Table of Contents

Determination of the Absolute Configuration - using silylated butyl glycosides

Introduction

The old way of determining the absolute configuration of sugars was to hydrolyse the polysaccharide, separate the monosaccharides by paper chromatography and then to measure the optical rotation for each of them. The sign of the rotation, compared to references, gave the absolute configuration. Normally, a positive rotation meant a D-sugar. Today, the sugars are reacted with an optically active alcohol to become one of two possible diastereoisomers.

Procedure

  1. Transfer 2 mg of dry polysaccharide or prehydrolyzed polysaccharide to a 13 x 100 mm screw-cap tube. If prehydrolyzed sample is used skip the hydrolysis and re-N-acetylate directly.
  2. Dissolve in 0.5 mL dry methanol. Add 35 µL of acetyl chloride, and bubble nitrogen through the solution for 30 sec and seal the tube.
  3. Solvolyze the sample for 24 h at 85°C.
  4. Evaporate the supernatant solution with a stream of nitrogen or air.
  5. If the polysaccharide contains aminosugars, re-N-acetylate with 25 µL of acetic anhydride for 4h at 20 °C. Evaporate with a stream of nitrogen or air.
  6. Dissolve the methyl glycosides in 0.2 mL of (+)-2-butanol. Add 15 µL of acetyl chloride, and bubble nitrogen through the solution for 30 sec and seal the tube.
  7. Butanolyze the sample for 8 h at 80 °C.
  8. If the polysaccharide contains aminosugars, re-N-acetylate with 25 µL of acetic anhydride for 4h at 20 °C. Evaporate with a stream of nitrogen or air.
  9. Trimethylsilylate at 20 °C for 30 min with 100 µL of Sigma SilA solution.
  10. Evaporate the solution with a stream of nitrogen and redissolve the sample in n-hexane. Filter the solution to a sample tube before injection.

References