Stereoselective Synthesis of Sugar Nucleotides Using Neighboring Group Participation

Shannon C. Timmons1, David L. Jakeman1

1 Dalhousie University, Halifax, Nova Scotia, Canada
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.7
DOI:  10.1002/0471142700.nc1307s31
Online Posting Date:  December, 2007
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A straightforward, efficient method for the chemical synthesis of sugar nucleotides derived from D‐mannose and L‐fucose precursors is described. This synthetic strategy involves the coupling of acylated glycosyl bromides with nucleoside 5′‐diphosphates, which enables the exploitation of neighboring group participation to exclusively prepare diastereomerically pure sugar nucleotides of desired 1,2‐trans anomeric configuration. This is the first stereoselective direct coupling approach to sugar nucleotide synthesis. Following deprotection using triethylamine and purification via C18 reversed‐phase ion‐pair chromatography, UDP‐ and GDP‐α‐D‐mannose as well as UDP‐ and GDP‐β‐L‐fucose were obtained in good yield in only four synthetic steps from D‐mannose and L‐fucose. Curr. Protoc. Nucleic Acid Chem. 31:13.7.1‐13.7.16. © 2007 by John Wiley & Sons, Inc.

Keywords: sugar nucleotides; glycosylation; glycosyl bromides; nucleoside 5′‐diphosphates; neighboring group participation; glycosyltransferases; oligosaccharides; glycoconjugates

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • D‐Mannose (S.1), ≥99% pure (Biochemical; for S.2)
  • Pyridine (py), ReagentPlus, ≥99% pure (Aldrich)
  • L‐Fucose (S.6), ≥99% pure (Sigma; for S.7)
  • Acetic anhydride (Ac 2O), ≥99% pure (Fluka; for S.2)
  • Benzoyl chloride (BzCl), 99% pure (Aldrich; for S.7)
  • Ethyl acetate (EtOAc), technical grade
  • Hexane, technical grade
  • Potassium permanganate (KMnO 4) TLC visualization solution (see recipe)
  • Dichloromethane (CH 2Cl 2), technical grade
  • 1 M aqueous hydrochloric acid (HCl)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Saturated aqueous sodium chloride (NaCl)
  • Sodium sulfate (Na 2SO 4)
  • Phosphorus tribromide (PBr 3), 97% pure (Aldrich)
  • Amberlite IR‐120 PLUS(H) ion‐exchange resin, free acid form (Alfa Aesar)
  • Methanol (MeOH), HPLC grade
  • Tetrabutylammonium hydroxide 30‐hydrate (Bu 4NOH), ≥98.0% pure (Fluka)
  • Uridine 5′‐diphosphate, sodium salt from Saccharomyces cerevisiae, 95% to 100% pure (Sigma; for S.4 and S.9)
  • Guanosine 5′‐diphosphate, sodium salt from Saccharomyces cerevisiae, ≥96% pure (Sigma; for S.5 and S.10)
  • Deuterium oxide (D 2O)
  • 3‐Å molecular sieves, 4‐ to 8‐mesh beads (Sigma)
  • Anhydrous acetonitrile (MeCN), 99.8% pure (Aldrich)
  • Nitrogen (or argon) gas, ultra high purity 5.0
  • Anhydrous triethylamine (Et 3N), 99.5% pure (Aldrich)
  • Triethylamine (Et 3N), ≥99.5% pure (Fluka)
  • Alkaline phosphatase, from calf intestine, buffered aqueous glycerol solution (Sigma)
  • 25 mM Tris·Cl buffer, pH 7.5 ( appendix 2A)
  • Tributylamine, ≥99.5% pure (Fluka)
  • HPLC buffer A: aqueous 12 mM tetrabutylammonium bromide (Bu 4NBr, 99% pure, Aldrich), 10 mM KH 2PO 4, and 5% (v/v) HPLC‐grade MeCN (pH 4)
  • HPLC buffer B: HPLC‐grade MeCN
  • Dry ice, crushed or pelleted
  • HPLC buffer C: 10 mM aqueous tributylammonium bicarbonate (TBAB) buffer, pH 6
  • HPLC buffer D: HPLC‐grade MeOH
  • Dowex 50W‐X8 cation‐exchange resin, Na+ form, 100‐ to 200‐mesh (Bio‐Rad)
  • Round‐bottom flasks (oven‐dried for condensation reaction) with rubber septa
  • Thin‐layer chromatography (TLC) plates: 250‐µm silica‐coated glass‐backed plates (SiliCycle)
  • 254‐nm UV lamp
  • Heat gun
  • Rotary evaporator equipped with a water aspirator
  • Vacuum/nitrogen (or argon) gas manifold
  • Disposable needle
  • Buchner funnel (fitted with a filter paper of coarse porosity)
  • Suction flask connected to water aspirator by vacuum tubing
  • Glass econo‐column chromatography columns (1.5 × 30–cm; Bio‐Rad)
  • Lyophilizer
  • Oil bath
  • Three‐way connector with septum
  • Gas bubbler
  • Double‐ended needle (cannula)
  • Condenser
  • Hot plate/stirrer equipped with temperature feedback probe
  • Sintered funnel with a coarse filter connected to a water aspirator
  • Agilent Zorbax 5‐µm Rx‐C18 (150 cm × 4.6–mm) column
  • 1‐L suction flask with rubber stopper and plastic tubing
  • Biotage SP1 automated flash chromatography instrument and 25M C18 column (15 × 25–mm)
  • Additional reagents and equipment for TLC ( appendix 3D), HPLC (unit 10.5), and reversed‐phase chromatography (unit 10.5)
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Literature Cited

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