Preparation of C5‐Functionalized Locked Nucleic Acids (LNAs)

Pawan Kumar1, Michael E. Østergaard1, Patrick J. Hrdlicka1

1 University of Idaho, Moscow, Idaho
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.43
DOI:  10.1002/0471142700.nc0443s44
Online Posting Date:  March, 2011
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Abstract

Relative to conventional locked nucleic acids (LNAs), C5‐functionalized LNAs with small entities at the C5‐position display markedly higher (1) duplex thermostability with complementary single‐stranded DNA/RNA targets, (2) target specificity, and (3) 3′‐exonuclease stability. C5‐Functionalized LNAs carrying a polarity‐sensitive fluorophore enable sensitive and efficient discrimination of single nucleotide polymorphisms (SNPs) under non‐stringent conditions. This unit describes protocols for chemical synthesis of an LNA uridine diol and corresponding C5‐functionalized LNA uridine phosphoramidites. A procedure for incorporation of C5‐functionalized LNA uridine phosphoramidites into oligodeoxyribonucleotides by automated DNA synthesis is also described. Curr. Protoc. Nucleic Acid Chem. 44:4.43.1‐4.43.22. © 2011 by John Wiley & Sons, Inc.

Keywords: antisense; bridged nucleic acid (BNA); conformationally restricted nucleotide; locked nucleic acid (LNA); nucleobase‐modified nucleotide; pyrene

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Unprotected LNA Uridine Diol
  • Basic Protocol 2: Preparation of C5‐Functionalized LNA Phosphoramidites
  • Basic Protocol 3: Synthesis, Isolation, and Characterization of Oligodeoxyribonucleotides Containing C5‐Functionalized LNA Building Blocks
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Unprotected LNA Uridine Diol

  Materials
  • 1,2‐Di‐O‐acetyl‐3‐O‐benzyl‐5‐O‐methanesulfonyl‐4‐C‐methanesulfonyloxymethyl‐D‐erythro‐pentofuranose ( S.1; Koshkin et al., ; unit 4.12)
  • Acetonitrile (Fisher, HPLC grade, 99.9%, stored over 3Å molecular sieves)
  • Uracil (Alfa Aesar, 99%)
  • N,O‐Bis(trimethylsilyl)acetamide (BSA, Alfa Aesar, 95%)
  • Argon gas
  • Trimethylsilyl trifluoromethanesulfonate (TMSOTf, Alfa Aesar, 99%)
  • Methanol (J.T. Baker, 99.8%)
  • Dichloromethane (CH 2Cl 2, Mallinckrodt Chemicals, AR grade, 99.5%)
  • Ethyl acetate (EtOAc, Fisher, 99%)
  • Petroleum ether (Mallinckrodt, AR)
  • TLC dipping reagent: 5% H 2SO 4 in ethanol
  • Saturated aqueous NaHCO 3 (prepared by saturating deionized water with food‐grade sodium bicarbonate)
  • Na 2SO 4, anhydrous (EMD, 99%)
  • Silica gel (Sorbent technologies, 0.040‐0.063 mm, 230‐400 mesh, porosity 60 Å)
  • 1,4‐Dioxane (Fisher, 99.9%)
  • NaOH (Fisher, 98%)
  • Citric acid (Fisher, crystalline, certified ACS)
  • Dimethylformamide (DMF, Alfa Aesar, HPLC grade, 99.7 %, septum‐capped)
  • Sodium benzoate (Acros Organics, 99%)
  • Tetrahydrofuran (THF, Fisher, HPLC, 99.9%, dried by distillation over Na, stored over 4Å molecular sieves)
  • Acetic acid (EMD, 99.7%)
  • 20% Pd(OH) 2/C (Alfa Aesar, batch no. L06R026)
  • Formic acid (J.T. Baker, 88% aq. solution, batch no. 2AA06260)
  • 500‐mL and 1‐L round‐bottom flasks
  • Rotary evaporator connected to vacuum pump (delivering ∼5 mbar max vacuum), chilled by recirculating ethylene glycol (down to −25°C)
  • Reflux condensers
  • Silicone oil bath (Alfa Aesar)
  • Silica‐coated aluminum‐backed TLC plates with fluorescent indicator (Sorbent Technologies, silica gel G F 254)
  • UV lamp, dual wavelength (254 and 365 nm)
  • Heat gun
  • 2‐L beaker
  • ∼2‐cm celite pad (∼10‐cm diameter, Celite 545, EMD)
  • Sintered glass funnels (10 × 8 cm)
  • 1‐L separating funnel
  • 2‐L conical flasks
  • 3‐inch (7.5‐cm) glass funnel with cotton plug
  • Glass chromatography columns, 6 × 40 and 3 × 30 cm
  • High‐performance pump delivering ∼10 mtorr vacuum
NOTE: Evaporation of solvents is carried out under reduced pressure at temperatures below 45°C.CAUTION: When preparing TLC dipping reagent, add H 2SO 4 drop‐wise to ice‐cold ethanol, as this is a highly exothermic reaction.

