====== Methylation analysis using (PMAAs) On phosphate containing LOS using dimsyl sodium ======
(CH<sub>3</sub>SOCH<sub>2</sub><sup>-</sup>-Na<sup>+</sup>)

===== Introduction =====

[[sop4|See previous]]

==== Reagents ====

  * Anhydrous hydrogen fluoride
  * Dimsyl sodium
  * Methyl iodide
  * Acetonitrile
  * Acetonitrile, 10% in water
  * Ethanol
  * Methanol
  * Sodium borohydride, 0.25 M in 1 M NH<sub>3(aq)</sub>
  * Ammonia 1 M
  * Acetic acid,10% in methanol
  * Acetic acid 50%
  * Acetic anhydride
  * Pyridine

[[chemicals|Full list of chemicals]]

==== Procedure ====

  - Transfer the dry sample (ca 1.5 mg) to a serum flask (5 mL). Add a 1 cm magnetic rod, flush the flask with N<sub>2</sub>, 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 H<sub>2</sub>O
  - Sep-Pak
    - Precondition: 10 mL EtOH, 2 x 2 mL H<sub>2</sub>O
    - Apply sample in DMSO/H<sub>2</sub>O 1:1
    - Rinse vial with 1 mL DMSO/H<sub>2</sub>O 1:1
    - Rinse SepPak: 8 mL H<sub>2</sub>O, 8 mL 15% CH<sub>3</sub>CN, keep the washings until you know where your sample is.
    - Elute methylated carbohydrate: 2 mL CH<sub>3</sub>CN , 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 CO<sub>2</sub>+ethanol or liquid N<sub>2</sub>, 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 NaBD<sub>4</sub> (NaBH<sub>4</sub>) in 1M NH<sub>3(aq)</sub> for 30 min at 20° (or 1M NH<sub>3(aq)</sub> in EtOH)
  - 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 Ac<sub>2</sub>O 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 H<sub>2</sub>O 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.

==== Comments ====

[[sop4|See previous notes]]