Carbocyclic C‐C Bond Formation: Intramolecular Radical Ring Closure to Yield Diastereomerically Pure (7′S‐Me‐ or 7′R‐Me‐) Carba‐LNA Nucleotide Analogs

Oleksandr Plashkevych1, Ram Shankar Upadhayaya2, Jyoti Chattopadhyaya1

1 Department of Cell and Molecular Biology, Uppsala University, Uppsala, 2 Bioimics AB, Uppsala
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.74
DOI:  10.1002/cpnc.29
Online Posting Date:  June, 2017
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Abstract

In light of the impressive gene‐silencing properties of carba‐LNA modified oligo DNA and RNA, both in antisense RNA and siRNA approaches, which have been confirmed as proof‐of‐concept for biochemical applications in post‐transcriptional gene silencing, we envision the true potential of carba‐LNA modifications to be revealed soon. Herein we provide detailed protocols for synthesis of carba‐LNA‐A, ‐G, ‐5‐MeC, and ‐T nucleosides on a medium/large scale (gram scale), as well as important guidelines for incorporation of these modified carba‐LNAs into DNA or RNA oligonucleotides. Creation of a stereoselective C‐C bond during the 5‐exo radical intramolecular cyclization involves trapping of a C2′ radical intermediate intramolecularly by the vicinal double bond of a C4′‐tethered ─CH2‐CH═CH2 group. All diastereomers of substituted carba‐LNAs are now available in pure form. The present procedure allows carba‐LNA to be commercialized for medicinal or biotechnological purposes. © 2017 by John Wiley & Sons, Inc.

Keywords: C‐C bond formation; carba‐LNA (carbocyclic Locked Nucleic Acid); conformationally constrained sugar modified nucleosides; nucleotides; oligonucleotides; siRNA; antisense approach

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Common Nucleoside Precursor Diacetate 8 as an α/β Anomeric Mixture
  • Basic Protocol 2: Nucleobase Coupling and Synthesis of Radical Cyclization Precursor 11(A‐D)
  • Basic Protocol 3: Radical Cyclization of Precursor 11 to Give Major (7′R‐ ME‐, 12(A‐D)) and Minor (7′S‐ME‐, 13(A‐D)) Isomers of Carba‐LNA
  • Support Protocol 1: Incorporation of Diastereomerically Pure (7′S‐Me or 7′R‐Me)‐Carba‐LNA‐A, ‐G, ‐meC, and ‐T Nucleosides Into Oligonucleotides
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of Common Nucleoside Precursor Diacetate 8 as an α/β Anomeric Mixture

