Synthesis and Characterization of Benzylidene Acetal–Type Bridged Nucleic Acids (BA‐BNA)

Tetsuya Kodama1, Kunihiko Morihiro2, Satoshi Obika2

1 Structural Biology Research Center and Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Aichi, 2 National Institute of Biomedical Innovation (NIBIO), Ibaraki, Osaka
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.31
DOI:  10.1002/0471142700.nc0131s58
Online Posting Date:  September, 2014
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Abstract

Benzylidene acetal‐type bridged nucleic acids (BA‐BNAs) have a bridged structure that cleaves upon exposure to appropriate external stimuli, which induces changes in oligonucleotide properties. In particular, duplex stability and resistance to enzymatic digestion vary depending on the incorporation number and/or position of BA‐BNAs. This unit describes the synthesis of some types of BA‐BNA thymine nucleosides and the corresponding BA‐BNA phosphoramidites, as well as the incorporation of BA‐BNA nucleosides into oligonucleotides. Moreover, typical procedures that induce property changes in each BA‐BNA are described. 6‐Nitroveratrylidene and 2‐nitrobenzylidene acetal type BA‐BNA respond to photoirradiation and subsequent thiol treatment. Benzylidene and 4‐nitrobenzylidene acetal type BA‐BNAs respond to acids and reducing agents, respectively. Every BA‐BNA is derivatized into 4′‐C‐hydroxymethyl RNA by hydrolysis of the acetal‐bridged structure. Curr. Protoc. Nucleic Acid Chem. 58:1.31.1‐1.31.22. © 2014 by John Wiley & Sons, Inc.

Keywords: property change; external stimuli; light‐trigger; acid‐trigger; redox‐change‐trigger; bridged nucleic acid (BNA); sugar‐modified nucleoside

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Benzylidene Acetal Type Bridged Nucleosides and Corresponding Phosphoramidites
  • Basic Protocol 2: Synthesis, Isolation, and Characterization of Oligonucleotides Containing BA‐BNs
  • Basic Protocol 3: External Stimuli–Induced Transformation of Oligonucleotides Containing BA‐BNAs
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of Benzylidene Acetal Type Bridged Nucleosides and Corresponding Phosphoramidites

  Materials
  • 1,2‐O‐Isopropylidene‐3‐O‐benzyl‐4‐C‐hydroxymethyl‐α‐D‐ribofuranose (1, KNC Laboratories)
  • N,N‐Dimethylformamide (DMF), anhydrous
  • 60% sodium hydride (NaH) in mineral oil
  • Benzyl bromide (BnBr) or benzyl chloride (BnCl)
  • Ethyl acetate (EtOAc)
  • Saturated aqueous sodium chloride (brine)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Silica gel (0.100‐mm Fuji Silysia PSQ‐100B)
  • n‐Hexane
  • Acetic acid (AcOH)
  • Acetic anhydride (Ac 2O)
  • Sulfuric acid (H 2SO 4), concentrated
  • Aqueous sodium hydrogen carbonate (NaHCO 3), saturated
  • Thymine
  • Acetonitrile (CH 3CN), anhydrous
  • N,O‐Bis(trimethylsilyl)acetamide (BSA, e.g., Tokyo Chemical Industry)
  • Trimethylsilyl trifluoromethanesulfonate (TMSOTf)
  • 20% (w/w) palladium hydroxide (Pd(OH) 2) on carbon wetted with 50% water (e.g., Wako Pure Chemical Industry)
  • Nitrogen (N 2) atmosphere (or argon atmosphere)
  • Hydrogen (H 2) atmosphere
  • Imidazole
  • 1,3‐Dichloro‐1,1,3,3‐tetraisopropyldisiloxane (TIPDSCl 2, e.g., Shin‐Etsu Chemical)
  • Tetrahydrofuran (THF), ice cold
  • Aqueous 40% methylamine, ice cold
  • 6‐Nitroveratraldehyde
  • 1,1,1,3,3,3‐Hexafluoro‐2‐propanol (HFIP)
  • Benzaldehyde
  • 2‐Nitrobenzaldehyde
  • Toluene, dehydrated
  • 4‐Nitrobenzaldehyde
  • Zinc chloride
  • 1 M tetrabutylammonium fluoride (TBAF) in THF (e.g., Tokyo Chemical Industry)
  • Pyridine, anhydrous
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl, e.g., Tokyo Chemical Industry)
  • Triethylamine (TEA)
  • Methanol (MeOH)
  • Dichloromethane (CH 2Cl 2)
  • N,N,N,N′‐Tetraisopropylphosphordiamidite (e.g., Tokyo Chemical Industry)
  • 0.25 M 4,5‐Dicyanoimidazole (DCI) in CH 3CN (e.g., SAFC Proligo)
  • Magnetic plate and stir bar
  • Rotary evaporator
  • Separatory funnels
  • Cotton‐plugged funnels
  • 30‐mL round‐bottom flasks
  • Kiriyama Rohto (Kiriyama)
  • No. 4 filter paper
  • Additional reagents and equipment for silica gel chromatography ( ) and thin‐layer chromatography (TLC; )

Basic Protocol 2: Synthesis, Isolation, and Characterization of Oligonucleotides Containing BA‐BNs

