Oligodeoxynucleotides Containing N1‐Methyl‐2′‐Deoxyadenosine and N6‐Methyl‐2′‐Deoxyadenosine

Sergey N. Mikhailov1, Edward N. Timofeev1, Mikhail S. Drenichev1, Ekaterina V. Efimtseva1, Piet Herdewijn2, Eric B. Roesch3, Marc M. Lemaitre4

1 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia, 2 Rega Instituut, Leuven, Belgium, 3 Glen Research Corporation, Sterling, Virginia, 4 ML‐Consulting, Purcellville, Virginia
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.36
DOI:  10.1002/0471142700.nc0436s38
Online Posting Date:  September, 2009
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes a simple and efficient synthesis of the phosphoramidite derivative of N1‐methyl‐2′‐deoxyadenosine from 2′‐deoxyadenosine. The synthesis starts with the monomethoxytritylation of 2′‐deoxyadenosine followed by methylation of 5′‐O‐protected nucleoside at N‐1. Subsequent N‐chloroacetylation leads to N6‐chloroacetyl‐N1‐methyl‐5′‐O‐(p‐anisyldiphenylmethyl)‐2′‐deoxyadenosine, which is finally converted to its 3′ phosphoramidite derivative. This phosphoramidite is used to incorporate N1‐methyl‐2′‐deoxyadenosine into synthetic oligonucleotides. N‐Chloroacetyl protection and controlled anhydrous deprotection conditions are used to avoid the Dimroth rearrangement. Curr. Protoc. Nucleic Acid Chem. 38:4.36.1‐4.36.19. © 2009 by John Wiley & Sons, Inc.

Keywords: Oligonucleotides; N1‐methyl‐2′‐deoxyadenosine; chloroacetyl protecting group; Dimroth rearrangement

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Protected N1‐Methyl‐2′‐Deoxyadenosine
  • Basic Protocol 2: Synthesis, Purification, and Characterization of Oligonucleotides Containing m1dA
  • Basic Protocol 3: Preparation of Oligodeoxynucleotides Containing N6‐Methyl‐2′‐Deoxyadenosine
  • Basic Protocol 4: Analysis of m1dA‐ and m6dA‐Modified Oligonucleotides
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of Protected N1‐Methyl‐2′‐Deoxyadenosine

  Materials
  • 2′‐deoxyadenosine monohydrate ( S.1; Acros)
  • Anhydrous pyridine (C 5H 5N)
  • Balloon of nitrogen or argon
  • Monomethoxytrityl chloride (MMTrCl)
  • Methanol (MeOH), reagent grade
  • Methylene chloride (CH 2Cl 2), reagent grade
  • Saturated solution of sodium bicarbonate (NaHCO 3)
  • Sodium sulfate anhydrous (Na 2SO 4)
  • Toluene, reagent grade
  • Silica gel (Kieselgel 60, 0.06‐ to 0.20‐µm, Merck)
  • Silica gel blue (Fluka)
  • N,N‐dimethylacetamide (DMA), reagent grade
  • Methyl iodide (MeI)
  • Sodium thiosulfate pentahydrate (Na 2S 2O 3), reagent grade
  • Diethyl ether (Et 2O), reagent grade
  • Hexane, reagent grade
  • Chloroacetic anhydride, 97% (Acros)
  • 2 M ammonia in methanol (NH 3/MeOH)
  • 1H‐Tetrazole, 99%
  • 4‐Å molecular sieves
  • Anhydrous acetonitrile
  • Bis(N,N‐diisopropylamino)‐2‐cyanoethoxyphosphine, 98% pure (Fluka)
  • Ethyl acetate (EtOAc), reagent grade
  • Triethylamine (Et 3N), reagent grade
  • 50‐, 250‐, and 500‐mL round‐bottom flasks with stoppers
  • Rotary evaporator equipped with a water aspirator
  • Magnetic stir bars
  • Magnetic plate
  • TLC‐plates: silica‐coated aluminum plates with fluorescent indicator (Merck silica gel 60F 254)
  • 254‐nm UV lamp
  • 250‐mL separatory funnel
  • 3 × 20–cm sintered glass chromatography columns, porosity 3
  • Vacuum oil pump
  • Stainless‐steel spatula
  • Vacuum desiccator
  • Glass filter (porosity 3)
  • Pasteur pipets
  • Additional reagents and equipment for thin‐layer chromatography ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Synthesis, Purification, and Characterization of Oligonucleotides Containing m1dA