  Materials
  • 3‐O‐benzyl‐4‐(hydroxymethyl)‐1,2‐O‐isopropylidene‐α‐D‐ribofuranose (1, CAS no. 63593‐03‐3), ≥99% pure (e.g., BOC Sciences, Santa Cruz Biotechnology, Carbosynth)
  • N,N‐dimethylformamide (DMF, CAS no. 68‐12‐2), anhydrous, ≥99.8% pure (e.g., Sigma Aldrich)
  • Sodium hydride (NaH, CAS no. 7646‐69‐7), 60% dispersion in mineral oil (e.g., Sigma Aldrich)
  • Benzyl bromide (BnBr, α‐bromotoluene; CAS no. 100‐39‐0), dry, ≥98%, (e.g., Sigma Aldrich)
  • Silica gel (Merck, 0.040 to 0.063 mm; CAS no. 112926‐00‐8; ≥230 mesh ASTM)
  • n‐Hexane (CAS no. 110–54–3), dry
  • Ethyl acetate (EtOAc, CAS no. 141‐78‐6), anhydrous, ≥99.8% pure (e.g., Sigma‐Aldrich)
  • Dichloromethane (DCM, CAS no. 75‐09‐2, dry, puriss. p.a., ACS reagent, reag. ISO), ≥99.9% by GC (e.g., Sigma Aldrich)
  • Dimethyl sulfoxide (DMSO, CAS no. 67‐68‐5), ≥99.9% (e.g., Sigma Aldrich)
  • Petroleum ether (CAS no. 101316‐46‐5), dry, puriss. p.a. ACS reagent, reag. ISO, low‐boiling‐point hydrogen‐treated naphtha, bp 40° to 60°C (≥90%; e.g., Sigma‐Aldrich)
  • Sodium sulfate (Na 2SO 4, CAS no. 7757‐82‐6), anhydrous, ≥99.0% pure (e.g., Sigma‐Aldrich)
  • Oxalyl chloride [ethanedioyl dichloride, (COCl) 2, CAS no. 79‐37‐8], ≥99% pure (e.g., Sigma Aldrich)
  • N,N‐Diisopropylethylamine (DIPEA or Hünig's base, CAS no. 7087‐68‐5) purified by redistillation, 99.5% (e.g., Sigma Aldrich)
  • Brine (saturated aqueous NaCl; CAS no. 7647‐14‐5), puriss. p.a., ≥99.5% (AT) (e.g., Sigma Aldrich)
  • Magnesium sulfate (MgSO 4, CAS no. 7487‐88‐9), ≥99.99% (e.g., Sigma Aldrich)
  • Methyl triphenylphosphonium bromide (CAS no. 1779‐49‐3), ≥98%, (e.g., Sigma Aldrich)
  • n‐Butyllithium (BuLi), 1.6 M in hexane solution (CAS no. 109‐72‐8; e.g., Sigma Aldrich)
  • Saturated aqueous solution of ammonium chloride (NH 4Cl; CAS no. 12125‐02‐9, e.g., for molecular biology, suitable for cell culture), ≥99.5% (Sigma Aldrich)
