A Simple Chemical Synthesis of Sugar Nucleoside Diphosphates in Water

Hidenori Tanaka1, Yayoi Yoshimura1, Ole Hindsgaul1

1 Carlsberg Laboratory, Copenhagen, Denmark
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.12
DOI:  10.1002/0471142700.nc1312s54
Online Posting Date:  October, 2013
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Abstract

Chemoenzymatic oligosaccharide synthesis is attractive since it eliminates the tedious multistep protection‐deprotection requirements of pure chemical synthesis. Chemoenzymatic synthesis using glycosyltransferases, however, requires not only the correct enzyme to control both regio‐ and stereospecificity, but also the glycosyl donor to provide the sugar that is added. This unit describes a simple synthesis of sugar‐nucleoside diphosphates (sugar‐NDPs), the type of glycosyl donor (e.g., UDP‐Glc, UDP‐Gal, ADP‐Glc) required by most glycosyltransferases, by using a chemical coupling reaction in water. The preparation of sugar‐NDPs by this method therefore does not require any skills in synthetic organic chemistry. Curr. Protoc. Nucleic Acid Chem. 54:13.12.1‐13.12.10. © 2013 by John Wiley & Sons, Inc.

Keywords: chemical synthesis; chemoenzymatic oligosaccharide synthesis; glycosyl‐transferase; pyrophosphate formation; sugar‐nucleoside diphosphate; sugar nucleotide

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Sugar‐NDPs in Water (D2O) Using ImIm Reagent
  • Basic Protocol 2: Use of Synthetic Crude UDP‐Gal as a Donor‐Substrate in a Galactosyltransferase Reaction
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Sugar‐NDPs in Water (D2O) Using ImIm Reagent

  Materials
  • 2‐Chloro‐1,3‐dimethylimidazolinium chloride (DMC, Sigma‐Aldrich)
  • Imidazole (Sigma‐Aldrich)
  • Deuterium oxide (D 2O, D: 99.9%, Cambridge Isotope Laboratories)
  • NMP disodium salt:
    • Uridine 5′‐monophosphate disodium salt (UMP, 22% water content, Carbosyth)
    • Adenosine 5′‐monophosphate disodium salt (AMP, 21% loss on drying, Carbosyth)
    • Guanosine 5′‐monophosphate disodium salt hydrate (GMP, 22% loss on drying, Sigma‐Aldrich)
  • Sugar 1‐phosphate:
    • α‐D‐Glucose 1‐phosphate dipotassium salt (Glc 1‐P, 12% water content, Carbosyth)
    • α‐D‐Galactose 1‐phosphate dipotassium salt pentahydrate (Gal 1‐P, Sigma‐Aldrich)
    • α‐D‐Mannose 1‐phosphate dipotassium salt (Man 1‐P, 0.02% loss on drying, Carbosyth)
  • 50 mM Tris(hydroxylmethyl)aminomethane hydrogen chloride (Tris·Cl) buffer, pH 8.0
  • 20 U/µL calf intestinal alkaline phosphatase (AP, Life Technologies)
  • MilliQ water
  • DEAE Sephacel ion‐exchange column (2.6 cm × 16 cm; GE Healthcare)
  • 30 and 400 mM ammonium acetate aqueous solution
  • 1.5‐mL microcentrifuge tubes
  • Magnetic stir bar
  • Magnetic stirrer
  • 15‐mL centrifuge tubes
  • 30°C incubator
  • ESI‐MS
  • 500‐mL round‐bottom flasks
  • Rotary evaporator
  • Lyophilizer

Basic Protocol 2: Use of Synthetic Crude UDP‐Gal as a Donor‐Substrate in a Galactosyltransferase Reaction

  Materials
  • 2‐Chloro‐1,3‐dimethylimidazolinium chloride (DMC, Sigma‐Aldrich)
  • Imidazole (Sigma‐Aldrich)
  • Uridine 5′‐monophosphate disodium salt (UMP, 22% water content, Carbosyth)
  • Deuterium oxide (D 2O, D: 99.9%, Cambridge Isotope Laboratories)
  • α‐D‐Galactose 1‐phosphate dipotassium salt pentahydrate (Gal 1‐P, Sigma‐Aldrich)
  • MilliQ water
  • GlcNAc‐TMR (Zhang et al., )
  • 4‐Morpholinepropanesulfonic acid (MOPS, Sigma)
  • Manganese (II) chloride tetrahydrate (MnCl 2, Sigma)
  • Bovine serum albumin (BSA, Sigma)
  • 0.65 µg/µL bovine β‐1,4‐galactosyltransferase (β‐1,4‐GalT, Sigma‐Aldrich)
  • Chloroform (CHCl 3, LAB‐SCAN)
  • Methanol (MeOH, LAB‐SCAN)
  • 2,5‐Dihydroxybenzoic acid (DHB, Sigma‐Aldrich)
  • Acetonitrile (ACN, LAB‐SCAN)
  • Trifluoroacetic acid (TFA, Sigma‐Aldrich)
  • 1.5‐mL microcentrifuge tubes
  • Magnetic stir bar and stirrer
  • 37°C incubator
  • Silica TLC plate (Merck)
  • Sep‐Pak C18 Plus Light Cartridge (Waters)
  • 20‐mL plastic syringe (Beckton Dickinson) connected to the Sep‐pak cartridge
  • Rotary evaporator (BUCHI) connected to a vacuum pump (KNF Lab)
  • Lyophilizer
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Figures

