Sugar analysis using anhydrous hydrogen fluoride

An alternative to hydrolysis of polysaccharides with dilute carboxylic acids in order to get monosaccharides, is solvolysis (reaction with solvent), with anhydrous hydrogen fluoride. The result of the solvolysis, after evaporation of the solvent, is a glycosyl fluoride which most often is directly hydrolysed to the monosaccharide upon addition of water as the last amount of HF is most difficult to evaporate. If, however, the hydrogen fluoride is completely removed the resultant glycosyl fluorides must be hydrolysed with acid, normally an hour reflux with 0.5 M TFA. The rest of the reaction sequence i.e. BH₄ -reduction, and acetylation, are as described for TFA hydrolysis. As hydrogen fluoride reacts with glass the solvolysis is often performed in a specially designed reaction apparatus made of Kel-F, a trifluorochloro-ethylene polymer. Teflon can also be used but the amount of liquid in the vessels can hardly be observed without a strong lamp. In Kel-F it can easily be observed, however. The use of anhydrous hydrogen fluoride is extremely hazardous and should normally be performed in the specially designed apparatus which is completely sealed. For complete solvolysis to the monomers usually room temperature reactions are used.
The main advantage with anhydrous hydrogen fluoride is that amides are not cleaved but glycosidic linkages are, i.e. the glycosidic linkage of a 2-acetamido-2-deoxy-hexoside can be quantitatively cleaved. When TFA is used amide hydrolysis is a competing reaction and the glycosidic linkage of the liberated 2-aminohexoside is not possible to cleave at all.

Flow scheme

Solvolysis

Hydrolysis

Reduction

Acetylation

Reagents

Anhydrous hydrogen fluoride
Trifluoroacetic acid (TFA), 0.5 or 2 M
Methanol
Sodium borohydride, 0.25 M in 1 M NH₄OH
Ammonia 1 M
Acetic acid, 10% in methanol
Acetic anhydride
Pyridine

Procedure

Transfer dried sample (ca 0.2 mg) to the HF reaction vessel together with a dry mini-size magnetic rod.
Option: Add internal standard, 50 µg of xylose or suitable sugar.
Cool with CO₂ +ethanol or liquid N₂ , the reaction vessel and heat, with a hair dryer, the HF supply vessel (Kel-F) until the required volume has distilled over (ca 0.5 mL). Leave for 3h 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 0.5M TFA and reflux for 1h. Evaporate to dryness, add 0.5 mL MeOH and evaporate again.
Optional: No hydrolysis.
Reduce with 0.3 mL fresh solution of NaBH₄ in NH₃ for 30 min at 20°
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 0.1 mL Ac₂O and 0.1 mL pyridine 100° 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 0.2 mL EtOAc, transfer into sample tube and concentrate to 25-50 µL.

Comments

For an exact weight of a sample of ca 0.2 mg take 1-2 mg and dissolve in 1 mL of water and take out appropriate volume.
The NaBH₄-solution needs to be made approximately twice per week. 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 identically except that NaBH₄ is substituted for NaBD₄ , 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.
Be careful so that no water is removed with the organic phase. Use holder for the Reacti-vial. Alternatively partition may be effected with the pasteur pipette.

Test substances, Standards

A mixture of Rha, Fuc, Ara, Rib, Xyl, Man, Gal, Glc, GlcNAc, GalNAc. (Take 0.5 mg each dissolve in 1 mL of H₂O , take 200 µL reduce and acetylate as described above. Freeze-dry the remainder.) Dissolve in 50 µL EtOAc.

Detection and Quantification

GLC with e.g. DB1 or DB225 fused silica columns. (100% methyl silicone or 95% methyl/5% phenyl silicone)