  • Diethyl ether (ether, ethyl ether , CAS no. 60‐29‐7), dry, puriss., meets analytical specification of Ph. Eur., BP, ≥99.5% (GC) (e.g., Sigma Aldrich)
  • Tetrahydrofuran (THF; CAS no. 109‐99‐9), anhydrous, ≥99.9%, inhibitor‐free (e.g., Sigma Aldrich)
  • Cyclohexane (CAS no. 110‐82‐7), dry, puriss., ≥99.5% (GC) (e.g., Sigma Aldrich)
  • 9‐Borabicyclo[3.3.1]nonane (9‐BBN), 0.5 M solution in THF (CAS no. 280‐64‐8; e.g., Sigma‐Aldrich)
  • Sodium hydroxide (NaOH; CAS no. 1310‐73‐2), pellets, ≥98% (e.g., Sigma‐Aldrich)
  • Hydrogen peroxide (H 2O 2) solution (CAS no. 7722‐84‐1), ≥30% (RT) solution in H 2O (e.g., Sigma‐Aldrich)
  • Acetic anhydride (CAS no. 108‐24‐7), ≥99.5% (e.g., Sigma Aldrich)
  • Acetic acid (CAS no. 64‐19‐7), ≥99.5% (e.g., Sigma Aldrich)
  • Trifluoromethanesulfonic acid (triflic acid, TfOH; CAS no. 1493‐13‐6, ≥99% (e.g., Sigma Aldrich)
  • Saturated solution of sodium bicarbonate (NaHCO 3; CAS no. 144‐55‐8, ≥98% (e.g., Sigma Aldrich)
  • Acetonitrile (CH 3CN; CAS no 75‐05‐8), dry, ≥99.5%, ACS reagent (e.g., Sigma Aldrich)
  • 1000‐ and 2000‐mL double‐ or triple‐neck round‐bottom flasks
  • Magnetic stir plate with stirring bar
  • Ice‐NaCl bath (1:1 mixture of ice and NaCl, temperature −10 to −5°C)
  • Dropping funnel
  • 1000‐mL separatory funnels
  • Chromatographic columns (30‐ to 50‐mm diameter)
  • Acetone–dry ice bath (technical grade or better acetone can be used, CAS no. 67‐64‐1, 99.5% pure; e.g., Bruce Scientific, Sigma Aldrich, etc.)
  • Rotary evaporator
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D; Meyers & Meyers, ) and column chromatography ( appendix 3E; Meyers, )
NOTE: All reagents obtained from commercial suppliers were used without further purification.NOTE: Solvents should be dried and degassed with N 2 when necessary.NOTE: Drying of any organic phase should be performed using Na 2SO 4 or MgSO 4 followed by filtration of the drying agent.

