Synthesis of 2′,4′‐Bridged Nucleosides Using a New Orthogonally Protected Sugar Synthon: 5‐O‐(tert‐Butyldiphenylsilyl)‐4‐C‐Hydroxymethyl‐1,2‐O‐Isopropylidene‐3‐O‐Napthyl‐α‐d‐Allofuranose

Punit P. Seth1, Thazha P. Prakash1

1 Isis Pharmaceuticals, Carlsbad, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.24
DOI:  10.1002/0471142700.nc0124s44
Online Posting Date:  March, 2011
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Abstract

2′,4′‐Bridged nucleic acids (BNA) provide unprecedented increases in the thermal stability of oligonucleotide duplexes. Locked nucleic acids (LNA), a representative member of the BNA class, have shown promise in antisense applications and as a diagnostic tool. While a number of BNA analogs have been reported in the literature, their evaluation in vivo has been stymied by difficulties in securing enough amidite to support extensive animal experiments. A bottleneck has been the ability to prepare a key mono‐protected allofuranose synthon that is the common starting material for all BNA analogs. This unit presents the synthesis of a novel orthogonally protected sugar synthon, 5‐O‐(tert‐butyldimethylsilyl)‐4‐C‐hydroxymethyl‐1,2‐O‐isopropylidene‐3‐O‐(2‐methylnaphthlene)‐α‐D‐allofuranose, which can be prepared in 100‐gram quantities without any chromatographic purification of intermediates or the final product. The utility of this new synthon is exemplified by the synthesis of BNA analogs containing uracil nucleobases, substituted 2′,4′‐bridges, and oxyamino functionalities that are otherwise more difficult to prepare using the 3′‐O‐benzyl protecting group strategy most commonly used in the literature. Curr. Protoc. Nucleic Acid Chem. 44:1.24.1‐1.24.23. © 2011 by John Wiley & Sons, Inc.

Keywords: constrained MOE (cMOE); N‐OMe‐2′‐amino BNA; Nap protecting group

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of 5′‐O‐TBDPS‐3′‐O‐Naphthyl‐4′‐C‐Hydroxymethyl Allofuranose
  • Basic Protocol 2: Preparation of R‐ and S‐Constrained Methoxyethyl (cMOE) Phosphoramidites
  • Basic Protocol 3: Preparation of N‐Methoxy‐2′‐Amino BNA Uracil Phosphoramidite
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of 5′‐O‐TBDPS‐3′‐O‐Naphthyl‐4′‐C‐Hydroxymethyl Allofuranose

  Materials
  • Nitrogen gas
  • 1,2:5,6‐Di‐O‐isopropylidene‐α‐D‐allofuranose (diacetone allofuranose)
  • 2‐Bromomethyl naphthalene (NapBr)
  • Dimethylformamide (DMF), anhydrous
  • Sodium hydride (NaH), 60% (w/w) in mineral oil
  • Hexanes, reagent grade
  • Glacial acetic acid
  • Ethyl acetate, reagent grade
  • Saturated sodium bicarbonate solution (NaHCO 3)
  • Brine
  • Sodium sulfate (Na 2SO 4), anhydrous
  • 1,4‐Dioxane, reagent grade
  • Sodium metaperiodate (NaIO 4)
  • Tetrahydrofuran (THF)
  • 35% aq. formaldehyde
  • 2 M sodium hydroxide (NaOH)
  • Dichloromethane (CH 2Cl 2), anhydrous
  • Triethylamine, anhydrous
  • 5% HCl
  • Diethyl ether
  • tert‐Butyldiphenylsilyl chloride (tert‐butyldiphenylchlorosilane)
  • 1‐ and 2‐L three‐neck round‐bottom flasks
  • 1‐ and 5‐L single‐neck round‐bottom flasks
  • Rubber septa
  • 18‐G disposable needles
  • 2‐L beaker
  • Sintered glass funnels with adaptor for vacuum line
  • Overhead stirrer
  • Rotary evaporator
  • Oil vacuum pumps
  • 1‐ and 2‐L separatory funnels
  • Cannula
  • 1‐, 5‐, 10‐, 20‐, and 50‐mL NORM‐JECT disposable syringes
  • Sonicator

Basic Protocol 2: Preparation of R‐ and S‐Constrained Methoxyethyl (cMOE) Phosphoramidites

