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.
- Anhydrous hydrogen fluoride
- Trifluoroacetic acid (TFA), 0.5 or 2 M
- Sodium borohydride, 0.25 M in 1 M NH4OH
- Ammonia 1 M
- Acetic acid, 10% in methanol
- Acetic anhydride
- 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
, 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
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.
- 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.
- 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.
which is performed identically except that NaBH4
is substituted for
, 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
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)