Basic Protocol 2: Preparation of C5‐Functionalized LNA Phosphoramidites

  Materials
  • LNA‐U diol S.6 (see protocol 1)
  • Iodine (EMD, 99.8%)
  • Ceric ammonium nitrate (CAN, Alfa Aesar, 99%)
  • Glacial acetic acid (EMD, 99.7%)
  • Methanol (J.T. Baker, 99.8%)
  • Dichloromethane (CH 2Cl 2, Mallinckrodt Chemicals, AR grade, 99.5%)
  • Silica gel (Sorbent technologies, 0.040‐0.063 mm, 230‐400 mesh, porosity 60 Å)
  • Anhydrous pyridine (Alfa Aesar, 99.5%, stored over 4Å molecular sieves)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl, Excel, 98%)
  • TLC dipping reagent: 5% H 2SO 4 in ethanol
  • Saturated aqueous NaHCO 3 (prepared by saturating deionized water with food‐grade sodium bicarbonate)
  • Na 2SO 4, anhydrous (EMD, 99%)
  • Dimethylformamide (DMF, Alfa Aesar, HPLC grade, 99.7 %, septum‐capped)
  • Tetrakistriphenylphosphine palladium [Pd(PPh 3) 4, TCI, 97%]
  • CuI (Alfa Aesar, 98%)
  • Triethylamine (Fisher, 99%, reagent grade)
  • Appropriate alkyne for C5 functionalization:
    • Trimethylsilylacetylene ( S.9a, Oakwood Products, 98%)
    • 2,2,2‐Trifluoro‐N‐(2‐propynyl)acetamide ( S.9c, prepared by N‐acetylation of commercially available propargylamine with trifluoroacetic anhydride, Trybulski et al., )
    • N‐(Prop‐2‐ynyl)pyrene‐1‐carboxamide ( S.9d, prepared from commercially available pyrene‐1‐carboxylic acid and propargylamine as described in Okamoto et al., , except that 1.1 equiv HATU is used as coupling reagent)
  • Isopropanol (i‐PrOH, JT Baker, ACS grade)
  • Chloroform (CHCl 3, JT Baker, ACS grade)
  • Brine (prepared by saturating deionized water with food‐grade non‐iodinized NaCl)
  • 1 M tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (∼5% water content, Acros Organics)
  • Tetrahydrofuran (THF, Fisher, HPLC, 99.9%, dried by distillation over Na, stored over 4Å molecular sieves)
  • Ethyl acetate (EtOAc, optional, Fisher, 99%)
  • Petroleum ether (optional)
  • Dichloroethane (DCE, Fisher, 99%)
  • N,N‐Diisopropylethylamine (DIPEA, Acros Organics, >98%, stored over 4Å molecular sieves)
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite (PCl reagent, Alfa Aesar, 95%)
  • 25‐, 250‐, and 500‐mL round‐bottom flasks
  • Argon balloon
  • Silica‐coated aluminum‐backed TLC plates with fluorescent indicator (Sorbent Technologies, silica gel G F 254)
  • Silicone oil bath (Alfa Aesar)
  • Rotary evaporator connected to vacuum pump (delivering ∼5 mbar max vacuum), chilled by recirculating ethylene glycol (down to −25°C)
  • Glass chromatography columns, 6 × 40, 3 × 30, 2 × 30, and 1.5 × 25 cm
  • High‐performance pump delivering ∼10 mtorr vacuum
  • Heat gun
  • 250‐mL and 1‐L separating funnels
  • 250‐mL and 1‐L conical flasks
  • Three‐way valve
  • 3‐inch (∼7.5‐cm) glass funnels equipped with a cotton plug
CAUTION: When preparing TLC dipping reagent, add H 2SO 4 drop‐wise to ice‐cold ethanol, as this is a highly exothermic reaction.NOTE: Evaporation of solvents is carried out under reduced pressure at temperatures below 45°C.

