Large‐Scale Preparation of Conjugated Oligonucleoside Phosphorothioates by the High‐Efficiency Liquid‐Phase (HELP) Method

Gian Maria Bonora1

1 University of Trieste, Trieste
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.27
DOI:  10.1002/0471142700.nc0427s22
Online Posting Date:  October, 2005
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Abstract

A new process for the preparation of large amounts of thioated oligonucleotides in a quasi‐classical solution condition is described. This method takes advantage of the use of polyethylene glycol as a soluble, inert support during synthesis. The easy purification of intermediates from a moderate excess of reagents allows very high coupling yields and, consequently, efficient production of the long oligonucleotide sequences required for pharmacological applications. The reaction of properly protected and activated oligonucleotides with high‐molecular‐weight polyethylene glycols allows a convenient procedure for the postsynthetic conjugation of those biopolymers in solution. The oligonucleotides are modified at the 5′ terminus using a liquid‐phase procedure with a linker carrying a terminal primary amino group to enhance its nucleophilic reactivity. The use of N, N′‐disuccinimidyl carbonate for the activation of the terminal OH groups of the PEG was preferred.

Keywords: liquid‐phase synthesis; polyethylene glycol; oligonucleotide; conjugated; phosphorothioate

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Phosphorothioated Oligonucleotides Via Help
  • Alternate Protocol 1: Preparation of Phosphorothioated Oligonucleotides Via Help Using Dimer Blocks
  • Basic Protocol 2: Preparation of 5′‐Aminoalkylated Phosphorothioate Oligonucleotides
  • Basic Protocol 3: 5′‐Conjugation of Phosphorothioated Oligonucleotides with High‐Molecular‐Weight Polyethylene Glycols: Activation with N,N′‐Disuccinimidyl Carbonate
  • Alternate Protocol 2: 5′‐Conjugation of Phosphorothioated Oligonucleotides with High‐Molecular‐Weight Polyethylene Glycols: Activation with p‐Nitrophenyl Chloroformate
  • Support Protocol 1: Purification and Analysis of Oligonucleotides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Phosphorothioated Oligonucleotides Via Help

  Materials
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐protected nucleoside (for attachment to support; Fig. ; Glen Research)
  • Pyridine, dried using 4‐Å  molecular sieves
  • 4‐Dimethylaminopyridine (DMAP)
  • Succinic anhydride, recrystallized from chloroform
  • Argon source
  • Chloroform
  • Ethanol (EtOH)
  • 60% perchloric acid
  • Toluene, anhydrous
  • Dichloromethane, anhydrous
  • 10% (w/v) citric acid, ice cold
  • Na 2SO 4, anhydrous
  • n‐Hexane
  • Diethyl ether, anhydrous
  • KOH pellets
  • Kieselgel 60, 70‐230 mesh (Merck)
  • Methanol (MeOH)
  • 1,2‐Dichloroethane (DCE), dried using 4‐Å  molecular sieves
  • N,N′‐Dicyclohexylcarbodiimide (DCC)
  • Polyethylene glycol monomethyl ether 10,000 (MPEG 10000; Nektar Therapeutics)
  • N‐Methylimidazole (NMI)
  • Acetone
  • Ethyl acetate
  • Iodine (I 2), crystalline
  • Methyl tert‐butyl ether (MTBE)
  • Acetonitrile (MeCN), dried using 4‐Å  molecular sieves
  • 2,6‐Lutidine
  • Acetic anhydride, distilled over anhydrous sodium acetate
  • 6% (w/v) trichloroacetic acid (TCA) in dry DCE (store at 4°C in a dark bottle)
  • 0.2 M base‐protected 5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐(2‐cyanoethyl‐N,N‐diisopropyl) nucleoside phosphoramidites (dABz‐CE, dCBz‐CE, dGiBu‐CE and dT‐CE; Glen Research) in MeCN (see recipe for phosphoramidites)
  • 0.5 M 1H‐tetrazole in MeCN (see recipe)
  • 0.5 M DDD in MeCN (see recipe)
  • Concentrated (∼30%) ammonium hydroxide
  • 80% (v/v) acetic acid
  • 250‐mL three‐neck round‐bottom flasks
  • Rubber septa
  • Calcium chloride drying tubes
  • Precoated silica gel 60 TLC sheets (5 × 10 cm) with fluorescent indicator (F 254; Merck)
  • UV lamp (UVP Mineralight lamp UVG‐11, 254 nm)
  • 25‐ and 250‐mL separatory funnels
  • Filter paper
  • Rotary evaporator with water aspirator as vacuum source
  • Sintered glass filters (Gooch G‐3 and G‐4)
  • Vacuum desiccator
  • 100 × 35–mm column for flash chromatography
  • 100‐mL and 250‐mL round‐bottom flasks
  • Spectrophotometer
  • Three‐way stopcock
  • High‐vacuum oil pump
  • Syringes and needles
  • Glass container with tight seal
  • 60°C water bath
  • Lyophilizer
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Alternate Protocol 1: Preparation of Phosphorothioated Oligonucleotides Via Help Using Dimer Blocks