Videos

Literature Cited

Literature Cited
  Cramer, F., Schaller, H., and Staab, H.A. 1961. Darstellung von Imidazoliden der Phosphorsäure. Chem. Ber. 94:1612‐1621.
  Isobe, T. and Ishikawa, T. 1999. 2‐Chloro‐1,3‐dimethylimidazolinium chloride. 1. A powerful dehydrating equivalent to DCC. J. Org. Chem. 64:6984‐6988.
  Mohamady, S. and Taylor, S.D. 2012. Rapid and efficient synthesis of nucleoside polyphosphates and their conjugates using sulfonyl imidazolium salts. Curr. Protoc. Nucl. Acid Chem. 51:13.11.1‐13.11.24.
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  Ramakrishna, B., Shah, P.S., and Qasba, P.K. 2001. α‐Lactalbumin (LA) stimulates milk β‐1,4‐galactosyltransferase I (β4Gal‐T1) to transfer glucose from UDP‐glucose to N‐acetylglucosamine: Crystal structure of β4Gal‐T1·LA complex with UDP‐Glc. J. Biol. Chem. 276:37665‐37671.
  Schmaltz, R.M., Hanson, S.R., and Wong, C.‐H. 2011. Enzymes in the synthesis of glycoconjugates. Chem. Rev. 111:4259‐4307.
  Simon, E.S., Grabowski, S., and Whitesides, G.M. 1990. Convenient syntheses of cytidine 5′‐triphosphate, guanosine 5′‐triphosphate, and uridine 5′‐triphosphate and their use in the preparation of UDP‐glucose, UDP‐glucuronic acid, and GDP‐mannose. J. Org. Chem. 55:1834‐1841.
  Tanaka, H., Yoshimura, Y., Jørgensen, M.R., Cuesta‐Seijo, J.A., and Hindsgaul, O. 2012. A simple synthesis of sugar nucleoside diphosphates by chemical coupling in water. Angew. Chem. Int. Ed. 51:11531‐11534.
  Tanaka, T., Huang, W.C., Noguchi, M., Kobayashi, A., and Shoda, S. 2009a. Direct synthesis of 1,6‐anhydro sugars from unprotected glycopyranoses by using 2‐chlror‐1,3‐dimethylimidazolium chloride. Tetrahedron Lett. 50:2154‐2157.
  Tanaka, T., Nagai, H., Noguchi, M., Kobayashi, A., and Shoda, S. 2009b. One‐step conversion of unprotected sugars to β‐glycosyl azides using 2‐chloroimidazolinium salt in aqueous solution. Chem. Commun. 23:3378‐3379.
  Tsukamoto, H. and Kahne, D. 2011. N‐Methyl‐imidazolium chloride‐catalyzed pyrophosphate formation: Application to the synthesis of lipid I and NDP‐sugar donors. Bioorg. Med. Chem. Lett. 21:5050‐5053.
  Wagner, G.K., Pesnot, T., and Field, R.A. 2009. A survey of chemical methods for sugar‐nucleotide synthesis. Nat. Prod. Rep. 26:1172‐1194.
  Wittmann, V. and Wong, C.‐H. 1997. 1H‐Tetrazole as catalyst in phosphomorpholidate coupling reactions: Efficient synthesis of GDP‐fucose, GDP‐mannose, and UDP‐galactose. J. Org. Chem. 62:2144‐2147.
  Zhang, Y., Le, X., Dovichi, N.J., Compston, C.A., Palcic, M.M., Diedrich, P., and Hindsgaul, O. 1995. Monitoring biosynthetic transformations of N‐acetyllactosamine using fluorescently labeled oligosaccharides and capillary electrophoretic separation. Anal. Biochem. 227:368‐376.
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