Synthesis of Morpholino Monomers, Chlorophosphoramidate Monomers, and Solid‐Phase Synthesis of Short Morpholino Oligomers

Jhuma Bhadra1, Sankha Pattanayak1, Surajit Sinha2

1 These authors contributed equally, 2 Department of Organic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.65
DOI:  10.1002/0471142700.nc0465s62
Online Posting Date:  September, 2015
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Abstract

Phosphorodiamidate morpholino oligomers (PMOs) are a highly capable class of synthetic antisense oligonucleotides that are used to study gene functions in in vitro and in vivo models. This unit describes the synthesis of exocyclic‐amine‐protected 7′‐hydroxy and 7′‐chlorophosphoramidate‐activated morpholino monomers of A, T, G, and C, together with their incorporation into short PMO oligomers by solid‐phase synthesis. Starting from ribonucleosides, the exocyclic‐amine‐protected 7′‐hydroxy monomers are prepared following a modified Summerton protocol, which consists of a periodate cleavage/Schiff base formation/reduction cycle. The exocyclic amine protections are installed at a later stage (except G) to avoid the use of costly exocyclic‐amine‐protected counterparts that give control over protecting group manipulation. The 7′‐hydroxy monomers with N‐Trit/N‐MMTr are then converted to the 7′‐chlorophosphoramidate morpholino monomers in one step employing a combination of lithium bromide and DBU. These chlorophosphoramidate monomers are finally assembled by solid‐support synthesis to obtain the short PMO oligomers. © 2015 by John Wiley & Sons, Inc.

Keywords: phosphorodiamidate morpholino oligonucleotides (PMO); morpholinos; oligonucleotides; antisense compounds; chlorophosphoramidate; solid‐phase synthesis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Chlorophosphoramidate‐Activated Morpholino Thymidine Monomer
  • Alternate Protocol 1: Synthesis of Chlorophosphoramidate‐Activated Morpholinocytidine Monomer
  • Alternate Protocol 2: Synthesis of Chlorophosphoramidate‐Activated Morpholinoadenosine Monomer
  • Alternate Protocol 3: Synthesis of Chlorophosphoramidate‐Activated Morpholino Guanosine Monomer
  • Basic Protocol 2: Synthesis, Isolation, and Characterization of 10‐MER Phosphorodiamidate Morpholino Oligonucleotide
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Chlorophosphoramidate‐Activated Morpholino Thymidine Monomer

  Materials
  • 5‐Methyluridine (1, Sigma Aldrich)
  • Dimethylformamide (DMF), anhydrous
  • Argon gas
  • Imidazole
  • tert‐Butyldiphenylsilyl chloride (TBDPSCl) ( Spectrochem )
  • Methanol (MeOH)
  • Ethyl acetate (EtOAc)
  • Ultrapure water
  • Brine (sat. aq. NaCl)
  • Sodium sulfate (Na 2SO 4)
  • Diethyl ether (ether)
  • Dichloromethane (DCM)
  • Sodium metaperiodate (NaIO 4)
  • Ammonium biborate tetrahydrate ((NH 4) 2B 4O 7.4H 2O)
  • Ninhydrine charring solution (1.5% weight in 3% AcOH‐n‐BuOH)
  • Celite 545 (Merck)
  • Sodium cyanoborohydride (NaCNBH 3)
  • 4 Å molecular sieves
  • Acetic acid (AcOH)
  • Saturated aqueous sodium bicarbonate solution (sat. aq. NaHCO 3)
  • Triethylamine (Et 3N), anhydrous
  • Trityl chloride (TrCl) (Sigma Aldrich)
  • Petroleum ether (PE)
  • 4‐Monomethoxytrityl chloride (MMTrCl) (Sigma Aldrich)
  • Tetrahydrofuran (THF), anhydrous
  • 1 M tetrabutylammonium fluoride (TBAF) in THF (Sigma Aldrich)
  • Lithium bromide (LiBr)
  • 1,8‐Diazabicycloundec‐7‐ene (DBU), anhydrous
  • N,N‐Dimethyl phosphoramidic dichloride (Sigma Aldrich)
  • Acetone
  • Acetonitrile (ACN), anhydrous
  • 100‐, 250‐, 500‐, and 1000‐mL round‐bottom flasks
  • 1‐, 2‐, 5‐, 10‐, 20‐, and 50‐ mL syringes
  • Magnetic stir plate and stir bar
  • Rotary evaporator equipped with vacuum pump
  • 100‐, 250‐, 500‐, and 1000‐mL separatory funnels
  • Büchner funnel
  • Long needle
  • Additional reagents and equipment for TLC and column chromatography ( appendix 3D)

