See the previous procedure for additional information.
- Anhydrous hydrogen fluoride
- Dimsyl sodium
- Methyl iodide
- Acetonitrile, 10% in water
- Sodium borohydride, 0.25 M in 1 M NH3(aq)
- Ammonia 1 M
- Acetic acid,10% in methanol
- Acetic acid 50%
- Acetic anhydride
- Transfer the dry sample (ca 1.5 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.
- From now on keep the flask in a fume hood due to odor and toxic fumes. Add 0.5 mL of dry DMSO by putting two needles through the septum. Stir the sample for 30 min.
- Add 0.25 mL dimsyl sodium. Stir at room temperature for at least 0.5 h.
- Freeze the sample and add 0.5 mL MeI. Stir when melted for ca 0.5 h. Excess pressure may have to be relieved shortly with needle.
- Remove septum and take away MeI by pushing a needle through the septum and applying vacuum. Dilute then with an equal volume of H2O
- Precondition: 10 mL EtOH, 2 x 2 mL H2O
- Apply sample in DMSO/H2O
- Rinse vial with 1 mL DMSO/H2O
- Rinse SepPak: 8 mL H2O
, 8 mL
, keep the washings until
you know where your sample is.
- Elute methylated carbohydrate: 2 mL CH3CN
, 2 mL EtOH into a
13 x 100 mm screw cap tube. Blow down the last 4 mL to dryness.
- Transfer dried sample (ca 0.2 mg) to the HF reaction vessel together with a dry mini-size magnetic rod. Dry the sample thoroughly in a dessicator and with a vacuum pump.
- Cool with CO2
+ethanol or liquid N2
, the reaction vessel and heat, with a blow dryer, the HF supply vessel (Kel-F) until the required volume has distilled over (ca 0.5 mL). Leave for 3 h at room temperature.
- Evaporate solvent by applying vacuum slowly to the reaction vessel and when it looks dry add 1 mL MeOH, evaporate. Transfer the reaction mixture with 0.5 mL 50% aqueous HOAc and heat at 80°C for 18 h. Evaporate to dryness, add 0.5 mL MeOH and evaporate again.
- Reduce with 0.3 mL fresh solution of NaBD4
) in 1M NH3(aq)
for 30 min at 20°C (or 1M NH3
- 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 at 80°C 2 h or overnight.
- Let the solution cool, evaporate solvent and add 1 mL toluene, evaporate.
- 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.
See previous notes