Basic Protocol 3: Synthesis, Isolation, and Characterization of Oligodeoxyribonucleotides Containing C5‐Functionalized LNA Building Blocks

  Materials
  • 5′‐O‐Dimethoxytrityl‐N4‐benzoyl‐2′‐deoxycytidine‐3′‐O‐succinoyl long‐chain alkylamino controlled‐pore glass (LCA‐CPG) support (500 Å pore size, Glen Research); nucleoside loading is batch‐dependent but provided by the vendor (typically ∼40 µmol/g)
  • Standard DNA phosphoramidites (dried over activated 3Å molecular sieves at least 24 hr):
    • N6‐Benzoyl‐2′‐deoxyadenosine phosphoramidite (dABz, Glen Research)
    • N4‐Benzoyl‐2′‐deoxycytidine phosphoramidite (dCBz, Glen Research)
    • N2‐Dimethylformamidine‐2′‐deoxyguanosine phosphoramidite (dGDMF, Proligo)
    • Thymidine phosphoramidite (dT, Glen Research)
  • Reagents for solid‐phase oligonucleotide synthesis (Glen Research):
    • Activator solution: 0.25 M 4,5‐dicyanoimidazole in anhydrous acetonitrile
    • Cap A: 9:1 (v/v) tetrahydrofuran (THF)/acetic anhydride
    • Cap B: 10% 1‐methylimidazole in 8:1 (v/v) THF/pyridine
    • Oxidizing solution: 0.02 M I 2 in THF/pyridine/H 2O
    • Deblocking mix: 3% trichloroacetic acid in CH 2Cl 2
  • Anhydrous acetonitrile (CH 3CN, Acros, 99.9%, HPLC grade, stored over 3Å molecular sieves)
  • C5‐Functionalized LNA uridine phosphoramidites S.11b‐d (see protocol 2)
  • Conc. aq. ammonia (28% to 30%, EMD)
  • Acetic acid (EMD, 99.7%)
  • 3 M sodium acetate (Acros, 99%,)
  • 5 M sodium perchlorate (Acros, 99%)
  • Acetone (EMD, 99.9%)
  • Mobile phase A: 0.05 M triethylammonium acetate (TEAA), pH 7 (DMTr‐ON, HPLC)
  • Mobile phase B: 25% H 2O in acetonitrile (DMTr‐ON, HPLC)
  • Matrix solution for MALDI‐TOF‐MS: 27.9 mg anthranilic acid in 500 µL acetonitrile and 600 µL aq. ammonium citrate (50 mM)
  • Expedite 8909 Nucleic Acid Synthesis System
  • Empty synthesis column, 0.2‐µmol scale (Glen Research)
  • 1‐ and 2‐mL plastic syringes
  • 2‐mL cryogenic vials (Corning)
  • 55°C incubator (model 10‐140 E, Quincy Lab)
  • 1.5‐mL microcentrifuge tubes (e.g., Fisher)
  • Speedvac evaporator (Centrivap centrifugal concentrator with cold trap model 7810010, Labconco)
  • Varian Prostar HPLC system equipped with:
    • XTerra MS C18 pre‐column (10 µm, 7.8 × 10 mm)
    • XTerra MS C18 column (10 µm, 7.8 × 150 mm)
    • Degasser (Uniflow, DG‐1300)
    • Two solvent delivery systems (Dynamax, Model SD‐200)
    • Fraction collector (Dynamax, Model FC‐1)
  • Additional reagents and equipment for automated DNA synthesis, including the trityl assay ( appendix 3C)
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Figures

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Literature Cited

Literature Cited
   Ahmadian, M., Zhang, P.M., and Bergstrom, D.E. 1998. A comparative study of the thermal‐stability of oligodeoxyribonucleotides containing 5‐substituted 2′‐deoxyuridines. Nucleic Acids Res. 26:3127‐3135.
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   Koch, T. and Ørum, H. 2008. Locked nucleic acid. In Antisense Drug Technology – Principles, Strategies, and Applications, 2nd ed (S.T. Crooke, ed.), p. 519‐564. CRC Press, Boca Raton, Fla.
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