Basic Protocol 2: Nucleobase Coupling and Synthesis of Radical Cyclization Precursor 11(A‐D)

  Materials
  • Crude diacetate 8 ( protocol 1)
  • N6‐benzoyladenine (CAS no. 4005‐49‐6), ≥99.0% pure (e.g., Sigma Aldrich)
  • N,O‐bis‐(trimethylsilyl)acetamide (BSA; CAS no. 10416‐59‐8), ≥99.8% pure (e.g., Sigma Aldrich)
  • Trimethylsilyl trifluoromethanesulfonate (TMSOTf, trimethylsilyl triflate, trifluoromethanesulfonic acid trimethylsilylester; CAS no. 27607‐77‐8), ≥99.0% pure (e.g., Sigma Aldrich)
  • Saturated aqueous solution of ammonium chloride (NH 4Cl; CAS no. 12125‐02‐9, e.g., for molecular biology, suitable for cell culture), ≥99.5% (Sigma Aldrich)
  • Ethyl acetate (EtOAc; CAS no. 141‐78‐6), anhydrous, ≥99.8% pure (e.g., Sigma Aldrich)
  • Magnesium sulfate (MgSO 4; CAS no. 7487‐88‐9), ≥99.99% (e.g., Sigma Aldrich)
  • Silica gel (Merck, 0.040 to 0.063 mm; CAS no. 112926‐00‐8; ≥230 mesh ASTM)
  • n‐Hexane (hexane; CAS no. 110‐54‐3), dry, >99%, (e.g., Sigma Aldrich)
  • Toluene, (CAS no. 108‐88‐3), dry, >99.7% (e.g., Sigma Aldrich)
  • N2‐acetyl‐O6‐diphenylcarbomoylguanine (CAS no. 112233‐74‐6), ≥99.0% pure (e.g., Carbosynth)
  • 1,2‐dichloroethane (ethylene chloride, ethylene dichloride; CAS no. 107‐06‐2 ), dry, >99.8%, (e.g., Sigma Aldrich)
  • Sodium sulfate (Na 2SO 4, CAS no. 7757‐82‐6), anhydrous, ≥99.0% pure (e.g., Sigma‐Aldrich)
  • Acetic anhydride (CAS no. 108‐24‐7), ≥99.5% (e.g., Sigma Aldrich)
  • Acetic acid (CAS no. 64‐19‐7), ≥99.5% (e.g., Sigma Aldrich)
  • Trifluoromethanesulfonic acid (triflic acid, TfOH; CAS no. 1493‐13‐6, ≥99% (e.g., Sigma Aldrich)
  • Compound 7 (see protocol 1, step 44)
  • Acetonitrile (CH 3CN)
  • N4‐benzoyl‐5‐methylcytosine (CAS no. 126354‐30‐1), ≥99.0% pure (e.g., Leancare Ltd., U.K.; Boc Sciences, U.S.A.)
  • Thymine (2,4‐dihydroxy‐5‐methylpyrimidine, 5‐methyluracil; CAS no. 65‐71‐4), ≥99.0% pure (e.g., Sigma Aldrich)
  • Dichloromethane (DCM; CAS no. 75‐09‐2, dry, puriss. p.a., ACS reagent, reag. ISO), ≥99.9% (GC) (e.g., Sigma‐Aldrich)
  • Tetrahydrofuran (THF; CAS no. 109‐99‐9), anhydrous, ≥99.9%, inhibitor‐free (e.g., Sigma Aldrich)
  • Sodium hydroxide (NaOH; CAS no. 1310‐73‐2), pellets, ≥98% (e.g., Sigma‐Aldrich)
  • Acetone (CAS no. 67‐64‐1), dry, ≥99.8% (e.g., Sigma Aldrich)
  • Methanol (CAS no. 67‐56‐1), ≥99.98% (Sigma‐Aldrich)
  • Ammonia (CAS no. 7664‐41‐7), anhydrous, ≥99.98% (Sigma‐Aldrich)
  • Pyridine (CAS no. 110‐86‐1), anhydrous, 99.8% (e.g., Sigma Aldrich)
  • O‐Phenyl chlorothionoformate (Phenyl thionochloroformate, CAS no. 1005‐56‐7), 99% (e.g., Sigma Aldrich)
  • Saturated aqueous solution of sodium bicarbonate (NaHCO 3; CAS no. 144‐55‐8, ≥98% (e.g., Sigma Aldrich)
  • Saturated aqueous solution of citric acid (CAS no. 77‐92‐9), anhydrous, ≥99.5% (e.g., Sigma Aldrich)
  • 4‐Dimethylaminopyridine (DMAP; CAS no. 1122‐58‐3), ≥99.0% (e.g., Sigma‐Aldrich)
  • Nitrogen gas (N2; CAS no. 7727‐37‐9), >99.999% pure (e.g., Sigma‐Aldrich)
  • 1000‐mL double‐ and triple‐neck round‐bottom flasks
  • Rubber septa
  • Parafilm M (Sigma‐Aldrich, cat. no. P7793, P7543, P7668, P6543)
  • 50ºC water bath
  • Reflux condenser
  • 1000 mL separatory funnel
  • Chromatographic columns (30‐ to 50‐mm diameter)
  • Magnetic stir plate with stirring bar
  • Balloon
  • 18‐G or 20‐G hypodermic needle
  • Vacuum pump
  • Heating mantle or water bath
  • 250‐mL and 500‐mL single‐neck round‐bottom flasks
  • 250‐mL and 500‐mL double‐ or triple‐neck round‐bottom flasks
  • Dropping funnel
  • Rotary evaporator
  • Additional reagents and equipment for column chromatography ( appendix 3E; Meyers, )

