Chemical Synthesis of Dinucleotide Cap Analogs

Anilkumar R. Kore1, Muthian Shanmugasundaram1

1 Bioorganic Chemistry Division, Life Technologies Corporation, Austin, Texas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.13
DOI:  10.1002/0471142700.nc1313s55
Online Posting Date:  December, 2013
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Abstract

This unit describes a reliable, efficient and general method for the synthesis of standard cap analog (mCAP), m7G[5′]ppp[5′]G, and anti–reverse cap analog (ARCA), m7,3′OG[5′]ppp[5′]G. The synthesis of required intermediate m7GDP or m27,3′OGDP has been achieved through regioselective methylation of the corresponding diphosphate using dimethyl sulfate under aqueous conditions. Then, the coupling reaction of m7GDP or m27,3′OGDP with ImGMP using ZnCl2/DMF system affords the corresponding cap analog in good yields. Curr. Protoc. Nucleic Acid Chem. 55:13.13.1‐13.13.12. © 2013 by John Wiley & Sons, Inc.

Keywords: cap analog; anti–reverse cap analog; regioselective methylation; eukaryotic mRNA; mRNA translation

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Guanosine 5′‐O‐monophosphate disodium salt hydrate from yeast, ≥99% (Aldrich)
  • Ammonium hydroxide (28% NH 3 in water; Aldrich)
  • DEAE Sepharose (GE Healthcare)
  • 2 M sodium chloride (NaCl; see recipe)
  • HPLC mobile phase A: 5 mM ammonium phosphate monobasic, pH 3.8 (see recipe)
  • HPLC mobile phase B: 750 mM ammonium phosphate monobasic, pH 3.7 (see recipe)
  • Imidazole (Aldrich)
  • Aldrithiol (Aldrich)
  • Triphenyl phosphine (Aldrich)
  • Triethylamine (Aldrich)
  • N,N‐Dimethylformamide (DMF), anhydrous 99.8% (Aldrich)
  • Argon gas cylinder
  • Sodium perchlorate (Aldrich)
  • Acetone
  • Guanosine 5′‐O‐diphosphate sodium salt (Aldrich)
  • Acetic acid
  • Dimethyl sulfate, ≥99.0% (Aldrich)
  • 1.0 M NaOH
  • Dichloromethane, technical grade (DCM; Aldrich)
  • Zinc chloride, anhydrous beads, 99.99% (Aldrich)
  • Ethylenediamine tetraacetic acid (EDTA) disodium salt, dihydrate, EDTA (Aldrich)
  • 3′‐O‐Me guanosine 5′‐O‐monophosphate free acid (Chemgenes)
  • Tris(tributylammonium) phosphate (Aldrich)
  • 4‐L conical flasks
  • pH meter (Thermo Fisher)
  • 10‐cm × 78.5–cm chromatography column
  • FPLC ÄKTA purifier (GE Healthcare)
  • Programmable gradient pump system
  • UV detector ranging from 254 to 280 nm
  • HPLC system (Waters) including:
    • Detector module
    • Hypersil SAX column (4.6 mm × 25 cm)
  • Rotary evaporator
  • High‐vacuum pump
  • 250‐mL round‐bottom flasks, oven dried (Chemglass)
  • Magnetic stirrer (VWR)
  • Teflon‐coated magnetic stirring bars (oval shaped, Aldrich)
  • Rubber septa for 24/40‐glass joints (Chemglass)
  • Vacuum/nitrogen (or argon) gas manifold
  • 500‐mL centrifuge bottles
  • Sorvall RC‐3B centrifuge
  • 5‐mL sealed glass syringe (Aldrich)
  • Disposable needles
  • 1‐L separatory funnel
  • Additional reagents and equipment for proton nuclear magnetic resonance (1H NMR and 31P NMR), and mass spectrometry.
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Figures

Videos

Literature Cited

Literature Cited
  Hata, T., Nakagawa, I., and Shimotohno, K. 1976. The synthesis of α,γ‐dinucleoside triphosphates. The confronted nucleotide structure found at the 5′‐terminus of eukaryote messenger ribonucleic acid. Chem. Lett.987‐990.
  Jemielity, J., Fowler, T., and Zuberek, J. 2003. Novel “anti‐reverse” cap analogs with superior translational properties. RNA9:1108‐1122.
  Kadokura, M., Wada, T., and Urashima, C. 1997. Efficient synthesis of γ‐methyl‐capped guanosine 5′‐triphosphate as a 5′‐terminal unique structure of U6 RNA via a new triphosphate bond formation involving activation of methyl phosphorimidazolidate using ZnCl2 as a catalyst in DMF under anhydrous conditions. Tetrahedron Lett. 38:8359‐8362.
  Kore, A.R., Charles, I., and Shanmugasundaram, M. 2008. Recent Developments in 5‐Terminal cap analogs: Synthesis and biological ramifications. Mini‐Rev. Org. Chem.5:179‐192.
  Kore, A.R., Charles, I., and Shanmugasundaram, M. 2010. Organic synthesis and improved biological properties of modified mRNA cap analogs. Curr. Org. Chem.14:1083‐1098.
  Mikkola, S., Salomaeki, S., and Zhang, Z. 2005. Preparation and properties of mRNA 5′‐cap structure. Curr. Org. Chem.9:999‐1022.
  Niedzwiecka, A., Marcotrigiano, J., and Stepinski, J. 2002. Biophysical studies of eIF4E cap‐binding protein: Recognition of mRNA 5′‐cap structure and synthetic fragments of eIF4G and 4E‐BP1 proteins. J. Mol. Biol.319:615‐635.
  Pasquinelli, A.E., Dahlberg, J.E., and Lund, E. 1995. Reverse 5′‐caps in RNAs made in vitro by phage RNA polymerases. RNA1:957‐967.
  Peng, Z.H., Sharma, V., and Singleton, S.F. 2002. Synthesis and application of a chain‐terminating dinucleotide mRNA cap analog. Org. Lett.4:161‐164.
  Sawai, H., Wakai, H., and Shimazu, M. 1991. Facile synthesis of cap portion of messenger RNA by Mn(II) ion‐catalyzed pyrophosphate formation in aqueous solution. Tetrahedron Lett.32:6905‐6906.
  Sawai, H., Shimazu, M., and Wakai, H. 1992. Divalent metal ion‐catalyzed pyrophosphate bond formation in aqueous solution: Synthesis of nucleotides containing polyphosphate. Nucleosides Nucleotides Nucleic Acids11:773‐785.
  Sawai, H., Wakai, H., and Nakamura‐Ozaki, A. 1999. Synthesis and reactions of nucleoside 5′‐diphosphate imidazolide. A nonenzymatic capping agent for 5′‐monophosphorylated oligoribonucleosides in aqueous solution. J. Org. Chem.64:5836‐5840.
  Stepinski, J., Waddell, C., and Stolarski, R. 2001. Synthesis and properties of mRNAs containing the novel “anti‐reverse” cap analogs 7‐methyl(3′‐O‐methyl)GpppG and 7‐methyl (3′‐deoxy)GpppG. RNA7:1486‐1495.
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