  Materials
  • Nitrogen gas
  • Oxalyl chloride
  • Dichloromethane (CH 2Cl 2), anhydrous
  • Dry ice/acetone bath
  • Dimethylsulfoxide, anhydrous
  • 5‐(tert‐Butyldiphenylsilyl)‐4‐C‐hydroxymethyl‐3‐O‐(2‐naphthyl)‐1,2‐O‐isopropylidene‐α‐D‐ribofuranose ( S.5; see protocol 1)
  • Triethylamine, anhydrous
  • 5% HCl
  • Saturated sodium bicarbonate solution (NaHCO 3)
  • Brine
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Magnesium turnings (Average size – 0.19 inch)
  • Mercury(II) chloride
  • Tetrahydrofuran (THF), anhydrous
  • Methoxymethyl bromide (MOMBr)
  • Saturated ammonium chloride solution
  • Ethyl acetate, reagent grade
  • Hexanes, reagent grade
  • 4‐Di(methylamino)pyridine (DMAP)
  • Methanesulfonyl chloride
  • Methanol (MeOH), reagent grade
  • Acetic acid, glacial
  • Acetic anhydride
  • Sulfuric acid, concentrated
  • p‐anisaldehyde TLC stain (see recipe)
  • Uracil
  • Acetonitrile, anhydrous
  • N,O‐Bis‐(trimethylsilyl)acetamide
  • Trimethylsilyl trifluoromethansulfonate (TMSOTf)
  • Potassium carbonate
  • Acetone, reagent grade
  • 2,3‐Dichloro‐5,6‐dicyano‐1,4‐benzoquinone (DDQ)
  • 10% (w/v) sodium bisulfite solution
  • Triethylamine trihydrofluoride
  • Triethylamine
  • Pyridine, anhydrous
  • 4,4′‐Dimethoxytrityl chloride
  • 1H‐Tetrazole
  • N‐Methylimidazole
  • 2‐Cyanoethyl (N,N,N′,N′‐tetraisopropyl)phosphordiamidite
  • 50‐, 200‐, 250‐, and 500‐mL round‐bottom flasks
  • 100‐ and 250‐mL three‐neck round‐bottom flasks
  • Rubber septa
  • 18‐ and 20‐G disposable needles
  • 1‐, 5‐, 10‐, 20‐, and 50‐mL NORM‐JECT disposable syringes
  • Double‐tipped cannulas
  • 125‐, 250‐, and 500‐mL separatory funnels
  • Vacuum pumps
  • Sintered glass funnels
  • Rotary evaporator
  • Constant temperature bath (–25°C)
  • 5 × 26–cm, 8 × 24–cm, and 10 × 31–cm glass columns
  • TLC plates: silica coated glass plate with fluorescent indicator 60 F 254
  • 254‐nm UV lamp
  • Oil bath
  • Reflux condenser
  • Heat gun
  • Vacuum oven (P 2O 5)
  • Additional reagents and equipment for column chromatography ( appendix 3E) and thin‐layer chromatography ( appendix 3D)

Basic Protocol 3: Preparation of N‐Methoxy‐2′‐Amino BNA Uracil Phosphoramidite

  Materials
  • Nitrogen gas
  • 5‐(tert‐Butyldiphenylsilyl)‐4‐C‐hydroxymethyl‐3‐O‐(2‐naphthyl)‐1,2‐O‐isopropylidene‐α‐D‐ribofuranose ( S.5; see protocol 1)
  • Acetic acid, glacial
  • Acetic anhydride
  • Sulfuric acid, concentrated
  • Ethyl acetate, reagent grade
  • Sodium bicarbonate solutions (NaHCO 3), saturated and 5% solution
  • Brine
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Hexanes, reagent grade
  • Uracil
  • Acetonitrile, anhydrous
  • N,O‐Bis‐(trimethylsilyl)‐acetamide
  • Trimethylsilyl trifluoromethansulfonate (TMSOTf)
  • 7 N methanolic ammonia
  • Methanol (MeOH), reagent grade
  • Dichloromethane (CH 2Cl 2), anhydrous
  • Pyridine, anhydrous
  • Methanesulfonyl chloride
  • Diazabicyclo[5.4.0]undec‐7‐ene (DBU)
  • 1,4‐Dioxane, reagent grade
  • 2 M aqueous sodium hydroxide (NaOH)
  • Di(methylamino)pyridine (DMAP)
  • Dry ice/ethanol bath
  • Trifluoromethanesulfonic anhydride
  • Dimethylformamide (DMF), anhydrous
  • N,N‐Diisopropylethylamine (DIPEA)
  • N‐Methoxylamine
  • 2,3‐Dichloro‐5,6‐dicyano‐1,4‐benzoquinone (DDQ)
  • Triethylamine
  • Tetrahydrofuran (THF), anhydrous
  • Triethylamine trihydrofluoride
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • 1H‐Tetrazole
  • N‐Methylimidazole
  • 2‐Cyanoethyl (N,N,N′,N′‐tetraisopropyl)phosphordiamidite
  • 5% HCl
  • 10% (w/v) sodium bisulfite
  • 250‐mL and 1‐L three‐neck round‐bottom flasks
  • 50‐mL, 250‐mL, 500‐mL, and 1‐L round‐bottom flasks
  • Rubber septa
  • 18‐ and 20‐G disposable needles
  • TLC plates: silica coated glass plate with fluorescent indicator 60 F 254
  • 254‐nm UV lamp
  • Rotary evaporator
  • Vacuum oil pumps
  • 125‐mL, 500‐mL, 1‐L, and 2‐L separatory funnels
  • Sintered glass funnels
  • 3 × 30–cm, 10 × 30–cm, and 5 × 20–cm glass columns
  • Reflux condenser
  • Vacuum oven (P 2O 5)
  • Double‐tipped cannulas
  • 1‐, 5‐, 10‐, 20‐, and 50‐mL NORM‐JECT disposable syringes
  • Constant temperature bath
  • 250‐mL pressure bottle
  • Additional reagents and equipment for column chromatography ( appendix 3E) and thin‐layer chromatography ( appendix 3D)
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Figures

