Time‐Dependent Thermocontrol of the Hydrophilic and Lipophilic Properties of DNA Oligonucleotide Prodrugs

Cristina Ausín1, Andrzej Grajkowski1, Jacek Cieślak1, Alexei Gapeev2, Serge L. Beaucage1

1 Food and Drug Administration, Bethesda, Maryland, 2 University of Maryland Baltimore County, Baltimore, Maryland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.42
DOI:  10.1002/0471142700.nc0442s43
Online Posting Date:  December, 2010
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Abstract

This unit describes the preparation of alkylthioalkylated and formamidoalkylated alcohols, an amidoalkylated alcohol, a hydroxylalkylated phosphoramidate, and their phosphoramidothioate derivatives, all of which have been identified as heat‐sensitive thiophosphate‐protecting groups in the development of thermolytic immunostimulatory DNA prodrugs. The alcohols are converted to their deoxyribonucleoside phosphoramidite derivatives, which are then used in the preparation of thermosensitive dinucleoside phosphorothioates. The thiophosphate‐protecting groups of these dinucleoside phosphorothioates presumably undergo thermolytic cyclodeesterification at elevated temperature under essentially neutral conditions to release the desired phosphorothioate diester function. On the basis of their thermolytic deprotection kinetics, one can identify those thiophosphate‐protecting groups that (i) may be useful for thiophosphate protection of CpG motifs of immunostimulatory DNA oligonucleotides (CpG ODNs); (ii) are suitable for protection of phosphodiester functions flanking the CpG motifs; and (iii) offer adequate protection of terminal phosphodiester functions against ubiquitous extracellular and intracellular exonucleases that may be found in biological environments. Curr. Protoc. Nucleic Acid Chem. 43:4.42.1‐4.42.31. © 2010 by John Wiley & Sons, Inc.

Keywords: oligonucleotide prodrugs; thiophosphate protecting groups; thermolytic deprotection; thermolytic conditions; solid‐phase oligonucleotide synthesis; CpG ODNs; lipophilicity; hydrophilicity

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis, Purification, and Characterization of Alkylthioalkylated Alcohols
  • Basic Protocol 2: Synthesis, Purification, and Characterization of Formamidoalkylated Alcohols
  • Basic Protocol 3: Synthesis, Purification, and Characterization of Hydroxyalkylated Phosphoramidate and Phosphoramidothioate Derivatives
  • Basic Protocol 4: Synthesis, Purification, and Characterization of Alkylthioalkylated, Formamidoalkylated, Phosphoramidated, and Phosphoramidothioated Deoxyribonucleoside Phosphoramidite Derivatives
  • Basic Protocol 5: Solid‐Phase Synthesis of Thermosensitive Dinucleoside Phosphorothioate Triesters and Their Conversion to Phosphorothioate Diesters Under Thermolytic Conditions
  • Basic Protocol 6: Automated Solid‐Phase Synthesis of a Heat‐Sensitive DNA Prodrug Model and Its Selective Thermolytic Deprotection
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis, Purification, and Characterization of Alkylthioalkylated Alcohols

