Synthesis of the Phosphoramidite Units for Benzene‐Glycol Nucleic Acid

Yusuke Maeda1, Nazuki Niwa1, Yoshihito Ueno1

1 Faculty of Applied Biological Science, Gifu University
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.42
DOI:  10.1002/cpnc.38
Online Posting Date:  September, 2017
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Abstract

Benzene‐glycol nucleic acid‐DNA chimeras form thermally and thermodynamically stable duplexes with complementary RNAs, and have base‐discriminating abilities. This unit describes the synthesis of four nucleoside analogs, an adenine, cytosine, thymine, and guanine analogs with base‐benzene‐glycol structure. The synthesis starts with conversion of (S)‐mandelic acid in arylboronic acid derivative, common intermediate. Nucleobase coupling of the intermediate and phosphitylation afford to phosphoroamidite units. © 2017 by John Wiley & Sons, Inc.

Keywords: amidite; benzene; DNA; glycol; RNA

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Benzene‐Glycol Adenine Analog
  • Basic Protocol 2: Synthesis of Benzene‐Glycol Cytosine Analog
  • Basic Protocol 3: Synthesis of Benzene‐Glycol Thymine Analog
  • Basic Protocol 4: Synthesis of Benzene‐Glycol Guanine Analog
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Benzene‐Glycol Adenine Analog

  Materials
  • (S)‐Mandelic acid
  • Ethanol (EtOH)
  • Concentrated sulfuric acid (H 2SO 4)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Ethyl acetate (EtOAc)
  • Brine
  • n‐Hexane
  • Silica gel (60 A°, 63 to 210 mesh; Kanto)
  • Argon
  • Sodium sulfate, anhydrous (Na 2SO 4)
  • Dichloromethane (CH 2Cl 2)
  • Silver trifluoromethanesulfonate (TfOAg)
  • Iodine (I 2)
  • Sodium hydrogen sulfite (NaHSO 3)
  • Sodium borohydride (NaBH 3)
  • Ammonium chloride (NH 4Cl)
  • N,N‐Dimethylformamide (DMF)
  • Trimethyl borate, imidazole
  • tert‐Butylchlorodimethylsilane (TBDMSCl)
  • Tetrahydrofran anhydrous (THF)
  • n‐Butyllithium (n‐BuLi)
  • Trimethyl borate
  • Hydrogen chloride (HCl)
  • Methanol (MeOH)
  • Copper(II)acetate monohydrate [Cu(OAc) 2·H 2O]
  • Adenine
  • Tetramethylethylenediamine (TEMED)
  • Pyridine
  • Benzoyl chloride (BzCl)
  • Tetrabutylammonium fluoride (TBAF)
  • 4,4′‐Dimethoxytrityl chloride (DMTrCl)
  • Chloroform (CHCl 3)
  • N,N‐Diisopropylethhylamine (DIPEA)
  • Chloro(2‐cyanoethoxy)(diisopropyamino)‐phosphine (i‐Pr 2NP(Cl)O(CH 2) 2CN)
  • 300‐, and 1000‐mL Round‐bottom flasks
  • Reflux condenser
  • Magnetic stirrer and stir bars
  • Rotary evaporator
  • Glass column
  • Vacuum pump
  • Funnels fitted with celite
  • Oil bath
  • Funnels fitted with cotton

Basic Protocol 2: Synthesis of Benzene‐Glycol Cytosine Analog

  Additional Materials (also see protocol 1)
  • Vacuum‐dried 6 (see protocol 1)
  • Cytosine
  • Funnels fitted with cotton

Basic Protocol 3: Synthesis of Benzene‐Glycol Thymine Analog

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • 3‐N‐Benzoylthimine
  • Concentrated NH 4OH
  • Funnels fitted with cotton

Basic Protocol 4: Synthesis of Benzene‐Glycol Guanine Analog

  Additional Materials (also see Basic Protocols protocol 11 and protocol 22)
  • 2‐[N,N‐bis(tert‐butoxycarbonyl)]‐6‐chloropurine
  • Ethylenediaminetetraacetic acid (EDTA)
  • 2‐Mercaptoethanol
  • Sodium methoxide (NaOMe)
  • N,N‐Dimethylformamide dimethyl acetal
  • Reflux condenser
  • 300‐mL Erlenmeyer flask
  • Funnels fitted with cotton
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Figures