Basic Protocol 3: Radical Cyclization of Precursor 11 to Give Major (7′R‐ ME‐, 12(A‐D)) and Minor (7′S‐ME‐, 13(A‐D)) Isomers of Carba‐LNA

  Materials
  • Radical reaction precursor 11 (a‐d) (synthesized as shown in protocol 2)
  • Nitrogen gas (N 2; CAS no. 7727‐37‐9), ≥99.999% pure (e.g., Sigma‐Aldrich)
  • Tributyltin hydride (Bu 3SnH; CAS no. 688‐73‐3), ≥97.0% (e.g., Sigma Aldrich)
  • 2,2′‐Azoisobutyronitrile (AIBN, α,α′‐azoisobutyronitrile, azobisisobutyronitrile; CAS no. 78‐67‐1), ≥98.0% (e.g., Sigma Aldrich)
  • Toluene (CAS no. 108‐88‐3, anhydrous), ≥99.9% pure (e.g., Sigma Aldrich)
  • Tetrachloromethane (CCl 4; CAS no. 56‐23‐5), ≥99.5% pure (e.g., Sigma Aldrich) Iodine (CAS no. 7553‐56‐2), ≥99.999% pure (e.g., Sigma Aldrich)
  • Ethyl acetate (EtOAc; CAS no. 141‐78‐6, anhydrous, ≥99.8% pure (e.g., Sigma Aldrich)
  • Saturated aqueous solution of potassium fluoride
  • Silica gel (Merck, 0.040 to 0.063 mm; CAS no. 112926‐00‐8; 230 mesh ASTM)
  • Methanol (MeOH, CAS no. 67‐56‐1), ≥99.98% (Sigma‐Aldrich)
  • Ammonia (CAS no. 7664‐41‐7), anhydrous, ≥99.98% (Sigma‐Aldrich)
  • Palladium hydroxide on carbon (Pd(OH) 2/C; CAS no. 12135‐22‐7), ≤50% water (e.g., Sigma Aldrich)
  • Ammonium formate (CAS no. 540‐69‐2), ≥99.0% (e.g., Sigma Aldrich)
  • Celite® 545 (SiO 2, CAS no. 68855‐54‐9), particle size 0.02 to 0.1 mm (e.g., Sigma Aldrich)
  • Pyridine (CAS no. 110‐86‐1), anhydrous, 99.8% (e.g., Sigma Aldrich)
  • Trimethylsilyl chloride (TMS‐Cl, chlorotrimethylsilane, TMCS, trimethylchlorosilane; CAS no. 75‐77‐4), ≥99.0% pure (e.g., Sigma Aldrich)
  • Benzoyl chloride (CAS no. 98‐88‐4), ≥99.9% (Sigma Aldrich)
  • Dichloromethane (DCM; CAS no. 75‐09‐2, dry, puriss. p.a., ACS reagent, reag. ISO), ≥99.9% (GC) (e.g., Sigma Aldrich)
  • n‐Hexane (CAS no. 110‐54‐3), dry
  • Sodium sulfate (Na 2SO 4, CAS no. 7757‐82‐6), anhydrous, ≥99.0% pure (e.g., Sigma‐Aldrich)
  • Formic acid (CAS no. 64‐18‐6), >96% (e.g., Sigma Aldrich)
  • Benzoic anhydride (benzaldehyde, bitter almond, CAS no. 100‐52‐7), purified by redistillation, ≥99.5% (e.g., Sigma Aldrich)
  • Sodium hydroxide (NaOH; CAS no. 1310‐73‐2), pellets, ≥98% (e.g., Sigma Aldrich)
  • Acetic acid (AcOH; CAS no. 64‐19‐7), ≥99.5% (e.g., Sigma Aldrich)
  • 250‐, 500‐ and 1000‐mL triple‐neck round‐bottom flasks, dry
  • Magnetic stir plate with stirring bar
  • Liebig or Vigreux condenser, dry
  • Dropping funnels
  • Heating mantle or oil bath
  • Vacuum pump
  • 60° and 55°C water baths
  • Chromatography columns (50 mm × 100 mm)
  • Reflux condenser
  • Additional reagents and equipment for column chromatography ( appendix 3E; Meyers, ), preparative HPLC (Upadhayaya et al., ), and thin‐layer chromatography (TLC; appendix 3D; Meyers & Meyers, )

Support Protocol 1: Incorporation of Diastereomerically Pure (7′S‐Me or 7′R‐Me)‐Carba‐LNA‐A, ‐G, ‐meC, and ‐T Nucleosides Into Oligonucleotides

  Materials
  • Synthesized nucleosides (Basic Protocols protocol 11 to protocol 33)
  • Hexane
  • Ethanol
  • 33% aqueous NH 3
  • CHIRALPAK‐IC (250 × 4.6) 5 micron ARD/K/7461 columns (Sigma‐Aldrich)
  • C‐18 reversed‐phase cartridge (SepPak, Waters, 0cat. no. 020515)
    • Additional reagents and equipment for preparative HPLC, preparation of phosphoramidites (Srivastava et al., ), oligonucleotide synthesis ( appendix 3C; Ellington & Pollard, ), denaturing polyacrylamide gel electrophoresis ( ; Albright & Slatko, ), and MALDI‐TOF mass spectrometry (unit 10.1; Castleberry, Chou, & Limbach, )
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Figures

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Literature Cited

Literature Cited
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