Table of Contents

Methylation analysis including carboxyl reduction of the methylated polysaccharide

Introduction

Acids e.g. uronic acids are not detectable in the usual methylation analysis as the uronic acid is converted to its sodium salt. One way to monitor the amount of uronic acid and to establish its mode of substitution is to reduce the methylated material whereby the methyl ester is reduced to a hydroxymethyl group (CO2Me→CH2OH. Two commonly used reagents are LiBH4 and Superhydride (Lithium triethyl borohydride). Both reagents are available as their deuterated forms also. The reagents are convenient to use because of the ease in workup. It is of utmost importance to have the sample dry as any amount of water will rapidly be converted, in the alkaline environment, to hydroxide ions. Thus, a 4-substituted glucuronic acid will end up as a 2,3-di-O-methyl-glucose in the methylation analysis (and analysed as acetylated alditol acetate). The reactions are mostly but far from always quantitative.
LiBH4 is highly hygroscopic and does not keep for long after opening. It can be prepared in situ from LiCl and NaBH4.1

Flow scheme

Methylation COOCH3-reduction Hydrolysis Reduction Acetylation

Reagents

Full list of chemicals

Procedure

  1. Transfer the dry sample (0.1-1 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.
  2. From now on keep the flask in a fume hood due to smell and poisonous fumes. Add 0.5 mL of dry DMSO and by putting two needles through the septum. Stir the sample for a couple of hours and then sonicate for 30 min. Repeat the stirring and sonication using the same times, if the sample is not dissolved. If still undissolved after 8 h continue anyway, or alternatively add another 0.5 mL DMSO and repeat dissolution procedure.
  3. Add 0.25-0.5 mL 2M CH3SOCH2-Na+. Stir at room temperature for at least 5 h or over night if analysis indicate undermethylation.
  4. Freeze the sample and add 0.25 mL MeI. Stir when melted for ca 1 h. Excess pressure may have to be relieved shortly with needle.
  5. Remove septum and take away MeI by pushing a needle through the septum and applying vacuum, or dry by using a stream of N2. A quick way of removing the MeI is to transfer the sample to a conical flask and to put it on an rotary evaporator. Dilute then with an equal volume of H2O.
  6. SepPak
    1. Precondition: 10 mL EtOH, 2 x 2 mL H2O
    2. Apply sample in DMSO/H2O 1:1
    3. Rinse vial with 1 mL DMSO/H2O 1:1
    4. Rinse SepPak: 8 mL H2O, 8 mL 15% CH3CN, keep the washings until you know where your sample is.
    5. Elute methylated carbohydrate: 2 mL CH3CN, 2 mL EtOH into a 13×100 mm screw cap tube. Blow down the last 4 mL to dryness.
  7. Dissolve in 0.4 mL dry tetrahydrofuran (THF) in a 13×100 mm screw cap tube and add 5 mg of LiBH4. Reflux at 70°C for 1 h.
    Alternatively:
    Add 0.5 mL of the Superhydride solution and let react at 0° for 1 h.
  8. Neutralise with 200 µL 10% acetic acid in methanol, evaporate to dryness and purify either with SepPak, or partition between 0.5 mL H2O and 0.5 mL CHCl3, take CHCl3 phase and evaporate to dryness, or dialyse and freeze dry.
  9. Hydrolyse in ~0.3 mL 2M TFA 120°C for 2h or 0.5M TFA 100°C over night
  10. Evaporate the solution to dryness by a stream of compressed air, add 0.5 mL MeOH, and evaporate. Repeat once.
  11. Reduce with 0.3 mL fresh solution of NaBH4 in NH3 for 30 min at 20°C.
  12. 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.
  13. Acetylate with 100 µL Ac2O and 100 µL pyridine 100°C 20 min. Add 50 µL of water if problems.
  14. Evaporate the solution and add 0.5 ml toluene, evaporate to dryness. Repeat once.
  15. 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 200 µL EtOAc, transfer into sample tube and concentrate to 25- 50 µL.

Comments

References

  1. - M. Linnerborg, R. Wollin and G. Widmalm, Eur. J. Biochem. 246 (1997) 565-573 PubMed 9208951
    1. J. Kollonitsch, O. Fuchs and V. Gábor, Nature 173 (1954) 125-126