  Materials
  • Sodium methyl mercaptide (Acros)
  • 6‐Chlorohexan‐1‐ol (Aldrich)
  • Chloroform (Fisher)
  • Anhydrous sodium sulfate (Fisher)
  • Methylene chloride (CH 2Cl 2; Fisher)
  • Silica gel (60 Å, 230 to 400 mesh; Merck)
  • Methanol (Fisher)
  • Phosphomolybdic acid (Aldrich)
  • Methyl 5‐chlorovalerate (Aldrich)
  • Diethyl ether and anhydrous diethyl ether (Et 2O; Fisher)
  • Dry argon gas cylinder (Matheson)
  • Lithium aluminum hydride (LiAlH 4; Aldrich)
  • Anhydrous tetrahydrofuran (THF; Acros)
  • Sulfuric acid (Aldrich)
  • Hexane (Fisher)
  • Ethyl acetate (EtOAc; American Bioanalytical)
  • 5‐Chloropentan‐1‐ol
  • Sodium ethoxide (Aldrich)
  • Ethanol and anhydrous ethanol (Aldrich)
  • 2‐Propanethiol (Aldrich)
  • 4‐Mercaptobutan‐1‐ol (Aldrich)
  • 1‐Trifluoromethyl‐1,3‐dihydro‐3,3‐dimethyl‐1,2‐benziodoxole (Kieltsch et al., )
  • Sodium hydroxide (NaOH; Aldrich)
  • Chloromethyl methyl ether (Aldrich)
  • 2‐(Methylthio)ethanol (Aldrich)
  • Anhydrous 1,2‐dimethoxyethane (Aldrich)
  • Sodium hydride, 60% dispersion in mineral oil (Aldrich)
  • Ethyl bromoacetate (Aldrich)
  • Potassium hydroxide (KOH; Fisher)
  • Dimethyl sulfoxide (DMSO; Acros)
  • Benzene (Aldrich)
  • Acetic anhydride (Acros)
  • 1,3‐Propanediol (Aldrich)
  • Amberlyst H+ (Sigma)
  • Hydrochloric acid (Fisher)
  • Anhydrous magnesium sulfate (Aldrich)
  • Ethylene glycol (Aldrich)
  • 1‐, 5‐, and 10‐mL plastic syringes (BD)
  • 10‐, 25‐, 100‐, 250‐, and 500‐mL round‐bottom flasks (Kontes)
  • Rubber septa for 14/20‐ and 24/40‐glass joints (Aldrich)
  • 100‐ and 250‐mL Erlenmeyer flasks (Kimax)
  • 50‐, 100‐, 250‐, and 500‐mL separatory funnels (Kontes)
  • 30‐, 60‐, and 100‐mm funnels (Nalgene)
  • Whatman no. 1 filter paper
  • Rotary evaporator (Büchi) connected to a vacuum pump (KNF)
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • 2.5 × 7.5–cm TLC plates precoated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • High‐vacuum oil pump (Edwards)
  • Reflux condensers equipped with calcium drying tubes (Kontes)
  • Dry ice/acetone bath
  • 30‐ and 60‐mL sintered glass funnels (coarse porosity, Kontes)
  • High‐vacuum distillation system (Kontes)
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 2: Synthesis, Purification, and Characterization of Formamidoalkylated Alcohols

  Materials
  • 2‐(Methylamino)ethan‐1‐ol (Aldrich)
  • Ethyl formate (Aldrich)
  • 3‐(Methylamino)propan‐1‐ol (Koepke et al., )
  • Ethanolamine (Aldrich), freshly distilled
  • Methylene chloride (CH 2Cl 2; Fisher)
  • Silica gel (60 Å, 230 to 400 mesh; Merck)
  • Methanol (Fisher)
  • Phosphomolybdic acid (Aldrich)
  • 25‐, 100‐, and 250‐mL round‐bottom flasks (Kontes)
  • Reflux condensers equipped with calcium drying tubes (Kontes)
  • Rotary evaporator (Büchi) connected to a vacuum pump (KNF)
  • High‐vacuum distillation system (Kontes)
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • 2.5 × 7.5–cm TLC plates precoated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 3: Synthesis, Purification, and Characterization of Hydroxyalkylated Phosphoramidate and Phosphoramidothioate Derivatives