  • 5′‐DMTr‐thymidylyl‐(3′→5′)‐thymidine (2‐cyanoethyl)thiophosphate (5′‐DMTr‐d(T PS(CE)T) dimer)
  • 0.2 M 5′‐DMTr‐d(GiBu PS(CE)GiBu)‐3′‐phosphoramidite in MeCN
  • Polyethylene glycol monomethyl ether 5000 (MPEG 5000; Nektar Therapeutics)
  • 250‐mL and 1‐L round‐bottom flasks
  • 1‐L three‐neck round‐bottom flasks

Basic Protocol 2: Preparation of 5′‐Aminoalkylated Phosphorothioate Oligonucleotides

  Materials
  • 5′‐DMTr‐oligonucleotide‐3′‐succ‐MPEG (see protocol 1)
  • 80% (v/v) acetic acid
  • Diethyl ether, anhydrous
  • Acetonitrile (MeCN), dried using 4‐Å  molecular sieves
  • 0.5 M 2‐[2‐(4‐monomethoxytrityl)aminoethoxy]ethyl‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite (Glen Research in MeCN)
  • 0.5 M 1H‐tetrazole in MeCN (see recipe)
  • Argon source
  • Methyl tert‐butyl ether (MTBE)
  • Ethanol (EtOH)
  • KOH pellets
  • TBHP solution: 80% tert‐butyl hydroperoxide in 3:2 (v/v) di‐tert‐butylperoxide/water (Fluka Sigma‐Aldrich)
  • Concentrated NH 4OH
  • Acetone
  • 0.03 M 2,4,6‐trinitrobenzenesulfonic acid (TNBS) in borate buffer, pH 9.3
  • Borate buffer, pH 9.3
  • 100‐mL round‐bottom flask
  • High‐vacuum oil pump
  • Lyophilizer
  • 100‐ml three‐neck round‐bottom flask and rubber septa
  • Syringes and needles
  • Sintered glass filters (Gooch G‐3 and G‐4)
  • Vacuum desiccator
  • Vessel with tight seal
  • 50°C oven
  • Refrigerated centrifuge
  • Spectrophotometer

Basic Protocol 3: 5′‐Conjugation of Phosphorothioated Oligonucleotides with High‐Molecular‐Weight Polyethylene Glycols: Activation with N,N′‐Disuccinimidyl Carbonate

  Materials
  • MPEG 5000 (Nektar Therapeutics)
  • Toluene, anhydrous
  • Pyridine, anhydrous
  • Dichloromethane (CH 2Cl 2), anhydrous
  • Acetonitrile (MeCN), anhydrous
  • N,N′‐Disuccinimidyl carbonate
  • Methyl tert‐butyl ether (MTBE)
  • Isopropanol, cold
  • Diethyl ether, anhydrous, cold
  • KOH pellets
  • Ethanol (EtOH)
  • Fully deprotected 5′‐aminoalkylated oligonucleotide (see protocol 3)
  • Na 2CO 3/NaHCO 3 buffer, pH 9
  • Chloroform (CHCl 3), anhydrous
  • Rotary evaporator
  • Vacuum desiccator
  • G‐3 and G‐4 sintered glass filters
  • High‐vacuum oil pump

Alternate Protocol 2: 5′‐Conjugation of Phosphorothioated Oligonucleotides with High‐Molecular‐Weight Polyethylene Glycols: Activation with p‐Nitrophenyl Chloroformate

  • p‐Nitrophenyl chloroformate
  • Triethylamine (TEA)
  • Ethanol (EtOH)

Support Protocol 1: Purification and Analysis of Oligonucleotides

  Materials
  • 0.1 and 3.0 M NaCl, pH 11.5 (see recipe)
  • Methanol
  • 0.05 M triethylammonium acetate (TEAA), pH 7.0 (see recipe)
  • 20:80 (v/v) TEAA/MeCN (see recipe)
  • 0.22‐µm sterile membrane filters
  • Sonicator
  • Centrifuge
  • Chromatography system and column(s):
    • Amersham Biosciences FPLC system with UV detector, MonoQ HR 5/5 ion‐exchange column, and PepRPC HR 5/5 reversed‐phase column
    • Gilson HPLC system with UV detector and 15 × 0.46–cm Progel‐TSK Oligo‐DNA‐RP column (Supelco)
    • Hewlett Packard series 1100 HPLC system with Lambda‐Max model 481 UV/vis detector and MEGA 2 integrator (Carlo Erba) and 15 × 0.46–cm Progel‐TSK Oligo‐DNA‐RP column (Supelco)
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Figures

Videos

Literature Cited

Literature Cited
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