analysis of polysaccharides. Contrary to the methylations performed with a strong base e.g. the dimsyl anion (CH3SOCH2-) where the methylation is performed after having made the polyanion, the alkylation with sodium hydroxide i.e. hydroxide anion is accomplished continuously as the polysaccharide reacts with the base and the methyl iodide. Dryness is as usual essential even if there is a methylation method that includes a large amount of water. The method often gives clean products, and is in general recommended only for oligosaccharides, not for polysaccharides. Often it is advantageous to reduce the oligosaccharides to oligosaccharide alditols before methylation, to reduce the risk of elimination reactions.
For the general introduction to the methodology read the section about methylation with dimsyl sodium.

Flow scheme

Methylation Hydrolysis Reduction Acetylation


Full list of chemicals


  1. Transfer the dried sample (0.1-1 mg, 0.5-5 µmol) to a conical reaction vessel (1 mL, Reacti-vial) fitted with a triangular magnetic rod and a cap. Add dry DMSO (50 µL), flush the vial with dry nitrogen, seal it and and keep it at room temperature. If there are solubility problems acetylate the sample under the conditions given below and then dissolve in DMSO.
  2. Remove the cap and add freshly powdered NaOH (120 mg) as quickly as possible, stir the resulting slurry for 20 minutes.
  3. Add methyl iodide (3 x 20 µL) with 10 min intervals.
  4. The methylated oligosaccharide is recovered, in the organic phase, after partition between CHCl3 and M sodium thiosulfate (1 mL each). Repeat with water.
  5. Hydrolyse in ~0.3 mL 2 M TFA at 120°C for 2h or in 0.5 M TFA at 100°C 12-16 h
  6. Evaporate the solutioon to dryness by a stream of compressed air, add 0.5 mL MeOH, and evaporate. Repeat once.
  7. Reduce with 0.3 mL fresh solution of NaBH4 in NH3(aq) for 30 min at 20°C.
  8. Quench with 0.5 mL 10% HOAc in MeOH, evaporate to dryness. Add 0.5 mL 10% HOAc in MeOH and evaporate to dryness. Repeat once or twice. Add 0.5 mL MeOH and evaporate to dryness. Repeat once or twice.
  9. Acetylate with 100 µL Ac2O and 100 µL pyridine 100°C 20 min. Add 50 µL of water if problems.
  10. Evaporate the solution and add 0.5 mL toluene, evaporate to dryness. Repeat once.
  11. Partition between 0.5 mL H2O and 0.5 mL EtOAc by stirring fast but not violently by using a triangular magnetic rod in a conical vial (Reacti-vial type) for a couple of minutes. Transfer the upper EtOAc phase to the old rinsed tube. Add another 0.5 mL EtOAc and extract. Repeat a third time. Concentrate to dryness dissolve in ca 0.2 mL EtOAc, transfer into sample tube and concentrate to 25 -50 µL.