Synthesis of Nonnatural Oligonucleotides Made Exclusively of Alkynyl C‐Nucleosides with Nonnatural Bases

Junya Chiba1, Masahiko Inouye1

1 Graduate School of Pharmaceutical Sciences, University of Toyama, Toyama
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.62
DOI:  10.1002/0471142700.nc0462s61
Online Posting Date:  June, 2015
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Abstract

This unit describes detailed procedures for the preparation of nonnatural C‐nucleosides comprising seven types of nonnatural nucleobases attached to 1′‐position of 2′‐deoxyribose through an acetylene bond with the β‐configuration. In addition, derivatization of these alkynyl C‐nucleosides into the corresponding phosphoramidites and the subsequent oligonucleotide synthesis are also presented. The processes are depicted in three parts. The first basic protocol deals with the synthesis of a key intermediate, 5‐O‐DMTr‐protected 1‐ethynyl‐2‐deoxy‐β‐D‐ribofuranoside. The second basic protocol mentions the procedures of the preparation of nonnatural C‐nucleosides by a palladium‐catalyzed coupling reaction of the ethynyl intermediate and halogen‐attached nonnatural nucleobases. The synthetic procedures of the corresponding nonnatural phosporamidites are also described. The third basic protocol presents the solid‐phase, automated synthesis of nonnatural oligonucleotides composed exclusively of the nonnatural C‐nucleotides. © 2015 by John Wiley & Sons, Inc.

Keywords: alkynyl C‐nucleosides; nonnatural nucleobases; oligonucleotides

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Alkynyl Deoxyribose Derivatives
  • Basic Protocol 2: Synthesis of Alkynyl C‐Nucleosides and Their Phosphoramidites
  • Basic Protocol 3: Solid‐Phase, Automated Synthesis of Nonnatural Oligonucleotides Composed Exclusively of Alkynyl C‐Nucleosides
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Alkynyl Deoxyribose Derivatives

  Materials
  • 2‐Deoxyribose (1)
  • Argon (or nitrogen) gas
  • Methanol (anhydrous MeOH, freshly distilled; keep dry with sieves)
  • 5% to 10% Hydrogen chloride/methanol reagent (5% to 10% HCl/MeOH; Tokyo Chemical Industry)
  • Sodium carbonate (Na 2CO 3)
  • Celite
  • Tetrahydrofuran (anhydrous THF; freshly distilled prior to use)
  • Potassium hydroxide (KOH; freshly be ground in powder prior to use)
  • Benzyl chloride (BnCl; Tokyo Chemical Industry)
  • Diethyl ether (Et 2O)
  • Silica gel (230 to 400 mesh; Merck)
  • n‐Hexane
  • Ethyl acetate (EtOAc)
  • 80% AcOH/20% H 2O solution (80% AcOH)
  • Distilled water
  • Saturated NaHCO 3 aqueous solution (sat. NaHCO 3)
  • Sodium sulfate (Na 2SO 4)
  • Trimethylsilyl acetylene (TMS acetylene; Shin‐Etsu Chemical)
  • 2.6 M n‐Butyl lithium in hexane (n‐BuLi; Kanto Chemical)
  • Saturated NH 4Cl aqueous solution (sat. NH 4Cl)
  • Dichloromethane (CH 2Cl 2)
  • Brine
  • Dicobalt octacarbonyl [Co 2(CO) 8; Kanto Chemical]
  • Boron trifluoride‐diethyl ether complex (BF 3•OEt 2; Tokyo Chemical Industry)
  • Magnesium sulfate (MgSO 4)
  • Iodine (I 2; Sigma‐Aldrich)
  • Saturated Na 2SO 3 aqueous solution (sat. Na 2SO 3)
  • Dichloromethane (anhydrous CH 2Cl 2; freshly distilled and kept dry with sieves)
  • 1.0 M Boron trichloride in CH 2Cl 2 (BCl 3/CH 2Cl 2; Aldrich)
  • Triethylamine (anhydrous Et 3N; freshly distilled and kept dry with sieves)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl; Wako Pure Chemical)
  • 1.0 M n‐Bu 4NF in THF (n‐Bu 4NF/THF; Aldrich)
  • Three‐neck flask, 500‐mL
  • Three‐way stopcock
  • Vacuum apparatus for filtration
  • Three neck flask, 1000‐mL
  • Pressure‐equalizing dropping funnel
  • Dimroth condenser
  • Oil bath
  • Chromatography columns
  • Round‐bottom flasks, 1000‐mL
  • Ultra cooling reactor
  • Two‐neck flasks, 200‐ and 300‐mL
  • Beakers, 2000‐mL
  • Fume hood
  • Thin‐layer chromatography (TLC) equipment