  Materials
  • N1‐methyl‐2′‐deoxyadenosine phosphoramidite S.5
  • Mild deprotection 2′‐deoxyribonucleoside phosphoramidites: dAPac, dGiPr‐Pac, dCAc, T (Glen Research)
  • Acetonitrile, anhydrous (amidite‐diluent grade; Glen Research)
  • 4‐Å molecular sieve beads
  • Reagents for oligonucleotide synthesis (Glen Research):
    • Activator
    • UltraMild cap mix A
    • Cap mix B
    • 0.02 M oxidizing solution
    • Deblocking mix
  • Mild deprotection synthesis columns (CPG 500 or dT LV‐200; Glen Research)
  • Dry argon or nitrogen source
  • 2 M ammonia in methanol (Aldrich, cat. no. 341428 or equivalent)
  • Buffer B: 50% CH 3CN in 0.05 M TEAA, pH 7.0
  • 2% trifluoroacetic acid (TFA)/water (Glen Research, cat. no. 60‐4040‐57)
  • Triethylamine
  • 2% LiClO 4 in acetone
  • Acetone
  • Buffer A: 0.05 M TEAA, pH 7.0
  • HPLC‐grade acetonitrile
  • 2.0 M triethylamine acetate, pH 7 (TEAA; Glen Research)
  • ABI model 394 automated DNA synthesizer
  • 10‐mL Luer‐tipped plastic syringes
  • Luer‐to‐tubing connector
  • 3‐mL conical glass vial (3‐mL Reacti‐Vial; Pierce/Thermo Fisher, cat. no. 13222) with Teflon caps
  • 1.5‐mL centrifuge tubes
  • 1.0‐mL pipets or Pasteur pipets
  • Hypersil ODS columns (5‐mm; 4.6 × 250–mm)
  • HPLC system with UV or UV/PDA‐detector
  • SpeedVac evaporator
  • Vortex
  • 0.2‐µm PVDF filters
  • UV‐spectrophotometer

Basic Protocol 3: Preparation of Oligodeoxynucleotides Containing N6‐Methyl‐2′‐Deoxyadenosine

  Materials
  • N1‐methyl‐2′‐deoxyadenosine phosphoramidite S.5
  • Acetonitrile, anhydrous
  • 4‐Å molecular sieve beads (2.4‐ to 4.8‐µm diameter; Fluka)
  • Appropriate solid support–filled column
  • Reagents for oligonucleotide synthesis (Glen Research):
    • Activator
    • Cap mix A
    • Cap mix B
    • Oxidizing solution
    • Deblocking mix
  • Standard 2′‐deoxyribonucleoside (dAbz, dGibu, dCbz, T) phosphoramidites (Glen Research)
  • Mobile phase B: 50% CH 3CN in 0.05 M triethylammonium acetate (TEAA), pH 7.0
  • Mobile phase A: 0.05M TEAA, pH 7.0
  • Trifluoroacetic acid
  • Triethylamine
  • 2% LiClO 4 solution in acetone
  • Acetone
  • ABI 3400 synthesizer
  • Synthesizer vials with screw caps
  • 55°C heating block
  • 1.5‐mL polypropylene microcentrifuge tubes
  • SpeedVac evaporator with water aspirator pump
  • 1‐mL plastic syringes
  • Syringe‐tip filter units with PVDF membranes (0.2‐µm pore size, 13‐mm diameter; Supelco)
  • Hypersil ODS HPLC column (5 µm; 4 × 250–mm)
  • HPLC system with UV‐detector
  • Vortex
  • UV‐spectrophotometer

