Table of Contents

Sugar analysis using alditol acetates\\ Hydrolysis with trifluoroacetic acid and morpholinoborane

Introduction

Acid labile sugars often degrade completely during hydrolysis. For that reason conditions have been worked out in which you can reduce during the hydrolysis to get the corresponding alditols. Two reagents are available, dicyanoborohydride and 4-methylmorpholinoborane. Only the latter is commercially available but is unfortunately easily degraded during the hydrolysis especially if the hydrolysis is performed in heat. If acid labile sugars are present as well as “normal” sugars, in a saccharide, a two step reduction is recommended using sodium borohydride in the second step.
Note! Kdo gives a 1:1 mixture of 3-deoxy-D-glycero-D-galacto- and 3-deoxy-D-glycero-D-talo-octono-1,4-lactones.

Flow scheme

Hydrolysis/Reduction BH4-reduction Acetylation

Reagents

Full list of chemicals

Procedure

  1. Reduction of the acid labile sugar. The sugar (1 equivalent) and morpholinoborane (10 eq.) in 0.5 M TFA is kept at 100° for 2h.
    E.g. 1 mg hexose (6 µmol) requires 7 mg morpholinoborane (60 µmol).
  2. The solution is evaporated to dryness and after addition of methanol again evaporated to dryness.
  3. Reduce with 0.3 mL fresh solution of NaBH4 in NH3 for 30 min at 20°.
  4. 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.
  5. Acetylate with TFA/Ac2O (0.2+0.2 mL) 10 min 100°.
  6. Partition between dichloromethane and water.

Test substances, Standards

Test your reagents with stachyose, aDGal(1→6)aDGal(1→6)aDGlc(1→2)bDFruf, which should give galactose, glucose and mannose (glucose and mannose from Fruf).