2.2 Experimental Procedures in Methylation Analysis

Preparation of dimsyl sodium (2M) in DMSO

Sodium hydride (50% suspension in oil; 5.0 g) is weighed into a 100 mL serum vial and suspended in dry light petroleum (25 mL). The hydride is allowed to settle and the liquide is decanted. This procedure is repeated twice and the washed sodium hydride is then dried by flushing with dry nitrogen. DMSO (dried over molecular sieve, 4 Å) (50 mL) is added to the vial which is sealed with a rubber septum and flushed with nitrogen via two injection needles. The vial is placed in an ultra-sonic bath and heated to 50-60°C. Hydrogen evolved is vented through an injection needle. After about 4 h hydrogen evolution ceases and the resulting greenish-grey opalescent solution of dimsyl sodium can be used for the permethylation reaction. The reagent can be stored in a refrigerator and is then stable for at least one month.

CAUTION: Sodium hydride as well as dimsyl sodium react violently with water and both are highly corrosive.

The methylation reaction

A dried sample(0.5 - 3.0 mg) of substrate (polysaccharide or glycoconjugate) is dissolved in dry DMSO (0.5 - 3.0 mL) contained in a serum vial (5 or 10 mL volume) which is sealed with a rubber septum. The vial is flushed with dry nitrogen via two injection needles. The dissolution of the sample is often facilitated by stirring using a small magnetic stirrer bar or by treatment in a ultra-sonic bath. 2M Dimsyl sodium in DMSO (0.5 -1.5 mL) is added dropwise via an injection needle using a syringe. The vial is agitated in an ultra-sonic bath at 20 - 25°C for 30 min and then left at room temperature overnight. The vial is cooled in an ice-water bath and methyl iodide (0.5 -1.5 mL) is added dropwise using a syringe. Excess pressure is released via a second needle. After ultra-sonic agitation at room temperature for oen hour the vial is opened and excess methyl iodide removed either by flushing with a stream of nitrogen or by evaporation in a rotatory evaporator (bath temperature below 40°C).

Work-up procedures

A. Polymeric substrate

The reaction mixture is poured into a dialysis bag (pre-boiled with distilled water, 3 x 30 min) and dialysed against running tap-water (≈20°C) overnight. The contents of the bag are then concentrated under reduced pressure to dryness in a 25 or 50 mL conical flask using a rotatory evaporator.

B. Oligomeric substrate

The reaction mixture is poured into water (5 mL) and the aqueous phase is exracted 4 times with methylene chloride (3 mL). The combined methylene chloride phases are washed with water (4 x 3 mL) and concentrated to dryness in a 25 or 50 mL conical flask using a rotary evaporator. The extractions are most conveniently carried out in centrifuge tubes (1.5 x 10 cm) which are agitated on a vibrational mixer to effect efficient mixing and then concentrated to facilitate phase separation.

Acid hydrolysis and transformation into alditol acetates

The methylated material is treated with 90% (v/v) aqueous formic acid (2 mL) at 100°C (boiling water bath) for one hour. (the reaction mixture is kept in a conical flask with a glass stopper.) The acid is removed in a rotary evaporator and the residue treated with 0.13 M aqueous sulfuric acid (3 mL) at 100°C for 16 h. The acid is neutralised with barium carbonate (small drops are checked on pH-paper; nitrogen is bubbled through the solution in order to avoid acidification due to dissolved carbon dioxide) and the solution is filtered. The filtered salts are washed with two 2 mL portionss of water. The combined filtrate is concentrated to ≈2 mL and sodium borohydride (or borodeuteride) (25 mg) is added. After two hours the solution is acidified to pH 3.5 with Dowex 50 (H⁺) ion exchange resin, filtered, concentrated to dryness and co-distilled with three portions (5 mL) of methanol. If the monomer mixture contains amino sugars, the acidification is instead effected with glacial acetic acid whereafter the mixture is concentrated.
The resulting alditols are acetylated with acetic anhydride and pyridine (distilled over phosphorus pentoxide) (1:1, 1 mL) at 100°C for one hour. The excess reagents are removed by co-distillation with toluene and the sample is transfered with methylene chloride to a small sample tube and concentrated. If acetic acid has been used for acidification, the sample in methylene chloride (2 mL) is extracted with three portions of water (1 mL) before concentration.

NOTE: If the mixture contains volatile components, e.g. a penta-O-methyl-hexitol or tri-O-methyl-6-deoxy-hexitol, prolonged evaporations should be avoided in order to minimize loss of such components.