Basic Protocol 4: Analysis of m1dA‐ and m6dA‐Modified Oligonucleotides

  Materials
  • Oligonucleotides with m1dA or m6dA modifications (see Basic Protocols protocol 22 and protocol 33)
  • 50 mM Tris⋅Cl (pH 7.5)/50 mM NaCl/7 mM MgCl 2
  • Snake venom phosphodiesterase (SVPDE, Amersham)
  • 0.5 M Tris⋅Cl, pH 9.0 ( appendix 2A)
  • Shrimp alkaline phosphatase (Promega)
  • Mobile phase B: 50% CH 3CN in 0.05 M triethylammonium acetate (TEAA), pH 7.0
  • Mobile phase A: 0.05 M triethylammonium acetate (TEAA), pH 7.0
  • 37°C thermostat
  • PVDF membrane (13‐mm diameter, 0.2‐µm pore size; Supelco)
  • Polypropylene 1.5‐mL microcentrifuge tubes
  • Hypersil ODS HPLC column (5 µm; 4 × 250–mm)
  • HPLC system with UV‐detector
  • MALDI‐TOF mass spectrometer
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Agris, P.F. 1996. The importance of being modified: Roles of modified nucleosides and Mg2+ in RNA structure and function. Prog. Nucleic Acid Res. Mol. Biol. 53:79‐129.
   Delaney, J.C. and Essigmann, J.M. 2004. Mutagenesis, genotoxicity, and repair of 1‐methyladenine, 3‐alkylcytosines. Proc. Natl. Acad. Sci. U.S.A. 101:14051‐14056.
   Jones, J.W. and Robins, R.K. 1963. Purine nucleosides. III. Methylation studies of certain naturally occurring purine nucleosides. J. Am. Chem. Soc. 85:193‐201.
   Helm, M., Giegé, R., and Florentz, C. 1999. A Watson‐Crick base‐pair‐disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNA. Biochemistry 38:13338‐13346.
   Höbartner, C. and Silverman, S.K. 2007. Recent advances in DNA catalysis. Biopolymers 87:279‐292.
   Kierzek, E. and Kierzek, R. 2003. The synthesis of oligoribonucleotides containing N6‐alkyladenosines and 2‐methylthio‐N6‐alkyladenosines via post‐synthetic modification of precursor oligomers. Nucleic Acids Res. 31:4461‐4471.
   Macon, J.B. and Wolfenden, R. 1968. 1‐Methyladenosine. Dimroth rearrangement and reversible reduction. Biochemistry 7:3453‐3458.
   Mikhailov, S.N., Rozenski, J., Efimtseva, E.V., Busson, R., Van Aerschot, A., and Herdewijn, P. 2002. Chemical incorporation of 1‐methyladenosine into oligonucleotides. Nucleic Acids Res. 30:1124‐1131.
   Mikhailov, S.N., Efimtseva, E.V., Rodionov, A.A., Shelkunova, A.A., Rozenski, J., Emmerechts, G., Schepers, G., Van Aerschot, A., and Herdewijn, P. 2005. Synthesis of RNA containing O‐β‐D‐ribofuranosyl‐(1′′‐2′)‐adenosine‐5′′‐O‐phosphate and 1‐methyladenosine, minor components of tRNA. Chem. Biodivers. 2:1153‐1163.
   Mishina, Y., Duguid, E.M., and He, C. 2006. Direct reversal of DNA alkylation damage. Chem. Rev. 106:215‐232
   Ratel, D., Ravanat, J.L., Berger, F., and Wion, D. 2006a. N6‐methyladenine: The other methylated base of DNA. Bioessays 28:309‐315.
   Ratel, D., Ravanat, J.L., Charles, M.P., Platet, N., Breuillaud, L., Lunardi, J., Berger, F., and Wion, D. 2006b. Undetectable levels of N6‐methyl adenine in mouse DNA: Cloning and analysis of PRED28, a gene coding for a putative mammalian DNA adenine methyltransferase. FEBS Lett. 580:3179‐3184.
   Reese, C.B., van Boom, J.H., de Leeuw, H.P.M., Nagel, J., and de Rooy, J.F.M. 1975. Synthesis of oligoribonucleotides preparation of ribonucleoside 2′‐acetal 3′‐esters by selective deacylation. J. Chem. Soc. Perkin Trans. I. 1:934‐942.
   Sedgwick, B. 2004. Repairing DNA‐methylation damage. Nature Rev. Mol. Cell. Biol. 5:148‐157.
   Silverman, S.K. 2008. Catalytic DNA (deoxyribozymes) for synthetic applications‐current abilities and future prospects. Chem. Commun. 30:3467‐3485.
   Takeuchi, Y., Tazawa, I., and Inoue, Y. 1982. Intramolecular stacking association of three dinucleoside monophosphates containing naturally‐occurring 1‐methyladenosine residue(s): m1ApA, Apm1A and m1Apm1A. Bull. Chem. Soc. Japan 55:3598‐3602.
   Ti, G.S., Gaffney, B.L., and Jones, R.A. 1982. Transient protection: Efficient one‐flask syntheses of protected deoxynucleosides. J. Am. Chem. Soc. 104:1316‐1319.
   Timofeev, E.N., Mikhailov, S.N., Zuev, A.N., Efimtseva, E.V., Herdewijn, P., Somers, R.L., and Lemaitre, M.M. 2007. Oligodeoxynucleotides containing 2′‐deoxy‐1‐methyladenosine and Dimroth rearrangement. Helv. Chim. Acta 90:928‐937.
   Voigts‐Hoffmann, F., Hengesbach, M., Kobitski, A., Van Aerschot, A., Herdewijn, P., Nienhaus, G.U., and Helm, M. 2007. A Methyl group controls conformational equilibrium in human mitochondrial tRNA Lys. J. Am. Chem. Soc. 129:13382‐13383.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library