Videos

Literature Cited

  Aboul‐Fadl, T. (2005). Antisense oligonucleotides: The state of the art. Current Medicinal Chemistry, 19, 2193–2214. doi: 10.2174/0929867054864859.
  Fluiter, K., Mook, O. R., Vreijling, J., Langkjaer, N., Højland, T., Wengel, J., & Baas, F. (2009). Filling the gap in LNA antisense oligo gapmers: The effects of unlocked nucleic acid (UNA) and 4′‐C‐hydroxymethyl‐DNA modifications on RNase H recruitment and efficacy of an LNA gapmer. Molecular bioSystems, 8, 838–843. doi: 10.1039/b903922h.
  Kurreck, J. (2003). Antisense technologies. Improvement through novel chemical modifications. European Journal of Biochemistry, 8, 1628–1644. doi: 10.1046/j.1432‐1033.2003.03555.x.
  Kole, R., Krainer, A. R., & Altman, S. (2012). RNA therapeutics: Beyond RNA interference and antisense oligonucleotides. Nature Reviews. Drug Discovery, 2, 125–140. doi: 10.1038/nrd3625.
  Manoharan, M. (2002). Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery, and mechanism of action. Antisense & Nucleic Acid Drug Development, 2, 103–128. doi: 10.1089/108729002760070849.
  Monia, B. P., Lesnik, E. A., Gonzalez, C., Lima, W. F., McGee, D., … Cook, P. D., & Freier, S. M. (1993). Evaluation of 2′‐modified oligonucleotides containing 2′‐deoxy gaps as antisense inhibitors of gene expression. The Journal of Biological Chemistry, 19, 14514–14522.
  Niwa, N., Ueda, K., & Ueno, Y. (2016). Synthesis of benzene‐glycol nucleic acids and their biophysical and biological properties. European Journal of Organic Chemistry, 14, 2435–2443. doi: 10.1002/ejoc.201600189.
  Niwa, N., Shimizu, S., Maeda, Y., Hiroakid, H., & Ueno, Y. (in press). Benzene‐Glycol Nucleic Acid (BGNA)‐DNA Chimeras: Synthesis, Binding Properties, and Ability to Elicit Human RNase H Activity. RSC Advance. in press.
  Schlegel, M. K., Peritz, A. E., Kittigowittana, K., Zhang, L., & Meggers, E. (2007). Duplex formation of the simplified nucleic acid GNA. ChemBioChem, 8, 927–932. doi: 10.1002/cbic.200600435.
  Schlegel, M. K., Xie, X., & Meggers, E. (2009). Insight into the High Duplex Stability of the Simplified Nucleic Acid GNA. Angewandte Chemie (International in English), 48, 960–963; Angew. Chem. 121: 978. doi: 10.1002/anie.200803472.
  Ueno, Y., Kato, T., Sato, K., Ito, Y., Yoshida, M., Inoue, T., … Kitade, Y. (2005). Synthesis and properties of nucleic acid analogues consisting of a benzene‐phosphate backbone. The Journal of Organic Chemistry, 70, 7925–7935. doi: 10.1021/jo050635m.
  Ueno, Y., Kawamura, A., Takasu, K., Komatsuzaki, S., Kato, T., Kuboe, S., … Kitade, Y. (2009). Synthesis and properties of a novel molecular beacon containing a benzene‐phosphate backbone at its stem moiety. Organic & Biomolecular Chemistry, 7, 2761–2769. doi: 10.1039/b901631g.
  Wu, H., Lima, W. F., Zhang, H., Fan, A., Sun, H., & Crooke, S. T. (2004). Determination of the role of the human RNase H1 in the pharmacology of DNA‐like antisense drugs. The Journal of Biological Chemistry, 17, 17181–17189. doi: 10.1074/jbc.M311683200.
  Zhang, L., Peritz, A., & Meggers, E. (2005). A simple glycol nucleic acid. Journal of the American Chemical Society, 127, 4174–4175. doi: 10.1021/ja042564z.
  Zhang, L., Peritz, A. E., Carroll, P. J., & Meggers, E. (2006). Synthesis of glycol nucleic acids. Synthesis, 645–653.
Key References
  Niwa, Ueda, & Ueno (2016). See above.
  Both articles discuss synthesis and properties of BGNA‐DNA chimeras.
  Niwa et al. (in press). See above.
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