Basic Protocol 2: Synthesis of Alkynyl C‐Nucleosides and Their Phosphoramidites

  Materials
  • 9 (see protocol 1)
  • Tris(dibenzylideneacetone)dipalladium(0)‐chloroform [Pd 2(dba) 3•CHCl 3; Aldrich]
  • Copper(I) iodide (CuI; Sigma Aldrich)
  • Triphenyl phosphine (PPh 3; Wako Pure Chemical)
  • Argon
  • Diisopropylamine (deoxygenated iPr 2NH, bubbled with argon for 10 min prior to use)
  • N,N‐Dimethylformamide (deoxygenated DMF; bubbled with argon for 10 min prior to use)
  • 5‐Iodo‐1‐methyluracil [T*‐I; prepared according to the procedure described in Robins et al. ( )]
  • Ethyl acetate (EtOAc)
  • Saturated NaCl aqueous solution (brine)
  • Magnesium sulfate (MgSO 4)
  • Silica gel (230‐400 mesh, Merck)
  • Methanol (MeOH)
  • Chloroform (CHCl 3)
  • 2‐Amino‐5‐bromopyrimidine [A*‐Br; prepared according to the procedure described in Paudler and Jovanovic ( )]
  • Bis(triphenylphosphine)palladium(II) dichloride [PdCl 2(PPh 3) 2; Tokyo Chemical Institute]
  • Triethylamine (deoxygenated Et 3N; bubbled with argon for 10 min prior to use)
  • 10% NaCl aqueous solution (10% NaCl)
  • Potassium carbonate (K 2CO 3)
  • Acetone
  • Dichloromethane (CH 2Cl 2)
  • 2,6‐Diamino‐5‐iodopyrimidine [D*‐I; prepared according to the procedure described in Shirato et al., )]
  • 1,1,1,3,3,3‐Hexamethyldisilazane [deoxygenated (Me 3Si) 2NH, bubbled with argon for 10 min prior to use]
  • Tetrahydrofuran (THF)
  • 1% Citric acid aqueous solution (1% citric acid)
  • 5% Na 2CO 3 aqueous solution (5% Na 2CO 3)
  • 5‐Iodo‐1‐methylisocytosine [C*‐I; prepared according to the procedure described in Wellington et al. ( )]
  • Sodium sulfate (Na 2SO 4)
  • Distilled water
  • 28% Ammonia aqueous solution (conc. NH 4OH)
  • 5‐Iodocytosine [iC*‐I; prepared according to the procedure described in Doi et al., ]
  • Saturated NaHCO 3 aqueous solution (sat. NaHCO 3)
  • Diethyl ether (Et 2O)
  • n‐Hexane
  • Benzene
  • N,N‐Dimethylformamide dimethylacetal [Me 2NCH(OMe) 2; Tokyo Chemical Institute]
  • Pyridine
  • 6‐Acetylamino‐3‐cyano‐5‐iodo‐2‐pyridone [G*‐I; prepared according to the procedure described in Shirato et al. ( )]
  • 5‐Iodoisocytosine [iG*‐I; prepared according to the procedure described in Doi et al., )]
  • Methanol (anhydrous MeOH; freshly distilled and kept dry with sieves)
  • N,N‐Dimethylformamide (anhydrous DMF; freshly distilled and kept dry with sieves)
  • Isobutyric anhydride [(iPrCO) 2O; Tokyo Chemical Institute]
  • Dichloromethane (anhydrous CH 2Cl 2; freshly distilled and kept dry with sieves)
  • N‐Ethyldiisopropylamine (for Peptide Synthesis) (iPr 2NEt, Wako Pure Chemical)
  • 2‐Cyanoethyl diisopropylchlorophosphoramidite [iPrNP(Cl)O(CH 2) 2CN; Wako Pure Chemical]
  • Methanol for HPLC
  • Acetonitrile for HPLC
  • Two‐neck flasks, 300‐ and 200‐, and 100‐, and 50‐mL
  • Three‐way stopcock
  • Vacuum apparatus
  • Oil bath
  • Chromatography columns
  • Dimroth condenser

Basic Protocol 3: Solid‐Phase, Automated Synthesis of Nonnatural Oligonucleotides Composed Exclusively of Alkynyl C‐Nucleosides

  Materials
  • Universal Support III (Glen Research)
  • 2 M Ammonia methanol solution (2 M NH 3/MeOH; Wako Pure Chemical)
  • 28% ammonia aqueous solution (conc. NH 4OH)
  • 5 mM ammonium formate
  • Column for DNA synthesis (Glen Research)
  • DNA synthesizer (Applied Biosystems Inc., model 392)
  • 1.5‐mL microcentrifuge tubes
  • Temperature‐controllable mixer (Thermomixer)
  • Membrane filter (pore size: 0.45 micro‐meter, diameter: 4 mm, Nacalai Tesque Inc.)
  • A, T, G, C phosphoramidites for DNA synthesis (Glen Research)
  • Reversed‐phase HPLC equipment
  • 5C 18‐AR‐II columns (4.6φ × 150 mm) or a Chemcobond 5‐ODS‐H columns
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Figures

