Table of Contents

Smith degradation

Introduction

The Smith degradation aims at selectively degrading a polysaccharide to either a polysaccharide with a smaller repeat or an oligosaccharide, from which structural information can be deduced. The procedure involves three steps, oxidation with periodate (IO4-), reduction to a polyalcohol with borohydride (BH4-) followed by hydrolysis with dilute acid under mild conditions, normally room temperature. During the hydrolysis step only acetals of oxidised sugar rings are supposed to be cleaved. The product may range from a monosaccharide glycoside to a polysaccharide. The following molecular fragments are susceptible to oxidation.

The following products are obtained from a terminal, a 2-substituted, a 4-substituted, a 6-substituted glucose residue after oxidation, reduction and mild acid hydrolysis.

A complete Smith degradation for a fictive polysaccharide would thus be

Reagents

Full list of chemicals

Procedure

  1. Dissolve the polysaccharide (10-50 mg) in acetate buffer, ca 1 mg/mL
  2. Add NaIO4 to a final concentration of 0.03M, leave in the dark (Al foil) in a refrigerator (4°C) for 72h.
  3. Destroy excess NaIO4 with ethylene glycol (100 µL per 10 µL buffer solution) leave for 2 h and put in dialysis bag under running tap water for >6h. Concentrate if diluted too much.
  4. Reduce the oxidised product with NaBH4 (3 mg per mg polysaccharide), leave for 3h, make pH 7 with 10% aqueous HOAc, dialyse under tap water and then distilled water. Evaporate to a small volume.
  5. Optional: check by methylation analysis that all periodate susceptible residues are oxidised. If not, do the oxidation-reduction sequence once more.
  6. Make the solution 0.5M with respect to CF3CO2H by adding the appropriate volume of acid. Leave for 24h at room temperature.
  7. Evaporate most of the acid, and make pH 7 by NaOH, purify by chromatography on Bio-Rad P2

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