Alternate Protocol 1: Synthesis of Chlorophosphoramidate‐Activated Morpholinocytidine Monomer

  Additional Materials (see protocol 1)
  • Cytidine (Aldrich)
  • Benzoyl chloride (BzCl)

Alternate Protocol 2: Synthesis of Chlorophosphoramidate‐Activated Morpholinoadenosine Monomer

  Additional Materials (also see protocol 1)
  • Adenosine (Aldrich)
  • Benzoyl chloride (BzCl)

Alternate Protocol 3: Synthesis of Chlorophosphoramidate‐Activated Morpholino Guanosine Monomer

  Additional Materials (also see protocol 1)
  • Guanosine, anhydrous (Aldrich)
  • Hexamethyldisilazane (HMDS) (Sigma Aldrich)
  • Isobutyric anhydride, anhydrous
  • Pyridine, anhydrous
  • Hexane

Basic Protocol 2: Synthesis, Isolation, and Characterization of 10‐MER Phosphorodiamidate Morpholino Oligonucleotide

  Materials
  • Polystyrene resin (Novasyn TG amino 0.26 mmol/g)
  • N‐Methyl pyrolidine (NMP), anhydrous
  • 6‐Aminocaproic acid
  • Pyridine, anhydrous
  • Argon
  • 4‐Monomethoxytitryl chloride (MMTrCl)
  • Ethyl acetate (EtOAc)
  • Ultrapure water
  • Brine
  • Sodium sulfate (Na 2SO 4)
  • Silica gel (100 to 200 mesh)
  • Petroleum ether (PE)
  • Triethylamine (Et 3N)
  • Hydroxybenzotriazole (HOBT)
  • O‐(Benzotriazol‐1‐yl)‐N,N,N′,N′‐tetramethyluronium hexafluorophosphate (HBTU)
  • N‐Ethyl morpholine (NEM), anhydrous
  • Dichloromethane (DCM), anhydrous
  • N,N‐Diisopropylethylamine (DIPEA), anhydrous
  • Distilled acetic anhydride, (Ac 2O)
  • 3‐Cyanopyridine (Sigma Aldrich)
  • Trifluoroethanol (TFE, Sigma Aldrich)
  • Trifluoroacetic acid (TFA, Sisco Research Laboratories)
  • Succinic anhydride
  • Dimethylaminopyridine (DMAP)
  • Methanol (MeOH)
  • Dichloromethane (DCM)
  • 30% aqueous ammonia (NH 4OH)
  • Acetone
  • D 2O
  • Sinapinic acid matrix, prepare fresh
  • Solid‐phase synthesis vessel
  • Vibromix (203 EVT, Technica)
  • Vortex
  • 25‐, 50‐, 100‐, and 250‐mL round‐bottom flasks
  • Rotary evaporator equipped with a vacuum pump
  • 0.5‐, 1.5‐, and 2‐mL microcentrifuge tubes (Eppendorf)
  • Teflon
  • 40°C water bath
  • Lyophilizer, SpeedVac concentrator (Thermo Scientific)
  • 15‐mL centrifuge tubes (Falcon)
  • Centrifuge
  • UV spectrophotometer (CARRY‐BIO)
  • SPOLAR C18 column (4.6‐mm id × 250 mm)
  • MALDI‐TOF machine (Bruker)
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Figures

Videos

Literature Cited

Literature Cited
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