  Materials
  • 2‐[(4,4′‐Dimethoxytrityl)oxy]ethan‐1‐ol ( S.16; Koizumi et al., ; Ferreira et al., )
  • 3‐[(4,4′‐Dimethoxytrityl)oxy]‐propan‐1‐ol ( S.17; Bannwarth et al., )
  • 4‐[(4,4′‐Dimethoxytrityl)oxy]butan‐1‐ol ( S.18; Greenberg et al., ; Chang et al., )
  • Dry N,N‐diisopropylethylamine (DIPEA; Aldrich)
  • Methylene chloride (CH 2Cl 2; Fisher) and anhydrous methylene chloride (Acros)
  • 2‐Cyanoethyl (N,N‐diisopropyl)phosphoramidochloridite (Aldrich)
  • 2‐Cyanoethyl (N,N‐diethyl)phosphoramidochloridite (Aldrich)
  • 2‐Cyanoethyl (N,N‐morpholinyl)phosphoramidochloridite (Aldrich)
  • Dry argon gas cylinder (Matheson)
  • 5.5 M tert‐Butyl hydroperoxide in decane (Aldrich)
  • Elemental sulfur (Aldrich)
  • Anhydrous sodium sulfate (Fisher)
  • Acetic acid (AcOH; Acros)
  • Chloroform (CHCl 3; Fisher)
  • Silica gel (60 Å, 230 to 400 mesh; Merck)
  • Methanol (Fisher)
  • Phosphomolybdic acid (Aldrich)
  • 100‐ and 250‐mL round‐bottom flasks (Kontes) with rubber septa (Aldrich)
  • Syringes (BD)
  • 100‐mL separatory funnels (Kontes)
  • 100‐mL Erlenmeyer flasks (Kimax)
  • 60‐mm funnels (Nalgene)
  • Whatman no. 1 filter paper
  • Rotary evaporator (Büchi) connected to a vacuum pump (KNF)
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • 2.5 × 7.5–cm TLC plates precoated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 4: Synthesis, Purification, and Characterization of Alkylthioalkylated, Formamidoalkylated, Phosphoramidated, and Phosphoramidothioated Deoxyribonucleoside Phosphoramidite Derivatives

  Materials
  • Desired alcohol(s) for derivatization ( S.1 S.15, S.19 S.25; see Basic Protocols protocol 11, protocol 22, and protocol 33)
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐bis(N,N‐diisopropylamino)phosphinyl‐2′‐ deoxythymidine ( S.26; unit 3.17)
  • Dry argon gas cylinder (Matheson)
  • Anhydrous acetonitrile (MeCN, Glen Research)
  • 1H‐Tetrazole (Glen Research), sublimed
  • Triethylamine (Et 3N, Aldrich)
  • Benzene (Aldrich) and anhydrous benzene (Acros)
  • Silica gel (60 Å, 230 to 400 mesh; EMD)
  • Hexane (Fisher)
  • 25‐mL round‐bottom flask (Kontes), flame dried
  • 100‐ and 250‐mL round‐bottom flasks (Kontes)
  • Rubber septa for 14/20‐ and 24/40‐glass joints (Aldrich)
  • 10‐mL plastic syringes (BD)
  • Rotary evaporator (Büchi) connected to a vacuum pump (KNF)
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • 2.5 × 7.5–cm TLC plates precoated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • Dry ice/acetone bath
  • High‐vacuum oil pump (Edwards)
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 5: Solid‐Phase Synthesis of Thermosensitive Dinucleoside Phosphorothioate Triesters and Their Conversion to Phosphorothioate Diesters Under Thermolytic Conditions

  Materials
  • Deblocking solution: 3% trichloroacetic acid (TCA) in dichloromethane (Glen Research)
  • Long‐chain alkylamine controlled‐pore glass (CPG) support loaded with 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxythymidine covalently bound through a 3′‐O‐hemisuccinate linker (Glen Research)
  • Acetonitrile (MeCN; Acros)
  • Dry argon gas cylinder (Matheson)
  • 0.45 M 1H‐tetrazole in MeCN (Glen Research)
  • 5′‐O‐DMTr‐2′‐deoxythymidine phosphoramidites ( S.27 S.48; see protocol 4)
  • 3H‐1,2‐Benzodithiol‐3‐one‐1,1‐dioxide (Glen Research)
  • Methylamine gas lecture bottle (Aldrich)
  • 2 M triethylammonium acetate (TEAA) buffer, pH 7.0 (Applied Biosystems)
  • Phosphate‐buffered saline (PBS, pH 7.4; Gibco)
  • 1‐, 3‐, and 10‐mL Luer‐tip syringes (B‐D)
  • Empty synthesis columns (Glen Research)
  • 20‐G hypodermic needles with Luer‐tip adapters
  • 250‐mL vacuum Erlenmeyer flask with rubber septa
  • Water aspirator or in‐house vacuum line
  • Tygon tubing (Fisher)
  • 4‐mL screw‐cap glass vials (Wheaton), one with rubber septum
  • 1‐mL gas‐tight glass syringe (Hamilton)
  • 250‐mL stainless steel pressure vessel (Parr Instruments)
  • 5‐µm Supelcosil LC‐18S HPLC column (25 cm × 4.6 mm, Supelco)
  • 37°C water bath
  • 90°C Heat block (VWR)

