Methylation with methyl iodide/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 this 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

Reagents

Sodium hydroxide
Methyl iodide
Acetonitrile
Acetonitrile, 10% in water
Ethanol
Chloroform
Sodium thiosulfate, 1 M
Trifluoroacetic acid, 2 M and 0.5 M
Methanol
Sodium borohydride
Ammonia 1 M
Acetic acid, 10% in methanol
Acetic acid
Acetic anhydride
Pyridine

Procedure

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.
Remove the cap and add freshly powdered NaOH (120 mg) as quickly as possible, stir the resulting slurry for 20 minutes.
Add methyl iodide (3 x 20 µL) with 10 min intervals.
The methylated oligosaccharide is recovered, in the organic phase, after partition between CHCl₃ and M sodium thiosulfate (1 mL each). Repeat with water.
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
Evaporate the solutioon to dryness by a stream of compressed air, add 0.5 mL MeOH, and evaporate. Repeat once.
Reduce with 0.3 mL fresh solution of NaBH₄ in NH₃_(aq) for 30 min at 20°C.
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.
Acetylate with 100 µL Ac₂O and 100 µL pyridine 100°C 20 min. Add 50 µL of water if problems.
Evaporate the solution and add 0.5 mL toluene, evaporate to dryness. Repeat once.
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.

Comments

The only way to handle small amounts is with a conical flask, the conical magnetic rod ensures stirring. The recipe can be doubled without any problems.
Sodium hydroxide pellets easily absorb H₂O and CO₂ from the air and should be handled quickly. A slurry of NaOH in DMSO has been recommended but found impractical by us.
Batchwise addition of methyl iodide is preferred since it is also gradually degraded.
Oligosaccharides larger than trisaccharides may be purified by SepPak. If the methylation mixture is only partitioned, dissolve the product in water and freeze dry.
Most methylated oligosaccharides are soluble in dilute TFA but if strange results are obtained treat the methylated material with 90% formic acid at 100°C for 1 h and evaporate. Continue with TFA as described. Too little liquid will give only drops on the wall and little in the bottom, so >0.3 mL.
Trace amounts of acid are almost impossible to get rid of but will not harm this step. Bubbles of hydrogen on the bottom normally indicate excess NaBH₄ but in ammonia they may be scarce. The solution of NaBH₄ in NH₄OH easily lasts a week. If not enough BH₄^- is present cyclic acetates will be formed which are fast-moving on the GLC column and with ^m/_z 115 and 157 as prominent peaks in the mass spectrum.
NaBD₄ -reduction, which is performed as described above except that NaBD₄ is used instead of NaBH₄ , and will give CHDOAc as the top fragment.
Acetic acid will convert the borohydride to boric acid which must be removed by acidic methanol as its methyl ester i.e. methyl borate. The procedure must be repeated. If boric acid is left it may form complexes with the alditols and cause underacetylation.
Be careful so that no water is removed with the organic phase. Use holder for the Reacti-vial. Alternatively partition may be effected with the pasteur pipette.

References