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Literature Cited

Literature Cited
   Abdur Rahman, S.M., Seki, S., Obika, S., Yoshikawa, H., Miyashita, K., and Imanishi, T. 2008. Design, synthesis, and properties of 2′,4′‐BNANC: A bridged nucleic acid analogue. J. Am. Chem. Soc. 130:4886‐4896.
   Graziewicz, M.A., Tarrant, T.K., Buckley, B., Roberts, J., Fulton, L., Hansen, H., Orum, H., Kole, R., and Sazani, P. 2008. An endogenous TNF‐α antagonist induced by splice‐switching oligonucleotides reduces inflammation in hepatitis and arthritis mouse models. Mol. Ther. 16:1316‐1322.
   Koshkin, A.A., Singh, S.K., Nielsen, P., Rajwanshi, V.K., Kumar, R., Meldgaard, M., Olsen, C.E., and Wengel, J. 1998. LNA (locked nucleic acids): Synthesis of the adenine, cytosine, guanine, 5‐methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerization, and unprecedented nucleic acid recognition. Tetrahedron 54:3607‐3630.
   Kumar, S., Hansen, M.H., Albæk, N., Steffansen, S.I., Petersen, M., and Nielsen, P. 2009. Synthesis of functionalized carbocyclic locked nucleic acid analogues by ring‐closing diene and enyne metathesis and their influence on nucleic acid stability and structure. J. Org. Chem. 74:6756‐6769.
   Lanford, R.E., Hildebrandt‐Eriksen, E.S., Petri, A., Persson, R., Lindow, M., Munk, M.E., Kauppinen, S., and Orum, H. 2010. Therapeutic silencing of microRNA‐122 in primates with chronic hepatitis C virus infection. Science 327:198‐201.
   Prakash, T.P., Siwkowski, A., Allerson, C.R., Migawa, M.T., Lee, S., Gaus, H.J., Black, C., Seth, P.P., Swayze, E.E., and Bhat, B. 2010. Antisense oligonucleotides containing conformationally constrained 2′,4′‐(N‐methoxy)aminomethylene and 2′,4′‐aminooxymethylene and 2′‐O,4′‐C‐aminomethylene bridged nucleoside analogues show improved potency in animal models. J. Med. Chem. 53:1636‐1650.
   Santhosh Kumar, T., Kumar, P., Sharma, P.K., and Hrdlicka, P.J. 2008. Optimized synthesis of LNA uracil nucleosides. Tetrahedron Lett. 49:7168‐7170.
   Seth, P.P., Allerson, C.R., Berdeja, A., Siwkowski, A., Pallan, P.S., Gaus, H., Prakash, T.P., Watt, A.T., Egli, M., and Swayze, E.E. 2010a. An exocyclic methylene group acts as a bioisostere of the 2′‐oxygen atom in LNA. J. Am. Chem. Soc. 132:14942‐14950.
   Seth, P.P., Vasquez, G., Allerson, C.A., Berdeja, A., Gaus, H., Kinberger, G.A., Prakash, T.P., Migawa, M.T., Bhat, B., and Swayze, E.E. 2010b. Synthesis and biophysical evaluation of 2′,4′‐constrained 2′O‐methoxyethyl and 2′,4′‐constrained 2′O‐ethyl nucleic acid analogues. J. Org. Chem. 75:1569‐1581.
   Singh, S.K., Kumar, R., and Wengel, J. 1998. Synthesis of novel bicyclo[2.2.1] ribonucleosides: 2′‐Amino‐ and 2′‐thio‐LNA monomeric nucleosides. J. Org. Chem. 63:6078‐6079.
   Straarup, E.M., Fisker, N., Hedtjarn, M., Lindholm, M.W., Rosenbohm, C., Aarup, V., Hansen, H.F., Orum, H., Hansen, J.B., and Koch, T. 2010. Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non‐human primates. Nucleic Acids Res. 8:7100‐7111.
   Veedu, R.N. and Wengel, J. 2009. Locked nucleic acid as a novel class of therapeutic agents. RNA Biol. 6:321‐323.
   Xia, J., Abbas, S.A., Locke, R.D., Piskorz, C.F., Alderfer, J.L., and Matta, K.L. 2000. Use of 1,2‐dichloro 4,5‐dicyanoquinone (DDQ) for cleavage of the 2‐naphthylmethyl (NAP) group. Tetrahedron Lett. 41:169‐173.
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