Basic Protocol 6: Automated Solid‐Phase Synthesis of a Heat‐Sensitive DNA Prodrug Model and Its Selective Thermolytic Deprotection

  Materials
  • Monomers and reagents for oligonucleotide synthesis:
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐(2‐cyanoethoxy)‐(N,N‐ diisopropylamino)phosphinyl‐2′‐deoxythymidine (Glen Research)
    • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐(2‐cyanoethoxy)‐(N,N‐ diisopropylamino)phosphinyl‐2′‐deoxyguanosine (Glen Research)
    • N4‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐(2‐methoxyethoxy)‐(N,N‐ diisopropylamino)phosphinyl‐2′‐deoxycytidine (prepared as described for S.33 in protocol 4; also see Ausín et al., )
    • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐[2‐(N‐formyl‐N‐methylamino)ethoxy]‐(N,N‐ diisopropylamino)phosphinyl‐2′‐deoxyguanosine (prepared as described for S.27 in protocol 4; also see Ausín et al., )
    • 0.45 M 1H‐tetrazole in MeCN (Glen Research)
    • 3H‐1,2‐Benzodithiol‐3‐one‐1,1‐dioxide (Glen Research)
    • Cap A solution: acetic anhydride in tetrahydrofuran/pyridine (Glen Research)
    • Cap B solution: 1‐methylimidazole in tetrahydrofuran (Glen Research)
    • Deblocking solution: 3% trichloroacetic acid in CH 2Cl 2 (Glen Research)
  • Acetonitrile (MeCN, Acros)
  • Anhydrous ammonia cylinder (Aldrich)
  • 2 M triethylammonium acetate (TEAA) buffer, pH 7.0 (Applied Biosystems)
  • Acetic acid (AcOH)
  • Phosphate‐buffered saline (PBS, pH 7.4, Gibco)
  • Loading buffer: 1:4 (v/v) 10× TBE, pH 8.3 ( appendix 2A) in formamide, containing 2 mg/mL bromphenol blue
  • Stains‐all (Aldrich)
  • Formamide (Fisher)
  • Automated DNA/RNA synthesizer (Applied Biosystems Model 392)
  • Synthesis columns with long‐chain alkylamine controlled‐pore glass (LCAA‐CPG, 500 Å) support loaded with 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxythymidine covalently bound through a 3′‐O‐hemisuccinate linker (Glen Research)
  • 250‐mL stainless steel pressure vessel (Parr Instruments)
  • High‐vacuum oil pump (Edwards)
  • Water aspirator or in‐house vacuum line
  • 1‐mL plastic syringes (BD)
  • 5‐µm Supelcosil LC‐18S reversed‐phase HPLC column (25 cm × 4.6 mm; Supelco)
  • 4‐mL screw‐capped glass vials (Wheaton), one with rubber septum
  • Heating block at 90°C (VWR)
  • 1.5‐mL microcentrifuge tubes
  • UV/Vis spectrophotometer equipped with a Peltier temperature control system (Hewlett‐Packard)
  • 1‐mL quartz cuvettes with Teflon caps
  • Additional reagents and equipment for automated DNA synthesis ( appendix 3C), RP‐HPLC (unit 10.5), and PAGE (unit 10.4 & appendix 3B)
NOTE: All ancillary reagents required for automated oligonucleotide synthesis were purchased from Glen Research and used as recommended by the manufacturer.
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Literature Cited

Literature Cited
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