Methylation analysis using PMAAs and dimsyl sodium

(CH₃SOCH₂^--Na⁺)

This is the classical analysis for establishing the linkage pattern of sugar residues. The method has several steps; conversion of the polysaccharide to the polyanion, methylation of all free alkoxy groups with methyl iodide, hydrolysis to monosaccharides, reduction to alditols and acetylation to give partially methylated alditol acetates (PMAAs). The three last steps are similar or identical to those in sugar analysis. The first alkylation step can be obtained in a variety of ways most of them relying on the dimsyl anion, CH₃SOCH₂^-, and some on the hydroxide anion. Thus, the finding of a 2,3,4,6-tetra-O-methyl-hexose in a hydrolysate, detected as the 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-hexitol derivative indicates a terminal hexose residue i.e. a 1-linked residue. Since position 5 is not methylated, the ring must be formed through that hydroxy group and the residue be pyranosidic i.e. have a 6-membered ring.
The presence of a 2,3,6-tri-O-methyl-hexose indicates either a 4-linked residue with a ring fused 1,5 i.e. a pyranosidic residue, or a 5-linked residue with a ring fused 1,4 i.e. a furanosidic residue. There are two ways to establish the presence of the possible furanosidic ring:

• partial acid hydrolysis which should result in cleavage of the furanosidic linkage before the pyranosidic linkages are cleaved.
• NMR - as furanosides tend to give signals at ca. 84 ppm.

Acetamido sugars are normally methylated on the nitrogen to give the N-methyl-acetamido sugar. Occasionally the acetamido sugars is not quantitatively methylated and two peaks are obtained in the GLC chromatogram, one for the N-methylated and one for the unmethylated. Uronic acids give, after hydrolysis and treatment with sodium borohydride, the sodium salts of the acid and these are not volatile. One of the most common problems encountered with methylation analysis is undermethylation i.e. hydroxy groups are not converted to OMe groups but remain as OH-groups.

• Dimsyl sodium (2M, frozen)
• Methyl iodide
• Acetonitrile
• Acetonitrile, 10% in water
• Ethanol
• Trifluoroacetic acid (TFA), 0.5 or 2 M
• Methanol
• Sodium borohydride, 0.25 M in NH_3(aq)
• Ammonia, 1M
• Acetic acid, 10% in methanol
• Acetic acid
• Acetic anhydride
• Pyridine

Full list of chemicals

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 odor and toxic fumes. Add 0.5 mL of dry DMSO using a syringe whilst releasing the pressure in the flask by inserting another neede 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 dimsyl sodium. Stir at room temperature for at least 5 h or over night if analysis indicates 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 through a 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. 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:
      1. 2 mL CH₃CN
      2. 2 mL EtOH into a 13 x 100 mm screw cap tube.
   6. Blow down the last 4 mL to dryness.
7. Hydrolyse in ~0.3 mL 2M TFA 120°C for 2h or 0.5M TFA 100°C over night.
8. Evaporate solvent and add 1 mL MeOH, evaporate.
9. Reduce with 0.3 mL fresh solution of NaBH₄ for 1 h at 20°C.
10. Quench with glacial HOAc, evaporate with
    • 2-3 x 0.5 mL 10% HOAc in MeOH (or less)
    • 2-3 x 0.5 mL MeOH
11. Acetylate with 100 µL Ac₂O and 100 µL pyridine 100°C 20 min. Add 50 µL of water if problems.
12. Let the solution cool, evaporate solvent and add 1 mL toluene, evaporate.
13. 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.

• Polysaccharide sample may be electrostatic and difficult to transfer due to adsorption to the glass. For an exact weight of a sample of 0.1-1 mg take ca 2 mg and dissolve in 1 mL of water and take out appropriate volume.
• Too much sonication may give rise to extra GLC peaks. Completely undissolved material should appear in the analysis as alditol acetates i.e. with no methyl groups. If acidic sugars are present it may be advantageous to start from the acid form rather that the sodium salt form because of the greater solubility of the acid form.
• 2M dimsyl sodium should be kept at -20°C, and when used, thawed until enough liquid is obtained, which is transferred with a syringe. The colour should be greenish or bluish, not brown or black. If the polysaccharides contains base labile sugars treatment by base for only five minutes, may give satisfactory results.
• As a test of excess base one drop of solution may be taken out and added to a crystal of triphenylmethane which turns red if dimsyl sodium present.
• The reaction between the polyanion and MeI is exothermic and the frozen mixture readily melts. If the sample is not frozen then the excess heat may decompose the sample and turn it black. If so start all over again.
• Sep-Pak is a small C18 column which retains the methylated polysaccharide. The adsorption of smaller oligosaccharides is less and molecules smaller than a tetrasaccharide are mostly not retained. Check however. The pretreatment with EtOH and H₂O is for cleaning and wetting. The Sep-Pak is fitted onto a 10 mL syringe with a regular straight flange.
• Most methylated polysaccharides are soluble in dilute TFA but if strange results are obtained treat the methylated material with 90% formic acid for 1 h and evaporate. Continue with TFA as described. Too little liquid will give only drops on the wall and little in the bottom, so use >0.3 mL.
• Use blow down equipment throughout, normally compressed air is dry enough to be used.
• Trace amounts of acid are almost impossible to get rid of but will not harm this step. Bubbles of hydrogen on the bottom normally indicate excess NaBH₄ but in ammonia they may be scarce. The solution of NaBH₄ in NH₃ easily lasts a week. If not enough BH₄^- is present cyclic acetates will be formed which are fast-moving on the GLC column and with m/z 115 and 157 as prominent peaks in the mass spectrum.
  NaBD₄-reduction, which is performed as described above except that NaBD₄ is used instead of NaBH₄, and will give CHDOAc as the top fragment.
• Acetic acid will convert the borohydride to boric acid which must be removed by acidic methanol as its methyl ester i.e. methyl borate. The procedure must be repeated. If boric acid is left it may form complexes with the alditols and cause underacetylation.
• Be careful so that no water is removed with the organic phase.

• CRC
• Methods
• Gula boken