A practical guide to structural analysis of carbohydrates

Methylation analysis with carboxyl reduction of the native polysaccharide

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Structural analysis
 The detection of sugars with carboxyl-function within the regular scheme of methylation analysis (methylation, hydrolysis, reduction, acetylation) presents a problem as the carboxyl group is converted to a non-volatile sodium carboxylate. There are two alternative ways of getting to neutral sugars, either carboxyl-reduction of the native polysaccharide, or carboxyl-reduction of the methylated polysaccharide. In both cases a CH2OH group is formed. The sugars will however appear as different derivatives. E. g. a 4-substituted glucuronic acid, if reduced before the methylation gives 2,3,6-tri-O-methyl-glucose, and if reduced after the methylation gives 2,3-di-O-methyl-glucose. The reduction of a native polysaccharide is mostly performed by coupling of the carboxyl group to a water-soluble carbodiimide at pH 4.75 followed by addition of NaBH4 . The intermediate may also be e. g. a lactone. The reduction of the complex is very much faster than alkaline hydrolysis of the complex.
The reaction is far from always quantitative. It may be advantageous to repeat the procedure up to a total of three times. Of course the acids can be analysed as e. g. methyl glycosides after treatment with methanolic hydrogen chloride. The disadvantage is that more than one derivative is formed and the difficulty in interpreting the mass spectra.

Flow scheme

COOH-reduction right arrow Methylation right arrow Hydrolysis right arrow Reduction right arrow Acetylation

Reagents

  • N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • 2-(4-Morpholino)-ethanesulfonic acid, MES
  • Hydrochloric acid, 4M
  • Dimsyl sodium (2M, frozen) or any other proper methylating reagent
  • Methyl iodide
  • Trifluoroacetic acid, TFA 0.5 or 2 M
  • Methanol
  • Sodium borohydride, 0.25M in 1M NH3(aq)
  • Ammonia, 1M
  • Acetic acid, 10% in methanol
  • Acetic acid
  • Acetic anhydride
  • Pyridine
  • Ethanol
  • Acetonitrile

Procedure

  1. Dissolve the polysaccharide (2 mg ca 12 µmol) containing a carboxylic acid in water (ca 2 mg in 1 mL=12 µmol). Add the carbodiimide (30 mg, 156 µmol), and adjust the pH to 4.75. Alternatively the compound MES may be used for buffering the pH.
  2. Add a few mL of 4 M HCl to adjust for the increase in pH that takes place when the protons are consumed. If MES is used no adjustment is needed.
  3. After one hour (1-1.5 h) add 10 mg of NaBH4 (1 µmol). Leave the reaction mixture for 1 hour.
  4. Make pH 7 with 4 M HCl, and dialyse or purify by gel filtration.
  5. Transfer the dry sample (0.5-1 mg) to a serum flask (5 mL) If the material is less than 0.5 mg, dissolve 1-2 mg in 1 mL of H2O and take out the volume corresponding to desired amount and freeze-dry in serum flask. Add a 1 cm magnetic rod and seal the flask with a rubber septum.
  6. From now on keep the flask in a fume hood due to smell and poison. Add 0.5 mL of dry DMSO and flush the flask with N2 by putting two needles through the septum and blowing N2 through one of them. 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.
  7. Add 0.25-0.5 mL 2M dimsyl sodium. Stir at room temperature for at least 5 h or over night if analysis indicates undermethylation.
  8. 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.
  9. Remove septum and blow off excess MeI and take away last traces of MeI with vacuum, or push two needles through the septum and blow air through. 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 .
  10. Sep-Pak
    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 x100 mm screw cap tube.
    6. Blow down the last 3 mL to dryness.
  11. Hydrolyse in ~0.3 mL 2M TFA 120°C for 2h or 0.5M TFA 100°C over night
  12. Evaporate solvent and add 1 mL MeOH, evaporate.
  13. Reduce with 0.3 mL fresh solution of NaBH4 for 1 h at 20°C.
  14. Quench with glacial HOAc, evaporate with
    1. 2-3 x 0.5 mL 10% HOAc in MeOH (or less)
    2. 2-3 x 0.5 mL MeOH
  15. Acetylate with 100 µL Ac2O and 100 µL pyridine 100°C 20 min. Add 50 µL of water if problems.
  16. Let the solution cool, evaporate solvent and add 1 mL toluene, evaporate.
  17. 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.

Comments

  • The correct pH is necessary for optimal coupling but the reaction will also work at a slightly different pH.
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