  Materials
  • BA‐BNs phosphoramidite(s) (10a‐d; see protocol 1)
  • Acetonitrile (CH 3CN), anhydrous
  • 2′‐Deoxyribonucleoside phosphoramidites (SAFC Proligo)
  • Argon (Ar)
  • 30% aqueous ammonia
  • Mobile phase A: 0.1 M triethylammonium acetate (TEAA), pH 7.0 (HPLC)
  • Mobile phase B: 50% CH 3CN in 0.1 M TEAA (HPLC)
  • Matrix solution I: 10 mg/mL 3‐hydroxypicolinic acid in water
  • Matrix solution II: 1 mg/mL diammonium hydrogen citrate in water
  • DNA synthesizer (e.g., Applied Biosystems Expedite 8909 Nucleic Acid Synthesis System)
  • 1‐mL syringes
  • 1.5‐mL screw‐capped tubes or vials
  • 55°C heating block
  • NAP‐10 column (GE Healthcare)
  • FD‐1000 lyophilizer (EYELA)
  • HPLC system with:
    • Column: 4.6 × 50–mm (analysis) or 10 × 50–mm (isolation) Waters X‐Bridge OST C18 column
    • Detector: 260 nm
  • Speedvac evaporator (TOMY)
  • Additional reagents and equipment for automated solid‐phase oligonucleotide synthesis ( ) and purification of oligonucleotides (units , , , & )

Basic Protocol 3: External Stimuli–Induced Transformation of Oligonucleotides Containing BA‐BNAs

  Materials
  • BA‐BNAs (a specific concentration solution prepared in protocol 2)
  • Reaction buffer: 25 mM sodium phosphate buffer (pH 7.2) or acidified buffer: 25 mM citrate‐phosphate buffer (pH 3.0 to 5.0)
  • Glutathione (GSH)
  • Cysteine (Cys)
  • Sodium dithionite (Na 2S 2O 4)
  • UV‐LED lamp (OMRON model no. ZUV‐C30H or ZUV‐L8H)
  • 37°C heating block
  • Additional reagents and equipment for HPLC (see protocol 2)
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Figures

Videos

Literature Cited

Literature Cited
   Amit, B. , Zehavi, U. , and Patchornik, A. 1974. Photosensitive protecting groups of amino sugars and their use in glycoside synthesis. 2‐Nitrobenzyloxycarbonylamino and 6‐nitroveratryloxycarbonylamino derivatives. J. Org. Chem. 39:192‐196.
   Bourget, C. , Trévisiol, E. , Bridon, I. , Kotera, M. , Lhommea, J. , and Laayounb, A. 2005. Biotin‐phenyldiazomethane conjugates as labeling reagents at phosphate in mono and polynucleotides. Bioorg. Med. Chem. 13:1453‐1461.
   Hari, Y. , Obika, S. , Ohnishi, R. , Eguchi, K. , Osaki, T. , Ohishi, H. , and Imanishi, T. 2006. Synthesis and properties of 2′‐O,4′‐C‐methyleneoxymethylene bridged nucleic acid. Bioorg. Med. Chem. 14:1029‐1038.
   Kasahara, Y. , Kitadume, S. , Morihiro, K. , Kuwahara, M. , Ozaki, H. , Sawai, H. , Imanishi, T. , and Obika, S. 2010. Effect of 3′‐end capping of aptamer with various 2′,4′‐bridged nucleotides: Enzymatic post‐modification toward a practical use of polyclonal aptamers. Bioorg. Med. Chem. Lett. 20:1626‐1629.
   Markiewicz, W.T. and Wiewiórowski, M. 1978. A new type of silyl protecting groups in nucleoside chemistry. Nucleic Acids Res. 1:s185‐s190.
   Mayer, G. and Heckel, A. 2006. Biologically active molecules with a “light switch.” Angew . Chem. Int. Ed. 45:4900‐4921.
   Mitsuoka, Y. Kodama, T. Ohnishi, R. Hari, Y. Imanishi, T. and Obika, S. 2009. A bridged nucleic acid, 2′,4′‐BNACOC: Synthesis of fully modified oligonucleotides bearing thymine, 5‐methylcytosine, adenine and guanine 2′,4′‐BNACOC monomers, and RNA‐selective nucleic acid recognition. Nucleic Acids Res. 37:1225‐1238.
   Morihiro, K. , Kodama, T. , Nishida, M. , Imanishi, T. , and Obika, S. 2009. Synthesis of light‐responsive bridged nucleic acid and changes in affinity with complementary ssRNA. Chem. Bio. Chem. 10:1784‐1788.
   Morihiro, K. , Kodama, T. , and Obika, S. 2011. Benzylidene acetal‐type bridged nucleic acids: Changes in properties upon cleavage of the bridge triggered by external stimuli. Chem. Eur. J. 17:7918‐7926.
   Nielsen, K. D. , Kirpekar, F. , Roepstorff, P. , and Wengel, J. 1995. Oligonucleotide analogues containing 4′‐C‐(hydroxymethyl)uridine: Synthesis, evaluation and mass spectrometric analysis. Bioorg. Med. Chem. 3:1493‐1502.
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