Videos

Literature Cited

Literature Cited
  Adamo, M.F.A. and Pergoli, R. 2007. Studies on the generation of unnatural C ‐nucleosides with 1‐alkynyl‐2‐deoxy‐D‐riboses. Org. Lett. 9:4443‐4446.
  Benner, S.A. 2004. Understanding nucleic acids using synthetic chemistry. Acc. Chem. Res. 37:784‐797.
  Chiba, J. and Inouye, M. 2010. Exotic DNA made of nonnatural bases and natural phosphodiester bonds. Chem. Biodivers. 7:259‐282.
  Chiba, J., Shirato, W., Yamade, Y., Kim, B.S., Matsumoto, S., and Inouye, M. 2012. Furanose ring conformations in a 1′‐alkynyl C ‐nucleoside and the dinucleotide. Tetrahedron 68:9045‐9049.
  Chiba, J., Takeshima, S., Mishima, K., Maeda, H., Nanai, Y., Mizuno, K., and Inouye, M. 2007. Artificial DNAs based on alkynyl C ‐nucleosides as a superior scaffold for homo‐ and heteroexcimer emissions. Chem. Eur. J. 13:8124‐8130.
  Doi, Y., Chiba, J., Morikawa, T., and Inouye, M. 2008. Artificial DNA made exclusively of nonnatural C ‐nucleosides with four types of nonnatural bases. J. Am. Chem. Soc. 130:8762‐8768.
  Egli, M. and Herdewijn, P. 2012. Chemistry and Biology of Artificial Nucleic Acids. Wiley‐VCH, Weinheim.
  Groebke, K., Hunjiker, J., Fraser W., Peng, L., Diederichsen, U., Zimmermann, K., Holzner, A., Leumann, C., and Eschenmoser, A. 1998. Pentose‐ and not hexose‐nucleic acids? Purine‐purine pairing in homo‐DNA: Guanine, isoguanine, 2.6‐diaminopurine, and xanthine. Helv. Chim. Acta 81:375‐474.
  Heinrich, D., Wanger, T., and Diederichsen, U. 2007. Synthesis and DNA incorporation of an ethynyl‐bridged cytosine C ‐nucleoside as guanosine surrogate. Org. Lett. 9:5311‐5314.
  Inouye, M., Doi, Y., Azuchi, J., Shirato, W., Chiba, J., and Abe, H. 2011. Hexamethyldisilazane‐promoted Sonogashira reaction of polyfunctionalized N ‐containing heterocycles. Heterocycles 82:1137‐1141.
  Krishnamurthy, R., Pitsch, S., Minton, M., Miculka, C., Windhab, N., and Eschenmoser, A. 1996. Pyranosyl‐RNA: Base pairing between homochiral oligonucleotide strands of opposite sense of chirality. Angew. Chem. Int. Ed. 35:1537.
  Krueger, A.T., Lu, H., Lee, A.H.F., and Kool, E.T. 2007. Synthesis and properties of size‐expanded DNAs: Toward designed, functional genetic systems. Acc. Chem. Res. 40:141‐150.
  Paudler, W.W. and Jovanovic, M.V. 1983. Bromination of some pyridine and diazine N ‐oxides. J. Org. Chem. 48:1064‐1069.
  Robins, M.J., Barr, P.J., and Giziewicz, J. 1982. Nucleic acid related compounds. 38. Smooth and high‐yield iodination and chlorination at C‐5 of uracil bases and p ‐toluyl‐protected nucleosides. Can. J. Chem. 60:554‐557.
  Shirato, W., Chiba, J., and Inouye, M. 2015. A firmly hybridizable, DNA‐like architecture with DAD/ADA‐ and ADD/DAA‐type nonnatural base pairs as an extracellular genetic candidate. Chem. Commun. 51:7043‐7046.
  Takase, M., Morikawa, T., Abe, H., and Inouye, M. 2003. Stereoselective synthesis of alkynyl C ‐2‐deoxy‐b‐D‐ribofuranosides via intramolecular Nicholas reaction: A versatile building block for nonnatural C ‐nucleosides. Org. Lett. 5:625‐628.
  Wellington, K.W., Ooi, H.C., and Benner, S.A. 2009. A convenient synthesis of N, N′‐dibenzyl‐2,4‐diaminopyrimidine‐2′‐deoxyribonucleoside and 1‐methyl‐2′‐deoxypseudocytidine. Nucleosides Nucleotides Nucleic Acids 28:275‐291.
  Winnacker, M. and Kool, E.T. 2013. Artificial genetic sets composed of size‐expanded base pairs. Angew. Chem. Int. Ed. 52:12498‐12508.
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