Methylation analysis including carboxyl reduction of the methylated polysaccharide

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

Dimsyl sodium (2M, frozen)
Methyl iodide
Acetonitrile (neat and 10% in water)
Ethanol
Tetrahydrofuran
LiBH₄ or Superhydride
Trifluoroacetic acid
Methanol
Sodium borohydride, 0.25 M in 1 M NH₃_(aq)
Ammonia, 1 M
Acetic acid 10% in methanol
Acetic acid
Acetic anhydride
Pyridine

Procedure

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.
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.
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.
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.
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 .
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 13x100 mm screw cap tube. Blow down the last 4 mL to dryness.
Dissolve in 0.4 mL dry tetrahydrofuran (THF) in a 13x100 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°C for 1 h.
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.
Hydrolyse in ~0.3 mL 2M TFA 120°C for 2h or 0.5M TFA 100°C over night
Evaporate the solution to dryness by a stream of compressed air, add 0.5 mL MeOH, and evaporate. Repeat once.
Reduce with 0.3 mL fresh solution of NaBH₄ in NH₃_(aq) for 30 min at 20°C.
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 100°C 20 min. Add 50 µL of water if problems.
Evaporate the solution and add 0.5 ml toluene, evaporate to dryness. Repeat once.
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

Polysaccharide samples may become static 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 due to increased solubility of the acid form.
Dimsyl sodium should be kept in the deep freezer, and when used, thawed until enough liquid is obtained, which is transferred with a syringe. The colour should be greenish or bluish, not brown, not black. Polysaccharides with base labile sugars may be treated for only five minutes with base, sometimes satisfactory results are obtained then.
As a test of excess base one drop of solution may be taken out and added to one crystal of triphenylmethane which turns red if dimsyl sodium present.
The reaction between the polyanion and CH₃I is exothermic and the frozen mixture readily melts. If sample is not frozen, 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.
Use an heating block as usual for the LiBH₄ -reduction.
Purification is recommended as just neutralisation will give a gel of NaOAc that is very difficult to clean from acid in the next step.
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 . 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. Must be repeated.
Be careful so that no water is coming with the organic phase.

References